Design, synthesis and cytotoxicity of cell death mechanism of rotundic acid derivatives

Article abstract of DOI:10.1016/j.bmcl.2013.03.005

In the present investigation, 16 new rotundic acid (RA) derivatives modified at the C-3, C-23 and C-28 positions were synthesized. The cytotoxicities of the derivatives were evaluated against HeLa, A375, HepG2, SPC-A1 and NCI-H446 human tumor cell lines b

Full text of DOI:10.1016/j.bmcl.2013.03.005

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2543–2547
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and cytotoxicity of cell death mechanism
of rotundic acid derivatives
d,f,
Yu-Fang He a,b, , Min-Lun Nan b, , Jia-Ming Sun e, Zhao-Jie Meng d, Wei Li c, , Ming Zhang
⇑
⇑
a School of Pharmaceutical Sciences, Jilin University, Changchun 130021, China
b Jilin Academy of Chinese Medicine Sciences, Changchun 130012, China
c College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun 130118, China
d Norman Bethune College of Medicine, Jilin University, Changchun 130021, China
e Development Center of Traditional Chinese Medicine and Bioengineering, Changchun University of Chinese Medicine, Changchun 130117, China
f Research and Development Center, Xiuzheng Pharmaceutical Group, Changchun 130012, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present investigation, 16 new rotundic acid (RA) derivatives modified at the C-3, C-23 and C-28
positions were synthesized. The cytotoxicities of the derivatives were evaluated against HeLa, A375,
HepG2, SPC-A1 and NCI-H446 human tumor cell lines by MTT assay. Among these derivatives, com-
pounds 4–7 exhibited stronger cell growth inhibitory than RA and compound 4 was found to be the best
Received 12 December 2012
Revised 22 February 2013
Accepted 1 March 2013
Available online 13 March 2013
inhibition activity on five human tumor cell lines with IC50 <10 lM. The apoptosis mechanism of com-
pound 4 in HeLa cells was investigated by western blot analysis. The results indicated that compound
4 could induce apoptosis through increasing protein expression of cleaved caspase-3 and Bax, and
decreasing protein expression of Bcl-2. In summary, the present work suggests that compound 4 might
serve as an effective chemotherapeutic candidate.
Keywords:
Rotundic acid
Derivative
Cytotoxicity
Apoptosis
Ó 2013 Elsevier Ltd. All rights reserved.
In the history of Traditional Chinese Medicine (TCM), medicinal
plants and their extracts were used to treat various diseases. Now-
adays, compounds derived from natural medicines with the unique
and diverse chemical entities still constituted a considerable re-
source for developing novel medicaments. Over the recent years,
a variety of biologically active constituents, including ginsenoside
Rg3,1–3 paclitaxel,4,5 platycodin D,6 triptolide,7 and flavone eupato-
rin,8 have been isolated from these sources and confirmed to have
anti-cancer activity in both experimental and epidemiologic inves-
tigations. The bark of Ilex rotunda Thumb., was early recorded in
‘Ling Nan Cai Yao Lu’, has been used for a long time as a TCM for
the treatment of colds, tonsillitis, pharyngitis, bone pain, acute gas-
troenteritis, and dysentery so on.9–11 Nowadays, this medical plant
is listed in Pharmacopoeia of the People’s Republic of China,
2010.12
duclitan,20 Nauclea officinalis.21 Although RA could be obtained
from the above plant resources, there were little reports on its bio-
activity. To date, in addition to the anti-tumor20,22 and lowering
blood pressure activity,23 no reports on other activities related to
RA were published. Our studies have proved that RA had the activ-
ity of prevention and treatment of cardiovascular disease and we
have applied two patents (one of the patent had been autho-
rized).24,25 More activities of RA need to be studied in the future.
