Synthesis of asiatic acid derivatives and their cytotoxic activity

Article abstract of DOI:10.1007/s00044-018-2176-y

Twenty-eight compounds, including 24 new (4–22, 25–26, and 28–30) and four known (2, 23, 24, and 27) have been synthesized starting from asiatic acid (1), which was isolated from Centella asiatica. The preparation procedure included the acetylation, hydrolyzation on the 2-, 3-, 23-, and the side chain hydroxyl, amino groups, as well as the amidation of the 28-carboxylic group. All the synthesized derivatives were structurally confirmed by ¹H, ¹³C NMR, and MS spectra. Besides, they were evaluated for their cytotoxicity against three cancer cell lines: KB (human carcinoma in the mouth), HepG2 (human hepatocellular carcinoma), and SK-LU-1 (human lung carcinoma). Eleven of the tested compounds, including nine newly synthesized and two known ones showed very strong activity against three tested cell lines with the IC₅₀ values ranging from 0.67 to 37.39 μM. Three compounds showed good activity against KB and HepG2 cell lines with the IC₅₀ values ranging from 1.95 to 32.12 μM. The activity against the KB and HepG2 cell lines was in general higher than that against the SK-LU-1 cell line. The acetylation of the hydroxyl groups in the A-ring and the OH or NH groups in the amide side chains strongly enhanced the activity of the compounds. In addition, five potent compounds (9, 12, and 15–17) were also evaluated for apoptosis-inducing activities. The ratio of apoptosis and necrotic cells increased when lung cancer cells were treated with these compounds. On the other hand, compound 9 increased caspase 3 activities (P < 0.05). Thus, these compounds induced lung cancer cell death by apoptosis and necrosis.

Full text of DOI:10.1007/s00044-018-2176-y

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2176-y
ORIGINAL RESEARCH
Synthesis of asiatic acid derivatives and their cytotoxic activity
Loc Tran Van1,2 Quynh Nhu Vo Thi
2,3 ●
Chien Tran Van
1,2 ●
Phuong Thao Tran Thi
1,2 ●
Ninh Pham Thi
1 ●
●
1 ●
1 ●
4 ●
2,4 ●
Sung Tran Van1,2
Thanh Nguyen Tuan Thu Ha Le Thi Nga Nguyen Thi Thao Do Thi
Received: 18 January 2018 / Accepted: 26 March 2018
©
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Twenty-eight compounds, including 24 new (4–22, 25–26, and 28–30) and four known (2, 23, 24, and 27) have been
synthesized starting from asiatic acid (1), which was isolated from Centella asiatica. The preparation procedure included the
acetylation, hydrolyzation on the 2-, 3-, 23-, and the side chain hydroxyl, amino groups, as well as the amidation of the 28-
1
13
carboxylic group. All the synthesized derivatives were structurally confirmed by H, C NMR, and MS spectra. Besides,
they were evaluated for their cytotoxicity against three cancer cell lines: KB (human carcinoma in the mouth), HepG2
(
human hepatocellular carcinoma), and SK-LU-1 (human lung carcinoma). Eleven of the tested compounds, including nine
newly synthesized and two known ones showed very strong activity against three tested cell lines with the IC50 values
ranging from 0.67 to 37.39 µM. Three compounds showed good activity against KB and HepG2 cell lines with the IC50
values ranging from 1.95 to 32.12 µM. The activity against the KB and HepG2 cell lines was in general higher than that
against the SK-LU-1 cell line. The acetylation of the hydroxyl groups in the A-ring and the OH or NH groups in the amide
side chains strongly enhanced the activity of the compounds. In addition, five potent compounds (9, 12, and 15–17) were
also evaluated for apoptosis-inducing activities. The ratio of apoptosis and necrotic cells increased when lung cancer cells
were treated with these compounds. On the other hand, compound 9 increased caspase 3 activities (P < 0.05). Thus, these
compounds induced lung cancer cell death by apoptosis and necrosis.
Graphical Abstract
Isolation
Centella asiatica
Cytotoxic activity
against KB, Hep G2,
SK-Lu-1 cell lines
Acetylation
Amidation
Hydrolyzation
Derivatives
-30
2
Electronic supplementary material The online version of this article
https://doi.org/10.1007/s00044-018-2176-y) contains supplementary
material, which is available to authorized users.
(
2
*
Loc Tran Van
tvloc61@gmail.com
Graduate University of Science and Technology, Vietnam
Academy of Science and Technology (VAST), Hanoi, Vietnam
3
4
Gio Linh High School, Gio Linh, Quang Tri Province, Vietnam
1
Institute of Chemistry, Vietnam Academy of Science and
Technology (VAST), 18 Hoang Quoc Viet, Cau Giay,
Hanoi, Vietnam
Institute of Biotechnology, Vietnam Academy of Science and
Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam

Medicinal Chemistry Research
●
●
●
●
Keywords Apoptosis Asiatic acid derivatives Acetylation Amidation Cytotoxicity
Introduction
different amides of asiatic acid by the reaction of asiatic acid
triacetate (2), which was obtained upon the treatment of 1
with acetic anhydride in pyridine for 12 h at room tempera-
ture. The yield was about 80% after a silica gel column
chromatography. The triacetate 2 was confirmed by its posi-
Centella asatica (L.) Urban [Apiaceae] is a tropical med-
icinal plant native to South and Southeast Asian countries
including Vietnam (Do et al. 2003). This plant was reported
to own a wide range of biological activities, such as anti-
oxidant, UV protection, and collagen synthesis (Hashim
et al. 2011), anticancer, brain- stimulating effects, and
wound-healing properties (Orhan 2012; Tabassum et al.
−
+
tive ESI-MS with the ion peaks at m/z 555 [M-CH COO ]
3
−
+
and 495 [M-2xCH COO ] , and its infrared (IR) spectrum
[1746.3 and 1237.0 (acetate), 1698.9 (–COOH)]. The H and
C NMR spectra of 2 showed beside the corresponding
3
1
1
3
2
013), and the anti-inflammatory, antiproliferative, and
signals in asiatic acid (1) clear signals of three acetyl groups at
δ 1.92, 1.96, and 2.12 (all s, 3H) and δ 170.83, 170.47, and
170.37. The signals of H-2β, H-3α, and H-23 were shifted to
proapoptotic potential on 1,2-dimethylhydrazine-induced
colon carcinogenesis in experimental rats (Siddique et al.
H
C
2
017). A review on the potential pharmacological activities
a lower field and appeared at δ 3.51 and 3.78 (each 1H, d,
H
of asiatic acid and its derivatives (Lv et al. 2018), and a
paper on the attenuation of the NO-production, and inhi-
bition of the inducible nitric oxide synthase (iNOS)
expression (Qian et al. 2018) have been recently published.
Phytochemical research shows that C. asiatica contains
triterpenes and triterpene glycosides as the main biological
active constituents (Hashim et al. 2011; Zheng and Qin
J = 11.5 Hz, H-23a and H-23b), 5.01 (1H, d, J = 10.5 Hz, H-
3α), and 5.09 (1H, dt, J = 4.5, 10.5 Hz, H-2β) in comparison
to the corresponding signals [δ 3.29 and 3.52 (each 1H, d,
H
J = 11.0 Hz, H-23), 3.38 (1H, d, J = 9.5 Hz, H-3α), and 3.72
(1H, dt, J = 4.5, 9.5 Hz, H-2β)] of compound 1. The spectral
data of compound 2 are in agreement with the literature
(Zhang et al. 2009). The triacetate 2 was then transformed to
chloride 3 by the reaction with oxalyl chloride at room tem-
perature. Compound 3 then reacted with different amines in
the presence of triethylamine to afford the corresponding
amides 4–13 with the yields of 75–92% after column chro-
matography. The structures of the compounds 4–13 were
evidenced by the ESI-MS spectra with the corresponding
2
007; James and Dubery 2009; Brinkhaus et al. 2000).
Among the triterpenoids, asiatic acid, its glycoside asiati-
coside, as well as madecassic acid and madecassoside are
given much attention due to their interesting bioactivity and
available amount in the plant. The structural and chemical
modification of these triterpenoides, and evaluation of bio-
logical activities of the obtained derivatives were reported
+
−
pseudomolecular ion peaks [M + H] or [M−H] , as well as
(
2
2
Zhao et al. 2007; Jing et al. 2016; Jeong 2006; Zhang et al.
009; Goncalves et al. 2016; Li et al. 2015; Dong et al.
004; Meng et al. 2018). However, there are not much
by the additional signals for the amide side chains in their
H and C NMR spectra. The signal of the 28-COOH group
1
13
in compound 2 (δC 183.68) was high-field shifted to δC
177.38–180.00 due to the amidation. Acetylation was done on
the hydroxyl and amino groups in the amide side chains of
compounds 4–7 and 13 to obtain the corresponding tetra-
acetyl derivatives 14–17 and the pentaacetate 18 with high
yield (75–80%) after column chromatography. The structures
of the derivatives 14–18 were confirmed by the ESI-MS
spectra of the corresponding compounds and by the signals of
studies about the amidation of the carboxylic group of
asiatic acid with amines and the biological evaluation of the
obtained amides. Herein, we report on the preparation of the
2
8-amide derivatives and the study of the cytotoxicity of 27
derivatives against three cancer cell lines: KB (human car-
cinoma in the mouth), HepG2 (human hepatocellular car-
cinoma), and SK-LU-1 (human lung carcinoma). Moreover,
five most active compounds were evaluated for apoptosis-
inducing activity. When lung cancer cells were treated with
these compounds, the ratio of apoptosis and necrotic cells
increased.
1
13
the additional acetyl groups in their H and C NMR
1
spectra. There were typical changes in the H NMR spectra
(see the Experimental section). Deacetylation of compounds
4–6 and 11 in methanolic potassium hydroxide at room
temperature afforded compounds 19–22 with the yields from
72 to 80% after silica gel column chromatography. Beside the
Results and discussion
corresponding pseudomolecular ion peaks in the ESI-MS
1
13
spectra, their H and C NMR spectra showed no more
signals for the acetyl groups. Accordingly, the resonance
signals of H-2β, H-3α, and H-23 are shifted to a high field and
appeared at the similar positions to those in the spectrum of
compound 1.
Chemistry
The synthetic approaches to asiatic acid (1) are presented in
Schemes 1 and 2. Scheme 1 was designed to synthesize

