Synthesis and cytotoxic activity of ursolic acid derivatives

Article abstract of DOI:10.5560/ZNB.2013-2261

Fourteen ursolic acid derivatives, among them four novel compounds, were synthesized by modification either at the C-3, C-28 or both positions. The cytotoxic activity of the obtained derivatives was evaluated against the four human cancer cell lines KB (human mouth epidermal carcinoma), HepG2 (human hepatocellular carcinoma), MCF7 (human breast carcinoma) and Lu (human lung carcinoma). As the result, compounds 7 and 8 were from two to three times more active than ursolic acid on all four tested cell lines. This is the first report on cytotoxic effects of the synthetized ursolic acid derivatives 4, 8, and 10-15.

Full text of DOI:10.5560/ZNB.2013-2261

Synthesis and Cytotoxic Activity of Ursolic Acid Derivatives
Dao Duc Thiena, Nguyen Thanh Tama, Dinh Gia Thienb, Nguyen Thi Hoang Anha, and
Tran Van Sunga
a
Institute of Chemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc
Viet road, Cau Giay, Hanoi, Vietnam
Le Quy Don High School, Tam Ky, Quang Nam, Vietnam
b
Reprint requests to Prof. Dr. Tran Van Sung. Fax: 0084 4 38361283.
E-mail: tranvansungvhh@gmail.com
Z. Naturforsch. 2013, 68b, 201 – 206 / DOI: 10.5560/ZNB.2013-2261
Received September 25, 2012
Fourteen ursolic acid derivatives, among them four novel compounds, were synthesized by mod-
ification either at the C-3, C-28 or both positions. The cytotoxic activity of the obtained derivatives
was evaluated against the four human cancer cell lines KB (human mouth epidermal carcinoma),
HepG2 (human hepatocellular carcinoma), MCF7 (human breast carcinoma) and Lu (human lung
carcinoma). As the result, compounds 7 and 8 were from two to three times more active than ursolic
acid on all four tested cell lines. This is the first report on cytotoxic effects of the synthetized ursolic
acid derivatives 4, 8, and 10–15.
Key words: Ursolic Acid Derivatives, Cytotoxic Activity
Introduction
The genus Eriobotrya (Rosaceae) contains about 26
Results and Discussion
Scheme 1 outlines the synthesis of ursolic acid
species. Only one of these species, Eriobotrya japon- derivatives 2–15. The hydroxyl group at C-3 of ur-
ica (Thunb.) Lindl. (loquat) was hitherto intensively solic acid (1) was acylated with acetic, succinic and
studied and contains many interesting chemical con- phthalic anhydrides to afford esters 2 (90%), 3 (60%)
stituents with biological activities. Our phytochemical and 4 (60%), respectively. Jones oxidation of ursolic
investigation of leaves of Eriobotrya poilanei J. E. Vid. acid yielded ketone 5 as the main product with a yield
growing in Vietnam showed the presence of ursolic of 65%. With the aim to introduce a nitrogen func-
acid in large amount (0.32% of dry leaves weight). tion to the ursolic acid skeleton, ketone 5 was trans-
Ursolic acid has been reported to possess a series of formed into ketoxime 6 and then to its acetyl prod-
biological activities such as antitumor, antimicrobial, uct 7 with good yield (62% for 6; 87% for 7). For
anti-inflammatory, hepatoprotective, and cardioprotec- the synthesis of derivatives with a nitrogen function at
tive properties [1]. A series of ursolic acid deriva- C-18 of ursolic acid, the 3-hydroxy group was acety-
tives and their biological activities have been previ- lated and the acetyl product 2 reacted with oxalyl chlo-
ously published [2, 3]. This article describes the isola- ride, then with the corresponding amines giving the
tion of ursolic acid from Eriobotrya poilanei’s leaves, amides 8, 9 and 11 (after hydrolysis of 9). The correla-
the synthesis of its derivatives, among them four are tions between the amide proton at δH = 6.00 ppm and
new compounds (8, 10, 14, 15), and their cytotoxic ac- the carbonyl carbon (δC = 178.5 ppm) and the methy-
tivity against the human cancer cell lines KB, HepG2, lene carbon (δC = 39.71 ppm) in the HMBC spectrum
MCF7, and LU. The cytotoxic activity test results show as well as the correlation between this proton and two
that ten derivatives exhibited activity on all four tested methylene protons at δH = 2.99 and 3.31 ppm (each 1
cancer cell lines, two of them, compounds 7 and 8, H, m) in the 1H-1H COSY spectrum of compound
are two to three times more active than ursolic acid 8 confirmed that the amidation took place at the pri-
itself.
