Synthesis and biological evaluation of oleanolic acid derivatives as antitumor agents

Article abstract of DOI:10.1080/10286020.2011.654110

Derivatives of oleanolic acid were synthesized and evaluated in vitro for their growth inhibition against human hepatocellular carcinoma cell line (HepG2) and colon cancer cell line (Col-02). Several derivatives exhibited moderate-to-good inhibitory activity, with 3 displaying the most promising inhibition [GI⁵⁰=1.75μM (HepG2), 0.71μM (Col-02)]. Structure-activity relationship analyses of these derivatives demonstrated that a 1-en-2-cyano-3-oxo in ring A and a nitro at C-17 were important in retention of the inhibition against HepG2 and Col-02 cells.

Full text of DOI:10.1080/10286020.2011.654110

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Synthesis and biological evaluation of
oleanolic acid derivatives as antitumor
agents
Lei Chen a , Jian-Bo Wu a , Fan Lei a , Shan Qian b , Li Hai a & Yong
Wu a
a Key Laboratory of Drug Targeting and Drug Delivery System of the
Education Ministry, Department of Medicinal Chemistry, West China
School of Pharmacy, Sichuan University, Chengdu, 610041, China
b Bioengineering College, Xihua University, Chengdu, 610039,
China
To cite this article: Lei Chen , Jian-Bo Wu , Fan Lei , Shan Qian , Li Hai & Yong Wu (2012): Synthesis
and biological evaluation of oleanolic acid derivatives as antitumor agents, Journal of Asian Natural
Products Research, 14:4, 355-363
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Journal of Asian Natural Products Research
Vol. 14, No. 4, April 2012, 355–363
Synthesis and biological evaluation of oleanolic acid derivatives
as antitumor agents
Lei Chena, Jian-Bo Wua, Fan Leia, Shan Qianb, Li Haia and Yong Wua*
aKey Laboratory of Drug Targeting and Drug Delivery System of the Education Ministry,
Department of Medicinal Chemistry, West China School of Pharmacy, Sichuan University,
Chengdu 610041, China; Bioengineering College, Xihua University, Chengdu 610039, China
b
(Received 11 October 2011; final version received 28 December 2011)
Derivatives of oleanolic acid were synthesized and evaluated in vitro for their growth
inhibition against human hepatocellular carcinoma cell line (HepG2) and colon cancer
cell line (Col-02). Several derivatives exhibited moderate-to-good inhibitory activity,
with 3 displaying the most promising inhibition [GI50 ¼ 1.75 mM (HepG2), 0.71 mM
(Col-02)]. Structure–activity relationship analyses of these derivatives demonstrated
that a 1-en-2-cyano-3-oxo in ring A and a nitro at C-17 were important in retention of
the inhibition against HepG2 and Col-02 cells.
Keywords: oleanolic acid; derivatives; synthesis; antitumor; inhibition ratio
1. Introduction
hot; meanwhile, some sequent findings
show very inspirited. For instance, 3-oxo-
OA was found to significantly inhibit the
growth of cancer cells derived from
different tissues, and had inhibitory effect
on melanoma in vivo [9], in which
2-cyano-3,12-dioxooleana-1,9(11)-dien-
28-oic acid (CDDO) is the most prominent
acid without any doubt. CDDO [15]
showed high inhibitory activity against
production of nitric oxide (NO) induced by
interferon-g in mouse macrophages
(IC50 ¼ 0.1 nM level) and also inhibited
proliferation. Moreover, the analogs of
CDDO even showed more potency than
their parent compound [16,17]. CDDO and
CDDO-methyl ester are currently in
clinical trial for the treatment of cancers,
which suggests that CDDOs have the
potential to be a viable therapy.
Oleanane-type pentacyclic triterpenoids
are abundant in plant kingdom [1,2].
Oleanolic acid (3b-hydroxy-olea-12-en-
28-oic acid, OA, 1) was firstly separated
from Oleaeurepaea, and exists widely in
almost 190 species of 60 branches of food,
medicinal herbs, and other plants in the
form of free acid or aglycones for
triterpenoid saponins [3]. It has been
investigated for anti-inflammatory activity
[4], protection of the liver against toxic
injury [5], induction of collagen synthesis
[6], and induction of differentiation in
leukemia or teratocarcinoma cells [7].
