Synthesis, antiproliferative and apoptosis-inducing effects of novel asiatic acid derivatives containing α-aminophosphonates

Article abstract of DOI:10.1039/c6ra11397d

A series of novel asiatic acid (AA) derivatives containing α-aminophosphonate were designed and synthesized as antitumor agents. In vitro antitumor activities of these compounds against five cancer cell lines (A549, Hct-116, T24, Spca-2 and SK-OV-3 cell) and a normal cell line (HUVEC cell) were evaluated, employing standard MTT assay. Antitumor activities screening result indicated that many target compounds displayed moderate to high levels of antitumor activities compared with AA, 5-fluorouracil (5-FU) and cisplatin, and exhibited much lower cytotoxicity against normal cell than 5-FU and cisplatin. In addition, the mechanism of representative compound 3d was preliminarily investigated by AO/EB staining, Hoechst 33258 staining, JC-1 mitochondrial membrane potential staining, flow cytometry and western blot. Compound 3d inducing apoptosis involved intracellular Ca²⁺ production, the loss of mitochondrial membrane potential and intracellular reactive oxygen species (ROS) production. Western blot analysis also demonstrated that compound 3d treatment triggered the mitochondrial apoptotic pathway, indicating by changing Bax/Bcl-2 ratios, cytochrome c release, and caspase-9 activation. Moreover, the cell cycle analysis showed that compound 3d may confine T24 cells in G₁/S phase mainly through the p53-dependent pathway. Together, these results implied a critical role of ROS, caspase-9 and p53 in compound 3d-inducing G₁/S arrest and apoptosis of T24 cells.

Full text of DOI:10.1039/c6ra11397d

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Graphical abstract
Synthesis, antiproliferative and apoptosisꢀinducing effects of
novel asiatic acid derivatives containing αꢀaminophosphonates
RiꢀZhen Huang, CaiꢀYi Wang, JianꢀFei Li, GuiꢀYang Yao, YingꢀMing Pan, ManꢀYi Ye, HengꢀShan
Wang, Ye Zhang
Improved in vitro
Cytotoxicity Activity
O
H
H
OEt
COOH
AcO
AcO
HO
HO
HN
P O
Cell Cycle Arrest
H
H
OEt
R
H
H
Induction of Apotosis
HO
AcO
AA
3d
A series of novel asiatic acid derivatives containing αꢀaminophosphonates was designed and
synthesized as antitumor agents. Representative compound 3d blocked the T24 cell cycle at G1/S
phase by the p53ꢀdependent pathway and induced apoptosis through mitochondrial pathway.
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DOI: 10.1039/C6RA11397D
Synthesis, antiproliferative and apoptosisꢀinducing effects of novel
asiatic acid derivatives containing αꢀaminophosphonates
RiꢀZhen Huanga,†, CaiꢀYi Wangb,†, JianꢀFei Lia, GuiꢀYang Yaoc, YingꢀMing Pana, ManꢀYi Yea,
HengꢀShan Wanga, *, Ye Zhanga,d,
*
a State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources
(Ministry of Education of China), School of Chemistry and Pharmaceutical Sciences of Guangxi
Normal University, Guilin 541004, PR China
b College of Chemical and Material Science, Hebei Normal University, Shijiazhuang 050024,
China
c Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering,
Southeast University, Nanjing 211189, PR China
d Department of Chemistry & Pharmaceutical Science, Guilin Normal College, Xinyi Road 15,
Guangxi 541001, PR China
* Corresponding author. State Key Laboratory Cultivation Base for the Chemistry and Molecular
Engineering of Medicinal Resources, School of Chemistry & Pharmaceutical Science of Guangxi
Normal University, Yucai Road 15, Guilin 541004, Guangxi, PR China.
Eꢀmail addresses: whengshan@163.com (H.ꢀS. Wang); zhangye81@126.com (Y. Zhang)
† Coꢀfirst author: These authors contributed equally to this work.
2 / 30

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Abstract
A series of novel asiatic acid (AA) derivatives containing αꢀaminophosphonate were designed
and synthesized as antitumor agents. In vitro antitumor activities of these compounds against
five cancer cell lines (A549, Hctꢀ116, T24, Spcaꢀ2 and SKꢀOVꢀ3 cell) and a normal cell line
(HUVEC cell) were evaluated, employing standard MTT assay. Antitumor activities screening
result indicated that many target compounds displayed moderate to high levels of antitumor
activities compared with AA, 5ꢀfluorouracil (5ꢀFU) and cisplatin, and exhibited much lower
cytotoxicy against normal cell than 5ꢀFU and cisplatin. In addition, the mechanism of
representative compound 3d was preliminarily investigated by AO/EB staining, Hoechst 33258
staining, JCꢀ1 mitochondrial membrane potential staining, flow cytometry and western blot.
Compound 3d inducing apoptosis involved intracellular Ca2+ production, the loss of mitochondrial
membrane potential and intracellular reactive oxygen species (ROS) production. Western blot
analysis also demonstrated that compound 3d treatment triggered the mitochondrial apoptotic
pathway, indicating by changing Bax/Bclꢀ2 ratios, cytochrome c release, and caspaseꢀ9 activation.
Moreover, the cell cycle analysis showed that compound 3d may confine T24 cells in G1/S phase
mainly through the p53ꢀdependent pathway. Together, these results implied a critical role of ROS,
caspaseꢀ9 and p53 in compound 3dꢀinducing G1/S arrest and apoptosis of T24 cells.
Keywords: Asiatic acid; αꢀAminophosphonate; Cytotoxicity; Apoptosis; Cell cycle
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1. Introduction
Natural products are widely used to treat human disease and have contributed to drug
discovery for at least a century [1−3]. Terpenes, which are produced by some plants used in
traditional medicine, have shown anticancer activity against various cancer cell lines in vitro [4,5].
The pentacyclic triterpenes are a class of terpenes with known biological and pharmacological
properties, including antiꢀinflammatory, antimicrobial, antiꢀHIV, antioxidant, hepatoprotective,
and analgesic activity [6−12]. Several naturally occurring pentacyclic triterpenes and their
derivatives can induce apoptosis in a wide variety of cancer cells, such as hepatoma, lung
carcinoma, neuroblastoma and colorectal carcinoma [13−15]. However, the disadvantage of using
many of these triterpenoids include toxicity and large differences of the effective concentrations
for various cellular effects [16]. Because of their promising biological properties, modification of
pentacyclic triterpenes to obtain agents with improved antitumor activity is of interest to
bioorganic chemists.
Asiatic acid (AA, 2α,3α,23ꢀtrihydroxyursꢀ12ꢀeneꢀ28ꢀoic acid) is a pentacyclic triterpene acid
isolated from Centalla asiatica, a herb used for several thousand years in Chinese traditional
medicine [16]. It has been reported to have a wide range of pharmacological activities and to offer
potential chemoprotection against cancer [17−20]. AA can induce apoptosis in human hepatoma
cells by enhancing intracellular calcium release and upꢀregulation of p53 expression [21]. AA has
also been shown to induce apoptosis in SKꢀMELꢀ2 human melanoma cells on increase of
intracellular reactive oxygen species (ROS) and enhanced expression of Bax, but not Bclꢀ2,
protein [22]. Structure–activity relationship studies have shown that the modification of Cꢀ2, Cꢀ3,
Cꢀ23, and Cꢀ28 of AA can improve its biological activities [23,24]. Furthermore, we have reported
that addition of an aniline group to AA at Cꢀ28 enhanced its anticancer activity and induced
apoptosis in human hepatoma cells resulting from production of ROS and increased expression of
Bclꢀ2 and its downstream proteins [25]. These results support continuing investigation of the
antitumor potential of AA derivatives, but the numbers of compounds that have been synthesized
and evaluated is still limited. Consequently, considerable effort is being applied to the design and
synthesis of novel AA derivatives to screen as antitumor drugs.
The addition of αꢀaminophosphonates (APAs) to some pharmacal core increases antitumor
activity, and many APA derivatives have been shown to strongly inhibit the growth of human
tumours [26−31]. Pharmacological studies have revealed that aminophosphonate derivatives can
induce apoptosis through a mitochondriaꢀdependent pathway [32] without significant toxicity to
normal cells, and we have confirmed that the addition of APAs to some potential pharmacal cores
led to improved mitochondriaꢀdependent antitumor activity [26–28]. One would expect that
adding APA moieties to an AA skeleton to lead to improved antitumor activity and multiple
apoptotic pathways. However, to the best of our knowledge, the synthesis and antitumor activity
of AA derivatives including APA moieties at Cꢀ28 have not been reported. Therefore, in the
present work, we introduced APA functional groups at Cꢀ28 of the AA skeleton and evaluated the
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in vitro cytotoxicity of the resulting compounds. We also investigated the mechanism of apoptosis
induced by compound 3d.
2. Results and discussion
2.1. Chemistry
29
20
21
22
30
13
O
H
19
18
HO
HO
12
O
17
H
11
25 26
28
H
1
24
COOH
16
(b)
(a)
9
14
HO
HO
H
10
2
8
15
H
6
O
HO
3
5
27
CH2OH
O
O
7
OH
4
H
OH
23
OH
AA
HO
OH
O
OH
CH3
OH OH
O
OH
O
R
H
OEt
H
COOH
H
COOCl
AcO
AcO
AcO
AcO
(c)
P O
HN
(d)
H
H
H
OEt
AcO
AcO
AcO
AcO
H
H
H
1
3aꢀ3r
2
AcO
(e)
O
H
O
O
O
H
H
OEt
H
COOCl
AcO
AcO
H
COOH
AcO
AcO
(f)
P O
HN
(g)
H
H
H
OEt
R
AcO
AcO
AcO
AcO
H
6i, 6n
AcO
5
4
Scheme 1. General synthetic route for compound 3−6. Reagents and conditions: (a) (i) NaOH,
MeOH, reflux; (ii) 2 mol/L HCl, MeOH (b) Ac2O, pyridine; (c) COCl2, CH2Cl2; (d) amine, apas,
CH2Cl2; (e) K2CrO7, AcOH; (f) COCl2, CH2Cl2; (g) amine, apas, CH2Cl2.
AA derivatives bearing APA structures (compounds 3 and 6) were synthesized as shown in
Scheme 1. The asiaticoside was isolated from the leaf of Centella asiatica (L.) Urba as previously
1
described [13], characterized by H nuclear magnetic resonance (NMR) and mass spectroscopy
and treated with NaOH in MeOH to yield the AA sodium salt. The AA sodium salt solution was
acidified in an iceꢀwater bath to give AA. AA was acetylated to yield
2α,3β,23ꢀtriacetoxyursꢀ12ꢀeneꢀ28ꢀoic acid (1). AA chloride was obtained by the condensation of
compound 1 with oxalyl chloride in the presence of triethylamine, and this intermediate was then
reacted with a variety of substituted αꢀaminophosphonates [28] to produce the desired compounds
3a−3r. 2α,3β,23ꢀtriacetoxyursꢀ11ꢀoxoꢀ12ꢀeneꢀ28ꢀoic (4) was synthesized by the treatment of 1 in
the presence of glacial acetic acid as previously described [33]. AA chloride was obtained by the
condensation of compound 4 with oxalyl chloride in the presence of triethylamine, and this
intermediate was then reacted with a variety of substituted αꢀaminophosphonates to give the
desired compounds 6i and 6n. All the new compounds were confirmed by spectroscopic methods,
including 1H NMR, 13C NMR and high resolution mass spectroscopy.
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DOI: 10.1039/C6RA11397D
2.2 Biological Activity
2.2.1 Cytotoxicity Test
The in vitro cytoxicity of the synthesized compounds was evaluated by
methylthiazoltetrazolium (MTT) assay in a panel of five human cancer cell lines including A549
adenocarcinomic human alveolar basal epithelial cells, SKꢀOVꢀ3 human ovarian cancer cells,
Spcaꢀ2 human lung cancer cells, T24 human bladder carcinoma cells and Hctꢀ116 human colon
tumour cells. The commercial anticancer drugs 5ꢀfluorouracil (5ꢀFU) and cisplatin were positive
controls. The assay results are shown in Table 1.