Medicinal Chemistry Research
Scheme 1 Reagents and conditions: a (CH3CO)2O, pyridine, rt, 12 h, 80%, b oxalyl chloride, DCM, rt, 24 h, c RNH2, DCM, rt, 24 h, (75–92%), d
AcCl, DMAP, DCM, 2 h, 75–80%, e KOH, MeOH, rt, 16 h, 72–80%, f acetyl chloride, DMAP, rt, 85%
In another way, compound 3 was treated with 30%
aqueous ammonia solution at room temperature to afford
the triacetyl amide 23 which was subsequently deacetylated
to afford compound 24. In an attempt to get the N-acetyl
derivative of amide 23 by reaction with acetyl chloride and
dimethylaminopyridine (DMAP) at room temperature, the
nitrile 25 was obtained instead of the desired N-acetyl
derivative. The nitrile 25 was formed due to the easy
dehydration of the primary amide group in the presence of
acetyl chloride and DMAP. Hydrolyzation of compound 25

Medicinal Chemistry Research
Scheme 2 Reagents and conditions: a 2,2-dimethoxypropane, TsOH.H2O, rt, 7 h, 88%, b succinic anhydride, DMAP, TEA, rt, 12 h, 74%, c
(
CH3CO)2O, pyridine, rt, 12 h, 82%, d 5% HCl, rt, 4 h, 90%
in methanolic potassium hydroxide solution afforded nitrile
3.54 (1H, d, J = 9.5 Hz, H-3) in comparison with δ 3.73
(1H, dt, J = 4.5, 10.5 Hz, H-2) and 3.40 (1H, d, J =
10.5 Hz, H-3) in compound 27. The carboxyl signals of
H
1
2
6. The H NMR spectrum of compound 23 contained the
signals of the –NH amide protons at δ 5.48 and 5.83 (each
2
H
1
13
1
H, brs). The H and C NMR spectroscopic data of
succinic residues appeared at δ 171.71 (COO–) and 178.04
C
1
3
compounds 23 and 24 are in good agreement with the lit-
erature (Zhang et al. 2009). The presence of a nitrile group
(COOH) in the C NMR spectrum of 28. The (−)-ESI-MS
spectrum of compound 29 contained a pseudomolecular ion
−
in compounds 25 and 26 was confirmed by the signals at δ
peak at m/z 569 [M–H] (100). The (+)-ESI-MS spectrum
C
13
1
22.94 and 126.41, respectively, in their C NMR, as well
of compound 30 showed a pseudomolecular ion peak at m/z
−
1
531 [M + H]+ (100). Compared to the H and C NMR
spectra of compound 29, the resonance signals from the
acetonide group disappeared in the spectra of compound 30.
1
13
as the absorption at 2228.8 cm in the IR spectrum. In
addition, this group was clearly supported by pseudomole-
+
cular ion peaks [M + Na] at m/z 618 and 492 in the (+)
ESI-MS spectra, respectively.
In Scheme 2, some changes in the A-ring of asiatic acid
Biological assay
(
1) were carried out to evaluate the cytotoxicity of the
obtained derivatives. Compound 1 was treated with 2,2-
dimethoxypropane in toluenesulfonic acid (TsOH) at room
temperature to afford compound 27 (88%). Compound 27
showed identical NMR spectra with those in the literature
The carboxyl and hydroxyl groups of the ursane triterpe-
noids are supposed to contribute remarkable biological
activities of this substance class. Our goal in this work is to
evaluate the cytotoxicity of the synthesized compounds
against three human cancer cell lines and one non-malignant
cell line using the MTT method (Tim 1983; Cos et al. 2006;
Scudiero et al. 1988). The obtained results are given in
Table 1.
(
Jeong et al. 2007a, b). The (−)-ESI-MS of 27 contained a
−
1
pseudomolecular ion peak at m/z 527 [M–H] (100). Its H
and C NMR spectra contained additional resonance sig-
1
3
nals of an acetonide group at δ 1.42 and 1.48 (each 3H, s);
H
δC 100.72. For obtaining a succinate derivative, which is
believed to be biologically active, compound 27 reacted
with succinic anhydride in DMAP and TEA at room tem-
perature to yield succinate 28. Besides, compound 27 was
acetylated to the acetyl derivative 29, which yielded the 2-
acetyl-asiatic acid (30) upon hydrolyzation. The structures
of compounds 27–30 were confirmed by spectroscopic
methods. The (−)-ESI-MS of compound 28 showed a
As shown in Table 1, eleven of the tested compounds (6,
8–10, 12, 14–17, and 23–24) exhibited very strong activity
against three cancer cell lines with the IC50 values ranging
from 0.67 to 37.39 µM. Among those, the five compounds
(9, 12, 15, 16, and 17) exhibited the strongest activities
against all tested cancer cell lines. Interestingly, those five
compounds only presented very moderate cytotoxic activ-
ities on NIH/3T3 normal cells, showing their cancerous
potential targeting. Compounds 5, 7, and 11 were at good
activities against the cell lines KB and HepG2 with the IC50
values ranging from 1.95 to 32.12 µM. Table 1 showed that
−
1
pseudomolecular ion peak at m/z 627 [M–H] (100). Its H
1
3
and C NMR spectra exhibited the corresponding reso-
nance signals at δ 5.03 (1H, dt, J = 4.5, 9.5 Hz, H-2) and
H

Medicinal Chemistry Research
Table 1 In vitro cytotoxic activity of synthesized asiatic acid derivatives
Compounds
IC50 (µM)
KB
Compounds
IC50 (µM)
KB
HepG2
65.57
SK-LU-1
n.d
NIH/3T3
n.d
HepG2
SK-LU-1
NIH/3T3
70.40 ±
1
2
61.93
36.32
16
17
1.34
1.11
1.17
2.51
4
.12
18.80 ±
.29
39.77
89.32
n.d
1.09
1.27
2
4
5
6
7
8
9
23.70
8.74
4.16
23.67
4.03
1.39
4.89
1.95
1.31
n.a
81.25
32.12
12.74
21.58
3.06
1.99
6.52
8.50
1.33
n.a
69.49
42.04
8.84
74.68
3.85
4.14
8.05
86.31
0.85
n.a
n.d
18
19
20
21
22
23
24
25
26
27
28
29
30
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.a
n.a
n.a
n.d
122.10
35.93
n.a
186.80
192.22
n.a
103.02
80.40
n.a
n.d
n.d
7.95 ± 0.78
n.d
0.67
0.82
9.30
1
1
1
1
1
1
0
1
2
3
4
5
3.51
2.75
17.54
33.29
42.62
142.41
125.78
34.05
151.85
n.d
29.18
38.93
33.86
94.62
20.46
50.94
31.48
42.22
109.13
203.82
28.40
144.36
21.05 ± 0.87
n.d
0.70
1.78
1.42
0.70
1.54
1.83
37.39
2.33
2.15
n.d
31.14 ± 2.61
1.54 ± 0.08
Ellipticine
Significant values are shown in bold
n.a considered as not active
n.d not determined
acetylation of the hydroxyl and the amino groups strongly
increased the cytotoxicity against the tested cell lines. The
transformation of the 28-COOH group of asiatic acid (1)
into the amides increased the cytotoxicity of the most tested
derivatives. The nitrile derivatives 25 and 26, where the
carboxyl group is lost, showed a moderate activity, only.
Our results are similar with the results in the published
literature for 11-oxoasiatic acid derivatives with substituted
aniline (Li et al. 2014; Meng et al. 2018).
Two models of cell death that are typically discussed are
necrosis and apoptosis (Fink and Cookson 2005). Apoptosis
is the programmed cell death which is characterized by the
change of morphological characteristics and the energy-
dependent cascade mechanism (Elmore 2007). The induc-
tion of apoptosis is one of the key characteristics of antic-
ancer drugs, as it plays an imperative role in the elimination
of damaged cells and the maintenance of homeostasis, and
many of the natural chemopreventive agents (Rasul et al.
normal cells (Table 1), were selected to measure for
apoptosis-inducing activities using lung cancer cells. These
compounds induced a change in cellular morphology at
different levels. When the cells were treated with different
compounds at the same concentrations, some cells showed
the hallmarks of apoptotic morphology, such as cell
shrinkage, chromatin condensation, or fragmentation.
Compound 9 presented the highest ratio with 9.85%
apoptotic cells and compound 12 showed the lowest ratio
with 4.52% of apoptotic cells (Fig. 1).
Beside the change of cell morphology, one of the early
events of apoptosis is the loss of phospholipid asymmetry
that leads to phosphatidylserine translocation from the
internal of the plasma membrane to the external of the
membrane (Elmore 2007). The translocation of phosphati-
dylserine occurs before the loss of cell membrane integrity
that is induced by the cell death in apoptosis or necrosis
process. On the other hand, phosphatidylserine has high
affinity with annexin V in the presence of calcium (Lizarbe
et al. 2013). Therefore, by using the fluorescent prober
Annexin V-FITC and propidium iodide, the cellular apop-
tosis population induced by treatment with compounds 9,
12, 15, 16, and 17 can be detected by the flow-cytometry
system. In flow-cytometry analysis of Annexin V-FITC and
propidium iodide double stain, four cell populations were
distinguished: the live cell at the lower-left quadrant
2
012). In the apoptotic process, the typical common
morphologies are cell shrinkage, nuclear and cytoplasmic
condensation, membrane blebbing, the fragmentation of
chromatin, and formation of pyknotic bodies of condensed
chromatin (Krysko et al. 2008; Fink and Cookson 2005).
These characteristics are usually used to determine apop-
totic cells under the microscope. Five compounds (9, 12,
1
5, 16, and 17), which had the strongest cytotoxic activity
−
−
on cancerous cells but very moderate activity on NIH/3T3
(Annexin V /PI ); early apoptosis cells at the lower-right

Medicinal Chemistry Research
Fig. 1 Effect of five compounds (9, 12, 15, 16, and 17) on nuclear
morphology after 24 h of treatment using Hoechst 33342 staining. The
SK-LU-1 cells that were treated with 0.5% DMSO (a) showed normal
oval shape of nucleases. Some cells treated with different tested
compounds 9, 12, 15, 16, and 17 (b–f, respectively) showed con-
densed or fragmented chromatin. The reference compound (camp-
tothecin) also induced apoptosis significantly (g)
Fig. 2 Effect of five compounds on induction of apoptosis in SK-LU-1 cells using Annexin V and PI staining. The cells were treated with 0.5%
DMSO (a) or different compounds 9, 12, 15, 16, and 17 at 16 µM (c–g, respectively)
+
−
quadrant (Annexin V /PI ); late apoptosis cells at the
activity, and compound 17 showed the lowest efficiency
than the others did. The number of necrotic cells also
increased (6.29–12.02%) when compared with negative
control (1.27%). Especially, compound 12 induced necrosis
up to 12.02%. These results suggested that five testing
compounds induced cell death through both apoptosis and
necrosis.
In order to clearly understand apoptotic mechanisms, the
effects of these compounds on caspase 3 activity were
examined. Apoptosis is an energy-dependent process that
+
+
upper-right quadrant (Annexin V /PI ); and necrotic cells
−
+
at the upper-left quadrant (Annexin V /PI ). As presented
in Fig. 2, compounds 9, 12, 15, 16, and 17 all induced
apoptosis after 24 h of treatment. As negative control, the
result showed that only 8.07% cells showed apoptotic
stages. Altogether, the ratio of both early and late apoptotic
cells increased, ranging from 11.37 to 21.23%. Compound
9
induced apoptosis more efficiently than the others.
Compounds 12, 15, and 16 displayed similar apoptosis

