Synthesis and antitumor activity evaluation of novel ursolic acid derivatives

Article abstract of DOI:10.1080/10286020.2015.1070830

Eleven novel ursolic acid (UA) derivatives were designed and synthesized with modification at positions of C-2, C-3, and C-28 of UA. Their structures were confirmed by MS, ¹H NMR, and elemental analysis. Their in vitro cytotoxicities against various cancer cell lines (HeLa, HepG2, and BGC-823) were evaluated by MTT assay. The results indicated that all compounds could inhibit cell proliferation of HeLa, HepG2, and BGC-823 cells. Among them, compounds I3 and I4 showed more potent cytotoxicity on these three tumor cells than gefitinib (positive control), worthy to be studied further.

Full text of DOI:10.1080/10286020.2015.1070830

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Synthesis and antitumor activity evaluation of
novel ursolic acid derivatives
Yan-Qiu Meng, Liang-Feng Zhang, Dong-Ying Liu, Li-Wei Liu, Yi Zhang & Min-
Jie Zhao
To cite this article: Yan-Qiu Meng, Liang-Feng Zhang, Dong-Ying Liu, Li-Wei Liu, Yi Zhang & Min-
Jie Zhao (2015): Synthesis and antitumor activity evaluation of novel ursolic acid derivatives,
Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2015.1070830
To link to this article: http://dx.doi.org/10.1080/10286020.2015.1070830
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Date: 12 November 2015, At: 07:01

Journal of Asian Natural Products Research, 2015
http://dx.doi.org/10.1080/10286020.2015.1070830
Synthesis and antitumor activity evaluation of novel ursolic acid
derivatives
Yan-Qiu Meng*, Liang-Feng Zhang, Dong-Ying Liu, Li-Wei Liu, Yi Zhang and
Min-Jie Zhao
Department of Pharmaceutical Engineering, Shenyang University of Chemical Technology,
Shenyang 110142, China
(Received 11 January 2015; final version received 6 July 2015)
Eleven novel ursolic acid (UA) derivatives were designed and synthesized with
modification at positions of C-2, C-3, and C-28 of UA. Their structures were confirmed
by MS, 1H NMR, and elemental analysis. Their in vitro cytotoxicities against various
cancer cell lines (HeLa, HepG2, and BGC-823) were evaluated by MTT assay. The
results indicated that all compounds could inhibit cell proliferation of HeLa, HepG2,
and BGC-823 cells. Among them, compounds I3 and I4 showed more potent
cytotoxicity on these three tumor cells than gefitinib (positive control), worthy to be
studied further.
Keywords: ursolic acid derivatives; triterpenoid; synthesized; antitumor activity
1. Introduction
the activities would be significantly
enhanced by introduced methoxycarbonyl,
carbonyl, or cyano-funtional groups.
Based on the reports that the ester
functionality at C-3 is essential for the
pharmacological activities of pentacyclic
triterpenes [18,19], a hydrogen donor
group at either C-3 position and/or C-28
position of UA is essential for the
cytotoxic activity [20]. Eleven UA deriva-
tives with substitution at positions of C-2,
C-3, and C-28 of UA have been syn-
thesized and their cytotoxic activities have
been evaluated in vitro against three
cancer cell lines (HeLa, HepG2, and
BGC-823). The results showed that
compound I3 has more potent cytotoxicity
on these three tumor cells than UA.
Ursolic acid (UA) is a pentacyclic
triterpene compound isolated from leaves,
berries, flowers, and fruits of many types
of medicinal plants such as Rosmarinus
officinalis, Etiobotrya japonica, and Gle-
cheoma hederaceae [1,2]. It has been
reported to possess a large range of
pharmacological activities, including anti-
tumor [3,4], anti-inflammatory [5,6], anti-
HIV [7], anti-allergic, anti-bacterial, anti-
viral [8], and anti-angiogenic effects [9] in
chick
chorioallantotic
membrane.
In recent years, a large number of literature
have reported that pentacyclic triter-
penoids show a certain effect on tumor
cells, and UA and their derivatives can
inhibit tumor cell growth, induce apoptosis
of tumor cells, and inhibit tumor angio-
genesis [10–15]. These effects of UA are
mediated through suppression of the
expression of lipoxygenase, COX-2,
MMP-9 [16], and iNOS, some of which
are genes regulated by NF-KB [17], and
2. Results and discussion
2.1. Chemical synthesis
UA was used as the raw material, and
eleven title compounds were designed and
*Corresponding author. Email: mengyanqiu@hotmail.com
q 2015 Taylor & Francis

2
Y.-Q. Meng et al.
