A-ring modified betulinic acid derivatives as potent cancer preventive agents

Article abstract of DOI:10.1016/j.bmcl.2013.12.041

Ten new 3,4-seco betulinic acid (BA) derivatives were designed and synthesized. Among them, compounds 7-15 exhibited enhanced chemopreventive ability in an in vitro short-term 12-O-tetradecanoylphorbol-13-acetate (TPA) induced Epstein-Barr virus early antigen (EBV-EA) activation assay in Raji cells. Specifically, analogs with a free C-28 carboxylic acid, including 7, 8, 11, and 13, inhibited EBV-EA activation significantly. The most potent compound 8 displayed 100% inhibition at 1 × 10³ mol ratio/TPA and 73.4%, 35.9%, and 8.4% inhibition at 5 × 10², 1 × 10 ², and 1 × 10 mol ratio/TPA, respectively, comparable with curcumin at high concentration and better than curcumin at low concentration. The potent chemopreventive activity of novel seco A-ring BAs (8 and 11) was further confirmed in an in vivo mouse skin carcinogenesis assay.

Full text of DOI:10.1016/j.bmcl.2013.12.041

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1005–1008
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A-ring modified betulinic acid derivatives as potent cancer
preventive agents
Hsin-Yi Hung a,e, Kyoko Nakagawa-Goto a,b, Harukuni Tokuda c, Akira Iida d, Nobutaka Suzuki c,
Ibrahim D. Bori a, Keduo Qian a, , Kuo-Hsiung Lee a,e,
⇑
⇑
a Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA
b Division of Pharmaceutical Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
c Department of Complementary and Alternative Medicine, Clinical R&D Graduate School of Medicine Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640, Japan
d Faculty of Agriculture, Kinki University, Nara 631-8505, Japan
e Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 401, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Ten new 3,4-seco betulinic acid (BA) derivatives were designed and synthesized. Among them, com-
pounds 7–15 exhibited enhanced chemopreventive ability in an in vitro short-term 12-O-tetradecanoyl-
phorbol-13-acetate (TPA) induced Epstein–Barr virus early antigen (EBV-EA) activation assay in Raji cells.
Specifically, analogs with a free C-28 carboxylic acid, including 7, 8, 11, and 13, inhibited EBV-EA activa-
tion significantly. The most potent compound 8 displayed 100% inhibition at 1 ꢀ 103 mol ratio/TPA and
73.4%, 35.9%, and 8.4% inhibition at 5 ꢀ 102, 1 ꢀ 102, and 1 ꢀ 10 mol ratio/TPA, respectively, comparable
with curcumin at high concentration and better than curcumin at low concentration. The potent chemo-
preventive activity of novel seco A-ring BAs (8 and 11) was further confirmed in an in vivo mouse skin
carcinogenesis assay.
Received 2 October 2013
Revised 6 December 2013
Accepted 10 December 2013
Available online 16 December 2013
Keywords:
Betulinic acid
seco A-ring
Chemopreventive
EBV-EA
Ó 2013 Elsevier Ltd. All rights reserved.
The concept of chemoprevention has grown over the past few
decades. Cancer is a multistage process, and the question of how
to stop the progression stage of its development has gradually
drawn increasing attention.7 The Epstein–Barr virus early antigen
(EBV-EA) activation assay was been established to quickly evaluate
chemopreventive activity in vitro,8 while a two-stage mouse skin
carcinogenesis assay can further assess in vivo activity.9,10 Devel-
opment of these biological assays has greatly facilitated research
on chemopreventive agents.
Betulinic acid (BA, Fig. 1) and its derivatives reportedly possess
various pharmacological functions, such as anti-cancer, anti-HIV,
anti-inflammatory, anti-malarial, and anti-bacterial effects. Prior
modifications of BA have focused mainly on the C-3 hydroxyl,
C-28 carboxylic acid, and C-30 allylic positions.1,2 Recently, we
introduced short fatty acids at the C-3 position of BA and the
resulting BA analogs demonstrated excellent cancer chemopreven-
tive activity in both EBV-EA activation and two-stage mouse
skin carcinogenesis assays.3 Akihisa et al. also reported that
compound 1 with a 3,4-seco lanostane structure exhibited inhibi-
tory effects against EBV-EA activation in Raji cells.4 In addition,
some limonoids [e.g. nomilin (2)] with an A-ring lactone showed
anti-proliferative effects on neuroblastoma cancer cells (SH-
SY5Y).5 The mechanism of action involved apoptosis induction,
cancer cell cycle arrest and aneuploidic effects.5 Furthermore,
other studies reported that 3,4-seco ursolic acid derivatives in-
duced cell cycle arrest and apoptosis in a human bladder cancer
cell line (NTUB1).6 Based on these discoveries, a group of novel
3,4-seco BA analogs 7–16 were designed to enhance the chemopre-
ventive activity. Herein, this Letter reports the design, synthesis
and biological evaluation of these novel compounds (see
Scheme 1).
3,4-seco BA analogs 7–16 were designed and synthesized
through oxepanone A-ring intermediates 5 and 6. A N-heptane
acetamide side chain was included in our analog design (9, 10
and 14–16), because this group enhanced the biological activity of
BA in our prior studies.11 Initially, betulin, a commercially available
pentacyclic triterpene, was oxidized with Jones reagent to provide
compound 3 with a ketone at C-3 and carboxylic acid at C-28. Bae-
yer–Villiger reaction of 3 using 3-chloroperbenzoic acid (mCPBA)
produced 5 with an oxepanone A-ring. Because prenyl-like groups
have played an important role in cancer preventive effects in our
prior studies,3 acid-catalytic lactone ring opening in MeOH was em-
ployed to produce the 4-methylene-3-methyl ester 7. Hydrolysis of
7 gave the corresponding dicarboxylic acid 8. Hydroboration–
oxidation of 7 produced the alcohol 11, which was esterified using
various acid anhydrides, such as 2,2-dimethylsuccinic and acetic
⇑
Corresponding authors. Address: Natural Products Research Laboratories,
Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC
27599-7568, USA. Tel.: +1 919 962 0066; fax: +1 919 966 3893 (K.-H. L.); tel.: +1
919 883 5306 (K.Q).
E-mail addresses: kdqian@unc.edu (K. Qian), khlee@unc.edu (K.-H. Lee).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.041

