Synthesis of novel oleanolic acid and ursolic acid in C-28 position derivatives as potential anticancer agents

Article abstract of DOI:10.1007/s12272-016-0868-8

A series of nitrogen-containing derivatives of oleanolic acid and ursolic acid were prepared by a modification at C-28 position via esterification with 2-hydroxyacetic acid followed by amidation with amines, such as piperazine, N-methylpiperazine, and alkane-1, 2-diamines, alkane-1, 4-diamines, alkane-1, 6-diamines. In vitro antiproliferative activities of the compounds prepared towards MCF-7, Hela and A549 cell lines were evaluated by a MTT method to show that OA-5a, OA-5b, OA-5c and UA-5a showed somewhat improved antiproliferative activities against MCF-7, Hela and A549 cells comparing to that of the positive control, gefitinib.

Full text of DOI:10.1007/s12272-016-0868-8

Arch. Pharm. Res.
DOI 10.1007/s12272-016-0868-8
RESEARCH ARTICLE
Synthesis of novel oleanolic acid and ursolic acid in C-28 position
derivatives as potential anticancer agents
Tian Tian1 Xinyu Liu1 Eung-Seok Lee3 Jingyang Sun1
•
•
•
•
Zhonghua Feng1 Longxuan Zhao1,2 Chunhui Zhao2
•
•
Received: 3 July 2016 / Accepted: 28 November 2016
Ó The Pharmaceutical Society of Korea 2017
Abstract A series of nitrogen-containing derivatives of
oleanolic acid and ursolic acid were prepared by a modi-
fication at C-28 position via esterification with 2-hydrox-
yacetic acid followed by amidation with amines, such as
piperazine, N-methylpiperazine, and alkane-1, 2-diamines,
alkane-1, 4-diamines, alkane-1, 6-diamines. In vitro
antiproliferative activities of the compounds prepared
towards MCF-7, Hela and A549 cell lines were evaluated
by a MTT method to show that OA-5a, OA-5b, OA-5c and
UA-5a showed somewhat improved antiproliferative
activities against MCF-7, Hela and A549 cells comparing
to that of the positive control, gefitinib.
Introduction
Cancer is a widespread, complex, and lethal disease.
Worldwide there were 14.1 million new cancer cases, 8.2
million deaths and 32.6 million people living with cancer
(within 5 years of diagnosis) in 2012 (WHO Media Centre
2015). Chemotherapy is the most widely used cancer
treatment, but is associated with serious side effects and
other problems (Alam and Lee 2016). Despite efforts to
develop small molecule anticancer drugs (Samadi et al.
2012; You et al. 2011; Dayyani et al. 2011; Hoelder et al.
2012) and drugs that target epidermal growth factor
receptor mutations (Sharma et al. 2007; Ciardiello and
Tortora 2008), their efficacy is limited. Thus, it is highly
desirable to develop new anticancer agents with improved
tumor selectivity, efficacy and safety.
Keywords Oleanolic acid Á Ursolic acid Á Antiproliferative
activity Á Structure–activity relationships
A major advancement during the last decade has been
the synthesis of derivatives of natural triterpenoids such as
oleanolic acid (OA). A literature survey revealed that the
activity of these pentacyclic triterpenoids is related to their
basic triterpenoid skeletal structure, and the attached
functional groups offer opportunities for chemical modifi-
cation and for improvement of activity (Shanmugam et al.
2012; Zhao et al. 2007, 2014; Meng et al. 2012). For
example, the OA derivative 2-cyano-3, 12-dioxo-oleana-1,
9(11)-dien-28-oic acid (CDDO, Fig. 1a), its C-28 methyl
ester (CDDO-Me, Fig. 1b) and C-28 imidazolide (CDDO-
Im, Fig. 1c) in clinical trials (Muthu et al. 2014) were more
potent than the parent compound in terms of anti-inflam-
matory and antitumor activities (Suh et al. 1999; Honda
et al. 2000). OA, a pentacyclic triterpenoid derived from
tropical medicinal plants, has various bioactivities, such as
antitumor (Liu 2005), anti-oxidant (Somova et al. 2003),
anti-inflammatory (Zhang and Shen 2011), hepatoprotec-
tive (Sheng and Sun 2011) and hypoglycemic (Ali et al.
Electronic supplementary material The online version of this
article (doi:10.1007/s12272-016-0868-8) contains supplementary
material, which is available to authorized users.
& Longxuan Zhao
lxzhao@lnnu.edu.cn
& Chunhui Zhao
zch@lnnu.edu.cn
1
School of Chemistry and Chemical Engineering, Liaoning
Normal University, Dalian 116-029, Liaoning, People’s
Republic of China
2
Liaoning Provincial Key Laboratory of Biotechnology and
Drug Discovery, Liaoning Normal University,
Dalian 116-029, Liaoning, People’s Republic of China
3
College of Pharmacy, Yeungnam University,
Kyongsan 712-749, Korea
123

T. Tian et al.
O
O
O
N
N
H
COOH
H
H
COOCH3
CN
O
CN
O
CN
O
O
H
H
H
CDDO (a)
CDDO-Im (c)
CDDO-Me (b)
Fig. 1 Structures of several reported anticancer agents whose functional groups offer opportunities for chemical modification
2002) activities. Ursolic acid (UA), its isomer, has similar
pharmacological properties (Shanmugam et al. 2013;
Yadav et al. 2010), and both contain hydroxyl and car-
boxylic acid groups, as shown in Fig. 2. OA and UA have
been shown to act at various stages of tumor development
to inhibit tumor initiation and promotion, as well as to
induce tumor cell differentiation and apoptosis (Tian et al.
2002; Zhang et al. 2008).
straight-chain diamine instead of piperazine ring to expect
improving anti-tumor activities. Ethylenediamine, butane-
diamine and hexanediamine were introduced at the C-28
position of OA and UA as shown in Fig. 4.
Materials and methods
General
The low water solubility of OA and UA, however,
results in low bioavailability. Efforts have been made to
improve their water solubility by chemical modification.
For instance, Ma and coworkers (Ma et al. 1999) found that
introduction of a moiety at the C-3 position of OA, such as
a carboxyl group, significantly improved the IC50 value.
Piperazine derivatives also possess anticancer properties.
Many currently notable anticancer agents, including ima-
tinib (Shah et al. 2004) and dasatinib (Johnson et al. 2005)
contain a piperazine ring as part of their molecular struc-
ture (Fig. 3). From previous studies in our group on pen-
tacyclic triterpenoids, related analogs of OA and UA were
shown to inhibit the growth of human non-small cell lung
cancer (NSCLC) A549 and PC9/G (acquired resistance to
gefitinib) cell lines in vitro (Chen et al. 2016; Zhao et al.
2015; Wang et al. 2013). Based on these results, we
modified OA and UA by introduction of an acetyl group at
the C-3 position, and attaching piperazine and N-methyl
piperazine moieties at the C-28 position via a glycolic acid
linker. A series of six OA and UA derivatives was designed
as shown in Fig. 4.
Synthesized compounds were purified on a silica gel col-
umn (200–300 mesh, Qingdao Marine Chemical Factory,
China) using petroleum ether/ethyl acetate or methylene
chloride/anhydrous methanol as eluent. Their structures
were confirmed using nuclear magnetic resonance (NMR)
on an AVANCE 500 MHz spectrometer (BRUKER,
Switzerland) in CDCl3. Chemical shifts are expressed in
ppm and the coupling constants (J) in Hz. Infrared (IR)
spectra were recorded in KBr pellets on a WGH-30
(Shanghai Yonggui Analysis Instrument co., Ltd.). Melting
points were determined on an X-5 fiber melting point
detector (temperature-controlled, Beijing Tektronix
Instrument Co., Ltd.). Mass spectra were recorded on a
GC-TOF high-resolution mass spectrometer (HR-MS) and
an Agilent 6540 RRLC/Q-TOF (Agilent, Santa Clara, Ca,
USA) MS. Most chemicals and solvents were purchased
from commercial sources. Further purification and drying
by standard methods were employed when necessary. All
the reagents and chemicals were of analytical grade or
chemically pure. The synthetic routes are presented in
Schemes 1 and 2.