mary amine group. If an aqueous ammonia solution
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D. D. Thien et al. · Synthesis and Cytotoxic Activity of Ursolic Acid Derivatives
30
29
19
18
21
20
17
12
26
22 OH
28
13
11
OH
O
25
14 H 16
1
4
9
2
O
a/ b/ c
8
10
15
H
3
5
27
7
RO
HO
6
2: R = COCH3
3: R = CO(CH2)2COOH
4: R = COC6H4COOH
H
1
23
24
d
OH
OH
OH
e
a
O
O
O
AcON
HON
O
6
7
5
H
OH
N
R1
O
1. f
2. g
O
R2
AcO
8: R1 = (CH2)4CH(NH2)CH3; R2 = OAc
9: R1 = (CH2)10COOCH3;
11: R1 = (CH2)10COOH;
R2 = OAc
R2 = OH
2
i
1. f
2. h
NH2
NH2
NH2
O
O
O
i
b/ c
AcO
HO
RO
10
13
14: R= CO(CH2)2COOH
15: R= COC6H4COOH
k
Reagents and reaction conditions
(a): (CH3CO)2O, pyridine, r. t., 24 h
(b): (CH2CO)2O, DMAP, pyridine, r. t. , 24 h
(c): C6H4(CO)2O, DMAP, pyridine, r. t. , 24 h
(d): CrO3, H2SO4, acetone, r. t., 2 h
(e): NH2OH.HCl, 60 oC, 4 h
(f): (COCl)2, CH2Cl2, 25 oC, 48 h
(g): R1NH2, TEA/CH2Cl2, r. t., 24 h
(h): NH3.H2O, 0 oC; r. t., 12 h
(i): NaOH 4N, THF/MeOH (1/1), r. t., 12 h
(k): CH3COCl, CH2Cl2, r. t., 2 h
CN
AcO
12
Scheme 1. Synthesis of ursolic acid derivatives.
was used instead of the amines, the amide 10 was ob- action of amide 10 with acetyl cloride after 2 hours
tained (85% yield) which was hydrolyzed to amide 13 at room temparature the dehydration product 12 was
and then to the acid amides 14 and 15 with the cor- obtained (76%) instead of an expected diacetyl prod-
responding diacid anhydrides. Interestingly, in the re- uct.
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D. D. Thien et al. · Synthesis and Cytotoxic Activity of Ursolic Acid Derivatives
203
Table 1. The cytotoxic activity of ursolic acid (1) and the syn- remaining synthetized derivatives. Our results indicate
thesized derivatives.
again a potential for the study of the structure-activity
relationship of ursolic acid derivatives.
Compound
IC50 (µg mL−1
)
KB
HepG2
MCF7
LU
1 (Ursolic acid)
2
3
4
5
7
8
10
11
12
13
14
15
Ellipticin
10.23
8.00
19.60
17.05
5.31
11.75
4.73
6.17
8.00
4.36
8.0
12.23
34.79
24.35
40.72
19.79
5.44
Experimental Section
27.50
21.07
19.73
25.36
3.43
General
4.32
4.90
4.31
5.03
FT-IR: Nicolet IMPACT 410. ESI-MS: AGILENT 1100
LC-MSD Trap spectrometer. HR-ESI-MS: Qstar pulsar
(Applied Bioystems). NMR: Bruker Avance 500 MHz.
Column chromatography (CC): silica gel (70–230 and
230 – 400 mesh, Merck). Thin layer chromatography (TLC):
DC-Alufolien 60 F254 (Merck).
4.78
4.96
> 128
11.60
> 128
62.92
26.0
> 128
17.90
> 128
53.46
9.14
> 128
14.44
> 128
43.44
44.58
44.62
0.72
> 128
17.56
> 128
71.23
33.57
49.31
0.68
6.39
0.51
8.00
0.79
Isolation of ursolic acid (1)
The leaves of Eriobotrya poilanei were collected in March
2009 in Bi Dup National Park, Nui Ba, Lam Dong province
of Vietnam, and identified by Dr. Nguyen Tien Hiep, Institute
of Ecology and Biological Resources, Vietnam Academy of
Science and Technology. A voucher specimen (VH4212) is
deposited at the Herbarium of this Institute.
