Med Chem Res (2014) 23:4631–4641
DOI 10.1007/s00044-014-1031-z
ORIGINAL RESEARCH
Enhancement of anti-bacterial and anti-tumor activities
of pentacyclic triterpenes by introducing exocyclic
a,b-unsaturated ketone moiety in ring A
Li R. Huang • Heng Luo • Xiao S. Yang • Lei Chen
Jian X. Zhang • Dao P. Wang • Xiao J. Hao
Received: 14 December 2013 / Accepted: 7 May 2014 / Published online: 23 May 2014
Ó Springer Science+Business Media New York 2014
Abstract Ursolic acid, oleanolic acid, glycyrrhetinic
acid, and betulinic acid, the representatives of pentacyclic
triterpenes, were modified by introducing 2-methylene-3-
oxo group as exocyclic a,b-unsaturated ketone moiety in
ring A. The anti-bacterial and anti-tumor activities of these
derivatives were assayed by comparing with the parent
compounds. Results indicated that pentacyclic triterpenes
carrying 2-methylene-3-oxo group in the ring A exhibited a
significant improvement in anti-bacterial activity that was
limited to Gram-positive bacteria Staphylococcus aureus
and Bacillus subtilis. The four derivatives also showed an
increased cytotoxicity against leukemia, lung, and breast
cancer cell lines in a dose-dependent manner in vitro. U2
and O2 compounds showed strong apoptotic activities to
lung carcinoma cell lines. The results for the first time
provided scientific evidence for improvement of anti-bac-
terial and anti-tumor activities of pentacyclic triterpenes
using derivatives of these compounds.
Keywords Pentacyclic triterpenes compounds 
2-Methylene-3-oxo group  Anti-bacteria  Anti-tumor 
Apoptosis
Li R. Huang and Heng Luo contributed equally to this work.
L. R. Huang  X. S. Yang
Ministry of Education Key Laboratory of Green Pesticide and
Ago-Bioengineering, Center for Research and Development of
Fine Chemicals of Guizhou University, Guiyang 550025, China
L. R. Huang  H. Luo  X. S. Yang (&)  L. Chen 
J. X. Zhang  D. P. Wang  X. J. Hao
The Key Laboratory of Chemistry for Natural Products of
Guizhou Province and Chinese Academy of Sciences,
Guiyang 550002, China
e-mail: gzcnp@sina.cn
MEDICINAL
CHEMISTRY
RESEARCH
•
Introduction
The pentacyclic triterpenes, as free, esters or glycosides
form, were widely distributed in many plants (Connolly
and Hil, 2008). These compounds possessed many biology
activities including anti-tumor, anti-bacteria, hepato-pro-
tective effect, depressurization, and hyperglycemia (Dzu-
bak et al., 2006; Liu, 2005; Girija et al., 2011; Wolska
et al., 2010; Long et al., 2013). However, poor solubility
and toxicity associated with haemolytic as well as cyto-
static have limited their development (Dzubak et al., 2006).
To solve this problem, various derivatives have been syn-
thesized through chemical modification (Lallemand et al.,
2011; Genet et al., 2010; Rao et al., 2008; Mallavadhani
et al., 2013). For example, compound 2-cyano-3, 12-di-
oxooleana-1, 9(11)-dien-28-oic acid (CDDO), which was
synthesized from oleanoic acid, has been used as a candi-
date drug for breast cancer treatment (Lapillonne et al.,
2003).
a,b-Unsaturated ketone moiety existed in many natural
products and synthesized compounds. It has attracted
extensive interest since compounds containing this moiety
might act as Michael acceptor to react with the sulfydryl
group of targeted protein or enzyme by the Michael addi-
tive reaction, resulting in their anti-tumor, anti-microbial,
and insecticidal activities (Dimmock et al., 1988; Elsub-
bagh et al., 2000; Schultz et al., 2005). Ethacrynic acid was
a diuretics, and its pharmacophoric group possessed a,b-