As shown in Table 1, the IC50 values of most of the conjugates were lower than that of AA and
5ꢀFU, indicating that the introduction of an APA moiety in the AA skeleton should lead to
improved cytotoxicity. Many of the target compounds, shown in bold in Table 1, had better
anticancer activity against the six cell lines than cisplatin, but all had lower cytotoxicity against
human umbilical vein endothelial cells (HUVEC) than cisplatin and 5ꢀFU, which indicated that
they were good candidate for antiꢀtumour drugs.
In most cases, halogen substituents in the 2ꢀ and 4ꢀposition of the benzene group of APA
moieties led, as in in 3d and 3f, to significant increase in potency compared with other analogues.
Compounds possessing a chloro group in the 2ꢀ and 4ꢀposition (3d and 3f) had increased potency
against T24 cells, with IC50 values of 6.59 and 8.93 ꢁM, respectively. 3e, with a chloro group in
the 3ꢀposition had an IC50 of 13.92 ꢁM in the same cell line. However, introduction of a
naphthalene substituent led, in 3p and 3q, to reduced potency, particularly against the A549 lung
cancer cell line, with IC50 values of 54.79 ꢁM and 46.33 ꢁM, respectively. Compound 3l, with a
methoxy substituent in the para position of the benzene ring of its APA moiety, also had significant
activity against SKꢀOVꢀ3 cells, with an IC50 of 10.26 ꢁM. Some of the compounds had strong
selective potency against Hctꢀ116 colon cancer cells and Spcaꢀ2 lung cancer cells, with IC50
values of 11.07 ꢁM and 10.10 ꢁM, respectively. The nearly identical cytotoxicity of compounds 6i
and 6n compared with compounds 3i and 3n, suggests the lack of importance of 11ꢀoxo for
biological activity.
The introduction of chloro as a highly electronegative centre seemed to improve the biological
properties of these compounds. Comparison with activities against normal HUVEC (Table 1)
revealed that the antiꢀproliferative activity of some compounds against cancer cells was much
higher than the activity against normal cells.
Table 1. IC50 values (ꢁM) of asiatic acid and its derivatives 3 and 6 towards five selected tumor
cell lines and normal cell lines for 48 h.
Compounds
IC50α(ꢁM)
Spcaꢀ2
A549
Hctꢀ116
T24
SKꢀOVꢀ3
HUVEC
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O
H
OEt
AcO
AcO
HN
P
O
H
OEt
21.77±1.12 25.22±1.45 16.19±2.91 10.10±4.01 31.26±0.31 >100
F
H
AcO
3a
3b
O
H
OEt
AcO
AcO
HN
P
O
H
OEt
27.15±0.77 28.74±0.23 32.03±1.00 45.78±2.11 34.64±0.51 >100
H
AcO
F
O
H
OEt
AcO
HN
P
O
H
OEt
AcO
AcO
20.18±1.19 18.19±2.32 20.78±2.12 29.73±1.54 25.24±0.15 >100
H
F
3c
3d
3e
O
H
H
H
OEt
AcO
AcO
HN
HN
HN
P
O
H
H
H
OEt
14.89±2.31 26.09±2.11
27.52±0.67 >100
6.59±0.16
15.01±1.95
Cl
H
AcO
O
OEt
AcO
AcO
P
O
OEt
29.80±2.52 28.01±1.03 13.92±0.92 18.02±0.99 35.47±1.96 >100
H
AcO
Cl
O
OEt
AcO
AcO
P
O
OEt
15.70±0.46 17.09±2.51 8.93±1.26
16.06±2.62 18.60±0.14 >100
H
AcO
Cl
3f
O
H
OEt
AcO
AcO
HN
P
O
H
OEt
12.27±1.20 24.91±2.54 14.88±1.98 22.61±1.97 26.95±0.84 >100
Br
H
AcO
3g
O
H
OEt
AcO
HN
P
O
H
OEt
26.28±0.68 15.20±1.56 20.24±1.63 24.13±2.19 21.17±0.36 >100
AcO
AcO
H
Br
3h
O
H
OEt
AcO
AcO
HN
P
O
H
OEt
17.59±1.70 16.97±1.49 17.93±0.77 27.76±0.97 18.07±0.47 >100
H
AcO
Br
3i
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DOI: 10.1039/C6RA11397D
O
H
OEt
AcO
AcO
HN
P
O
H
OEt
25.99±0.52 37.87±2.54 31.93±3.34 26.98±0.77 22.63±1.72 >100
H
AcO
3j
O
H
OEt
AcO
HN
P
O
H
27.71±0.55 32.64±1.20 25.24±0.55 23.80±1.09 19.37±0.53 >100
OEt
AcO
AcO
H
3k
O
H
OEt
AcO
HN
P
O
H
OEt
12.36±4.42 28.54±1.36 17.83±4.54 20.28±1.07 10.26±0.69 >100
47.31±1.12 11.07±2.96 24.11±0.66 31.43±2.09 40.13±0.32 >100
35.73±1.34 27.42±0.97 16.65±1.85 22.75±1.26 42.29±0.79 >100
AcO
AcO
H
3l
O
H
OEt
AcO
HN
P
O
H
OEt
AcO
AcO
H3CO
H
3m
O
H
OEt
AcO
HN
P
O
H
OEt
AcO
AcO
H
H3CO
3n
O
H
OEt
AcO
AcO
HN
P
O
H
OEt
45.57±0.45 25.09±3.55 18.11±0.33 27.70±1.98 38.20±0.83 >100
H
AcO
OCH3
3o
3p
O
H
OEt
AcO
AcO
HN
P
O
H
54.79±2.27 41.02±1.29 43.80±1.03 46.14±1.23 40.14±0.12 >100
OEt
H
AcO
O
H
OEt
AcO
AcO
HN
P
O
H
OEt
46.33±2.08 38.97±0.61 49.10±0.11 48.72±0.54 46.22±0.27 >100
H
AcO
3q
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DOI: 10.1039/C6RA11397D
O
O
H
OEt
AcO
AcO
HN
P
O
H
37.92±1.18 22.98±0.90 28.92±0.53 39.39±2.35 30.87±0.18 >100
OEt
H
AcO
3r
O
O
H
OEt
AcO
AcO
HN
P
O
H
OEt
23.35±1.52 18.85±1.69 13.80±1.90 16.76±1.48 19.70±0.11 >100
H
AcO
Br
6i
O
O
H
OEt
AcO
HN
P
O
H
OEt
27.12±3.01 28.94±0.78 21.89±1.65 25.11±0.99 20.12±1.13 >100
AcO
AcO
H
6n
AA
37.81±2.16 29.35±1.39 33.72±1.25 39.44±0.87 35.65±0.98 >100
34.47±1.90 23.50±2.34 40.14±2.14 44.34±1.90 27.23±0.16 56.00±4.0
13.48±0.37 10.43±0.47 30.05±0.39 15.83±0.54 15.60±1.68 9.68±1.15
5ꢀFU
Cisplatin
αIC50 values are presented as the mean ± SD (standard error of the mean) from three separated
experiments.
2.2.2 Investigation of Cell Cycle Distribution
The mechanism of the antiꢀproliferative effect of the AA derivatives was investigated by flow
cytometric assay of the cell cycle distribution of T24 cells 48 h following treatment with 0, 2.5 or
5 ꢁM of each analogue. As shown in Fig. 1, treatment of T24 cells with compound 3d at each
concentration enhanced cell cycle arrest at the G1 phase, resulting in concomitant increase in the
population in the G1 phase (to 46.48% and 56.57%) compared with the control cells (38.51%).
The Sꢀphase population of T24 cells decreased by 48.27% and 36.97% compared with the control
cells (57.65%). These results suggested that compound 3d arrested the cell cycle of T24 cells in
the G1 stage.
The molecular events involved in cellular responses to the effective compound were
investigated, and the expression of regulatory proteins implicated in G1 arrest, including p21,
cyclin A, p27, cyclin E1, and cyclin D1 were evaluated. The cell cycle regulatory proteins that
control the G1 to Sꢀphase transition are cyclins and cyclinꢀdependent kinases (CDKs). The activity
of cyclin Dꢀdependent CDK4 is detected first in midꢀG1 phase and then increases as cells
approach the G1/S boundary. Cyclin E is expressed periodically with maximum levels occurring
near the G1/S transition, binding to a different catalytic subunit, CDK2. The CDK inhibitors are
tumourꢀsuppressor proteins that interact with distinct cyclinꢀCDK complexes and thereby inhibit
the activity of enzymes such as p21 and p27, which regulate the G1 to Sꢀphase transition of the
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cell cycle [34–36]. Flow cytometry data clearly show that cell cycle arrest occurred at G1 and
possibly had an effect on cell cycle regulatory proteins. The levels of cyclins (cyclin A, cyclin D1
and cyclin E), CDKs (CDK2, and CDK4) and CDK inhibitors (p53, p16, p21 and p27) in T24
cells treated for 48 h with 2.5 ꢁM, 5 ꢁM and 10 ꢁM 3d were determined by western blot assay(Fig.
2). The T24 cell lysates revealed that 3d reduced the levels of CDK2, CDK4, cyclin E and cyclin
D1 compared with controls, and synchronously increased the levels of p21 and p27 in a
concentrationꢀdependent manner, indicating that the cells were effectively arrested at the G1 phase
of the cell cycle. However, 3d treatment did not alter the levels of cyclin A in T24 cells.
(a)
(b)
(c)
Fig. 1. Cell cycle analysis of compound 3d treated T24 cells. T24 cells were treated with different
concentrations (0, 5 and 10 ꢁM) of compound 3d for 48 h to determine DNA fluorescence and cell
cycle phase distribution.
Control
2.5 µM
8
5 µM
10 µM
6
4
2
0
(a)
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Control
2.5 µM
5 µM
1.2
0.9
0.6
0.3
0.0
10 µM
(b)
Fig. 2. Effect of asiatic acid derivatives on the expression of cyclins and associated proteins. T24
cells were treated with compound 3d different concentrations for 48 h. Western blot analysis was
carried out with antibodies against (cyclin D1, cyclin A and cyclin E); CDK2, CDK4 and βꢀactin
was used as loading control.
2.2.3. Apoptosis Assay by Hoechst 33258 Staining
Apoptosis is a key pathway leading to cell death and is characterized by chromatin
condensation and fragmented nuclei. It was considered of interest to investigate the
apoptosisꢀinducing effect of compound 3d by Hoechst 33258 staining of T24 bladder cancer cells.
Cells treated with 10 ꢁM and 20 ꢁM 3d for 24 h were stained with Hoechst 33258; cells not
treated with 3d were 24 h controls. The results (Fig. 3) showed that T24 control cells not treated
with compound 3d were normally stained blue (in the web version). Cells treated with 3d for 12
and 24 h displayed strong blue fluorescence and had characteristic apoptotic morphologies. The
observations revealed that compound 3d induced apoptosis of T24 cells.
(a)
(b)
(c)
Fig. 3. Effects of compound 3d on morphological changes of T24 cells after staining with Hoechst
33258 dye. (a) The cells not treated with 3d were used as control, (b, c) compound 3d treated T24
cells at concentrations of 10 and 20 ꢁM, respectively.
2.2.4. Apoptosis Assay by Acridine Orange/Ethidium Bromide (AO/EB) Staining
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To further characterize the cell apoptosis induced by compound 3d, AO/EB staining was
carried out to evaluate the accompanying changes in morphology. The cytotoxicity of compound
3d was evaluated in T24 cells following treatment with 10 ꢁM and 20 ꢁM for 24 h. T24 cells not
treated with 3d were 24 h controls. The results (Fig. 4) showed that at both concentrations, the
morphology of 3dꢀtreated T24 cells had changed significantly. The cell nuclei were stained yellow
green or orange, and the morphology showed pycnosis, membrane blebbing and cell budding
characteristic of apoptosis. The nearly complete absence of redꢀstained cells showed that 3d
treatment was associated with very low cytotoxicity. The results thus demonstrated that compound
3d induced apoptosis with low cytotoxicity, and were consistent with the results of Hoechst 33258
staining.