1006
H.-Y. Hung et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1005–1008
O
HOOC
H
OAc
H
O
COOH
HOOC
28
H
O
O
O
O
3
H
4
O
HO
H
2: Nomilin
1
Betulinic Acid (BA)
Figure 1. structures of betulinic acid, 3,4-seco lanostane compound and nomolin.
anhydrides, to provide 12 and acetate 13, respectively. For the prep-
aration of N-heptane acetamide analogs, the related carboxylic
acids 3 and 13 were treated with oxalyl chloride and heptane-1,7-
diamine followed by acetic anhydride to obtain 4 and 14, respec-
tively. Analogs 6, 9 and 10 were prepared from 4 through the same
procedure as for the preparation of 5, 7 and 8. The methyl ester and
acetate on 14 were hydrolyzed to produce 15, which was treated
with dimethylsuccinic anhydride to provide 16.
control. As seen in the results, 3,4-seco BA analogs showed sub-
stantial chemopreventive activity. Four compounds 7, 8, 11, and
13 significantly inhibited EBV-EA activation, showing 100% inhibi-
tion at the highest tested concentration. All four compounds con-
tained a C-28 carboxylic acid and showed better activity than
corresponding compounds with
a C-28-N-heptane acetamide
(compare 7/9, 8/10, 13/14). Three additional compounds with a
C-28-N-heptane acetamide (compare 6, 15, 16) were also less ac-
tive. The most active compound 8 also contained a C-3 carboxylic
acid and C-4 methylene in addition to the C-28 carboxylic group.
It displayed 100% inhibition at 1 ꢀ 103 mol ratio/TPA, and 73.4%,
Compounds 6–16 were evaluated in an in vitro EBV-EA inhibi-
tion assay and the results are shown in Table 1. Bevirimat
and BA were also evaluated and curcumin was used as a positive
H
H
H
O
H
a, b
O
d
R
H
H
O
OH
H
R
O
H
3
H
4
H
HO
O
H
H
betulin
3
4
5 R = OH
R = OH
R = NH(CH2)7NHCOCH3
c
6
R = NH(CH2)7NHCOCH3
H
H
O
O
O
f
O
e
H
R1
H
R2
R3
3
R1
R2
H
H
4
H
H
1
2
7
11 R1 = OH, R2 = OMe, R3 = OH
R = OH, R = OMe
g
8 R1 = OH, R2 = OH
O
h
O
9
12 R1 = OH, R2 = OMe, R3 =HO
1
2
R = NH(CH2)7NHCOCH3, R = OMe
O
g
10 R1 = NH(CH2)7NHCOCH3, R2 = OH
H
H
i
O
O
R1
O
g
O
H
H
R
MeO
HO
H
H
AcO
R2
H
H
15 R1 = NH(CH2)7NHCOCH3, R2 = OH
13
14
R = OH
R = NH(CH2)7NHCOCH3
O
h
c
1
2
HO
16
R = NH(CH2)7NHCOCH3, R =
O
O
Scheme 1. Synthesis of 3,4-seco betulinic acid derivatives. Reagents and conditions: (a) Jone’s oxidation, (b) H2,THF,Pd/C, HOAc, (c) (1) (CO)2Cl2, NH2(CH2)7NH2, CH2Cl2, (2)
Ac2O, DMAP, pyridine, r.t. (d) mCPBA, CH2Cl2, (e) H2SO4, MeOH, (f) (1) BH3SMe2,(2) NaOH, H2O2, (g) 4N NaOH/THF and MeOH, (h) 2,2-dimethylsuccinic anhydride, DMAP,
pyridine, 160 ºC, (i) Ac2O, DMAP, pyridine, r.t.

