Synthesis and antitumor activity evaluation of new asiatic acid derivatives

Article abstract of DOI:10.1080/10286020.2012.699961

Twelve novel asiatic acid (AA) derivatives were designed and synthesized. Their structures were confirmed using NMR, MS, and IR spectra. Their in vitro cytotoxicities on various cancer cell lines (HeLa, HepG2, BGC-823, and SKOV3) were evaluated by the 3-(

Full text of DOI:10.1080/10286020.2012.699961

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Synthesis and antitumor activity
evaluation of new asiatic acid
derivatives
Yan-Qiu Meng a , Yun-Yun Li a , Feng-Qing Li a , Yan-Ling Song a
Hai-Feng Wang a , Hong Chen b & Bo Cao b
,
a Department of Pharmaceutical Engineering, Shenyang University
of Chemical Technology, Shenyang, 110142, China
b Department of Pharmacognosy, Medical College of Chinese
People's Armed Police Forces, Tianjin, 300162, China
Published online: 28 Aug 2012.
To cite this article: Yan-Qiu Meng , Yun-Yun Li , Feng-Qing Li , Yan-Ling Song , Hai-Feng Wang , Hong
Chen & Bo Cao (2012): Synthesis and antitumor activity evaluation of new asiatic acid derivatives,
Journal of Asian Natural Products Research, 14:9, 844-855
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Journal of Asian Natural Products Research
Vol. 14, No. 9, September 2012, 844–855
Synthesis and antitumor activity evaluation
of new asiatic acid derivatives
Yan-Qiu Menga*, Yun-Yun Lia, Feng-Qing Lia, Yan-Ling Songa, Hai-Feng Wanga,
Hong Chenb and Bo Caob
aDepartment of Pharmaceutical Engineering, Shenyang University of Chemical Technology,
Shenyang 110142, China; Department of Pharmacognosy, Medical College of Chinese People’s
Armed Police Forces, Tianjin 300162, China
b
(Received 20 December 2011; final version received 31 May 2012)
Twelve novel asiatic acid (AA) derivatives were designed and synthesized. Their
structures were confirmed using NMR, MS, and IR spectra. Their in vitro cytotoxicities
on various cancer cell lines (HeLa, HepG2, BGC-823, and SKOV3) were evaluated by
the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Most of
the derivatives were found to have stronger cell growth inhibitory activity than AA.
Among them, compounds 5–8 and 11 with substituted amide group at C-28 exhibited
more potent cytotoxicity than AA, Gefitinib, and etoposide (positive control).
Keywords: asiatic acid derivatives; triterpenoid; synthesized; antitumor activity
1. Introduction
either C-3 position and/or C-28 position of
ursolic acid is essential for the cytotoxic
activity [16]. A series of AA derivatives
with substitution at positions of C-2, C-3,
C-23, and C-28 of AA have been
synthesized, and their cytotoxic activities
have been evaluated in vitro on four
cancer cell lines (HeLa, HepG2, SKOV3,
and BGC-823). The results showed that
acetylation of the C-2, C-3, C-23 alcohol,
together with a substituted amino group at
C-28, resulted in derivatives having stron-
ger cell growth inhibitory activity than AA.
Asiatic acid (AA, 2a,3b,23-trihydroxyurs-
12-ene-28-oic acid), a member of the ursane
family of pentacyclic triterpenoids found in
Centalla asiatica, can be easily prepared
fromhydrolysisofasiaticoside.AAhasbeen
traditionally used as a tonic in skin diseases
and leprosy [1–3]. In addition, AA, like
other triterpenes, has been reported to
possess other biological effects including
antitumor [4–6], anti-inflammation [7],
hepatoprotective effect [8], anti-depression,
and anti-Alzheimer’s disease [9,10].
In previous studies, we have reported
that some ursane triterpenoid derivatives
such as boswellic acid and ursolic acid
derivatives show strong biological activi-
ties [11–13]. Based on the reports that the
acid moiety at C-17 and ester functionality
at C-3 are essential for pharmacological
activities of pentacyclic triterpenes
[14,15], and a hydrogen donor group at
2. Results and discussion
2.1 Chemistry
AA was used as the lead compound, and
the structure modification was done at the
positions C-2, C-3, C-23, and C-28. The
synthetic pathways are shown in Scheme 1.
Compound 1 was prepared by reaction of
AA with aceticanhydride intetrahydrofuran
*Corresponding author. Email: mengyanqiu@hotmail.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2012.699961
http://www.tandfonline.com

Journal of Asian Natural Products Research
845
Scheme 1. Conditions and reagents: (a) Ac2O, DMAP (cat.), THF, rt. (b) (COCL)2, Et3N, CH2Cl2,
rt. (c) 4-Tert-butylphenol/4-nitrophenol/amines, Et3N, CH2Cl2, rt. (d) K2Cr2O7/AcOH, reflux 1068C.
(e) NH(C2H5)/morpholine, Et3N, rt. (f) BrCH2CH2Br, K2CO3, DMF, 08C. (g) H2O, K2CO3, DMF, rt.
(THF) in the presence of 4-dimethylamino-
pyridine in good yield, then it was treated
with oxalyl chloride to give compound 2.
This intermediate was then condensed with
the appropriate amino and phenol com-
pounds in the presence of triethylamine to
givethecompounds3–11.Compound1was
converted to a,b-unsaturated ketone 1* by
the oxidation with K2Cr2O7 in the presence
of acetic acid [17]. According to the same
method for 3–11, compounds 12 and 13
were synthesized from 2* with amines. a,b-
Unsaturated ketone 1* with 1,2-dibro-
moethane in the presence of K2CO3 in
DMF gave 2-bromoethyl ester, and with
H2O gave 15. The target compounds were
purified on a silica gel column with
petroleum ether–ethyl acetate (or acetone)
as eluents, and their structures were
confirmed by NMR, MS, and IR spectra.