In our previous study,26,27 the results showed that a few of new
amino acid derivatives showed stronger cytotoxicities than RA. In
order to find the compounds with better cytotoxicities, we contin-
ued to carry out chemical modification and antitumor activity of
RA. Since the nitrogen-containing organic compounds had high
biological activities, which play an important role on the chemical
research, so many researchers carried out research in this area.28 In
the work described herein, we focus on increasing the cytotoxicity
of RA. As shown in Figure 1, due to higher steric hindrance, 19-OH
is difficult to modified, Therefore, RA can be modified easily at C-3,
C-23 and C-28 position. In the present study, we intend to modify
RA to obtain better derivatives, their cytotoxicities are determined
on the five human tumor cell lines including A-375 (human malig-
nant melanoma cells), SPC-AI (human lung adenocarcinoma), Hela
(cervical cancer cells), HepG2 (hepatoma cells) and NCI-H446
(small cell lung cancer). The derivatives with low IC50 value will
be further evaluated to explore its mechanism on the traditional
apoptosis signal pathway.
Rotundic acid (3b,19a,23-trihydroxy-urs-12-en-28-oic acid,
RA), a pentacyclic triterpene acid, is the major component isolated
from the dry bark of I. rotunda. In addition to existence in
aquifoliaceae plants,13–16 RA also was isolated from Mussaenda
Pubescens,17 Guettarda platypoda,18 Olea europaea,19 Planchonella
⇑
Corresponding author. Tel.: +86 431 65680041; fax: +86 431 65680011.
E-mail addresses: liwei7727@126.com (W. Li), zhangming99@jlu.edu.cn (M.
Zhang).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.005

2544
Y.-F. He et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2543–2547
Recently, the antitumor mechanism mainly focused on their ef-
fect on trigger apoptosis. Apoptosis,29 known as programmed cell
death, is closely related to many anticancer reagents. It has been
broadly accepted that mitochondria play an important role during
drug-induced apoptosis in cancer cells. Many of the stimulus
including anticancer drug that triggered apoptosis on the mito-
chondria, which respond to proapoptotic signals by releasing cyto-
chrome C, which belongs to an effective catalyst of apoptosis.
Members of the Bcl-2 family include either proapoptotic (Bax) or
antiapoptotic (Bcl-2) function,could lead to mitochondrial death
signaling through cytochrome C release. Normally, Bcl-2 is local-
HO
19
28
OH
O
3
23 OH
HO
Figure 1. Structure of rotundic acid (RA).
HO
HO
HO
OH
O
a
OH
Cl
b
O
O
CH COO
CH COO
3
3
HO
OOCCH
2
OOCCH
3
3
OH
1
3
c
f
HO
HO
O
O
CH COO
3
OOCCH
CH COO
16-17 3
3
OOCCH
3
4-7
CH2
17:R =
2
16:R2=
4:R1=CH2CH2
5:R1=CH(CH3)CH2
g
CH
2
(NH)
HO
1
7:R1=
6
:R =CH CH CH
2
2
2
d
O
e
HO
HO
OH
HO
18-19
CH2
18:R2
2
O
19:R =
O
=
H CCOO
3
HO
OOCCH
3
OH
8-11
8:R1=CH2CH2
12-15
12:R1=CH2CH2
13:R1=CH(CH3)CH2
9:R1=CH(CH3)CH2
CH
CH
2
2
(NH)
(NH)
1
1
11:R1=
15:R1=
10:R =CH CH CH
14:R =CH CH CH
2
2
2
2
2
2
Scheme 1. Synthesis of RA derivatives. Reagents and conditions: (a) pyridine, acetic oxide, 80 °C, 16 h, 69.9%; (b) CH2Cl2, oxalyl chloride, rt, 20 h, 77.8–80.5%; (c) CH2Cl2,
amino alcohols, rt, 1.5 h, 66.5–77.5%; (d) 1 mol/l NaOH in 60% methanol, 100 °C, 8 h, 88.4–90.1%; (e) CH2Cl2, acetic oxide, 4-DMAP, rt, 4 h, 79.5–90.1%; (f) CH2Cl2, Et3N, Ar-R2-
NH2, rt, 1.5 h, 71.8–72.5%; (g)1 mol/l NaOH in 60% methanol, 100 °C, 6 h, 86.8–90.2%.

