The synthesis of ursolic acid derivatives with cytotoxic activity and the investigation of their preliminary mechanism of action

Article abstract of DOI:10.1016/j.bmc.2008.11.036

Nineteen ursolic acid derivatives (15 novel compounds) modified at the C-3 and the C-28 positions were synthesized. The cytotoxic activity of the derivatives was evaluated against HeLa, BGC-823 and SKOV3 cells by MTT assay. Inducing apoptosis and affecting cell cycle distribution by the derivatives in HeLa cells were assessed by flow cytometry and DNA fragmentation. Compounds 10b and 11b were particularly active to inhibit HeLa cells growth through inducing apoptosis and arresting cell cycle progression. The typical 'sub-G1 peak' and DNA ladder formation were checked and cell cycle was arrested at the S phase in a dose-dependent manner.

Full text of DOI:10.1016/j.bmc.2008.11.036

Bioorganic & Medicinal Chemistry 17 (2009) 848–854
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The synthesis of ursolic acid derivatives with cytotoxic activity
and the investigation of their preliminary mechanism of action
a
a
b
b
b
Yan-Qiu Meng a, , Dan Liu , Ling-Li Cai , Hong Chen , Bo Cao , Yi-Zheng Wang
*
a Department of Pharmaceutical Engineering, Shenyang Institute of Chemical Technology, Jing Ji Kai Fa Que 11 street, Shenyang 110142, PR China
b Department of Pharmacognosy, Medical College of Chinese People’s Armed Police Forces, Tianjin 300162, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Nineteen ursolic acid derivatives (15 novel compounds) modified at the C-3 and the C-28 positions were
synthesized. The cytotoxic activity of the derivatives was evaluated against HeLa, BGC-823 and SKOV3
cells by MTT assay. Inducing apoptosis and affecting cell cycle distribution by the derivatives in HeLa cells
were assessed by flow cytometry and DNA fragmentation. Compounds 10b and 11b were particularly
active to inhibit HeLa cells growth through inducing apoptosis and arresting cell cycle progression. The
typical ‘sub-G1 peak’ and DNA ladder formation were checked and cell cycle was arrested at the S phase
in a dose-dependent manner.
Received 3 September 2008
Revised 12 November 2008
Accepted 13 November 2008
Available online 20 November 2008
Keywords:
Ursolic acid derivatives
Triterpene
Ó 2008 Elsevier Ltd. All rights reserved.
Anti-tumor activity
1. Introduction
results in derivatives having stronger cell growth inhibitory than
ursolic acid (IC50 values of compounds 10a, 11a and 12a, 10.83
Ursolic acid (UA, 3b-hydroxy-urs-12-en-28-oic acid, 1), a penta-
cyclic triterpene acid, exists abundantly in the plant kingdom and
as constituents of medicinal herbs. UA has been reported to display
a remarkable spectrum of biochemical activities to influence pro-
cesses that are dysregulated during cancer development. These
include inhibition of tumorigenesis, tumor promotion, invasion,
metastasis, angiogenesis and induction of tumor cell differentia-
tion.1–10 In addition, Ya-Ling Hsu et al. have reported that UA
inhibits the cell proliferation of human lung cancer cell line A549
and provided a molecular understanding for this effect.11 Mecha-
nistic studies revealed that UA blocked the cell cycle progression
in the G1 phase and UA treatment resulted in the triggering of
apoptosis as determined by DNA fragmentation assay.11
l
M, 5.44
lM and 1.63 l
M, respectively15).
Following on these previous findings, we now report the design
and synthesis of UA-amino acid (or related amino alcohol com-
pound) conjugates and preliminary structure-activity relation-
ship (SAR) study of their cytotoxic activity. The present studies also
explore preliminary mechanisms of action of UA–amino alcohol
conjugates’ antiproliferative effects: compounds 10b, 11b may
act through inducing apoptosis and blocking cell cycle in S phase.
2. Results and discussion
2.1. Chemistry
The pleuripotent anti-tumor activities of UA have stimulated
active research in this field. We think it is very important to probe
novel ursane triterpenoids structures with a view to biological
testing. Based on the reports that the acid moiety at C-17 and ester
functionality at C-3 are essential for pharmacological activities of
pentacyclic triterpenes12,13, and a hydrogen donor group at either
C-3 position and/or C-28 position of ursolic acid is essential for
the cytotoxic activity14, a series of UA derivatives have been syn-
thesized and their cytotoxic activities have been evaluated against
HeLa cells. The results showed that acetylation of C-3 alcohol
together with coupling an amino alcohol or benzylamine at C-28
The synthetic routes of UA derivatives are outlined in Scheme 1.
Ursolic acid 1 was first converted to its 3-O-acetate 2, which was
then treated with oxalyl chloride to give the 28-acyl chloride 3.14
This intermediate was then condensed with the appropriate amino
compound (methyl ester of serine, threonine, tyrosine, glycine, iso-
leucine and phenylalanine; 2-aminoethanol, 2-amino-1-propanol;
o-aminophenyl-carbinol) in the presence of triethylamine to give
the compounds 4a–12a. Saponification of compounds 4a–9a gave
the corresponding N-[3b-hydroxy-urs-12-en-28-oyl]-amino acids
compounds 4b–9b. Compounds of 10b–12b were obtained by
re-acetylating compounds 10a–12a, respectively. The structures
and high purity of the target compounds were confirmed by appli-
cation of mp, Infrared (IR), mass spectra (MS), 1H NMR and 13C
NMR.