2.2 Biological activity
In this experiment, cells of HeLa, HepG2,
and BGC-823 were the model chosen to
determine the cytotoxic activity of AA,
most of its derivatives, Gefitinib, and Vp-
16 (a positive control). Antiproliferative
effects were identified with 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide. Each experiment was
repeated at least three times. The results
are summarized in Table 1.
As shown in Table 1, compounds 3–6
were tested in HeLa, BGC823, and SKOV3
three cell lines and showed better inhibitory
activity than AA except for compound 3.
Compounds 5 and 6 presented more
significant inhibition to HeLa and BGC-
823 cell lines than Gefitinib and Vp-16 at a
concentration of 10 mmol/l, while the
inhibitory activity of compound 4 against

846
Y.-Q. Meng et al.

Journal of Asian Natural Products Research
847
both the cell lines was similar to that of Vp-
16, higher than that of Gefitinib. It is evident
that compounds 7, 8, and 11 exhibited good
inhibitory activity on HeLa, HepG2, and
BGC-823celllineshigherthanGefitiniband
Vp-16. Furthermore, IC50 values of com-
pound 6 (0.65 ^ 0.32 and 0.12 ^ 0.26);
compound7(1.77 ^ 0.71, 2.88 ^ 0.25, and
1.45 ^ 0.27), and compound 11 (1.14 ^
0.02, 4.79 ^ 3.07, and 1.51 ^ 0.25) were
significantly lower than Vp-16 and Gefiti-
nib. Especially, compounds 6, 7, and 11
presented strong inhibition on the HeLa and
BGC-823 cell lines. Among them, IC50
values of compounds 7 and 11 were
approximate half of Vp-16 and 10% of
Gefitinib on HeLa cells; IC50 value of
compound 11 was one quarter of Gefitinib
on HepG2 cells (Table 1).
and their antitumor activities on HeLa,
HepG2, BGC-823, and SKOV3 cell lines
were evaluated. Among them, compounds
5–8 and 11 showed the most significant
antiproliferative effects on tumor cells.
As shown, our data suggested that most of
AA conjugates with amino alcohol, aniline
or aniline with electron-donating group
substituted at C-28 such as compounds 4, 5,
8, and 11 resulting in stronger cytotoxic
activities than AA against the three cancer
cell lines; AA conjugated with electron-
withdrawing substituted group at C-28 such
as compounds 9 and 10 might increase
cytotoxic activity. It was suggested that 11-
carbonyl group was not the necessary group
of cytotoxic activity of AA derivatives
according to the activities of compounds 6
and 12. In addition, structure–activity
relationship study of the modified com-
pounds was carried out such that amino
group-substituted C-28 might be important
to the inhibitory activity of tumor cell
growth.
The nearly identical cytotoxicity of
compounds 4 and 15 (similar to Vp-16)
suggested the unimportance of 11-oxo for
biological activity. However, compound 3
with ester function was found to have
relatively lower antitumor effect than other
compounds. Modification of AA into
amide group at C-28 position increased
the antitumor activity such as in com-
pounds 5–8 and 11, and alkyl side chains
at C-28 amide chain might be important to
the inhibitory activity of tumor cell
growth.
4. Experimental
4.1 Material and methods
Melting points were determined in capillary
tubes on a Buchi B-545 melting point
apparatus produced by Broker Corporation
(Flawil, Switzerland) and are uncorrected.
NMR spectra were recorded on Bruker
ARX-300MHz spectrometers from Bruker
Corporation (Ettlingen, Germany) and the
solvent is CDCl3, using trimethylsilyl as an
internal standard. ESI-MS were recorded on
Thermo-Finnigan LCQ equipment from
Thermo Finnigan (San Francisco, CA,
USA). HR-ESI-MS were recorded on
micrOTOF-Q II equipment from BRUKER
OPTICS (Ettlingen, Germany). Thin-layer
chromatography was carried out with
GF254, column chromatography 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.
As demonstrated in Figure 1, HeLa and
HepG2 cells were treated with each
compound at different concentrations for
12 h. As shown in (C) and (E), exposure to
1 mM compounds 11 and 7 for 12 h induced
HeLa cells fragmented, compared with the
0.1% DMSO (A). As shown in (D) and (F),
exposure to 10 mM of compounds 11 and 7,
the fragmented cells were increased.
The treatment of compound 11 (4 mM)
exhibited similar cytotoxicity to Gefitinib
(20 mM), as shown in (I) and (H).
3. Conclusion
In this paper, 12 AA derivatives with the
modification of the functional groups of
C-2, C-3, C-23, and C-28 were prepared,

848
Y.-Q. Meng et al.
Table 2. The 13C NMR spectral data of some novel compounds 6–13.
