Rational design and synthesis of topoisomerase i and II inhibitors based on oleanolic acid moiety for new anti-cancer drugs

Article abstract of DOI:10.1016/j.bmc.2013.11.034

Semisynthetic reactions were conducted on oleanolic acid, a common plant-derived oleanane-type triterpene. Ten rationally designed derivatives of oleanolic acid were synthesized based on docking studies and tested for their topoisomerase I and IIα inhibitory activity. Semisynthetic reactions targeted C-3, C-12, C-13, and C-17. Nine of the synthesized compounds were identified as new compounds. The structures of these compounds were confirmed by spectroscopic methods (1D, 2D NMR and MS). Five oleanolic acid analogues (S2, S3, S5, S7 and S9) showed higher activity than camptothecin (CPT) in the topoisomerase I DNA relaxation assay. Four oleanolic acid analogues (S2, S3, S5 and S6) showed higher activity than etoposide in a topoisomerase II assay. The results indicated that the C12-C13 double bond of the oleanolic acid skeleton is important for the inhibitory activity against both types of topoisomerases, while insertion of a longer chain at either position 3 or 17 increases the activity against topoisomerases by various degrees. Some of the synthesized compounds act as dual inhibitors for both topoisomerase I and IIα.

Full text of DOI:10.1016/j.bmc.2013.11.034

Bioorganic & Medicinal Chemistry 22 (2014) 211–220
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Rational design and synthesis of topoisomerase I and II inhibitors
based on oleanolic acid moiety for new anti-cancer drugs
Ahmed Ashour a,b, Saleh El-Sharkawy b,c, Mohamed Amer b, Fatma Abdel Bar b, Yoshinori Katakura d,
Tomofumi Miyamoto e, Nozomi Toyota a, Tran Hai Bang a, Ryuichiro Kondo a, Kuniyoshi Shimizu a,
⇑
a Department of Agro-Environmental Sciences, Faculty of Agriculture, Kyushu University, Fukuoka, Japan
b Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
c Department of Pharmacognosy, Faculty of Pharmacy, Delta University for Science and Technology, Egypt
d Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan
e Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Semisynthetic reactions were conducted on oleanolic acid, a common plant-derived oleanane-type triter-
pene. Ten rationally designed derivatives of oleanolic acid were synthesized based on docking studies and
tested for their topoisomerase I and IIa inhibitory activity. Semisynthetic reactions targeted C-3, C-12, C-
13, and C-17. Nine of the synthesized compounds were identified as new compounds. The structures of
these compounds were confirmed by spectroscopic methods (1D, 2D NMR and MS). Five oleanolic acid
analogues (S2, S3, S5, S7 and S9) showed higher activity than camptothecin (CPT) in the topoisomerase
I DNA relaxation assay. Four oleanolic acid analogues (S2, S3, S5 and S6) showed higher activity than eto-
poside in a topoisomerase II assay. The results indicated that the C12–C13 double bond of the oleanolic
acid skeleton is important for the inhibitory activity against both types of topoisomerases, while insertion
of a longer chain at either position 3 or 17 increases the activity against topoisomerases by various
Received 8 October 2013
Revised 15 November 2013
Accepted 16 November 2013
Available online 25 November 2013
Keywords:
Oleanolic acid
Topoisomerase I and II
Inhibitor
SARS
degrees. Some of the synthesized compounds act as dual inhibitors for both topoisomerase I and IIa.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Topoisomerases are universal and present in eukaryotes,
archaebacteria and eubacteria.4 They control DNA topology by
cleaving and rejoining DNA strands and play an important role in
regulation of the physiological function of genome.5 Beyond their
normal functions, topoisomerases are important targets, especially
in the treatment of human cancer.5 They are essential enzymes
that relax DNA supercoiling inside cells during several processes
like replication, recombination, and transcription. The opening of
duplex DNA and separation of its two strands during transcription
and replication generate supercoiling (torsional tension) on both
sides of the open DNA segment. Excessive positive supercoiling
tightens the DNA and prevents further strand separation, thereby
stalling the polymerase. Negative supercoiling behind the poly-
merases, on the other hand, tends to extend DNA strand separation
and facilitate the formation of abnormal nucleic acid structures.
Topoisomerases prevent the formation of such potentially deleteri-
ous structures by removing free supercoiling.1 There are two major
classes of topoisomerases, type I and type II, that are distinguished
by the number of DNA strands that they cleave and the mechanism
by which they alter the topological properties of the genetic
material.6
Inhibition of topoisomerases is one of the most important
mechanisms of anticancer drugs.1 Pentacyclic triterpenes such as
boswellic, betulinic, ursolic and oleanolic acids are reported to in-
hibit topoisomerases I and IIa by competing with DNA for topoiso-
merase binding through direct interaction with the enzyme
preventing topoisomerase-DNA complex formation in both topoi-
somerases. The structure–activity relationship suggests that the
general pentacyclic ring structure is an important system for topo-
isomerase inhibitory activity. Carboxylation of the pentacyclic ring
structure has also been suggested to be necessary for topoisomer-
ase inhibition.2 Our previous work3 resulted in isolation of olean-
olic (Fig. 1) and betulinic acid as pentacylic triterpene from
unused parts of Hibiscus sabdariffa. Based on a literature review,
betulinic acid was found to be tested against topoisomerase inhibi-
tion, while the current literature included no reports of oleanolic
acid derivatives as topoisomerase inhibitors. As such, oleanolic
acid was suggested as a backbone for rational design of specific
topoisomerase inhibitors.
This work was conducted to facilitate the design of new
analogues from oleanolic acid with enhanced activity against
⇑
Corresponding author. Tel.: +81 92 642 3002.
E-mail address: shimizu@agr.kyushu-u.ac.jp (K. Shimizu).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.034

212
A. Ashour et al. / Bioorg. Med. Chem. 22 (2014) 211–220
30
means that these compounds can act as dual inhibitors for topoiso-
29
19
merase I and II. This ability is very useful for cancer inhibition, as
specific topoisomerase I and II inhibitors are able to cause perma-
nent DNA damage.
20
17
21
22
12
26
18
13
14
27
Given that both enzymes are good targets, it would be desirable
to jointly inhibit them, but use-limiting toxicity of sequential or
simultaneous combinations of topoisomerase I and II poisons in-
clude severe to life-threatening neutropenia and anemia. Further-
more, the emergence of resistance phenomena to topoisomerase
I inhibitors is often accompanied by a concomitant rise in the level
of topoisomerase II expression and vice versa, leading to the failure
of clinical therapies.10 In this regard, a single compound able to in-
hibit both topoisomerase I and II may present the advantage of
improving antitopoisomerase activity, with reduced toxic side ef-
fects, with respect to the combination of the two inhibitors. These
compounds will therefore be synthesized and their activities
against topoisomerase I and II will be confirmed using an in vitro
assay.
11
25
10
28
16COOH
1
9
6
2
15
8
3
4
7
5
HO
23
24
Figure 1. Structure of oleanolic acid.
topoisomerase inhibition using computer-assisted molecular mod-
eling techniques.
2. Results and discussion
2.2. Semisynthesis of oleanolic analogues (S1–S10)
2.1. Molecular modeling and docking
Compounds S1–S10 were prepared (Schemes 1 and 2) after hav-
ing a preliminary idea about their activity using the molecular
modeling as mentioned before.