Medicinal Chemistry Research
2
Experimental
1
1
1
1
.8
.6
.4
.2
1
General
0
4
8
µM
µM
µM
Reagents, except asiatic acid which was isolated from C.
asiatica, were purchased from Aldrich and Merck with
analytical grade and used without further purification. Sol-
vents for column chromatography were distilled before
using. IR spectra were recorded on an IMPACT 410 Nicolet
spectrometer. ESI-MS spectra were measured on 1100
0
0
0
0
.8
.6
.4
.2
0
16 µM
.5 µM
2
1
13
Agilent LC/MS ion Trap. NMR spectra ( H, C, DEPT,
compound 9 compound 12 compound 15 compound 16 compound 17 camptothecin
HSQC, and HMBC) were recorded on a Bruker Avance
Fig. 3 Effect of compounds 9, 12, 15, 16, and 17 at different con-
centrations on the activities of caspase 3 in SK-LU-1 cells. Values
represent mean ± SD of three independent experiments
1
5
00 MHz with TMS as the internal standard for H and
1
3
solvent signal for C. Coupling constants (J) are given in
Hz. The multiplicities of signals are abbreviated as the
following: singlet (s), broad (br), doublet (d), triplet (t),
doublet of triplets (dt), and multiplet (m). Thin-layer chro-
matographies were performed on a pre-coated silica gel 60
F254 (Merck) and were visualized under UV light (λmax
254 nm) and stained with a solution of 1% (w/w) vanillin in
H SO . Column chromatography was performed on silica
involves the activation of caspase groups, such as caspase 2,
8
, 9, and 10 (apoptosis initiator caspases) or caspase 3, 6,
and 7 (apoptosis effector caspases) (Lavrik and Krammer
009; Elmore 2007). Among them, caspase 3 is a frequently
2
activated death protease, catalyzing the specific cleavage of
many key cellular proteins (Porter and Jänicke 1998).
Caspase 3 is also required for some typical hallmarks of
apoptosis, such as chromatin condensation and DNA frag-
mentation in many cell types (Lakhani et al. 2006).
Therefore, the caspase 3 activity is one of the indicators of
apoptosis induction. As the results show in Fig. 3, the
caspase 3 activity increased after compound 9 treatment at
2
4
gel 300–400 mesh (Merck). Melting points (in °C) were
determined on a Boetius apparatus (Germany). Flow-
cytometry analysis apoptosis was done with the NOVO-
CYTE flow cytometer (Acea Bioscience Inc., San Diego,
CA 92121, USA).
8
0
µM and 16 µM in comparison with negative control (P <
.05). Compounds 15, 16, and 17 slightly induced caspase 3
Synthesis
activity at 16 µM (compounds 15 and 16) or 8 µM (com-
pound 17) compared with negative control but there is no
statistical significance. Compound 12 failed to induce cas-
pase 3 at all tested concentrations for 24 h. On the contrary,
this compound cleavaged caspase 3 at 4, 8, and 16 µM (P <
Isolation of asiatic acid (1)
Ground dried Centella asiatica whole plant (1800 g) was
extracted with MeOH three times (3 × 5 L) at 80 °C for 2 h.
The filtrate was concentrated under reduced pressure to
0
.05). Thus, compounds 9, 12, 15, 16, and 17 again proved
yield a residue (330 g), which was suspended with H O
2
that they induced cell death through apoptosis and necrosis
pathways.
(0.8 L) and partitioned by n-hexane and EtOAc to obtain the
corresponding residues of 40 and 50 g. The aqueous phase
was then concentrated to dryness and redissolved in MeOH,
followed by treatment with 10% NaOH at 80 °C for 2 h.
Afterward, the solution was cooled to 10 °C with ice-water
bath and acidified with 5% HCl to pH ≈4. The precipitated
solid was collected and subjected to a silica gel column
chromatography eluting with a gradient solvent of DCM/
MeOH (95/5 → 90/10) to furnish asiatic acid (1) (6.8 g,
0.37% w/w) as a white powder; mp 222–224 °C. IR (KBr)
νmax 1459, 1690 (–COOH), 2866, 2930, and 3411 (–OH)
Conclusion
Twenty-four new (4–22, 25, 26, and 28–30) and four (2,
2
3, 24, and 27) known derivatives of asiatic acid (1) iso-
lated from the plant Centella asiatica were synthesized and
evaluated for their cytotoxic activity against three cancer
cell lines: KB, HepG2, and Lu-1. The results showed that
acetylation of the hydroxy and amino groups and/or ami-
dation of the carboxyl group strongly increased the cyto-
toxic activity of the synthesized compounds. Five of the
most active derivatives (compounds 9, 12, and 15–17) were
evaluated for apoptosis-inducing activities. As a result,
these compounds induced lung cancer cell death by apop-
tosis and necrosis.
−
1
−
1
cm . ESI-MS m/z 487 [M–H] . H NMR (CD OD,
3
500 MHz) δ = 0.72 (3H, s), 0.87 (3H, s), 0.91 (3H, d, J =
6.5 Hz), 0.99 (3H, brs), 1.07 (3H, s), 1.16 (3H, s), 2.23 (1H,
d, J = 11.5 Hz, H-18), 3.29 (1H, d, J = 11.0 Hz, H-23a),
3.38 (1H, d, J = 9.5 Hz, H-3), 3.52 (1H, d, J = 11.0 Hz, H-
23b), 3.72 (1H, dt, J = 4.5, 9.5 Hz, H-2), and 5.26 (1H, t, J
1
3
= 3.5 Hz, H-12); C NMR (CD OD, 125 MHz) δ = 181.63
3