d (ppm)
6
7
8
9
10
11
12
13
C1
C2
C3
C4
C5
C6
C7
C8
39.0
73.4
74.9
46.7
49.8
17.9
35.5
43.9
65.3
39.1
24.2
125.7
138.0
42.2
27.8
23.4
37.8
52.6
39.7
39.1
30.5
42.0
14.0
69.7
17.3
20.9
23.4
172.6
17.0
20.8
47.6
47.6
39.0
73.4
74.8
47.5
47.8
17.9
37.3
43.8
65.3
39.1
24.8
138.3
140.0
42.5
27.8
23.4
37.8
53.0
39.8
39.6
30.9
41.9
14.0
69.9
17.2
20.9
23.2
177.7
17.0
20.8
47.6
39.1
73.1
74.8
47.7
48.5
17.9
37.1
43.9
65.3
39.1
25.0
135.6
140.3
42.2
27.8
23.2
37.8
54.3
39.7
39.9
30.9
41.9
13.9
69.9
17.3
20.9
23.7
175.9
17.1
20.8
39.1
73.3
74.7
47.6
48.7
17.8
37.2
44.9
65.2
39.1
25.4
140.3
148.4
42.7
27.5
23.4
37.8
54.3
39.9
39.6
30.8
42.1
13.9
69.9
17.2
20.9
23.2
174.8
17.1
20.8
39.0
73.2
74.8
47.4
47.4
17.9
37.0
44.8
65.3
39.1
25.4
138.0
140.7
42.5
27.9
23.6
37.8
55.0
39.4
39.1
30.9
42.0
13.8
69.8
17.2
20.9
23.8
175.6
16.9
20.7
39.1
73.3
74.8
47.6
47.6
17.9
37.1
43.8
65.4
39.1
24.8
140.1
148.5
42.5
27.8
23.3
37.8
53.2
39.7
39.6
30.7
42.0
13.9
69.6
17.3
20.9
23.5
178.3
17.1
20.7
39.2
73.6
75.0
47.5
48.5
17.4
37.1
43.8
65.5
39.1
198.6
130.6
164.2
42.6
27.9
23.4
37.8
55.2
39.8
39.7
30.6
42.3
13.9
69.6
17.2
20.8
23.4
174.3
17.0
20.8
44.1
44.1
39.0
73.4
75.9
47.4
47.6
17.8
37.8
43.8
65.3
39.1
198.8
130.4
162.4
42.2
28.0
23.2
37.8
54.6
39.6
39.2
30.5
42.7
13.9
69.1
17.2
20.9
23.7
176.3
16.9
20.8
48.6
48.6
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
CH–N
Ph
137.5
127.5
127.5
128.0
128.0
127.5
119.7
119.7
129.5
129.5
135.5
117.7
21.3
170.8
170.5
170.4
21.2
122.0
122.0
125.6
129.7
135.3
128.1
129.6
112.9
112.9
120.6
130.7
138.6
114.0
114.0
126.0
126.0
123.2
129.1
CH3–Ph
CH3COZ
170.9
170.5
170.4
21.1
170.8
170.5
170.4
21.2
170.8
170.8
170.8
21.2
170.6
170.6
170.6
21.6
170.8
170.5
170.4
21.2
170.3
170.4
170.4
21.2
170.8
170.4
170.4
21.0
CH3COZ
21.1
21.1
21.1
21.1
21.1
21.1
21.1
21.1
21.3
21.0
21.2
21.0
21.2
21.3
21.1
21.2
N–CH2CH3
CH2ZOZ
12.8
12.8
12.7
12.7
66.2
66.2

Journal of Asian Natural Products Research
849
Figure 1. The morphological feature changes in tumor cells. HeLa cells were treated with: (A) 0.1%
DMSO; (B) 10 mM of Vp-16; (C) 1 mM of compound 11; (D) 10 mM of compound 11; (E) 1 mmol/l
of compound 7; and (F) 10 mM of compound 7, respectively; HepG2 cells were treated with:
(G) 0.1% DMSO; (H) 20 mM of Gefitinib; (I) 4 mM of compound 11, and then all HepG2 cells were
stained with Giemsa.
4.2 Preparation of the compounds
0.91 (s, 3H, CH3) (4 £ CH3), 0.80 (d, 6H,
J ¼ 12.0Hz, CH3 £ 2). ESI-MS: m/z 632.5
[M þ H2O]þ. HR-ESI-MS: m/z 615.3925
[M þ H]þ (calcd for C36H55O8, 615.3923).
4.2.1 2a,3b,23-Triacetoxyurs-12-ene-
28-oic acid (1)
To a solution of AA (100mg, 0.20mmol)
in THF (5ml) was added acetic anhydride
(567mg, 6.00mmol) and a small amount of
4-dimethylaminopyridine. The resultant
mixture was stirred at room temperature
for 2 h. Then, the solvent was removed by
evaporation under reduced pressure, the
resultant residue was added water and
dispersed. Then, it was filtered and the filter
cake was washed with water to pH 7. The
crude product was purified by column
chromatography (petroleum ether–acetic
ether: 3/1) to give 1 as a white solid
(105mg, 85.50%); m.p. 150.4–152.08C. IR
4.2.2 4-(Tert-butyl) phenyl-2a,3b,23-
triacetoxyurs-12-en-28-oate (3)
To a solution of compound 1 (100mg,
0.16 mmol) in dichloromethane (5 ml) was
slowly added oxalyl chloride (82 mg,
0.64 mmol). The resultant mixture was
stirred at room temperature for 24h. Then,
the solvent was removed by evaporation
under reduced pressure, and cyclohexane
(2 ml £ 3) was added to the residue,
concentrated to dryness to give 2. Com-
pound 2 (0.16 mmol) was dissolved
in dichloromethane, then basified to pH
8–9 with triethylamine. Then, 4-tert-
butyl-phenol (96 mg, 0.64 mmol) was
added. The resultant mixture was stirred at
room temperature for 3 h. After the solvent
was removed by evaporation under reduced
pressure, the resultant residue was dispersed
inwater, thenacidifiedtopH3–4withdilute
hydrochloric acid, then filtered and filter
v
max (KBr, cm21): 3421, 2951, 2923, 1747,
1
1697, 1457, 1370, 1235, 1045; H NMR
(300MHz, CDCl3, d ppm): 5.29 (t-like, 1H,
H-12), 5.21–5.10 (m, 2H, H-2/3), 3.90 (d,
1H, J ¼ 12.0 Hz, H-23), 3.61 (d, 1H,
J ¼ 12.0Hz, H-23), 2.41 (1H, s, H-9), 2.19
(s, 3H, CH3CO), 2.04 (s, 3H, CH3CO), 1.96
(s, 3H, CH3CO) (3 £ CH3CO), 1.21 (s, 3H,
CH3), 1.14 (s, 3H, CH3), 1.00 (s, 3H, CH3),

850
Y.-Q. Meng et al.
