The cytotoxic activity of ursolic acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2005.01.001

Ursolic acid and 2α-hydroxyursolic acid isolated from apple peels were found to show growth inhibitory activity against four tumor cell lines, HL-60, BGC, Bel-7402 and Hela. Structural modifications were performed on the C-3, C-28 and C-11 positions of ursolic acid and the cytotoxicity of the derivatives was evaluated. The SAR revealed that the triterpenes possessing two hydrogen-bond forming groups (an H-donor and a carbonyl group) at positions 3 and 28 exhibit cytotoxic activity. The configuration at C-3 was found to be important for the activity. Introduction of an amino group increased the cytotoxicity greatly. A 3β-amino derivative was 20 times more potent than the parent ursolic acid. The 28-aminoalkyl dimer compounds showed selective cytotoxicity.

Full text of DOI:10.1016/j.ejmech.2005.01.001

European Journal of Medicinal Chemistry 40 (2005) 582–589
www.elsevier.com/locate/ejmech
The cytotoxic activity of ursolic acid derivatives
Chao-Mei Ma a,b, Shao-Qing Cai a,*, Jing-Rong Cui a, Rui-Qing Wang a, Peng-Fei Tu a,
Masao Hattori c, Mohsen Daneshtalab b,*
a School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Haidian District, Beijing 100083, China
b School of Pharmacy, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3V6
c Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan
Received 2 November 2004; received in revised form 23 December 2004; accepted 7 January 2005
Available online 08 February 2005
Abstract
Ursolic acid and 2a-hydroxyursolic acid isolated from apple peels were found to show growth inhibitory activity against four tumor cell
lines, HL-60, BGC, Bel-7402 and Hela. Structural modifications were performed on the C-3, C-28 and C-11 positions of ursolic acid and the
cytotoxicity of the derivatives was evaluated. The SAR revealed that the triterpenes possessing two hydrogen-bond forming groups (an
H-donor and a carbonyl group) at positions 3 and 28 exhibit cytotoxic activity. The configuration at C-3 was found to be important for the
activity. Introduction of an amino group increased the cytotoxicity greatly. A 3b-amino derivative was 20 times more potent than the parent
ursolic acid. The 28-aminoalkyl dimer compounds showed selective cytotoxicity.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Apple peel; Triterpene; Ursolic acid derivative; Anti-tumor; Cytotoxic activity
1. Introduction
which is closely related mechanistically to carcinogenesis can
destroy functional normal tissues [12–16].
However, when compared to betulinic acid derivatives,
ursolic acid derivatives have not been thoroughly explored
for their cytotoxic activity. In order to find biologically more
active derivatives of this naturally occurring triterpene, we
selected apple peel extract as the source of these triterpenes.
The present article describes the isolation and cytotoxicity of
triterpenes from apple peels and the structural modification
of ursolic acid.
Triterpenes, especially, ursolic acid, oleanolic acid and
betulinic acid exist abundantly in the plant kingdom. These
triterpenes and their derivatives have been reported to have
interesting bioactivity, such as anti-HIV [1–4], inhibition of
HIV protease [5] and cytotoxicity to tumor cell lines [6–8].
Renewed interest in the cytotoxic activity of triterpenes has
come from the findings that betulinic acid exhibited selective
cytotoxicity against melanoma and had apoptosis induction
properties [9]. Considerable structural modification has been
performed on betulinic acid and potentially important deriva-
tives, which may be developed as anti-tumor drugs, have been
produced [10,11]. Ursolic acid and its esterified derivatives
have also been reported to show significant cytotoxicity
against some tumor cell lines [7,8]. Some oleanane and ursane
triterpenoids with modified ringsA and C have been reported
to have high inhibitory activity against nitric oxide produc-
tion. This suggests the potential of these compounds as can-
cer chemopreventive drugs, as excessive production of NO,
2. Chemistry
The ethyl acetate extract of the peel of apples (Malus
pumila Mill) was subjected to column chromatography over
Silica gel, ODS and HPLC to obtain six compounds. These
compounds were identified as ursolic acid (1), 2a-
hydroxyursolic acid (2), euscaphic acid (3), uvanol (4), 2a,
3a-dihydroxy-urs-12-en-28-oic acid (5), 2a, 3a-dihydroxy-
olean-12-en-28-oic acid (6), by analyzing and comparing their
spectral data with those reported in literature [17] (Fig. 1).