H.-Y. Hung et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1005–1008
1007
Table 1
Two of the most active compounds, 8 and 11, were further
evaluated in a mouse skin carcinogenesis assay (Fig. 1). Mouse skin
papillomas were induced by DMBA and promoted by TPA. Inhibi-
tory effects of 8 and 11 were monitored and determined over
20 weeks by both the percentages of papilloma-bearing mice
(Fig. 2A) and the average numbers of papillomas/mouse (Fig. 2B).
Although skin papillomas still appeared, their occurrence was
delayed for three weeks in both treated groups compared with
the control group. At weeks 8 and 11, 50% and 100% of mice in
the positive control group bore papillomas, while only 0–10% and
10–20% of mice in the 8- and 11-treated groups displayed papillo-
mas. The average numbers of papillomas/mouse at week 8 and 11
were 1.2 and 2.7 for the positive control group, but only 0 and 0.4,
respectively, for the 8-treated group. The in vivo results are quite
promising and consistent with the in vitro data.
The structure–activity relationship (SAR) trends are summa-
rized as follows. The 3,4-seco structural feature can significantly
increase chemopreventive activity. A C-3 carboxylic acid is better
than a methyl ester. A C-4 methylene is better than hydroxymethyl
or acetoxymethyl groups. A C-28 carboxylic acid is considerably
better than N-heptane acetamide. Among all new compound, 7,
8, 11, and 13 significantly inhibited TPA induced EBV-EA
activation.
EBV-EA inhibition abilitya of betulinic acid derivatives
Compd
Percentage EBV-EA positive cells
Compound concentration (mol ratio/TPAb)
d
1000
500
100
10
IC50
6
7
8
9
10
11
12
13
14
15
16
BA
15.3 0.4 (60)c
0.0 0.5 (70)
0.0 0.4 (70)
7.1 0.6 (60)
6.7 0.5 (60)
0.0 0.4 (70)
3.7 0.5 (70)
0.0 0.5 (70)
7.8 0.6 (60)
6.6 0.5 (60)
11.3 0.4 (60)
7.9 0.4 (60)
6.1 0.3 (60)
0.0 0.2 (60)
61.4 1.4
29.1 1.5
26.6 1.5
54.4 1.6
50.2 1.5
31.6 1.3
36.4 1.6
33.8 1.5
56.0 1.6
52.1 1.4
57.2 1.6
37.2 1.2
36.1 1.3
22.8 1.2
83.2 2.1
66.9 2.5
65.1 2.4
77.2 2.5
76.3 2.4
68.1 2.3
71.3 2.5
68.8 2.3
79.4 2.3
77.5 2.5
81.3 2.3
75.1 2.3
74.2 2.2
81.7 2.5
100 0.3
94.2 0.4
91.6 0.5
100 0.3
100 0.2
96.3 0.4
100 0.4
98.2 0.5
100 0.4
100 0.4
100 0.2
100 0.6
96.5 0.9
100 0.5
523
311
295
479
460
323
386
335
490
471
501
403
396
341
Bevirimat
Curcumine
a
b
c
For experimental details, see Ref. 13.
TPA concentration is 20 ng/mL (32 pmol/mL).
Relative ratio of EBV-EA activation with respect to the control without test
compound (100%). Values in parentheses are viability percentages of Raji cells.
d
The molar ratio of compound, relative to TPA, required to inhibit 50% of control
activated with 32 pmol TPA.
e
Positive control.
In this study, new BA derivatives were designed, synthesized
and evaluated for cancer chemopreventive activity. Most of the
newly synthesized compounds showed significant cancer preven-
tion effects. In an in vitro EBA-EA assay, 8 was the most potent
derivative with comparable inhibitory ability to curcumin, a
known chemopreventive agent, at high concentration and better
inhibitory ability at low concentration. Compounds 8 and 11 de-
layed occurrence of papillomas in an in vivo mouse skin carcino-
genesis assay. These results provided convincing evidence that
3,4-seco modification can greatly enhance the chemopreventive
activity of BA analogs.
35.9% and 8.4% inhibition at 5 ꢀ 102, 1 ꢀ 102, and 1 ꢀ 10 mol ratio/
TPA, respectively, with an IC50 value of 295 mol ratio/TPA. Even at
the lowest concentration (1 ꢀ 10 mol ratio/TPA), 8 exhibited great-
er inhibitory activity than curcumin, a known cancer chemopre-
ventive agent. This result may be because the C-5 isopropenyl
group is similar to a prenyl group, which has been an effective
modification in cancer prevention research.3 Considering the C-3
substituent, compound 8 with a C-3 carboxylic acid was slightly
more active than 7 with a C-3 methyl ester. Finally, regarding C-
4 substituents, analog 7 with a C-4 methylene was slightly more
potent than 11 and 13 with hydroxymethyl and acetoxymethyl,
respectively.
Acknowledgments
This work was supported in part by NIH Grant CA177584-01
from the National Cancer Institute and AI-33066-22 from the
National Institute of Allergy and Infectious Diseases awarded to
K.-H. Lee.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
12.041.
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Figure 2. Inhibitory effects of compounds 8 and 11 on DMBA-TPA mouse skin
carcinogenesis. Tumor formation in all mice was initiated with DMBA (390 nmol)
and promoted with TPA (1.7 nmol) twice weekly beginning 1 week after initiation.
(A) Papilloma percentage in mice. (B) Average number of papillomas/mouse. (Ç)
Control TPA alone; (4) TPA + compound 11 (85 nmol); (j) TPA + compound 8
(85 nmol). After 20 weeks of promotion, a significant difference in the number of
papillomas/mouse between the treated groups and the control group was evident
(p < 0.05).