* Corresponding author. Tel.: +86 2489383903; fax: +86 2489383760.
E-mail addresses: mengyanqiu@hotmail.com, meng@kemi.dtu.dk (Y.-Q. Meng).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.036

Y.-Q. Meng et al. / Bioorg. Med. Chem. 17 (2009) 848–854
849
a
COOH
b
COOH
COCl
HO
H3CCOO
H3CCOO
1
3
2
d
c
CONHR'COOCH3
CONHR''OH
H3CCOO
10a-12a
4a-9a
H3CCOO
5a: R'=CH(CHOHCH3)
4a: R'=CH(CH2OH)
6a: R'=CH(CH2C6H4-p-OH)
8a: R'=CH(CH(CH3)C2H5)
f
10a: R''=(CH2)2
7a: R'=CH2
9a: R'=CH(CH2C6H5)
11a: R''=CH(CH3)CH2
12a: R''=C6H4(o-CH2)
e
CONHR''OCOCH3
CONHR'COOH
10b-12b
H3CCOO
HO
4b-9b
10b: R''=(CH2)2
4b: R'=CH(CH2OH)
5b: R'=CH(CHOHCH3)
7b: R'=CH2
11b: R''=CH(CH3)CH2
12b: R''=C6H4(o-CH2)
6b: R'=CH(CH2C6H4-p-OH)
8b: R'=CH(CH(CH3)C2H5)
9b: R'=CH(CH2C6H5)
Scheme 1. Reagents and conditions: (a) and (f) (CH3CO)2O, DMAP, THF, rt; (b) (COCl)2, CH2Cl2, rt; (c) NH2-R0 CO2CH3, Et3N, rt; (d) NH2-R00OH, Et3N, rt; (e) NaOH, CH3OH, THF,
40 °C.
2.2. Biological activity
in our previous study, incorporation of an anilino group at C-28
in UA-acetate to give N-[3b-acetoxy-urs-12-en-28-oyl]-aniline re-
sulted in less activity than 12a15, while compound 12b was slightly
more active than N-[3b-acetoxy-urs-12-en-28-oyl]-aniline. These
observations suggested that the electron-releasing group at ben-
zene ring might be important factor on activity. In order to get a
deeper understanding, many more derivatives containing a ben-
zene ring with different substituent groups are being prepared in
our laboratory.
We conclude that both the 3-O-acetyl group and a 28-amido
group might be important for improving the tumor cell growth
inhibitory activity. Compounds 4a, 5a, 6a, 10a, 11a and 12a with
free hydroxyl group at the C-28 amide side chain showed similar
inhibitory activity as compounds 7a, 10b and 11b without the
hydroxyl group on the amide side chain, suggesting that a hydroxyl
group at the C-28 amide side chain could not influence the antipro-
liferative effect.
2.2.1. Cytotoxic activity
In this experiment, HeLa cell lines were the model chosen to
determine the cytotoxic activity of UA, its derivatives and pacli-
taxol (a positive control). Some compounds were also tested in
two other tumorigenic human cell lines, SKOV3 and BGC-823 cells.
Antiproliferative effects were identification with tetrazolium dye
assay (MTT). Each experiment was repeated at least three times.
The results are shown in Table 1.
As shown in Table 1, esterification of the 3-OH of ursolic acid to
yield compound 2 resulted in increasing inhibitory activity against
HeLa cell lines. Significant further improvement of the cell growth
inhibition to HeLa cell lines was achieved when the selected amino
acid methyl ester was coupled to the C-28. Compounds 4a–7a, 9a
(IC50 values of 4a–7a, 4.30, 6.86, 9.04, 4.86
inhibition in the HeLa cell lines model at concentrations of
10 M. (Compounds 4b–7b, 9b with the free 3-OH group, the
lM) presented strong
l
saponification products of 4a–7a, 9a had lower anti-tumor effects
than compounds 4a–7a, 9a but were still more active than the par-
ent UA). Similar results had been seen in our previous study.15 It is
interesting to note that among other UA-amino acid conjugates
tested, compounds 8a and 8b showed similar HeLa cell lines activ-
ity to that of UA. The reduced of 8a and 8b might be explained by a
steric hindrance due to the presence of the sec-butyl on the C-28
amide side chain.
2.2.2. Investigation of apoptosis and cell cycle distribution
It has been reported that UA induces apoptosis and growth inhi-
bition at the G1 phase of cell cycle in certain cancer cell systems.16
Compounds 10b and 11b were selected for characterizing the
mechanisms in inhibiting growth of UA derivatives using flow
cytometric analysis of HeLa cells.