(a)
(b)
(c)
Fig. 4. Compound 3d induced apoptotic in T24 cells were determined by AO/EB staining and
were photographed via fluorescence microscopy. (a) Not dealt with compound 3d was used as
control at for 24 h, (b, c) dealt with compound 3d for 24 h at concentrations of 10 and 20 ꢁM,
respectively.
2.2.5. Apoptosis Assay by Flow Cytometry
Following evaluation of the anticancer activity of 3d and its effect on the cell cycle, we assayed
its ability to trigger apoptosis in T24 cells by flow cytometry (Fig. 5). Untreated T24 cells
(controls) or T24 cells treated with 10.0 ꢁM or 20.0 ꢁM compound 3d for 24 h were double
stained with fluorescein isothiocyanate (FITC)ꢀannexin V and propidium iodide (PI) and analysed
by flow cytometry. This assay allows permits the differentiation of live cells (annexin V–/PI–),
early apoptotic cells (annexin V+/PI–), late apoptotic cells (annexin V+/PI+), and necrotic cells
(annexin V–/PI+). Treatment of T24 cells with 10.0 ꢁM compound 3d (Fig. 5a, b) led to an
increase in the number of apoptotic cells, from 7.39% in controls to 14.69% in treated cells (i.e.,
10.2% early apoptotic cells and 4.49% late apoptotic cells). Treatment was also accompanied by a
decrease in the percentage of live cells; 89.2% in controls and 81.5% in treated cells. Moreover,
the effect on the apoptosis rate increased with concentration, and after treatment with 20 ꢁM
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compound 3d, 30.85% of the cells were apoptotic, a significantly higher percentage than observed
in the controls. The results further demonstrated the apoptosis induced by compound 3d in
addition to its inhibition of cell proliferation.
(a)
(b)
(c)
Fig. 5. Flow cytometry analysis of cells stained with Annexin VꢀFITC and PI. (a) The T24 cells
not treated with 3d were used as control, (b, c) compound 3d treated T24 cells for 24 h at
concentration of 10 and 20 ꢁM, respectively.
2.2.6. Increase of Intracellular ROS Level Induced in T24 Cells by Compound 3d
ROS are a byꢀproduct of normal metabolism, and include free radicals such as the superoxide
anion, hydroxyl and lipid radicals, as well as nonꢀradical oxidizing species such as hydrogen
peroxide, peroxynitrite and singlet oxygen. They often cause cellular damage and lead to cell
death and tissue injury, especially at high concentrations. The generation of intracellular ROS may
be responsible for the induction of apoptosis [37,38]. We studied the loss of mitochondrial
transmembrane potential, and the resulting generation of ROS caused by compound 3d in vitro, by
fluorescence microscopy using the fluorescent probe 2,7ꢀdichlorofluorescein diacetate. T24 cells
treated with compound 3d exhibited more intense fluorescence in the cytoplasm than untreated
control cells. Under the same experimental procedures applied to 3dꢀtreated cells, the fluorescence
detected in controls was weak and uniformly distributed within the cells. Figure 6 shows strong
green fluorescence (in the web version) in T24 cells treated with compound 3d, indicating that 3d
significantly induced apoptosis of T24 cells. An increase of ROS might thus be an early mediator
in compound 3dꢀinduced apoptosis. These findings are consistent with an effect of compound 3d
on mitochondrial function and accumulation of ROS as cues for the induction of apoptosis.
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(a)
(b)
(c)
Fig. 6. Compound 3d affected the levels of intracellular ROS in T24 cells. The T24 cells not
treated with 3d were used as control, (b, c) Compound 3d treated T24 cells at concentrations of 10
and 20 ꢁM, respectively.
2.2.7. Compound 3dꢀinduced Loss of Mitochondrial Membrane Potential (ꢂΨm) in T24 Cells
(a)
(b)
(c)
Fig. 7. Effects of compound 3d on morphological changes and mitochondrial membrane potential
(ꢂψm) in T24 cells. (a) Not dealt with compound 3d was used as control at for 24 h, (b, c) dealt
with compound 3d at concentration of 20 ꢁM for 12 and 24 h , respectively.
Mitochondria play a central role in the life and death of cells and are known to be a major
source and target of oxidative stress. Damage to mitochondrial integrity and the consequent loss of
mitochondrial membrane potential are early events in the initiation and activation of apoptotic
cascades [39]. To further investigate the apoptosisꢀinducing effect of compound 3d, the
fluorescent probe JCꢀ1 was used to design and detect the changes of mitochondrial membrane
potential. T24 cells treated with different concentrations of 3d for 24 h were stained with JCꢀ1;
cells not treated with 3d were used as 24 h controls. The results in Fig. 7 show that control cells
not treated with compound 3d were normally red (in the web version), while 3dꢀtreated cells
showed strong green fluorescence and had typical apoptotic morphologies at both 12 and 24 h
after treatment. These results indicate that apoptosis is the primary mode of cell death induced by
compound 3d.
2.2.8. Intracellular Ca2+ Release
Calcium is a ubiquitous second messenger involved in many cellular processes, including
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regulation of transcription, metabolism, proliferation and cell death. Because of its multiple effects,
and because calcium overload can induce cell apoptosis, the intracellular Ca2+ concentration is
tightly regulated. Mitochondria can be considered as a firewall that controls Ca2+ concentration in
the cell and in cytoplasmic microdomains by tuning the frequency of oscillatory Ca2+ signals and
by blunting the spread of cytosolic Ca2+ waves. We found that compound 3d disrupted
mitochondrial function and induced apoptosis. To determine the role of calcium signalling in
3dꢀinduced apoptosis, T24 cells were treated with 3d for 24 h, and Ca2+ was detected by
fluorescence microscopy using Fluoꢀ3/AM, a calcium indicator dye. As shown in Fig. 8, 3d
treatment resulted in a doseꢀdependent elevation of intracellular Ca2+ concentration. The results
suggest that 3dꢀinduced apoptosis was associated with an increase in Ca2+concentration.
(a)
(b)
(c)
Fig. 8. Compound 3d caused the levels of intracellular Ca+ elevation in T24 cells. The T24 cells
not treated with 3d were used as control, (b, c) Compound 3d treated T24 cells at concentrations
of 10 and 20 ꢁM, respectively.
2.2.9. Caspaseꢀdependent Apoptosis in T24 Cells
To confirm the molecular mechanisms of apoptosis, we investigated the effects of compound 3d
on expression of proteins needed for mitochondriaꢀmediated apoptosis. Cancer cells can limit or
evade apoptosis in a number of ways, which often include perturbation of the Bclꢀ2 intrinsic
apoptotic pathway. Members of the Bclꢀ2 family play important regulatory roles both as proꢀ and
as antiꢀapoptotic proteins. Accordingly, the Bclꢀ2/Bax ratio is more important in the regulation of
apoptosis than the individual levels of each Bclꢀ2 family protein. To further verify that 3d
promotes apoptosis in T24 cells, we assayed the expression of Bax, Bclꢀ2 and cytochrome c
proteins following treatment with 3d. As shown in Fig. 9a, treatment of T24 cells with 3d led to an
increase in Bax expression and decrease in Bclꢀ2 expression followed by the release of
mitochondrial cytochrome c into the cytosol. These results indicate that 3d induced apoptosis by
regulating the levels of the Bclꢀ2 family proteins, Bax and Bclꢀ2.
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Control
5 µM
10 µM
20 µM
12
10
8
2.0
1.5
1.0
0.5
0.0
Control
5 µM
10 µM
20 µM
6
4
2
0
(a)
(b)
18
15
12
9
Control
5 µM
10 µM
20 µM
6
3
0
p53
(c)
Fig. 9. Effects of compound 3d on the level of cytochrome c, Bclꢀ2, Bax, Caspaseꢀ9, Caspaseꢀ3
and p53. T24 cells were treated with of compound 3d for 24 h at concentrations of 0, 5, 10, 20 ꢁM,
respectively. Equal amount of protein was loaded on SDSꢀPAGE gel for western blot analysis as
described in experimental section. βꢀactin was used as an internal control.
It is wellꢀknown that proteins of the Bclꢀ2 family play a pivotal role in apoptosis by interfering
with caspases, which are the key effectors of programmed cell death. The caspase cascade is
initiated by the proteolysis of inactive procaspases, and it is propagated by the cleavage of
downstream caspases and substrates such as poly (ADPꢀribose) polymerase (PARP) cleavage. To
determine whether caspases are involved in 3dꢀinduced apoptosis, we assayed caspaseꢀ9, ꢀ3 and
PARP expression in western blots. As shown in Fig. 9b, the expression of caspaseꢀ9, ꢀ3 and PARP
in compound 3dꢀtreated T24 cells was significantly higher than that in control cells. These
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observations suggest that compound 3d may exert proꢀapoptotic effects through
a
mitochondriaꢀmediated pathway and caspase cascade.
p53, a crucial tumour suppressor protein with a key role in many cellular processes, is one of
the most relevant cellular components related to cancer growth. Depending on the cellular context,
accumulation of p53 in response to various stresses including DNA damage induces cell cycle
inhibition, senescence and apoptosis [40]. p53 protein is an attractive target of anticancer therapy
because it can be functionally activated to eradicate tumours. Western blot analysis revealed a
doseꢀdependent increase of p53 expression in T24 cells treated with 3d compared with expression
in controls (Fig. 9c). In line with our other observations, the effect of 3d treatment on these cell
cycle regulators was doseꢀdependent. These results indicate that enhanced p53 expression was
likely involved in the mechanism of 3dꢀinduced G1/S cell cycle arrest.
In summary, we successfully synthesized a series of AA–APA conjugates and evaluated their
cytotoxicity in a panel of cancer cell lines. The target compounds exhibited remarkable anticancer
activity with low cytotoxicity against normal HUVEC. The cell apoptosisꢀinducing activity of
representative compound 3d in T24 cells revealed that the anticancer activity of this compound
depended on apoptosis of cancer cells via regulation of Bclꢀ2 family members, activation of
caspaseꢀ9 and caspaseꢀ3 and subsequent cleavage of PARP. Cell cycle analysis confirmed that
compound 3d inhibited proliferation by G1 phase arrest that depended on expression of p53
protein. In light of our results, the rational design of AA derivatives containing APA moieties
represents a promising basis for the development of new anticancer agents.
3. Experimental Section
3.1. Chemistry
All chemicals were reagent grade and are commercially available. NMR spectra were
measured on a BRUKER AVANCE AV500 spectrometer using tetramethyl silane, (TMS) as the
internal standard. The mass spectra were obtained on a BRUKER ESQUIRE HCT spectrometer.
Melting points were determined using an Xꢀ4 apparatus and were uncorrected. All chemical
reagents including the aromatic aldehyde and solvents were analytical grade. GelRed nucleic acid
stain was purchased from Biotium.
3.1.1 General procedure for compound 1 (2α, 3β, 23ꢀtriacetoxyursꢀ12ꢀeneꢀ28ꢀoic acid)
Acetic anhydride (0.5 mL, 5.0 mmol) was added to a solution of AA (200 mg, 0.4 mmol) in
pyridine (10 mL) and the mixture was stirred at 20oC for 8 h. After dilution with ethyl acetate (25
mL), the mixture was washed with aqueous 1 M HCl (10 mL×5), saturated CuSO4 (15 mL×2) and
saturated NaCl solution (20 mL). The organic phase was dried over anhydrous sodium sulfate.