Y.-F. He et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2543–2547
2545
izes to the mitochondria and blocks the activation of proapoptotic
proteins, such as Bax, to the mitochondria. When the cancer under-
went chemotherapy, proapoptotic Bax can be upregulated in re-
sponse to sensing DNA damage, Bax in turn stimulates
mitochondria to release cytochrome C. Cytochrome C is the major
inducer of caspase activation downstream of mitochondria. Finally,
it will activate caspase-3 to be cleaved and execute apoptosis.
As reported in our previous investigation, RA was isolated and
purified from I. rotunda,26 its purity was P98% (HPLC assay) and
the extraction yield of RA was much higher up to 100 mg/g. In this
Letter, the synthetic routes of RA derivatives are outlined in
Scheme 1. RA (1) was first converted to its 3,23-O-diacetate (2),
which was then treated with oxalyl chloride to give the 28-acyl
chloride (3).30 This intermediate was then reacted with the appro-
priate amino alcohols (2-amino ethanol, 2-amino-1-propanol, 3-
amino-1-propanol, 2-amino-benzyl alcohol) in the presence of tri-
mor cell lines with IC50 values of 11.41, 23.24, 13.09, 6.85 and
7.11 M, respectively. Significant further improvement of the cell
l
growth inhibition on the five cell lines was achieved when the se-
lected amino alcohols was coupled to the C-28 to yield compounds
4–7. However, compounds 8–11, hydrolyzation product of corre-
sponding compounds 4–7, were found to exhibit lower anti-tumor
effect than compounds 4–7, and the anti-tumor activity of com-
pounds 8–11 were even weaker than it’s lead compound RA.
Though compounds 12–15 (esterification product of corresponding
compounds 4–7) showed higher anti-tumor activity than com-
pounds 8–11, they showed slightly lower anti-tumor activity com-
pared to compounds 4–7, the activity on A375 and Hela, even as
same as compounds 4–7. The results suggested that 3,23-O-diace-
tate group might be important to the inhibitory activity of tumor
cell growth and free 3,23-OH might be detrimental to the inhibi-
tory activity. In addition, from the above results we can speculate
the reason that the activity of compounds 4–7 higher than 12–15
might be that compounds 4–7 has free hydroxyl group at the C-
28 amide side chain. Because of the existence of free hydroxyl
groups, together with the 3,23-O-diacetate group, may change es-
ter water partition coefficient of compounds 4–7. As for com-
pounds 16–19, the anti-tumor activity was not satisfactory as
expected. Especially compounds 18–19, there was almost no inhib-
itory activity on the five tumor cell lines at all. However, the inhib-
itory activity of 16–17 was significantly higher than its hydrolysis
product compounds 18–19. The result also gave the evidence that
3,23-O-diacetate group should be the essential group to increase
the activity of RA.
ethylamine to give the compounds N-[3b,23-diacetoxy-19
a
-hydro-
xy-urs-12-en-28-oyl]-2-amino alcohols (compounds
4–7).
Hydrolyzation of compounds 4–7 gave the corresponding N-
[3b,19 ,23-thrihydroxy-urs-12-en-28-oyl]-2-amino alcohols
(compounds 8–11). Compounds N-[3b,23-diacetoxy-19 -hydro-
a
a
xy-urs-12-en-28-oyl]-2-amino alcohol acetates (compounds 12–
15) were obtained by re-acetylating compounds 4–7 in the pres-
ence of 4-dimethyl aminopyridine (4-DMAP), respectively. Com-
pound 3 was treated with phenylamine or benzylamine in the
presence of triethylamine to give the N-[3b,23-diacetoxy-19a-hy-
droxy-urs-12-en-28-oyl]-aromatic amine (compounds 16–17),
hydrolyzation of compounds 16–17 to gave the corresponding N-
[3b,19
a
,23-thrihydroxy-urs-12-en-28-oyl]-2-aromatic amine (18–
From the Table 1, we can also notice that the anti-tumor activity
of compound 4 is the highest among these compounds. The IC50 va-
lue of compound 4 was significantly less than RA treatment group
19). The structures of these synthesized compounds were con-
firmed by Infrared (IR), Mass spectra (HR-ESI-MS), 1H NMR and
13C NMR assay.31–34 The all 16 compounds obtained here were syn-
thesized and reported for the first time, the purity of all these 16
compounds were above 98% with higher yield (HPLC).