Compounds 1–4, which were obtained in relatively large
amounts, were tested for their cytotoxicity. Ursolic acid (1)
* Corresponding authors. Tel.: +1 709 777 6958; fax: +1 709 777 7044.
E-mail address: mohsen@pharm.mun.ca (M. Daneshtalab).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.01.001

C.-M. Ma et al. / European Journal of Medicinal Chemistry 40 (2005) 582–589
583
and 2a-hydroxyursolic acid (2), with a b-hydroxyl group at
C-3 and a carboxyl group at C-17, were found to show cyto-
toxicity against all the four tumor cell lines tested (HL-60,
BGC, Bel-7402 and Hela). Compound 3, whose structure is
different from 2 due to the configuration of C-3 hydroxyl
group, did not show cytotoxicity even at 100 µg/ml. Com-
pound 4, with a hydroxymethyl group at C-17, did not show
cytotoxicity either. Ursolic acid (1) was obtained in the larg-
est amount and thus was used as starting material for struc-
ture modification.
Ursolic acid and its methyl ester (obtained by treating urso-
lic acid with diazomethane) were oxidized with pyridinium
chlorochromate (PCC) to give the 3-oxo compounds, 10 and
11. Treatment of the 3-oxo compounds with NH2OH in pyri-
dine yielded the hydroxyimino compounds 12 and 13. Reduc-
tion of 13 with TiCl3 and NaCNBH3 followed by column
chromatography yielded two 3-amino compounds, 14 and 15
1
(Scheme 1). The large coupling constants of H-3 in the H
NMR of 14 [d 2.38 (1H, dd, J = 12.0, 5.7 Hz)] compared
with that of 15 [d 2.67 (1H, br. s)] indicated that H-3 is a
orientated in 14 and b orientated in 15 (i.e. compound 14 is
3b-amino compound and 15 is its 3a isomer) in Scheme 1.
Acetyl ursolic acid methyl ester was refluxed with CrO3 in
acetic acid to give the 11-oxo compound, 16. Alkaline
hydrolysis of 16 yielded the 3-hydroxy-11-oxo derivative, 17,
as depicted in Scheme 2.
Compounds 19, 21 and 23 were synthesized via the route
outlined in Scheme 3. Ursolic acid was first converted to its
acetyl ester, which was then treated with oxalyl chloride to
give the corresponding acid chloride. Condensation of 1,9-
diaminononane or diethylenetriamine with acetyl ursolic acid
Fig. 1. Triterpenes isolated from apple peels.
Scheme 1. Synthesis of ursolic acid derivatives with different substituents at C-3.

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C.-M. Ma et al. / European Journal of Medicinal Chemistry 40 (2005) 582–589
chloride followed by chromatography, produced compounds
19, 21 and 23. Saponification of these compounds yielded
compounds 18, 20 and 22, respectively, in Scheme 3.
3. Biological activity
The cytotoxicity of all compounds was assessed against
different cancer cell lines, as described in Section 6.
4. Results and discussion
The structures of the synthesized compounds are depicted
in Fig. 2 and the corresponding in vitro cytotoxicity results
are summarized in Table 1.
The ursolic acid methyl ester (7) showed similar activity
as that of ursolic acid on all cell lines tested. The lack of cyto-
Scheme 2. Synthesis of C-11 oxidized ursolic acid.
Scheme 3. Synthesis of ursolic acid derivatives with substituents at C-17.

C.-M. Ma et al. / European Journal of Medicinal Chemistry 40 (2005) 582–589
585
Table 1
Cytotoxicity of ursolic acid derivatives
Compound
ED50 (µg/ml)
Bel-7402
HL-60
72.0
BGC
Hela
49.4
55.0
1
53.7
45.0
2
49.0
55.9
54.8
3
4
5
> 100.0
> 100.0
nt
> 100.0
> 100.0
nt
> 100.0
> 100.0
nt
> 100.0
> 100.0
nt
6
nt
nt
nt
nt
7
54.8
54.1
41.5
51.8
8
9
55.9
100.0
> 100.0
60.0
58.4
50.0
> 100.0
19.5
> 100.0
58.5
> 100.0
76.5
10
11
12
13
14
15
16
17
> 100.0
53.3
> 100.0
53.3
> 100.0
38.1
> 100.0
50.1
88.3
71.2
58.0
64.3
2.0
2.5
1.7
2.4
79.5
> 100.0
> 100.0
> 100.0
BGC
39.6
55.2
>100.0
51.1
> 100.0
> 100.0
MDA-MB-435
90.0
> 100.0
> 100.0
HL-60
35.0
18
19
20
21
22
23
> 100.0
> 100.0
> 100.0
> 100.0
30.0
> 100.0
< 1.0a
< 1.0a
5.0
> 100.0
> 100.0
> 100.0
8.0
5.0
35.0
95.0
nt: not tested.
a Cytotoxicity only changed a little in the concentration range of
1–10 µg/ml.