Dried leaves (1800 g) were extracted exhaustively with
methanol-water = 85 : 15 at room temperature. The organic
solvent was evaporated under reduced pressure, and the
aqueous solution was successively extracted with n-hexane,
ethyl acetate and n-butanol. Evaporation of these extracts
yielded 9.4, 50 and 130 g of a residue, respectively. The ethyl
acetate extract (50 g) was purified on a silica gel column elut-
ing with increasing polarity of n-hexane/ethyl acetate and
then ethyl acetate/methanol to furnish 5.8 g of 1 (0.32% of
the dried plant material). The spectral data of 1 are in good
agreement with those reported for ursolic acid [7].
Ursolic acid (1) and its twelve synthetized deriva-
tives have been tested against four human cancer cell
lines: human mouth epidermal carcinoma (KB), hu-
man hepatocellular carcinoma (Hep-G2), human breast
carcinoma (MCF-7), and human lung carcinoma (LU).
The results show that, except compounds 10 and 12, all
other compounds were active against all 4 tested can-
cer cell lines with different IC50 values (Table 1). Espe-
cially compounds 7 and 8, where the nitrogen function
has been introduced at the position C-3, the cytotoxi-
city was from 2 to 3 times higher than that of ursolic
acid itself against all tested cell lines. Besides these
two derivatives (7 and 8) there are some other good
active ones, for example compound 2 against Hep-G2
(IC50 = 4.73 µg mL−1) and 5 against KB and Hep-G2
(IC50 = 5.31 and 4.36 µg mL−1, respectively).
Bioactivity assays
It has been reported that 3-oxo-ursolic acid (5)
and 3β-O-acetylursolic acid (2) possess cytotoxic ac-
tivity against HONE-1 (human nasopharyngeal car-
cinoma), KB, and HT29 (colorectal carcinoma) can-
cer cell lines [4]. Among the components isolated
Bioactivity assays were carried out in the Institute of
Chemistry, Vietnam Academy of Science and Technology
(VAST), Hanoi, Vietnam. Cytotoxic assays were performed
according to Likhiwitayawuid et al. [8] and Skehan et al. [9]
from the dichloromethane extract of the dried fruits at different concentrations in 96-well plates. The Hep-G2,
KB and MCF-7 cell lines were maintained in the RPMI-
1640 culture medium with 10% fetal bovine serum (FBS).
The LU cell line was maintained in DMEM culture medium
with 10% fetal bovine serum (FBS).
of Chaenomeles speciosa (Sweet) Nakai (Rosaceae),
3β-O-acetyl ursolic acid showed the highest activity
against both prostaglandin-H-synthase isoenzymes [5].
3β-Succinoyl-urs-12-en-28-oic acid (3) exhibited cy-
totoxicity against NTUB1 (human bladder cancer cell
line) with IC50 = 8.65 µM [6]. This is the first re-
port about the cytotoxicity against MCF7 and LU
cancer cell lines of 3β-O-acetylursolic acid (2) and
3-acetoxyimino-urs-12-en-28-oic acid (7). Until now
3β-Acetoxy-urs-12-ene-28-oic acid (2)
Ursolic acid (1) was treated with acetic anhydride and
pyridine at r. t. overnight and worked up as usual to give
acetyl-ursolic acid (2). The NMR spectroscopic data of 2 are
there were no investigations on the cytotoxicity of the in good agreement with those of acetyl-ursolic acid [10].
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D. D. Thien et al. · Synthesis and Cytotoxic Activity of Ursolic Acid Derivatives
3β-Succinoyl-urs-12-en-28-oic acid (3) and
3β-phthaloyl-urs-12-en-28-oic acid (4)
60 ◦C for 4 h. After cooling to r. t., the reaction mixture was
evaporated and extracted with ethyl acetate (3 times). The or-
ganic phase was washed with water, dried over Na2SO4, fil-
tered, concentrated and chromatographed on a silica gel col-
umn (dichloromethane-methanol = 98 : 2) to furnish 29 mg
(62%) of 6 as a colorless powder. The NMR spectroscopic
data of compounds 5 and 6 were identical with published
data [3].