Since antitumor activity was significantly increased after
introducing piperazinyl moiety to C-28 position (Shah et al.
2004; Johnson et al. 2005), alternatively we selected
Chemistry
30
29
30
29
Synthesis and characterization of oleanolic acid
derivatives
COOH
28
COOH
28
3b-Acetoxyolean-12-ene-28-oic acid (OA-1)
3
3
HO
HO
A solution of OA (3.0 g, 6.57 mmol) and DMAP
(963.2 mg, 7.88 mmol) dissolved in anhydrous THF, stir-
ring 3 h at room temperature, then adding acetic anhydride
UA
OA
Fig. 2 Structure of OA and UA
123

Synthesis of novel oleanolic acid and ursolic acid in C-28 position derivatives…
Fig. 3 The representative
diamine moiety compounds
N
HN
N
N
HO
N
H
N
Cl
O
N
S
HN
N
N
N
HN
H3C
O
N
diamine moiety
N
CH3
diamine moiety
b Dasatinib
a Imatinib
R2 R3
R2 R3
R1
lead compounds
lead compounds
R1
O
O
O
O
NH2
N
N
H
Oglycolic linkage
Oglycolic linkage
NH
AcO
AcO
diamine moiety
diamine moiety
R1 = H, R2 = R3 = CH3 , Oleanolic acid (OA)
R1 = R2 = CH3, R3 = H, Ursolic acid (UA)
d Target compound in scheme 2
c Target compound in scheme 1
Fig. 4 Strategy for design of
OA and UA derivatives
(1.6 mL, 9.71 mmol) to mixture, stirring 2 h at room tem-
perature. The solvent was evaporated under reduced pres-
sure to remove THF. The residue was extracted with ethyl
acetate and water, washing to neutral with saturated NaCl
solution. The organic layer was dried over anhydrous mag-
nesium sulfate. Filtration and evaporation of solvent at
reduced pressure gave light yellow solid, which was purified
by silica gel chromatography with a gradient elution of ethyl
acetate-petroleum ether (1:10, v/v) to yield a white solid
22.86, 21.26, 18.17, 17.14, 16.64, 15.36; IR (KBr) mmax
2932, 2861 (CH), 1724(C=O), 1256 (C–O–C) cm-1
.
Acetic acid 3b-(olean-12-ene-28-chlorocarbonyl) ester
(OA-2) and 2-(3b-Acetoxyolean-12-ene- 28-acetoxy) acetic
acid (OA-3)
To a stirred solution of OA-1 (4.0 g, 8.02 mmol) and oxalyl
chloride (2.7 mL, 32.08 mmol) in anhydrous methylene
chloride, the reaction mixture was stirred overnight at room
temperature. The solvent was evaporated under reduced
pressure to remove methylene chloride, gave light yellow
solid (OA-2), which was used directly in subsequent reac-
tion without further purification due to its hydroscopic
property. A solution of glycolic acid (1.2 g, 16.04 mmol)
and TEA (11.2 mL, 80.21 mmol) in anhydrous THF was
stirred 7 h at room temperature. Reduced pressure
1
(2.42 g, 73%), m.p.259.5–261.5 °C; H-NMR (500 MHz,
CDCl3) d: 5.27 (s, 1H, H-12), 4.49 (t, J = 6.8 Hz, 1H, H-3),
2.81 (dd, J1 = 13.7 Hz, J2 = 4.2 Hz, 1H, H-18), 2.04 (s,
3H, CH3CO–), 1.13, 0.94, 0.93, 0.90, 0.87, 0.85, 0.75 (each
s,3H); 13C-NMR (125 MHz, CDCl3) d: 184.22, 171.01,
143.59, 122.54, 80.93, 55.30, 47.55, 46.54, 45.83, 41.54,
40.91, 39.29, 38.07, 37.68, 36.99, 33.79, 33.04, 32.53,
32.44, 30.64, 28.03, 27.66, 25.89, 23.57, 23.51, 23.38,
123

T. Tian et al.
R
R
3
R
R
3
2
2
R
R
1
1
R
R
3
R
R
3
R
R
3
2
2
2
R
R
R
1
1
1
O
OH
ii
OH
i
O
iii
iv
Cl
O
O
OH
O
N
O
O
O
O
N
AcO
CH
3
HO
OA-6 or UA-6
OA-1 or UA-1
OA-2 or UA-2
OA-3 or UA-3
OA or UA
R
R
3
R
R
3
2
v
2
R
R
1
1
vi
O
O
O
O
N
N
O
N
O
NH
Bn
OA-7 or UA-7
OA-8 or UA-8
Reagents and conditions: i) Ac2O, DMAP, THF, rt; ii) (COCl)2, CH2Cl2, rt; iii) HOCH2COOH, TEA, rt; iv) N-methyl piperazine, EDCI,
DMAP, CH2Cl2, 0 °C to rt; v) Benzylpiperazine, EDCI, DMAP, CH2Cl2, 0 °C to rt; vi) 10 % Pd/C, H2, anhydrous ethanol, rt.
Scheme 1 Synthesis of OA and UA Piperazine Derivatives
Scheme 2 Synthesis of OA and
UA Diamine Derivatives
Reagents and conditions: i) N-Boc-Diamine, EDCI, DMAP, CH2Cl2, 0 °C to rt; ii) TFA, CH2Cl2, 0 °C to rt.
distillation to remove the solvent, the reaction mixture was
extracted with ethyl acetate and water, washing to neutral
with saturated NaCl solution. The organic layer was dried
over anhydrous magnesium sulfate. Filtration and evapo-
ration of solvent at reduced pressure gave light yellow solid,
which was purified by silica gel chromatography with a
gradient elution of ethyl acetate-petroleum ether (1:5, v/v)
to yield a white solid (1.37 g, 31%), m.p.244.5–258.7 °C;
1H-NMR (500 MHz, CDCl3) d: 5.29 (t, J = 3.5 Hz, 1H,
H-12), 4.64 (d, J = 16.3 Hz, 1H, –COOCH2COOH), 4.49
(dd, J1 = 10.0 Hz, J2 = 8.2 Hz, 1H, H-3), 2.87 (dd,
J1 = 13.7 Hz, J2 = 4.3 Hz, 1H, H-18), 2.04 (s, 3H, CH3-
CO–), 0.92 (s, 6H, CH3–), 1.13, 0.90, 0.86, 0.72 (each
s,3H); 13C-NMR (125 MHz, CDCl3) d: 176.96, 171.93,
171.10, 143.49, 122.52, 80.99, 60.72, 59.93, 55.31, 47.55,
46.81, 45.90, 41.75, 41.28, 39.35, 38.15, 37.69, 36.93,
33.85, 33.02, 32.69, 32.19, 30.65, 28.04, 27.59, 25.75,
23.60, 23.41, 23.11, 21.27, 18.22, 16.89, 16.68, 15.38; IR
(KBr) mmax 2944, 2866 (CH), 1734(C=O), 1248 (C–O–C)
cm-1
.