Following filtration and evaporation of solvent at reduced pressure, the crude product was purified
by column chromatography (petroleum ether : acetic ether = 3:1) to yield compound 1 as a white
o
1
solid. Yield 85.5%. m.p. 151.2~154.6 C. H NMR (500 MHz, CDCl3) δ 5.19 (t, J = 3.2 Hz, 1H,
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Hꢀ12), 5.11 (td, J = 10.9, 4.6 Hz, 1H, Hꢀ2), 5.04 (d, J = 10.3 Hz, 1H, Hꢀ3), 3.80 (d, J = 11.8 Hz,
1H, Hꢀ23), 3.54 (d, J = 11.9 Hz, 1H, Hꢀ23), 2.14 (d, J = 11.3 Hz, 1H, Hꢀ9), 2.04 (s, 3H, CH3CO),
1.98 (s, 3H, CH3CO), 1.94 (s, 3H, CH3CO), 1.89−1.11 (triterpene’s H, 19H), 1.05 (s, 3H, CH3ꢀ27),
1.03 (s, 3H, CH3ꢀ24), 0.90 (s, 3H, CH3ꢀ25), 0.83 (s, 3H, CH3ꢀ26), 0.80 (d, J = 6.4 Hz, 3H,
CH3ꢀ29), 0.71 (d, J = 6.4 Hz, 3H, CH3ꢀ30). 13C NMR (126 MHz, CDCl3) δ 184.04, 170.89,
170.54, 170.43, 138.03, 125.19, 74.77, 69.91, 65.21, 60.41, 52.39, 47.88, 47.53, 47.43, 43.66,
41.87, 39.45, 38.94, 38.75, 37.77, 36.61, 32.37, 30.54, 27.83, 23.90, 23.43, 23.29, 21.17, 21.09,
20.89, 20.79, 17.81, 17.01, 16.93, 14.19, 13.90; ESIꢀHRMS m/z Calc for C36H54O8 [M−H]ꢀ :
613.37459, founded: 613.37483.
3.1.2 General procedure for the preparation of compounds 3
Oxalyl chloride (1.2 mL) at 0oC was added dropwise to a solution of compound 2 (100.00 mg,
0.14 mmol) in CH2Cl2, and the mixture was stirred at room temperature for 8 h. The solvent was
evaporated at reduced pressure. CH2Cl2 (10 ml × 3) was added to the mixture, with vacuum
concentration. The mixture was the allowed to react with individual APAs (0.56 mmol) with
stirring at room temperature for 4 h. After dilution with ethyl acetate (25 mL), the mixture was
washed with water (20 mL × 3) and the organic phase was dried over anhydrous sodium sulfate.
Following filtration and evaporation of solvent at reduced pressure, the residue was purified by
column chromatography to yield a white solid.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ2ꢀfluorophenyl)methꢀyl}phosphonate
o
(3a). Yield 65.5%. m.p. 135.7~139.0 C. 1H NMR (500 MHz, CDCl3) δ 7.55 (d, J = 7.2 Hz, 1H,
ArꢀH), 7.40–7.27 (m, 2H, ArꢀH), 7.17−7.12 (m, 1H, ArꢀH), 6.51–6.21 (m, 1H, NH), 5.82−5.67 (m,
1H, Hꢀ11), 5.30−5.20 (m, 1H, Hꢀ3), 5.16–5.10 (m, 1H, Hꢀ2), 5.04 (m, 1H, PꢀCH), 4.23–4.10 (m,
2H, ꢀOCH2), 3.40–3.75 (m, 2H, ꢀOCH2), 3.73–3.42 (m, 2H, Hꢀ23), 2.04 (s, 3H, COCH3), 2.01(s,
3H, COCH3), 1.97 (s, 3H, COCH3), 1.96–1.10 (m, triterpene’s H, 21H), 1.33 (t, J =5 Hz, 6H,
CH3×2), 1.10 (s, 3H, CH3ꢀ27), 1.04 (s, 3H, CH3ꢀ24), 1.00 (s, 3H, CH3ꢀ26), 0.95 (d, J = 5.5 Hz, 3H,
CH3ꢀ30), 0.88 (s, 3H, CH3ꢀ25), 0.82 (d, J = 3.3 Hz, 3H, CH3ꢀ29). 13C NMR (126 MHz, DMSO) δ
176.2, 170.7, 170.4, 170.3, 139.1, 138.2, 129.7, 129.2, 128.9, 126.9, 125.7, 125.2, 74.7, 69.8, 65.2,
63.1, 62.8, 54.1, 53.6, 47.9, 47.6, 43.7, 42.3, 42.1, 41.8, 39.6, 39.3, 38.9, 38.8, 32.5, 32.2, 30.7,
29.6, 27.6, 24.2, 23.2, 21.1, 21.0, 20.8, 20.7, 17.8, 17.6, 16.9, 16.8, 16.3, 15.9, 13.8. ESIꢀHRMS
m/z Calc for C47H69FNO10P [M+H]+ : 858.47159 founded: 858.46844.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ3ꢀfluorophenyl)metꢀhyl}phosphonate
o
(3b). Yield 67.5%. m.p. 133.4~135.6 C. 1H NMR (500 MHz, CDCl3) δ 7.53 (d, J = 7.2 Hz, 1H,
ArꢀH), 7.38–7.26 (m, 2H, ArꢀH), 7.15−7.10 (m, 1H, ArꢀH), 6.49–6.19 (m, 1H, NH), 5.80−5.65 (m,
1H, Hꢀ11), 5.28−5.19 (m, 1H, Hꢀ3), 5.14–5.08 (m, 1H, Hꢀ2), 5.02 (m, 1H, PꢀCH), 4.21–4.09 (m,
2H, ꢀOCH2), 3.88–3.75 (m, 2H, ꢀOCH2), 3.71–3.42 (m, 2H, Hꢀ23), 2.02 (s, 3H, COCH3), 1.99 (s,
3H, COCH3), 1.95 (s, 3H, COCH3), 1.94–1.08 (m, triterpene’s H, 21H), 1.31 (t, J =5 Hz, 6H,
CH3×2), 1.07 (s, 3H, CH3ꢀ27), 1.02 (s, 3H, CH3ꢀ24), 0.98 (s, 3H, CH3ꢀ26), 0.93 (d, J = 5.5 Hz, 3H,
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CH3ꢀ30), 0.86 (s, 3H, CH3ꢀ25), 0.80 (d, J = 3.3 Hz, 3H, CH3ꢀ29). 13C NMR (126 MHz, CDCl3) δ
176.2, 170.7, 170.4, 170.3, 139.1, 138.2, 135.1, 133.0, 129.4, 127.5, 125.7, 125.2, 74.7, 69.8, 65.2,
63.1, 62.8, 54.2, 50.5, 47.9, 47.6, 43.7, 42.3, 42.1, 41.8, 39.6, 39.3, 38.9, 38.8, 32.5, 32.2, 30.7,
29.6, 27.7, 24.2, 23.2, 21.0, 21.0, 20.8, 20.7, 17.8, 17.6, 16.9, 16.8, 16.4, 15.9, 13.8. ESIꢀHRMS
m/z Calc for C47H69FNO10P [M+H]+ : 858.47159 founded: 858.46891.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ4ꢀfluorophenyl)metꢀhyl}phosphonate
(3c). Yield 68.1%. m.p. 133.2~135.1 oC. 1H NMR (500 MHz, CDCl3) δ 7.39−7.32 (m, 2H, ArꢀH),
7.02−6.98 (m, 2H, ArꢀH), 6.67−6.43 (m, 1H, NH), 5.51−5.33 (m, 1H, Hꢀ11), 5.26 (dd, J = 11.3,
9.4 Hz, 1H, Hꢀ3), 5.15−5.08 (m, 1H, Hꢀ2), 5.04 (dd, J = 14.6, 10.3 Hz, 1H, PꢀCH), 4.15−4.02 (m,
2H, ꢀOCH2), 3.92−3.77 (m, 2H, ꢀOCH2), 3.73−3.50 (m, 2H, Hꢀ23), 2.10−1.10 (m, triterpene’s H,
21H), 2.04 (s, 3H, COCH3), 2.00 (s, 3H, COCH3), 1.96 (s, 3H, COCH3), (3×CH3CO), 1.30 (td, J =
7.1 Hz, 6H, CH3×2), 1.07 (s, 3H, CH3ꢀ27), 1.04 (s, 3H, CH3ꢀ24), 1.00 (s, 3H, CH3ꢀ26), 0.94 (d, J
= 4.6 Hz, 3H, CH3ꢀ30), 0.86 (s, 3H, CH3ꢀ25), 0.83 (d, J = 3.2 Hz, 3H, CH3ꢀ29). 13C NMR (126
MHz, CDCl3) δ 176.50, 170.8, 170.4, 170.3, 139.2, 138.1, 130.9, 130.5, 129.7, 125.5, 125.2, 115.6,
74.8, 69.9, 65.3, 62.9, 62.7, 54.1, 50.4, 48.0, 47.6, 43.8, 42.3, 42.2, 41.8, 39.6, 39.5, 38.9, 38.8,
32.5, 32.4, 30.8, 29.6, 27.6, 24.3, 23.2, 21.1, 21.0, 20.8, 20.7, 17.8, 17.7, 17.0, 16.7, 16.3, 16.1.