To design more active analogues of oleanolic acid as topoisomer-
ase inhibitors, a computer-aided molecular modeling study was car-
Reaction of m-chloroperoxybenzoic acid with oleanolic acid
afforded product S1. 1H and 13C NMR data of S1 suggested the dis-
appearance of the double bond of oleanolic acid. 13C NMR is the
same as oleanolic acid except that two signals of double bond
are replaced by two signals at dc 90.6 and 76.4. 1H NMR spectrum
of S1 showed one oxygenated proton at dH 3.89 (H-12) which is dif-
fer from oleanolic acid. This proton correlated with carbon signal at
dc 76.4 (C-12) in HMQC spectrum and showed HMBC correlations
with the carbon signals at dc 42.3 (C-14), 44.6 (C-9), 51.1 (C-18),
90.5 (C-13). These correlations confirmed the position of this pro-
ton. The FAB–MS of this compound (positive-ion mode) displayed
peak at m/z = 473 [M+H]+ while negative-ion mode displayed peak
at m/z at 471 [MꢀH]+ suggesting a molecular formula C30H48O4.
ried out within the active site of the human topoisomerase I and II
starting with the crystal structure of the enzyme bound to DNA (PDB
1t8i and 1bgw for topoisomerase I and II
, respectively).7,8 The active
a,
a
site of topoisomerase enzymes is an arginine- and lysine-rich pocket
with condensed positive charge that allows electrostatic interactions
with the negatively charged DNA-phosphate backbone.9 The C-28
COOH group of oleanolic acid represents the exclusive binding phar-
macophore, generating a hydrogen-bonding interaction with the
structural skeleton, but it is too short to bridge to the other side of
the enzyme (Fig. 2A), so our study depends mostly on elongation of
the chain at the substituents at positions 3 and 17 (Fig. 2B). Based
on this idea, several analogues of oleanolic acid were proposed
(Fig. 3). The tautomeric forms of these designed structures were cal-
culated using Marvin Suite (ChemAxon, Budapest, Hungary) at assay
Comparing the previous data (13C, 1H NMR, ½a D24
ꢁ
and mass) with
similar published data,11 compound S1 was determined to be
oleanderolide.
pH and then docked to topoisomerase I and IIaactive sites. The struc-
tures of the topoisomerase I and II inhibitors camptothecin (CPT) and
etoposide, respectively, were used as reference molecules in docking
and modeling studies (Fig. 4).
The proposed molecules were docked to topoisomerase I and II
active sites. The results are shown in Table 1. From these results, it
was found that the proposed structures have a good ability to inhi-
bit both topoisomerase I and II. It was also found that some struc-
tures have high binding affinity to both types of enzymes, which
Carbamoylation of oleanolic acid by reaction with different iso-
cyanates afforded compounds S2–S9. Down field shift of the H-3
and C-3 signals in S2 (+1.30 and +5.6 ppm, respectively) compared
to that of the starting material (oleanolic acid) suggested a possible
carbomylation at C-3. The NMR data (Table 2) further supported
this fact and were closely comparable to those of oleanolic acid
with additional C-3-O-[N-(benzyl) carbomyl] moiety signals.
(A)
(B)
Figure 2. Detailed view of docked structure oleanolic acid (A) and S2 (B) as one example of the proposed structures with the corresponding interacting amino acids of
topoisomerase I binding sites.

A. Ashour et al. / Bioorg. Med. Chem. 22 (2014) 211–220
213
O
C
6''
3''
HO
S1
7''
2''
O
5''
4''
NH
COOH
1''
7'
8'
O
HO
1'
O
6'
O
O
HN
HN
3'
2'
5' 4'
O
S2
S3
29
20
30
19
21
22
6''
3''
12
7''
2''
18
11
13
5''
4''
17
1
9
COOH
NH
16
COOH
14
2
8
7
10
1''
15
O
7'
5''
6'
6''
4
5
3
2'
5'
1'
O
HN
6
1'
O
HO
O
4''
3'
HN
1''
23
24
O
3'' 2''
4'
3'
2'
4'
S6
S4
S5
29
20
30
6''
7''
29
20
30
5''
3''
8''
9''
6''
5''
O
S
19
O
NH
21
22
19
10''
21
22
12
4''
18
17
11
13
12
1''
2''
18
17
4''
11
9
13
1''
NH
6'
7'
3'
O
1
9
7'
8'
2''
1
COOH
16
O
S
3'' 7'
6'
8'
O
2'
14
2
H
8
O
9'
10
14
2
1' O
O
5'
6'
5'
15
8
7
N
9'
10
15
4
10' HN
7
11'
5
3
4
1'
10'HN
O
11'
5
4'
3
6
1'
O
6
2'
23
24
5'
2'
23
24
3'
O
S9
4'
O
3'
4'
S8
S7
29
20
3
19
21
22
12
18
17
11
9
13
1
COOH
16
14
2
3
8
7
10
5'
6'
15
4
4'
5
O
6
1' S
O
3'
23
24
2'
O
S10
Figure 3. Oleanolic acid analogues designed for molecular modeling.
Protons and carbons assignments were further confirmed using
HMQC spectrum. The carbonyl carbon at dc 157.1 was assigned
to C-10 on the basis of its 2J-HMBC correlation with the broad sin-
glet H-20 (dH 4.50). Proton H-20 also showed 2J-HMBC with the qua-
ternary C-30 (dc 138.4) and 3J-HMBC correlation with the methine
carbon C-40 and C-80 (dc 127.4). The proton signal H-40/80 (dH
7.26) showed HMBC correlation with the methine carbon C-60 (dc
127.5) while H-50/70 signal (dH 7.32) showed a correlation with
the quaternary carbon C-30 (dc 138.4). The FAB–MS of this com-
pound (negative-ion mode) displayed peak at m/z at 588 [MꢀH]+
suggesting a molecular formula C38H55NO4. Thus compound S2
was proved to be 3-O-[N-(benzyl) carbamoyl]-oleanolic acid.
Down field shift of the H-3 and C-3 signals in S3 (+1.30 and
+5.6 ppm, respectively) compared to that of the starting material
(oleanolic acid) suggested a possible carbomylation at C-3. The
13C NMR data (Table 2) showed an upfield shift of the quaternary
carbon C-28 (ꢀ7.9 ppm) compared to that of oleanolic acid, sug-
gesting a possible reaction at this position. The NMR data further
supported this fact and were closely comparable to those of olean-
olic acid with additional C-3-O-[N-(benzyl)carbomyl] moiety and
17b-O-[N-(benzyl)carbomyl] moiety signals. Protons and carbons
assignments were further confirmed using HMQC spectrum. The
carbonyl carbon at dc 154.8 was assigned to C-10 on the basis of
and 3J-HMBC correlation with the methine carbon C-40 and C-80 (dc
128.3). The proton doublet H-40/80 (dH 7.34) showed HMBC correla-
tion with the methine carbon C-60 (dc 126.9) while H-50/70 (dH 7.22)
signal showed a correlation with the quaternary carbon C-30 (dc
138.4). The carbonyl carbon at dc 172.9 was assigned to C-28 on
2
the basis of its J-HMBC correlation with the broad singlet H-100
(dH 4.98). Proton H-100 also showed 2J-HMBC with the quaternary
C-200 (dc 138.7) and 3J-HMBC correlation with the methine carbon
C-300 and C-700 (dc 128.6). The proton signal H-300/700 (dH 7.33)
showed HMBC correlation with the methine carbon C-500 (dc
127.4). EI-MS spectrum showed molecular ion peak at m/z 678
[M]+. This suggests the empirical formula to be C45H62N2O3. Thus
compound S3 was proved to be 3-O-[N-(benzyl)carbamoyl]-17b-
O-[N-(benzyl)carbomyl]-oleanolic acid.