2.2.2.1. Apoptosis. The DNA fluorescence histograms of PI-stained
cells showed low DNA stainability of compounds 10b or 11b-trea-
ted, apoptotic cells, which resulted in a distinct, quantifiable region
below the G1 peak. In contrast, the G1 peak predominated in control
cells (Fig. 1A). The apoptotic cells increased up to 35.3% and 32.5%
UA-amino alcohol conjugates 10a–12a was acetylated to give
compounds 10b–12b. Compared to 10a and 11a, compounds
10b, 11b showed more potent anti-tumor activity in HeLa cell lines
(IC50 values of 10b, 11b, 1.13, 2.62
10b and 11b also showed improved anti-tumor activity in SKOV3
(IC50 values of 10b and 11b 6.09 M, 2.24 M) and BGC-823 cell
lines (IC50 value of 11b 8.30 M). Interestingly, compound 12b is
an exception, exhibiting less activity than compound 12a, as seen
lM). Additionally, compounds
(10
l
M) after incubation in compounds 10b or 11b of concentra-
M for 24 h. Quantification of dose depen-
l
l
tions higher than 2.5 l
l
dency was done by monitoring the amount of nuclei with
sub-diploid DNA content with flow cytometry (Fig. 1B). Meanwhile,

850
Y.-Q. Meng et al. / Bioorg. Med. Chem. 17 (2009) 848–854
Table 1
Inhibitory activity of UA and derivatives on the HeLa, SKOV3 and BGC-823 cells proliferation
Compounda
Inhibition rate%b
BGC-823
IC50 (l
M)
c
HeLa
SKOV3
HeLa
SKOV3
BGC-823
UA (1)
2
4a
5a
6a
7a
8a
9a
10a
11a
12a
4b
5b
6b
7b
8b
9b
12.36 3.01
37.84 6.28
59.90 8.79
55.92 4.29
51.53 5.09
61.69 10.08
15.89 6.79
32.86 8.62
50.34 5.71
58.38 6.23
77.52 12.09
16.84 3.09
21.96 4.15
23.58 6.08
15.58 2.59
16.79 3.68
27.47 5.36
73.50 12.03
69.34 9.35
30.53 3.91
9.06 2.02
10.20 1.02
nt
15.72 2.81
nt
41.59 7.08
17.85 3.18
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
>10
>10
>10
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
>10
>10
nt
>10
nt
>10
>10
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
ntd
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
4.30 1.01
6.86 0.98
9.04 1.21
4.86 0.81
>10
>10
10.82 1.09
5.44 0.68
1.63 0.32
>10
>10
>10
>10
>10
>10
nt
5.01 1.28
nt
54.80 10.12
70.54 12.56
1.19 1.02
nt
nt
nt
>10
10b
11b
12b
Paclitaxol
42.05 6.22
56.32 8.58
13.0 1.89
1.13 0.09
2.62 1.25
>10
6.09 0.92
2.24 0.56
>10
8.30 1.21
>10
0.0189 0.00257
0.001 0.0005
0.016 0.0037
a
Compounds 2, 10a, 11a and 12a are known compounds.
b
c
Inhibitory percentage of cells treated with each compound at a concentration of 10
The agent concentration that inhibited HeLa cells growth by 50%.
Not tested.
lM for 96 h.
d
Figure 1. (A) Demonstration of apoptosis by flow cytometric analysis. (1) Untreated Hela cells. (2)–(4) and (5)–(7) Appearance of cells with sub-diploid DNA content after
exposed to increasing concentrations of compounds 10b and 11b (2.5, 5.0, 10.0
lM). (B) Dose-dependent induction of apoptosis by 10b and 11b.
agarose gel electrophoresis showed typical DNA fragmentation
pattern of apoptosis (Fig. 2). DNA fragmentation caused by 10b or
11b was also dose dependent, the intensity of DNA fragments in-
The percentage of cells arrested in S phase increased with the
increase of concentration. The curve of S arrest following treatment
for 24 h with increasing doses of 10b is shown in Figure 3B. The
creased when the amounts of 10b or 11b (2.5–5.0
increased.
lM) were
percentage of cells in S peaked when cells were exposed to
2.5 M of 10b. The dose-dependent curve of S arrest following
l
treatment for 24 h with increasing doses of 11b is shown in Fig-
ure 3C. The percentage of cells in S peaked when cells were ex-
posed to10 lM of 11b.
2.2.2.2. Cell cycle distribution. To evaluate the possible role of
cell cycle arrest in UA derivatives-caused growth inhibition, HeLa
cell lines were treated with compounds 10b and 11b. Cell cycle
distribution was evaluated by flow cytometric analysis after stain-
ing of cellular DNA with propidium iodide at the different concen-
trations. This indicated that 10b and 11b induced an accumulation
in S phase of cell cycle. After treatment with 2.5–10 lM of 10b and
11b for 24 h, the number of cells in S phase was higher than that in
untreated cells (Fig. 3A).
3. Conclusion
In summary, our data suggest that (1)Acetylation of C-3 alcohol
together with coupling an amino acid methyl ester or amino alco-
hol acetate at C-28 results in derivatives having stronger antiprolif-
erative ability. (2) Six compounds (4a, 5a, 6a, 7a, 10b, 11b) showed

Y.-Q. Meng et al. / Bioorg. Med. Chem. 17 (2009) 848–854
851
triethylamine at room temperature for 6 h, the reactant was parti-
tioned in 3 mL water, then treated with 2 N HCl to pH 3. CH2Cl2
was removed under vacuum to precipitate white solid, then
filtered. The filter was washed with water to pH 6, and dried. The
crude was purified on a silica gel column with petroleum ether
and ethyl acetate to yield a white powder.