cake was washed with water to pH 7. The
residue was purified by column chromatog-
raphy (petroleum ether–acetic ether: 8/1) to
give 3 as a white solid (25 mg, 20.95%);
m.p. 183.6–185.88C. IR vmax (KBr, cm21):
2912, 1735, 1630, 1611, 1509, 1390, 1360,
1120; 1H NMR (300MHz, CDCl3, d ppm):
7.39(d, 1H, J ¼ 9.0 Hz, Ar–H), 7.36(d, 1H,
J ¼ 9.0 Hz, Ar–H), 6.95(d, 1H, J ¼ 9.0 Hz,
Ar–H), 6.92 (d, 1H, J ¼ 9.0 Hz, Ar–H),
5.34 (t-like, 1H, H-12), 5.20–5.09 (m, 2H,
H-2/3), 3.88 (d, 1H, J ¼ 12.0Hz, H-23),
3.60 (d, 1H, J ¼ 12.0Hz, H-23), 2.40(1H, s,
H-9), 2.11 (s, 3H, CH3CO), 2.04 (s, 3H,
CH3CO), 2.00 (s, 3H, CH3CO)
(3 £ CH3CO), 1.31 (s, 9H, CH3 £ 3), 1.21
(s, 3H, CH3), 1.14 (s, 3H, CH3), 1.00 (s, 3H,
CH3), 0.91 (s, 3H, CH3) (4 £ CH3), 0.80 (d,
6H, J ¼ 12.0Hz, CH3 £ 2); ESI-MS: m/z
764.1 [M þ H2O]þ; HR-ESI-MS: m/z
747.4817 [M þ H]þ (calcd for C46H66O8,
747.4819).
[M þ H]þ (calcd for C38H59NO8,
658.4393).
4.2.4 N-[2a,3b,23-Triacetoxy-urs-12-
ene-28-oyl]-3-aminopropanol (5)
According to the same method for 3,
compound 5 was prepared from 1 (100mg,
0.16 mmol) and 3-amino-1-propanol
(600mg, 8.10mmol) through the corre-
sponding acyl chloride (2). The solid was
purified by column chromatography (pet-
roleumether–aceticether:2.5/1)togive5as
a white solid (50 mg, 43.03%); m.p. 151.0–
152.08C. IR vmax (KBr, cm21): 3418, 2946,
1
1736, 1633, 1526, 1454, 1373, 1247; H
NMR (300MHz, CDCl3, d ppm): 6.21 (s,
1H, NH), 5.33 (t-like, 1H, H-12), 5.10–5.07
(m, 2H, H-2/3), 3.85 (d, 1H, J ¼ 12.0Hz, H-
23), 3.59(d, 1H, J ¼ 12.0Hz, H-23), 3.54(t,
2H, J ¼ 6.0 Hz, CH2O), 3.47–3.38 (m, 1H,
NCH), 3.19–3.08(m, 1H, NCH), 2.70–2.59
(m, 2H, NHCH2CH2), 2.54 (t, 1H,
J ¼ 12.0Hz, H-9), 2.09 (s, 3H, CH3CO),
2.03 (s, 3H, CH3CO), 1.99 (s, 3H, CH3CO)
(3 £ CH3CO), 1.47 (s, 3H, CH3), 1.10 (s,
3H, CH3), 0.96 (s, 3H, CH3), 0.90 (s, 3H,
CH3) (4 £ CH3), 0.81 (d, 6H, J ¼ 12.0Hz,
CH3 £ 2). ESI-MS: m/z: 672.5 [M þ H]þ.
HR-ESI-MS: m/z 672.4522 [M þ H]þ
(calcd for C39H61NO8, 672.4521).
4.2.3 N-[2a,3b,23-Triacetoxy-urs-12-
ene-28-oyl]-2-amino-1-ethanol (4)
According to the same method as for 3,
compound 4 was prepared from 1 (100mg,
0.16 mmol)
and
2-amino-1-ethanol
(600mg, 1.00mmol) through the corre-
sponding acyl chloride (2). The solid was
purified by column chromatography (pet-
roleumether–aceticether:2.5/1)togive4as
a white solid (60 mg, 55.89%); m.p. 149.0–
151.08C. IR vmax (KBr, cm21): 3421, 2922,
4.2.5 N-(2a,3b,23-Triacetoxyurs-12-
ene-28-oyl)diethyl-amine (6)
According to the same method for 3,
compound 6 was prepared from 1 (100mg,
0.16 mmol) and diethylamine (116 mg,
1.6 mmol) through the corresponding acyl
chloride (2). The solid was purified by
column chromatography (petroleum ether–
acetic ether: 4/1) to give 6 as a white solid
(38 mg, 35.50%); m.p. 118.2–120.08C. IR
1
1736, 1637, 1530, 1457, 1312, 1249; H
NMR (300MHz, CDCl3, d ppm): 6.36 (s,
1H, NH), 5.35 (t-like, 1H, H-12), 5.18–5.07
(m, 2H, H-2/3), 3.86(d, 1H, J ¼ 12.0Hz, H-
23), 3.69 (t, 2H, J ¼ 6.0 Hz, CH2O), 3.59 (d,
1H, J ¼ 12.0Hz, H-23), 3.48–3.42 (m, 1H,
NCH), 3.31–3.22 (m, 1H, NCH), 2.10 (s,
3H, CH3CO), 2.04 (s, 3H, CH3CO), 2.00 (s,
3H, CH3CO) (3 £ CH3CO), 1.50 (s, 3H,
CH3), 1.12 (s, 3H, CH3), 0.97 (s, 3H, CH3),
0.90 (s, 3H, CH3) (4 £ CH3), 0.85 (d, 6H,
J ¼ 12.0Hz, CH3 £ 2). ESI-MS: m/z 658.6
[M þ H]þ. HR-ESI-MS: m/z 658.4399
v
max (KBr, cm21): 2942, 1735, 1635, 1528,
1456, 1373, 1246; 1H NMR (300MHz,
CDCl3, d ppm): 5.27 (t-like, 1H, H-12),
5.18–5.08 (m, 2H, H-2/3), 3.88 (d, 1H,
J ¼ 12.0 Hz, H-23), 3.58 (d, 1H,
J ¼ 12.0Hz, H-23), 3.30–3.24 (m, 4H, N-
CH2 £ 2), 2.12 (s, 3H, CH3CO), 2.10 (s, 3H,

Journal of Asian Natural Products Research
851
CH3CO), 1.99 (s, 3H, CH3CO)
(3 £ CH3CO), 1.94–1.92 (m, 3H,
CH3 £ 2), 1.14 (s, 3H, CH3), 1.11 (s, 3H,
CH3), 0.96 (s, 3H, CH3), 0.90 (s, 3H, CH3)
(4 £ CH3), 0.83 (d, 6H, J ¼ 12.0 Hz,
CH3 £ 2); for 13C NMR (300 MHz,
CDCl3, d ppm) spectral data, see Table 1.