Chemical shift of the H-3 and C-3 signals in S4 is nearly the
same compared to that of the starting material (oleanolic acid)
suggested no carbomylation reaction occur at C-3 while the 13C
NMR data (Table 2) showed an upfield shift of the quaternary car-
bon C-28 (ꢀ7.9 ppm) compared to that of oleanolic acid, suggest-
ing a possible reaction at this position. The NMR data further
supported this fact and were closely comparable to those of olean-
olic acid with additional 17b-O-[N-(benzyl)carbomyl] moiety sig-
nals. Protons and carbons assignments were further confirmed
using HMQC spectrum. The carbonyl carbon at dc 172.9 was as-
signed to C-28 on the basis of its 2J-HMBC correlation with the
2
its J-HMBC correlation with the broad singlet H-20 (dH 4.52). Pro-
ton H-20 also showed 2J-HMBC with the quaternary C-30 (dc 138.4)

214
A. Ashour et al. / Bioorg. Med. Chem. 22 (2014) 211–220
Figure 4. Detailed view of docked structures of camptothecin and etoposide with topoisomerase I and II respectively (A) binding sites of CPT, (B) complex structure of CPT
with topoisomerase I, (C) binding sites of etoposide, (D) complex structure of etoposide with topoisomerase II.
broad singlet H-100 (dH 4.42). Proton H-100 also showed 2J-HMBC
Table 1
with the quaternary C-200 (dc 138.7) and 3J-HMBC correlation with
Comparison of energy scores for different compounds with topoisomerase I and II
the methine carbon C-300 and C-700 (dc 127.5). The proton signal H-
Topoisomerase I
Eintra Escore
15.2
Topoisomerase II
300/700 showed HMBC correlation with the methine carbon C-500 (dc
127.4). The proton signal H-400/600 showed HMBC correlation with
the methine carbon C-200 (dc 138.7). EI-MS spectrum showed
molecular ion peak at m/z 545 [M]+. This suggests the empirical
formula to be C37H55NO2. Thus compound S4 was proved to be
17b-O-[N-(benzyl)carbomyl]-oleanolic acid.
Einter
Einter
Eintra
Escore
Camptothecin
Etoposide
Oleanolic acid
ꢀ114.9
—
ꢀ99.7
—
—
—
—
—
ꢀ8.5
5.1
ꢀ5.3
ꢀ8.8
ꢀ7.3
1.7
2.7
0.8
7.2
ꢀ163.8
ꢀ116.9
ꢀ124.6
ꢀ156.7
5.5
ꢀ158.3
ꢀ125.9
ꢀ119.4
ꢀ173.7
ꢀ162.9
ꢀ136.2
ꢀ128.5
ꢀ176.1
ꢀ181.2
ꢀ206.4
ꢀ191.5
ꢀ156.4
ꢀ87.6
ꢀ96.1
ꢀ89.8
ꢀ130.3
ꢀ8.9
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
ꢀ95.09
ꢀ124.9
ꢀ139.1
ꢀ115.9
ꢀ129.6
ꢀ146.0
ꢀ163.9
ꢀ164.2
ꢀ103.5
ꢀ120.5
5.2
17.0
ꢀ1.8
ꢀ4.3
The 1H and 13C NMR data (Table 2) of S5 suggested close simi-
larity to oleanolic acid with an additional C-3-O-[(N-allyl)carb-
omyl] moiety. This suggestion was further confirmed by HMQC
and HMBC spectrum. The nitrogenated methylene carbon C-20 (dc
43.4) was assigned based on HMBC correlation with H-40a and H-
40b. The olefinic methine carbon C-30 (dc 134.8) showed HMBC cor-
relation with H-40 and H-20. Also the olefinic methylene carbon
showed HMBC correlation with H-20. All of these assignments con-
firmed the allyl side chain. The FAB–MS of this compound (nega-
tive-ion mode) displayed peak at m/z at 538 [MꢀH]+ suggesting a
molecular formula C34H53NO4. Thus compound S5 was proved to
be 3-O-[N-(allyl)carbamoyl]-oleanolic acid.
Down field shift of the H-3 of S6 compared to that of oleanolic
acid confirming biphenyl carbamoylation at C-3. Due to the steric
effect of the bulky biphenyl carbamoyl group12, the 13C NMR spec-
trum revealed line broadening of three carbon signals for C-3, C-10,
and C-30,70. Assignment of these carbons was based on extensive
analysis of HMBC and HMQC data. The biphenyl system was fur-
ther confirmed via HMBC correlation of the proton H-30, 70 (dH
ꢀ147.9 ꢀ161.1
ꢀ123.3 ꢀ131.9
ꢀ127.8 ꢀ133.8
ꢀ143.3 ꢀ173.4
ꢀ163.1 ꢀ184.1
5.31
ꢀ2.71
2.9
ꢀ157.0
ꢀ205.9
ꢀꢀ0.5
ꢀ23.3
ꢀ8.7
ꢀ30.7
ꢀ134.2 ꢀ168.2
ꢀ123.2 ꢀ147.7
ꢀ2.7
O
HO
S1
O
C
H
COOH
m-CPBA
H
3
HO
HO
H
OA
Scheme 1. Preparation of compound S1.

A. Ashour et al. / Bioorg. Med. Chem. 22 (2014) 211–220
215
30
29
20
17
21
22
19
12
26
18
15
13
14
11
25
28
16COOH
1
H
9
6
2
3
R2
10
8
27
R1-N=C=O
4
7
5
O
H
HO
23
O
24
R1
N
H
H
1`
OA
R2
R1
S2 a
S3 a
S5 c
COOH
b
N=C=O
COOH
S6
S7
d
e
COOH
f
S8 e
S9 g
COOH
h
NH
H
H
H
O
H
COOH
6`
HO
H
O
H
O
S
O
S10
S4
1`
H
3`
7`
O
7`
5``
5``
3``
6`
9`
2`
2`
4`
1``
O
5`
2`
3`CH2
28
NH
1``
5`
28
NH
5`
4`
3``
2`
4`e
f
c
a
b
d
O
7` O
2`
5``
3``
O
O
S
O
5`
g
h
28
S
N
H
1``
Scheme 2. Preparation of compound S2–S10.
7.46) with the quaternary carbon signal at dc 140.6 (C-100) and the
proton H-200,600 (d 7.57) with the quaternary carbon C-20 (dc 137.4).
The FAB-MS of this compound (negative-ion mode) displayed peak
at m/z at 650 [MꢀH]+ suggesting a molecular formula C43H57NO4.
Thus, the structure of S6 was determined to be 3-O-[N-(biphe-
nyl)-p-carbamoyl]-oleanolic acid.
carbamoyl moiety.12 The FAB–MS of this compound (positive-ion
mode) displayed peak at m/z = 751 [M+H]+ while negative-ion
mode displayed peak at m/z at 749 [MꢀH]+ suggesting a molecular
formula C51H62N2O3. Thus, the structure of S7 was proved to be 3-
O-[N-(1-naphthyl)-carbamoyl]-17-b-[N-(1-naphthyl)-carbamoyl]-
oleanolic acid.