29
20
30
21
19
12
13
17 22
11
18
14
25
5
R3
C
O
26
COR3
COOH
f
COOH
28
g
a
1
OHC
O
2
3
16
9
6
10
4
8
15
27
O
O
7
HO
II1~6
2a~2f
UA-1
UA
b
24 23
COCl
e
C
O
R2
c
COOH
d
COOH
R
R
1ON
1ON
R
1ON
=
~
HON
I2 I5
1
I1
UA-2
H
H
N
,
,
,
,
R2
-COCH3
=
=
=
=
=
=
=
=
;
;
I
O
;
;
;
I1. R1
I2. R1
R2
3. R1
R2
I
4. R1
R2
N
I5. R1
-COCH3
-COCH3
-COCH3
-COCH3
OCH2
N
HOH2C
H
N
,
=
=
=
=
=
=
II6. R3
;
;
;
II4. R3
-OCH(CH
)
3
2
;
II1. R3
II2. R3
H
II3. R3
II5. R3
-OCH3
and
-OC2
-OCH2CH2CH3
e.
;
R2H
f.
N
O
5
,
,
,
,
: a.
or
c.
or
;
;
;
Jone's reagent
NH OH·HCl
Ac
DMAP, THF;
Et
CH Cl
3N,
R I R3Br K2CO3 DMF
conditions
b.
g.
d.
2O,
Reagents
Py
(COCl)2
2
2
2
3
.
,
R
reagent,
Vilsmeier
CH2Cl2
3H;
(COCl)2
Scheme 1. Synthetic routes of target compounds.
synthesized by the structure modification
at the positions C-2, C-3, and C-28 of UA.
The synthetic routes of the title com-
pounds are shown in Scheme 1. Com-
pound UA-1 was synthesized by the
reaction of UA with Jone’s reagent in
acetone, then it was treated with NH2-
OH·HCl to give compound UA-2. UA-2
was converted to compound I1 by the
reaction with Ac2O in the presence of 4-
dimethylaminopyridine (DMAP) in tetra-
hydrofuran (THF). This intermediate was
then condensed with the appropriate
amino and phenol compounds in the
presence of triethylamine to give the
compounds I2–I5. The reaction of UA-1
with bromoalkane in the presence of
K2CO3 in N,N-dimethylformamide
(DMF) gave carboxylic ester, then with
Vilsmeier reagent gave compounds II1–
II6. The title compounds were purified on a
silica gel column with petroleum ether–
ethyl acetate as eluents, and their struc-
1
tures were confirmed by MS, H NMR,
and elemental analysis, the results are
summarized in Tables 1 and 2.
Table 1. MS and elemental analysis data of target compounds.
Elemental analysis/found (calculated)
Compound
ESI-MS (m/z)
C
H
N
O
I1
I2
I3
I4
510.5[M 2 H]2
603.8[M þ Naþ]þ
587.1[M þ H]þ
617.4[M þ H]þ
602.3[M þ H]þ
497.6[M þ H]þ
511.2[M þ H]þ
525.9[M þ H]þ
552.7[M þ H]þ
558.3[M þ H]þ
525.6[M þ H]
74.92(75.11)
74.40(74.44)
77.93(77.77)
76.04(75.93)
77.65(77.83)
77.29(77.38)
77.49(77.60)
77.67(77.82)
76.22(76.18)
79.71(79.67)
77.59(77.82)
9.47(9.65)
9.75(9.72)
9.41(9.28)
9.28(9.15)
9.17(9.21)
9.66(9.74)
9.61(9.87)
9.83(9.99)
9.71(9.68)
9.17(9.22)
9.76(9.99)
2.68(2.74)
4.96(4.82)
4.72(4.77)
4.51(4.54)
2.36(2.33)
—
12.57(12.51)
11.31(11.02)
8.25(8.18)
10.11(10.37)
10.67(10.63)
12.71(12.88)
12.64(12.53)
12.25(12.19)
11.53(11.60)
8.34(8.60)
I5
II1
II2
II3
II4
II5
II6
—
—
2.46(2.54)
2.28(2.51)
—
12.03(12.19)

Journal of Asian Natural Products Research
3
Table 2. 1H NMR spectral data of target compounds.