Medicinal Chemistry Research
(
–COOH, C-28), 139.81 (C-12), 126.66 (C-11), 78.17 (C-
N-(2α,3β,23-Triacetoxyurs-12-ene-28-oyl)-n-heptyldiamine
+
3
4
3
), 69.68 (C-2), 66.31 (C-23), 54.34 (C-18), 48.16, 48.03,
4.18 (2C), 43.38, 40.79, 40.40 (2C), 38.97, 38.10, 33.63,
1.76, 29.15, 25.30, 24.46, 24.15 (2C), 21.58, 19.06, 17.85
(4) White solid. Yield: 85%. ESI-MS m/z 727 [M + H] .
1
H NMR (CD OD, 500 MHz) δ = 0.85 (3H, s), 0.94 (6H,
3
brs), 0.99 (3H, d, J = 5.5 Hz), 1.16 (3H, s), 1.71 (3H, s),
1.99 (3H, s), 2.04 (3H, s), 2.08 (3H, s), 2.17 (1H, d, J =
(
2C), 17.67 (2C), and 13.93.
1
0.5 Hz, H-18), 2.69 (2H, t, J = 7.0 Hz, 2H-1′), 3.13 (2H, t,
J = 7.0 Hz, 2H-7′), 3.66 (1H, d, J = 12.0 Hz, H-23a), 3.86
1H, d, J = 12.0 Hz, H-23b), 5.05 (1H, d, J = 10.5 Hz, H-3),
5.20 (1H, dt, J = 4.5, 11.0 Hz, H-2), and 5.36 (1H, t-line, H-
Synthesis of 2,3,23-triacetyl asiatic acid (2)
(
A stirred solution of asiatic acid (1.46 g, 3 mmol) in pyridine
1
3
(
10 mL) was treated with acetic anhydride (10 mL) at room
12); C NMR (CD OD, 125 MHz) δ = 180.00 (C-28),
3
temperature for 12 h. The reaction mixture was then diluted
172.43, 172.21 (2C), 140.26 (C-13), 126.47 (C-12), 76.30
(C-3), 71.04 (C-2), 66.04 (C-23), 54.11 (C-18), 44.79,
43.34, 43.21, 40.97, 40.93 (2C), 40.80, 40.58, 40.27, 39.02,
38.78, 33.59, 31.89, 30.70, 30.24, 29.93, 29.22, 28.90,
27.96, 27.49, 25.11, 24.40, 23.99, 21.56 (C2), 20.97, 20.75,
20.72 (2C), 18.93, 17.94, 17.72, 17.49, and 14.25.
with EtOAc (40 mL), washed successively with H O, 1 N
2
HCl, and the saturated aqueous solution of NaHCO . The
3
solvent was evaporated under reduced pressure, and the
residue was chromatographed on silica gel column
(
n-hexane/EtOAc, 2/1) to yield 2 (1.47 g, 80%), as a white
−
+
solid; mp 160–163 °C. ESI-MS m/z 555 [M-CH COO ] .
3
IR (KBr) νmax 1237 (–CO-acetate), 1698 (–COOH), 1746
N-(2α,3β,23-Triacetoxyurs-12-ene-28-oyl)-n-nonadiamine
−
1
1
+
(
–CO-ester), 2875, and 2953 cm . H NMR (CDCl3,
(5) White powder. Yield: 75%. ESI-MS m/z 755 [M + H] .
1
5
0
00 MHz) δ (ppm): 0.69 (3H, s), 0.78 (3H, d, J = 6.5 Hz),
.81 (3H, s), 0.86 (3H, d, J = 6.0 Hz), 1.00 (3H, s), 1.03
H NMR (CD OD, 500 MHz) δ = 0.84 (3H, s), 0.94 (3H,
3
s), 0.94 (3H, d, J = 6.0 Hz), 0.99 (3H, d, J = 6.0 Hz), 1.16
(3H, s), 1.17 (3H, s), 2.00 (3H, s), 2.04 (3H, s), 2.04 (3H, s),
2.08 (3H, s), 2.17 (1H, d, J = 10.5 Hz, H-18), 2.94 (2H, t, J
= 7.5 Hz, 2H-1′), 3.12–3.14 (2H, m, 2H-9′), 3.66 (1H, d, J
= 12.0 Hz, H-23a), 3.87 (1H, d, J = 12.0 Hz, H-23b), 5.06
(1H, d, J = 10.5 Hz, H-3), 5.18 (1H, dt, J = 3.5, 10.5 Hz, H-
(
3H, s), 1.92 (3H, s, –CH –CO), 1.96 (3H, s, –CH –CO),
3
3
2
3
.02 (3H, s, –CH –CO), 2.12 (1H, d, J = 11.0 Hz, H-18),
.51 (1H, d, J = 11.5 Hz, H-23a), 3.78 (1H, d, J = 11.5 Hz,
3
H-23b), 5.01 (1H, d, J = 10.5 Hz, H-3), 5.09 (1H, dt, J =
13
4
.5, 10.5 Hz, H-2), and 5.17 (1H, t-line, H-12); C NMR
1
3
(
CDCl , 125 MHz) δ = 183.68 (C-28), 170.83 (CH CO–),
2), and 5.36 (1H, t, J = 3.5 Hz, H-12); C NMR (CD OD,
3
3
3
1
1
5
3
2
1
70.48 (CH CO–), 170.37 (CH CO–), 138.01 (C-12),
125 MHz) δ = 180.01 (C-28), 172.48, 172.33, 172.24,
140.36 (C-13), 132.40 (C-12), 76.28 (C-3), 71.12 (C-2),
66.25 (C-23), 54.19 (C-18), 44.78, 43.37, 43.23, 40.96,
40.85, 40.69, 40.27, 40.18, 39.05, 38.77, 33.60, 31.89,
30.56, 30.41, 30.34, 30.23, 30.13, 28.91, 28.72, 28.27,
27.51, 25.13, 24.96, 24.43, 23.98, 21.55, 20.99, 20.74,
3
3
25.18 (C-12), 74.77 (C-3), 69.88 (C-2), 65.21 (C-23),
2.40 (C-18), 47.86, 47.52, 47.44, 43.67, 41.86, 39.44,
8.93, 38.73, 37.75, 36.58, 32.36, 31.87, 30.52, 29.64,
9.30, 27.82, 23.90, 23.39, 23.27, 22.64, 21.17, 21.04,
7.50, 16.88, 14.08, and 13.86.
20.71, 18.94, 17.99, 17.70, 17.54, 14.38, and 14.24.
General procedure for the synthesis of 2,3,23-
triacetylasiatic acid-28-amides (4–13)
N-(2α,3β,23-Triacetoxyurs-12-ene-28-oyl)-n-decadiamine
+
(
6) Brown powder. Yield: 76%. ESI-MS m/z 769 [M + H] .
1
To a solution of 2 (307 mg, 0.5 mmol, 1 equiv.) in anhy-
H NMR (CD OD, 500 MHz) δ = 0.85 (3H, s), 0.94 (3H,
3
drous dichloromethane (DCM, 15 mL), oxalyl chloride
s), 0.94 (3H, d, J = 6.5 Hz), 0.99 (3H, d, J = 6.0 Hz), 1.16
(3H, s), 1.17 (3H, s), 2.00 (3H, s), 2.04 (3H, s), 2.08 (3H, s),
2.17 (1H, d, J = 11.0 Hz, H-18), 2.94 (2H, t, J = 7.5 Hz,
2H-1′), 3.14–3.11 (2H, m, 2H-10′), 3.66 (1H, d, J =
12.0 Hz, H-23a), 3.87 (1H, d, J = 12.0 Hz, H-23b), 5.06
(1H, d, J = 10.5 Hz, H-3), 5.20 (1H, dt, J = 4.5, 10.5 Hz, H-
(
3.0 mmol, 6 equiv.) in DCM (2 mL) was added. After
being stirred at room temperature for 24 h, the solvent and
excess of oxalyl chloride were removed under reduced
pressure. The residue was redissolved in anhydrous DCM
(
(
(
20 mL), followed by the addition of triethylamine
3.0 mmol, 6 equiv.) and a solution of monoamine
0.75 mmol, 1.5 equiv.) or diamine compounds (1.2 mmol,
1
3
2), and 5.36 (1H, t, J = 3.5 Hz, H-12); C NMR (CD OD,
3
125 MHz) δ = 179.98 (C-28), 172.43, 172.26, 172.19,
140.36 (C-13), 126.46 (C-12), 76.30 (C-3), 71.07 (C-2),
66.09 (C-23), 54.19 (C-18), 44.82, 43.38, 43.23, 41.09,
40.96, 40.84, 40.73, 40.27, 39.05, 38.76, 33.61, 31.90,
30.68, 30.52, 30.50, 30.44, 30.33, 29.46, 28.52, 28.29,
27.56, 26.19, 25.14, 24.43, 23.98, 23.66, 21.55, 20.97,
20.74, 20.71, 18.94, 18.01, 17.71, 17.54, 14.40, and 14.26.
4
.0 equiv.) in DCM (2 mL). After stirring for further 20 h,
the reaction mixture was quenched with DCM (30 mL) and
washed with 5% HCl. The organic phase was dried over
Na SO and evaporated to dryness. The pure products 4–13
2
4
were obtained by purification on silica gel column chro-
matography using n-hexane/EtOAc as the eluent.