In recent years, it was found that OA
had marked antitumor effects and exhib-
ited cytotoxic activity toward many cancer
cell lines [6,8–14]. For example, OA has
been demonstrated to have antihepatocel-
lular carcinoma activity in humans without
apparent side effects [8]. For the good
activities, its modification becomes very
During the structural modification of
OA, we found that nitroethylene (vinyl)
derivatives (2) showed strong inhibition
*Corresponding author. Email: wyong@scu.edu.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.654110
http://www.tandfonline.com

356
L. Chen et al.
Figure 1. The chemical structures of compounds 1–3.
(Scheme 1). The 1H NMR spectral data of
against HepG2 and Col-02 in vitro unex-
pectedly. Encouraged from these findings,
we designed, synthesized serials of novel
OA derivatives, and also evaluated their
growth inhibitions against human hepato-
cellular carcinoma cell line (HepG2) and
colon cancer cell line (Col-02), which are
related to the most frequent cancers in the
world [18]. On the design of the OA
derivatives, we retained the nitroethylene
at C-17 and combined it with the 1-en-2-
cyano-3-oxo in ring A as CDDOs, except-
ing that the two groups could function in
coordination with each other. Finally, 15
oleanane derivatives were synthesized and
the evaluation result showed that several
derivatives exhibited moderate-to-good
inhibitory activity, with 3 displaying the
best activity [GI50 ¼ 1.75 mM (HepG2),
0.71 mM (Col-02)]. We have also discussed
the potential structure–activity relation-
ship (SAR) of these compounds and the
implication of these findings (Figure 1).
ketone 2 displayed three olefinic hydro-
gens at d 7.20, 6.93, and 5.35, respectively.
The IR spectrum showed the olefinic
hydrogen absorption bands at 3110,
2949, and 1524 cm21, suggesting the
formation of nitrovinyl group. Elemental
analysis and ESI-MS confirmed the
structure of ketones 2 and 11.
Subsequently, we designed a variety of
new OA derivatives with modified ring
A. Similarly, deprotection of ketal 7 yielded
the ketone compound. Hydroxymethylene
12 was prepared by the formulation of the
ketone compound with ethyl formate in the
presence of sodium methoxide in benzene.
Isoxazole 13 was prepared by the conden-
sation of 12 with hydroxylamine. Cleavage
of the isoxazole moiety of 13 with sodium
methoxide gave nitrile 14. Enone 15 was
prepared by 2,3-dichloro-5,6-dicyano-
1,4-benzoquinone (DDQ) oxidation of 14
in benzene [20]. Similarly, oxidation of 15
with PCC furnished aldehyde 16, which was
converted to 3 with CH3COONH4 and
CH3NO2 (Scheme 2).
2. Results and discussion
First, the modifications on C-17 of OA
were carried out. Jone’s oxidation of 1
furnished ketone 4, which was reacted
with bromoethane and K2CO3 in dry DMF
to give ethyl oleanate 5 in a high yield.
Acetalization of 5 with ethylene glycol
gave 6, followed by the reduction in
LiAlH4 afford to alcohol 7 [19]. Oxidation
of 7 with pyridinium chlorochromate
(PCC) afforded aldehyde 8, which was
then converted to 9 with CH3COONH4
and CH3NO2. Compound 9 was reduced to
10 using LiAlH4. Deprotection of 6 and 7
yielded ketones 2 and 11, respectively
The inhibitions against HepG2 and Col-
02 of OA (1) and its synthetic derivatives
were evaluated in vitro (Table 1). The keto
derivative 4 is a little more active than 1.
Acetylation of the C-3 keto group (6–8)
diminished the inhibitory activities com-
pared to 4, indicating that C-3 keto groups
contributed to inhibition. Introducing
nitro group at C-17 of OA increased the
potency by three times compared to 1
[2: GI50 ¼ 1.30 mM (HepG2), 4.18 mM
(Col-02)]. These results indicated that the
nitro moiety was important for the retention

Journal of Asian Natural Products Research
357
Scheme 1. Synthesis of 2 and 4–11. Reagents and conditions: (a) PCC, CH2Cl2, r.t.; (b) EtBr,
K2CO3, DMF, r.t.; (c) HOCH2CH2OH, TsOH, toluene, ref.; (d) LiAlH4, THF, r.t.; (e) PCC, CH2Cl2,
r.t.; (f) CH3COONH4, CH3NO2, toluene, 708C; (g) LiAlH4, THF, r.t.; and (h) 2 mol/l dilute
hydrochloric acid, THF, 1008C.