Compound 1H NMR (CDCl3)
I1
d 11.0 (s, 1H, COOH), 5.35–5.29 (m, 1H, H-12), 2.26 (d, J ¼ 8.6 Hz, 1H, H-18), 2.18
(s, 3H, CH3COON), 1.38–1.34 (m, 1H, H-19), 1.32–1.29 (m, 1H, H-20), 1.27 (t,
J ¼ 7.5 Hz, 1H, H-5), 1.20 (t, J ¼ 7.3 Hz, 1H, H-9), 1.15 (s, 3H, CH3), 1.12 (s, 3H,
CH3), 1.05 (s, 3H, CH3), 0.96 (s, 3H, CH3), 0.89 (d, J ¼ 7.0 Hz, 3H, CH3), 0.85 (d,
J ¼ 7.1 Hz, 3H, CH3), 0.79 (s, 3H, CH3)
I2
d 5.33 (t, J ¼ 3.5 Hz, 1H, H-12), 3.66 (t, J ¼ 8.5 Hz, 2H, NCH2CH2aO), 3.62 (t,
J ¼ 8.5 Hz, 2H, NCH2CH2bO), 3.48 (t, J ¼ 8.0 Hz, 2H, NCH2aCH2O), 3.41 (t,
J ¼ 8.0 Hz, 2H, NCH2bCH2O), 3.00 (d, J ¼ 7.5 Hz, 1H, H-18), 2.15 (s, 3H,
CH3COON), 1.41–1.39 (m, 1H, H-19), 1.37–1.33 (m, 1H, H-20), 1.28 (t, J ¼ 7.5 Hz,
1H, H-5), 1.22 (t, J ¼ 7.0 Hz, 1H, H-9), 1.13 (s, 3H, CH3), 1.09 (s, 3H, CH3), 1.04 (s,
3H, CH3), 0.93 (s, 3H, CH3), 0.86 (d, J ¼ 7.5 Hz, 3H, CH3), 0.81 (d, J ¼ 7.0 Hz, 3H,
CH3), 0.77 (s, 3H, CH3)
I3
d 8.51 (s, 1H, NH), 7.53–7.21 (m, 5H, Ph), 5.31 (t, J ¼ 4.0 Hz, 1H, H-12), 2.43 (d,
J ¼ 8.3 Hz, 1H, H-18), 2.32 (s, 3H, CH3COON), 1.46–1.42 (m, 1H, H-19), 1.38–1.35
(m, 1H, H-20), 1.30 (t, J ¼ 7.0 Hz, 1H, H-5), 1.23 (t, J ¼ 7.1 Hz, 1H, H-9), 1.10 (s, 3H,
CH3), 1.06 (s, 3H, CH3), 1.02 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.89 (d, J ¼ 7.5 Hz, 3H,
CH3), 0.83 (d, J ¼ 7.0 Hz, 3H, CH3), 0.76 (s, 3H, CH3)
I4
d 8.62 (s, 1H, NH), 7.68–7.26 (m, 4H, Ph), 5.34 (t, J ¼ 3.6 Hz, 1H, H-12), 5.27 (s, 1H,
OH), 4.64 (s, 2H, CH2OH), 2.50 (d, J ¼ 7.6 Hz, 1H, H-18), 2.21 (s, 3H, CH3COON),
1.43–1.40 (m, 1H, H-19), 1.38–1.35 (m, 1H, H-20), 1.26 (t, J ¼ 7.5 Hz, 1H, H-5), 1.21
(t, J ¼ 7.0 Hz, 1H, H-9), 1.08 (s, 3H, CH3), 1.01 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.89 (s,
3H, CH3), 0.85 (d, J ¼ 8.1 Hz, 3H, CH3), 0.80 (d, J ¼ 7.5 Hz, 3H, CH3), 0.75 (s, 3H,
CH3)
I5
d 7.52–7.21 (m, 5H, Ph), 5.29 (t, J ¼ 3.6 Hz, 1H, H-12), 4.55 (s, 2H, CH2O), 2.53 (d,
J ¼ 7.5 Hz, 1H, H-18), 2.20 (s, 3H, CH3COON), 1.44–1.40 (m, 1H, H-19), 1.36–1.33
(m, 1H, H-20), 1.24 (t, J ¼ 7.0 Hz, 1H, H-5), 1.18 (t, J ¼ 6.5 Hz, 1H, H-9), 1.10 (s, 3H,