Medicinal Chemistry Research
N-(2α,3β,23-Triacetoxyurs-12-ene-28-oyl)-n-propanolamine
23.25, 21.18, 21.06, 20.85, 20.76 (2C), 17.87, 17.13 (2C),
17.05, 16.43, 14.15, and 13.89.
(
7) White solid. Yield: 81%; mp 121–123 °C. ESI-MS m/z
+
6
1
5
72 [M + H] . IR (KBr) ν
1037, 1237 (–CO-acetate),
max
−
1 1
629, 1740, 2867, 2923, and 3381 cm . H NMR (CDCl ,
N-(2α,3β,23-Triacetoxyurs-12-ene-28-oyl)-imidazol (10)
3
+
1
00 MHz) δ = 0.79 (3H, s), 0.86 (3H, d, J = 6.5 Hz), 0.89
White powder. Yield: 84%. ESI-MS m/z 664 [M] . H
(
3H, s), 0.96 (3H, brs), 1.10 (3H, s), 1.11 (3H, s), 1.99 (3H,
s), 2.03 (3H, s), 2.08 (3H, s), 3.11–3.16 (1H, m, H-1′a),
.53–3.63 (3H, m, H-1′b, 2H-3′), 3.59 (1H, d, J = 12.0 Hz,
H-23a), 3.84 (1H, d, J = 12.0 Hz, H-23b), 5.08 (1H, d, J =
0.0 Hz, H-3), 5.16 (1H, dt, J = 4.5, 10.0 Hz, H-2), 5.32
1H, t-line, H-12), and 6.18 (1H, t, J = 5.5 Hz,
NMR (CDCl , 500 MHz) δ = 0.70 (3H, s), 0.89 (3H, s),
3
0.91 (3H, d, J = 6.0 Hz), 1.01 (3H, d, J = 6.0 Hz), 1.09 (3H,
s), 1.12 (3H, s), 1.98 (3H, s), 2.03 (3H, s), 2.08 (3H, s), 2.46
(1H, d, J = 11.0 Hz, H-18), 3.59 (1H, d, J = 11.5 Hz, H-
23a), 3.82 (1H, d, J = 11.5 Hz, H-23b), 5.06 (1H, d, J =
10.0 Hz, H-3), 5.16 (1H, dt, J = 4.5, 10.5 Hz, H-2), 5.25
(1H, brs, H-12), 7.03 (1H, s, H-2′), 7.54 (1H, s, H-1′), 8.26
3
1
(
1
3
–
NH–amide); C NMR (CD OD, 125 MHz) δ = 179.42
3
1
3
(
C-28), 170.73, 170.42, 170.30, 139.76 (C-13), 125.16 (C-
(1H, s, H-3′); C NMR (CDCl , 125 MHz) δ = 174.67 (C-
3
12), 74.77 (C-3), 69.86 (C-2), 65.25 (C-23), 59.26 (C-3′),
53.72 (C-18), 47.81, 47.57, 47.49, 43.76, 42.45, 41.90,
39.71, 39.58, 39.01, 37.74, 37.45, 36.09, 32.41, 32.21,
30.80, 27.72, 24.72, 23.42, 23.13, 21.15, 21.00, 20.78,
20.70, 17.83, 17.15, 17.00, 16.81, and 13.85.
28), 170.82, 170.43, 170.40, 137.14 (C-13), 137.08 (C-3′),
129.74 (C-1′), 126.16 (C-12), 117.94 (C-2′), 74.85 (C-3),
69.88 (C-2), 65.33 (C-23), 54.18 (C-18), 47.56 (2C), 43.82,
41.16, 41.93, 39.52, 39.29, 38.72, 37.78 (2C), 35.58, 32.36,
30.41, 27.71, 25.02, 23.48, 23.41, 21.07, 21.04, 20.87,
20.78, 17.82, 17.06 (2C), 16.77, 13.91.
N-(2α,3β,23-Triacetoxyurs-12-ene-28-oyl)-hydrazide (8)
+
Brown powder. Yield: 72%. ESI-MS m/z 629 [M + H] . IR
N-(2α,3β,23-Triacetoxyurs-12-ene-28-oyl)-methylhydrazide
+
(
(
KBr) νmax 1043, 1235 (–CO-acetate), 1377, 1476, 1742
acetate), 2929, and 3418 (–NH ) cm . H NMR (CDCl ,
(11) White powder. Yield: 68%. ESI-MS m/z 643 [M + H] .
−
1
1
1
H NMR (CDCl , 500 MHz) δ = 0.75 (3H, s), 0.86 (3H, d,
2
3
3
5
00 MHz) δ = 0.77 (3H, s), 0.85 (3H, d, J = 6.5 Hz), 0.89
J = 6.0 Hz), 0.89 (3H, s), 0.94 (3H, d, J = 6.5 Hz), 1.08
(
3H, s), 0.94 (3H, d, J = 6.5 Hz), 1.05 (3H, s), 1.08 (3H, s),
(3H, s), 1.10 (3H, s), 1.98 (3H, s), 2.02 (3H, s), 2.09 (3H, s),
1
1
.98 (3H, s), 2.02 (3H, s), 2.09 (3H, s), 2.24 (1H, d, J =
1.5 Hz, H-18), 3.58 (1H, d, J = 12.0 Hz, H-23a), 3.86 (1H,
3.17 (3H, s, –NH–CH ), 3.57 (1H, d, J = 12.0 Hz, H-23a),
3
3.86 (1H, d, J = 12.0 Hz, H-23b), 4.12 (0.5H, q, J = 7.0 Hz,
–NH–CH ), 4.36 (0.5H, q, J = 7.0 Hz, –NH–CH ), 5.08
d, J = 12.0 Hz, H-23b), 5.09 (1H, d, J = 10.5 Hz, H-3), 5.16
1H, dt, J = 4.5, 11.0 Hz, H-2), and 5.25 (1H, t, J = 3.0 Hz,
3
3
(
(1H, d, J = 10.5 Hz, H-3), 5.16 (1H, dt, J = 4.0, 10.5 Hz, H-
2), and 5.22 (1H, t-line, H-12).
1
3
H-12); C NMR (CDCl , 125 MHz) δ = 177.38 (C-28),
3
1
7
4
3
2
1
70.81, 170.40, 170.33, 138.31 (C-13), 124.96 (C-12),
4.87 (C-3), 69.91 (C-2), 65.29 (C-23), 52.82 (C-18),
7.82, 47.62, 47.51, 43.79, 42.06, 41.89, 39.60, 39.05,
8.83, 37.78, 36.56, 32.55, 30.65, 29.66, 29.33, 27.87,
4.08, 23.37, 23.32, 22.66, 21.15, 21.04, 20.83, 17.89,
7.02, 16.94, 14.20, 14.08, and 13.90.
N-(2α,3β,23-Triacetoxyurs-12-ene-28-oyl)-isopropylamine
(12) White solid. Yield: 74%; mp = 252–253 °C. ESI-MS
+
m/z 656.4 [M + H] . IR (KBr) ν
1042, 1232, 1639,
max
−
1 1
1739, 2923, and 3412 cm . H NMR (CDCl , 500 MHz) δ
3
= 0.76 (3H, s), 0.84 (3H, d, J = 6.0 Hz), 0.88 (3H, s), 1.09
(3H, d, J = 6.5 Hz), 1.10 (3H, s), 1.12 (3H, s), 1.31 (3H, s),
N-(2α,3β,23-Triacetoxyurs-12-ene-28-oyl)-glycine
ethyl
1.94 (3H, s), 1.99 (3H, s), 2.02 (3H, s), 3.65 (1H, d, J =
12.5 Hz, H-23a), 3.79 (1H, d, J = 12.0 Hz, H-23b), 3.92
(1H, hept, J = 7.0 Hz, H-1′), 4.93 (1H, d, J = 10.0 Hz, H-3),
5.16 (1H, dt, J = 10.5 Hz, H-2), and 5.49 (1H, brs, H-12);
ester (9) Brown solid. Yield: 78%; mp 251–251 °C. ESI-
+
1
MS m/z 700 [M + H] . H NMR (CDCl , 500 MHz) δ =
3
0
.72 (3H, s), 0.87 (3H, d, J = 6.0 Hz), 0.86 (3H, s), 0.96
1
3
(
3H, brs), 1.09 (3H, s), 1.10 (3H, s), 1.29 (3H, t, J = 7.0 Hz,
C NMR (CDCl , 125 MHz) δ = 176.56 (C-28), 170.32,
3
H-2″), 1.98 (3H, s), 2.02 (3H, s), 2.08 (3H, s), 3.58 (1H, d,
J = 12.0 Hz, H-23a), 3.84 (1H, d, J = 12.0 Hz, H-23b), 3.83
170.25, 169.91, 138.72 (C-13), 124.91 (C-12), 74.30 (C-3),
69.41 (C-2), 65.02 (C-23), 54.01 (C-18), 47.68, 47.42,
46.43, 45.78, 43.09, 41.19, 39.52, 39.65, 38.97, 37.38,
34.58, 31.48, 30.65, 28.98, 27.68, 27.12, 25.45, 24.34,
23.49, 23.01, 22.84, 22.55, 21.59, 20.93, 20.74, 17.02,
14.56, 14.54, and 13.92.
(
1H, dd, J = 4.0, 18.5 Hz, H-1′a), 4.05 (1H, dd, J = 5.0,
8.5 Hz, H-1′b), 4.21 (2H, q, J = 7.0 Hz, H-1″), 5.08 (1H,
d, J = 10.5 Hz, H-3), 5.15 (1H, dt, J = 5.0, 11.0 Hz, H-2),
1
5
–
.41 (1H, t, J = 3.0 Hz, H-12), and 6.48 (1H, t, J = 4.0 Hz,
1
3
NH–amide); C NMR (CDCl , 125 MHz) δ = 170.47
3
(
(
4
3
3AcO), 139.21 (C-13), 125.78 (C-12), 74.83 (C-3), 69.89
C-2), 65.29 (C-23), 61.48 (C-1″), 53.68 (C-18), 47.72,
7.64, 43.79, 42.34, 41.93, 41.74, 39.69, 39.61, 39.01,
7.76, 36.96, 32.22, 30.90, 30.81, 27.74, 24.80, 23.48,
N,N-(2α,3β,23-Triacetoxyurs-12-ene-28-oyl)-ethanolamine
(13) White solid. Yield: 76%; mp = 121–123 °C. IR (KBr)
−
1
νmax 1391, 1468, 1605, 1647, 2923, and 3356 cm . ESI-
+
1
MS m/z 702.4 [M + H] . H NMR (CD OD, 500 MHz) δ
3

Medicinal Chemistry Research
=
(
0.86 (3H, s), 0.93 (3H, d, J = 6.0 Hz), 0.94 (3H, s), 1.00
3H, brs), 1.16 (3H, s), 1.17 (3H, s), 1.99 (3H, s), 2.04 (3H,
s), 2.08 (3H, s), 2.43 (1H, d, J = 11.0 Hz), 3.66 (1H, d, J =
1.5 Hz, H-23a), 3.86 (1H, d, J = 12.0 Hz, H-23b), 3.72
s), 2.03 (3H, s), 2.08 (3H, s), 3.00 (1H, m, H-1′a), 3.30 (1H,
m, H-1′b), 3.23 (2H, q, J = 6.5 Hz, H-7′), 5.08 (1H, d, J =
10.0 Hz, H-3), 5.16 (1H, dt, J = 4.5, 10.0 Hz, H-2), 5.30
(1H, brs, H-12), 5.61 (1H, brs, 1′–NH–amide), and 5.85
1
1
3
(
4H, brs, 2H-2′), 5.04 (1H, d, J = 10.5 Hz, H-3), 5.21 (1H,
(1H, t, J = 5.0 Hz, 7′–NH–); C NMR (CDCl , 125 MHz)
3
1
3
dt, J = 5.0, 10.5 Hz, H-2), and 5.24 (1H, brs, H-12);
C
δ = 177.84 (C-28), 170.82, 170.51, 170.37, 140.08 (C-13),
124.96 (C-12), 74.79 (C-3), 69.94 (C-2), 65.29 (C-23),
53.96 (C-18), 47.70, 47.62, 47.53, 43.79, 42.55, 41.95,
39.78, 39.61, 39.54, 39.32, 39.10, 37.80, 37.26, 32.32,
30.89, 29.53, 29.23, 28.75, 27.79, 26.88, 26.72, 24.80,
23.49, 23.35, 23.56, 21.22, 21.09, 20.87, 20.78, 17.89,
17.21, 17.09, 16.95, and 15.93.
NMR (CD OD, 125 MHz) δ = 179.00 (C-28), 172.48,
3
1
6
4
72.27, 172.20, 76.41 (C-3), 71.07 (C-2), 66.28 (C-3),
0.87 (C-2′), 52.73 (C-18), 44.88, 43.23 (2C), 40.87 (2C),
0.13, 39.07 (2C), 31.64, 24.37 (2C), 21.63 (2C), 20.96
(
(
2C), 20.74 (2C), 20.71 (2C), 18.97, 18.09 (2C), 17.79
2C), 17.51, and 14.24 (2C).
General procedure for the synthesis of the amides 14–18
To a stirred solution of the corresponding amides (4–7, 13)
N-(2α,3β,23-Triacetoxyurs-12-ene-28-oyl)-N-acetyl-n-nona-
diamine (16) Brown powder. Yield: 78%. IR (KBr) νmax
1114, 1256, 1500, 1637, 1723, 2873, 2935, 3161, and
−
1
− 1
(
(
1.0 equiv.) and DMAP (0.01 mol%) in dry CH Cl
20 mL), acetyl chloride (2 equiv.) was added. The resulting
3393 cm . ESI-MS m/z 795 [M–H] . H NMR (CDCl ,
2
2
3
500 MHz) δ = 0.79 (3H, s), 0.87 (3H, d, J = 6.5 Hz), 0.90
(3H, s), 0.96 (3H, brs), 1.10 (3H, s), 1.11 (3H, s), 1.97 (3H,
s), 1.99 (3H, s), 2.03 (3H, s), 2.08 (3H, s), 3.01 (1H, m, H-
1′a), 3.29 (1H, m, H-1′b), 3.22 (2H, m, H-9′), 3.59 (1H, d,
J = 12.0 Hz, H-23a), 3.85 (1H, d, J = 12.0 Hz, H-23b), 5.09
(1H, d, J = 10.5 Hz, H-3), 5.15 (1H, dq, J = 4.0, 10.5 Hz,
H-2), 5.31 (1H, brs, H-12), 5.88 (t, J = 5.0 Hz, –NH–amide
solution was stirred for further 2 h, and then quenched with
aqueous solution of 10% NaHCO . The organic phase was
3
washed with H O, brine solution, and dried over Na SO .
2
2
4
After removal of the solvent, the residue was subjected to a
silica gel column chromatography eluting with n-hexane/
EtOAc to yield the pure acetylated products. The products
1
13
13
were characterized based on ESI-MS, H, and C-NMR
analysis.
C-1′), and 5.96 (1H, brs, 9′–NH–amide); C NMR (CDCl3,
125 MHz) δ = 177.66 (C-28), 170.68, 170.45, 170.23,
170.01, 140.06 (C-13), 124.81 (C-12), 74.61 (C-3), 69.90
N-(2α,3β,23-Triacetoxyurs-12-ene-28-oyl)-n-propylamine
acetate (14) White powder. Yield: 82%; mp = 218–220 °C.
(C-2), 65.15 (C-23), 53.85 (C-18), 47.57, 47.48, 47.40,
43.64, 42.43, 41.83, 39.65, 39.57, 39.48, 39.31, 38.98,
37.68, 37.12, 32.21, 30.78, 29.49, 29.34, 29.13, 29.10,
29.08, 27.66, 27.00, 26.81, 24.65, 23.37, 23.17, 23.04,
21.11, 20.98, 20.75, 20.66, 17.77, 17.10, 16.97, 16.84, and
13.82.
IR (KBr) ν
1238, 1369, 1527, 1632, 1741, 2923, and
max
−
1
− 1
3
381 cm . ESI-MS m/z 712 [M–H] . H NMR (CDCl ,
3
5
00 MHz) δ = 0.76 (3H, s), 0.87 (3H, d, J = 6.5 Hz), 0.89
(
3H, s), 0.96 (3H, brs), 1.10 (3H, s), 1.11 (3H, s), 1.98 (3H,
s), 2.02 (3H, s), 2.07 (3H, s), 2.08 (3H, s), 3.09 (1H, m, H-
1
3
′a), 3.39 (1H, m, H-1′b), 3.58 (1H, d, J = 11.5 Hz, H-23a),
.85 (1H, d, J = 11.5 Hz, H-23b), 4.10 (2H, t, J = 6.0 Hz,
N-(2α,3β,23-Triacetoxyurs-12-ene-28-oyl)-N-acetyl-n-deca-
diamine (17) Brown powder. Yield: 75%. IR (KBr) νmax
1237, 1371, 1503, 1554, 1467, 1723, 2864, 3029, 3236, and
H-3′), 5.08 (1H, d, J = 10.5 Hz, H-3), 5.16 (1H, dt, J = 4.0,
−
1
+ 1
1
5
1
1
6
4
3
2
1
0.5 Hz, H-2), 5.33 (1H, t-line, H-12), and 6.05 (1H, t, J =
3554 cm . ESI-MS m/z 833 [M + Na] . H NMR (CDCl ,
3
.0 Hz, –NH–amide); 13C NMR (CDCl , 125 MHz) δ =
500 MHz) δ = 0.79 (3H, s), 0.87 (3H, d, J = 6.0 Hz), 0.89
(3H, s), 0.96 (3H, brs), 1.10 (3H, s), 1.11 (3H, s), 1.97 (3H,
s), 1.99 (3H, s), 2.03 (3H, s), 2.08 (3H, s), 3.02 (1H, m, H-
1′a), 3.28 (1H, m, H-1′b), 3.22 (2H, m, H-10′), 3.59 (1H, d,
J = 12.0 Hz, H-3), 3.85 (1H, d, J = 12.0 Hz, H-23b), 5.09
(1H, d, J = 10.0 Hz, H-3), 5.15 (1H, dt, J = 4.5, 10.0 Hz, H-
2), 5.30 (1H, t, J = 3.0 Hz, H-12), 5.71 (1H, brs, 10′-NH-
3
77.90 (C-28), 171.12, 170.73, 170.40, 170.29, 139.81 (C-
3), 125.08 (C-12), 74.81 (C-3), 69.86 (C-2), 65.28 (C-23),
2.05 (C-3′), 53.82 (C-18), 47.71, 47.60, 47.52, 43.78,
2.71, 41.92, 39.69, 39.60, 39.06, 37.78, 37.33, 36.21,
2.30, 30.20, 29.65, 28.63, 27.76, 24.69, 23.44, 23.17,
2.64, 21.01, 20.92, 20.80, 20.72, 17.87, 17.15, 17.00,
6.92, and 13.88.
1
3
acetyl), and 5.85 (1H, t, J = 5.0 Hz, 1′-NH–amide);
C
NMR (CDCl , 125 MHz) δ = 177.74 (C-28), 170.78,
3
N-(2α,3β,23-Triacetoxyurs-12-ene-28-oyl)-N-acetyl-n-heptyl-
170.53, 170.31, 170.01, 140.18 (C-13), 124.88 (C-12),
74.72 (C-3), 69.93 (C-2), 65.24 (C-23), 53.97 (C-18),
47.66, 47.57, 47.47, 43.75, 42.52, 41.90, 39.74, 39.65,
39.57, 39.42, 39.07, 37.75, 37.19, 32.29, 30.85, 29.59,
29.43, 29.37 (2C), 29.24, 29.18, 27.74, 27.08, 26.85, 24.74,
diamine (15) Brown powder. Yield: 82%. IR (KBr) ν
max
−
1
1
242, 1642, 1747, 2858, 2938, 3183, 3392, and 3557 cm .
−
1
ESI-MS m/z 767.5 [M–H] . H NMR (CDCl , 500 MHz) δ
3
=
0.79 (3H, s), 0.87 (3H, d, J = 6.5 Hz), 0.89 (3H, s), 0.96
(
3H, brs), 1.09 (3H, s), 1.11 (3H, s), 1.98 (3H, s), 1.99 (3H,