Of the two 3-amino isomers, the 3b-form was 20 times
more potent cytotoxic than the 3a-form (14 vs. 15), confirm-
ing that the configuration at C-3 is an important factor on the
activity. The 3a-amino derivative exhibited somewhat selec-
tive cytotoxicity, showing similar cytotoxicity on HL-60, Bel-
7402 and Hela cell lines but little toxicity against BGC cell
line, compared with ursolic acid.
Significant improvement of cytotoxicity against HL-60 cell
line was observed when amino alkyl groups were introduced
to position 28 (20–23), whether or not the 3-OH group was
free or acetylated. In the case of dimeric compounds with a
secondary amino alky group (20, 21), selective cytotoxicity
was observed only against HL-60 cell lines. The dimeric com-
pounds (18, 19) with amide but no amino alkyl groups in
position 28 showed only a little cytotoxicity, while the
3-acetylated analogue (19) was inactive.
It is interesting to note that the concentration dependent
cytotoxicity patterns against HL-60 cell lines are quite simi-
lar among the dimers with a free secondary amino group (20,
21). While the cytotoxicity of both compounds increases obvi-
ously when the concentration is increased from 10 to
100 µg/ml, there is only a little change in the pattern of cyto-
toxicity at concentrations between 1 and 10 µg/ml. In con-
trast, the cytotoxicity of compounds with a free primary amino
group (22, 23) drops sharply when concentration changes
from 10 to 1 µg/ml. These phenomena may suggest that the
mechanism of cytoxicity of the dimeric compounds (20, 21)
is different from that of the monomer compounds.
Fig. 2. Structures of ursolic acid derivatives for anti-tumor assay.
toxicity of compound 4, with a hydroxyl group but no carbo-
nyl group at C-17, suggests that a carbonyl group at position
17 is necessary for the cytotoxicity. Introduction of an addi-
tional oxo group to position 11 did not improve the cytotoxic
activity (16 vs. 9), (17 vs. 7).
Among the C-3 modified derivatives, acetylursolic acid (8)
was slightly less active than ursolic acid against BGC cell
line. Esterification of both the 3-OH and 28-COOH of urso-
lic acid to yield compound 9 resulted in loss of cytotoxic activ-
ity. Compared to ursolic acid, the 3-oxo derivative (10) dem-
onstrated more potent cytotoxicity against HL-60 and similar
cytotoxicity against other cell lines tested. However, when
the 28-COOH of the 3-oxo compound was methylated (11),
the cytotoxicity once again was lost. The C-3 hydroxyimino
derivatives (12 and 13) showed a cytotoxicity comparable to
those of ursolic acid and ursolic acid methyl ester against all
of the four cell lines. This evidence suggests that a hydrogen
donor group at either position 3 and/or 28 is essential for the
cytotoxic activity.

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5. Conclusions
tra were obtained on a Bruker AVANCE 500 MHz or on a
JEOL JNM-AL 300 NMR spectrometer with TMS as inter-
nal standards. APCI and ESI mass spectra were measured on
an Agilent 1100 series LC/MSD system. HRFAB-MS were
measured on a JEOL JMS-700 apparatus with a resolution of
5000 and with m-nitrobenzyl alcohol as matrix. Silica gel used
in column chromatography was provided by Tsingtao Marine
Chemistry Co. Ltd. (200–300 mesh) or Sigma-Aldrich Com.
(Silica gel, Davisil, 100–200 mesh). Aluminum oxide (neu-
tral, –150 mesh) and octadecyl-functionalized silica gel (ODS)
were obtained from Sigma-Aldrich. The Organic solvents for
chromatography were obtained from Fisher Scientific. Chemi-
cals for chemical synthesis were obtained from Sigma-
Aldrich. Semi-preparative HPLC was carried out on a sys-
tem Gold HPLC systems with an Agilent Zorbax SB-
C18 column (9.4 × 250 mm) and methanol–water (80–
100%) as mobile phase.