ESI-MS: m/z 670.6 [M þ H]þ. HR-ESI-
MS: m/z 670.4723 [M þ H]þ (calcd for
C40H63N1O7, 670.4721).
a white solid (62 mg, 53.08%); m.p. 255.0–
257.58C. IR vmax (KBr, cm21): 3497, 2828,
1
1686, 1610, 1486, 1397, 1106; H NMR
(300MHz, CDCl3, d ppm): 7.58 (s, 1H,
NH), 7.34 (d, 2H, J ¼ 4.0 Hz Ar–H), 7.11
(d, 2H, J ¼ 4.0 Hz, Ar–H), 5.49 (t-like, 1H,
H-12), 5.17–5.07 (m, 2H, H-2/3), 3.85 (d,
1H, J ¼ 12.0 Hz, H-23), 3.59 (d, 1H,
J ¼ 12.0 Hz, H-23), 2.31 (s, 3H, Ar–
CH3), 2.09 (s, 3H, CH3CO), 2.03 (s, 3H,
CH3CO), 2.00 (s, 3H, CH3CO)
(3 £ CH3CO), 1.14 (s, 3H, CH3), 1.07 (s,
3H, CH3), 0.99 (s, 3H, CH3), 0.92 (s, 3H,
CH3) (4 £ CH3), 0.74 (d, 6H, J ¼ 9.0 Hz,
CH3 £ 2); for 13C NMR (300MHz, CDCl3,
d ppm) spectral data, see Table 1. ESI-MS:
m/z 704.5 [M þ H]þ; HR-ESI-MS: m/z
4.2.6 N-(2a,3b,23-Triacetoxyurs-12-
ene-28-oyl)benzylamine (7)
According to the same method for 3,
compound 7 was prepared from 1 (100mg,
0.16 mmol) and phenylamine (171 mg,
1.6 mmol) through the corresponding acyl
chloride (2). The solid was purified by
column chromatography (petroleum ether–
acetic ether: 4/1) to give 7 as a white solid
(61 mg, 54.23%); m.p. 137.9–139.78C. IR
704.4519
[M þ H]þ
(calcd
for
C43H61N1O7, 704.4521).
v
max (KBr, cm21): 3498, 2826, 1684, 1612,
4.2.8 N-(2a,3b,23-Triacetoxyurs-12-
ene-28-oyl)-4-chloro-3-fluoroaniline (9)
1487, 1398, 1108; 1H NMR (300MHz,
CDCl3, d ppm): 7.35–7.24 (m, 5H, Ar–H),
6.11 (s, 1H, NH), 5.23 (t-like, 1H, H-12),
5.16–5.07(m, 2H, H-2/3), 4.53, 4.20(d, 1H,
J ¼ 12.0 Hz NHCH2), 3.86 (d, 1H,
J ¼ 12.0 Hz, H-23), 3.59 (d, 1H,
J ¼ 12.0Hz, H-23), 2.09 (s, 3H, CH3CO),
2.04 (s, 3H, CH3CO), 1.99 (s, 3H, CH3CO)
(3 £ CH3CO), 1.09 (s, 3H, CH3), 1.08 (s,
3H, CH3), 0.96 (s, 3H, CH3), 0.90 (s, 3H,
CH3) (4 £ CH3), 0.73 (d, 6H, J ¼ 12.0Hz,
CH3 £ 2); for 13C NMR (300MHz, CDCl3,
d ppm) spectral data, see Table 1. ESI-MS:
m/z 704.5 [M þ H]þ; HR-ESI-MS: m/z
According to the same method as for 3,
compound 9 was prepared from 1 (100mg,
0.16 mmol) and 4-chloro-3-fluoroaniline
(144 mg, 1.6 mmol) through the corre-
sponding acyl chloride (2). The solid was
purified by column chromatography (pet-
roleum ether–acetic ether: 5/1) to give 9 as
a white solid (80 mg, 67.48%); m.p. 159.8–
161.48C. IR vmax (KBr, cm21): 3496, 2827,
1
1686, 1610, 1486, 1397, 1103; H NMR
(300MHz, CDCl3, d ppm): 7.74 (s, 1H,
Ar–H), 7.62 (s, 1H, NH), 7.18–7.07 (m,
2H, Ar–H), 5.49 (t-like, 1H, H-12), 5.17–
5.07 (m, 2H, H-2/3), 3.85 (d, 1H,
J ¼ 12.0 Hz, H-23), 3.59 (d, 1H,
J ¼ 12.0Hz, H-23), 2.09 (s, 3H, CH3CO),
2.03 (s, 3H, CH3CO), 2.00 (s, 3H, CH3CO)
(3 £ CH3CO), 1.14 (s, 3H, CH3), 1.08 (s,
3H, CH3), 1.00 (s, 3H, CH3), 0.92 (s, 3H,
CH3) (4 £ CH3), 0.71 (d, 6H, J ¼ 9.0 Hz,
CH3 £ 2); for 13C NMR (300MHz, CDCl3,
d ppm) spectral data, see Table 1. ESI-MS:
m/z 742.4 [M þ H]þ. HR-ESI-MS: m/z
764.3102 [M þ Na]þ (calcd for C42H57Cl1-
F1N1O7, 764.3100).