3b-Acetoxyolean-12-ene-28-oic acid [1-(2-N-Boc-amino-
ethylamino)-1-oxo]ethyl ester (OA-4a)
To a stirred solution of OA-3 (250.12 mg, 0.45 mmol), N-
Boc-ethylenediamine (86.29 mg, 0.54 mmol) and DMAP
(65.85 mg, 0.54 mmol) in anhydrous methylene chloride
was added EDCI (344.23 mg, 1.80 mmol) which dissolved
in anhydrous methylene chloride at 0 °C. The reaction
mixture was stirred for 5 min, and then at room temperature
for 3 h. The reaction mixture was extracted with methylene
chloride and water, washing to neutral with saturated NaCl
solution. The organic layer was dried over anhydrous
magnesium sulfate. Filtration and evaporation of solvent at
reduced pressure gave light yellow solid, which was puri-
fied by silica gel chromatography with a gradient elution of
123

Synthesis of novel oleanolic acid and ursolic acid in C-28 position derivatives…
ethyl acetate-petroleum ether (1:2, v/v) to yield a white
solid (244.02 mg, 78%), m.p.200.4–200.7 °C; 1H-NMR
(500 MHz, CDCl3) d: 6.68 (s, 1H, –CONH–), 5.34 (s, 1H,
H-12), 4.86 (s, 1H, –NH-Boc), 4.66 (d, J = 15.6 Hz, 1H, –
OCH2CO–), 4.50–4.47 (m, 1H, H-3), 4.35 (d, J = 15.6 Hz,
1H, –OCH2CO–), 3.53–3.49 & 3.35–3.25 (m, 4H, –
NHCH2CH2NH–), 2.88 (dd, J1 = 13.7 Hz, J2 = 3.5 Hz,
1H, H-18), 2.04 (s, 3H, CH3CO–), 1.44 (s, 9H, –C(CH3)3),
1.15, 0.95, 0.89, 0.88, 0.86, 0.85, 0.69 (each s,3H); 13C-NMR
(125 MHz, CDCl3) d: 176.19, 170.92, 168.00, 156.47,
144.38, 122.29, 80.81, 79.61, 62.81, 55.26, 47.43, 46.94,
45.72, 41.78, 41.48, 40.36, 40.01, 39.27, 38.06, 37.64, 36.88,
33.72, 32.97, 32.54, 32.25, 30.62, 29.63, 28.30, 27.99, 27.54,
26.87, 25.83, 23.60, 23.44, 23.37, 23.13, 21.22, 18.12, 16.89,
16.62, 15.31; IR (KBr) mmax 3428 (NH), 2932, 2861 (CH),
1734, 1681 (C=O), 1253 (C–O–C) cm-1. HR-MS calcd for
C41H66N2O7 [M?Na]? 721.4870, found 721.4879.
mmol) according to the same procedure used in the
preparation of OA-4a, to give OA-4b (276.6 mg, 85%) as a
white solid: m.p.112.8–113.4 °C; 1H-NMR (500 MHz,
CDCl3) d: 6.23 (s, 1H, –CONH–), 5.29 (t, J = 3.2 Hz, 1H,
H-12), 4.63 (d, J = 15.5 Hz, 1H, –OCH2CO–), 4.47 (s, 1H, –
NH-Boc), 4.50–4.47 (m, 1H, H-3), 4.37 (d, J = 15.5 Hz, 1H,
–OCH2CO–), 3.39–3.34 & 3.28–3.21 (m, 2H, –NHCH2-
CH2CH2CH2NH–), 3.14 (d, J = 5.7 Hz, 2H, –NHCH2-
CH2CH2CH2– NH–), 2.85 (dd, J1 = 13.6 Hz, J2 = 4.1 Hz,
1H, H-18), 2.06 (s, 3H, CH3CO–), 1.44 (s, 9H, –C(CH3)3),
1.15, 0.95, 0.94, 0.92, 0.86, 0.85, 0.68 (each s, 3H); 13C-
NMR (125 MHz, CDCl3) d: 176.12, 170.97, 167.41, 155.96,
144.65, 122.20, 80.81, 62.96, 55.28, 47.44, 47.00, 45.72,
41.85, 41.61, 39.31, 38.85, 38.10, 37.67, 36.90, 33.69, 32.95,
32.53, 32.46, 30.65, 28.41, 28.02, 27.51, 27.00, 26.90, 25.88,
23.57, 23.47, 23.40, 23.28, 21.26, 18.14, 16.93, 16.65, 15.32;
IR (KBr) mmax 3421 (NH), 2940, 2869 (CH), 1734 (C=O),
1254 (C–O–C) cm-1. HR-MS calcd for C43H70N2O7
[M?H]? 727.5183, found 727.5192.
3b-Acetoxyolean-12-ene-28-oic acid [1-(2-amino-
ethylamino)-1-oxo]ethyl ester (OA-5a)
3b-Acetoxyolean-12-ene-28-oic acid [1-(4-amino-
butylamino)-1-oxo]ethyl ester (OA-5b)
To a stirred solution of OA-4a (200.12 mg, 0.29 mmol) in
anhydrous methylene chloride was added TFA (0.85 mL,
11.45 mmol) at 0 °C. The reaction mixture was stirred for
2 h at room temperature. After the reaction, with 0.1 mol
L-1 NH3ÁH2O adjust pH to 9 under the ice water bath. The
reaction mixture was extracted with methylene chloride and
water. The organic layer was dried over anhydrous mag-
nesium sulfate. Filtration and evaporation of solvent at
reduced pressure gave light yellow solid, which was purified
by silica gel chromatography with a gradient elution of
methylene chloride-anhydrous methanol (5:1 v/v) to yield a
white solid (131.26 mg, 77%), m.p.134.9–135.4 °C; 1H-NMR
(500 MHz, CDCl3) d: 7.07 (t, J = 5.6 Hz, 1H, –CONH–),
5.30–5.29 (m, 1H, H-12), 4.62 (d, J = 15.5 Hz, 1H, –OCH2-
CO–), 4.50–4.47 (m, 1H, H-3), 4.42 (d, J = 15.5 Hz, 1H, –
OCH2CO–), 3.58–3.46 (m, 4H, –NHCH2CH2NH2), 3.08(s,
2H, –NH2), 2.84 (dd, J1 = 13.5 Hz, J2 = 3.7 Hz, 1H, H-18),
2.04 (s, 3H, CH3CO–), 1.14, 0.92, 0.91, 0.91, 0.86, 0.85, 0.69
(each s, 3H); 13C-NMR (125 MHz, CDCl3) d: 176.67, 170.97,
169.65, 144.29, 122.24, 80.82, 62.62, 55.28, 47.44, 46.97,
45.76, 41.82, 41.45, 40.35, 39.29, 38.42, 38.11, 37.67, 36.89,
33.73, 32.99, 32.59, 32.27, 30.61, 28.03, 27.55, 25.82, 23.53,
23.42, 23.17, 21.26, 18.16, 16.85, 16.67, 15.32; IR (KBr) mmax
3429 (NH), 2949, 2869 (CH), 1736, 1680 (C=O), 1254 (C–O–
C) cm-1. HR-MS calcd for C36H58N2O5 [M?H]? 599.4346,
found 599.4351.
The OA-4b (200.00 mg, 0.27 mmol) was reacted with TFA
(0.82 mL, 11.00 mmol) according to the same procedure
used in the preparation of OA-5a, to give OA-5b
(121.89 mg, 71%) as a white solid: m.p.106.9–107.7 °C;
1H-NMR (500 MHz, CDCl3) d: 6.40 (s, 1H, –CONH–), 5.29
(t, J = 3.3 Hz, 1H, H-12), 4.64 (d, J = 15.5 Hz, 1H, –
OCH2CO–), 4.50–4.47 (m, 1H, H-3), 4.37 (d, J = 15.5 Hz,
1H, –OCH2CO–), 3.39–3.23 (m, 4H, –NHCH2CH2CH2-
CH2NH2), 2.86 (d, J = 4.0 Hz,1H, H-18), 2.83–2.81 (m,
2H, –NH2), 2.04 (s, 3H, CH3CO–), 1.15, 0.94, 0.92, 0.91,
0.86, 0.85, 0.69 (each s, 3H); 13C-NMR (125 MHz, CDCl3)
d:176.23, 170.79, 167.66, 144.63, 122.18, 80.80, 62.87,
55.28, 47.44, 46.99, 45.75, 41.85, 41.57, 40.83, 39.31, 38.79,
38.10, 37.67, 36.90, 33.71, 32.96, 32.54, 32.44, 30.65, 29.67,
28.02, 27.51, 26.90, 25.87, 23.58, 23.47, 23.41, 23.26, 21.26,
18.14, 16.93, 16.66, 15.32; IR (KBr) mmax 3429 (NH), 2941,
2870 (CH), 1735, 1672 (C=O), 1247 (C–O–C) cm-1. HR-
MS calcd for C38H62N2O5 [M?H]?627.4659, found
627.4662.