Scheme 2. Synthesis of 3 and 12–16. Reagents and conditions: (a) 2 mol/l dilute hydrochloric acid,
THF, 1008C; (b) HCOOCH2CH3, CH3ONa, toluene, r.t.; (c) NH2OH.HCl, EtOH/Ether, ref.;
(d) CH3ONa, Ether/MeOH, r.t.; (e) DDQ, toluene, ref.; (f) PCC, DCM, i.b.; and (g) CH3NO2,
NH4OAc, toluene, ref.

358
L. Chen et al.
Table 1. Growth inhibition rates and GI50 values of compounds 1–16 against human
hepatocellular carcinoma and colon cancer cell lines.
HepG2
Col-02
Compounds
Growth %a
GI50 (mg/ml)
Growth %
GI50 (mg/ml)
1
2
3
4
5
6
7
8
9
104.82
33.78
5.89
ND
1.30
1.75
ND
ND
ND
ND
ND
ND
ND
ND
ND
9.71
ND
ND
5.31
0.05
93.20
242.78
212.63
71.34
ND
4.18
0.71
ND
ND
ND
ND
ND
ND
ND
ND
9.16
ND
ND
ND
1.44
0.03
73.11
134.94
124.66
133.87
139.57
50.39
96.73
90.55
73.00
46.92
105.58
50.79
27.42
5.36
92.76
105.04
124.71
76.19
59.03
10
11
12
13
14
15
16
Taxolb
108.68
111.37
10.36
62.17
61.95
45.96
3.46
28.28
Note: ND, not determined.
a The maximum inhibition concentration is 10 mg/ml.
b Taxol was used as the positive control, of which the maximum inhibition concentration is 10 mg/ml.
of inhibitory activities. Several OA nitro
derivatives (2, 3, 9) with modified ring A
exhibited moderate-to-good inhibitory
activity, with 3 displaying the best activity
[GI50 ¼ 1.75mM (HepG2), 0.71mM (Col-
02)]. SAR analyses of these derivatives
indicated that 1-en-2-cyano-3-oxo in ring A
and a nitro at C-17 produced inhibition in
coordination with each other as anticipated.
Both the structures were important in the
retention of inhibition against HepG2 and
Col-02.
as promising drugs are ongoing in our
laboratories, and the results will be reported
in due course.
3. Experimental
3.1 General experimental procedures
Melting points were measured on a YRT-3
melting point apparatus (Tianda Tianfa
Technology Co., Ltd, Tianjin, China) and
are uncorrected. Optical rotations were
determined on a PerkinElmer model 241
polarimeter (PerkinElmer, Inc., CA, USA).
IR spectra were obtained on a PerkinElmer
983 (PerkinElmer, Inc., CA, USA). Elemen-
tal analyses were carried out by Atlantic
Microlab (Atlanta, GA, USA). NMR spectra
were recorded on a Varian INOVA 400
(Varian, Palo Alto, CA, USA) and on a
Bruker AC-E200. Chemical shifts (d) are
expressed in ppm, tetramethylsilane used as
theinternalreference,andcouplingconstants
(J) are expressed in Hz. Mass spectra were
recorded on an Agilent 1946B ESI-MS
instrument (Agilent Technologies, Inc.,
In summary, 15 oleanolic derivatives
with modified ring A and substitutions at C-
17 were synthesized and biologically eval-
uatedagainst HepG2 and Col-02. Among the
three series of compounds, 3 [GI50
¼
1.75 mM (HepG2), 0.71 mM (Col-02)]
exhibited more potent antitumor activity
than the parent compound 1. SAR analysis
showed that a 1-en-2-cyano-3-oxo in ring A
and a nitro at C-17 were important in
retentionoftheinhibitionagainstHepG2and
Col-02.Basedontheseresults,furtherdesign
and biological evaluation of OA derivatives

Journal of Asian Natural Products Research
359
Santa Clara, CA, USA). TLC was performed
using precoated silica gel GF254 (0.2 mm;
Chemical Industry Institute, Yantai, China),
and column chromatography was performed
using silica gel (100–200 mesh; Qingdao
Haiyang Chemical Co., Ltd, Qingdao,
China).
CH3NO2 (150 ml) was added toluene
(2ml). The mixture was stirred at 708C for
overnight and concentrated to remove
CH3NO2. The residue was dispersed in
CH2Cl2 and washed with aqueous NaHCO3
and water and dried over sodium sulfate,
filtered, and concentrated to afford yellow
solid,andtheresultingresiduewassubjected
to a silica gel column chromatography
(PE:EtOAc ¼ 80:1) to give 9 (1.2 g, 69%)
Oleanic acid (1) was purchased from
Huakang Chemical Company, Inc.