CH3), 1.05 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.87 (s, 3H, CH3), 0.83 (d, J ¼ 7.5 Hz, 3H,
CH3), 0.79 (d, J ¼ 7.5 Hz, 3H, CH3), 0.72 (s, 3H, CH3)
II1
II2
d 9.68 (s, 1H, CHO), 5.33 (t, J ¼ 3.1 Hz, 1H, H-12), 3.76 (s, 3H, COOCH3), 3.10 (t,
J ¼ 7.0 Hz, 1H, H-2), 2.52 (d, J ¼ 6.0 Hz, 1H, H-18), 1.42–1.38 (m, 1H, H-19), 1.32–
1.29 (m, 1H, H-20), 1.21 (t, J ¼ 6.2 Hz, 1H, H-5), 1.15 (t, J ¼ 7.8 Hz, 1H, H-9), 1.08 (s,
3H, CH3), 1.03 (s, 3H, CH3), 0.98 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.88 (d, J ¼ 6.5 Hz,
3H, CH3), 0.83 (d, J ¼ 7.1 Hz, 3H, CH3), 0.77 (s, 3H, CH3)
d 9.66 (s, 1H, CHO), 5.31 (t, J ¼ 3.5 Hz, 1H, H-12), 4.08–4.05 (m, 2H, COOCH2CH3),
3.12 (t, J ¼ 7.0 Hz, 1H, H-2), 2.50 (d, J ¼ 7.0 Hz, 1H, H-18), 1.48–1.44 (m, 1H, H-19),
1.35–1.32 (m, 1H, H-20), 1.25 (t, J ¼ 8.7 Hz, 3H, COOCH2CH3), 1.20 (t, J ¼ 6.5 Hz,
1H, H-5), 1.13 (t, J ¼ 7.0 Hz, 1H, H-9), 1.06 (s, 3H, CH3), 1.01 (s, 3H, CH3), 0.97 (s,
3H, CH3), 0.93 (s, 3H, CH3), 0.86 (d, J ¼ 7.5 Hz, 3H, CH3), 0.82 (d, J ¼ 8.0 Hz, 3H,
CH3), 0.75 (s, 3H, CH3)
II3
d 9.68 (s, 1H, CHO), 5.32 (t, J ¼ 3.5 Hz, 1H, H-12), 4.24–4.21 (m, 1H, COOCH), 3.11
(t, J ¼ 7.0 HZ, 1H, H-2), 2.52 (d, J ¼ 6.5 Hz, 1H, H-18), 1.50–1.47 (m, 1H, H-19),
1.44–1.41 (m, 1H, H-20), 1.20 (d, J ¼ 8.0 Hz, 3H, COOCH(CH3a)2), 1.17 (d,
J ¼ 8.0 Hz, 3H, COOCH(CH3b)2), 1.14 (t, J ¼ 7.3 Hz, 1H, H-5), 1.10 (t, J ¼ 7.0 Hz,
1H, H-9), 1.05 (s, 3H, CH3), 1.01 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.91 (s, 3H, CH3),
0.87 (d, J ¼ 7.5 Hz, 3H, CH3), 0.80 (d, J ¼ 8.0 Hz, 3H, CH3), 0.73 (s, 3H, CH3)
d 9.67 (s, 1H, CHO), 5.30 (t, J ¼ 3.5 Hz, 1H, H-12), 3.65 (t, J ¼ 8.5 Hz, 2H,
NCH2CH2aO), 3.62 (t, J ¼ 8.5 Hz, 2H, NCH2CH2bO), 3.50 (t, J ¼ 8.0 Hz, 2H,
NCH2aCH2O), 3.46 (t, J ¼ 8.0 Hz, 2H, NCH2bCH2O), 3.10 (t, J ¼ 7.0 HZ, 1H, H-2),
2.50 (d, J ¼ 7.5 Hz, 1H, H-18), 1.51–1.48 (m, 1H, H-19), 1.46–1.42 (m, 1H, H-20),
1.19 (t, J ¼ 8.0 Hz, 1H, H-5), 1.13 (t, J ¼ 7.0 Hz, 1H, H-9), 1.09 (s, 3H, CH3), 1.04 (s,
3H, CH3), 0.93 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.85 (d, J ¼ 7.0 Hz, 3H, CH3), 0.81 (d,
J ¼ 8.0 Hz, 3H, CH3), 0.74 (s, 3H, CH3)
II4
(Continued)

4
Y.-Q. Meng et al.