In the present study, five kinds of human tumor cell lines
including A-375, SPC-AI, Hela, HepG2 and NCI-H446 were the
model chosen to determine the cytotoxicity of RA (as a positive
control) and its derivatives 4–19. Antiproliferative effects were
determined with MTT assay.35 Each experiment was repeated at
least three times. The results are shown in Table 1.
(<10
lM) on the five cell lines with IC50 values of 5.49, 3.61, 2.83,
4.40, and 6.67
lM, respectively. These results indicated that com-
pound 4 can be the candidate for the development of new antican-
cer drug. To explore its elementary anticancer effect, the apoptosis
signal related protein level will be tested in the following
experiment.
As seen in above experiment, result about the cytotoxicity of
compound 4 was excited. The IC50 is much lower in compound 4
treatment for 24 h compared to RA group and other derivatives.
In this experiment, we treated Hela cell with compound 4 at 10,
As shown in Table 1, compound 2 (esterification of the 3-OH,
23-OH of RA) had stronger inhibitory activity against the five tu-
20 and 40
l
M for 24 h, Cisplatin (10
l
g/ml) was used as positive
Table 1
The IC50 values of RA and its derivatives on human cancer cell lines (
lM)
Compound
IC50
Hela
A375
SPC-A1
HepG2
NCI-H446
1
2
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
16.58 1.22
11.41 0.99
5.49 1.02*
4.77 3.00*
3.28 0.88*
3.71 0.75*
21.43 2.01
35.48 1.96
12.47 2.01
18.97 0.69
4.17 0.12*
5.10 0.89
10.06 1.61*
9.12 0.66*
17.74 1.03
13.71 1.00
70.79 0.85
>100a
83.95 3.28
23.24 1.26
3.61 1.59*
12.88 3.55
13.36 2.11
8.09 1.46*
>100a
31.92 2.01
13.09 0.88
2.83 1.01*
11.35 3.26
6.97 1.97*
3.32 0.48*
>100a
28.84 1.29
17.74 1.55
12.27 0.86
5.39 0.37*
5.30 0.36
4.31 0.91*
4.27 2.33*
15.78 2.01
19.18 2.23
23.17 0.76
28.05 1.12
7.33 0.68
6.85 0.63*
4.40 0.97*
7.30 0.89*
5.49 1.23*
4.45 1.03*
45.60 2.03
55.09 2.02
77.27 2.13
>100a
11.40 2.32
7.11 0.45
6.67 1.12*
5.51 0.78*
5.08 1.26*
7.03 0.92*
24.72 1.87
39.81 2.45
20.89 2.26
13.96 1.12
18.62 0.66
14.69 1.33
12.82 1.83
69.69 2.01
24.83 0.75
26.92 1.09
70.79 0.78
>100a
>100a
83.18 1.89
>100a
9.37 0.69*
18.85 1.77
11.96 0.93
13.21 1.55
16.21 1.08
12.10 2.06
>100a
4.44 0.48*
9.12 1.25
11.96 1.28
12.35 2.78
22.23 2.26
19.50 0.99
64.65 0.69
>100a
>100a
Notes: Data are represented in mean SD; n = 3.
a
IC50 values more than 100
lM are indicated as >100.
*
P <0.05 versus RA and IC50 value <10
lM.