(Sichuan, China). HepG2 and Col-02 were
supplied by Di Ao Pharmaceutical Group
(Chengdu, SiChuan, China). Positive con-
trolTaxolwas supplied byShanghaiNatural
Bio-engineering Co. Ltd. (Shanghai,
China).
20
as a white solid. M.p.: 140–1428C; ½aꢀD
þ46.8 (c ¼ 0.5, CH2Cl2); IR (KBr) ymax
3112, 2950, 2866, 1640, 1525, 1462, 1383,
1
1346, 1239, 1113 cm21; H NMR (CD3Cl,
400 MHz) d 7.20 (1H, d, J ¼ 13.6 Hz, H-
28), 6.94 (1H, d, J ¼ 13.6 Hz, H-29), 5.35
(1H, t, J ¼ 3.6 Hz, H-12), 3.94 (4H, m,
ZOCH2CH2OZ), 2.33 (1H, dt, J ¼ 13.6,
4.0 Hz, H-18), 2.22 (2H, dt, J ¼ 12.8,
4.0 Hz, H-11), 1.14 (3H, s, H-26), 0.95
(3H, s, H-27), 0.93 (3H, s, H-25), 0.92, 0.91,
0.85, 0.73 (each 3H, 12H, s, H-23, H-24,
H-30, H-31); ESI-MS: m/z 526 [M þ H]þ;
Elemental analysis: Found: C, 75.47%; H,
9.88%; N, 2.54%; calcd for C33H51NO4: C,
75.39%; H, 9.78%; N, 2.66%.
3.2 Synthesis of compounds 2, 3, and
8–16
3.2.1 Olean-12-en-28-aldehyde-3-
spiro[10, 20]dioxolane (8)
To a solution of 7 [18] (2.0 g, 4.0 mmol)
in CH2Cl2 (12 ml) was added PCC (2.6 g,
12 mmol). The mixture was stirred at room
temperature for 4 h. The solution was
washed with water, dried over sodium
sulfate, filtered, and concentrated, and the
resulting residue was subjected to a silica
gel column chromatography [petroleum
ether (PE):EtOAc ¼ 5:1] to give 8 (1.4 g,
75%) as a white solid. M.p.: 242–2448C;
3.2.3 Olean-12-en-28-(20-amino-ethyl)-
3-spiro[10, 20]dioxolane (10)
To
a suspension of LiAlH4 (0.5 g,
20
½aꢀD þ36.3 (c ¼ 0.5, CH2Cl2); IR (KBr)
12.7mmol) in absolute THF (10ml) was
added the solution of 9 (2.0g, 2.8 mmol) in
THF (10 ml) dropwise. The mixture was
stirred at room temperature overnight to
remove THF. The resulting residue was
subjected to a silica gel column chroma-
tography (PE:EtOAc ¼ 5:1) to give 10
(779.3mg, 56%) as a white solid. M.p.:
y
max 2950, 2862, 2801, 2701, 1724, 1462,
1383, 1202, 1111 cm21; 1H NMR (CD3Cl,
400 MHz): d 9.40 (1H, s, H-28), 5.33 (1H,
t, J ¼ 3.6 Hz, H-12), 3.94 (4H, m,
ZOCH2CH2OZ), 2.62 (1H, dd, J ¼ 14.0,
4.4 Hz, H-18), 1.14 (3H, s, H-26), 0.95
(3H, s, H-27), 0.93 (3H, s, H-25), 0.92,
0.91, 0.85, 0.73 (each 3H, 12H, s, H-23, H-
24, H-29, H-30); ESI-MS: m/z 483
[M þ H]þ; Elemental analysis: Found: C,
79.76%; H, 10.29%; calcd for C32H50O4:
C, 79.62%; H, 10.44%.
20
78–808C; ½aꢀD þ27.7 (c ¼ 0.5, CH2Cl2);
IR (KBr) ymax 3445, 2924, 2862, 1463,
1
1382, 1202, 1109, 1057cm21; H NMR
(CD3Cl, 400 MHz): d 5.15 (1H, t, J ¼
3.2 Hz, H-12), 3.92 (4H, m, ZOCH2CH2-
OZ), 3.58 (2H, q, J ¼ 7.2 Hz, H-29), 2.71
(1H, m, H-18), 2.54 (2H, br s, ZNH2), 2.53
(2H, m, H-11), 1.83 (2H, m, H-28),
1.09(3H, s, H-26), 0.96(3H, s, H-27), 0.95
(3H, s, H-25), 0.91, 0.84 (each 3H, 6H, s,
3.2.2 Olean-12-en-28-(20-nitro-vinyl-)
-3-spiro [10, 20]dioxolane (9)
To a solution of 8 (1.6 g, 3.4 mmol) and
CH3COONH4 (1.5 g, 19.5 mmol) in

360
L. Chen et al.
H-23, H-24); ESI-MS: m/z 498 [M þ H]þ;
Elemental analysis: Found: C, 79.49%; H,
11.29%; N, 2.78%; calcd for C33H55NO2:
C, 79.62%; H, 11.14%; N, 2.81%.