The 1H NMR of S7 spectrum showed a downfield shift of H-3
(+1.44) compared to oleanolic acid, suggesting carbamoylation at
C-3. The 13C NMR data (Table 2) showed an upfield shift of the qua-
ternary carbon C-28 (ꢀ6.9) compared to that of oleanolic acid, sug-
gesting a possible reaction at this position. Careful assignment of
13C NMR spectrum revealed line broadening of several carbon sig-
nals. These carbon signals were identified through both HMQC and
HMBC spectra. In HMQC spectrum, the carbamoylation position (C-
3), showed a cross peak of H-3 (dH 4.56) and a broad carbon signal
at dc 83.0. Similarly, the proton signals at dH 7.88 showed cross
peaks in HMQC with broad carbon signals at dc 120.8 (C-60). Proton
H-40 and H-50 confirmed this assignment through their HMBC cor-
relations with C-50 and C-60, respectively. HMBC spectrum revealed
third and fourth very broad quaternary carbon signal at dc 134.0
(C-20), which was correlated with both H-40 and H-90 and broad
carbon signal at dc 132.8 (C-100), which was correlated with both
H-800 and H-300. The carbonyl carbon C-10 was assigned to be the
fifth broad signal at dc 157.0. The line broadening of these carbons
may be attributed to the steric effect of the bulky naphthyl
The 1H NMR spectrum of S8 showed a downfield shift of H-3
(+1.44) suggesting a possible carbamoylation at C-3. Careful
assignment of 13C NMR spectrum revealed line broadening of five
carbon signals. These carbon signals were identified through both
HMQC and HMBC spectra. In HMQC spectrum, the carbamoylation
position (C-3), showed a cross peak of H-3 (dH 4.56) with a broad
carbon signal at dc 83.5. Similarly, the proton signals at dH 7.64
and 7.90 showed cross peaks in HMQC with broad carbon signals
at dc 124.0 and 121.0 (C-50 and C-60). COSY spectrum was used
for confirmation of the assignment of the aromatic protons. Proton
H-40 and H-50 confirmed this assignment through their HMBC cor-
relations with C-50 and C-60, respectively. HMBC spectrum revealed
a fourth very broad quaternary carbon signal at dc 134.2 (C-20),
which was correlated with both H-40 and H-90. The carbonyl carbon
C-10 was assigned to be the fifth broad signal at dc 156.7. The line
broadening of these carbons may be attributed to the steric effect
of the bulky naphthyl carbamoyl moiety.12 The FAB–MS of this
compound (positive-ion mode) displayed peak at m/z = 626
[M+H]+ while negative-ion mode displayed peak at m/z at 624

216
A. Ashour et al. / Bioorg. Med. Chem. 22 (2014) 211–220
Table 2
NMR data (Table 2) showed an upfield shift of the quaternary car-
13C NMR (150 MHz) data of compounds S2–S10
bon C-28 compared to that of oleanolic acid, suggesting a possible
reaction at this position. The reaction of organic acids’ COOH
groups with sulfonyl isocyanates to produce N-acylsulfonylamides
was previously documented.13 The assignment of the protons and
carbons of the two aromatic moieties were determined based on
HMQC and HMBC data. Proton H-40,60 (dH 7.47) showed a correla-
tion with the quaternary carbon at dc 137.2 (C-20), while protons
H-30, 70 (dH 7.87) showed correlation with the aromatic carbon at
dc 140.6 (C-50). Proton H-300, 500 (dH 7.49) showed a correlation with
the aromatic carbon at dc 139.2 (C-100), while protons H-200, 600 (dH
7.96) showed a correlation with the aromatic carbon at dc 140.5
(C-400). EI-MS spectrum showed molecular ion peak at m/z 778
[M]+. This suggests the empirical formula to be C43H58N2O7S2. Thus
compound S9 was proved to be 3-O-[N-(phenylsulfonyl)-carbam-
oyl-17b-N-(phenylsulfonyl)amide]-oleanolic acid.
Reaction of benzene sulfonyl chloride with oleanolic acid
afforded compound S10. The 1H NMR showed five aromatic pro-
tons H-20,60 (dH 7.91), H-30,50 (dH 7.51), and H-40 (dH 7.61). The
13C NMR data (Table 2) showed three methine carbons at dc
127.6 (C-20,60), dc 129.0 (C-30,50) and 133.3 (C-40) in addition to
quaternary one at dc 137.9 (C-10), confirming the benzene sulfonyl
moiety. HMBC correlations of the methyl singlets (H-23 and H-
24) with the downfield oxymethine carbon signals at dc 91.2 (C-
3), confirming benzenesulfonation at C-3. Further confirmation
of assignment of protons and carbons was obtained using HMQC
and HMBC spectrum. COSY spectrum was used for confirmation
of the assignment of the aromatic protons. The FAB–MS of this
compound (negative-ion mode) displayed peak at m/z at 595
[MꢀH]+ suggesting a molecular formula C36H52O5S. Thus com-
pound S10 was proved to be 3-O-(benzenesulfonyloxy)-oleanolic
acid.