3b-Acetoxyolean-12-ene-28-oic acid [1-(6-N-Boc-amino-
hexylamino)-1-oxo]ethyl ester (OA-4c)
The OA-3 (267.29 mg, 0.48 mmol) was connected with N-
Boc-1, 6-hexanediamine hydrochloride (145.62 mg,
0.57 mmol) according to the same procedure used in the
preparation of OA-4a, to give OA-4c (215.33 mg, 60%) as a
white solid: m.p.90.3–90.7 °C; 1H-NMR (500 MHz,
CDCl3)d: 6.20(s, 1H, –CONH–), 5.29–5.28 (m, 1H, H-12),
4.63 (d, J = 15.5 Hz, 1H, –OCH2 CO–), 4.50–4.47 (m, 2H,
3b-Acetoxyolean-12-ene-28-oic acid [1-(4-N-Boc-amino-
butylamino)-1-oxo]ethyl ester (OA- 4b)
The OA-3 (250.03 mg, 0.45 mmol) was connected with
N-Boc-1, 4-diaminobutane hydrochloride (121.13 mg, 0.54
123

T. Tian et al.
H-3 9 1 & –NH-Boc 9 1), 4.37 (d, J = 15.5 Hz, 1H, –
OCH2CO–), 3.38–3.31 & 3.26–3.19 (m, 2H, –NHCH2CH2-
CH2CH2CH2CH2NH–), 3.10 (d, J = 6.3 Hz, 2H, –NHCH2–
CH2CH2CH2CH2CH2NH–), 2.85 (dd, J1 = 13.6 Hz, J2 =
4.0 Hz, 1H, H-18), 2.04 (s, 3H, CH3CO–), 1.44 (s, 9H, –
C(CH3)3), 1.15, 0.95, 0.94, 0.92, 0.86, 0.85, 0.68 (each s,3H);
13C-NMR (125 MHz, CDCl3) d: 176.09, 170.95, 167.27,
155.96, 144.64, 122.18, 80.79, 79.04, 62.96, 55.26, 47.43,
46.98, 45.73, 41.83, 41.59, 40.38, 39.30, 38.99, 38.09, 37.66,
36.89, 33.68, 32.94, 32.50, 32.45, 30.64, 29.98, 29.55, 28.40,
28.01, 27.48, 26.89, 26.47, 26.31, 25.88, 23.56, 23.46, 23.38,
23.26, 21.24, 18.13, 16.91, 16.65, 15.31; IR (KBr) mmax 3414
(NH), 2973, 2931 (CH), 1736, 1671 (C=O), 1253 (C–O–C)
cm-1. HR-MS calcd for C45H74N2O7 [M?H]? 755.5496,
found 755.5503.
3H). 13C-NMR (125 MHz, CDCl3) d: 177.06, 170.91,
165.18, 143.68, 122.30, 80.87, 60.90, 55.28, 54.79, 54.40,
47.52, 46.86, 45.90, 45.86, 44.38, 41.69, 41.27, 39.33,
38.10, 37.66, 36.91, 33.83, 33.03, 32.61, 32.32, 30.65,
29.01, 28.01, 27.62, 25.78, 23.66, 23.49, 23.04, 21.24,
18.20, 16.96, 16.65, 15.35; IR (KBr) mmax 2939, 2869
(CH), 1735, 1681 (C=O), 1241 (C–O–C) cm-1. HR-MS
calcd for C39H62N2O5 [M?H]? 639.4659, found 639.4667.
3b-Acetoxyolean-12-ene-28-oic acid [1-(4-benzyl-1-
piperazinyl)-1-oxo]ethyl ester (OA-7)
The OA-3 (226.6 mg, 0.4 mmol) was connected with N-
benzylpiperazine (70.50 mg, 0.4 mmol) according to the
same procedure used in the preparation of OA-4a, to give
OA-7 (198.1 mg, 76%) as a white solid: m.p.81.5–82.3 °C;
1H-NMR (500 MHz, CDCl3) d: 7.73–7.19 (m, 5H, Ph-),
5.22 (t, J = 3.4, 1H, H-12), 4.64–4.55 (m, 2H, –OCH2CO–
), 4.43 (m, 1H, H-3), 3.55–2.38 (m, 8H, piperazineH-2 9 2
& H-3 9 2 & H-5 9 2 & H-6 9 2), 3.45 (s, 2H, PhCH2–),
2.83 (dd, J1 = 13.7, J2 = 4.2, 1H, H-18), 1.98 (s, 3H,
CH3CO–), 1.07, 0.87, 0.86, 0.84, 0.80, 0.79, 0.66 (s, each
3H). 13C-NMR (125 MHz, CDCl3) d: 177.07, 170.95,
165.14, 143.72, 137.51, 129.06, 128.34, 127.30, 122.31,
80.90, 62.84, 61.00, 55.30, 52.89, 52.55, 47.54, 46.88,
45.93, 44.66, 41.89, 41.70, 41.28, 39.35, 38.12, 37.68,
36.93, 33.85, 33.05, 32.63, 32.35, 30.57, 28.03, 27.64,
25.80, 23.69, 23.51, 23.40, 23.05, 21.26, 18.21, 16.98,
16.66, 15.36; IR (KBr) mmax 2946, 2877 (CH), 1734, 1679
3b-Acetoxyolean-12-ene-28-oic acid [1-(6-amino-
hexylamino)-1-oxo]ethyl ester (OA-5c)
The OA-4c (141.20 mg, 0.19 mmol) was reacted with TFA
(0.56 mL, 7.49 mmol) according to the same procedure
used in the preparation of OA-5a, to give OA-5c (58.2 mg,
79%) as a white solid: m.p.103.7–104.2 °C; 1H-NMR
(500 MHz, CDCl3) d: 6.25 (d, J = 5.4 Hz, 1H, –CONH–),
5.30–5.29 (m, 1H, H-12), 4.63 (d, J = 15.6 Hz, 1H, –
OCH2CO–), 4.50–4.47 (m, 1H, H-3), 4.36 (d, J = 15.6 Hz,
1H, –OCH2CO–), 3.38–3.31 (m, 2H, –NHCH2CH2CH2-
CH2CH2CH2NH2), 3.26–3.21 (m, 2H, –NHCH2CH2CH2-
CH2CH2CH2NH2), 2.86 (d, J = 4.0 Hz, 1H, H-18),
2.83–2.81 (m, 2H, –NH2), 2.04 (s, 3H, CH3CO–), 1.15,
0.94, 0.92, 0.91, 0.86, 0.85, 0.69 (each s, 3H); 13C-NMR
(125 MHz, CDCl3) d: 176.15, 170.98, 167.53, 144.66,
122.20, 80.81, 62.89, 55.27, 47.44, 47.00, 45.74, 41.85,
41.59, 39.31, 38.92, 38.10, 37.67, 36.90, 33.69, 32.96,
32.52, 32.46, 30.65, 29.37, 28.02, 27.49, 26.27, 25.96,
25.88, 23.56, 23.47, 23.39, 23.27, 21.26, 18.14, 16.92,
16.66, 15.31; IR (KBr) mmax 3437 (NH), 2940, 2862 (CH),
1735, 1672 (C=O), 1254 (C–O–C) cm-1. HR-MS calcd for
C40H66N2O5 [M?H]? 655.4972, found 655.4979.
(C=O), 1254 (C–O–C) cm-1
.
HR-MS calcd for
C45H66N2O5 [M?H]? 715.4972, found 715.4977.