3.6 Hz, H-2), 2.36 (1H, dq, J ¼ 16.0,
3.6 Hz, H-20), 1.85–2.03 (4H, m, H-11,
H-22), 1.15 (3H, s, H-26), 1.09 (3H, s, H-
27), 1.04 (3H, s, H-25), 1.04, 0.92, 0.92,
0.80 (each 3H, 12H, s, H-23, H-24, H-29,
H-30).
3.2.4 3-Oxo-12-en-28-(20-nitro-vinyl)-
olean (2)
According to the reported method [19],
compound 2 was obtained as a white solid
from dioxolane 9, yield 78%. M.p.: 98–
3.2.5 2-Hydroxymethylene-3-oxoolean-
12(13)-en-28-ol (12)
20
1008C; ½aꢀD þ87.3 (c ¼ 0.5, CH2Cl2); IR
A solution of 7 (5.76 g, 12.0 mmol) in 2 M
HCl (60 ml) and THF (100 ml) was refluxed
for 1 h, and then cooled to room tempera-
ture. The organic layer was separated and
the aqueous layer was extracted with
EtOAc. The combined organic layer was
washed with brine, dried over Na2SO4,
condensed to give an amorphous solid.
Without any purification, the solid and
NaOMe (6.43 g, 119 mmol) were dissolved
in dry toluene (150 ml) and stirred at room
temperature for 10 min. Then ethyl formate
(8.28 g, 119 mmol) was dropped into the
solution, which was stirred at room
temperature for 3 h. The mixture was diluted
with toluene, washed by turns with 5%
aqueous HCl solution, saturated aqueous
NaHCO3 solution and brine, dried over
Na2SO4, and filtered. The filtrate was
evaporated in vacuo to give a brown oil,
which was purified by flash column
chromatography (PE:EtOAc ¼ 40:1) to
give 12 (4.25 g, 76.20%) as an amorphous
(KBr) ymax 3110, 2949, 2866, 1704, 1641,
1
1524, 1462, 1347, 1256 cm21. H NMR
(CD3Cl, 400 MHz): d 7.20 (1H, d,
J ¼ 14.0 Hz, H-28), 6.93 (1H, d, J ¼
14.0 Hz, H-29), 5.35 (1H, t, J ¼ 3.6 Hz,
H-12), 2.57 (1H, m, H-18), 2.36 (2H, m,
H-11), 1.16 (3H, s, H-26), 1.09 (3H, s,
H-27), 1.06 (3H, s, H-25), 1.05, 0.93, 0.90,
0.84 (each 3H, 12H, s, H-23, H-24, H-30,
H-31); 13C NMR (CDCl3, 100 MHz) d
217.5 (C, C-3), 151.9 (CH, C-28), 143.2
(C, C-13), 139.7 (CH, vCHNO2), 123.3
(CH, C-12), 55.3 (C, C-17), 47.5 (CH, C-
5), 46.8 (C, C-4), 46.0 (CH, C-9), 43.6
(CH2, C-19), 41.8 (CH, C-18), 39.6 (C, C-
14), 39.2 (CH, C-1), 39.0 (C, C-8), 36.7
(C, C-10), 34.1 (CH2, C-21), 34.0 (CH2,
C-22), 33.7 (C, C-2), 32.9 (CH2, C-7), 31.9
(CH2, C-15), 30.7 (C, C-20), 27.1 (CH2,
C-16), 26.3 (CH3, C-30), 26.1 (CH3, C-
29), 25.8 (CH3, C-27), 23.6 (CH3, C-24),
23.4 (CH3, C-23), 21.5 (CH2, C-11), 19.3
(CH2, C-6), 16.7 (CH3, C-26), 15.0 (CH3,
C-25); ESI-MS: m/z 482 [M þ H]þ;
Elemental analysis: Found: C, 77.36%;
H, 9.86%; N, 2.85%; calcd for
C31H47NO3: C, 77.29%; H, 9.83%; N,
2.91%.