4.2.1. Methyl N-[3b-acetoxy-urs-12-en-28-oyl]-2-amino-3-
hydroxypropionate (4a)
Compound 2 was reacted by using general procedure with ser-
ine methyl ester to get compound 4a. Eluted by petroleum ether/
ethyl acetate (V/V) = 3:1. Yield 55.4%; mp 182–184 °C; IR (KBr):
3454, 1736, 1639, 1511, 1247 cmꢁ1 1H NMR (300 MHz, DMSO-
;
d6):d 7.17 (1H, d, J = 7.2 Hz, NH), 5.19 (s, 1H, H-12), 4.97 (1H, t,
OH), 4.39 (t, 1H, J = 6.3 Hz, H-3), 4.29–4.26 (m, 1H, NHCH), 3.59
(m, 2H, CH2OH) 3.59 (m, 3H, OCH3), 1.99 (s, 3H, CH3CO), 1.04 (s,
3H, CH3), 0.92 (s, 3H, CH3), 0.89 (d, 3H, J = 6.2 Hz, CH3), 0.84 (s,
3H, CH3),0.83(d, 3H, J = 6.3 Hz, CH3) 0.81 (s, 3H, CH3), 0.65 (s, 3H,
CH3); 13C NMR (CDCl3, 75 MHz): d 178.1 (C-28), 173.8 (–COOCH3),
172.0 (CH3CO), 138.8 (C-13), 126.8 (C-12), 81.9 (C-3), 63.4
(CH2OH); ESI-MS m/z: 600.3 (M+H)+.
Figure 2. Electrophoresis analysis of apoptosis in Hela cells treated with 10b and
11b: (A) 5.0 M 10b, (B) 2.5 M 10b, (C) untreated Hela cells, (D) marker, (E)
2.5 M 11b, (F) 5.0 M 11b.
l
l
l
l
significant anti-tumor activity against the HeLa cell lines in the
preliminary pharmacological test, and compounds 10b, 11b also
showed improved anti-tumor activity in SKOV3 and BGC-823 cells.
(3) The C-3 free hydroxyl might decrease the inhibitory activity,
while the free hydroxyl at amide side chain might not influence
the activity. (4) Too many alkyl side chains at C-28 amide chain
can decrease the inhibitory activity, such as compounds 8a and
8b. (5) Preliminary mechanisms study demonstrated that com-
pounds 10b, 11b may inhibit cell growth through inducing apopto-
sis and arresting cell cycle progression at the S phase. These results
indicated that UA–amino alcohol conjugates may share some
similarities in their mechanism of action with HeLa cell lines, but
this should be more deeply investigated. The finding of these
new active leads provides a powerful incentive for further research
on the chemical modifications and structure-activity relationships
of ursolic acid and other triterpenoid acids.
4.2.2. Methyl N-[3b-acetoxy-urs-12-en-28-oyl]-2-amino-3-
hydroxybutyrate (5a)
Compound 2 was reacted by using general procedure with thre-
onine methyl ester to get compound 5a. Eluted by petroleum
ether/ethyl acetate (V/V) = 6:1; Yield: 41.2%; mp 123–125 °C; IR
(KBr): 3424, 2949, 1737, 1655, 1508, 1456, 1373, 1246 cmꢁ1 1H
;
NMR (300 MHz, CDCl3): d 6.61 (br, 1H, NH), 5.39 (s, 1H, H-12),
4.52 (br, 1H, H-3), 4.52 (br, 1H, NHCH), 4.18 (br, 1H, CHOH), 3.74
(s, 3H, OCH3), 2.04 (s, 3H, CH3CO), 1.09 (s, 3H, CH3), 1.00 (s, 3H,
CH3), 0.96 (d, 3H, J = 6.5 Hz, CH3), 0.93 (s, 3H, CH3),0.89 (d, 3H,
J = 6.6 Hz, CH3), 0.85 (s, 3H, CH3), 0.69 (s, 3H, CH3); 13C NMR (CDCl3,
75 MHz): d 177.1 (C-28), 172.6 (–COOCH3), 171.0 (CH3CO), 138.5
(C-13), 126.1 (C-12), 81.4 (C-3), 67.4 (CH2OH), 59.0 (NHCH); ESI-
MS m/z: 614.4 (M+H)+.
4.2.3. Methyl N-[3b-acetoxy-urs-12-en-28-oyl]-2-amino-3-(p-
hydroxy)-phenyl propionate (6a)
4. Experimental
4.1. Chemicals
Compound 2 was reacted by using general procedure with tyro-
sine methyl ester to get compound 6a. Eluted by petroleum ether/
ethyl acetate (V/V) = 3:1; Yield: 44.6%; mp > 300 °C; IR(KBr): 3410,
2949, 1736, 1635, 1616, 1516, 1451, 1369, 1246, 830 cmꢁ1 1H
;
UA was purchased from China Chengdu Scholar Bio-Tech. Co.
Ltd., with a over 98% purity. Other reagents were bought from com-
mercial suppliers in an analytic grade without further purification
unless otherwise noted. The melting points were determined on an
electrically heated X-4 digital visual melting point apparatus and
were uncorrected. IR spectra were recorded on Thermo Nicolet
470FT spectrometer. 1H NMR, 13C NMR spectra were recorded on
a BRUKER ARX-300MHz spectrometer at room temperature, and
chemical shifts were measured in ppm downfield from TMS as
internal standard. Mass spectra were recorded on Thermo-Finni-
gan LCQ equipment, in positive Electron Spray Ionization (ESI)
mode and are reported as m/z.