Table 2 – continued
Compound 1H NMR (CDCl3)
II5
d 9.68 (s, 1H, CHO), 8.49 (s, 1H, NH), 7.50–7.23 (m, 5H, Ph), 5.32 (t, J ¼ 3.1 Hz, 1H,
H-12), 3.15 (t, J ¼ 7.0 Hz, 1H, H-2), 2.53 (d, J ¼ 7.6 Hz, 1H, H-18), 1.49–1.45 (m, 1H,
H-19), 1.41–1.38 (m, 1H, H-20), 1.21 (t, J ¼ 8.0 Hz, 1H, H-5), 1.11 (t, J ¼ 7.0 Hz, 1H,
H-9), 1.08 (s, 3H, CH3), 1.05 (s, 3H, CH3), 0.99 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.83 (d,
J ¼ 7.5 Hz, 3H, CH3), 0.79 (d, J ¼ 8.5 Hz, 3H, CH3), 0.76 (s, 3H, CH3)
d 9.66 (s, 1H, CHO), 5.30 (t, J ¼ 3.8 Hz, 1H, H-12), 4.08 (t, 2H, J ¼ 7.6 Hz,
COOCH2CH2CH3), 3.10 (t, J ¼ 7.0 Hz, 1H, H-2), 2.51 (d, J ¼ 7.5 Hz, 1H, H-18),
1.82–1.78 (m, 2H, COOCH2CH2CH3), 1.43–1.40 (m, 1H, H-19), 1.38–1.34 (m, 1H,
H-20), 1.22 (t, J ¼ 6.5 Hz, 1H, H-5), 1.15 (t, J ¼ 7.0 Hz, 1H, H-9), 1.07 (s, 3H, CH3),
1.03 (s, 3H, CH3), 0.98 (s, 3H, CH3), 0.95 (t, J ¼ 8.0 Hz, 3H, COOCH2CH2CH3), 0.91
(s, 3H, CH3), 0.86 (d, J ¼ 7.5 Hz, 3H, CH3), 0.82 (d, J ¼ 8.0 Hz, 3H, CH3), 0.77 (s, 3H,
CH3)
II6
2.2. Bioassays
HeLa, HepG2, and BGC-823 cell lines
were 58.5%, 45.4%, 58.3%, while the
inhibitory rates of compound I4 against
these cells were 46.7%, 39.2%, 50.1%,
respectively, significantly higher than that
of UA, similar to the clinical medicine
Gefitinib against HepG2 cells, signifi-
cantly higher than Gefitinib against HeLa
and BGC-823 cell lines. Modification of
UA into acetyl oxyimino at C-3 position
and amide group at C-28 position could
improve the antitumor activity. The
inhibitory activity of compound II5 on
these kinds of tumor cells was higher than
Antitumor activities of the title compounds
were evaluated in vitro by the MTT
method against HeLa, HepG2, and BGC-
823 cell lines with UA and Gefitinib as the
positive control. The results are summar-
ized in Table 3.
As shown in Table 3, all the target
compounds showed better inhibitory
activity on HeLa, HepG2, and BGC-823
cell lines. Especially compounds I3 and I4
presented more significant inhibition to
these kinds of tumor cells, and the
inhibitory rates of compound I3 against
Table 3. Inhibitory activity of the target compounds on the HeLa, HepG2, and BGC-823 cells
proliferation.a
Inhibition rate (%)
IC50 (mmol L21
)
Compound
HeLa
HepG2
BGC-823
HeLa
HepG2
BGC-823
UA
I1
I2
I3
I4
I5
II1
II2
II3
II4
II5
II6
Gefitinib
12.3
25.9
31.3
58.5
46.7
28.1
16.2
18.7
21.5
29.3
31.7
24.5
42.6
11.8
12.8
25.2
45.4
39.2
26.6
24.2
24.8
20.7
33.1
35.6
23.9
46.2
10.2
30.3
34.2
58.3
50.1
36.5
20.1
20.4
25.2
32.6
34.5
30.2
46.0
.50
.50
32.6
9.25
13.8
.50
.50
.50
.50
49.3
33.4
.50
17.1
.50
.50
.50
21.2
23.7
.50
.50
.50
.50
39.5
36.0
.50
20.7
.50
40.6
35.2
8.06
9.15
32.9
.50
.50
.50
37.6
34.7
40.7
19.3
a Inhibitory percentage of cells treated with each compound at a concentration of 1025 mol l21 for 72 h.

Journal of Asian Natural Products Research
compound UA, so the introduced electron- serum,
100 U ml21
5
penicillin,
withdrawing group (carbonyl group) at C-
2 position of UA could also increase the
antitumor activity. Thus, reasonable modi-
fication of UA into amide group at C-28
position increased the antitumor activity
such as in compounds I3, I4, and II5; alkyl
side chains at C-3 such as alkanoyloxy
imino chain, and electron-withdrawing
group at C-2 position such as formyl
group might be important to the inhibitory
activity of tumor cell growth.
100 mg ml21 streptomycin) was obtained
from Shanghai Bioleaf Biotech Co., Ltd
(Shanghai, China). Tetrazolium bromide
salt (MTT), proteinase K (Proteinase K),
and bovine serum albumin were obtained
from Beyotime Institute of Biotechnology
(Shanghai, China).
Tumor cells were cultivated on Sanyo
MCO-175 carbon dioxide incubator from
NAPCO Corporation (Gunmaken, Japan).
SW-CJ-2FD Double Single surface clean
bench was obtained from Suzhou Purifi-
cation equipment Co., Ltd (Suzhou,
China); Olympus IX70 inverted micro-
scope from Olympus Corporation (Shin-
3. Conclusion
In this paper, eleven UA derivatives with
the modification at C-2, C-3, and C-28
positions were prepared, and their anti-
tumor activities on HeLa, HepG2, and
BGC-823 cell lines were evaluated.