The above results indicate that apple peel can serve as a
useful source of bioactive compounds, such as ursolic acid,
which can be modified to more potent bioactive derivatives.
Ursolic acid derivatives possessing two hydrogen-bond form-
ing groups (an H-donor and a carbonyl group) at positions
3 and 28 exhibit cytotoxic activity. The configuration at
C-3 was found to be important for the activity. Compounds
with a b-orientated hydrogen-bond forming group at C-3 ex-
hibit more potent cytotoxicity than the a-counterpart. Intro-
duction of an amino-bearing group in either C-3 or C-28 posi-
tion could greatly increase the cytotoxicity.
Ursolic acid has been reported to have significant cyto-
toxic activity [6–8,18]. A study on mechanism revealed that
ursolic acid blocked the cell cycle progression in the G1 phase
and that ursolic acid treatment resulted in the triggering of
apoptosis as determined by a DNA fragmentation assay [18].
The present work has provided more potent cytotoxic deriva-
tives of ursolic acid, such as the 3-amino and 28-aminoalkyl
compounds. These compounds are now undergoing further
mechanistic and pharmacologic studies for the possibility of
development as anticancer drugs. The SARS described herein
can serve as the basis for the synthesis of more potent usolic
acid derivatives as anti-tumor drugs.
6.2.1. Methylation and acetylation of ursolic acid
Methyl ursolate (7) was prepared by treating ursolic acid
with diazomethane in ether. Ursolic acid (1) was treated with
acetic anhydride and pyridine (1:1) at room temperature over-
night and worked up as usual to get acetyl ursolic acid (8).
Compound 8 was treated with diazomethane get compound
9.
6.2.2. 3-Oxo-urs-12-en-28-oic acid (10)
6. Experimental protocols
To a solution of ursolic acid (1.9 g, 4.1 mmol) in acetone–
CH2Cl2 (10 ml) was added PCC (2.7 g, 12.5 mmol). After
being stirred at room temperature for 8 h, the mixture was
concentrated and partitioned with H2O and CHCl3. The CHCl3
layer was concentrated and purified by silica gel column chro-
matography eluted with n-hexane–acetone (95:5) to give 10.
Yield 57%; amorphous powder; 1H NMR (CDCl3) d 0.81 (3H,
s, CH3), 0.84 (3H, d, J = 6.6 Hz, CH3), 0.93 (3H, d, J = 6.6 Hz,
CH3), 1.01 (3H, s, CH3), 1.03 (3H, s, CH3), 1.07 (6H, s, CH3),
2.18 (1H, d, J = 12.0 Hz, H-18), 2.36 (1H, m, H2a), 2.50 (1H,
m, H2b), 5.24 (1H, t-like, H-12); MS (EI): 454 [M]+ (5), 408
(10), 248 (100), 203 (67), 133 (75).
6.1. Extraction and Isolation
The apple peels were cut from the fruit of M. pumila Mill
purchased in April, 2002 from the basement of the Lang Qiu
Yuan Supermarket, Xue Yuan Road, Beijing, China.
The commercially obtained apple, (27 kg), was peeled to
obtain the fresh apple peel. It was extracted with ethyl acetate
by supersonication three times (2 h per time). After being
concentrated in vacuo, the extract was defatted by petroleum
ether (125 ml × 8) under supersonication and the white pre-
cipitate was collected to get the triterpene mixture (25 g).
The triterpene mixture was separated by silica gel column
chromatography, eluted with hexane–chloroform–acetone to
get 43 fractions. Fr. 1–3 was purified with ODS eluted with
H2O–MeOH to get 4 (10 mg); Ursolic acid (1, 19 g) was
obtained from Fr. 4–24. Fraction 25–34 was purified with
ODS eluted with H2O–MeOH and purified with HPLC to
obtain 6 (2 mg) and 5 (3 mg); Fraction 35–40 was purified
with ODS eluted with H2O–MeOH to obtain 3 (200 mg); Frac-
tion 41–43 was purified with ODS eluted with H2O–MeOH
and purified with HPLC to obtain compound 2 (30 mg). These
compounds were identified by comparing their spectra data
with the literature [17].