unsaturated ketone moiety. The anti-cancer mechanism of
ethacrynic acid resulted from a Michael additive reaction
between the sulfydryl group of glutathione (GSH) and a,b-
unsaturated ketone group, which inhibited the activity of
GSH (Awasthi et al., 1993; Townsend and Tew, 2003).
Reddy et al. have found that parthenin exhibited a higher
anti-cancer activity due to its a,b-unsaturated ketone
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moiety, via inhibition of NF-kB (Reddy et al., 2011). In
addition, a series of chalcones derivatives containing a,b-
unsaturated ketone were shown to demonstrate anti-fungal
activity against Candida albicans strains (Bag et al., 2009).
Nevertheless, compounds containing exocyclic double
bond a,b-unsaturated ketone moiety were less common in
natural products while exhibited stronger biology activity.
For example, arkomycin contains a exocyclic double bond
a,b-unsaturated ketone moiety that shows strong anti-
microbial and inhibitory effects on the proliferation and
progression of Erlich ascites tumors (EAT) in mice (Bo-
eckman et al., 1980).
Studies on structure–activity relationship of the kaurane
diterpenes (oridonin and lasiokaurin) have revealed that the
exocyclic double bond a,b-unsaturated ketone moiety of
these compounds was responsible for their anti-cancer
activity. However, the deoxidation of the exocyclic double
bond led to inhibition of this anti-cancer activity (Hueso-
Falcón et al., 2010). Furthermore, another study demon-
strated that strong anti-tumor activity of the eriocalyxin B
was because of a,b-unsaturated ketone moieties in ring A
and D (Zhao et al., 2007). Nevertheless, few studies
reported structure and activities of pentacyclic triterpenes
containing exocyclic double bond a,b-unsaturated ketone
moiety. Johns and his partners have synthesized the com-
pound methyl 2-methylene-3-oxo-olean-12-en-28-oate for
the first time without examining its pharmacological
activity (Johns et al., 1983).
In this paper, four derivatives of pentacyclic triterpenes
(U2, O2, G2, and B2) were synthesized by introducing
2-methylene-3-oxo group as exocyclic a,b-unsaturated
ketone moiety in ring A of the parent compounds ursolic
acid (UA), oleanoic acid (OA), glycyrrhetintic acid (GA),
and betulinic acid (BA). The structure of these four novel
derivatives was determined by spectroscopic techniques
IR, NMR, and MS. Experimental examination of these
compounds demonstrated significant improvement in anti-
bacterial and anti-tumor activities when compared with the
parent compounds. The ability of the novel compounds to
induce apoptosis was confirmed by Hoechst 33258 dye and
DNA laddering techniques. These results provided further
foundation for improving the activity of pentacyclic tri-
terpenes for cancer and disease prevention.
Results and discussion
Chemistry
The synthetic pathways of the target compounds (B2, U2,
O2, and G2) are detailed in Scheme 1, and their structures
were determined using Infrared (IR), mass spectra (MS),
and NMR spectral data analyses. Four intermediates 3-keto
derivatives (B1, U1, O1, and G1) were obtained by the
oxidation reaction with Jones reagent (Leal et al., 2013) in
good yields. Other oxidation reagents such as PCC (Wen
et al., 2006) and KMnO4 (Esteb et al., 2003) were not used
due to lower yields or complicated processes. The modified
compounds showed a similar sharp band at about
1,700 cm-1 by IR spectrum and a carbonyl signal at about
217 ppm by the 13C NMR spectrum, which indicated the
presence of the ketone group in the structures. In