A mixture of ursolic acid (1) (0.1 mmol), succinic
(0.5 mmol) or phthalic anhydrides (0.5 mmol) and 4-
dimethylaminopyridine (DMAP, 0.3 mmol) in pyridine
(5 mL) was stirred at 60 ◦C. After stirring for 8 h, water was
added, and the mixture was extracted with EtOAc. The or-
ganic phase was neutralized with 1N HCl solution, washed
with water, dried, evaporated and chromatographed on a sil-
ica gel column (CH2Cl2-MeOH = 95 : 5) to furnish 32 mg of
3 or 35 mg of 4 as colorless solids (60%).
3-Acetoxyimino-urs-12-en-28-oic acid (7)
A mixture of 6 (30 mg, 0.064 mmol) and acetic anhydride
(0.1 mL) in pyridine (1 mL) was stirred at r. t. After 2 h, the
reaction mixture was concentrated in vacuo to dryness and
then purified using a silica gel column, eluted with n-hexane-
ethyl acetate = 4 : 1 to furnish 28 mg (87%) of 7 as a color-
less amorphous powder. – IR (KBr): ν = 2947, 2869, 1774,
1692, 1623, 1459, 1371, 1211 cm−1. – 1H NMR (500 MHz,
CDCl3): δ = 0.80 (3 H, s, CH3), 0.86 (3H, d, 6.3 Hz, CH3),
0.94 (3H, d, 6.3 Hz, CH3), 1.02 (3H, s, CH3), 1.07 (3H, s,
CH3), 1.12 (3H, s, CH3), 2.18 (3H, s, -COCH3), 2.19 (1H,
d, 11.5 Hz, H-18), 2.30 (1H, m, H-2a), 2.93 (1H, m, H-2b),
5.25 ppm (1H, m, 12-H).
Compound 3: colorless powder.
– MS ((+)-ESI):
m/z = 555 [M–H]−, 579 [M+Na]+ (C34H52O6). – 1H NMR
(500 MHz, CDCl3 + CD3OD): δ = 0.80 (3H, s, CH3), 0.82
(3H, d, 6.2 Hz, CH3), 0.86 (3H, s, CH3), 0.87 (3H, s, CH3),
0.94 (3H, d, 6.0 Hz, CH3), 0.95 (3H, s, CH3), 1.09 (3H,
s, CH3), 4.52 (1H, dd, 7.0, 9.0 Hz, 3-H), 5.24 ppm (1H, t-
like, 12-H). – 13C NMR (125 MHz, CDCl3 + CD3OD):
δg 15.35q, 16.59q, 16.77q, 16.89q, 18.07t, 21.04q, 23.17t,
23.41q, 24.04t, 27.88q, 28.74t, 28.82t, 28.91t, 29.52t, 32.80t,
36.68t, 36.75s, 37.61t, 38.12s, 38.77t, 38.95d, 39.37d,
41.92s, 47.34s, 47.73d, 51.76s, 52.61d, 55.19d, 81.44d,
125.35d, 138.04s, 172.33s, 173.12s, 181.31s ppm.
Compounds 8, 9 and 10 were synthesized as following:
To a solution of 2 (49 mg, 0.1 mmol) in CH2Cl2 (3 mL) was
added oxalyl chloride (0.1 mL) at r. t. After 48 h, the mixture
was evaporated in vacuo to yield 2a.
Compound 4: colorless powder.
– MS ((+)-ESI):
m/z = 603 [M–H]−, 627 [M+Na]+ (C38H52O6). – 1H NMR
(500 MHz, CDCl3 + CD3OD): δ = 0.82 (3H, s, CH3), 0.87
(3H, d, 6.0 Hz, CH3), 0.92 (3H, s, CH3), 0.94 (3H, d, 8.0 Hz,
CH3), 0.97 (3H, s, CH3), 1.11 (3H, s, CH3), 1.26 (3H, s,
CH3), 4.72 (1H, dd, 3.7, 11.3 Hz, 3-H), 5.25 (1H, t-like,
12-H), 7.50 – 7.72 ppm (4H, m). – 13C NMR (125 MHz,
CDCl3 + CD3OD): δ = 15.32q, 16.70q, 16.76q, 16.90q,
18.09t, 21.03q, 23.19t, 23.40q, 24.08t, 27.90t, 28.02q,
29.56t, 30.56t, 32.84t, 36.70s, 36.77t, 37.82s, 38.20t, 38.79d,
38.97d, 39.38s, 41.96s, 47.37d, 47.70s, 52.69d, 55.35d,
82.61d, 125.26d, 128.45d, 128.62d, 130.05d (× 2), 130.83s,
131.92s, 138.13s, 168.21s (× 2), 180.66s ppm.