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H.-Y. Hung et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1005–1008
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suspension. The EBV-EA inducing cells were stained by the means of an
indirect immunofluorescence technique. In each assay, at least 500 cells were
counted, and the number of stained cells (positive cells) was recorded.
Triplicate assays were performed for each compound. The average EBV-EA
induction of the test compound was expressed as a ratio relative to the control
experiment (100%), which was carried out with n-butyric acid (4 mM) plus TPA
(32 pM). EBV-EA induction was ordinarily around 35%. The viability of treated
Raji cells was assayed by the Trypan blue staining method. The cell viability of
the TPA positive control was greater than 80%. Therefore, only compounds that
induced less than 80% (% of control) of the EBV-active cells (those with a cell
viability of more than 60%) were considered able to inhibit the activation
caused by promoter substances. Student’s t-test was used for all statistical
analysis.
11. Huang, L.; Ho, P.; Lee, K. H.; Chen, C. H. Bioorg. Med. Chem. 2006, 14, 2279.
12. In vitro EBV-EA activation experiments: EBV-EA positive serum from a patient
with nasopharyngeal carcinoma (NPC) was a gift from Professor H. Hattori,
Department of Otorhinolaryngology, Kobe University. The EBV genome
carrying lymphoblastoid cells (Raji cells derived from Burkitt’s lymphoma)
were cultured in 10% fetal bovine serum (FBS) in RPMI-1640 medium (Sigma
R8758, USA). Spontaneous activation of EBV-EA in our subline of Raji cells was
less than 0.1%. The inhibition of EBV-EA activation was assayed using Raji cells
(virus non-producer type) as described below. The cells were incubated at
37 °C for 48 h in 1 mL of medium containing n-butyric acid (4 mM), TPA
[32 pM = 20 ng in 2
test compounds dissolved in 2
l
L dimethyl sulfoxide (DMSO)] and various amounts of the
L of DMSO. Smears were made from the cell
l