NMR (300 MHz, DMSO-d6): d 9.14 (s, 1H, Ar-OH), 7.39 (d, 1H,
J = 7.4 Hz, NH), 7.01 (d, 2H, J = 8.3 Hz, Ar-H), 6.62 (d, 2H,
J = 8.28 Hz, Ar-H), 5.02 (s, 1H, H-12), 4.39–4.38 (m, 1H, H-3), 4.22
(br, 1H, NHCH), 3.58 (s, 3H, OCH3), 2.85–2.82 (m, 2H, Ar-CH2),
1.99 (s, 3H, CH3CO), 0.95 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.83 (d,
3H, J = 6.0 Hz, CH3), 0.81(s, 3H, CH3), 0.78 (d, 3H, J = 6.1, CH3),
0.75 (s, 3H, CH3), 0.70 (s, 3H, CH3); 13C NMR (CDCl3, 75 MHz): d
176.8(C-28), 171.7 (–COOCH3), 170.1 (CH3CO), 158.7 (Ar-C 40),
138.2 (C-13), 131.8, 130.9 (Ar-C20, 60), 129.4 (Ar-C10), 125.8 (C-
12), 118.2, 117.8 (Ar-C 30, 50), 81.0(C-3); ESI-MS m/z: 676.3 (M+H)+.
4.2.4. Methyl N-[3b-acetoxy-urs-12-en-28-oyl]-2-amino acetate
(7a)
4.2. General procedure for the synthesis of N-[3b-acetoxy-urs-
12-en-28-oyl]-amino acid methyl-esters (4a–9a)
Yield: 51.6%; mp 82–84 °C ;IR (KBr): 3421, 1736, 1656, 1519,
1460, 1384, 1246 cmꢁ1 1H NMR(300 MHz, CDCl3): d 6.52 (br, 1H,
;
NH), 5.40 (t-like, 1H, H-12), 4.49 (t, 1H, J = 7.9 Hz, H-3), 4.11 (dd,
1H, J2 = 18.7 Hz, J3 = 5.5 Hz, NHCHaCO), 3.84 (dd, 1H, J2 = 18.6 Hz,
J3 = 3.5 Hz, 1H, NHCHbCO), 3.76 (s, 3H, OCH3), 2.04 (s, 3H, CH3CO),
1.09 (s, 3H, CH3), 0.98 (s, 3H, CH3), 0.96 (d, J = 6.3 Hz, 3H, CH3), 0.93
(s,3H, CH3), 0.89 (d, 3H, J = 6.2 Hz CH3), 0.85 (s, 3H, CH3), 0.71 (s,
3H, CH3); 13C NMR (CDCl3, 75 MHz): d 176.5 (C-28), 170.7 (CH3CO),
169.8 (–COOCH3), 138.4 (C-13), 126.0 (C-12), 80.5 (C-3); ESI-MS m/
z: 570.3 (M+H)+.
To a solution of compound 2 (0.20 mmol) in 3 mL CH2Cl2 was
added oxalyl chloride (0.07 mL) and stirred at room temperature
for 24 h. The mixture was concentrated to dryness under reduced
pressure. Cyclohexane (2ꢀ 2 mL) was added to the residue, then
the concentrated to dryness to yield crude 3-O-acetylursolyl chlo-
ride 3. To a CH2Cl2 (4 mL) solution of 3 was added glycine methyl
ester (0.80 mmol). The reaction mixture was stirred in presence of

852
Y.-Q. Meng et al. / Bioorg. Med. Chem. 17 (2009) 848–854
Figure 3. Effect of compounds 10b and 11b on cell cycle distribution. (A) Cell cycle fraction of HeLa cells grown exposed to 0.1% DMSO (1) and 2.5, 5.0, 10.0
11b (2–4 and 5–7). (B) and (C) effect results of Hela cells exposed for 24 h to concentration of compounds 10b and 11b varying from 2.5 to 10.0 M.
lM of 10b and
l
4.2.5. Methyl N-[3b-acetoxy-urs-12-en-28-oyl]-2-amino-3-
methyl-valerate (8a)
0.85 (s, 3H, CH3), 0.66 (s, 3H, CH3); 13C NMR (CDCl3, 75 MHz): d
177.5 (C-28), 172.5 (–COOCH3), 171.0 (CH3CO), 138.4 (C-13),
126.4 (C-12), 80.9 (C-3), 56.2 (C-5), 55.3 (C-18), 53.9 (NHCH),
51.9 (OCH3); ESI-MS m/z: 626.5 (M+H)+.
Compound 2 was reacted by using general procedure with iso-
leucine methyl ester to get compound 8a. Eluted by petroleum
ether/ethyl acetate (V/V) = 10:1, Yield: 64.9%; mp 196–198 °C;
IR(KBr): 3419, 2927, 1735, 1652, 1511, 1457, 1370, 1251 cmꢁ1
;
4.2.6. Methyl N-[3b-acetoxy-urs-12-en-28-oyl]-2-amino-3-
phenylpropionate (9a)
Compound 2 was reacted by using general procedure with
phenylalanine methyl ester to get compound 9a. Eluted by petro-
leum ether/ethyl acetate (V/V) = 10:1; Yield: 43.4%; mp > 300 °C;
1H NMR (300 MHZ, CDCl3): d 6.46 (br, 1H, NH), 5.38 (s, 1H, H-12),
4.50 (t-like, 1H, H-3), 4.42 (m, 1H, NHCH), 3.70 (s, 3H, OCH3),
2.05 (s, 3H, CH3COO), 1.08 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.93 (d,
3H, J = 6.4 Hz, CH3), 0.90 (s, 3H, CH3), 0.88 (d, 3H, J = 6.3 Hz, CH3),

Y.-Q. Meng et al. / Bioorg. Med. Chem. 17 (2009) 848–854
853
IR (KBr): 3414, 2948, 1736, 1666, 1497, 1456, 1369, 1246,
701 cmꢁ1 1H NMR (300 MHz, CDCl3): d 7.26–7.28 (m, 3H, Ar-H),
75 MHz): d 175.8 (C-28), 173.2 (–COOH), 138.4 (C-13), 126.0 (C-
12), 78.5 (C-3); ESI-MS m/z: 514.3 (M+H)+.