3b-Acetoxyolean-12-ene-28-oic acid [1-(1-piperazinyl)-1-
oxo]ethyl ester (OA-8)
A solution of OA-7 (160.0 mg, 0.2 mmol) dissolved in
anhydrous ethanol (20 mL), adding 10% Pd/C (25.0 mg)
under the stirring. Stirring in the hydrogen atmosphere for
24 h. Filtration and evaporation of solvent at reduced
pressure gave light yellow solid, which was purified by
silica gel chromatography with a gradient elution of
methylene chloride-anhydrous methanol (10:1, v/v) to
yield a white solid (102.0 mg, 73%), m.p.68.5–69.1 °C;
1H-NMR (500 MHz, CDCl3) d: 5.28 (t, J = 3.5, 1H,
H-12), 4.72–4.62 (m, 2H, –OCH2CO–), 4.49 (m, 1H, H-3),
3.63–2.41 (m, 8H, piperazineH-2 9 2 & H-3 9 2 &
H-5 9 2 & H-6 9 2), 2.90 (dd, J1 = 13.9, J2 = 4.4, 1H,
H-18), 2.05(s, 3H, CH3CO–), 1.14, 0.93, 0.92, 0.88, 0.87,
0.86, 0.74(s, each 3H). 13C-NMR (125 MHz, CDCl3) d:
177.06, 170.94, 165.11, 143.71, 122.30, 80.90, 60.97,
55.29, 52.72, 52.18, 47.54, 46.87, 45.92, 44.60, 41.81,
41.70,41.28, 39.34, 38.12, 37.67, 36.92, 33.85, 33.04,
32.63, 32.34, 30.66, 28.02, 27.63, 25.79, 23.68, 23.51,
3b-Acetoxyolean-12-ene-28-oic acid [1-(4-methyl-1-
piperazinyl)-1-oxo]ethyl ester (OA-6)
The OA-3 (100.0 mg, 0.2 mmol) was connected with N-
methyl piperazine (0.1 mmL, 0.2 mmol) according to the
same procedure used in the preparation of OA-4a, to give
OA-6 (93.0 mg, 81%) as a white solid: m.p.88.5–89.8 °C;
1H-NMR (500 MHz, CDCl3) d: 5.28(t, J = 3.5, 1H, H-12),
4.70(d, J = 14.0, 1H, –OCH2CO–), 4.62(d, J = 14.0, 1H,
–OCH2CO–), 4.50–4.47(m, 1H, H-3), 2.87–2.89(m, 1H,
H-18), 3.61–2.39 (m, 8H, piperazineH-2 9 2 & H-3 9 2 &
H-5 9 2 & H-6 9 2), 2.30 (s, 3H, –N–CH3), 2.04 (s, 3H,
CH3CO–), 1.13, 0.93, 0.92, 0.90, 0.86, 0.85, 0.84 (s, each
123

Synthesis of novel oleanolic acid and ursolic acid in C-28 position derivatives…
23.40, 23.05, 21.25, 18.21, 16.97, 16.66, 15.35; IR (KBr)
mmax 2939, 2873 (CH), 1735, 1667 (C=O), 1241 (C–O–C)
cm-1. HR-MS calcd for C38H60N2O5 [M?H]? 625.4502,
found 625.4507.
21.25, 21.08, 18.14, 17.01, 16.97, 16.69, 15.46; IR (KBr)
mmax 3421 (NH), 2933, 2870 (CH), 1737, 1686 (C=O),
1246 (C–O–C) cm-1. HR-MS calcd for C41H66N2O7
[M?H]? 721.4870, found 721.4877.
3b-Acetoxyurs-12-ene-28-oic acid [1-(2-amino-
ethylamino)-1-oxo]ethyl ester (UA-5a)
Synthesis and characterization of ursolic acid derivatives
3b-Acetoxyurs-12-ene-28-oic acid (UA-1), Yield: 72%,
1
The UA-4a (102.91 mg, 0.15 mmol) was reacted with TFA
(0.44 mL, 5.89 mmol) according to the same procedure
used in the preparation of OA-5a, to give UA-5a (63.4 mg,
72%) as a white solid: m.p.104.2–104.7 °C; 1H-NMR
(500 MHz, CDCl3) d: 6.84 (d, J = 5.3 Hz, 1H, –CONH–),
5.28 (s, 1H, H-12), 4.63 (d, J = 15.5 Hz, 1H, –OCH2CO–),
4.50–4.47 (m, 1H, H-3), 4.39 (d, J = 15.5 Hz, 1H, –
OCH2CO–), 3.66–3.50 (m, 2H, –NHCH2CH2NH2),
3.49–3.36 (m, 2H, –NHCH2CH2NH2), 2.97 (d, J = 5.0 Hz,
2H, –NH2), 2.24 (d, J = 11.2 Hz, 1H, H-18), 2.04 (s, 3H,
CH3CO–), 1.09, 0.96, 0.93, 0.88, 0.86, 0.85, 0.70 (each
s,3H); 13C-NMR (125 MHz, CDCl3) d: 176.24, 170.96,
168.48, 139.03, 125.34, 80.83, 62.80, 55.29, 52.91, 48.36,
47.39, 42.15, 40.83, 40.18, 39.51, 39.09, 38.80, 38.27,
37.67, 36.85, 36.65, 32.82, 30.53, 28.06, 27.88, 26.90,
24.37, 23.54, 23.28, 21.26, 21.09, 18.15, 17.02, 16.95,
16.70, 15.47; IR (KBr) mmax 3429 (NH), 2933, 2865 (CH),
1735, 1680 (C=O), 1246 (C–O–C) cm-1. HR-MS calcd for
C36H58N2O5 [M?H]? 599.4346, found 599.4353.
m.p.186.2–187.0 °C; H-NMR (500 MHz, CDCl3) d: 5.26
(t, J = 3.5 Hz, 1H, H-12), 4.49 (m, 1H, H-3), 2.18 (d,
J = 11.6 Hz, 1H, H-18), 2.04 (s, 3H, CH3CO–), 1.07, 0.96,
0.95, 0.88, 0.86, 0.85, 0.77 (s, each 3H); 13C-NMR
(125 MHz, CDCl3) d: 184.35, 171.00, 143.60, 122.54,
80.93, 55.30, 47.55, 46.54, 45.83, 41.53, 40.90, 39.28,
38.06, 37.68, 36.99, 33.79, 33.04, 32.53, 32.44, 30.64,
28.03, 27.66, 25.89, 23.57, 23.51, 23.38, 22.85, 21.26,
18.16, 17.17, 16.64, 15.36; IR (KBr) mmax: 2927, 2869
(CH), 1741, 1689 (C=O), 1241 (C–O–C) cm-1. Acetic
acid 3b-(urs-12-ene-28 -chlorocarbonyl) ester (UA-2)
and 2-(3b-Acetoxyurs-12-ene-28-acetoxy) acetic acid
(UA-3), Yield: 29%, m.p.122.1–122.7 °C; 1H-NMR
(500 MHz, CDCl3) d: 5.26 (t, J = 3.5, 1H, H-12), 4.58 (d,
J = 6.0, 2H, –CH2COOH), 4.51–4.48 (m, 1H, H-3), 2.25
(d, J = 11.3, 1H, H-18), 2.05 (s, 3H, CH3CO–), 1.08, 0.96,
0.94, 0.87, 0.86, 0.85, 0.74 (s, each 3H); 13C-NMR
(125 MHz, CDCl3) d: 176.71, 171.87, 171.07, 137.97,
125.75, 80.97, 59.86, 55.31, 52.78, 48.23, 47.50, 42.10,
39.57, 39.08, 38.79, 38.33, 37.68, 36.87, 36.42, 32.97,
30.62, 28.07, 27.97, 26.91, 24.24, 23.55, 23.44, 23.30,
3b-Acetoxyurs-12-ene-28-oic acid [1-(4-N-Boc-amino-
butylamino)-1-oxo]ethyl ester (UA- 4b)
21.27, 21.14, 18.20, 17.01, 16.72, 15.51; IR (KBr) mmax
:
2927, 2863 (CH), 1730 (C=O), 1247 (C–O–C) cm-1
Procedures reference the OA ones.
.