704.4523
[M þ H]þ
(calcd
for
C43H61N1O7, 704.4521).
4.2.7 N-(2a,3b,23-Triacetoxyurs-12-
ene-28-oyl)p-toluidine (8)
According to the same method for 3,
compound was prepared from
8
1
(100 mg, 0.16 mmol) and p-toluidine
(171 mg, 1.6 mmol) through the corre-
sponding acyl chloride (2). The solid was
purified by column chromatography (pet-
roleum ether–acetic ether: 5/1) to give 8 as

852
Y.-Q. Meng et al.
4.2.9 N-(2a,3b,23-Triacetoxyurs-12-
ene-28-oyl)-3,4-di-chloroaniline (10)
3H, CH3), 1.12 (s, 3H, CH3), 1.00 (s, 3H,
CH3) (4 £ CH3), 0.81 (d, 6H, J ¼ 9.0 Hz,
CH3 £ 2); for 13C NMR (300MHz, CDCl3,
d ppm) spectral data, see Table 1. ESI-MS:
m/z 705.5 [M þ H]þ; HR-ESI-MS: m/z
According to the same method for 3,
compound 10 was prepared from 1
(100mg, 0.16mmol) and 3,4-dichloroani-
line (230mg, 1.6 mmol) through the corre-
sponding acyl chloride (2). The solid was
purified by column chromatography (pet-
roleum ether–acetic ether: 5/1) to give 10 as
a white solid (86 mg, 71.00%); m.p. 129.6–
131.18C. IR vmax (KBr, cm21): 3492, 2825,
727.4299
[M þ Na]þ
(calcd
for
C42H60N2O7, 727.4293).
4.2.11 2a,3b,23-Triacetoxyurs-11-oxo-
12-ene-28-oic acid (1*)
A solution of 1 (50.0 mg, 0.08mmol) and
K2Cr2O7 · 2H2O (75 mg, 0.25 mmol) in
10ml of acetic acid was refluxed for 5 h.
The mixture was cooled to 208C and diluted
withH2O(5 ml),thenextractedwithCH2Cl2
(3 £ 5 ml). The organic phase was neutral-
ized with saturated NaHCO3 solution to pH
7–8, washed with water (5 ml £ 2), and
saturated NaCl solution (5 ml). The organic
phase was dried over anhydrous magnesium
sulfate. Filtration and evaporation of solvent
atreduced pressureyielded light green solid,
which was purified by silica gel chromatog-
raphy (petroleum ether–ethyl acetate: 2/1)
to yield a white solid (41.6 mg, 81.1%).
m.p. 288.4–289.68C. IR vmax (KBr, cm21):
3446, 1745, 1661, 922, 803, 744; 1H NMR
(300MHz, CDCl3, d ppm): 5.37 (s, 1H, H-
12), 5.28–5.19 (m, 2H, H-2/3), 3.90 (d, 1H,
J ¼ 12.0 Hz, H-23), 3.61 (d, 1H,
J ¼ 12.0Hz, H-23), 2.41 (1H, s, H-9), 2.11
(s, 3H, CH3CO), 2.04 (s, 3H, CH3CO), 1.96
(s, 3H, CH3CO) (3 £ CH3CO), 1.37 (s, 3H,
CH3), 1.28 (s, 3H, CH3), 1.21 (s, 3H, CH3),
0.97 (s, 3H, CH3) (4 £ CH3), 0.80 (d, 6H,
J ¼ 12.0Hz, CH3 £ 2). ESI-MS: m/z 629.5
[M þ H]þ; HR-ESI-MS: m/z 629.3723
[M þ H]þ (calcd for C37H55NO9,
629.3721).
1
1685, 1613, 1486, 1390, 1105; H NMR
(300MHz, CDCl3, d ppm): 7.80 (s, 1H, Ar–
H), 7.67 (s, 1H, NH), 7.35 (d, 1H,
J ¼ 9.0 Hz, Ar–H), 7.21 (d, 1H,
J ¼ 9.0 Hz, Ar–H), 5.50 (t-like, 1H, H-
12), 5.17–5.07 (m, 2H, H-2/3), 3.85 (d, 1H,
J ¼ 12.0 Hz, H-23), 3.59 (d, 1H,
J ¼ 12.0Hz, H-23), 2.09 (s, 3H, CH3CO),
2.03 (s, 3H, CH3CO), 2.00 (s, 3H, CH3CO)
(3 £ CH3CO), 1.14 (s, 3H, CH3), 1.08 (s,
3H, CH3), 1.00 (s, 3H, CH3), 0.92 (s, 3H,
CH3) (4 £ CH3), 0.70 (d, 6H, J ¼ 9.0 Hz,
CH3 £ 2); for 13C NMR (300MHz, CDCl3,
d ppm) spectral data, see Table 1. ESI-MS:
m/z 758.4 [M þ H]þ; HR-ESI-MS: m/z
780.3410 [M þ Na]þ (calcd for C42H57Cl2-
F1N1O7, 780.3408).