6.2.3. 3-Oxo-urs-12-en-28-oic acid methyl ester (11)
Methyl ursolate was reacted in the same fashion with PCC
as above to get compound 11. Yield 90%; amorphous pow-
1
der; H NMR (CDCl3) d 0.80 (3H, s, CH3), 0.87 (3H, d,
J = 6.6 Hz, CH3), 0.95 (3H, d, J = 6.6 Hz, CH3), 1.04 (3H, s,
CH3), 1.05 (3H, s, CH3), 1.08 (6H, s, CH3), 2.25 (1H, d,
J = 12.0 Hz, H-18), 2.38 (1H, m, H2a), 2.55 (1H, m, H2b),
3.63 (3H, s, CH3O-28), 5.28 (1H, t-like, H-12); MS (EI): 468
[M]+ (10), 453 (4), 408 (12), 393 (10), 262 (80), 203 (93),
133 (100).
6.2.4. 3-Hydroxyimino-urs-12-en-28-oic acid (12)
A solution of 10 (420 mg, 0.9 mmol) and hydroxylamine
hydrochloride (145 mg, 2.5 mmol) in pyridine (2 ml) was
heated for four hour at 50 °C. After cooling to room tempera-
ture, the reaction mixture was concentrated under vacuum to
6.2. Chemistry
Optical rotation was measured with an AA-10R polarim-
eter manufactured by the OpticalActivity Co. Ltd. NMR spec-

C.-M. Ma et al. / European Journal of Medicinal Chemistry 40 (2005) 582–589
587
dryness, then purified with RP-18 (MeOH, 90–100%) to
obtain 12. Yield 49%; colorless powder; 1H NMR (CDCl3) d
0.82 (3H, s, CH3), 0.88 (3H, d, J = 6.6 Hz, CH3), 0.96 (3H, d,
J = 6.6 Hz, CH3), 1.02 (3H, s, CH3), 1.05 (3H, s, CH3), 1.08
(3H, s, CH3), 1.16 (3H, s, CH3), 2.22 (1H, d, J = 12.0 Hz,
H-18),), 2.26 (1H, m, H-2a), 3.04 (1H, m, H-2b), 5.28 (1H,
t-like, H-12); Negative APCI-MS: 468 (M+-H, 100%).
6.2.7. 3-Acetoxy-11-oxo-urs-12-en-28-oic acid methyl ester
(16)
Acetyl ursolic acid methyl ester (200 mg, 0.39 mmol) was
refluxed with CrO3 (50 mg, 0.5 mmol) in acetic acid (7 ml)
for 2 h. The reaction mixture was partitioned with water and
CHCl3. The CHCl3 layer was chromatographed on silica gel
eluted with hexane–CHCl3 to obtain the 11-oxo compound,
16. Yield 60%; colorless powder; 1H NMR (CDCl3): d 0.87
(9H, br, 3 × CH3), 0.90 (3H, s, CH3), 0.97 (3H, d, J = 6.3 Hz,
CH3), 1.15 (3H, s, CH3), 1.29 (3H, s, CH3), 2.05 (3H, s,
–OCOCH3), 2.32 (1H, s, H-9), 2.41 (1H, d, J = 11.1 Hz,
H-18), 2.79 (1H, dt, J = 14.0, 3.5 Hz, H-1a), 3.63 (3H, s,
COOCH3), 4.51 (1H, dd, J = 4.8, 11.1 Hz, H-3), 5.60 (1H, br.
H-12). EI-MS m/z 526 (M+, 10%), 317 (75%), 189 (70%),
119 (70%), 43 (100%).
6.2.5. 3-Hydroxyimino-urs-12-en-28-oic acid methyl ester
(13)
A solution of 11 (366 mg, 0.78 mmol) and hydroxylamine
hydrochloride (250 mg, 3.6 mmol) in pyridine (5 ml) was
heated for 2 h at 50 °C. After cooling to room temperature,
the reaction mixture was diluted with CH2Cl2 (75 ml) and
washed with 10% HCl (3 × 50 ml). The CH2Cl2 layer was
dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The product was crystallized from diethyl ether and
CH2Cl2 to obtain 13.Yield 90%; colorless powder; 1H NMR
(CDCl3) d 0.80 (3H, s, CH3), 0.87 (3H, d, J = 6.6 Hz, CH3),
0.96 (3H, d, J = 6.6 Hz, CH3), 1.02 (3H, s, CH3), 1.04 (3H, s,
CH3), 1.06 (3H, s, CH3), 1.17 (3H, s, CH3), 2.18 (1H, m,
H-2a), 2.25 (1H, d, J = 12.0 Hz, H-18), 3.03 (1H, m, H-2b),
3.62 (3H, s, CH3O-28), 5.27 (1H, t, J = 3.0 Hz, H-12); MS
(EI): 483 [M]+ (4), 406 (7), 262 (87), 203 (100), 133 (96).