The UA-3 (250.11 mg, 0.36 mmol) was connected with N-
Boc-1, 4-diaminobutane hydrochloride (119.81 mg,
0.54 mmol) according to the same procedure used in the
preparation of OA-4a, to give UA-4b (232.5 mg, 71%) as a
white solid: m.p.99.7–100.4 °C; 1H-NMR (500 MHz,
CDCl3) d: 6.23 (s, 1H, –CONH–), 5.24 (s, 1H, H-12), 4.65
(d, J = 15.5 Hz, 1H, –OCH2CO–), 4.59 (s, 1H, –NH-Boc),
4.50–4.47 (m, 1H, H-3), 4.30 (d, J = 15.5 Hz, 1H, –
OCH2CO–), 3.39–3.34 & 3.27–3.20 (m, 2H, –NHCH2-
CH2CH2 CH2NH–), 3.13 (d, J = 6.0 Hz, 2H, –NHCH2-
CH2CH2CH2NH–), 2.21 (d, J = 11.2 Hz, 1H, H-18), 2.03
(s, 3H, CH3CO–), 1.43 (s, 9H, –C(CH3)3), 1.09, 0.96, 0.92,
0.86, 0.85, 0.83, 0.69 (each s, 3H); 13C-NMR (125 MHz,
CDCl3) d: 175.89, 170.94, 167.47, 155.96, 139.47, 125.14,
80.78, 79.17, 62.87, 55.25, 53.00, 48.36, 47.35, 42.18,
40.11, 39.48, 39.09, 38.81, 38.23, 37.64, 36.82, 36.71,
32.76, 30.47, 28.38, 28.03, 27.79, 27.49, 27.01, 24.42,
23.55, 23.48, 23.22, 21.24, 21.05, 18.10, 17.03, 16.94,
16.68, 15.42; IR (KBr) mmax 3437 (NH), 2932, 2861 (CH),
1734, 1648 (C=O), 1246 (C–O–C) cm-1. HR-MS calcd for
C43H70N2O7 [M?H]? 727.5183, found 727.5191.
3b-Acetoxyurs-12-ene-28-oic acid [1-(2-N-Boc-amino-
ethylamino)-1-oxo]ethyl ester (UA-4a)
The UA-3 (200.03 mg, 0.36 mmol) was connected with N-
Boc-ethylenediamine (69.06 mg, 0.43 mmol) according to
the same procedure used in the preparation of OA-4a, to
give UA-4a (142.7 mg, 85%) as
a
white solid:
1
m.p.119.2–119.8 °C; H-NMR (500 MHz, CDCl3) d: 6.69
(s, 1H, –CONH–), 5.31 (s, 1H, H-12), 4.87 (s, 1H, –NH-
Boc), 4.67 (d, J = 15.7 Hz, 1H, –OCH2CO–), 4.50–4.47
(m, 1H, H-3), 4.31 (d, J = 15.7 Hz, 1H, –OCH2CO–),
3.53–3.50 & 3.33–3.24 (m, 4H, –NHCH2CH2NH–), 2.27
(d, J = 11.0 Hz, 1H, H-18), 2.04 (s, 3H, CH3CO–), 1.44 (s,
9H,–C(CH3)3), 1.09, 0.95, 0.93, 0.88, 0.85, 0.83, 0.70 (each
s, 3H); 13C-NMR (125 MHz, CDCl3) d: 176.03, 170.95,
168.14, 156.49, 139.23, 125.33, 80.84, 79.63, 62.78, 55.29,
52.84, 48.36, 47.39, 42.16, 40.35, 40.06, 39.50, 39.12,
38.76, 38.23, 37.66, 36.85, 36.57, 32.81, 30.53, 28.44,
28.33, 28.05, 27.68, 26.90, 24.33, 23.57, 23.50, 23.25,
123

T. Tian et al.
3b-Acetoxyurs-12-ene-28-oic acid [1-(4-amino-
butylamino)-1-oxo]ethyl ester (UA-5b)
in the preparation of OA-5a, to give UA-5c (79.6 mg, 71%)
1
as a white solid: m.p.92.5–92.9 °C; H-NMR (500 MHz,
CDCl3) d: 6.24 (t, J = 5.6 Hz, 1H, –CONH–), 5.24 (d,
J = 3.3 Hz, 1H, H-12), 4.65 (d, J = 15.5 Hz, 1H, –OCH2-
CO–), 4.50–4.47 (m, 1H, H-3), 4.31 (d, J = 15.5 Hz, 1H, –
OCH2CO–), 3.45–3.37 & 3.26–3.20 (m, 4H, –NHCH2CH2-
CH2CH2– CH2CH2NH2), 2.78 (s, 2H, –NH2), 2.21 (d,
J = 11.1 Hz, 1H, H-18), 2.04 (s, 3H, CH3CO–), 1.09, 0.96,
0.92, 0.88, 0.85, 0.83, 0.70 (each s, 3H); 13C-NMR
(125 MHz, CDCl3) d: 175.91, 170.95, 167.46, 139.44,
125.17, 80.79, 62.86, 55.26, 53.01, 48.37, 47.37, 42.19,
39.50, 39.10, 38.97, 38.84, 38.26, 37.65, 36.84, 36.73, 32.77,
30.49, 29.66, 29.50, 28.04, 27.81, 26.42, 26.19, 24.43, 23.57,
23.49, 23.24, 21.25, 21.07, 18.12, 17.05, 16.95, 16.70, 15.44;
IR (KBr) mmax 3429 (NH), 2941, 2862 (CH), 1735, 1671
(C=O), 1253 (C–O–C) cm-1. HR-MS calcd for C40H66N2O5
[M?H]?655.4972, found 655.4977.
The UA-4b (121.60 mg, 0.17 mmol) was reacted with TFA
(0.50 mL, 6.69 mmol) according to the same procedure used
in the preparation of OA-5a, to give UA-5b (84.7 mg, 81%)
as a white solid: m.p.106.3–106.6 °C; 1H-NMR (500 MHz,
CDCl3) d: 6.42–6.40 (m, 1H, –CONH–), 5.25–5.24 (m, 1H,
H-12), 4.66 (d, J = 15.5 Hz, 1H, –OCH2CO–), 4.50–4.47
(m, 1H, H-3), 4.31 (d, J = 15.5 Hz, 1H, –OCH2CO–),
3.58–3.52 (m, 2H, –NHCH2CH2 CH2CH2NH2), 3.37–3.35
& 3.26–3.24 (m, 2H, –NHCH2CH2CH2CH2NH2), 2.84 (s,
2H, –NH2), 2.22 (d, J = 11.2 Hz, 1H, H-18), 2.04 (s, 3H,
CH3CO–), 1.09, 0.96, 0.92, 0.88, 0.86, 0.85, 0.70 (each
s,3H); 13C-NMR (125 MHz, CDCl3) d: 176.01, 170.95,
167.80, 139.45, 125.15, 80.79, 62.78, 55.26, 52.95, 48.37,
47.36, 42.19, 40.687, 39.50, 39.10, 38.80, 38.73, 38.25,
37.65, 36.83, 36.69, 32.79, 30.49, 28.19, 28.04, 27.82, 26.90,
24.40, 23.54, 23.49, 23.24, 21.25, 21.07, 18.12, 17.02, 16.96,
16.69, 15.43; IR (KBr) mmax 3429 (NH), 2933, 2870 (CH),
1734, 1672 (C=O), 1247 (C–O–C) cm-1. HR-MS calcd for
C38H62N2O5 [M?H]?627.4659, found 627.4667.
3b-Acetoxyurs-12-ene-28-oic acid [1-(4-methyl-1-
piperazinyl)-1-oxo]ethyl ester (UA-6)
The UA-3 (100.0 mg, 0.2 mmol) was connected with
methyl piperazine (0.1 mL, 0.2 mmol) according to the
same procedure used in the preparation of OA-4a, to give
UA-6 (84.63 mg, 74%) as a white solid: m.p.82.1–82.7 °C;
1H-NMR (500 MHz, CDCl3) d: 5.24 (t, J = 3.4, 1H, H-12),
4.67 (d, J = 14.0, 1H, –OCH2CO–), 4.61 (d, J = 14.0, 1H,
–OCH2CO–), 4.51–4.48 (m, 1H, H-3), 3.61–2.39 (m, 8H,
piperazineH-2 9 2 & H-3 9 2 & H-5 9 2 & H-6 9 2),
2.30 (s, 3H, –N–CH3), 2.27 (d, J = 11.6, 1H, H-18), 2.06 (s,
3H, CH3CO–), 1.08, 0.95, 0.94, 0.86, 0.85, 0.75(s, each 3H).