13.9. ESIꢀHRMS m/z Calc for C47H69FNO10P [M+H]+ : 858.47159 founded: 858.46698.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ2ꢀchlorophenyl)metꢀhyl}phosphonate
(3d). Yield 64.9%. m.p. 141.3~144.6 oC. 1H NMR (500 MHz, CDCl3) δ 7.39−7.33 (m, 2H, ArꢀH),
7.27–7.20 (m, 2H), 6.91−6.79 (m, 1H, NH), 5.82−5.72 (m, 1H, Hꢀ11), 5.20–5.13(m, 1H, Hꢀ3),
5.12–5.06 (m, 1H, Hꢀ2), 5.09−5.06 (m, 1H, PꢀCH), 4.19–4.09 (m, 2H, ꢀOCH2), 3.89−3.72 (m, 2H,
ꢀOCH2), 3.73–3.47 (m, 2H, Hꢀ23), 2.03 (s, 2H, COCH3), 1.99 (s, 3H, COCH3), 1.96 (s, 3H,
COCH3), 1.95–1.09 (m, triterpene’s H, 21H), 1.34−1.28 (m, 6H, CH3×2), 1.06 (s, 3H, CH3ꢀ27),
1.02 (s, 1H, CH3ꢀ24), 0.99 (s, 3H, CH3ꢀ26), 0.94 (d, J = 4.1 Hz, 3H, CH3ꢀ30), 0.86 (s, 3H,
CH3ꢀ25), 0.81 (s, 3H, CH3ꢀ29). 13C NMR (126 MHz, CDCl3) δ 176.8, 170.8, 170.5, 170.3, 139.1,
138.2, 133.2, 129.7, 129.2, 126.9, 125.7, 125.2, 74.8, 69.9, 65.2, 63.1, 62.8, 54.1, 47.9, 47.6, 47.4,
43.8, 42.3, 42.1, 41.9, 39.6, 39.3, 38.9, 32.5, 32.2, 30.7, 29.6, 27.7, 24.5, 23.4, 23.1, 21.1, 21.0,
20.8, 20.7, 17.8, 17.7, 16.9, 16.8, 16.4, 15.9, 13.9. ESIꢀHRMS m/z Calc for C47H69ClNO10P
[M+H]+ : 874.44204 founded: 874.43933.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ3ꢀchlorophenyl)metꢀhyl}phosphonate
(3e). Yield 68.3%. m.p. 132.9~135.7 oC. 1H NMR (500 MHz, CDCl3) δ 7.35 (d, J = 15.0 Hz, 1H,
ArꢀH), 7.25 (m, 3H, ArꢀH), 6.70–6.44 (m, 1H, NH), 5.49−5.33 (m, 1H, Hꢀ11), 5.28–5.22 (m, 1H,
Hꢀ3), 5.15−5.10 (m, 1H, Hꢀ2), 5.06−5.01 (m, 1H, PꢀCH), 4.18–4.00 (m, 2H, ꢀOCH2), 3.95–3.77
(m, 2H, ꢀOCH2), 3.63–3.51 (m, 2H, Hꢀ23), 1.93−1.01 (m, triterpene’s H, 21H), 2.03 (s, 3H,
COCH3), 1.99 (s, 3H, COCH3), 1.96 (s, 3H, COCH3), 1.30 (t, J = 7.1 Hz, 6H, CH3×2), 1.07 (s, 3H,
CH3ꢀ27), 1.05 (s, 3H, CH3ꢀ24), 0.94 (s, 3H, CH3ꢀ26), 0.90 (d, J = 3.4 Hz, 3H, CH3ꢀ30), 0.86 (s,
3H, CH3ꢀ25), 0.83 (d, 3H, J = 4.3 Hz, CH3ꢀ29). 13C NMR (126 MHz, CDCl3) δ 176.6, 170.8,
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170.4, 170.3, 139.0, 136.9, 134.5, 129.8, 128.3, 127.0, 126.1, 125.5, 74.8, 69.8, 65.2, 63.0, 62.8,
54.2, 50.8, 48.0, 47.4, 43.7, 42.3, 41.9, 41.8, 39.6, 39.4, 38.9, 32.5, 32.4, 30.7, 29.6, 27.6, 24.3,
23.4, 23.1, 21.1, 21.0, 20.8, 20.7, 17.8, 17.7, 17.0, 16.8, 16.4, 16.1, 13.9. ESIꢀHRMS m/z Calc for
C47H69ClNO10P [M+H]+ : 874.44204 founded: 874.43604.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ4ꢀchlorophenyl)metꢀhyl}phosphonate
o
1
(3f). Yield 65.6%. m.p. 139.2~140.8 C. H NMR (500 MHz, CDCl3) δ 7.33 (d, J = 8.3 Hz, 2H,
ArꢀH), 7.28 (d, J = 2.8 Hz, 2H, ArꢀH), 6.69–6.42 (m, 1H, NH), 5.49–5.33 (m, 1H, Hꢀ11),
5.28−5.21 (m, 1H, Hꢀ3), 5.13−5.10 (m, 1H, Hꢀ2), 5.06−5.03 (m, 1H, PꢀCH), 4.13–4.06 (m, 2H,
ꢀOCH2), 3.92–3.75 (m, 2H, ꢀOCH2), 3.57–3.51 (m, 1H, Hꢀ23), 2.03 (s, 3H, COCH3), 2.00 (s, 3H,
COCH3), 1.96 (s, 3H, COCH3), 1.95–1.00 (m, triterpene’s H, 21H), 1.30 (t, J = 7.0 Hz, 6H,
CH3×2), 1.08–1.05 (m, 6H, CH3ꢀ24/27), 0.94 (s, 3H, CH3ꢀ26), 0.90 (d, J = 3.9 Hz, 3H, CH3ꢀ30),
0.86 (s, 3H, CH3ꢀ25), 0.83 (d , J = 4.1 Hz, 3H, CH3ꢀ29). 13C NMR (126 MHz, CDCl3) δ 176.5,
170.8, 170.4, 170.3, 139.2, 138.2, 133.6, 130.2, 129.2, 128.8, 125.5, 125.3, 74.8, 69.8, 65.2, 63.0,
62.7, 54.1, 50.6, 48.0, 47.9, 43.7, 42.3, 41.9, 39.6, 39.4, 38.9, 38.8, 32.5, 32.4, 30.7, 29.6, 27.6,
23.4, 23.2, 21.1, 21.0, 20.8, 20.7, 17.8, 17.7, 17.0, 16.9, 16.7, 16.4, 16.3, 13.9. ESIꢀHRMS m/z
Calc for C47H69ClNO10P [M+H]+ : 874.44204 founded: 874.43893.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ2ꢀbromophenyl)metꢀhyl}phosphonate
o
(3g). Yield 68.9%. m.p. 147.3~150.3 C. 1H NMR (500 MHz, CDCl3) δ 7.53 (d, J = 8.0 Hz, 2H,
ArꢀH), 7.38–7.25 (m, 2H, ArꢀH), 7.16−7.09 (m, 1H, NH), 5.79−5.67 (m, 1H, Hꢀ11), 5.19−5.13 (m,
1H, Hꢀ3), 5.12−5.06 (m, 1H, Hꢀ2), 5.06−5.01 (m, 1H, PꢀCH), 4.22−4.09 (m, 2H, ꢀOCH2),
3.89−3.76 (m, 2H, ꢀOCH2), 3.56−3.39 (m, 2H, Hꢀ23), 2.03 (s, 2H, COCH3), 2.00 (s, 3H, COCH3),
1.96 (s, 3H, COCH3), 1.96−1.10 (m, triterpene’s H, 21H), 1.35ꢀ1.30 (m, 6H, CH3×2), 1.06 (s, 3H,
CH3ꢀ27), 1.03 (s, 3H, CH3ꢀ24), 0.99 (s, 3H, CH3ꢀ26), 0.94 (d, J = 4.9 Hz, 3H, CH3ꢀ30), 0.86 (s,
3H, CH3ꢀ25), 0.81 (d, J = 4.9 Hz, 3H, CH3ꢀ29). 13C NMR (126 MHz, CDCl3) δ 176.3, 170.9,
170.6, 170.4, 138.2, 135.1, 133.1, 129.5, 129.2, 127.6, 125.8, 125.3, 74.8, 69.9, 65.3, 63.3, 62.9,
53.7, 50.6, 48.00, 47.7, 43.8, 42.3, 42.2, 41.9, 39.6, 39.0, 37.8, 32.5, 32.3, 30.8, 27.7, 24.6, 24.3,
23.5, 21.2, 21.1, 20.9, 20.8, 17.9, 17.7, 17.1, 16.9, 16.5, 16.0, 13.9. ESIꢀHRMS m/z Calc for
C47H69BrNO10P [M+H]+ :918.39152 founded: 918.38855.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ3ꢀbromophenyl)metꢀhyl}phosphonate
(3h). Yield 62.9%. m.p. 136.2~139.7 oC. 1H NMR (500 MHz, CDCl3) δ 7.50 (d, J = 13.6 Hz, 1H,
ArꢀH), 7.41 (t, J = 10.0 Hz, 1H, ArꢀH), 7.32 (dd, J = 15.6, 7.7 Hz, 1H, ArH), 7.18 (t, J = 7.8 Hz,
1H, ArꢀH), 6.68 −6.44 (m, 1H, NH), 5.48–5.32 (m, 1H, Hꢀ11), 5.28–5.22 (m, 1H, Hꢀ3), 5.13 (td, J
= 11.0, 4.5 Hz, 1H, Hꢀ2), 5.06−5.01 (m, 1H, PꢀCH), 4.16–4.02 (m, 2H, ꢀOCH2), 3.95−3.80 (m, 2H,
ꢀOCH2), 3.67–3.52 (m, 2H, Hꢀ23), 2.04 (s, 3H, COCH3), 2.00 (s, 3H, COCH3), 1.97 (s, 3H,
COCH3), 1.95–1.09 (m, triterpene’s H, 21H), 1.31 (t, J = 7.1 Hz, 6H, CH3×2), 1.05 (s, 3H,
CH3ꢀ27), 1.01 (s, 3H, CH3ꢀ24), 0.95 (s, 3H, CH3ꢀ26), 0.91 (d, 3H, J = 3.5 Hz, CH3ꢀ30), 0.86 (s,
3H, CH3ꢀ25), 0.83 (d, J = 3.6 Hz, 3H, CH3ꢀ29). 13C NMR (126 MHz, CDCl3) δ 176.6, 170.8,
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170.4, 170.3, 139.0, 138.1, 137.2, 131.6, 130.1, 127.5, 126.6, 125.5, 74.8, 69.9, 65.3, 63.0, 62.9,
54.2, 49.9, 48.1, 47.9, 43.8, 42.3, 41.9, 39.7, 39.5, 38.9, 37.8, 32.6, 32.4, 30.8, 27.6, 24.8, 23.4,
23.2, 21.1, 21.1, 20.9, 20.8, 17.9, 17.7, 17.0, 16.9, 16.5, 16.2, 16.1, 13.9. ESIꢀHRMS m/z Calc for
C47H69BrNO10P [M+H]+ :918.39152 founded: 918.38776.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ4ꢀbromophenyl)metꢀhyl}phosphonate
(3i). Yield 66.5%. m.p. 142.1~144.6 oC. 1H NMR (400 MHz, CDCl3) δ 7.43 (dd, J = 8.2, 3.4 Hz,
2H, ArꢀH), 7.28−7.22 (m, 2H, ArꢀH), 6.71−6.42 (m, 1H, NH), 5.48−5.31 (m, 1H, Hꢀ11),
5.26−5.18 (m, 1H, Hꢀ3), 5.12 (td, J = 10.9, 4.5 Hz, 1H, Hꢀ2), 5.03 (dd, J = 13.0, 10.4 Hz, 1H,
PꢀCH), 4.16−4.00 (m, 2H, ꢀOCH2), 3.92 − 3.76 (m, 2H, ꢀOCH2), 3.74 − 3.48 (m, 2H, Hꢀ23), 2.04
(s, 3H, CH3CO), 1.99 (d, J = 2.7 Hz, 3H, CH3CO), 1.95 (s, 3H, CH3CO), (3×CH3CO), 1.93−0.99
(m, triterpene’s H, 21H), 1.29 (t, J = 7.1 Hz, 6H, CH3×2), 1.07 (s, 3H, CH3ꢀ27), 1.04 (s, 3H,
CH3ꢀ24), 0.93 (s, 3H, CH3ꢀ26), 0.91 (d, J = 4.5 Hz, 3H, CH3ꢀ30), 0.86 (s, 3H, CH3ꢀ25), 0.84 (d, J
= 3.4 Hz, 3H, CH3ꢀ29). 13C NMR (101 MHz, CDCl3) δ 176.5, 170.7, 170.4, 170.3, 139.1, 138.2,
134.1, 131.6, 130.5, 129.6, 125.5, 125.3, 74.8, 69.8, 65.2, 63.0, 62.7, 54.1, 50.8, 48.0, 47.9, 43.7,
42.2, 41.8, 39.6, 39.4, 38.9, 38.8, 32.5, 32.4, 30.7, 29.6, 27.6, 23.4, 23.1, 21.1, 21.0, 20.8, 20.7,
17.8, 17.6, 17.0, 16.9, 16.8, 16.3, 16.1, 13.9. ESIꢀHRMS m/z Calc for C47H69BrNO10P [M+H]+ :
918.39152 founded: 918.39420.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ2ꢀmethylphenyl)metꢀhyl}phosphonate
(3j). Yield 61.9%. m.p. 130.9~134.1 oC. 1H NMR (500 MHz, CDCl3) δ 7.45−7.32 (m, 1H, ArꢀH),
7.20–7.10 (m, 3H, ArꢀH), 6.81− 6.47 (m, 1H, NH), 5.86–5.56 (m, 1H, Hꢀ11), 5.29 (d, J = 80.5 Hz,