6.2.8. 3-Hydroxy-11-oxo-urs-12-en-28-oic acid methyl
ester (17)
Compound 16 was treated with 4 N NaOH (1.2 ml) in
CH3OH–THF (1:1.5, 5 ml) at room temperature overnight.
The solution was neutralized with 2 N HCl, removed the
organic solvent and then extracted with EtOAc to get com-
pound 17.Yield 90%; colorless powder; 1H NMR (CDCl3): d
0.75 (3H, s, CH3), 0.82 (3H, d, J = 6.3 Hz, CH3), 0.86 (3H, s,
CH3), 0.93 (3H, d, J = 6.3 Hz, CH3), 0.95 (3H, s, CH3), 1.07
(3H, s, CH3), 1.23 (3H, s, CH3), 2.26 (1H, s, H-9), 2.37 (1H,
d, J = 11.1 Hz, H-18), 2.75 (1H, dt, J = 14.0, 3.5 Hz, H-1a),
3.18 (1H, dd, J = 5.8, 10.3 Hz, H-3), 3.57 (3H, s, COOCH3),
5.59 (1H, br. H-12). EI-MS m/z 484 (M+, 13%), 317 (74%),
189 (70%), 119 (78%), 43 (100%).
6.2.6. 3b-Amino-urs-12-en-28-oic acid methyl ester (14)
and 3␣-amino-urs-12-en-28-oic acid methyl ester (15)
Sodium cyanoborohydride (0.29 g, 9 mmol) was added to
a methanol solution of 13 (200 mg, 0.41 mmol) and ammo-
nium acetate (0.37 g, 6.8 mmol) under nitrogen atmosphere.
The solution was cooled in ice water and 15% aqueous tita-
nium trichloride (1.2 ml, 1.4 mmol) was added dropwise over
20 min. The mixture was stirred at room temperature for 12 h
then adjusted with 2 N sodium hydroxide to pH 10. The aque-
ous solution was extracted with CH2Cl2, and the organic layer
was washed with distilled water and concentrated to dryness.
The crude products were subjected to neutral Al2O3 chroma-
tography, and eluted with MeOH/CHCl3 (0–20%) to give 14
6.2.9. General synthetic procedure for compounds 18–23
Compounds 18–23 were synthesized via the route out-
lined in Scheme 3. To a solution of compound 8 (1.3–
1.6 mmol) in 10 ml CHCl3 was added oxalyl chloride (3 ml)
and stirred at room temperature for 48 h. The mixture was
concentrated to dryness under reduced pressure. Hexane (3 ×
5 ml) was added to the residue, then concentrated to dryness.
To a CH2Cl2 (4 ml) solution of 1,9-diaminononane
(1.28 mmol) or diethylenetriamine (1.7 mmol) was added the
above acid chloride (1.27 mmol for diaminononane or
1.6 mmol for diethylenetriamine). The reaction mixture was
stirred at room temperature overnight. The reaction mixture
was concentrated and chromatographed on ODS to get com-
pounds 19, 21 and 23.
(150 mg) and 15 (28 mg).
25
Compound 14: yield 78%, amorphous powder [a]D
+
5.0° (c = 0.87, CHCl3): 1H NMR (CDCl3) d 0.71 (3H, s, CH3),
0.79 (3H, d, J = 6.2 Hz, CH3), 0.83 (3H, s, CH3), 0.87 (3H, d,
J = 6.0 Hz, CH3), 0.92 (3H, s, CH3), 1.00 (3H, s, CH3), 1.18
(3H, s, CH3), 2.16 (1H, d, J = 11.1 Hz, H-18), 2.38 (1H, dd,
J = 12.0, 5.7 Hz, H-3), 3.54 (3H, s, CH3O-28), 5.18 (1H,
t-like, H-12); MS (EI): 469 [M]+ (25), 454 (13), 413 (10),
Compounds 19, 21 and 23 were treated with 4 N NaOH
(1.2 ml) in CH3OH–THF (1:1.5, 5 ml) at room temperature
overnight. The solutions were neutralized with 2 N HCl,
recovered the organic solvent and then extracted with EtOAc
to get compounds 18, 20 and 22.