;
7.11–7.08 (m, 2H, Ar-H), 6.39 (br, 1H, NH), 5.24 (s, 1H, H-12),
4.47 (t-like, 1H, H-3), 4.13–4.11 (m, 1H, NHCH), 3.67 (s, 3H,
OCH3), 3.12–3.05 (m, 2H, Ar-CH2), 2.04 (s, 3H, CH3CO), 1.05
(s, 3H, CH3), 0.98 (s, 3H, CH3), 0.94 (d, 3H, J = 6.2 Hz, CH3), 0.89
(s, 3H, CH3), 0.85 (d, 3H, J = 6.3 Hz, CH3), 0.84 (s, 3H, CH3), 0.60
(s, 3H, CH3); 13C NMR (CDCl3, 75 MHz): d 175.8 (C-28), 171.8
(–COOCH3), 170.2 (CH3CO), 138.5 (C-13), 136.8 (Ar-C10), 129.1
(Ar-C30, 50), 128.5 (Ar-C20, 60), 126.6 (Ar-C40), 125.8(C-12), 80.6
(C-3). ESI-MS m/z: 660.4 (M+H)+.
4.3.5. N-[3b-Hydroxy-urs-12-en-28-oyl]-2-amino-3-methyl-
valeric acid (8b)
Yield: 60.1%; mp 257–259 °C; IR(KBr): 3417, 2927, 1726, 1636,
1510, 1458, 1383 cmꢁ1 1H NMR (300 MHZ, CDCl3): d 6.56 (br, 1H,
;
NH), 5.37 (s, 1H, H-12), 4.45 (m, 1H, NHCH), 3.23 (m, 1H, H-3), 1.09
(m, 3H, CH3), 0.98 (m, 3H, CH3), 0.95 (d, 3H, J = 6.4 Hz, CH3),0.92 (s,
3H, CH3), 0.88 (d, 3H, J = 6.4 Hz, CH3), 0.77 (s, 3H, CH3), 0.71 (s, 3H,
CH3); 13C NMR (CDCl3, 75 MHz): d 178.9 (C-28), 173.8 (–COOH),
138.4 (C-13), 126.3 (C-12), 78.7 (C-3), 56.3 (C-5), 54.8 (C-18),
53.7 (NHCH);ESI-MS m/z: 626.5 (M+H)+.
4.3. General procedure for the synthesis of N-[3b-Hydroxy-urs-
12-en-28-oyl]-amino acids (4b–9b)
4.3.6. N-[3b-Hydroxy-urs-12-en-28-oyl]-2-amino-3-phenylpro-
pionic acid (9b)
A solution of 4a (or 5a–9a) with aqueous NaOH (4 N) in CH3OH/
THF was stirred for 3.5 h at 40 °C and concentrated in vacuo. The
residue was suspended in water treated with 2 N HCl to pH 3. Or-
ganic solvent was removed under vacuum to precipitate white so-
lid, filtered. The filter was washed with water to pH 6, and dried to
give a white powder.
Yield:73.1%; mp > 300 °C; IR(KBr): 3419, 2926, 1636, 1604,
1509, 1455, 1391, 700 cmꢁ1 1H NMR (300 MHz, CDCl3): d 7.26–
;
7.23 (m, 5H, Ar-H), 6.44 (br, 1H, NH), 5.07 (1H, t-like, H-12), 4.36
(m, 1H, NHCH), 3.18 (m,1H, H-3), 3.18 (m, 1H, OH), 2.98–2.96 (m,
2H, Ar-CH2), 0.98 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.90(d, 3H,
J = 6.2 Hz, CH3) 0.87 (s, 3H, CH3), 0.84 (d, 3H, J = 6.1 Hz, CH3), 0.79
(s, 3H, CH3), 0.76 (s, 3H, CH3); 13C NMR (CDCl3, 75 MHz): d 176.9
(C-28), 173.6 (–COOH), 138.5 (C-13), 136.8 (Ar-C10), 130.1 (Ar-C30,
50), 129.3 (Ar-C20, 60), 126.2 (Ar-C40), 125.6(C-12), 78.5(C-3);
ESI-MS m/z: 604.5 (M+H)+.
4.3.1. N-[3b-Hydroxy-urs-12-en-28-oyl]-2-amino-3-hydroxy
propionic acid (4b)
Yield: 88.0%; mp 190–192 °C; IR(KBr): 3407, 2927, 1736, 1633,
1512, 1457, 1388 cmꢁ1; 1H NMR (300 MHz, DMSO-d6): d 12.62 (br,
1H, COOH), 6.98 (d, 1H, J = 6.6 Hz, NH), 5.29 (s, 1H, H-12), 4.88 (br,
1H, NHCH), 4.29 (br, 1H, CHaOH), 4.17 (br, 1H, CHbOH), 2.99 (s, 1H,
H-3), 1.04 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.92 (d, 3H, J = 6.4 Hz,
CH3), 0.88(s, 3H, CH3), 0.84 (d, 3H, J = 6.4 Hz, CH3), 0.67 (s, H,
CH3), 0.64 (s, 3H, CH3); 13C NMR (CDCl3, 75 MHz): d 178.1 (C-28),
174.8 (–COOH), 138.7 (C-13), 126.8 (C-12), 79.2 (C-3), 63.4
(CH2OH). ESI-MS m/z: 544.3 (M+H)+.