Medicinal Chemistry Research
2
1
3.45, 23.29, 23.11, 21.18, 21.06, 20.83, 20.74, 17.85,
7.18, 17.05, 16.91, and 13.90.
(CD OD, 125 MHz) δ = 179.91 (C-28), 140.32 (C-13),
126.74 (C-12), 78.16 (C-3), 69.59 (C-2), 66.34 (C-23),
3
54.19 (C-18), 48.13, 47.96, 44.09 (2C), 43.47 (2C), 42.49,
N,N-(2α,3β,23-Triacetoxyurs-12-ene-28-oyl)-acetoxy-etha-
nol amine (18) White powder. Yield: 80%; mp =
40.89 (2C), 40.83, 40.71, 40.24, 38.95, 38.72, 33.59, 33.53,
31.94, 30.75, 30.59, 30.51, 30.33, 28.91, 28.31, 28.05,
25.19, 24.51, 24.45, 21.66, 19.04, 18.12, 17.78, and 13.98.
+
1
1
90–192 °C. ESI-MS m/z 808 [M + Na] . H NMR
(
(
2
CDCl , 500 MHz) δ = 0.81 (3H, s), 0.94 (6H, brs), 1.00
3H, d, J = 5.0 Hz), 1.16 (3H, s), 1.17 (3H, s), 1.99 (3H, s),
.04 (3H, s), 2.07 (6H, s), 2.08 (3H, s), 2.43 (1H, d, J =
0.5 Hz, H-18), 3.66 (1H, d, J = 11.5 Hz, H-23a), 3.86 (1H,
3
N-(2α,3β,23-Trihydroxyurs-12-ene-28-oyl)-n-decadiamine
+
(21) White powder. Yield: 54%. ESI-MS m/z 643 [M + H] .
1
1
H NMR (CD OD, 500 MHz) δ = 0.72 (3H, s), 0.83 (3H,
3
d, J = 12.0 Hz, H-23b), 4.25 (4H, brs, 2H-2′), 5.05 (1H, d,
s), 0.93 (3H, d, J = 6.5 Hz), 0.99 (3H, d, J = 6.5 Hz), 1.07
(3H, s), 1.16 (3H, s), 2.16 (1H, d, J = 10.0 Hz, H-18), 2.74
(2H, t, J = 7.0 Hz, H-1′), 3.13 (2H, t, J = 7.0 Hz, H-10′),
J = 10.0 Hz, H-3), 5.21 (1H, dt, J = 4.5, 10.5 Hz, H-2), and
5
.26 (1H, brs, H-12).
3.29 (1H, d, J = 11.0 Hz, H-23a), 3.80 (1H, d, J = 11.0 Hz,
General procedure for the synthesis of the compounds 19–
H-23b), 3.53 (1H, d, J = 11.0 Hz, H-3), 3.73 (1H, dt, J =
13
2
2
4.5, 11.0 Hz, H-2), and 5.36 (1H, t, J = 3.5 Hz, H-12);
C
NMR (CD OD, 125 MHz) δ = 180.00 (C-28), 140.36 (C-
3
The amides 4–6, and 11 (1 equiv.) in MeOH (15 mL) were
treated with 4 N aqueous KOH (2 mL) at room temperature
for 16 h. The reaction mixture was concentrated under
reduced pressure, acidified with 5% HCl to pH≈4, and
extracted with DCM (3 × 20 mL). The combined organic
13), 126.76 (C-12), 78.17 (C-3), 69.61 (C-2), 66.34 (C-23),
54.23 (C-18), 48.14, 47.97, 44.11, 43.49, 41.97 (2C), 40.91,
40.85, 40.74, 40.26, 38.98, 38.74, 33.54, 32.12 (2C), 31.94,
30.73, 30.65 (2C), 30.54, 30.32, 28.93, 28.32, 27.89, 25.21,
24.52, 24.12, 21.62, 19.05, 18.14, 17.78, 17.75, and 13.96.
phase was washed with aqueous NaHCO 10% and dried
3
over Na SO . The pure products 19–22 were obtained by
N-(2α,3β,23-Trihydroxyurs-12-ene-28-oyl)-methylhydrazide
(22) White powder. Yield: 75%. ESI-MS m/z 517.4 [M +
2
4
purification on silica gel column chromatographies
CH Cl /MeOH, 95/5) and characterized by the analysis of
+
1
(
H] . H NMR (CDCl , 500 MHz) δ = 0.77 (3H, s), 0.88
2
2
3
1
13
their IR, MS, H, and C-NMR spectra.
(3H, s), 0.89 (3H, d, J = 6.0 Hz), 0.99 (3H, d, J = 6.5 Hz),
1.10 (3H, s), 1.47 (3H, s), 2.06 (1H, d, J = 10.0 Hz, H-18),
N-(2α,3β,23-Trihydroxyurs-12-ene-28-oyl)-n-heptadiamine
3.34 (1H, d, J = 11.5 Hz, H-23a), 3.54 (1H, d, J = 11.5 Hz,
H-23b), 4.16 (0.5H, q, J = 7.0 Hz, –NH–), 4.24 (0.5H, q, J
= 7.0 Hz, –NH–), 5.06 (1H, d, J = 10.5 Hz, H-3), 5.37 (1H,
dt, J = 4.0, 10.5 Hz, H-2), and 5.42 (1H, t-line, H-12).
+
(
19) White powder. Yield: 70%. ESI-MS m/z 601 [M + H] .
1
H NMR (CD OD, 500 MHz) δ = 0.72 (3H, s), 0.83 (3H,
3
s), 0.93 (3H, d, J = 5.0 Hz), 0.98 (3H, d, J = 5.0 Hz), 1.06
(
(
3H, s), 1.16 (3H, s), 2.16 (1H, d, J = 10.0 Hz, H-18), 2.86
2H, t, J = 10.0 Hz, H-1′), 3.14 (2H, m, H-7′), 3.29 (1H, d,
2α,3β,23-Triacetoxyurs-12-ene-28-amide (23) To a solu-
tion of 2 (307 mg, 0.5 mmol, 1 equiv.) in anhydrous DCM
(15 mL), oxalyl chloride (3.0 mmol, 6 equiv.) in DCM
(2 mL) was added. After being stirred at room temperature
for 24 h, the solvent and excess of oxalyl chloride were
removed under reduced pressure. The residue was redis-
solved in DCM (20 mL), followed by an addition of 30%
ammonia aqueous solution (2 mL). After stirring for further
20 h, the solvent was evaporated under reduced pressure.
Pure product 23 (270 mg, 88%) was obtained by purifica-
tion on silica gel column chromatography (n-hexane/
EtOAc; 3/1). Brown powder; mp 148–149 °C. IR (KBr)
νmax 1244, 1376, 1462, 1662, 1740, 2874, 2929, 3220, and
J = 10.0 Hz, H-23a), 3.38 (1H, d, J = 10.0 Hz, H-23b), 3.53
(
1H, d, J = 10.0 Hz, H-3), 3.72 (1H, dt, J = 5.0, 10.0 Hz, H-
13
2
1
1
4
3
2
1
), and 5.36 (1H, t, J = 5.0 Hz, H-12); C NMR (CD OD,
3
25 MHz) δ = 180.10 (C-28), 140.32 (C-13), 126.80 (C-
2), 78.20 (C-3), 69.64 (C-2), 66.35 (C-23), 54.21 (C-18),
4.12 (2 C), 43.49 (2 C), 41.18, 40.92, 40.84, 40.61, 40.30,
8.98, 33.54, 31.92, 30.24, 29.99, 29.90, 28.92, 28.01,
7.57, 25.20, 24.50, 24.09, 21.57 (2C), 19.04, 18.08, 17.72,
7.70 (2C), and 13.92.
N-(2α,3β,23-Trihydroxyurs-12-ene-28-oyl)-n-nonadiamine
+
(
20) White powder. Yield: 58%. ESI-MS m/z 629 [M + H] .
1
−1
+ 1
H NMR (CD OD, 500 MHz) δ = 0.73 (3H, s), 0.83 (3H,
3467 cm . ESI-MS m/z 615 [M + H] . H NMR (CDCl ,
3
3
s), 0.93 (3H, d, J = 6.5 Hz), 0.99 (3H, d, J = 6.0 Hz), 1.07
500 MHz) δ = 0.87 (3H, s), 0.90 (3H, d, J = 8.0 Hz), 0.91
(3H, s), 0.96 (3H, brs), 1.10 (3H, s), 1.11 (3H, s), 1.98 (3H,
s), 2.03 (3H, s), 2.04 (3H, s), 3.58 (1H, d, J = 12.0 Hz, H-
23a), 3.85 (1H, d, J = 12.0 Hz, H-23b), 5.08 (1H, d, J =
10.0 Hz, H-3), 5.16 (1H, dt, J = 4.5, 10.0 Hz, H-2), 5.32
(
(
3
2
1
3H, s), 1.16 (3H, s), 2.16 (1H, d, J = 10.5 Hz, H-18), 2.66
2H, t, J = 7.0 Hz, H-1′), 3.13 (2H, t, J = 6.5 Hz, H-9′),
.29 (1H, d, J = 9.5 Hz, H-23a), 3.38 (1H, d, J = 9.5 Hz, H-
3b), 3.53 (1H, d, J = 11.0 Hz, H-3), 3.72 (1H, dt, J = 4.5,
13
1.0 Hz, H-2), and 5.36 (1H, brs, H-12); C NMR
(1H, t, J = 3.5 Hz, H-12), 5.48 (1H, brs, –NH ), and 5.83
2