N-(3β-Acetoxy-urs-12-en-28-oyl)-2,6-diaminohexane (8)
A solution of 2a in CH2Cl2 (5 mL) was added to a mix-
ture of NH2(CH2)4CH(CH3)NH2 (58 mg, 0.5 mmol), tri-
ethylamine (15 mg) and CH2Cl2 (10 mL) at r. t. After stir-
ring overnight, the reaction mixture was diluted with CH2Cl2
(30 mL) and washed with water. The organic layer was
dried over Na2SO4, evaporated, purified by silicagel column
chromatography using dichloromethane-methanol = 100 : 5
as eluent to give 42 mg of 8 as a colorless powder,
70%. – MS ((+)-ESI): m/z = 595 [M–H]−. – HRMS ((+)-
ESI): m/z = 597.49961 (calcd. 597.49952 for C38H65N2O3,
[M+H]+). – 1H NMR (500 MHz, CDCl3): δ = 0.77 (3H,
s, CH3), 0.86 (3H, d, 5.0 Hz, CH3), 0.87 (3H, s, CH3),
0.88 (3H, s, CH3), 0.95 (3H, s, CH3), 0.96 (3H, d, 6.2 Hz,
CH3), 0.96 (3H, s, CH3), 1.09 (3H, s, CH3), 2.05 (3H, s,
OCOCH3), 2.71 [1H, m, -CH(CH3)-NH2], 2.99 (1H, m,
-NH-CH2-), 3.31 (1H, m, -NH-CH2-), 4.50 (1H, dd, 5.0,
10.0 Hz, 3-H), 5.31 (1H, t-like, 12-H), 6.00 ppm (1H, br s,
NH). – 13C NMR (125 MHz, CDCl3): δ = 15.58q, 16.74q,
16.98q, 17.30q, 17.51q, 18.17t, 21.25q, 21.32q, 23.23q,
23.43t, 23.55t, 24.89t, 26.70t, 27.85t, 28.08q, 30.91t, 31.48t,
32.70t, 36.86s, 37.26t, 37.70s, 38.33t, 39.09d, 39.41d,
39.58s, 39.71t, 39.79d, 42.52s, 47.09s, 47.48t, 47.68d,
3-Oxo-urs-12-en-28-oic acid (5)
To a solution of ursolic acid (1) (46 mg, 0.1 mmol) in
acetone (10 mL) were added CrO3 (100 mg, 1.0 mmol) and
H2SO4 (20%, 10 mL). The reaction mixture was stirred at
r. t. for 4 h, then concentrated in vacuo and extracted with
ethyl acetate (50 mL). The organic phase was washed with
water (2 × 20 mL), dried over Na2SO4, filtered and concen-
trated in vacuo, then purified by silica gel column chromatog-
raphy (n-hexane-ethyl acetate = 7 : 1) to give 30 mg (65%)
of 5 as a colorless solid. – IR (KBr): ν = 3423, 3174, 2932,
1697, 1460, 1387, 1275, 1030 cm−1
3-Hydroxyimino-urs-12-en-28-oic acid (6)
.
A mixture of 5 (46 mg, 0.1 mmol) and NH2OH·HCl (40% 53.87d, 55.24d, 80.85d (C-3), 125.49d (C-12), 140.05s (C-
in water, 1 mL) in pyridine/ethanol (1/1.2 mL) was heated at 13), 171.02s (OCOCH3), 178.5s ppm (NH-C=O).
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D. D. Thien et al. · Synthesis and Cytotoxic Activity of Ursolic Acid Derivatives
205
N-(3β-Acetoxy-urs-12-en-28-oyl)-11-aminoundecanoic acid with water, dried over Na2SO4, filtered and evaporated in
methyl ester (9)
vacuo. Purification of the crude product on a silica gel col-
umn (n-hexane-ethyl acetate = 2 : 1) furnished 30 mg of 11,
while chloroform-methanol = 4 : 1 yielded 35 mg of 13.