a b
COOH COOH COOH

HO O O

BA B1 B2

R2 R3 R2 R3 R2 R3
R1 R1 R1

X a X b X
R4 R4 R4

HO O O

UA: R1=CH3, R2=CH3, R3=H, R4=COOH, X=CH2 U1: R1 =CH3 , R2=CH3, R3=H, R4=COOH, X=CH2 U2: R1 =CH3 , R2=CH3, R3=H, R4=COOH, X=CH2
OA: R1=H, R2 =CH3 , R3 =CH3 , R4 =COOH, X=CH2 O1: R1=H, R2=CH3, R3 =CH3 , R4 =COOH, X=CH2 O2: R1=H, R2=CH3, R3 =CH3 , R4 =COOH, X=CH2
GA: R1=H, R2 =CH3 , R3 =COOH, R4 =CH3 , X=CO G1: R1=H, R2=CH3, R3 =COOH, R4 =CH3 , X=CO G2: R1=H, R2=CH3, R3 =COOH, R4 =CH3 , X=CO

Scheme 1 Reagents and conditions: a Jones reagent, acetone, 0 °C, 2 h; b method A: paraformaldehyde, dimethylamine hydrochloride, acetic
acid, DMF, 100 °C, 1.5 h; method B: KOH, formaldehyde, refluxing for 3 h

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Fig. 1 Representative pictures of inhibitory zones of tested com- 0.5 McFarland. Sterile filter paper disks were impregnated with 30 lL
pounds against three bacteria. The data showed the inhibition of the of tested compounds (100 lM), dried and then placed on each plate.
test organisms, namely E. coli (a), B. subtilis (b), and S. aureus (c). After incubation for 24 h at 37 °C, the diameter of the inhibition zone
Luria Broth agar plates were seeded with bacteria at concentration of was measured. Tests were performed in triplicate

preparation of the target compounds (B2, U2, O2, and G2), spectrum of compound U2 showed two singlets at d 6.00
it was easy to consider employing the aldol condensation and 5.16 ppm, which are characteristic signals for the
reaction with formaldehyde under base condition, but this exocyclic methylene protons. Furthermore, the 13C NMR
method led to low yields (about 40 %) and was abandoned spectrum of U2 showed a pair of double bond signals at d
(Method B). In order to increase the reaction yields, 141.8 ppm (C, C-2) and 123.7 ppm (CH2, C-31) suggesting
Mannich reaction of 3-keto derivatives with paraformal- the presence of 2-methylene. The same signals were also
dehyde and dimethylamine hydrochloride was adopted to observed in compounds O2, G2, and B2.
generate intermediate Mannich base compounds. Hoffman
elimination reaction then followed to generate the target Improved anti-bacterial activity by the pentacyclic
compounds with middle yields (above 70 %) (Method A). triterpenes derivatives
The structure assigned for 2-methylene-a,b-unsaturated
group was supported by comparing with 3-keto compound The anti-bacterial activities of four derivatives and parent
using the 1H NMR and 13C NMR spectra. The 1H NMR compounds are summarized in Fig. 1; Tables 1 and 2. In
Kirby-Bauer test, at concentration of 100 lM, G2 exhib-
Table 1 Anti-bacterial activity of compounds against three strains at ited the best inhibitory activity against Staphylococcus
concentration of 100 lM in vitro aureus (S. aureus) and Bacillus subtilis (B. subtilis) of four
Compound Inhibition rate (%)
derivatives (Fig. 1). The results showed that all compounds
tested had growth inhibitory activity against Gram-positive
E. coli ATCC B. subtilis ATCC S. aureus ATCC bacteria including S. aureus and B. subtilis, but not to
25922 6051 25923
Gram-negative bacteria Escherichia coli (E. coli)
UA NA 47.55 ± 2.20 58.97 ± 5.12 (Table 1), which was consistent with previous reports
U2 NA 98.75 ± 2.57** 99.87 ± 0.80**
OA 19.19 ± 3.25 23.81 ± 5.00 36.34 ± 4.10 Table 2 The MIC values of the anti-bacterial activity of the deriv-
O2 9.2 ± 4.04 98.21 ± 0.98## 97.58 ± 2.99## atives against B. Subtilis and S. Aureus strains in vitro
GA 6.95 ± 1.48 48.87 ± 3.45 43.97 ± 3.03 Compound MIC (lM)
G2 NA 96.09 ± 4.1544 97.57 ± 3.2644
B. subtilis ATCC 6051 S. aureus ATCC 25923
BA NA 20.58 ± 2.55 16.75 ± 1.68
B2 NA 35.01 ± 4.39* 41.46 ± 5.05** U2 80.00 ± 4.45 60.00 ± 2.15
Ampicillin – – 99.34 ± 3.26 O2 80.00 ± 3.10 50.00 ± 5.02
Streptomycin 94.12 ± 4.40 97.05 ± 3.88 – G2 45.00 ± 4.66 35.00 ± 2.44
Values were mean ± standard deviation of three independent Ampicillin – 5.00 ± 1.22
experiments. The bacteria were seeded in 96-well microculture plates Streptomycin 10.00 ± 2.08 –
at concentration of 1 9 105 CFU/mL in Luria Broth. Tested com-
Values were mean ± standard deviation of three independent
pounds and positive compound solution were then added to the well
experiments. A series of different concentrations of tested compounds
to obtain the final concentration of 100 lM. The growth inhibitory
were added to 96-well microculture plates containing the bacteria
activity was measured in an ELISA plate reader at 570 nm after
culture at concentration of 1 9 105 CFU/mL. The plates were mea-
cultures shaken on a vibrating platform at 37 °C for 20 h
sured in an ELISA plate reader at 570 nm after cultures shaken on a
NA no activity; ‘‘–’’ not tested vibrating platform at 37 °C for 20 h. The MIC values were the lowest
*, #, 4, * P \ 0.05; **, ##, 44, ** P \ 0.01 compared with UA, OA, concentration of compound whose absorbance was comparable with
GA, BA, respectively. Same as the below results the negative control