4.4. General procedure for the synthesis of N-[3b-acetoxy-urs-
12-en-28-oyl]-amino alcohol acetates (10b–12b)
A solution containing compound 3 and ethanolamine (or 2-ami-
no-1-propanol, 3-amino-1-propanol, 2-aminophenylcarbinol,) was
stirred in the presence of tri-ethylamine for 1.5 h at room temper-
ature. The reactant was partitioned in 3 mL water, then treated
with 2 N HCl to pH 3. CH2Cl2 was removed under vacuum to pre-
cipitate white solid, then filtered. The filter was washed with water
to pH 6, and dried. The crude was purified on a silica gel column
with petroleum ether and ethyl acetate to yield a white powder
10a–12a. Compounds 10a–12a was acetylated with two equiva-
lence of acetic anhydride in anhydrous THF in presence of
4-(dimethylamino)-pyridine to give compounds 10b–12b.
4.3.2. N-[3b-Hydroxy-urs-12-en-28-oyl]-2-amino-3-hydroxy-
butyric acid (5b)
Yield: 74.5%; mp 207–210 °C; IR(KBr): 3417, 2927, 1715, 1609,
1513, 1459, 1391 cmꢁ1 1H NMR (600 MHz, CDCl3): d 7.04 (br, 1H,
;
NH), 5.39 (s, 1H, H-12), 4.28 (br, 1H, CHOH), 4.28 (br, 1H, NHCH),
3.22–3.20 (m, 1H, H-3), 1.09 (s, 3H, CH3), 0.98 (s, 3H, CH3), 0.95
(d, 3H, J = 6.2 Hz, CH3), 0.88 (s, 3H, CH3), 0.86 (d, 3H, J = 6.2 Hz,
CH3), 0.76 (s, 3H, CH3), 0.71 (s, 3H, CH3); 13C NMR (CDCl3,
75 MHz): d 177.2 (C-28), 176.5 (–COOH), 138.5 (C-13), 126.2 (C-
12), 79.5 (C-3), 67.4 (CH2OH), 60.0 (NHCH); ESI-MS: 558.4 (M+H)+.
4.4.1. N-[3b-Acetoxy urs-12-en-28-oyl]-2-aminoethanol acetate
(10b)
Yield: 29.7%; mp 83–85 °C; IR(KBr): 3418, 2947, 1736, 1652,
1521, 1458, 1384, 1245; 1H NMR (300 MHz, CDCl3): d 6.17 (br,
1H, NH), 5.31(s, 1H, H-12), 4.49 (t-like, 1H, H-3), 4.15–4.08 (m,
2H, CH2OCOCH3), 3.58 (br, 1H, NHCHa), 3.34 (br, 1H, NHCHb),
2.08 (s, 3H, OCOCH3), 2.04 (s, 3H, CH3COO), 1.09 (s, 3H, CH3),
0.94 (s, 6H, CH3ꢀ2), 0.89–0.87 (m, 9H, CH3ꢀ3), 0.77 (s, 3H,
CH3); 13C NMR (CDCl3): d 179.5(C-28), 171.7(CH3COO), 138.7(C-
13), 125.6 (C-12), 80.9 (C-3), 64.8 (CH2OCOCH3); ESI-MS: 584.4
(M+H)+.
4.3.3. N-[3b-Hydroxy-urs-12-en-28-oyl]-2-amino-3-(p-hydroxy)-
phenylpropionic acid (6b)
Yield: 78.3%; mp > 300 °C; IR(KBr): 3401, 2927, 1733, 1633,
1616, 1516, 1454, 1377, 830 cmꢁ1 1H NMR(300 MHz, DMSO-d6):
;
d 9.13 (s, 1H, Ar-OH), 7.14 (s, 1H, NH), 6.98 (s, 2H, Ar-H), 6.61 (s,
2H, Ar-H), 5.04 (s, 1H, H-12), 4.27 (s, 1H, NHCH), 4.15 (s, 1H, H-
3), 2.94 (br, 2H, Ar-CH2), 1.09 (s, 3H, CH3), 0.99 (s, 3H, CH3), 0.95
(d, 3H, J = 6.5 Hz, CH3), 0.89 (s, 3H, CH3), 0.79 (d, 3H, J = 6.4 Hz,
CH3), 0.72 (s, 3H, CH3), 0.67 (s, 3H, CH3); 13C NMR (CDCl3,
75 MHz): d 175.8(C-28), 174.7(–COOH), 157.7(Ar-C 40), 138.2(C-
13), 131.5, 130.4(Ar-C 20, 60), 129.4(Ar-C10), 125.8(C-12), 118.0,
117.4(Ar-C 30, 50), 78.6(C-3); ESI-MS: 620.4(M+H)+.