411 (11), 262 (70), 203 (95), 133 (100).
25
Compound 15: yield 15%, amorphous powder [a]D
+
7.1° (c = 0.39, CHCl3): 1H NMR (CDCl3) d 0.71 (3H, s, CH3),
0.82 (3H, d, J = 6.2 Hz, CH3), 0.86 (3H, s, CH3), 0.90 (6H, s,
2 × CH3), 0.91 (3H, d, J = 6.2 Hz, CH3), 1.09 (3H, s, CH3),
2.20 (1H, d, J = 11.1 Hz, H-18), 2.67 (1H, br, H-3), 3.58 (3H,
s, CH3O-28), 5.22 (1H, t-like, H-12); MS (EI): 469 [M]+ (30),
454 (17), 413 (12), 411 (17), 262 (74), 203 (93), 133 (100).
6.2.9.1. N1,N9-Bis (3b-hydroxy-urs-12-en-28-oyl)-1,9-
diaminononane (18). Yield 96% from 19; amorphous pow-
1
der; H NMR (CDCl3): d 0.80 (6H, s, CH3), 0.88 (6H, d,
J = 6.5 Hz CH3), 0.92 (6H, d, J = 6.5 Hz, CH3), 0.94 (6H, s,
CH3), 0.96 (6H, s, CH3), 1.00 (6H, s, CH3), 1.11 (6H, s, CH3),

588
C.-M. Ma et al. / European Journal of Medicinal Chemistry 40 (2005) 582–589
3.00 (2H, m, –CONHCHa–), 3.23 (2H, dd, J = 4.0, 11.0 Hz,
H-3′), 3.31 (2H, m., –CONHCHb–), 5.31 (2H, br. H-12′), 5.91
(2H, t-like, –CONH–); Positive APCI-MS m/z 1036.8 (M +
H+, 10%), 282 (100); Positive HR-FAB-MS: 1035.8826 ([M
br. –NH2 and –CH2NH2), 2.99 (1H, m., –CONHCHa–), 3.29
(1H, m., –CONHCHb–), 4.47 (1H, dd, J = 5.5, 10.0 Hz, H-3′),
5.28 (1H, br. H-12′), 5.88 (1H, br. –CONH–); Positive
+
HR-FAB-MS: 639.5471 ([M + H]+, C41H71N2O3 ; Calc.
+
+ H]+, C69H115N2O4 ; Calc. 1035.8862).
639.5468).
6.2.9.2. N1,N9-Bis (3b-acetoxy-urs-12-en-28-oyl)-1,9-
diaminononane (19). Yield 26%; amorphous powder; H
6.3. Cells and cytotoxicity assay
1
NMR (CDCl3): d 0.79 (6H, s, CH3), 0.88 (12H, br., CH3),
0.93 (6H, d, J = 6.0 Hz, CH3), 0.97 (12H, s, CH3), 1.10 (6H,
s, CH3), 2.07 (6H, s, –OCOCH3), 3.01 (2H, m, –CONH-
CHa–), 3.32 (2H, m., –CONHCHb–), 4.51 (2H, dd, J = 5.5,
10.0 Hz, H H-3′), 5.32 (2H, br. H-12′), 5.90 (2H, t-like,
–CONH–); NegativeAPCI-MS m/z 1153.8 (M + Cl–, 100%);
Cell lines. The cell lines used were: Human leukemia can-
cer cell line (HL-60); human gastric cancer cell line (BGC-
823); human breast cancer cell line (MDA-MB-435); human
cervical cell line (Hela), and human hepatocellular carci-
noma cell line (Bel-7402).
The in vitro HL-60 tumor cell assay was carried out accord-
ing to the procedure reported by Twentyman and Luscombe
[19]; BGC-823, Bel-7402, and Hela tumor cell assays were
carried out by a sulforhodamine B (SRB) assay method [20].
A cell suspension in the culture medium (4 × 104 cells per
ml) was inoculated to each well of 96-well microtiter plate.
One day after plating, a control plate at time zero was made.