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(Wolska et al., 2010; Fontanay et al., 2008). Similarly, Gram-positive bacteria (P \ 0.05). The results indicated
Kurek and co-workers have demonstrated that OA and UA that anti-bacterial activities of parent compounds were
could affect the peptidoglycan metabolism, change cell improved by introducing 2-methylene-3- ketone moiety in
morphology, and enhance the autolysis of the bacterial ring A. The U2, O2, and G2 showed a potent anti-bacterial
cells (Kurek et al., 2010). The peptidoglycan is the main activity against Gram-positive bacteria at 100 lM, which
composition in cell wall of Gram-positive bacteria and parallel with control ampicillin and streptomycin com-
plays an important role in maintaining regular function of pounds. The B2 compound showed a lower activity than
cell membrane. others. Since the ring E of B2 is structurally different from
Our results, however, found a significant difference the U2, O2, and G2, we concluded that the difference in
(P \ 0.01) in the extent of inhibition of U2, G2, and O2 by anti-bacterial activity of four derivatives must resist within
comparing with their corresponding parent compounds. In the ring E. BA and B2 indeed were lupane-type compounds
addition, B2 was significantly inhibited the growth of with structure similar to hopan-type molecules located
Fig. 2 Effects of derivatives on
tumor cell survival. Cells were
treated with different
concentrations of tested
compounds for 48 h, and then
cell survival was determined by
MTT assay. a, b, and c showed
K562, A549, and MCF-7 cells
treated with the indicated
compounds. The results
represent the mean ± standard
deviation of three independent
experiments.*P \ 0.05,
**
P \ 0.01 compared with
control