13C-NMR (125 MHz, CDCl3) d: 176.85, 170.91, 165.21,
138.08, 125.50, 80.87, 60.84, 55.27, 54.78, 54.40, 52.73,
48.25, 47.46, 45.86, 44.38, 42.03, 41.58, 39.55, 39.06,
38.73, 38.28, 37.65, 36.84, 36.55, 32.90, 30.64, 28.04,
27.98, 24.19, 23.52, 23.44, 23.27, 21.24, 21.14, 18.18,
17.05, 17.00, 16.70, 15.48; IR (KBr) mmax 2927, 2863 (CH),
1734 (C=O), 1247 (C–O–C) cm-1. HR-MS calcd for
C39H62N2O5 [M?H]?639.4659, found 639.4664.
3b-Acetoxyurs-12-ene-28-oic acid [1-(6-N-Boc-amino-
hexylamino)-1-oxo]ethyl ester (UA-4c)
The UA-3 (300.00 mg, 0.54 mmol) was connected with N-
Boc-1, 6-hexanediamine hydrochloride (163.4 mg,
0.65 mmol) according to the same procedure used in the
preparation of OA-4a, to give UA-4c (273.5 mg, 67%) as a
white solid: m.p.90.6–91.0 °C; 1H-NMR (500 MHz,
CDCl3) d: 6.19 (s, 1H, –CONH–), 5.25–5.24 (m, 1H, H-12),
4.65 (d, J = 15.5 Hz, 1H, –OCH2CO–), 4.50–4.47 (m, 2H,
H-3 9 1 & –NH-Boc 9 1), 4.31 (d, J = 15.5 Hz, 1H, –
OCH2CO–), 3.38–3.31 & 3.26–3.19 (m, 2H, –NHCH2CH2-
CH2CH2CH2CH2NH-), 3.10 (d, J = 6.4 Hz, 2H, –NHCH2-
CH2CH2CH2– CH2CH2NH–), 2.22 (d, J = 11.2 Hz, 1H,
H-18), 2.04 (s, 3H, CH3CO–), 1.46 (s, 9H, –C(CH3)3), 1.09,
0.97, 0.92, 0.88, 0.86, 0.85, 0.70 (each s,3H); 13C-NMR
(125 MHz, CDCl3) d: 175.87, 170.94, 167.35, 155.96,
139.47, 125.06, 80.79, 79.05, 62.92, 55.26, 53.03, 48.37,
47.37, 42.19, 40.39, 39.50, 39.10, 38.98, 38.85, 38.26, 37.65,
36.84, 36.73, 32.76, 30.48, 29.99, 29.59, 28.40, 27.80, 26.89,
26.47, 26.31, 24.44, 23.57, 23.49, 23.23, 21.24, 21.06, 18.12,
17.05, 16.94, 16.70, 15.44; IR (KBr) mmax 3398 (NH), 2941,
2862 (CH), 1737 (C=O), 1246 (C–O–C) cm-1. HR-MS calcd
for C45H74N2O7 [M?H]? 755.5496, found 755.5505.
3b-Acetoxyurs-12-ene-28-oic acid [1-(4-benzyl-1-
piperazinyl)-1-oxo]ethyl ester (UA-7)
The UA-3 (600.0 mg, 1.1 mmol) was connected with
benzylpiperazine (138.2 mg, 1.4 mmol) according to the
same procedure used in the preparation of OA-4a, to give
UA-7 (249.4 mg, 64%) as a white solid: m.p.88.6–89.2 °C;
1H-NMR (500 MHz, CDCl3) d: 7.32–7.26 (m, 5H, Ph-),
5.24 (t, J = 3.4, 1H, H-12), 4.66 (d, J = 14.0, 1H, –
OCH2CO–), 4.59 (d, J = 14.0, 1H, –OCH2CO–),
4.51–4.48 (m, 1H, H-3), 3.61–2.43 (m, 8H, piperazineH-
2 9 2 & H-3 9 2 & H-5 9 2 & H-6 9 2), 3.50 (s, 2H,
PhCH2–), 2.27 (d, J = 11.3, 1H, H-18), 2.04 (s, 3H,
3b-Acetoxyurs-12-ene-28-oic acid [1-(6-amino-
hexylamino)-1-oxo]ethyl ester (UA-5c)
The UA-4c (135.60 mg, 0.17 mmol) was reacted with TFA
(0.51 mL, 6.69 mmol) according to the same procedure used
123

Synthesis of novel oleanolic acid and ursolic acid in C-28 position derivatives…
CH3CO–), 1.42, 1.10, 1.08, 0.95, 0.93, 0.87, 0.85(s, each
3H). 13C-NMR (125 MHz, CDCl3) d: 176.80, 170.86,
165.13, 138.07, 129.08, 128.32, 127.34, 125.47, 80.84,
62.74, 60.89, 55.24, 52.80, 52.69, 52.45, 48.22, 47.43,
44.55, 42.00, 41.74, 39.61, 39.04, 38.70, 38.25, 37.62,
36.82, 36.53, 32.87, 30.61, 28.02, 27.95, 26.85, 24.17,
23.50, 23.42, 23.24, 21.21, 21.12, 18.15, 17.03, 16.99,
16.67, 15.45; IR (KBr) mmax 2919, 2869 (CH), 1734, 1671
microplate reader (Multiskan Ascen, Thermo Fisher Sci-
entific, Waltham, MA) was employed to determine optical
density (OD) at 570 nm. Dose response curves were plot-
ted for the samples and the IC50 values were calculated as
the concentrations of the test compounds resulting in 50%
reduction of absorption compared with the control cells.
The data represented the mean SD of three independent
experiments in which each compound concentration was
tested in three replicate wells.
(C=O), 1247 (C–O–C) cm-1
.
HR-MS calcd for
C45H66N2O5 [M?H]? 715.4972, found 715.4980.
3b-Acetoxyurs-12-ene-28-oic acid [1-(1-piperazinyl)-1-
oxo]ethyl ester (UA-8)
Results and discussion
Synthesis and characterization
The UA-7 (100.0 mg, 0.2 mmol) was reacted with 10% Pd/
C (25.0 mg) according to the same procedure used in the
preparation of OA-8, to give UA-8 (68.0 mg, 46%) as a
white solid: m.p.83.1–84.3 °C; 1H-NMR (500 MHz,
CDCl3) d: 5.17 (s, 1H, H-12), 4.60 (d, J = 14.0, 1H, –
OCH2O–), 4.54 (d, J = 14.0, 1H, –OCH2O–), 4.44–4.41
(m, 1H, H-3), 3.55–2.34 (m, 8H, piperazineH-2 9 2 &
H-3 9 2 & H-5 9 2 & H-6 9 2), 2.20 (d, J = 11.3, 1H,
H-18), 1.97 (s, 3H, CH3CO–), 1.03, 1.02, 1.00, 0.88, 0.87,
0.80, 0.68 (s, each 3H). 13C-NMR (125 MHz, CDCl3) d:
176.90, 170.93, 165.19, 138.10, 125.52, 80.88, 60.84,
55.28, 52.74, 52.58, 52.23, 52.05, 48.28, 47.47, 42.04,
39.56, 39.07, 38.75, 38.29, 37.67, 36.86, 36.57, 32.91,
30.65, 28.05, 27.99, 24.21, 23.54, 23.45, 23.29, 21.25,
21.15, 18.19, 17.07, 17.02, 16.71, 15.49; IR (KBr) mmax
A series of eighteen novel OA and UA derivatives were
designed and synthesized. For the preparation of OA and UA
piperazine derivatives, synthesis of the compounds designed
was quite straight-forward (Scheme 1). After protection of
3-OH by acetylation, OA and UA derivatives were esterified
with 2-hydroxyacetic acid, and which were amidated with
N-substituted piperazine in the presence of EDCI to obtain
OA-6, -7, -8, and UA-6, -7, -8 in total yield of 12.6–22.6 and
13.9–29.4%, respectively. Similarly, OA-4, OA-5, UA-4,
and UA-5 were prepared (Scheme 2). The compounds were
1
characterized by IR, H-NMR, 13C-NMR and HR-MS. IR
spectra showed typical absorption peaks for the functional
1
groups present. H-NMR and 13C-NMR spectra were as
expected. Compounds OA-5a, OA-5b, OA-5c, OA-6, OA-
8, UA-5a, UA-5b, UA-5c, UA-6, and UA-8 were charac-
terized by GC-TOF HR-MS, while OA-4a, OA-4b, OA-4c,
OA-7, UA-4a, UA-4b, UA-4c and UA-7 were characterized
by 6540 RRLC/Q-TOF HR-MS. The HR-MS measured
values coincided with theoretical values. IR, 1H-NMR, 13C-
NMR and HR-MS data of newly synthesized compounds are
summarized in the ‘‘Chemistry’’ Section.