1H, Hꢀ3), 5.21–5.00 (m, 2H, Hꢀ2, PꢀCH), 4.17−4.04 (m, 2H, ꢀOCH2), 3.88–3.76 (m, 2H, ꢀOCH2),
3.65–3.51 (m, 2H, Hꢀ23), 2.40 (s, 3H, ArꢀCH3), 2.03 (s, 3H, COCH3), 1.99 (s, 3H, COCH3), 1.96
(s, 3H, COCH3), 1.96–1.06 (m, triterpene’s H, 21H), 1.35–1.26 (m, 6H, CH3×2), 1.05 (s, 3H,
CH3ꢀ27), 0.94 (s, 3H, CH3ꢀ24), 0.86 (s, 3H, CH3ꢀ26), 0.85 (d, J = 5.0 Hz, 3H, CH3ꢀ30), 0.81 (s,
3H, CH3ꢀ25), 0.80 (d, J = 3.3 Hz, 3H, CH3ꢀ29). 13C NMR (126 MHz, CDCl3) δ 176.5, 170.8,
170.5, 170.4, 138.9, 138.3, 134.5, 133.5, 130.5, 128.0, 126.2, 125.5, 74.9, 69.9, 65.2, 62.8, 62.4,
54.3, 50.6, 47.9, 47.5, 43.8, 42.2, 41.9, 39.7, 39.3, 38.9, 37.6, 32.5, 32.4, 30.8, 27.7, 24.7, 24.3,
23.2, 21.1, 21.1, 20.8, 20.8, 19.6, 17.9, 17.7, 17.0, 16.8, 16.5, 16.0, 15.9, 13.9. ESIꢀHRMS m/z
Calc for C48H72NO10P [M+H]+ : 854.49666 founded: 854.49109.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ3ꢀmethylphenyl)metꢀhyl}phosphonate
(3k). Yield 68.5%. m.p. 134.8~136.9 oC. 1H NMR (500 MHz, CDCl3) δ 7.25 − 7.20 (m, 2H, ArꢀH),
7.10−7.07 (m, 2H, ArꢀH), 6.66 − 6.42 (m, 1H, NH), 5.47−5.33 (m, 1H, Hꢀ11), 5.27 − 5.22 (m, 1H,
Hꢀ3), 5.15 − 5.07 (m, 1H, Hꢀ2), 5.03 (dd, J = 17.7, 10.3 Hz, 1H, PꢀCH), 4.11−4.05 (m, 2H,
ꢀOCH2), 3.91 − 3.75 (m, 2H, ꢀOCH2), 3.73−3.50 (m, 2H, Hꢀ23), 2.29 (s, 3H, ArꢀCH3), 2.02 (s, 3H,
COCH3), 1.99 (s, 3H, COCH3), 1.95 (s, 3H, COCH3), (3×CH3CO), (3×CH3CO), 2.08−1.10 (m,
triterpene’s H, 21H), 1.29 (td, J = 8.3, 5.8 Hz, 6H, CH3×2), 1.04 (d, J = 3.7 Hz, 6H, CH3ꢀ24/27),
0.98 (s, 3H, CH3ꢀ26), 0.92 (d, , J = 3.9 Hz, 3H, CH3ꢀ30), 0.85 (s, 3H, CH3ꢀ25), 0.81 (d, J = 4.7 Hz,
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3H, CH3ꢀ29). 13C NMR (126 MHz, CDCl3) δ 176.3, 170.8, 170.4, 170.3, 139.1, 138.2, 132.6,
131.7, 129.2, 128.6, 127.7, 125.4, 74.8, 69.8, 65.2, 62.7, 62.5, 54.1, 50.7, 48.6, 47.4, 43.7, 42.2,
41.8, 39.6, 39.3, 38.8, 37.7, 32.5, 32.3, 30.7, 27.6, 24.6, 24.3, 23.1, 21.1, 21.0, 20.8, 20.7, 19.9,
17.8, 17.6, 17.0, 16.9, 16.6, 16.2, 16.1, 13.8. ESIꢀHRMS m/z Calc for C48H72NO10P [M+H]+ :
854.49666 founded: 854.49299.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ4ꢀmethylphenyl)metꢀhyl}phosphonate
(3l). Yield 64.3%. m.p. 138.1~141.6 oC. 1H NMR (400 MHz, CDCl3) δ 7.24 (dd, J = 8.5 Hz, 2H,
ArꢀH), 7.10 (d, J = 7.6 Hz, 2H, ArꢀH), 6.71 − 6.42 (m, 1H, NH), 5.51 − 5.33 (m, 1H, Hꢀ11), 5.29 −
5.23 (m, 1H, Hꢀ3), 5.12 (m, 1H, Hꢀ2), 5.07 − 5.00 (m, 1H, PꢀCH), 4.15 − 4.02 (m, 2H, ꢀOCH2),
3.84−3.76 (m, 2H, ꢀOCH2), 3.57−3.51 (m, 2H, Hꢀ23), 2.30 (s, 3H, ArꢀCH3), 2.05 (s, 3H, COCH3),
2.00 (s, 3H, COCH3), 2.15−1.10 (m, triterpene’s H, 21H), 1.96 (s, 3H, COCH3), (3×CH3CO), 1.33
− 1.26 (m, 6H, CH3×2), 1.07 (s, 3H, CH3ꢀ27), 1.02 (s, 3H, CH3ꢀ24), 0.99 (s, 3H, CH3ꢀ26), 0.94 (d,
J = 4.5 Hz, 3H, CH3ꢀ30), 0.86 (s, 3H, CH3ꢀ25), 0.83 (d, J = 3.5 Hz, 3H, CH3ꢀ29). 13C NMR (126
MHz, CDCl3) δ 175.2, 170.7, 170.4, 170.1, 138.0, 132.3, 131.4, 130.4, 129.5, 129.2, 128.2,
128.0, 74.9, 68.9, 65.2, 62.9, 62.7, 53.4, 50.1, 47.3, 47.1, 43.7, 42.4, 41.8, 39.1, 38.8, 38.5, 37.6,
32.5, 32.4, 30.4, 28.0, 24.4, 24.0, 23.2, 21.1, 21.0, 20.9, 20.7, 20.5, 18.2, 17.6, 17.3, 17.0, 16.5,
16.4, 16.1, 13.8. ESIꢀHRMS m/z Calc for C48H72NO10P [M+H]+ :854.49666 founded: 854.48822.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ2ꢀmethoxyphenyl)ꢀmethyl}phosphonate
(3m). Yield 69.0%. m.p. 132.3~135.2 oC. 1H NMR (500 MHz, CDCl3) δ 7.32−7.20 (t, 2H, ArꢀH),
6.92−6.87 (m, 1H, ArꢀH), 6.84 (m, 1H, NH), 5.78−5.67 (m, 1H, Hꢀ11), 5.16–5.11 (m, 1H, Hꢀ3),
5.10–5.08 (m, 1H, Hꢀ2), 5.03−5.00 (m, 1H, PꢀCH), 4.15–4.01 (m, 2H, ꢀOCH2), 3.90–3.75 (m, 5H,
ꢀOCH2, ArꢀOCH3), 3.57−3.50 (m, 1H, Hꢀ23), 2.02 (s, 2H, COCH3), 1.98 (s, 3H, COCH3), 1.95 (s,
3H, COCH3), 1.95–1.06 (m, triterpene’s H, 21H), 1.28 (m, 6H, CH3×2), 1.05–0.99 (m, 6H,
CH3ꢀ27/24), 0.98 (s, 3H, CH3ꢀ26), 0.93 (d, J = 6.3 Hz, 3H, CH3ꢀ30), 0.84 (s, 3H, CH3ꢀ25), 0.80 (d,
J = 4.4 Hz, 3H, CH3ꢀ29). 13C NMR (126 MHz, CDCl3) δ 176.1, 170.8, 170.5, 170.3, 157.3, 138.8,
129.3, 125.4, 125.2, 123.3, 120.8, 111.0, 74.8, 69.9, 65.2, 62.6, 62.4, 55.7, 54.1, 47.9, 47.4, 43.7,
42.2, 41.8, 39.6, 39.3, 38.9, 37.6, 32.5, 32.2, 30.8, 27.6, 24.6, 24.3, 23.2, 23.0, 21.1, 21.0, 20.8,
20.7, 17.8, 17.7, 17.0, 16.8, 16.4, 16.1, 15.9, 13.9. ESIꢀHRMS m/z Calc for C48H72NO11P [M+H]+ :
870.49158 founded: 870.48547.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ3ꢀmethoxyphenyl)ꢀmethyl}phosphonate
(3n). Yield 65.7%. m.p. 131.2~134.8 oC. 1H NMR (500 MHz, CDCl3) δ 7.20 (td, J = 7.9, 3.2 Hz,
1H, ArꢀH), 6.97 − 6.88 (m, 2H, ArꢀH), 6.79 (t, J = 8.2, 6.7 Hz, 1H), 6.68 − 6.44 (m, 1H, NH), 5.48
− 5.32 (m, 1H, Hꢀ11), 5.30 − 5.24 (m, 1H, Hꢀ3), 5.15 − 5.08 (m, 1H, Hꢀ2), 5.03 (dd, J = 16.2, 10.3
Hz, 1H, PꢀCH), 4.14 − 4.01 (m, 2H, ꢀOCH2), 3.94−3.79 (m, 2H, ꢀOCH2), 3.77 (d, J = 4.2 Hz, 3H,
ArꢀOCH3), 3.73−3.53 (m, 2H, Hꢀ23), 2.04 (d, J = 12.5 Hz, 3H, COCH3), 1.99 (d, J = 2.7 Hz, 3H,
COCH3), 1.96 (d, J = 2.6 Hz, 3H, COCH3), 1.30 (t, J = 7.1 Hz, 6H, CH3×2), 1.07 (s, 3H, CH3ꢀ27),
1.04 (s, 3H, CH3ꢀ24), 1.00 (s, 3H, CH3ꢀ26), 0.94 (d, J = 5.3 Hz, 3H, CH3ꢀ30), 0.85 (s, 3H,
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CH3ꢀ25), 0.82 (d, J = 9.0 Hz, 3H, CH3ꢀ29). 13C NMR (126 MHz, CDCl3) δ 176.4 (Cꢀ28), 170.7
(COCH3), 170.4 (COCH3), 170.3 (COCH3), 159.7 (ArꢀC), 139.0 (Cꢀ13), 129.6 (ArꢀC), 125.5
(Cꢀ12), 121.0 (ArꢀC), 120.2 (ArꢀC), 114.5 (ArꢀC), 113.5 (ArꢀC), 74.8 (Cꢀ3), 69.8 (Cꢀ2), 65.2
(Cꢀ23), 62.7 (ꢀOCH2), 62.6 (ꢀOCH2), 55.2 (ꢀOCH3), 54.1 (PꢀCH), 50.3 (Cꢀ18), 48.0 (Cꢀ9), 47.8
(Cꢀ5), 43.7 (Cꢀ4), 42.3 (Cꢀ1), 41.9 (Cꢀ17), 41.8 (Cꢀ10), 39.60 (Cꢀ8), 39.3 (Cꢀ19), 38.9 (Cꢀ14),
38.8 (Cꢀ20), 32.5 (Cꢀ7), 32.4 (Cꢀ22), 30.7 (Cꢀ16), 29.6 (Cꢀ15), 27.6 (Cꢀ21), 23.4 (Cꢀ11), 23.1
(Cꢀ27), 21.1 (Cꢀ29), 21.0 (COCH3), 20.8 (COCH3), 20.7 (COCH3), 17.8 (Cꢀ25), 17.7 (Cꢀ30), 17.0
(Cꢀ26), 16.8 (Cꢀ6), 16.7 (Cꢀ24), 16.2 (CH3), 16.1 (CH3). ESIꢀHRMS m/z Calc for C48H72NO11P
[M+H]+ : 870.49158 founded: 870.48761.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ4ꢀmethoxyphenyl)ꢀmethyl}phospꢀ
honate (3o). Yield 61.9%. m.p. 133.4~135.8 oC. 1H NMR (500 MHz, CDCl3) δ 7.30–7.27 (m, 2H,
ArꢀH), 6.83 (dd, J = 8.7, 3.3 Hz, 2H, ArꢀH), 6.63–6.50 (m, 1H, NH), 5.46−5.33 (m, 1H, Hꢀ11),
5.28−5.23 (m, 1H, Hꢀ3), 5.16−5.08 (m, 1H, Hꢀ2), 5.06−5.01 (m, 1H, PꢀCH), 4.15–4.00 (m, 2H,
ꢀOCH2), 3.91–3.79 (m, 2H, ꢀOCH2), 3.77 (s, 3H, ArꢀOCH3), 3.70–3.51 (m, 2H, Hꢀ23), 2.03 (s, 3H,
COCH3), 2.00 (s, 3H, COCH3), 1.96 (s, 3H, COCH3), 1.95–1.08 (m, triterpene’s H, 21H), 1.30 (t,
J = 7.0 Hz, 6H, CH3×2), 1.06–1.03 (m, 6H, CH3ꢀ27/24), 1.00 (s, 3H, CH3ꢀ26), 0.94 (d, J = 5.9 Hz,
3H, CH3ꢀ30), 0.85 (s, 3H, CH3ꢀ25), 0.83 (d, J = 7.4 Hz, 3H, CH3ꢀ29). 13C NMR (126 MHz, CDCl3)
δ 176.3, 170.8, 170.4, 170.3, 159.4, 139.2, 138.2, 130.0, 129.1, 126.8, 125.4, 114.0, 74.8, 69.9,
65.3, 62.7, 62.5, 55.3, 54.1, 47.9, 47.5, 43.8, 42.3, 41.9, 39.6, 39.4, 38.8, 37.7, 32.5, 32.4, 30.8,
27.6, 24.6, 24.3, 23.4, 23.1, 21.1, 21.0, 20.8, 20.7, 17.8, 17.7, 17.0, 16.9, 16.6, 16.3, 16.1,
13.9.ESIꢀHRMS m/z Calc for C48H72NO11P [M+H]+ :870.49158 founded: 870.48566.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀphenyl)methyl}phꢀosphonate
(3p).