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within the bacterial membranes, which are products of 35.00 (±2.44) lM, and another a,b-unsaturated group in
bacterial metabolism (Fontanay et al., 2008). Thus, the BA/ ring C may contribute to its activity.
B2 and its derivatives may lack an anti-bacterial activity
due to structural similarity with the lupan-type compounds. Improved cell cytotoxic activity by the pentacyclic
Both U2 and O2 demonstrated an equal inhibitory triterpenes derivatives
activity against B. subtilis with a minimum inhibitory
concentration (MIC) of 80.00 lM, but different inhibitory The cytotoxic activity of the derivatives was analyzed on
effect to S. aureus with MICs of 60.00 (±2.15) lM and several cancer cell lines at different concentrations using
50.00 (±5.02) lM, respectively. The difference in inhibi- MTT assay which determines cell viability (Fig. 2). These
tory activity of U2 and O2 might be due to the position of results demonstrated a significant difference in the inhibi-
methyl group in ring E, which indicated the importance of tory ability of the compounds, summarized as follow: there
structure configuration for biological activity. G2 had the was a large difference in the level of inhibition between U2
most inhibitory activity in four derivatives against B. and O2 on human leukemic cell lines (K562) at lower
subtilis and S. aureus, with MICs of 45.00 (±4.66) lM and concentration (P \ 0.01); G2 and B2 exhibited a signifi-
cant inhibitory effect (P \ 0.01) at concentrations higher
than 1.56 lM, when compared with controls (Fig. 2a).
Similar inhibitory results were also observed for human
Table 3 Result of growth inhibitory activity of parent compounds lung carcinoma cell lines (A549) (Fig. 2b). In breast cancer
and their derivatives to three cancer cell lines in vitro cell lines (MCF-7), the cytotoxicity effect was observed for
Compound IC50(lM) U2, O2, and G2 compounds (P \ 0.01) at doses from 5.00
to 50.00 lM, but no significant difference was detected for
K562 A549 MCF-7
B2 at concentration 10.00–5.00 lM (Fig. 2c). Overall, the
UA 31.58 ± 0.22 26.65 ± 2.21 28.36 ± 1.22 cytotoxic activity of the four derivatives on the three
U2 25.70 ± 0.31* 10.49 ± 2.34** 14.53 ± 1.89** cancer cell lines was increased with concentrations of the
OA [200 [200 [200 tested compounds, indicating the dose-dependent manner
O2 28.71 ± 1.30## 15.09 ± 0.26## 19.53 ± 2.45## of the inhibitory response.
GA 85.57 ± 1.23 96.18 ± 4.33 71.96 ± 0.48 Analyzing the cytotoxic activity of the derivatives and
G2 62.44 ± 1.1944 83.75 ± 0.184 83.05 ± 0.824 their parent compounds (Table 3) failed to detect a ex-
BA 108.84 ± 1.52 82.13 ± 0.33 85.70 ± 0.12 tremely significant increase in cytotoxic activity between B2
B2 100.98 ± 2.03 97.76 ± 1.17* 95.79 ± 2.07* and BA, which was consistent with the anti-bacterial activity
Adriamycin 1.06 ± 1.42 0.51 ± 1.14 0.40 ± 0.88 results. This data further suggested that the structure of ring
E may be crucial for the cytotoxic activity. G2 compound
Values were mean ± standard deviation of three independent
experiments. The indicated cell lines were incubated with serial displayed some selectivity: demonstrated a better inhibitory
dilutions of the compounds for 48 h. Cell viability (%) was measured effect on proliferation of K562 and A549 cells than GA, but
by MTT assay and used for determination of IC50. The experiments exhibited an opposite result on MCF-7 cells. It was impor-
were carried out in triplicate cultures. All compounds were dissolved tant noting that O2 exhibited about 100-fold higher anti-
in DMSO, and diluted with culture medium containing 0.1 % DMSO,
respectively. The control cells treated with culture medium containing tumor activity than parent compound OA, indicating the
0.1 % DMSO contribution of 2-methylene-a,b-unsaturated ketone moiety

Fig. 3 IC50 values of four

derivatives for three cancer cell
lines **P \ 0.01, compared U2
for three cells

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Fig. 4 Photograph of A549 cells treated with O2 or U2 for 48 h at concentration of 20 lM. Original magnification 9100

Fig. 5 Result from the Hoechst 33258 dye test (9200). A549 cells were treated with O2 or U2 for 48 h at concentration of 20 lM. Cells were
fixed in 4 % paraformaldehyde and then stained with Hoechst 33258 for fluorescence microscope observation

in ring A to its anti-tumor activity. Further, quantitative

analysis of the inhibitory effect of the four derivatives on
three cancer cell lines by calculating IC50 values (Fig. 3)
concluded that U2 and O2 were better inhibitors than G2
and B2.