Medicinal Chemistry Research
(
(
1H, brs, –NH2); 13C NMR (CDCl , 125 MHz) δ = 181.01
C-28), 170.80, 170.46, 170.36, 139.85 (C-13), 125.18 (C-
33.90, 30.95, 29.66, 26.46, 24.51, 23.57, 21.17, 19.03,
18.19, 17.80, 17.42, and 13.95.
3
1
4
3
2
1
2), 74.78 (C-3), 69.90 (C-2), 65.27 (C-23), 54.19 (C-18),
7.95, 47.58, 47.51, 43.77, 42.49, 41.91, 39.70, 39.44,
9.03, 37.77, 37.12, 32.32, 30.81, 27.77, 24.75, 23.43,
3.12, 21.17, 21.09, 21.05, 20.84, 20.75, 17.86, 17.15,
7.04, and 13.90.
2α,3β,23-Trihydroxyurs-12-ene-17-nitrile (26) Compound
25 (595 mg, 1 mmol) in MeOH (10 mL) was treated with
4 N KOH (2 mL) at room temperature for 16 h. The reaction
mixture was concentrated under reduced pressure, acidified
with 5% HCl to pH ≈4, and extracted with DCM (3 ×
20 mL). The combined organic phase was washed with 10%
NaHCO and dried over Na SO . The pure product 26 was
2
α,3β,23-Trihydroxyurs-12-ene-28-amide (24) 2,3,23-Tria-
cetyl asiatic acid-28-amides 23 (1 equiv.) in MeOH (10 mL)
3
2
4
were treated with 4 N KOH (2 mL) at room temperature for
obtained in 74% yield (368 mg) by purification on silica gel
1
6 h. The reaction mixture was concentrated under reduced
column chromatography (CH Cl /MeOH, 95/5) as a white
2
2
pressure, acidified with 5% HCl to pH ≈4, and extracted
with DCM (3 × 20 mL). The combined organic phase was
washed with 10% NaHCO and dried over Na SO . The
powder. IR (KBr) ν 1251, 1471, 1553, 1633, 1741, 2235,
max
2861, and 2936 cm . ESI-MS m/z 505 [M + HCl] . H
−
1
–
1
NMR (CD OD, 500 MHz) δ = 0.74 (3H, s), 0.90 (3H, d, J
3
2
4
3
pure product 24 was obtained by purification on silica gel
= 6.5 Hz), 0.98 (3H, d, J = 6.5 Hz), 1.10 (3H, s), 1.11 (3H,
s), 1.18 (3H, s), 3.30 (1H, d, J = 11.0 Hz, H-23a), 3.38 (1H,
d, J = 11.0 Hz, H-23b), 3.54 (1H, d, J = 11.0 Hz, H-3), 3.74
(1H, dt, J = 4.5, 11.0 Hz, H-2), and 5.42 (1H, t, J = 3.5 Hz,
column chromatography (CH Cl /MeOH, 95/5). Brown
2
2
powder. IR (KBr) ν 1235, 1373, 1464, 1664, 1738, 2925,
max
−
1
+
1
and 3477 cm . ESI-MS m/z 488 [M + H] . H NMR
1
3
(
CD OD, 500 MHz) δ = 0.72 (3H, s), 0.90 (3H, s), 0.93
H-12); C NMR (CD OD, 125 MHz) δ = 138.61 (C-13),
3
3
(
1
=
(
2
1
1
4
3
1
3H, d, J = 7.0 Hz), 0.99 (3H, d, J = 5.5 Hz), 1.06 (3H, s),
128.32 (C-12), 126.41 (–CN, C-28), 78.14 (C-3), 69.66 (C-
2), 66.27 (C-23), 56.76 (C-18), 48.16, 48.08, 44.13, 43.69,
41.21, 40.93, 39.96 (2 C), 39.89, 38.97, 37.62, 33.90,
30.95, 29.66, 26.46, 24.51, 23.57, 21.17, 19.03, 18.19,
17.80, 17.42, and 13.95.
.17 (3H, s), 2.10 (1H, d, J = 10.0 Hz, H-18), 3.29 (1H, d, J
11.0 Hz, H-23a), 3.37 (1H, d, J = 11.0 Hz, H-23b), 3.52
1H, d, J = 11.0 Hz, H-3), 3.72 (1H, dt, J = 4.5, 11.0 Hz, H-
13
), and 5.36 (1H, t, J = 3.5 Hz, H-12); C NMR (CD OD,
3
25 MHz) δ = 183.59 (C-28), 140.15 (C-13), 126.94 (C-
2), 78.21 (C-3), 69.68 (C-2), 66.35 (C-23), 54.48 (C-18),
9.85, 48.02, 44.12, 43.49, 40.85, 40.77, 40.32, 38.98,
8.71, 33.54, 31.91, 28.97, 25.29, 24.49, 24.08 (2C), 21.58,
9.05, 17.88, 17.71, 17.68 (2C), and 13.91.
2α-Hydroxy-3β,23-isopropylidenedioxy-urs-12-ene-28-oic
acid (27) To a stirred solution of asiatic acid (1) (2.44 g,
5 mmol) and TsOH.H O (10 mg, 0.05 mmol) in DMF
2
(20 mL), 2,2-dimethoxypropane (580 mg, 5.5 mmol) was
added dropwise and kept further for 5 h at room tempera-
ture. The reaction mixture was neutralized with 5% NaOH
to pH ≈8 and extracted with ethyl acetate (2 × 100 mL). The
2
α,3β,23-Triacetoxyurs-12-ene-17-nitrile (25) To a stirred
solution of 23 (307 mg, 0.5 mmol) and DMAP (6 mg;
.05 mmol) in dry CH Cl2 (10 mL), acetyl chloride (2
0
combined organic phase was washed with H O (3 × 50 mL),
2
2
equiv.) was added. The resulting solution was stirred for
dried over Na SO , and evaporated under reduced pressure.
2
4
further 2 h, and then quenched with 10% NaHCO aqueous
Compound 27 was obtained in 88% yield (2.32 g) by pur-
ification on a silica gel column chromatography (CH Cl /
3
solution. The organic phase was washed with H O, brine
2
2
2
solution, and dried over Na SO . After removal of the
MeOH, 20/1) as a brown solid; mp: 195–197 °C. IR (KBr)
νmax 1033, 1190, 1391, 1454, 1552, 1648, 1689, 2871,
2939, 3199, and 3466 cm . ESI-MS m/z 527 [M–H] . H
2
4
solvent, the residue was subjected to a silica gel column
chromatography eluting with n-hexane/EtOAc to yield pure
product 25 (252 mg, 85%) as a white solid; mp: 215–217 °C.
IR (KBr) νmax 1032, 1230, 1370, 1460, 1739, 2228 (–CN),
−
1
– 1
NMR (CD OD, 500 MHz) δ = 0.68 (3H, s), 0.91 (3H, d, J
3
= 6.5 Hz), 0.93 (3H, brs), 1.08 (6H, s), 1.27 (3H, s), 1.42
(3H, s), 1.48 (3H, s), 2.23 (1H, d, J = 11.0 Hz, H-18), 3.40
(1H, d, J = 10.5 Hz, H-3), 3.50 (1H, d, J = 10.5 Hz, H-23a),
3.56 (1H, d, J = 10.5 Hz, H-23b), 3.73 (1H, dt, J = 4.5,
−
1
+
1
and 2931 cm . ESI-MS m/z 618 [M + Na] . H NMR
CDCl , 500 MHz) δ = 0.84 (3H, d, J = 6.5 Hz), 0.91 (3H,
(
3
s), 0.94 (3H, d, J = 6.5 Hz), 1.09 (6H, s), 1.17 (3H, s), 1.98
3H, s), 2.03 (3H, s), 2.09 (3H, s), 3.59 (1H, d, J = 11.5 Hz,
H-23a), 3.87 (1H, d, J = 11.5 Hz, H-23b), 5.09 (1H, d, J =
13
(
10.5 Hz, H-2), and 5.26 (1H, br t, J = 3.5 Hz, H-12);
C
NMR (CD OD, 125 MHz) δ = 181.43 (C-28), 139.69 (C-
3
1
5
1
0.5 Hz, H-3), 5.18 (1H, dt, J = 4.5, 10.5 Hz, H-2), and
13), 126.47 (C-12), 100.72 (C-31), 83.03 (C-3), 73.66 (C-
23), 66.15 (C-2), 54.30 (C-18), 52.27 (C-5), 43.31, 40.84,
40.39, 40.36, 39.22, 38.29, 38.04, 33.68, 31.75, 30.74,
30.09 (2C), 29.18, 25.29, 24.28, 24.24 (2C), 21.60, 19.65,
18.52, 18.38, 17.67 (2C), and 14.09.
.37 (1H, brs, H-12); 13C NMR (CDCl , 125 MHz) δ =
3
70.38 (C-28), 136.92 (C-13), 126.92 (C-12), 122.41
(
–CN), 78.14 (C-3), 69.66 (C-2), 66.27 (C-23), 56.76 (C-
8), 44.13, 43.69, 41.21, 40.93, 39.96, 39.89, 38.97, 37.62,
1