o
1
Yield 66.2%. m.p. 129.8~132.1 C. H NMR (500 MHz, CDCl3) δ 7.35 (td, J = 8.2, 3.9 Hz, 2H,
ArꢀH), 7.32−7.26 (m, 3H, ArꢀH), 6.74−6.49 (m, 1H, NH), 5.47 − 5.31 (m, 1H, Hꢀ11), 5.31 − 5.21
(m, 1H, Hꢀ3), 5.14 − 5.07 (m, 1H, Hꢀ2), 5.05 − 4.99 (m, 1H, Hꢀ2, PꢀCH), 4.16 − 4.03 (m, 2H,
ꢀOCH2), 3.89 − 3.75 (m, 2H, ꢀOCH2), 3.68 − 3.46 (m, 2H, Hꢀ23), 2.04 (d, J = 8.0 Hz, 3H,
COCH3), 1.99 (d, J = 2.7 Hz, 3H, COCH3), 1.95 (d, J = 2.3 Hz, 3H, COCH3), 1.32 − 1.26 (m, 6H,
CH3×2), 1.05 (s, 3H, CH3ꢀ27), 1.00 (m, 3H, CH3ꢀ24), 0.99 (s, 3H, CH3ꢀ26), 0.93 (d, J = 6.1 Hz,
3H, CH3ꢀ30), 0.84 (d, J = 1.9 Hz, 3H, CH3ꢀ25), 0.81 (d, J = 4.8 Hz, 3H, CH3ꢀ29). ESIꢀHRMS m/z
Calc for C47H70NO10P [M+H]+ : 840.48101 founded: 840.47461.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ1ꢀnaphthyl)methyl}ꢀphosphonate (3q).
o
1
Yield 61.2%. m.p. 141.2~144.8 C. H NMR (500 MHz, CDCl3) δ 7.88–7.83 (m, 4H, ArꢀH),
7.56–7.48 (m, 3H, ArꢀH), 6.82– 6.63 (m, 1H, NH), 5.74−5.53 (m, 1H, Hꢀ11), 5.39–5.31 (m, 1H,
Hꢀ3), 5.18−5.12 (m, 1H, Hꢀ2), 5.09−4.98 (m, 1H, PꢀCH), 4.23–4.06 (m, 2H, ꢀOCH2), 3.96–3.72
(m, 2H, ꢀOCH2), 3.75–3.49 (m, 2H, Hꢀ23), 2.09 (s, 3H, COCH3), 2.04(s, 3H, COCH3), 2.01 (s, 3H,
COCH3), 1.99–1.12 (m, triterpene’s H, 21H), 1.39−1.32 (m, 6H, CH3×2), 1.08 (s, 3H, CH3ꢀ27),
1.01 (s, 3H, CH3ꢀ24), 0.99 (s, 3H, CH3ꢀ26), 0.99 (d, J = 6.7 Hz, 3H, CH3ꢀ30), 0.91 (s, 3H,
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CH3ꢀ25), 0.87 (d, J = 8.2 Hz, 3H, CH3ꢀ29). 13C NMR (126 MHz, CDCl3) δ 176.7, 170.8, 170.5,
170.3, 138.1, 132.3, 131.6, 128.9, 128.6, 126.7, 126.4, 125.9, 125.7, 125.1, 125.0, 123.5, 74.8,
69.8, 65.2, 62.9, 62.5, 54.1, 48.0, 47.3, 43.7, 42.2 41.8, 39.6, 39.4, 38.9, 37.7, 32.5, 32.2, 30.8,
27.6, 24.6, 24.3, 23.4, 23.0, 21.1, 21.0, 20.8, 20.7, 17.8, 17.5, 16.9, 16.5, 15.9, 15.7, 15.4, 13.8.
ESIꢀHRMS m/z Calc for C51H72NO10P [M+H]+ : 890.49666 founded: 890.49374.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ12ꢀeneꢀ28ꢀoyl]ꢀ2ꢀnaphthyl)methyl}ꢀphosphonate (3r).
o
1
Yield 69.0%. m.p. 137.1~140.2 C. H NMR (500 MHz, CDCl3) δ 7.84–7.78 (m, 4H, ArꢀH),
7.52–7.45 (m, 3H, ArꢀH), 6.81–6.56 (m, 1H, NH), 5.71−5.44 (m, 1H, Hꢀ11), 5.35–5.28 (m, 1H,
Hꢀ3), 5.14−5.03 (m, 1H, Hꢀ2), 5.03−4.94 (m, 1H, PꢀCH), 4.19– 4.06 (m, 2H, ꢀOCH2), 3.93–3.73
(m, 2H, ꢀOCH2), 3.67–3.46(m, 2H, Hꢀ23), 2.01 (s, 3H, COCH3), 1.98 (s, 3H, COCH3), 1.96 (s, 3H,
COCH3), 1.95–1.08 (m, 21H), 1.35−1.31 (m, 6H, CH3×2), 1.00 (s, 3H, CH3ꢀ27), 0.98 (s, 3H,
CH3ꢀ24), 0.95−0.97 (m, 6H, CH3ꢀ26/30), 0.87 (s, 3H, CH3ꢀ25), 0.84 (d, J = 8.2 Hz, 3H, CH3ꢀ29).
13C NMR (126 MHz, CDCl3) δ 176.6, 170.8, 170.5, 170.3, 139.0, 138.2, 133.2, 133.0, 132.2,
128.4, 127.9, 127.6, 126.5, 126.4, 126.1, 125.4, 74.8, 69.9, 65.3, 62.9, 62.7, 54.2, 48.0, 47.6, 43.8,
42.2, 41.8, 39.7, 39.2, 38.9, 37.7, 32.4, 32.3, 30.8, 27.6, 24.7, 24.3, 23.4, 23.0, 21.1, 21.0, 20.8,
20.7, 17.7, 17.5, 17.0, 16.8, 16.5, 16.1, 16.0, 13.8. ESIꢀHRMS m/z Calc for C51H72NO10P
[M+H]+ : 890.49666 founded: 890.49476.
3.1.3 General procedure for compound 4 (2α, 3β, 23ꢀTriacetoxyursꢀ11ꢀoxoꢀ12ꢀeneꢀ28ꢀoic acid )
A solution of 1 (100 mg, 0.16 mmol) and K2Cr2O7·2H2O (150 mg, 0.5 mmol) in 20 mL of
acetic acid was refluxed for 5 h. The mixture was cooled to 20oC and neutralized with 10%
NaHCO3 to pH 7−8, diluted with ethyl acetate (20 mL), washed with water (10 mL × 5) and
saturated NaCl solution (15 mL). The organic phase was dried over anhydrous sodium sulfate.
Following filtration and evaporation of solvent at reduced pressure, the crude product was purified
by silica gel chromatography with a gradient elution of CH2Cl2/MeOH (30:1, v:v) to yield a white
o
1
solid (125 mg). Yield 85.5%. m.p. 191.2~194.8 C. H NMR (400 MHz, CDCl3) δ 5.72 (s, 1H,
Hꢀ12), 5.27−5.21 (m, 1H, Hꢀ2), 5.00 (d, J = 10.2 Hz, 1H, Hꢀ3), 3.78 (d, J = 11.8 Hz, 1H, Hꢀ23),
3.55 (d, J = 11.9 Hz, 1H, Hꢀ23), 3.16 (dd, J = 12.8, 4.5 Hz, 1H, Hꢀ19), 2.39 (s, 1H, Hꢀ9),
2.21−1.06 (triterpene’s H, 17H), 2.05 (s, 3H, CH3CO), 1.98 (s, 3H, CH3CO), 1.93 (s, 3H, CH3CO)
(3×CH3CO), 1.31 (s, 3H, CH3ꢀ27), 1.22 (s, 3H, CH3ꢀ24), 0.97 (d, J = 6.1 Hz, 3H, CH3ꢀ29), 0.89 (d,
J = 6.4 Hz, 3H, CH3ꢀ30), 0.85 (s, 6H, CH3ꢀ25/26). 13C NMR (101 MHz, CDCl3) δ 199.05,
182.78, 170.84, 170.54, 170.25, 163.15, 130.58, 74.89, 69.03, 65.25, 61.12, 52.52, 47.50, 47.38,
44.64, 44.18, 43.76, 41.96, 38.60, 38.53, 37.71, 36.01, 32.48, 30.25, 28.33, 23.56, 21.04, 20.99,
20.93, 20.70, 20.80, 19.07, 17.80, 17.04, 16.97, 13.90; ESIꢀHRMS m/z Calc for C42H56FNO8
[M+H]+ : 629.36841, founded: 629.36667.
3.1.4 General procedure for the preparation of compounds 6
Oxalyl chloride (1.2 mL) at 0oC was added dropwise to a solution of compound 4 (100.00 mg,
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0.14 mmol) in CH2Cl2, and the mixture was stirred at room temperature for 8 h. The solvent was
evaporated at reduced pressure, and CH2Cl2 (10 ml × 3) was added to the mixture, with vacuum
concentration. The mixture was allowed to react with individual APAs (0.56 mmol) at room
temperature for 4 h with stirring. After dilution with ethyl acetate (25 mL), the mixture was
washed with water (20 mL × 3) and the organic phase was dried over anhydrous sodium sulfate.
Following filtration and evaporation of solvent at reduced pressure, the residue was purified by
column chromatography to yield a white solid.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ11ꢀoxoꢀ12ꢀeneꢀ28ꢀoyl]ꢀ4ꢀbromophꢀenyl)methyl}phosph
o
1
onate (6i). Yield 62.0%. m.p. 157.1~160.0 C. H NMR (500 MHz, CDCl3) δ 7.42 (dd, J = 11.2,
8.5 Hz, 2H, ArꢀH), 7.27– 7.19 (m, 2H, ArꢀH), 6.69 (d, J = 47.9 Hz, 1H), 5.62 (s, 1H, Hꢀ12),
5.41−5.28 (m, 1H, Hꢀ3), 5.25 (m, 1H, Hꢀ2), 5.00 (dd, J = 10.2, 7.0 Hz, 1H, PꢀCH), 4.15–4.02 (m,
2H, ꢀOCH2), 3.92–3.78 (m, 2H, ꢀOCH2), 3.69–3.52 (m, 2H, Hꢀ23), 2.05 (s, 3H, CH3CO), 1.99 (s,
3H, CH3CO), 1.94 (s, 3H, CH3CO), 1.93−1.30 (m, 19H), 1.30 (t, J = 12.9, 6.5 Hz, 6H, CH3×2),
1.14 (s, 3H, CH3ꢀ27), 1.08 (s, 3H, CH3ꢀ24), 1.06 (s, 3H, CH3ꢀ26), 0.96 (d, J = 5.0 Hz, 3H,
CH3ꢀ30), 0.88 (s, 3H, CH3ꢀ25), 0.86 (d, J = 5.2 Hz, 3H, CH3ꢀ29). 13C NMR (126 MHz, CDCl3) δ
198.1, 175.7, 170.7, 170.4, 170.0, 162.6, 134.8, 133.9, 131.9, 131.7, 130.4, 130.3, 130.0, 129.8,
74.9, 68.9, 65.2, 63.1, 62.9, 61.0, 52.9, 50.5, 47.4, 44.1, 43.7, 41.9, 39.1, 38.8, 38.6, 37.6, 32.5,
30.4, 27.9, 24.4, 24.0, 20.9, 20.9, 20.8, 20.7, 18.5, 18.3, 17.6, 17.0, 16.9, 16.5, 16.4, 16.1, 13.8.