As shown, our data suggested the trans-
formation of the hydroxyl at C-3 and the
carboxyl at C-28 of UA play a key role to
improve the antitumor activity, and
reasonable modifications at C-3 and C-28
of UA have a greater impact on the
antitumor activity of UA. It will be the
guiding significance for the next structure
design and optimization, and lay a good
foundation for the future research and
development of high efficiency and low
toxicity of anticancer drugs.
juku, Tokyo,
Japan); BIO-RAD
MODEL680 microplate reader from
North-West Medical Base Co. (St. Peters-
burg, Russia); TGL-16B low-speed desk-
top centrifuge from ShangHai Anting
Scientific Instrument Factory (Shanghai,
China). Thin-layer chromatography (TLC)
was carried out with GF254, column
chromatograph with silica gel (200–
300 mesh) obtained from Qing-dao Marine
Chemical Factory (Qingdao, China). The
reagents were all of analytical grade or
chemically pure.
4.2. Preparation of the compounds
4.2.1. 3-Oxoursolic acid (UA-1)
To a solution of UA (50 mg, 0.11 mmol) in
acetone was titrated Jone’s reagent
(0.2 ml, freshly prepared) below 08C, and
the mixture was warmed to room tem-
perature gradually, and stirred for 1 h. The
end of reaction was monitored by TLC
(eluent: petroleum ether–ethyl acetate;
3:1; the chromogenic agent was 10%
sulfuric acid ethanol solution). Then,
isopropanol (4 ml) was added to the
mixture and stirred for 30 min at room
temperature to quench the oxidation
resistance. A small amount of saturated
brine was added and then extracted with
ethyl acetate for three times. The organic
phases were combined and dried with
4. Experimental
4.1. Reagents and instruments
Melting points were determined in capil-
lary tubes on a Bu¨chi B-540 melting point
apparatus produced by Broker Corporation
(Flawil, Switzerland) and are uncorrected.
NMR spectra were recorded on Bruker
ARX-300 MHz spectrometers from Bruker
Corporation (Ettlingen, Germany) and the
solvent is CDCl3, using trimethylsilane as
an internal standard. ESI-MS were
recorded on Thermo-Finnigan LCQ equip-
ment from Thermo Finnigan (San Fran-
cisco, CA, USA). RPMI-1640 culture
medium (containing 10% fetal bovine

6
Y.-Q. Meng et al.
anhydrous sodium sulfate overnight, evap-
orated under reduced pressure to give a
white solid powder UA-1, with a yield of
70.8%. m.p. 193.7–195.18C; ESI-MS: m/z
455.2 [M þ H]þ.
a yield of 76.3%. m.p. 153.2–164.78C;
ESI-MS: m/z 483.2 [M þ H]þ.
4.2.5. 3-Oxoursolic acid isopropyl ester
(2c)
According to the same method for
compound 2a, compound 2c was prepared
from UA-1 (100 mg, 0.22 mmol) and
K2CO3 (30.0 mg, 0.22 mmol), and five
drops of isopropyl bromide in DMF. The
crude product was purified by column
chromatography on silica gel with pet-
roleum ether–ethyl acetate (10:1), to
obtain compound 2c, with a yield of
66.8%. m.p. 143.6–145.28C; ESI-MS: m/z
497.6 [M þ H]þ.
4.2.2. 3-Oximeursolic acid (UA-2)
UA-1 (50 mg, 0.11 mmol) was allowed to
react with hydroxylamine hydrochloride
(100 mg, 1.44 mmol) in pyridine, and the
mixture was refluxed at 1158C for 1.5 h,
then poured into ice water. A large amount
of white precipitate was produced, filtered
and the cake was washed with water, dried
to give a white solid UA-2, with a yield of
82.96%. m.p. 215.6–218.18C; ESI-MS: m/
z 468.2 [M 2 H]2.
4.2.6. 3-Oxoursolic acid morpholine
amide (2d)
4.2.3. 3-Oxoursolic acid methyl ester
(2a)
According to the same method for
compound I2, compound 2d was prepared
from 3-oxoursolic acid chloride (100 mg,
0.21 mmol) and 5 ml of morpholine in
DMF. The crude product was purified by
column chromatography on silica gel with
petroleum ether–ethyl acetate (6:1), to
obtain compound 2d, with a yield of
53.5%. m.p. 122.7–124.58C; ESI-MS: m/z
524.7 [M þ H]þ.
UA-1 (100 mg, 0.22 mmol) was dissolved
in DMF, then K2CO3 (30.0 mg,
0.22 mmol) and three drops of iodo-
methane were added at room temperature.
The reaction end was monitored by TLC.