Compound 11: colorless powder, yield 95%. – IR (KBr):
ν = 3405, 2923, 2874, 1717, 1632, 1530, 1459, 1383, 1263,
1034 cm−1. – 1H NMR (500 MHz, CDCl3): δ = 0.78 (s, 3H,
CH3), 0.87 (3H, d, 6.5 Hz, CH3), 0.93 (3H, d, 7.0 Hz, CH3),
0.99 (6H, s, CH3), 1.07 (3H, s, CH3), 1.26 (3H, s, CH3), 2.32
(2H, t, 7.5 Hz, -CH2-COOH), 3.02 (1H, br s), 3.22 (1H, m),
3.28 (1H, m, 3-H), 5.31 (1H, br s, 12-H), 5.93 ppm (1H, br s,
-CONH-).
Compound 13: colorless powder, yield 76%. – MS ((+)-
ESI): m/z = 454 [M–H]− (C30H49O2N). – IR (KBr): ν =
3498, 3413, 3194, 2933, 2876, 1671, 1601, 1460, 1376,
1200, 1036 cm−1. – 1H NMR (500 MHz, CDCl3): δ = 0.78
(3H, s, CH3), 0.85 (3H, s, CH3), 0.87 (3H, d, 6.5 Hz, CH3),
0.92 (3H, d, 7.4 Hz, CH3), 0.96 (3H, s, CH3), 0.99 (3H, s,
CH3), 1.11 (3H, s, CH3), 3.22 (1H, dd, 4.6, 11.1 Hz, 3-H),
5.31 (1H, t-like, 12-H), 5.78, 5.89 ppm (each 1H, br s, NH2).
Compound 2a (51 mg) in CH2Cl2 (5 mL) and 50 L of tri-
ethylamine were dropped slowly into a solution of 22 mg
(0.1 mmol) of NH2(CH2)10COOCH3 in CH2Cl2 (3 mL)
and stirred at r. t. for 12 h. The mixture was then diluted
with CH2Cl2 (30 mL), washed with water (20 mL), dried
over Na2SO4, evaporated in vacuo and chromatographed
over a silica gel column (n-hexane-ethyl acetate = 6 : 1) to
give 60 mg of 9 as a colorless powder, 87%. – 1H NMR
(500 MHz, CDCl3): δ = 0.84 (3H, s, CH3), 0.88 (3H, d,
6.4 Hz, CH3), 0.95 (3H, s, CH3), 1.05 (3H, d, 5.6 Hz, CH3),
1.06 (3H, s, CH3), 1.09 (3H, s, CH3), 1.11 (3H, s, CH3),
2.30 (2H, t, 7.5 Hz, -CH2-COOCH3), 2.56, 3.03 (each 1H,
m, -CONH-CH2), 3.28 (1H, m, 3-H), 3.66 (3H, s, -COOCH3)
5.33 (1H, br s, 12-H), 5.88 ppm (1H, t, J = 5.5 Hz, -CONH-).
3β-Acetoxy-urs-12-en-28-carboxamide (10)
A solution of 2a (94 mg) in THF (10 mL) was slowly
added to aqueous NH3 (25%, 10 mL) at 0 ◦C. The reac-
tion mixture was stirred at r. t. overnight and then con-
centrated. After addition of ethyl acetate, the solution was
washed with 1 N HCl, neutralized with NaHCO3, washed
with water, dried, and evaporated in vacuo. The residue was
chromatographed on a silica gel column (n-hexane-ethyl ac-
etate = 3 : 1) to afford 77 mg (85%) of compound 10 as
a colorless powder. – IR (KBr): ν = 3477, 3173, 2947,
2869, 1730, 1674, 1607, 1458, 1377, 1251, 1038 cm−1. –
HRMS ((+)-ESI): m/z = 498.39461 (calcd. 498.39472 for
C32H52NO3, [M+H]+). – 1H NMR (500 MHz, CDCl3):
δ = 0.85 (3H, s, CH3), 0.86 (3H, d, 5.5 Hz, CH3), 0.87 (3H,
s, CH3), 0.88 (3H, d, 6.8 Hz, CH3), 0.95 (3H, s, CH3), 0.96
(3H, s, CH3), 1.10 (3H, s, CH3), 2.05 (3H, s, OCOCH3), 4.50
(1H, dd, 5.5, 10.0 Hz, 3-H), 5.30 (1H, t-like, 12-H), 5.90 ppm
(2H, s, NH2). – 13C NMR (125 MHz, CDCl3): δ = 15.53q,
16.68q, 17.11q, 17.23q, 18.12t, 21.18q, 21.26q, 23.18q,
23.35t, 23.50t, 24.81t, 27.84t, 28.03q, 30.84t, 32.70t, 36.83s,
37.11t, 37.65s, 38.28t, 39.04d, 39.38s, 39.71d, 42.43s,
47.44d, 47.91s, 54.21d, 55.22d, 80.81d (C-3), 125.62d (C-
12), 139.74s (C-13), 170.95s (OCOCH3), 181.36s ppm (C-
28).