C
S2
37.7
S3
38.0
S4
37.9
S5
37.9
S6
37.9
S7
37.9
S8
38.0
S9
37.8
S10
38.2
1
2
3
4
5
6
7
8
9
27.4
84.6
38.0
55.2
18.1
32.6
39.4
47.5
36.8
23.4
27.4
84.6
38.2
55.3
18.1
32.6
39.4
47.4
36.8
23.4
27.5
81.5
38.2
55.4
18.2
33.0
39.4
47.5
36.9
23.5
27.7
81.4
38.0
55.3
18.2
33.1
39.3
47.5
36.9
23.4
27.7
83.4
38.1
55.4
18.2
33.1
39.3
47.6
37.0
23.4
27.4
83.0
38.2
55.3
18.1
32.9
39.5
47.9
36.8
23.6
27.7
83.5
38.1
55.4
18.2
32.5
39.3
47.5
37.0
23.4
27.6
85.1
37.8
55.2
18.1
32.4
39.2
47.4
36.9
22.8
27.6
91.2
38.5
55.5
18.3
32.5
39.2
47.5
36.8
23.3
10
11
12 122.8 122.8 123.0 122.5 122.6 123.1 122.5 122.4 122.4
13 143.2 143.2 143.2 143.6 143.6 144.9 143.6 143.6 143.6
14
15
16
17
18
19
20
21
22
23
24
25
26
27
41.7
27.8
23.6
46.7
41.2
45.7
30.6
33.6
33.0
27.8
16.8
17.1
15.2
25.8
41.7
27.8
23.6
47.5
41.2
45.7
30.7
33.6
33.0
28.0
16.8
17.2
15.3
25.8
41.8
28.0
23.6
47.5
41.3
45.7
30.7
33.6
32.6
28.0
16.7
17.1
15.3
25.8
41.5
28.0
23.6
46.5
40.9
45.8
30.7
33.8
32.4
28.0
17.1
16.7
15.4
25.9
41.6
23.9
22.9
46.5
41.0
45.8
30.7
33.8
32.4
28.1
17.1
16.8
15.4
25.9
43.0
23.8
23.5
47.5
42.3
46.9
30.7
34.2
32.4
28.1
17.0
16.7
15.4
25.9
41.6
23.9
22.9
46.5
41.0
45.9
30.6
33.8
32.4
28.1
16.7
17.1
15.4
25.9
41.5
23.6
22.6
46.5
40.9
45.8
30.6
33.7
31.6
27.9
16.6
17.1
15.4
25.9
41.6
24.8
22.9
46.5
41.0
45.8
30.6
33.8
32.4
27.9
17.1
16.3
15.3
25.9
28 179.9 172.9 172.9 184.1 183.4 176.6 183.6 171.4 182.9
29
30
31.4
22.9
31.4
23.0
31.4
23.9
32.5
23.9
32.5
23.6
33.0
23.7
33.0
23.6
33.0
23.3
33.0
23.5
1z 157.1 154.8
156.6 154.8 157.0 156.7 150.7 137.9
43.4 137.4 134.0 134.2 137.2 127.6
134.8 119.5 125.7 125.7 127.9 129.0
115.9 127.7 125.7 125.7 129.3 133.3
136.1 124.8 124.0 140.6 129.0
127.7 120.8 121.0 129.3 127.6
119.5 126.0 126.1 127.9
126.1 125.9
20
44.8 45.0
30 138.4 138.4
40 127.4 128.3
50 128.6 126.7
60 127.5 126.9
70 128.6 126.7
80 127.4 128.3
90
2.3. Topoisomerase I-DNA relaxation assay14
128.9 128.7
132.7 132.7
134.1 134.1
Inhibition of the topoisomerase function constitutes a useful
strategy for the identification of potential antitumor agents. Olean-
olic acid derivatives were tested using a topoisomerase I-DNA
relaxation assay and compared to the known natural topoisomer-
ase I inhibitor CPT. Reaction mixtures were analyzed by agarose
gel electrophoresis. Figure 5A and Table 3 show the catalytic inhi-
bition of topoisomerase I by oleanolic acid and its derivatives (S1–
S10), as shown in lane 5–14 at 100 lM in comparison to 100 lM
camptothecin (lane 4). Compound S4 (Fig. 4A, lane 8) showed topo-
isomerase I inhibitory activity comparable to CPT with ICCPT100
100
110
1
46.7
45.1
140.6 132.8
126.8 125.9
128.8 125.9
127.0 119.9
128.8 119.3
126.8 126.0
126.0
139.2
129.4
129.3
140.5
129.3
129.4
200
300
400
500
600
7’’
800
900
1000
138.7 138.7
128.6 127.5
127.5 128.6
127.4 127.4
127.5 128.6
128.6 127.5
128.7
124.8
126.5
100
lM (the concentration of the compound exhibiting activity
similar to that of 100
lM CPT is defined as ICCPT100). Compounds
S2, S3, S5, S7 and S9 (Fig. 4A, lane 6, 7, 9, 11 and 13) showed topo-
isomerase I inhibitory activities toward relaxation of supercoiled
DNA with activity higher than that of CPT itself with ICCPT100 values
[MꢀH]+ suggesting a molecular formula C41H55NO4. Thus, the
structure of S8 was proved to be 3-O-[N-(1-naphthyl)-carbam-
oyl]-oleanolic acid.
of 16.13, 25.4, 15.23, 17.4 and 23.1
lM, respectively, compared to
The down field shift of H-3 and C-3 signals in S9 versus those of
oleanolic acid (ICCPT100 265
lM). We used ICCPT100 in order to find a
oleanolic acid suggested a possible carbamoylation at C-3. The 13
C
15 14 13 12 11 10
9
8
7 6 5 4 3 2 1
16 15 14 13 12 11 10
9 8 7 6 5 4 3 2 1
Lanes
Relaxed DNA
Supercoiled DNA
A
B
Figure 5. (A) Catalytic inhibition of topoisomerase I by oleanolic acid derivatives. Lane 1, supercoiled DNA alone (250 ng); lane 2, same as lane 1 + 0.5% DMSO; lane 3, same as
lane 1 + topoisomerase I (2 U); lane 4, topoisomerase I (2 U) + 100 M camptothecin and DNA. Lane 5–14, topoisomerase I (2 U) + DNA and 100 M oleanolic acid analogues:
l
l
lane 5 (S1); lane 6 (S2); lane 7 (S3); lane 8 (S4); lane 9 (S5); lane 10 (S6); lane 11 (S7); lane 12 (S8); lane 13 (S9); lane 14 (S10); lane 15 (oleanolic acid); lane 16 (relaxed DNA).
(B) Catalytic inhibition of topoisomerase II by oleanolic acid derivatives. Lane 1, supercoiled DNA alone (250 ng); lane 2, same as lane 1 + 0.5% DMSO; lane 3, same as lane
1 + topoisomerase II (2 U); lane 4, topoisomerase I (2 U) + 100
lM etoposide and DNA. Lane 5-14, topoisomerase I (2 U) + DNA and 100 lM oleanolic acid analogues: lane 5
(S1); lane 6 (S2); lane 7 (S3); lane 8 (S4); lane 9 (S5); lane 10 (S6); lane 11 (S7); lane 12 (S8); lane 13 (S9); lane 14 (S10); lane 15 (oleanolic acid).

A. Ashour et al. / Bioorg. Med. Chem. 22 (2014) 211–220
217
Table 3
Kyushu University. TLC was performed on aluminum sheets pre-
coated with 0.2-mm silica gel 60 F254 (Merck). Plates were devel-
oped in a solvent mixture of n-hexane-ethyl acetate (9:1 and 8:2,
v/v), and the developed chromatograms were visualized under
254-nm UV light and the spots were made visible by spraying with
vanillin/H2SO4 reagent before warming in an oven preheated to
110 °C for 5 min. Silica gel 60 (Wako), 75–150 mesh, was used
for column chromatography.
Topoisomerase I and II inhibitory activity of oleanolic acid and its analogues (S1–S10)
Compound Topoisomerase Topoisomerase Topoisomerase Topoisomerase
I inhibitiona
(100 M)
I inhibition
II inhibitiona
M)
II inhibition
relative to
etoposideb
l
relative to CPTb (100
l
Oleanolic 6.5
acid
0.84
5.9
0.74
S1
S2
S3
S4
S5
S6
S7
S8
S9
6.0
0.77
1.64
1.42
0.98
1.57
0.79
1.55
0.40
1.36
0.54
1
7.7
8.9
9.6
8.5
10.0
9.6
8.5
8.2
7.2
7.3
ND
7.9
0.97
1.12
1.21
1.07
1.26
1.21
1.07
1.03
0.91
0.92
ND
12.7
11.0
7.6
12.1
6.1
12.0
3.1
10.5
4.2
3.2. Plant material
Oleanolic acid was isolated and purified from a methanolic ex-
tract of unused parts of Hibiscus sabdariffa grown in Egypt.3
3.3. Preparation of compound S115
S10
CPT
7.7
To a solution of oleanolic acid (90 mg, 0.2 mmol) in methylene
chloride (2.5 mL) was added 70% m-chloroperoxybenzoic acid (m-
CPBA) (59 mg, 0.24 mmol). The solution was stirred for 1 h at room
temperature and monitored by TLC [silica gel, petroleum ether–
EtOAc (90:10)]. The reaction was stopped by addition of 10% aque-
ous sodium sulfite (10 mL). The aqueous solution was extracted
twice with methylene chloride. The combined organic layers were
washed with saturated sodium bicarbonate, and then dried under
vacuum. The product was further purified by chromatography on
silica gel column previously packed in petroleum ether and isocrat-
ically eluted with 10% ethyl acetate in petroleum ether. The efflu-
ents, 10 mL fractions, were monitored by TLC on silica gel plates,
and similar fractions were pooled. Sub-fractions 22–24 afforded
compound S1 (13 mg, 13%) and its purity was confirmed by TLC
plate [Rf = 0.35, petroleum ether–EtOAc (75:25)].