20
solid. M.p.: 86–888C; ½aꢀD þ96.0 (c ¼ 0.5,
CH2Cl2); IR (KBr) ymax 3426, 2947, 2924,
2864, 1634, 1584, 1460, 1362, 1051 cm21
;
1H NMR (CDCl3, 400 MHz): d 14.92 (1H, s,
HO-C ¼ ), 8.58 (1H, s, H-C ¼ O), 5.25 (1H,
t, J ¼ 3.6 Hz, H-12), 3.57 (1H, d,
J ¼ 10.8 Hz, CH2OH), 3.23 (1H, d,
J ¼ 10.8 Hz, CH2OH), 2.29 (2H, dd,
J ¼ 14.4, 3.2Hz, H-1), 1.20 (3H, s, H-26),
1.19 (3H, s, H-27), 1.13 (3H, s, H-25), 1.00,
0.93, 0.90, 0.89 (each 3H, s, H-23, H-24, H-
29, H-30); ESI-MS: m/z 469 [M þ H]þ;
Elemental analysis: Found: C, 79.31%; H,
10.26%; calcd for C31H48O3: C, 79.44%; H,
10.32%.
Compound 11 was prepared according
to the same procedure described for 2.
20
M.p.: 153–1558C; ½aꢀD þ88.8 (c ¼ 0.5,
CH2Cl2); IR (KBr) ymax 2950, 2928, 2859,
2707, 1708, 1459, 1382, 1160, 1113cm21
;
1H NMR (CDCl3, 400 MHz) d 9.40 (1H, s,
H-28), 7.73 (1H, s, H-1), 5.37 (1H, t,
J ¼ 3.6 Hz, H-12), 2.63 (1H, dd, J ¼ 13.2,
4.0 Hz, H-18), 2.55 (1H, dt, J ¼ 16.0,

Journal of Asian Natural Products Research
361
3.2.6 Isoxazolo[4, 5-b]olean-12(13)-
en-28-ol (13)
CH2Cl2); IR (KBr) ymax 3422, 2948, 2926,
2866, 2206, 1718, 1627, 1460, 1382, 1367,
1267, 1047 cm21
;
1H NMR (CDCl3,
A solution of 12 (150mg, 0.32mmol) in
EtOH (10 ml) and water (4ml) was added
hydroxylamine hydrochloride (222.37 mg,
3.2 mmol). The mixture was heated under
reflux for 1 h and concentrated. Water was
added and the mixture was extracted with
EtOAc. The combined organic layers were
washed with water and brine twice,
respectively,driedoverNa2SO4 andfiltered.
The filtrate was evaporated in vacuo to give
a solid, which was purified by flash column
chromatography (PE:EtOAc ¼ 30:1) to
give 13 (110mg, 73.81%) as an amorphous
400 MHz): d 5.88 (1H, OH-3), 5.23
(1H, t, J ¼ 3.6 Hz, H-12), 3.56 (1H, d,
J ¼ 10.8 Hz, CH2OH), 3.22 (1H, d,
J ¼ 10.8 Hz, CH2OH), 1.26 (3H, s, H-
26), 1.17 (3H, s, H-27), 1.08 (3H, s, H-25),
0.98, 0.97, 0.89, 0.88 (each 3H, s, H-23, H-
24, H-29, H-30); ESI-MS: m/z 466
[M þ H]þ; Elemental analysis: Found: C,
79.87%; H, 10.13%; N, 3.06%; calcd for
C31H47NO2: C, 79.95%; H, 10.17%; N,
3.01%.
20
solid.M.p.:81–838C; ½aꢀD þ91.1(c ¼ 0.5,
3.2.8 2-Cyano-3-oxoolean-1, 12(13)-
dien-28-ol (15)
CH2Cl2); IR (KBr) ymax 3425, 2947, 2864,
1640, 1482, 1460, 1382, 1367, 1050cm21
;
A mixture of 14 (600mg, 1.29mmol) and
DDQ(439.26 mg, 1.95mmol)indrytoluene
(50 ml) was heated under reflux for 8 h and
filtered. The insoluble matter was washed
with EtOAc and the combined organic
layers were washed with 5% aqueous NaOH
solution and brine, respectively, dried over
Na2SO4, and filtered. The filtrate was
evaporated in vacuo to give a dark oil,
which was purified by flash column
chromatography (PE:EtOAc ¼ 10:1) to
give 15 (447.5mg, 74.82%) as a light
yellow amorphous solid. M.p.: 161–1628C;
1H NMR (CDCl3, 400 MHz) d 7.99 (1H, s,
H-C ¼ N), 5.26 (1H, t, J ¼ 3.6 Hz, H-12),
3.57 (1H, d, J ¼ 10.8Hz, CH2OH), 3.24
(1H, d, J ¼ 10.8Hz, CH2OH), 1.32 (3H, s,
H-26), 1.24 (3H, s, H-27), 1.19 (3H, s, H-
25), 1.00, 0.91, 0.90, 0.89 (each 3H, s, H-23,
H-24, H-29, H-30); ESI-MS: m/z 466
[M þ H]þ; Elemental analysis: Found: C,
79.89%; H, 10.23%; N, 3.09%; calcd for
C31H47NO2: C, 79.95%; H, 10.17%; N,
3.01%.