3β-Acetoxy-urs-12-en-17-nitrile (12)
A solution of acetyl cloride (0.3 mL) in CH2Cl2 (5 mL)
was dropped into a solution of 10 (0.2 mmol) in CH2Cl2
(10 mL) and DMAP (0.5 mmol) at r. t. After stirring for
2 h at r. t., 10 mL of 1 N HCl was added. The organic
layer was washed with saturated aqueous NaHCO3, dried
and chromatographed on a silica gel column (CH2Cl2-
MeOH = 100 : 3) to furnish 75 mg (76%) of 12 as a col-
orless solid. – MS ((+)-ESI): m/z = 502 [M+Na]+. –
HRMS ((+)-ESI): m/z = 502.36555 (calcd. 502.36610 for
C32H49NO2Na, [M+Na]+). – 1H NMR (500 MHz, CDCl3):
δ = 0.84 (3H, d, 6.5 Hz, CH3), 0.87 (3H, s, CH3), 0.88 (3H,
s, CH3), 0.94 (3H, d, 7.5 Hz, CH3), 0.99 (3H, s, CH3), 1.08
(6H, s, 2× CH3), 2.05 (3H, s, OCOCH3), 4.50 (1H, dd, 7.0,
9.3 Hz), 5.36 ppm (1H, t-like, 12-H). – 13C NMR (125 MHz,
CDCl3): δ = 15.64q, 16.74q, 16.93q, 17.40q, 18.19t, 20.85q,
21.28q, 23.03q, 23.34t, 23.55t, 25.42t, 28.09q, 28.54t,
29.99t, 33.31t, 36.45s, 36.88s, 37.69s, 38.42t, 38.68t, 39.68d,
39.78d, 42.30s, 47.52s, 47.52d, 55.32d, 55.36d, 80.85d (C-
3), 125.00s (CN), 127.32d (C-12), 136.75s (C-13), 170.99s
ppm (OCOCH3).
N-(3β-Hydroxy-urs-12-ene-28-oyl)-11-amino undecanoic
acid (11) and 3β-hydroxy-urs-12-ene-28-carboxamide (13)
3β-Succinoyl-urs-12-en-28-carboxamide (14) and
3β-phthaloyl-urs-12-en-28-carboxamide (15)
Compounds 11 and 13 were obtained through the hydroly-
Compound 13 was converted into 14 and 15 by the same
sis of 9 and 10, respectively. A 4 N NaOH solution (0.5 mL) manner and with the same reagents as for 3 and 4, respec-
was added to a mixture of 9 or 10 (0.05 mmol in 5 mL of tively.
THF/CH3OH = 1/1) at r. t. After stirring for 15 h, the reac-
Compound 14: colorless powder, yield 60%. – IR
tion mixture was evaporated in vacuo. The residue was dis- (KBr): ν = 3458, 3201, 2922, 2865, 1735, 1713, 1641,
solved in water, neutralized with 2 N HCl to pH = 7 and ex- 1577, 1454, 1385, 1267, 1213, 1000 cm−1. – MS ((+)-
tracted with CH2Cl2 (50 mL). The organic layer was washed ESI): m/z = 556 [M+H]+; 554 [M–H]−. – HRMS ((+)-
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206
D. D. Thien et al. · Synthesis and Cytotoxic Activity of Ursolic Acid Derivatives
ESI): m/z = 556.40019 (calcd. 556.40020 for C34H54NO5, CH3), 0.91 (3H, d, 6.1 Hz, CH3), 0.91 (3H, s, CH3), 0.92 (3H,
[M+H]+). – 1H NMR (300 MHz, CDCl3): δ = 0.81 (3H, s, CH3), 1.06 (3H, s, CH3), 4.61 (1H, dd, 4.1, 11.0 Hz, 3-H),
s, CH3), 0.85 (3H, d, 8.5 Hz, CH3), 0.89 (3H, s, CH3), 5.22 (1H, t-like, 12-H), 6.60, 6.67 (each 1H, s, NH2), 7.54