After completion of reaction, the mixture
was extracted with ethyl acetate and
washed with saturated brine, dried over
anhydrous magnesium sulfate. The sol-
vent was evaporated under reduced
pressure to give a white solid. The crude
product was purified by column chroma-
tography on silica gel with petroleum
ether–ethyl acetate (10:1) to obtain
compound 2a, with a yield of 80.1%. m.
p. 166.8–168.18C; ESI-MS: m/z 469.5
[M þ H]þ.
4.2.7. 3-Oxoursolic acid aniline (2e)
According to the same method for
compound I2, compound 2e was prepared
from 3-oxoursolic acid chloride (100 mg,
0.21 mmol) and 6 ml of aniline in DMF.
The crude product was purified by column
chromatography on silica gel with pet-
roleum ether–ethyl acetate (8:1) to obtain
compound 2e, with a yield of 61.2%. m.
p. 128.3–129.58C; ESI-MS: m/z 530.4
[M þ H]þ.
4.2.4. 3-Oxoursolic acid ethyl ester (2b)
According to the same method for
compound 2a, compound 2b was prepared
from UA-1 (100 mg, 0.22 mmol), K2CO3
(30.0 mg, 0.22 mmol), and five drops of
bromoethane in DMF. The crude product
was purified by column chromatography
on silica gel with petroleum ether–ethyl
acetate (10:1) to obtain compound 2b, with
4.2.8. 3-Oxoursolic acid propyl ester (2f)
According to the same method for
compound 2a, compound 2f was prepared
from UA-1 (100 mg, 0.22 mmol) and

Journal of Asian Natural Products Research
7
K2CO3 (30.0 mg, 0.22 mmol) and four
drops of isopropyl bromide in DMF. The
crude product was purified by column
chromatography on silica gel with pet-
roleum ether–ethyl acetate (10:1), to
obtain compound 2f, with a yield of
60.3%. m.p. 138.7–140.18C; ESI-MS: m/z
497.3 [M þ H]þ.
2 mol l21 HCl to pH 3–4, CH2Cl2 was
removed under vacuum to precipitate
white solid. After filtration, the cake was
washed with water to pH 7 and dried with
anhydrous sodium sulfate. The crude was
purified on a silica gel column with
petroleum ether–ethyl acetate (15:1) to
give a white powder I2, with a yield of
36.0%. m.p. 129.8–131.28C.
4.2.9. 3-Acetoxyimineursolic acid (I1)
4.2.11. 3-Acetoxyimineursolic acid
aniline amide (I3)
To a stirred solution of UA-2 (50.0 mg,
0.11 mmol) in THF, pyridine (0.26 ml),
acetic anhydride (0.340 g, 3.3 mmol), and
a small amount of DMAP were added at
room temperature, the reaction end was
monitored by TLC (eluent:petroleum
ether–ethyl acetate (3:1), and the chromo-
genic agent was 10% sulfuric acid ethanol
solution). The resultant residue was parti-
tioned in 3 ml water, then treated with
2 mol l21 HCl to pH 3–4, and white
precipitate was produced, filtered and the
cake was washed with water to pH 7 and
dried with anhydrous sodium sulfate. The
crude was purified on a silica gel column
chromatography (petroleum ether–ethyl
acetate; 10:1) to give compound I1 as a
white solid, with a yield of 73.8%, m.
p. 89.5–91.48C.
According to the same method for
compound I2, compound I3 was prepared
from I1 (50.0 mg, 0.099 mmol) and aniline
(20.0 mg, 0.21 mmol) through the corre-
sponding acyl chloride (1). The solid was
purified by column chromatography (pet-
roleum ether–ethyl acetate; 10:1) to give
compound I3 as a white powder, with a
yield of 36.5%. m.p. 94.4–96.78C.
4.2.12. 3-Acetoxyimineursolic acid-2-
hydroxymethyl aniline (I4)
According to the same method for
compound I2, compound I4 was prepared
from I1 (50.0 mg, 0.099 mmol) and 2-
aminophenyl-methanol
(30.5 mg,
0.24 mmol) through the corresponding
acyl chloride (1). The solid was purified
by column chromatography (petroleum
ether–ethyl acetate; 10:1) to give com-
pound I4 as a white solid, with a yield of
38.9%. m.p. 78.4–80.68C.