Etoposide ND
ND
1
a
Topoisomerase inhibitory activity was analyzed by measuring the intensity of
supercoiled DNA using imageJ software, with the positive control drug CPT (topo-
isomerase I assay) or etoposide (topoisomerase II assay).
b
Values were standardized by dividing the value of intensity of each compound
by the value of CPT or etoposide in topoisomerase I and II, respectively.
method to standardize the activity of these compounds due to the
difficulty of determining the IC50 value.
2.4. Topoisomerase II-DNA relaxation assay14
Oleanolic acid derivatives were tested using a topoisomerase II
DNA relaxation assay alongside etoposide as a positive control.
Reaction mixtures were analyzed by agarose gel electrophoresis.
Figure 5B and Table 3 show the catalytic inhibition of topoisomer-
ase II by oleanolic acid derivatives (S1–S10), as shown in lanes 5–
3.4. Preparation of carbamates (compounds S2–S9)16
14 at 100 lM in comparison to 100 lM etoposide (lane 4).
Compounds S1, S4, S7, and S8 (Fig. 5B, lane 5, 8, 11 and 12)
showed topoisomerase II inhibitory activity comparable to that of
To a solution of oleanolic acid (100 mg) in toluene (3 mL), an
equivalent amount of benzyl isocyanate (29.0 mg) or allyl isocya-
nate (18.3 mg) or 4-biphenyl isocyanate (42.9 mg) or 1-naphthyl
isocyanate (37.2 mg) or benzene sulfonyl isocyanate (40.7 mg),
etoposide with ICetoposide 100
lM (the concentration of the com-
pound exhibiting activity similar to that of 100
lM etoposide is de-
were added and the solutions were separately mixed with 15 lL
fined as ICetoposide). Compounds S2, S3, S5 and S6 (Fig. 5B, lane 6, 7,
9 and 10) showed topoisomerase II inhibitory activities toward
relaxation of supercoiled DNA higher than that of etoposide itself
of triethylamine and refluxed for 1 h. The reactions were moni-
tored by TLC using petroleum ether–EtOAc (80:20). Water
(10 mL) was then added and each reaction product was extracted
with ethyl acetate (3 ꢂ 10 mL). The ethyl acetate extracts were
dried over anhydrous sodium sulfate and evaporated under vac-
uum at 40 °C.
with ICetopside of 68.03, 56.99, 52.98 and 46.52
lM, respectively,
compared to oleanolic acid (ICetoposide 275.8 M).
l
From these results, we have concluded that the double bond be-
tween C-12 and 13 is important for anticancer activity, as compound
S1 has lower activity against topoisomerase I and slightly higher
activity against topoisomerase II. In contrast, insertion of a nonpolar
moiety at C-3 or C-17 significantly increased the activity against both
topoisomerase I and II, with the exception of compounds S8 and S10,
which showed lower activity against topoisomerase I despite inser-
tion of a longer chain. We do not have a full explanation for the
behavior of these two compounds, as our study did not include con-
sideration of the kinetic mechanism and focused only on the devel-
opment of active inhibitory compounds against topoisomerase
enzymes. The mechanism by which these compounds inhibits topo-
isomerase may be examined in our future study.
3.5. Purification of compounds S2–S4
Upon monitoring the reaction using TLC in case of benzyl isocy-
anate, it was found that it gives three products more non polar
than oleanolic acid. The product was further purified by chroma-
tography on silica gel column previously packed in petroleum
ether and isocratically eluted with 10% ethyl acetate in petroleum
ether. The effluents, 10 mL fractions, were monitored by TLC on sil-
ica gel plates using solvent system petroleum ether–EtOAc (80:20),
and similar fractions were pooled. Sub-fractions 37–39 afforded
compound S2 [Rf = 0.62, 23 mg, 23%], Sub-fractions 45–48 afforded
compound S3 [Rf = 0.55, 25 mg, 25%], Sub-fractions 52–61 afforded
compound S4 [Rf = 0.50, 11 mg, 11%] and their purity was con-
firmed by TLC plate.
3. Materials and methods
3.1. General experimental procedure
3.6. Purification of compound S5
Optical rotations were determined on JASCO, DIP-730 digital
polarimeter. The 1H, 13C, HMBC and HMQC NMR spectra were ana-
lyzed on JEOL JNM ECA at 600 MHz for 1H and 150 MHz for 13C
NMR spectra. FAB–MS and EI-MS were conducted at Kasuga-shi,
TLC of the crude reaction mixture in case of allyl isocyanate re-
vealed the presence of one product more non polar than oleanolic
acid. The product was further purified by chromatography on silica

218
A. Ashour et al. / Bioorg. Med. Chem. 22 (2014) 211–220
gel column previously packed in petroleum ether and isocratically
eluted with 5% ethyl acetate in petroleum ether. The effluents,
10 mL fractions, were monitored by TLC on silica gel plates, and
similar fractions were pooled. Sub-fractions 28–31 afforded com-
pound S5 [Rf = 0.71, petroleum ether–ethyl acetate (80:20),
50 mg, 50%].
3.12. Molecular modeling and docking
Molegro Virtual Docker (MVD, Molegro ApS, Aarhus, Denmark)
is an integrated environment for studying and predicating how li-
gands interact with macromolecules. The identification of ligand
binding modes is done by iteratively evaluating a number of candi-
date solutions (ligand conformations) and estimating the energy of
their interaction with the macromolecule. MVD requires a three-
dimensional structure of both protein and ligand.
3.7. Purification of compound S6
TLC of the crude reaction mixture in case of 4-biphenyl isocya-
nate revealed the presence of one product more non polar than ole-
anolic acid. The product was further purified by chromatography
on silica gel column previously packed in petroleum ether and iso-
cratically eluted with 5% ethyl acetate in petroleum ether. The
effluents, 10 mL fractions, were monitored by TLC on silica gel
plates, and similar fractions were pooled. Sub-fractions 21–26
afforded compound S6 [Rf = 0.62, petroleum ether–ethyl acetate
(80:20), 10 mg, 10%].
The MolDock scoring function (MolDock Score) used by MDV is
derived from the piecewise linear potential (PLP) scoring functions
originally proposed by Gehlhaar et al., 18and later extended by
Yang and Chen.19 The MolDock scoring function further improves
these scoring functions with a new hydrogen bonding term and
new charge schemes. The docking scoring function, Escore, is de-
fined by the following energy terms;
Escore ¼ Einter þ Eintra
where: Einter is the ligand–protein interaction energy.
Eintra is the internal energy of ligand.