4.2.10 N-(2a,3b,23-Triacetoxyurs-12-
ene-28-oyl)-phenylhydrazine (11)
According to the same method for 3,
compound 11 was prepared from 1
(100mg, 0.16mmol) and phenylhydrazine
(69 mg, 0.64mmol) throughthe correspond-
ing acyl chloride (2). The solid was purified
by column chromatography (petroleum
ether–acetic ether: 3/1) to give 11 as a
white solid (49 mg, 43.50%); m.p. 186.6–
188.28C. IR vmax (KBr, cm21): 3492, 2825,
1
4.2.12 N-[2a,3b,23-Triacetoxy-urs-11-
oxo-12-ene-28-oyl]-diethylamine (12)
1685, 1613, 1486, 1390, 1105; H NMR
(300MHz,CDCl3,dppm):7.69(s,1H,NH),
7.25–6.85 (m, 5H, Ar–H), 5.45 (t-like, 1H,
H-12), 5.19–5.09 (m, 2H, H-2/3), 3.87 (d,
1H, J ¼ 12.0 Hz, H-23), 3.61 (d, 1H,
J ¼ 12.0Hz, H-23), 2.10 (s, 3H, CH3CO),
2.04 (s, 3H, CH3CO), 2.00 (s, 3H, CH3CO)
(3 £ CH3CO), 1.27 (s, 3H, CH3), 1.13 (s,
Compound 1* (54.5 mg, 0.09mmol) was
allowedtoreactwithdiethylamine(25.3 mg,
0.35mmol) by using general procedure to
get compound 12 (22.4 mg, 56.28%). The
reaction mixture was stirred at room
temperaturefor4 h.Theresiduewaspurified

Journal of Asian Natural Products Research
4.2.14 2-Bromoethyl-2a,3b,23-
triacetoxy-11-oxo-urs-12-en-28-oate (14)
853
by column chromatography (petroleum
ether–ethyl acetate: 3/2). m.p. 237.3–
240.08C; IR vmax (KBr, cm21): 2971, 2930,
2872, 1744, 1651, 1614, 1473, 1390, 1234,
1035; 1H NMR (300MHz, CDCl3, d ppm):
5.67 (t-like, 1H, H-12), 5.34–5.26 (m, 1H,
H-2), 5.05 (d, 1H, J ¼ 12.0Hz, H-3), 3.85
(d, 1H, J ¼ 12.0Hz, H-23), 3.57 (d, 1H,
J ¼ 12.0Hz, H-23), 3.28–3.22 (m, 4H, N–
CH2 £ 2), 2.36 (s, 1H, H-9), 2.09 (s, 3H,
CH3CO), 2.01 (s, 3H, CH3CO), 1.95 (s, 3H,
CH3CO) (3 £ CH3CO), 1.90–1.88 (m, 6H,
CH3 £ 2), 1.29 (s, 3H, CH3), 1.28 (s, 3H,
CH3), 1.12 (s, 3H, CH3), 1.00 (s, 3H, CH3)
(4 £ CH3), 0.87 (d, 6H, J ¼ 6.0 Hz,
CH3 £ 2); for 13C NMR (300 MHz,
CDCl3, d ppm) spectral data, see Table 1.
ESI-MS: m/z 684.5 [M þ H]þ; HR-ESI-
MS: m/z 684.4567 [M þ H]þ (calcd for
C40H61N1O8, 684.4566).
Compound 1* (100 mg, 0.16mmol) and
potassium carbonate (0.045g, 0.32mmol)
were dissolved in DMF (4ml), and then 1,2-
dibromoethane (0.060g, 0.32mmol) was
added and the solution was stirred at 08C for
2–3 h. The reaction mixture was filtered,
and the cake was washed with ethyl acetate
(4 £ 15ml). The organic phase was neutral-
ized by 1N HCl (2 £ 10ml), washed with
saturated sodium chloride solution, and
dried over anhydrous magnesium sulfate.
Filtration and evaporation of solvent at
reduced pressure gave the title compound as
a white solid (81 mg, 68.94%). m.p. 234.7–
236.98C. IR vmax (KBr, cm21): 2932, 2873,
1740, 1659, 1045, 563; ESI-MS: m/z 814.3
[M þ 2K]þ. HR-ESI-MS: calcd for
C38H55Br1O9 Hþ [M þ H]þ: 735.3123,
found 735.3122.