20
½aꢀD þ76.1 (c ¼ 0.5, CH2Cl2); IR (KBr)
3.2.7 2-Cyano-3-oxoolean-12(13)-
en-28-ol (14)
ymax 3447, 2949, 2868, 2235, 1687, 1461,
1385, 1224, 1050cm21; 1H NMR (CDCl3,
400 MHz) d 7.75 (1H, s, H-1), 5.36 (1H, t,
J ¼ 3.6 Hz, H-12),3.57(1H,d,J ¼ 10.8Hz,
CH2OH), 3.21 (1H, d, J ¼ 10.8 Hz,
CH2OH), 1.24 (3H, s, H-26), 1.22 (3H, s,
H-27), 1.19 (3H, s, H-25), 1.15, 1.05, 0.91,
0.89 (each 3H, s, H-23, H-24, H-29, H-30);
ESI-MS: m/z 464 [M þ H]þ; Elemental
analysis: Found: C, 80.41%; H, 9.83%; N,
3.05%; calcd for C31H45NO2: C, 80.30%; H,
9.78%; N, 3.02%.
A solution of 13 (100 mg, 0.215 mmol) in
MeOH (4 ml) and Et2O (8 ml) in an ice
bath was added NaOMe (46.44 g,
0.86 mmol). The mixture was stirred at
room temperature for 6 h, and then
concentrated. Water was added and the
mixture was extracted with DCM. The
combined organic layers were washed by
turns with 5% aqueous HCl solution,
saturated aqueous NaHCO3 solution and
brine, dried with Na2SO4, and filtered. The
filtrate was evaporated in vacuo to give a
solid, which was purified by flash column
chromatography (PE:EtOAc ¼ 20:1) to
give 14 (85 mg, 85.00%) as a white solid.
3.2.9 2-Cyano-3-oxoolean-1, 12(13)-
dien-28-aldehyde (16)
A solution of 15 (792.1 mg, 1.71 mmol) in
DCM (30 ml) was dropped into the
20
M.p.: 150–1528C; ½aꢀD þ101.8 (c ¼ 0.5,

362
L. Chen et al.
solution of PCC (735.3 mg, 3.42 mmol) in
DCM (30 ml) and stirred at room tem-
perature for 0.5 h and filtered. The
insoluble matter was washed with DCM.
The combined organic layers were washed
with brine twice, dried over Na2SO4, and
filtered. The filtrate was evaporated in
vacuo and purified by flash column
chromatography (PE:EtOAc ¼ 60:1) to
give 16 (410 mg, 51.93%) as a white
dried over Na2SO4, and filtered. The
filtrate was evaporated in vacuo and
purified by flash column chromatography
(PE:EtOAc ¼ 40:1) to give 3 (52 mg,
47.50%) as an amorphous solid.