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Y.-F. He et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2543–2547
Figure 2. Alteration of protein expression of Bax, Bcl-2, cytochrome C in Hela cells after 24 h compound 4 treatment. (A) Protein expression of Bcl-2; (B) protein expression of
Bax; (C) the ratio of Bax to Bcl-2; (D) protein expression of cytochrome C; (E) presented compound 4, C presented cisplatin, ⁄P <0.05 compared to control group, ⁄⁄P <0.01
compared to control group.
treatment group. The protein expression of Bax, Bcl-2, cytochrome
C and caspase-3 were detected by western blot. As shown in Fig-
ure 2, the antiapoptosis effect of compound 4 presented dose
dependent manners after 24 h treatment, Compound 4 could de-
crease the protein expression of Bcl-2, a antiapoptotic member of
Bcl family, and increase a proapoptotic protein Bax level, the ratio
of Bax to Bcl-2 was remarkable increased in a dose dependent
manner. The key protein of mithochondial related apoptosis signal,
cytochrome C in the cytoplasm also dramatically increased follow-
ing the compound 4 treatment. All these result indicated that com-
pound 4 might block the expression of Bcl-2, which could localize
to the mitochondria and block the activation of proapoptotic pro-
teins, such as Bax, to the mitochondria. The ratio of Bax to Bcl-2
has been used in many studies as the evaluation the level of apop-
tosis. The present study, compound 4 increased this ration by ele-
vated Bax expression and depressed Bcl-2 expression finaly trigger
the cytochrome C release from mitochondrial. It has been broadly
accepted that the alteration on Bcl-2, Bax was closely related to
Figure 3. Alteration of protein expression of caspase-3 and cleaved caspase-3 in Hela cells after 24 h compound 4 treatment. (A) Protein expression of caspase-3; (B) protein
expression of cleaved caspase-3, E presented compound 4; (C) presented cisplatin, ⁄P <0.05 compared to control grup, ⁄⁄P <0.01 compared to control group.

Y.-F. He et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2543–2547
2547
stimulation of apoptosis, the effector of their alteration presented
Supplementary data
as the cytochrome C releasing, which will trigger the cleave of
executor caspase, caspase-3. As shown in Figure 3, the protein
expression of caspase-3 was significantly elevated by compound
4 treatment, the cleaved protein expression of caspase-3 remark-
abley increased as well. It has been broadly reported that caspases
are widely expressed in an inactive proenzyme form in most cells
and once activated can often activate other procaspases. Caspase-3
among the caspase family is considered to be the most important
of the executioner caspases. Caspases-3 is cysteine proteases, using
the sulfur atom in cysteine to cleave polypeptide chains. And the
cleaved capase-3 is the active form when the apoptosis was trig-
gered. The cleaved caspase-3 will finally induce the DNA fragmen-
tation in cancer cell. Therefore, the elevation of cleaved caspase-3
is very important for the compound which affects cancer growth
by increasing apoptosis. Our result showed that compound 4 could
increase the protein level of cleaved caspase-3 in a dose dependent
manner. Taken together, compound 4 has an active pharmacologi-
cal effect by promoting apoptosis in Hela cells.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
03.005. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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we can conclude that 3,23-O-diacetate group might be important
to the inhibitory activity of tumor cell growth and free 3,23-OH
might be detrimental to the inhibitory activity. In view of the best
activity of compound 4, we further carried out the investigation on
the anticancer mechanism on Hela cells. The results indicated that
compound 4 could suppress the growth of Hela human cells, and
induce apoptosis by increasing the protein expression of cleaved
caspase-3and Bax, and decreasing the protein expression of Bcl-2.
Therefore, compound 4 may serve as a potential lead compounds
for new anticancer drug development.
In summary, our study provided a powerful incentive for fur-
ther research on the chemical modifications, and laid the founda-
tion for further study of RA derivatives and their structure-
activity relationships. Moreover, our study also provided theoreti-
cal basis on finding safe and effective anti cancer lead compounds
from the natural medicine.
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This work was supported by Jilin Science & Technology Devel-
opment Plan (SC201102129 and 20116045). We also thank Profes-
sor Quancheng Zhao at the Jilin Academy of Chinese Medicine
Sciences for the project design and suggestion.
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