3.8. Purification of compounds S7 and S8
It is given by negative charge. The more negative the energy
score (kcal/mol), more is the binding affinity.
TLC of the crude reaction mixture in case of 1-naphthyl isocya-
nate revealed the presence of two products more non polar than
oleanolic acid. The product was further purified by chromatogra-
phy on silica gel column previously packed in petroleum ether
and isocratically eluted with 5% ethyl acetate in petroleum ether.
The effluents, 10 mL fractions, were monitored by TLC on silica
gel plates using solvent system petroleum ether–EtOAc (80:20),
and similar fractions were pooled. Sub-fractions 22–26 afforded
compound S7 [Rf = 0.67, 4 mg, 0.04%], Sub-fractions 29–40 afforded
compound S8 [Rf = 0.46, 23 mg, 23%].
The crystal structures of topoisomerase I and II were down-
loaded from RCSB Protein Data Bank (PDB 1t8i, for topoisomerase
I7) or (PDB 1bgw, for topoisomerase II8). The structure in PDB for-
mat was prepared using MVD. Using MVD 6.0, the water molecules
were removed and co-factor was incorporated into the protein
structure was automatically prepared. The structures of the topoi-
somerase I and II inhibitors camptothecin (CPT) and etoposide,
respectively, were used as reference molecules in docking and
modeling studies. The three-dimensional structures of compounds
(ligands) were first constructed using Chemsketch 12.0 software
[Cambridge Soft corporation, USA], then they were energetically
minimized by using Chimera 1.6.2 software with RMS gradient of
0.10 and then they were saved as mol2 format. Each of prepared
ligands was submitted separately to Id Target server, and Gastei-
ger–Hückel charges to calculate the atomic charges of uploaded
ligands.
3.9. Purification of compound S9
TLC of the crude reaction mixture in case of benzenesulfonyl
isocyanate revealed the presence of one product more polar than
oleanolic acid. The product was further purified by chromatogra-
phy on silica gel column previously packed in petroleum ether
and isocratically eluted with 10% ethyl acetate in petroleum ether.
The effluents, 10 mL fractions, were monitored by TLC on silica gel
plates, and similar fractions were pooled. Sub-fractions 73–90
afforded compound S9 [Rf = 0.38, petroleum ether–ethyl acetate
(80:20), 60 mg, 60%].
The binding potential between topoisomerase I, II and mole-
cules were evaluated by MVD. Scores are stated as binding free en-
ergy (Escore kcal/mol). The more negative the energy score (kcal/
mol), more is the binding affinity.
3.13. Inhibition of DNA relaxation by topoisomerase I enzyme
3.10. Preparation of compound S1017
Topoisomerase I drug screening kit (TopoGen, Inc., Port Orange,
FL) was used to determine the inhibitory activity of S1–S10 to
block or reduce topoisomerase I DNA relaxation activity. For this
assay, 250 ng of supercoiled plasmid DNA was added to the assay
buffer (10 mM Tris–HCl, pH 7.9, 1 mM EDTA, 150 mM NaCl, 0.1%
BSA, 0.1 mM spermidine, and 5% glycerol), followed by tested com-
To a solution of oleanolic acid (100 mg) in pyridine (3 mL), ben-
zenesulfonyl chloride (40 mg) was added, and the mixture was
stirred for 48 h at room temperature. The reaction was monitored
by TLC using petroleum ether–ethyl acetate (80:20). The reaction
was stopped by addition of water and extracted with EtOAc
(3 ꢂ 10 mL). The combined EtOAc extract was dried over anhy-
drous sodium sulfate and evaporated under vacuum.
pound or camptothecin at a final concentration of 100 lM. Equal
concentrations of DMSO were maintained in each reaction mixture
so as not to produce solvent mediated inhibition of topoisomerase I
activity. Topoisomerase I (2 U) was added to the assay mixture, and
reactions were carried out at 37 °C for 30 min, then terminated by
addition of 1% SDS (sodium dodecyl sulfate). The reaction mixtures
3.11. Purification of compound S10
TLC of the reaction mixture revealed the presence of one prod-
uct. The product was further purified by chromatography on silica
gel column previously packed in petroleum ether and isocratically
eluted with 5% ethyl acetate in petroleum ether. The effluents,
10 mL fractions, were monitored by TLC on silica gel plates, and
similar fractions were pooled. Sub-fractions 33–42 afforded com-
pound S10 [Rf = 0.43, petroleum ether–ethyl acetate (80:20),
12 mg, 12%].
were digested with proteinase K (50 lg/mL) for 15 min at 37 °C.
DNA was separated by electrophoresis in a 1% agarose gel in TAE
buffer (40 mM Tris–acetate, 1 mM EDTA) at 1 V/cm for 1–2 h at
room temperature. The agarose gels were stained with ethidium
bromide (0.5 lg/mL) and extensively destained in water, and the
DNA bands were visualized by transillumination with UV light
and quantified using imageJ software.

A. Ashour et al. / Bioorg. Med. Chem. 22 (2014) 211–220
219
3.14. Inhibition of decatenation of kDNA by topoisomerase II
enzyme
3.14.6. 3-O-[N-(Biphenyl)-p-carbamoyl]-oleanolic acid (S6)
White amorphous solid, ½a D24
ꢁ
+27.5 (0.08, CHCl3); 1H NMR (CD3-
Cl3) d: 4.52 (br s, H-3), 5.28 (br s, H-12), 0.96 (s, H-23), 0.76 (s, H-
24), 0.93 (s, H-25), 0.98 (s, H-26), 1.15 (s, H-27), 0.96 (s, H-29), 0.91
(s, H-30), 7.46 (m, H-30, 70), 7.53 (m, H-40, 60), 7.57 (m, H-200, 600),
7.41 (m, H-300, H-500), 7.31 (m, H-400); 13C NMR (Table 2).
Compounds S1–S10 were screened for the ability to inhibit the
decatenation of kDNA using a Topoisomerase II assay kit (TopoGen,
Inc., Port Orange, FL). Briefly, 100 M of each compound or etopo-
l
side was incubated with kinetoplast DNA (kDNA) and 2 U topoiso-
merase II enzyme for 30 min at 37 °C. The reaction mixtures were
3.14.7. 3-O-[N-(1-Naphthyl)-carbamoyl]-17-b-[N-(1-naphthyl)-
digested with proteinase K (50
l
g/mL) for 15 min at 37 °C. DNA
carbamoyl]-oleanolic acid (S7)
was separated by electrophoresis in a 1% agarose gel in TAE buffer
(40 mM Tris–acetate, 1 mM EDTA) at 1 V/cm for 1–2 h at room
temperature. The agarose gels were stained with ethidium bro-
mide (0.5 lg/mL) and extensively destained in water, and the
DNA bands were visualized by transillumination with UV light
Brown amorphous solid, ½a D24
ꢁ
+14.8 (0.027, CHCl3); 1H NMR
(CD3Cl3) d: 4.56 (br s, H-3), 5.32 (br s, H-12), 0.92 (s, H-23), 0.66
(s, H-24), 0.94 (s, H-25), 0.97 (s, H-26), 1.15 (s, H-27), 1.01 (s, H-
29), 0.81 (s, H-30), 7.45 (m, H-30, 40), 7.62 (m, H-50), 7.88 (m, H-
60, 90), 7.46 (m, H-70, 80), 7.47 (m, H-200, 300), 7.85 (m, H-400, H-800),
8.22 (m, H-500), 7.49 (m, H-600, H-700); 13C NMR (Table 2).
and quantified using imageJ software.