ESIꢀHRMS m/z Calc for C47H67BrNO11P [M+H]+ : 932.37079 founded: 932.36475.
O,O’Diethyl{Nꢀ[2α,3β,23ꢀTriacetoxyursꢀ11ꢀoxoꢀ12ꢀeneꢀ28ꢀoyl]ꢀ4ꢀmethylpheꢀnyl)methyl}phosp
honate (6n). Yield 65.1%. m.p. 161.2~164.8 oC. 1H NMR (500 MHz, CDCl3) δ 7.21 (dd, J = 28.2,
6.7 Hz, 2H, ArꢀH), 7.08 (dd, J = 11.3, 8.0 Hz, 2H, ArꢀH), 6.71−6.60 (m, 1H, NH), 5.61 (d, J = 7.0
Hz, 1H, Hꢀ12), 5.51−5.36 (m, 1H, Hꢀ3), 5.27−5.22 (m, 1H, Hꢀ2), 4.99 (dd, J = 10.2, 8.1 Hz, 1H,
PꢀCH), 4.12−4.02 (m, 2H, ꢀOCH2), 3.88−3.76 (m, 2H, ꢀOCH2), 3.64−3.51 (m, 2H, Hꢀ23), 2.3 (s,
3H, ArꢀCH3), 2.04 (s, 3H, CH3CO), 1.98 (s, 3H, CH3CO), 1.93 (s, 3H, CH3CO), 1.92−1.06 (m,
19H), 1.31−1.28 (m, 6H, CH3×2), 1.21 (s, 3H, CH3ꢀ27), 1.03 (s, 3H, CH3ꢀ24), 1.01 (s, 3H,
CH3ꢀ26), 0.95 (d, J = 6.3 Hz, 3H, CH3ꢀ30), 0.85 (s, 3H, Cꢀ25), 0.84 (d, J = 3.7 Hz, 3H, CH3ꢀ29).
13C NMR (126 MHz, CDCl3) δ 198.0, 175.1, 170.7, 170.4, 170.1, 162.7, 132.3, 131.3, 130.3,
129.4, 129.2, 128.2, 128.0, 74.8, 68.9, 65.1, 62.8, 62.7, 61.0, 53.4, 50.0, 47.2, 44.2, 43.7, 41.8,
39.1, 38.5, 37.6, 36.6, 32.4, 32.3, 30.4, 27.7, 24.3, 23.9, 21.1, 21.0, 20.8, 20.7, 18.4, 18.1, 17.6,
17.3, 17.0, 16.5, 16.4, 16.1, 13.8. ESIꢀHRMS m/z Calc for C48H70NO11P [M+H]+ : 868.47593
founded: 868.47266.
3.2. Biological Assays
3.2.1. Cytotoxicity of AA Derivatives
A549 human nonꢀsmall cell lung carcinoma cells, T24 human bladder carcinoma cells, Hctꢀ116
human colorectal carcinoma cells, SKꢀOVꢀ3 human ovarian carcinoma cells, HUVEC and Spcaꢀ2
human lung adenocarcinoma cells were all obtained from the Institute of Biochemistry and Cell
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Biology, China Academy of Sciences. They were cultured in a humidified, 5% CO2 atmosphere at
37°C and maintained in monolayer culture in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), 100 mg/mL streptomycin and 100 mg/mL
penicillin. Chemosensitivity was assessed with a 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyl
tetrazolium bromide (MTT) assay. Briefly, exponentially growing cells were seeded into 96ꢀwell
plates and treated with the indicated concentrations of AA derivatives for 48 h, and then 10 mL of
MTT (10 mg/mL) was added. After incubation for 4 h at 37oC, the purple formazan crystals (a
reduced form of MTT) generated in viable cells were dissolved by adding 100 ꢁL DMSO to each
well. The plates were swirled gently for 10 min to dissolve the precipitate, and quantified by
measuring the optical density of the plates at 490 nm using a plate reader (TECAN infinite
M1000). Each concentration was repeated in three wells and the same experimental conditions
were maintained for all testing procedures. The MTT assays were repeated three times for each
cell line.
3.2.2. Hoechst 333258 Staining
Cells grown on a sterile cover slips in sixꢀwell tissue culture plates were treated with test
compounds for the indicated time. The culture medium containing the compounds was removed,
and the cells were fixed in 4% paraformaldehyde for 10 min. After washing twice with phosphate
buffered saline (PBS), the cells were stained with 0.5 mL of Hoechst 33258 (Beyotime) for 5 min
and again washed twice with PBS. Nuclear staining was observed with
a Nikon
ECLIPSETE2000ꢀS fluorescence microscope at 350 nm excitation and 460 nm emission
wavelengths.
3.2.3. AO/EB Staining
Cells were seeded at a concentration of 5×104 cell/mL in a volume of 2 mL on sterile cover slips
in sixꢀwell tissue culture plates. Following incubation, the medium was removed and replaced
with fresh medium plus 10% FBS and supplemented with compound 3d. After treatment, cover
slips with cell monolayers were inverted on a glass slide with 20 ꢁL of AO/EB stain (100 mg/mL).
Fluorescence was read on a Nikon ECLIPSETE2000ꢀS fluorescence microscope (OLYMPUS Co.,
Japan).
3.2.4. Mitochondrial Membrane Potential Staining
Mitochondrial depolarization was assayed in T24 cells using a JCꢀ1 probe. Briefly, cells
cultured in sixꢀwell plates after the indicated treatment were incubated with an equal volume of
JCꢀ1 staining solution (5 ꢁg/mL) at 37oC for 20 min and rinsed twice with PBS. Mitochondrial
membrane potentials were monitored by determining the relative amounts of dual emissions from
mitochondrial JCꢀ1 monomers or aggregates using a Nikon ECLIPSETE2000ꢀS fluorescent
microscope. Mitochondrial depolarization was indicated by an increase in the green/red
fluorescence intensity ratio.
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3.2.5. Apoptosis Analysis
Apoptosis was assayed by annexin VꢀFITC and PI. Cells were seeded at 2×106/well in 10%
FBS–DMEM into sixꢀwell plates and treated with test compounds for 24 h. The cells were then
washed twice with cold PBS and resuspended in 1×binding buffer (0.1 M pH 7.4 Hepes/NaOH,
1.4 M NaCl, 25 mM CaCl2) at a concentration of 1×106 cells/mL. A 100 ꢁL volume of the solution
(1×105 cells) was transferred to a 5 mL culture tube; 5 ꢁL of FITC Annexin V (BD, Pharmingen)
and 5 µL PI were added to each tube. The cell suspension was gently vortexed and incubated for
30 minutes at room temperature (25oC) in the dark, and then 200 ꢁl PBS was added to each tube.
The apoptosis assay was carried out by flow cytometry (FACSVerse, BD, USA) at 488 nm
excitation. The lower left quadrant included viable cells (annexin V–PI–); lower right quadrant
included early apoptotic cells (annexin V+/PI–); upper right quadrant included late apoptotic cells
(annexin V+/PI+); and the upper left quadrant included necrotic cells (annexin V–/PI+). The
percentage of PI+ and/or Annexin VꢀFITC+ cells inside the quadrants was reported.
3.2.6. Cell Cycle Analysis
Cell cultures were treated with the indicated concentrations of compound 3d and after 48 h
incubation, the cells were washed twice with iceꢀcold PBS, fixed and permeabilized with iceꢀcold
70% ethanol at −20oC overnight. The cells were treated with 100 ꢁg/mL RNase A at 37oC for 30
min after washing with iceꢀcold PBS, and finally stained with 1 mg/mL PI in the dark at 4oC for
30 min. Cell cycle analysis was performed by flow cytometry (FACSVerse, BD, USA) at an
excitation of 488 nm and an emission of 620 nm.
3.2.7. ROS Assay
T24 cells were seeded into sixꢀwell plates, and following treatment, were incubated with 10
mM DCFHꢀDA (Beyotime, Haimen, China) dissolved in cellꢀfree medium for 30 min at 37oC and
in the dark. They were then washed three times with PBS. Cellular fluorescence was measured
with a Nikon ECLIPSETE2000ꢀS fluorescence microscope at 485 nm excitation and 538 nm
emission.
3.2.8. Calcium Analysis
To monitor the effect of AA APA derivatives on calcium release, T24 cells were seeded into
sixꢀwell plates, and loaded with 5 mM of the membraneꢀpermeable calcium indicator Fluoꢀ3
acetoxymethyl ester (Beyotime, Haimen, China) in PBS buffer for 40 min at 37oC. After loading
with the Fluoꢀ3 dye, cells were washed with PBS and suspended in Caꢀfree PBS containing 5 mM
EGTA. Fluoꢀ3 was excited by argon laser light at 488 nm; fluorescence was measured at 515 nm,
and quantified with a Nikon ECLIPSETE2000ꢀS fluorescence microscope.
3.2.9. Western Blot Assay
The western blot procedure was performed as described previously [3]. T24 cells were collected
after treatment with compound 3d (20 ꢁM) for 0, 6, 12 or 24 h and then lysed in iceꢀcold lysis
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buffer (1% sodium dodecyl sulfate in 25 mM pH 7.5 Tris–HCl, 4 mM EDTA, 100 mM NaCl, 1
mM phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin and 10 mg/mL soybean trypsin inhibitor).
Wholeꢀcell lysates were centrifuged at 12,000×g for 5 min. Thereafter, the protein concentration
was determined with a bicinchoninic acid protein assay kit (Beyotime Co, China). An aliquot of
cell lysate (40–50 ꢁg) was fractionated by SDSꢀPAGE on 12% polyacrylamide gels for 2 h and
transferred to polyvinylidene difluoride membranes. After blocking with 5% nonꢀfat dry milk in
PBSꢀt for 1 h at room temperature, the membranes were incubated with βꢀactin, cytochrome c,
caspaseꢀ9, caspaseꢀ3, Bax or Bclꢀ2 antibodies (Bioworld Technology Inc, USA) overnight at 4oC,
washed with trisꢀbuffered saline and Tween 20, and then incubated with horseradish
peroxidaseꢀconjugated secondary antibodies for 1 h at room temperature. Proteins were detected
by electrochemiluminescence, Thermo Fisher Scientific, USA) and analysed by Image J software.
3. 2. 10 Statistical Analysis
Data are expressed as mean ± SD for three different determinations. Statistical significance was
analyzed by oneꢀway ANOVA. Mean separations were performed using the least significant
difference method. P<0.05was defined as statistically significant.
Acknowledgments
This study was supported by the National Natural Science Foundation of China (No. 81260472
21362002, 21501032 and 21431001), Guangxi Natural Science Foundation of China (No.
2014GXNSFDA118007 and 2014GXNSFBA118050), the Project of Outstanding Young Teachers'
Training in Higher Education Institutions of Guangxi’, the State Key Laboratory Cultivation Base
for the Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Science and
Technology of China (CMEMR2014ꢀB14), the Innovation Program for Graduate Students in
Jiangsu Province (KYLX_0162), the Fundamental Research Funds for the Central Universities,
Special Research Found for the Doctoral Program of Higher Education(NO. 20134504110002)
and the Foundation of Ministry of Education Innovation Team (NO. IRT1225).
Conflict of Interest
The authors declare no conflict of interest.
Author Contributions
All authors of this paper have directly participated in the planning or drafting of this
manuscript and have read and approved the final version submitted.
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