4.2.10. 3-Acetoxyimineursolic acid
morpholine amide (I2)
To a solution of compound I1 (50.0 mg,
0.099 mmol) in dichloromethane was
added excess oxalyl chloride and the
resultant mixture was stirred at room
temperature for 20 h, the solvent and
unreacted oxalyl chloride were removed
under vacuum to give 3-acetoxyimineur-
solic acid chloride 1. Compound 1 was
dissolved completely in dichloromethane,
then treated with triethylamine to pH 9–
10, stirred for 5 min, added morpholine
(20.8 mg, 0.15 mmol), and reacted at room
temperature. The reaction end was mon-
itored by TLC. The resultant residue was
partitioned in 2 ml water, then treated with
4.2.13. 3-Acetoxyimineursolic acid
benzyl ester (I5)
According to the same method for
compound I2, compound I5 was prepared
from I1 (50.0 mg, 0.099 mmol) and benzyl
alcohol (25.6 mg, 0.24 mmol) through the
corresponding acyl chloride (1). The solid
was purified by column chromatography
(petroleum ether–ethyl acetate; 13:1) to
give compound I5 as a white solid, with a
yield of 56.8%. m.p. 83.4–84.68C.

8
Y.-Q. Meng et al.
4.2.14. 2-Formyl-3-oxoursolic acid
methyl ester (II1)
from 2d (50.0 mg, 0.095 mmol). The crude
product was purified by column chroma-
tography (petroleum ether–ethyl acetate;
20:1) to give compound II4 as a white
solid, with a yield of 40.3%. m.p. 101.3–
102.68C.
To a solution of compound 2a (50.0 mg,
0.107 mmol) in dichloromethane was
added Vilsmeier reagent (freshly pre-
pared) in an ice bath, and the mixture
was stirred for 3 h at room temperature.
Then a small amount of solid sodium
acetate was added with stirring. The
reaction end was monitored by TLC. The
obtained reaction was treated with 0.3%
NaHCO3 to pH 7 and extracted with ethyl
acetate, dried with anhydrous magnesium
sulfate, and the solvent was distilled under
reduced pressure to give the crude product
which was purified on a silica gel column
with petroleum ether–ethyl acetate (15:1)
to give a white solid II1, with a yield of
52.7%, m.p. 163.7–165.28C.
4.2.18. 2-Formyl-3-oxoursolic acid
aniline (II5)
According to the same method for
compound II1, compound II5 was prepared
from 2e (50.0 mg, 0.094 mmol). The crude
product was purified by column chroma-
tography (petroleum ether–ethyl acetate;
15:1) to give compound II5 as a white
solid, with a yield of 38.7%. m.p. 112.5–
114.38C.
4.2.19. 2-Formyl-3-oxoursolic acid
propyl ester (II6)
4.2.15. 2-Formyl-3-oxoursolic acid ethyl
ester (II2)
According to the same method for
compound II1, compound II6 was prepared
from 2f (50.0 mg, 0.101 mmol). The crude
product was purified by column chroma-
tography (petroleum ether–ethyl acetate;
15:1) to give compound II6 as a white
solid, with a yield of 50.1%. m.p. 137.1–
139.58C.
According to the same method for
compound II1, compound II2 was prepared
from 2b (50.0 mg, 0.062 mmol). The crude
product was purified by column chroma-
tography (petroleum ether–ethyl acetate;
10:1) to give compound II2 as a white
solid, with a yield of 56.2%, m.p. 175.1–
177.58C.
4.3. Antitumor bioassays
4.2.16. 2-Formyl-3-oxoursolic acid
isopropyl ester (II3)
Listed anticancer drug gefitinib was used
as the positive control medicine; the
antitumor activities of the 11 target
compounds were evaluated by MTT
assay in tumor cells including human
cervical cancer cells (HeLa), human
hepatoma cells (HepG2), and human
gastric carcinoma cells (BGC-823). Log-
arithmic growth phase tumor cells were
cultured in 96-well culture plate, each well
100 ml (containing 4000 cells approxi-
mately), and 1025 mol l21 of test sub-
stance was added to the control group. To
the negative control group and the
treatment group was added an equal
volume of solvent, cultured in an incubator
According to the same method for
compound II1, compound II3 was prepared
from 2c (50.0 mg, 0.101 mmol). The crude
product was purified by column chroma-
tography (petroleum ether–ethyl acetate;
10:1) to give compound II3 as a white
solid, with a yield of 45.6%. m.p. 150.5–
152.38C.
4.2.17. 2-Formyl-3-oxoursolic acid
morpholine amide (II4)
According to the same method for
compound II1, compound II4 was prepared

Journal of Asian Natural Products Research
9
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Disclosure statement
No potential conflict of interest was reported
by the authors.
[13] D. Liu, Y.Q. Meng, J. Zhao, and
L.G. Chen, Chem. Res. Chin. Univ. 24,
42 (2008).
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Funding
This work was supported by the Natural
Science Foundation of China [No. 21372156],
Program for Liaoning Excellent Talents in
University [No. LR2013017], Program of
Science and Technology in Shenyang of
China [No. F13-316-1-47].
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