3.14.1. Oleanderolide (S1)
3.14.8. 3-O-[N-(1-Naphthyl)-carbamoyl]-oleanolic acid (S8)
Colorless amorphous solid, ½a D24
ꢁ
+74.5 (0.2, CHCl3); 1H and 13C
Brown amorphous solid, ½a D24
ꢁ
+48.4 (0.33, CHCl3); 1H NMR (CD3-
NMR were compared with the published literature data.11
Cl3) d: 4.56 (br s, H-3), 5.28 (br s, H-12), 0.93 (s, H-23), 0.76 (s, H-
24), 0.94 (s, H-25), 0.97 (s, H-26), 1.14 (s, H-27), 1.07 (s, H-29), 0.84
(s, H-30), 7.44 (m, H-30, 40), 7.64 (m, H-50), 7.90 (m, H-60),7.50 (m,
H-70, 80), 7.85 (m, H-90); 13C NMR (Table 2).
3.14.2. 3-O-[N-(Benzyl)carbamoyl]-oleanolic acid (S2)
Colorless amorphous solid, ½a D24
ꢁ
+12.5 (0.2, CHCl3); 1H NMR
(CD3Cl3) d: 4.42 (br s, H-3), 5.28 (br s, H-12), 0.91 (s, H-23), 0.70
(s, H-24), 0.85 (s, H-25), 0.71 (s, H-26), 1.11 (s, H-27), 0.90 (s, H-
29), 0.76 (s, H-30), 4.50 (br s, H-20), 7.26 (m, H-40, 80), 7.32 (m, H-
50, 70), 7.27 (m, H-60); 13C NMR (Table 2)
3.14.9. 3-O-[N-(Phenylsulfonyl)-carbamoyl-17b-N-
(phenylsulfonyl)amide]-oleanolic acid (S9)
White amorphous solid, ½a D24
ꢁ
+32.5 (0.2, CHCl3); 1H NMR (CD3-
Cl3) d: 4.56 (br s, H-3), 5.28 (br s, H-12), 0.91 (s, H-23), 0.71 (s,
H-24), 0.88 (s, H-25), 0.77 (s, H-26), 1.10 (s, H-27), 0.89 (s, H-29),
0.80 (s, H-30), 7.87 (d, J = 9.0, H-30, 70), 7.47 (m, H-40, 60), 7.49 (m,
H-50),7.96 (d, J = 9.0, H-200, 600), 7.49 (m, H-300, 500), 7.51 (m, H-400);
13C NMR (Table 2).
3.14.3. 3-O-[N-(Benzyl)carbamoyl]-17b-O-[N-
(benzyl)carbomyl]-oleanolic acid (S3)
Colorless amorphous solid, ½a D24
ꢁ
+48.6 (0.33, CHCl3); 1H NMR
(CD3Cl3) d: 4.42 (br s, H-3), 5.28 (br s, H-12), 0.92 (s, H-23), 0.70
(s, H-24), 0.86 (s, H-25), 0.71 (s, H-26), 1.14 (s, H-27), 0.90 (s, H-
29), 0.77 (s, H-30), 4.52 (br s, H-20), 7.34 (m, H-40, 80), 7.22 (m, H-
50, 70), 7.28 (m, H-60), 4.98 (br s, H-100), 7.33 (m, H-300, 700), 7.24
(m, H-400, 600), 7.25 (m, H-500); 13C NMR (Table 2).
3.14.10. 3-O-(Benzenesulfonyloxy)-oleanolic acid (S10)
White amorphous solid, ½a D24
ꢁ
+59.3 (0.133, CHCl3); 1H NMR
(CD3Cl3) d: 4.28 (br s, H-3), 5.25 (br s, H-12), 0.80 (s, H-23), 0.72
(s, H-24), 0.79 (s, H-25), 0.87 (s, H-26), 1.15 (s, H-27), 0.89 (s, H-
29), 0.90 (s, H-30), 7.91 (d, J = 7.2, H-20, 60), 7.51 (m, H-30, 50),
7.61 (m, H-40);13C NMR (Table 2).
3.14.4. 17b-O-[N-(Benzyl)carbomyl]-oleanolic acid (S4)
Colorless amorphous solid, ½a D24
ꢁ
+60.3 (0.33, CHCl3); 1H NMR
(CD3Cl3) d: 3.30 (br s, H-3), 5.30 (br s, H-12), 0.92 (s, H-23), 0.67
(s, H-24), 0.88 (s, H-25), 0.85 (s, H-26), 1.15 (s, H-27), 0.93 (s, H-
29), 0.79 (s, H-30), 4.42 (br s, H-100), 7.28 (m, H-300, 700), 7.32 (m,
H-400, 600), 7.29 (m, H-500);13C NMR (Table 2).
4. Conclusion
Ten rationally designed analogues of oleanolic acid were syn-
thesized on the basis of molecular modeling studies and tested
for their topoisomerase I and IIa inhibitory activity. Semisynthetic
reactions targeted C-3, C-28, C-12 and C-13 in oleanolic acid
(Fig. 6). It was found that compounds S2, S3, S5, S7 and S9 showed
greater topoisomerase I inhibitory activity than CPT. Compounds
S2, S3, S5 and S6 showed greater activity than etoposide against
topoisomerase II inhibition. Considering the previous results, we
3.14.5. 3-O-[N-(Allyl)carbamoyl]-oleanolic acid (S5)
White amorphous solid, ½a D24
ꢁ
+28.5 (0.133, CHCl3); 1H NMR
(CD3Cl3) d: 4.38 (br s, H-3), 5.26 (br s, H-12), 0.92 (s, H-23), 0.81
(s, H-24), 0.83 (s, H-25), 1.07 (s, H-26), 1.12 (s, H-27), 0.90 (s, H-
29), 0.74 (s, H-30), 3.80 (br s, H-20), 5.38 (br s, H-30), 5.12 (d,
J = 10.2, H-40a), 5.19 (dd, J = 16.8, 1.2, H-40b); 13C NMR (Table 2).
Double bond at this
position is essential for
antitopoisomerase activity
30
20
29
21
22
19
12
18
15
17
13
14
11
25
10
26
28
Replacement of substituent
with a lipophilic moiety while
keeping the carboxyl group
enhance the inhibitory activity
16COOH
1
9
6
2
8
27
3
4
7
5
HO
24
23
Replacement
substituent with a lipophilic
moiety enhances the
inhibitory activity
of
the
Figure 6. Summarized structure–activity relationships of oleanolic acid with regard to topoisomerase inhibition.

220
A. Ashour et al. / Bioorg. Med. Chem. 22 (2014) 211–220
7. Staker, B.; Feese, M.; Cushman, M.; Pommier, Y.; Zembower, D.; Stewart, L.;
Burgin, A. J. Med. Chem. 2005, 48, 2336.
have concluded that we have four compounds (S2, S3, S5 and S7)
that have high inhibitory activities towards both topoisomerase I
and II, which suggests that these compounds can act as dual inhib-
itors for both enzymes, thus leading to the prevention of cancer.
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Acknowledgment
The Egyptian Government is acknowledged for the fellowship
support to Ahmed Adel Ashour.
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