4.2.13 N-[2a,3b,23-Triacetoxy-urs-11-
oxo-12-ene-28-oyl]-morpholine (13)
4.2.15 2-(Hydroxyl)ethyl-2a,3b,23-
triacetoxy-11-oxo-urs-12-en-28-oate (15)
Compound 1* (100.00 mg, 0.14 mmol) was
allowed to react with morpholine (49.0 mg,
0.56mmol) by using general procedure to
get compound 13 (38.0 mg, 36.61%). The
reaction mixture was stirred at room
temperaturefor5 h.Theresiduewaspurified
by column chromatography (petroleum
ether–ethyl acetate: 2.8/1). m.p. 138.8–
140.78C; IR vmax (KBr, cm21): 2955, 2872,
1746, 1661, 1620, 1460, 1369, 798, 757; 1H
NMR (300MHz, CDCl3, d ppm): 5.63 (s,
1H, H-12), 5.30–5.27 (m, 1H, H-2), 5.09 (d,
1H, J ¼ 12.0 Hz, H-3), 3.85 (d, 1H,
J ¼ 12.0Hz, H-23), 3.64–3.57 (m, 8H,
N(CH2CH2)2O), 3.57 (d, 1H, J ¼ 12.0Hz,
H-23), 2.10 (s, 3H, CH3CO), 2.02 (s, 3H,
CH3CO), 1.95 (s, 3H, CH3CO)
(3 £ CH3CO), 0.98 (s, 3H, CH3), 0.94 (s,
3H, CH3), 0.89 (s, 3H, CH3), 0.87 (s, 3H,
CH3) (4 £ CH3), 0.78 (d, 6H, J ¼ 9.0 Hz,
CH3 £ 2); for 13C NMR (300MHz, CDCl3,
d ppm) spectral data see Table 1. ESI-MS:
m/z 730.5 [M þ Na]þ; HR-ESI-MS: m/z
A solution of 14 (100mg, 0.14mmol) in
10ml of 95% alcohol was refluxed, then the
solution was cooled to room temperature
and filtered. The residue was successively
washed with ethyl acetate. The organic
phase was washed with saturated sodium
bicarbonate and saturated sodium chloride
solution, and dried over anhydrous mag-
nesium sulfate. Filtration and evaporation of
solvent at reduced pressure gave the title
compound as a white solid (47 mg, 49.89%)
(petroleum ether–ethyl acetate: 4/1),
m.p. 129.7–132.68C. IR vmax (KBr, cm21):
3556, 2949, 2875, 1741, 1718, 1648, 1035,
901, 799, 745; 1H NMR (300MHz, CDCl3,
d ppm): 5.37 (s, 1H, H-12), 5.29–5.18 (m,
2H, H-2/3), 4.22–4.15 (m, 4H, ZOCH2-
CH2OZ), 3.90 (d, 1H, J ¼ 12.0Hz, H-23),
3.61(d, 1H, J ¼ 12.0Hz, H-23), 2.41(1H, s,
H-9), 2.11 (s, 3H, CH3CO), 2.04 (s, 3H,
CH3CO), 1.96 (s, 3H, CH3CO)
(3 £ CH3CO), 1.37 (s, 3H, CH3), 1.28 (s,
3H, CH3), 1.21 (s, 3H, CH3), 0.97 (s, 3H,
CH3) (4 £ CH3), 0.80 (d, 6H, J ¼ 12.0Hz,
730.4525
[M þ Na]þ
(calcd
for
C40H59N1O9, 730.4526).

854
Y.-Q. Meng et al.
CH3 £ 2). ESI-MS: m/z 673.5 [M þ H]þ;
HR-ESI-MS: m/z 673.4519 [M þ H]þ
(calcd for C38H56O10, 673.4521).
[4] B.C. Park, K.O. Bosire, E.S. Lee,
Y.S. Lee, and J.A. Kim, J. Cancer Lett.
218, 81 (2005).
[5] Y.L. Hsu, P.L. Kuo, L.T. Lin, and
C.C. Lin, J. Pharmacol. Exp. Ther. 313,
333 (2005).
4.3 Biological activity assays
[6] Y.S. Lee, D.R. Jin, E.J. Kowon,
E.J. Kwon, S.H. Park, E.S. Lee,
T.C. Jeong, D.H. Nam, K. Huh, and
J.A. Kim, J. Cancer Lett. 186, 83 (2002).
[7] Y.M. Fan, L.Z. Xu, J. Gao, Y. Wang,
X.H. Tang, X.N. Zhao, and Z.X. Zhang,
Fitoterapia 75, 253 (2004).
[8] J. Gao, X.H. Tang, H. Dou, Y.M. Fan,
X.N. Zhao, and Q. Xu, J. Pharm.
Pharmacol. 56, 1449 (2004).
[9] S.S. Jew, C.H. Yoo, D.Y. Lim, H. Kim,
I. Mook-Jung, M.W. Jung, H. Choi,
Y.H. Jung, H. Kim, and H.G. Park,
J. Bioorg. Med. Chem. Lett. 10, 119
(2000).
[10] I. Mook-Jung, J.E. Shin, S.H. Yun,
K. Huh, J.Y. Koh, H.K. Park, S.S. Jew,
and M.W. Jung, J. Neurosci. Res. 58, 417
(1999).
Tumor cells (200 ml per well) were cultured
in 96-well plates in Roswell Park Memorial
Institute (RPMI) 1640 with 10% fetal calf
serum under 5% CO2 and 378C. After 24h,
the appropriate test compound was added
with different indicated concentrations of
1025, 1026, 1027, and 1028 mol/l, respect-
ively, for another 72-h incubation. Then,
dimethyl-2-thiazolyl-2,5-diphenyl-2H-tet-
razolium-bromide was added, and absor-
bancewasmeasuredonanELISAreaderata
wavelength of 490 nm. Each testwas carried
out three times. The concentration of
compounds which gives 50% growth
inhibition corresponds to the IC50.
The results are summarized in Table 1.
[11] Y.Q. Meng, L.X. Zhao, Z. Wang, D. Liu,
and Y.K. Jing, J. Chin. Chem. Lett. 16,
867 (2005).
[12] D. Liu, Y.Q. Meng, J. Zhao, and
L.G. Chen, Chem. Res. Chin. Univ. 24,
42 (2008).
[13] Y.Q. Meng, D. Liu, L.L. Cai, H. Chen,
B. Cao, and Y.Z. Wang, J. Bioorg. Med.
Chem. 17, 848 (2009).
Acknowledgments
The authors thank the Project-sponsored by
SRF for ROCS (2008) and Natural Science
Foundation of Liaoning Province of China
(No. 20042009).
[14] Y. Kashiwada, J. Chiyo, Y. Ikeshiro,
T. Nagao, H. Okabe, L.M. Cosentino,
K. Fowke, S.L.M. Natschke, and
K.H. Lee, Chem. Pharm. Bull. 48, 1387
(2000).
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Journal of Asian Natural Products Research
855
R1
C
O
R
H
AcO
AcO
AcO
H
3-15
R1 = H, or = O
The synthesis of AA derivatives with cytotoxic activity against various cancer cell lines
(HeLa, HepG2, BGC-823, and SKOV3) is reported.
Yan-Qiu Meng, Yun-Yun Li, Feng-Qing Li, Yan-Ling Song, Hai-Feng Wang, Hong Chen
and Bo Cao
Synthesis and antitumor activity evaluation of new asiatic acid derivatives
844–855