2933, 2863 (CH), 1735, 1681 (C=O), 1247 (C–O–C) cm-1
.
HR-MS calcd for C38H60N2O5 [M?H]?625.4502, found
625.4509.
Biology
The antiproliferative activities of MCF-7 (human breast
cancer cells), Hela (human cervical carcinoma cells) and
A549 (human NSCLC cells) cells under different treat-
ments of synthesized compounds were determined by the
MTT colorimetric assay. Briefly, cells were cultured on
RPMI-1640 medium supplemented with 10% fetal bovine
serum, 100 U/mL penicillin, and 100 lg/mL streptomycin
at 37 °C in humidified atmosphere with 5% CO2. For the
cytotoxicity tests, cells were plated at a density of 1 9 104
cells per well in 96-well plates in complete medium and
cultured for 24 h, and media replaced with RPMI-1640,
10% FBS with or without pentacyclic triterpenoids
derivatives. Test compounds were dissolved in DMSO at
final concentrations less than 0.1%, prior to addition to cell
culture assays. Following incubation at 37 °C for 48 h, 10
lL MTT (5 mg/mL) was added to each well and incubated
for 4 h. After careful removal of the medium, 150 lL
DMSO was added to each well and shaken carefully. A
Anticancer activity
We determined the antiproliferative activity of the two
pentacyclic triterpenoids (OA and UA) and 18 analogs
against MCF-7 cell lines, and then tested the six most
active compounds against HeLa and A549 cell lines.
Gefitinib was used as the positive control and the results
are summarized in Table 1.
Further analysis clearly revealed that strong antiprolifera-
tive activities were observed when various R groups were
introduced at the C-28 position of OA and UA. Introduction
of
a piperazine moiety such as in OA-6 (IC50
64.08 4.88 lM), OA-8 (IC50 40.27 4.88 lM), UA-6
(IC50 61.25 4.88 lM), and UA-8 (IC50 56.85 4.88 lM)
gave much stronger growth inhibitory effects than the lead
compounds OA and UA, which had IC50 values[ 100,
123

T. Tian et al.
Structure–activity relationships
Table 1 IC50 Values of OA and UA Derivatives on MCF-7, Hela,
and A549 Cells Proliferation
The IC50 value for OA-4a, OA-4b and OA-4c were sig-
nificantly higher than those for OA-5a, OA-5b and OA-5c.
Also, OA and UA derivatives containing primary amine
groups were more active than those containing secondary
or tertiary amines. The antiproliferative activities of the
secondary amine compounds were better than those of the
tertiary amines. For example the IC50 value of OA-7 and
UA-7 is higher than OA-8 and UA-8. In summary, the
order of antiproliferative activity in diamine compounds is
primary amine [ secondary amine [ tertiary amine.
The antiproliferative activity against MCF-7 cell lines
revealed a clear preference for activity when the R’ group
was an ethylenediamine, which might indicate that a more
water-soluble catenulate diamine group at the C-28 posi-
tion contributed to the potency of the target compounds.
For example, the IC50 value of compound OA-5a was
clearly lower than that of OA-8. In summary, introduction
of a group with a catenulate structure may confer better
antiproliferative activity in OA and UA derivatives, sup-
ported by the better antiproliferative activity compared to
those containing a cyclic piperazine group.
Sample
M/W
–
IC50(lM)
MCF-7
IC50(lM)
Hela
IC50(lM)
A549
Gefitinib
OA
17.83 7.85 15.40 4.65 11.02 3.27
456.70 [100
[100
[100
OA-4a
OA-4b
OA-4c
OA-5a
OA-5b
OA-5c
OA-6
698.49 [100
–
–
726.52 [100
–
–
754.55 59.27 4.88
598.43 9.19 0.82
626.47 7.48 0.33
654.50 7.61 0.33
638.47 64.08 4.88
691.09 66.5 4.88
600.97 40.27 4.88
456.70 [100
–
–
8.56 0.53
12.72 0.79
8.64 0.88
9.87 0.64
6.29 1.64
6.87 0.78
–
–
OA-7
–
–
OA-8
–
–
UA
[100
[100
UA-4a
UA-4b
UA-4c
UA-5a
UA-5b
UA-5c
UA-6
698.49 [100
–
–
726.52 [100
–
–
754.55 [100
–
–
598.43 8.45 0.26
626.47 9.21 0.73
8.37 0.11
10.06 1.39
10.35 2.72 15.14 0.66
654.50 12.91 0.71 17.21 6.92 22.14 2.33
638.47 61.25 4.88
691.09 [100
600.97 56.85 4.88
–
–
–
–
–
–
Increasing carbon chain length, exemplified by OA-5a,
OA-5b and OA-5c, had no significant impact on the
antiproliferative activity of OA and UA derivatives.
UA-7
UA-8
nd Not determined
MCF-7 human breast cancer cells, Hela human cervical carcinoma
cells, A549 human NSCLC cells, Gefitinib positive control for
antiproliferative activity
Conclusion
Each data represents mean S.D. From three different experiments
performed in triplicate
A series of pentacyclic triterpenoid derivatives bearing
diamine moieties were synthesized and investigated for
their cytotoxic potential against MCF-7, HeLa and A549
cells. Most of the compounds showed significant antipro-
liferative activity compared to their parent compounds. In
particular, OA-5c showed the highest inhibitory activity
against three human cancer cell lines. Furthermore, OA-5a
has the best prospects for development as a drug, since it
has significant anticancer activity, is low-cost and is easily
purified. Therefore, OA-5a is considered worthy of further
investigation as a new drug candidate.
against MCF-7 cancer cells. However, their intermediates,
OA-7 and UA-7, displayed relatively weak cytotoxicity with
IC50 values of 66.5 4.88 lM and [100 lM, respectively.
The derivatives containing ethylenediamine, butanedi-
amine and hexanediamine moieties showed the strongest
cytotoxic activity against three human cancer cell lines, fol-
lowed by compounds UA-5b and UA-5c. In particular, IC50
values for OA-5a, OA-5b, OA-5c and UA-5a exhib-
ited\10 lM and these compounds were twice as active
against MCF-7 and HeLa cell lines (IC50 9.19 0.82/
8.56 0.53, 7.48 0.33/8.64 0.88, 7.61 0.33/6.29
1.64 and 8.45 0.26/8.37 0.11 lM, respectively) than
positive control, gefitinib (IC50 15.40 4.65 lM). However,
their intermediate products OA-4a, OA-4b, UA-4a, UA-4b
and UA-4c displayed poor cytotoxicity against the cancer cell
lines with IC50 values[ 100 lM.
Acknowledgements This work was supported by the National Nat-
ural Science Foundation of China (Nos. 21102067).
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