20
M.p. .2308C (decomp); ½aꢀD þ69.3
(c ¼ 0.5, CH2Cl2); IR (KBr) ymax 3112,
2949, 2923, 2857, 2230, 1689, 1520, 1464,
1382, 1349, 1014 cm21; 1H NMR (CDCl3,
400 MHz): d 7.73 (1H, s, H-1), 7.20 (1H, d,
J ¼ 14.0 Hz, H-28), 6.93 (1H, d, J ¼
14.0 Hz, H-29), 5.41 (1H, t, J ¼ 3.6 Hz),
2.42 (1H, dd, J ¼ 13.8, 4.4 Hz, H-18), 1.23
(3H, s, H-26), 1.21 (3H, s, H-27), 1.17 (3H,
s, H-25), 1.14, 0.94, 0.91, 0.89 (each 3H, s,
H-23, H-24, H-29, H-30); 13C NMR
(CDCl3, 100 MHz) d 197.9 (C, C-3),
169.5 (CH, C-1), 151.6 (CH, C-28),
143.7 (C, C-13), 139.5 (CH, vCHNO2),
122.0 (CH, C-12), 114.8 (C, CN), 113.8
(C, C-2), 52.3 (C, C-17), 45.7 (CH, C-5),
44.7 (C, C-14), 43.7 (CH2, C-19), 42.0 (C,
C-4), 41.0 (CH, C-18), 40.5 (C, C-10),
40.4 (C, C-8), 38.9 (CH, C-9), 33.8 (CH2,
C-21), 33.6 (CH2, C-22), 32.8 (CH2, C-7),
31.6 (CH2, C-16), 30.6 (C, C-20), 29.6
(CH2, C-15), 27.7 (CH3, C-30), 26.8 (CH3,
C-29), 25.8 (CH3, C-27), 25.6 (CH2, C-
11), 23.3 (CH3, C-24), 23.2 (CH3, C-23),
18.5 (CH2, C-6), 17.7 (CH3, C-25), 17.2
(CH3, C-26); ESI-MS: m/z 505 [M þ H]þ;
Elemental analysis: Found: C, 76.23%; H,
8.82%; N, 5.51%; calcd for C32H44NO3:
C, 76.15%; H, 8.79%; N, 5.55%.
20
solid. M.p. .2008C (decomp); ½aꢀD
þ58.5 (c ¼ 0.5, CH2Cl2); IR (KBr) ymax
2922, 2856, 2697, 2234, 1725, 1691, 1464,
1383, 1220, 1005 cm21; 1H NMR (CDCl3,
400 MHz) d 9.39 (1H, s, H-28), 7.73 (1H,
s, H-1), 5.42 (1H, t, J ¼ 3.6 Hz, H-12),
2.68 (1H, dd, J ¼ 13.8, 4.4 Hz, H-18), 1.22
(3H, s, H-26), 1.21 (3H, s, H-27), 1.16 (3H,
s, H-25), 1.14, 0.94, 0.93, 0.84 (each 3H,
s, H-23, H-24, H-29, H-30); 13C NMR
(CDCl3, 100 MHz) d 207.0 (C, C-3), 198.0
(CH, C-28), 170.0 (CH, C-1), 143.7
(C, C-13), 121.7 (CH, C-12), 114.9
(C, CN), 113.8 (C, C-2), 52.5 (C, C-17),
49.1 (CH, C-5), 45.2 (CH2, C-19), 44.8
(C, C-14), 42.0 (C, C-4), 41.0 (CH, C-9),
40.5 (C, C-10), 40.5 (C, C-8), 40.3 (CH,
C-18), 33.0 (CH2, C-21), 32.1 (CH2, C-7),
30.6 (C, C-20), 29.6 (CH2, C-15), 27.7 (CH2,
C-22), 27.5 (CH3, C-30), 26.5 (CH3, C-29),
25.3 (CH3, C-27), 23.3 (CH2, C-11), 23.2
(CH2, C-16), 21.8 (CH3, C-24), 21.6 (CH3,
C-23), 18.6 (CH2, C-6), 17.9 (CH3, C-25),
17.5 (CH3, C-26); ESI-MS: m/z 462
[M þ H]þ; Elemental analysis: Found: C,
80.57%; H, 9.30%; N, 3.01%; calcd for
C31H43NO2: C, 80.65%; H, 9.39%; N,
3.03%.
3.3 Cytotoxic assay in vitro
The cytotoxicity of individual compound
screened was determined by the sulforho-
damine B assay using the cytotoxicity
detection kit (Roche Diagnostics, Shang-
hai, China) according to the manufac-
turer’s instructions. The method described
here has been optimized for the cytotoxic
screening of compounds to adherent cell in
a 96-well microtiter plate. After an
incubation period, cell monolayers are
fixed with 10% (w/v) trichloroacetic acid
and stained for 30 min, after which the
3.2.10 2-Cyano-3-oxo-28-(b-nitro)
vinylolean-1,12(13)-diene (3)
A solution of 16 (100 mg, 0.217 mmol) and
NH4OAc (333.5 mg, 4.34 mmol) in
CH3NO2 (15 ml) and toluene (2 ml) was
heated under reflux for 6 h and concen-
trated. Water was added and the mixture
was extracted with DCM. The combined
organic layers were washed with brine,

Journal of Asian Natural Products Research
363
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excess dye is removed by washing
repeatedly with 1% (v/v) acetic acid.
The protein-bound dye is dissolved in
10 mM Tris base solution for oleanolic
acid derivatives (OD) determination at
510 nm using
a
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