In the presence or absence of compounds, the cells were incu-
bated for a further 48 h in a 5% CO2 incubator. Cells were
fixed with 50 µl of 20% trichloroacetic acid solution for 1 h
at 4 °C and plates were washed five times with tap water and
air-dried. A 50 µl of SRB solution (0.4 in 1% acetic acid) was
added and the staining was done at room temperature for
30 min. The residual dye was washed out with 1% acetic acid
and air-dried. To each well, Tris buffer solution (10 mM, pH
10.5) was added. Optical density (OD) was measured with a
microtiter plate reader at 540 nm. Growth inhibition was cal-
culated as follows:
Positive HR-FAB-MS: 1119.9033 ([M + H]+, C73H119N2O6 ;
+
Calc. 1119.9074).
6.2.9.3. N1,N5-Bis (3b-hydroxy-urs-12-en-28-oyl)-diethy-
lenetriamine (20). Yield 95% from 21; amorphous powder;
1H NMR (CDCl3): d 0.78 (6H, s, CH3), 0.89 (6H, d,
J = 6.0 Hz, CH3), 0.92 (12H, br., CH3), 0.96 (6H, s, CH3),
1.00 (6H, s, CH3), 1.11 (6H, s, CH3), 2.83 (4H, m.
–CH2NHCH2–), 3.22 (4H, m, H-3′, overlapped with –CON-
HCHa–), 3.48 (2H, m, –CONHCHb–), 5.34 (2H, br., H-12′),
6.49 (2H, br. –CONH–); Positive API-MS m/z 981.0 (M +
H+, 100%), 1003.0 (M + Na+, 70%); Positive HR-FAB-MS:
+
980.8181 ([M + H]+, C64H106N3O4 ; Calc. 980.8189).
6.2.9.4. N1,N5-Bis (3b-acetoxy-urs-12-en-28-oyl)-diethy-
lenetriamine (21). Yield 60%; amorphous powder; 1H NMR
(CDCl3): d 0.76 (6H, s, CH3), 0.85 (6H, s, CH3), 0.87 (6H, s,
CH3), 0.89 (6H, d, J = 6.0 Hz, CH3), 0.94 (6H, d, J = 6.0 Hz,
CH3), 0.97 (6H, s, CH3), 1.10 (6H, s, CH3), 2.03 (6H, s,
–OCOCH3), 2.76 (4H, br. –CH2NHCH2–), 3.18 (2H, br.,
–CONHCHa–), 3.45 (2H, br., –CONHCHb–), 4.49 (2H, dd,
J = 5.3, 10.3 Hz, H-3′), 5.30 (2H, br. H-12′), 6.31 (2H, br.
–CONH–); NegativeAPCI-MS m/z 1098.7 (M + Cl–, 100%);
% inhibition = [1 – (OD value for treated cells/OD value
for untreated cells)] 100%
Acknowledgements
+
Positive HR-FAB-MS: 1064.8380 ([M + H]+, C68H110N3O6 ;
The authors are grateful to Dr. Sunao Takeda of Central
Research Laboratories, SSP Co., Ltd. Japan, for measure-
ment of HR-FAB-MS. The authors would also like to extend
their appreciation to Dr. Norio Nakamura of Institute of Natu-
ral Medicine, Toyama Medical and Pharmaceutical Univer-
sity for his unfailing advice during the synthesis of triterpene
derivatives.
Calc. 1064.8400).
6.2.9.5. N1-(3b-hydroxy-urs-12-en-28-oyl)-1,9-diaminon-
onane (22). Yield 95% from 23; amorphous powder; 1H NMR
(CDCl3): d 0.76 (3H, s, CH3), 0.87 (3H, d, J = 6.0 Hz, CH3),
0.92 (3H, d, J = 6.0 Hz, CH3), 0.94 (3H, s, CH3), 0.97 (3H, s.,
CH3), 1.00 (3H, s., CH3), 1.10 (3H, s, CH3), 2.89 (2H, br.
–NH2), 2.99 (2H, m, –CH2NH2), 3.21 (1H, dd, J = 5.0,
10.5 Hz, H-3′), 3.29 (2H, m., –CONHCH2), 5.31 (1H, br.
H-12′), 5.93 (1H, br. –CONH–); PositiveAPCI-MS m/z 597.5
(M + H+, 100%); Positive HR-FAB-MS: 597.5384 ([M + H]+,
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