4.4.2. N-[3b-Acetoxy-urs-12-en-28-oyl]- 2-amino-1-propanol
acetate (11b)
Yield: 44.3%; mp 77–80 °C; IR(KBr): 3263, 2925, 1725, 1693,
1459, 1378, 1223, 1189; 1H NMR (300 MHz, CDCl3): d 5.91 (d,
J = 7.7 Hz, 1H, NH), 5.27 (s, 1H, H-12), 4.49 (t, 1H, J = 7.8 Hz, H-3),
4.24 (br, 1H, NHCH), 4.10–3.97 (m, 2H, CH2OCOCH3), 2.09 (s, 3H,
OCOCH3), 2.04 (s, 3H, CH3COO), 1.14 (d, 3H, J = 6.6 Hz,
NHCH(CH3)CH2OCOCH3), 1.09 (s, 3H, CH3), 0.94 (s, 6H, CH3ꢀ2),
0.86–0.85 (m, 9H, CH3ꢀ3), 0.80 (s, 3H, CH3); 13C NMR (CDCl3): d
179.1 (C-28), 171.3 (CH3COO), 138.6 (C-13), 125.8 (C-12), 81.49
(C-3), 71.7 (CH2OCOCH3); ESI-MS:598.4 (M+H)+.
4.3.4. N-[3b-Hydroxy-urs-12-en-28-oyl]-2-amino acetic acid (7b)
Yield: 85.6%; mp 162–164 °C; IR(KBr): 3441, 2926, 1750, 1639,
1527, 1456, 1413 cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 7.26 (s,
;
1H, NH), 5.21 (s, 1H, H-12), 3.43 (br, 1H, CHaCOOH), 3.00 (br, 1H,
H-3), 3.00 (br, 1H, CHbCOOH), 1.04 (s, 3H, CH3), 0.96 (s, 3H, CH3),
0.91 (d, 3H, J = 6.4 Hz, CH3), 0.89 (s, 3H, CH3), 0.84 (d, 3H,
J = 6.3 Hz, CH3), 0.71 (s, 3H, CH3), 0.66 (s, 3H, CH3); 13C NMR (CDCl3,

854
Y.-Q. Meng et al. / Bioorg. Med. Chem. 17 (2009) 848–854
4.4.3. N-[3b-Acetoxyurs-12-en-28-oyl]-2-aminophenyl-carbinol
acetate (12b)
two times with PBS, then resuspended in 0.5 ml of extraction buf-
fer (100 mmol/L NaCl, 10 mmol/L Tris–HCl pH 8.0, 25 mmol/L
EDTA pH 8.0, 0.1 mg/ml Proteinase K) at 50 °C for 12 h. DNA
was extracted with an equal volume of phenol saturated/chloro-
form/isoamyl alcohol (25:24:1) and extracted again with a combi-
nation of chloroform/isoamyl alcohol (24:1), then centrifuged at
12,000g for 30 min. Precipitated DNA was analysed on a 2.0%
agarose gel.
Yield: 30.3%; mp 238–240 °C; 1H NMR(300 MHz, CDCl3): d 8.35
(s, 1H, NH), 7.86 (d, 1H, J = 8.3 Hz, Ar-H), 7.34–7.33 (m, 2H, Ar-H),
7.11 (t, 1H, J = 7.4 Hz, Ar-H), 5.36 (s, 1H, H-12), 5.05 (s, 2H, CH2OH),
4.48 (t-like, 1H, H-3), 2.10 (s, 3H, OCOCH3), 2.04 (s, 3H, CH3COO),1.13
(s, 3H, CH3), 0.99 (s, 3H, CH3), 0.94–0.91 (m, 6H, CH3ꢀ2), 0.86–0.84
(m, 6H, CH3ꢀ2), 0.80 (s, 3H, CH3); 13C NMR (CDCl3): d 178.5 (C-28),
170.7 (CH3COO), 140.1 (C-10-Ar), 138.5 (C-13), 132.5 (C-20-Ar), 128.8
(C-30, 50-Ar), 126.1 (C-40-Ar), 125.4 (C-12), 121.2 (C-60-Ar), 81.0
(C-3), 65.8 (CH2 OCOCH3); ESI-MS: 646.4 (M+H)+.
Acknowledgments
The authors would like to thank Natural Science Foundation of
Liaoning Province of China (No. 20042009), Science and Technol-
ogy Foundation of Shenyang City of China (No. 20050785) for their
financial support; The authors also like to expressly thank Dr. M.J.
Calverley, at the Department of Chemistry, Technical University of
Denmark, for his constructive criticism and great interest in this
work.
4.5. Biological activity assays
4.5.1. Cell culture
All human tumor cells were cultured in RPMI 1640 medium sup-
plementedwith10%foetalbovineserumandpenicillin/streptomycin.
A humidified incubator was set at 37.8 °C; the air contained 5% CO2.
4.5.2. Antiproliferative effects
Supplementary data
Antiproliferative effects were identification with tetrazolium
dye assay (MTT). Cells were detached by trypsinisation, seeded at
1.0–2.0 ꢀ 103 cells/well in a 96-well microtitre plate overnight,
and treated with different concentrations of drugs in RPMI
1640with 10% foetal bovine serum. Finally, 50 mL of MTT solution
(1mg/mL in RPMI 1640) was added to each well and incubated at
37.8 °C for 4 h. The MTT-formazan formed by metabolically viable
cells was dissolved in 150 mL of DMSO, and monitored by a micro-
plate reader at dual-wavelength of 490 nm; IC50 was defined as the
drug concentrations that inhibited the cell number to 50% after
96 h. Each experiment was repeated at least three times.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.11.036.
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4.5.4. Assessment of apoptosis by DNA fragmentation
Assessment of apoptosis by DNA fragmentation: HeLa cells
incubated with 10b or 11b for 24 h were collected and washed