Medicinal Chemistry Research
2
α-Succinoyl-3β,23-isopropylidenedioxy-urs-12-ene-28-oic
The solvent was removed, and the residue was dissolved in
H O (15 mL), followed by extraction with CH Cl (3 ×
acid (28) To a stirred solution of 27 (264 mg, 0.5 mmol)
2
2
2
and triethylamine (1 mmol) in dry CH Cl (10 mL) at room
30 mL). The combined organic phase was washed with
2
2
temperature, succinic anhydride (60 mg, 0.6 mmol) was
added. After 12 h, the reaction mixture was diluted with
ethyl acetate (20 mL), washed with 1 N HCl, and followed
brine solution and dried over Na SO . Product 30 (477 mg,
90%) was obtained by purification on a silica gel column
chromatography (n-hexane/EtOAc; 7/3) as a white solid;
2
4
by an aqueous solution of 10% NaHCO . The solvent was
removed, and the residue was chromatographed on a silica
mp: 195–197 °C. IR (KBr) ν
1740, 2393, and 3533 cm . ESI-MS m/z 531 [M + H] . H
1042, 1240, 1374, 1464,
max
3
−
1
+ 1
gel column (n-hexane/EtOAc, 2/1) to yield pure product 28
NMR (CD OD, 500 MHz) δ = 0.75 (3H, s), 0.87 (3H, s),
3
(
1
232 mg, 74%) as a white solid; mp: 194–196 °C. νmax 965,
0.90 (3H, d, J = 6.0 Hz), 0.99 (3H, d, J = 6.0 Hz), 1.12 (3H,
−
1
044, 1165, 1391, 1459, 1648, 1713, 2931, and 3450 cm .
s), 1.16 (3H, s), 2.07 (3H, s, CH –CO), 2.22 (1H, d, J =
3
–
1
ESI-MS m/z 627 [M–H] . H NMR (CD OD, 500 MHz) δ
=
1
3
9
11.5 Hz, H-18), 3.31 (1H, H-23a overlap), 3.64 (1H, d, J =
10.5 Hz, H-23b), 3.54 (1H, d, J = 11.0 Hz, H-3), 5.03 (1H,
dt, J = 4.5, 11.5 Hz, H-2), and 5.25 (1H, t, J = 3.5 Hz, H-
3
0.76 (3H, s), 0.84–0.86 (6H, m), 0.94–0.95 (3H, overlap),
.09–1.10 (6H, overlap), 1.50 (3H, s, H-32), 1.52 (3H, s, H-
3), 2.57–2.71 (4H, m, H-35, H-36), 3.54 (1H, d, J =
.5 Hz, H-3), 3.48 (1H, d, J = 10.5 Hz, H-23a), 3.53 (1H, d,
1
3
12); C NMR (CD OD, 125 MHz) δ = 181.55 (C-28),
3
173.05 (–CO), 139.83 (C-13), 126.55 (C-12), 74.73 (C-3),
74.24 (C-2), 65.81 (C-23), 54.34 (C-18), 47.93, 44.92,
44.55, 43.39, 40.82, 40.41, 40.39, 39.12, 38.08, 33.57,
31.76, 30.72, 29.17, 25.30, 24.42, 24.17 (2C), 21.56, 21.32,
19.01, 17.80, 17.67, 17.48, and 13.88.
J = 10.5 Hz, H-23b), 5.03 (1H, dt, J = 4.5, 9.5 Hz, H-2),
and 5.23 (1H, brs, H-12); 13C NMR (CDCl , 125 MHz) δ =
3
1
1
2
4
3
2
84.02 (C-28), 178.04 (C-37), 171.71 (C-34), 138.00 (C-
3), 125.38 (C-12), 99.41 (C-31), 78.89 (C-3), 72.67 (C-
3), 69.13 (C-2), 52.67 (C-18), 51.15 (C-5), 47.95, 47.58,
4.68, 42.02, 39.63, 39.00, 38.36, 38.32, 37.50, 36.58,
2.32 (2C), 30.56, 29.70, 28.99, 27.91, 23.96, 23.73, 23.71,
1.14, 19.22, 17.77, 17.45, 16.99, 16.67, and 13.51.
Cytotoxic assay
Human cell lines, KB (CCL-17TM), HepG2 (HB-
8065TM), and LU (HTB-57TM), were supplied from the
2
α-O-acetyl-3β,23-isopropylidenedioxy-urs-12-ene-28-oic
American Type Culture Collection and maintained in Dul-
becco's Modified Eagle's Medium with 10% fetal bovine
serum. Cytotoxicity of asiatic acid derivatives against
HepG2, KB, and LU human cancer cell lines was evaluated
by the MTT method (Tim 1983; Cos et al. 2006; Scudiero
acid (29) To a stirred solution of compound 27 (264 mg,
0
0
acetate (40 mL) was added. The organic phase was washed
with 1 N HCl, aqueous solution of NaHCO , and brine
.50 mmol) in pyridine (5 mL), acetic anhydride (76 mg,
.74 mmol) was added. After keeping for further 12 h, ethyl
4
et al. 1988). Briefly, 6 × 10 cells/mL were seeded in 96-
3
solution. Removal of the solvent and subsequent purifica-
tion on a silica gel column chromatography (n-hexane/
EtOAc, 2/1) afforded pure product 29 (234 mg, 82%) as a
white solid; mp: 155–157 °C. IR (KBr) νmax 868, 967, 1033,
well microplates and preincubated. Then, five different
concentrations (128.0, 32.0, 8.0, 2.0, and 0.5 μg/mL) of
each tested compound in DMSO were added to each well.
Each concentration was tested in triplicate. After incubation
−
1
1
243, 1373, 1460, 1694, 1737, 2867, and 2942 cm . ESI-
at 37 °C in 5% CO for 48 h, MTT (0.2 mg/mL) was added
2
–
1
MS m/z 569 [M–H] . H NMR (CDCl , 500 MHz) δ = 0.75
to each well and incubated for another 4 h. Then the liquid
in the well was removed, and DMSO (200 μL) was added to
each well. Ellipticine was used as positive control. The
absorbance was recorded on a microplate reader at the
wavelength of 570 nm. Cell viability was measured, and
IC50 values were calculated based on the Raw data software.
3
(
1
(
1
(
2
3H, s), 0.84 (3H, d, J = 6.5 Hz), 0.94 (3H, d, J = 6.0 Hz),
.09 (6H, s), 1.11 (3H, s), 1.41 (3H, s), 1.43 (3H, s), 2.01
3H, s), 2.18 (1H, d, J = 11.5 Hz, H-18), 3.47 (1H, d, J =
0.5 Hz, H-23a), 3.53 (1H, d, J = 10.5 Hz, H-23b), 3.54
1H, d, J = 10.5 Hz, H-3), 4.99 (1H, dt, J = 4.5, 10.5 Hz, H-
13
), and 5.23 (1H, t, J = 3.0 Hz, H-12); C NMR (CDCl3,
1
25 MHz) δ = 183.15 (C-28), 170.62 (CH –CO–), 137.92
Hoechst staining
3
(
C-13), 125.39 (C-12), 99.37 (C-31), 78.96 (C-3), 72.68 (C-
2
4
3
2
3), 68.73 (C-2), 52.53 (C-18), 51.17 (C-5), 47.92, 47.62,
4.83, 42.02, 39.60, 39.05, 38.81, 38.32, 37.49, 36.65,
2.36, 30.61, 29.75, 29.70, 27.98, 24.03, 23.64, 23.22,
1.29, 21.15, 19.22, 17.69, 17.42, 16.93, and 13.54.
SK-LU-1 cells were seeded on the 35-mm plate at 5 × 104
cells/mL density and incubated overnight before treatment
with different compounds. After 24 h of incubation, cells
were fixed with 4% formaldehyde for 30 min, followed by
staining with Hoechst 33342 (0.5 mg/mL). The cells were
observed under the inverted fluorescence microscope
(Zeiss) using the 350-nm excitation wavelength and 461-nm
emission wavelength. The cell with a fraction or brighter
2
α-Acetoxy-3β,23-dihydroxy-urs-12-ene-28-oic acid (30)
Compound 29 (570 mg, 1 mmol) in MeOH (10 mL) was
treated with 1 N HCl (2 mL) at room temperature for 4 h.

Medicinal Chemistry Research
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Conflict of interest The authors declare that they have no conflict of
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