0.92 (3H, d, 6.2 Hz, CH3), 0.95 (3H, s, CH3), 0.96 (3H, s, (3H, m), 7.70 ppm (1H, d, 2.6 Hz). – 13C NMR (125 MHz,
CH3), 1.10 (3H, s, CH3), 2.64 (4H, br s, H-20 and H-30), [D6]DMSO): δ = 15.08q, 16.62q, 16.82q, 16.97q, 17.71t,
4.52 (1H, dd, 6.0, 10.0 Hz, 3-H), 5.30 (1H, br s, 12-H), 21.02q, 22.60t, 22.80t, 23.14q, 23.59t, 27.36t, 27.86q,
5.93, 6.78 ppm (each 1H, br s, NH2). – 13C NMR (75 MHz, 30.32s, 30.44t, 32.48t, 36.40s, 36.89t, 37.40s, 37.73t, 38.41d,
CDCl3): δ = 15.71q, 16.90q, 17.16q, 17.23q, 18.19t, 21.19q, 38.86d, 41.61s, 46.60d, 46.84s, 52.15d, 54.80d, 81.38d,
23.24q, 23.35t (× 2), 24.61t, 27.81t, 28.11q, 29.24t, 29.67t, 124.37d (× 2), 127.75d, 128.57d, 130.36d, 132.75s, 138.50s,
30.74t, 32.52t, 36.83s, 37.11t, 37.72s, 38.04t, 39.00d, 39.33s, 138.50s, 167.21s, 178.98s (× 2) ppm.
39.66d, 42.28s, 47.20d, 47.79s, 53.94d, 55.09d, 81.25d,
Acknowledgement
125.73d, 139.50s, 171.84s, 176.32s, 182.81s ppm.
Compound 15: colorless powder, yield 60%. – IR (KBr):
ν = 3519, 3392, 2968, 2919, 1717, 1627, 1570, 1454,
1280, 1134 cm−1. – MS ((+)-ESI): m/z = 602 [M–H]−. –
HRMS ((+)-ESI): m/z = 604.40031 (calcd. 604.40020 for
C38H54O5N, [M+H]+). – 1H NMR (500 MHz, [D6]DMSO):
δ = 0.77 (3H, s, CH3), 0.82 (3H, d, 5.0 Hz, CH3), 0.82 (3H, s,
We would like to thank the National Foundation for Sci-
ence and Technology Development of Vietnam (NAFOS-
TED) for financial support (project no. 104.01.128.09). Fur-
thermore we thank Mr. Dang Vu Luong for NMR mea-
surements and Dr. Nguyen Tien Hiep for plant determina-
tion.
[1] J. Liu, J. Ethnopharmacol. 1995, 49, 57 – 68.
[2] Y.-Q. Meng, D. Liu, L.-Li Cai, H. Chen, B. Cao,
Y.-Z. Wang, Bioorg. Med. Chem. 2009, 17, 848 –
854.
[3] C.-M. Ma, S.-Q. Cai, J.-R. Cui, R.-Q. Wang, P.-F. Tu,
M. Hattori, M. Daneshtalab, Eur. J. Med. Chem. 2005,
40, 582 – 589.
[4] Y.-M. Chiang, J.-Y. Chang, C.-C. Kuo, C.-Y. Chang,
Y.-H. Kuo, Phytochemistry 2005, 66, 495 – 501.
[5] E. Reininger, R. Bauer, Nat. Prod. Commun 2008, 3,
1991 – 1994.
[6] H.-Y. Tu, A.-M. Huang, B.-L. Wei, K.-H. Gan, T.-C.
Hour, S.-C. Yang, Y.-S. Pu, C.-N. Lin, Bioorg. Med.
Chem. 2009, 17, 7265 – 7272.
[7] M. Goretii, V. Silva, Molecules 2008, 13, 2482 – 2487.
[8] K. Likhitwitayawuid, C. K. Angerhofer, G. A. Cordell,
J. M. Pezzuto, J. Nat. Prod. 1993, 56, 30 – 33.
[9] P. Skehan, R. Storeng, D. Scudiero, A. Monks, M. J.
Mahon, D. Vistica, J. T. Warren, H. Bokesch, S. Ken-
ney, M. R. Boyd, J. Natl. Cancer Inst. 1990, 82,
1107 – 1112.
[10] A. V. Tkachev, A. Yu. Denisov, Tetrahedron 1994, 50,
2591 – 2598.
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