Apoptosis study by morphological observation

and DNA laddering

A549 cells were treated with 20 lM of compounds U2 and

O2 for 48 h and observed for morphological changes by
fluorescence microscope. Comparing to the untreated cells,
A549 cells treated with U2 or O2 demonstrated the signs of
apoptosis including shrinkage, lowered ability of adhesion
to the plates, and increased cell debris in the medium
(Fig. 4). In addition, the effects of two compounds on
nuclear chromatin condensation of A549 cells were Fig. 6 Induction of apoptosis by DNA laddering. A549 cells were
assayed by Hoechst 33258 dye (Fig. 5). As expected, the treated with 20 lM U2 or O2 for 24 h. DNA was collected, analyzed
cells showed a significant morphological change in nuclear on a agarose gel, and photographed by gel imaging system
chromatin. The nucleus of the untreated cells was less
bright with more homogeneous color than the U2- and O2-
treated cells suggesting chromatin condensation, which is
the hallmark of apoptosis. and O2 (Fig. 6). The results of morphological observation
The induced apoptosis by U2 and O2 compounds on and DNA laddering demonstrated that U2 and O2 were
A549 cells were tested by DNA laddering. Result revealed able to induce apoptosis of A549 cells, indicating DNA
increased DNA fragmentation in the cells treated with U2 damaging nature of these drugs.

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Conclusion
Four novel derivatives were obtained by introducing
2-methylene-3-oxo group as exocyclic a,b-unsaturated
ketone moiety in ring A of UA, OA, GA, and BA,
respectively. Compared with the parent compounds,
derivatives exhibited higher anti-bacterial activities to
Gram-positive bacteria and increased cytotoxic/DNA
damaging activity to three cancer cell lines in vitro. These
results indicated that introduction of a moiety ring to
pentacyclic triterpenes could potentially improve their anti-
bacterial and anti-tumor activities.
Experimental
General
Melting points were uncorrected (Leica hot stage micro-
scope), NMR spectra were recorded on the Varian Nova
spectrometers (USA) using Me4Si as internal standard, IR
spectra were recorded using potassium bromide disks on a
Perkin-Elmer FT-IR spectrometer (USA), and MS spectra
were carried out on a VG Auto Spec-3000 spectrometer
(VG, Manchester, England). TLC was performed on silica
gel (Merck 5554, detection by UV absorption).
General procedure for oxidating C-3 hydroxy group
to ketone in the ring A of four acids
According to the literature (Qian et al., 2010), the parent
compound (2.00 mmol) was dissolved in 100 mL acetone,
stirred for 15 min at 0 °C, and then Jones reagent (0.78 mL,
2.1 mmol) was added dropwise to the mixture. The mixture
was continuously stirred at 0 °C until the starting material
was not observed by thin layer chromatography (TLC)
detection. To the final, the mixture added 500 mL colder
water; precipitation was then carried out by vacuum filtra-
tion and dried. The crude products were purified by silica gel
chromatography (petroleum ether-acetone).
3-Oxo-urs-12-en-28-oic acid (U1)
UA was reacted by general procedure to obtain compound
U1 as colorless crystals. Yield 95.0 %; mp 280–282 °C
(Kashiwada et al., 2000, mp: 282–285 °C); IR (KBr) tmax:
2958 (CH3), 2922 (CH2), 1730 (C=O, C28), 1704 (C=O, C3),
1455 (CH), 744 (C=CH) cm-1; 1H NMR (400 MHz,
CDCl3): d ppm 5.25 (s, 1H, C12-H), 2.58 (m, 1H, C2a-H),
2.39 (m, 1H, C2b-H), 2.20 (d, 1H, J = 11.2 Hz, C18-H),
1.08, 1.08, 1.05, 1.02, 0.82 (each s, 15H, 5 9 CH3), 0.96 (d,
3H, J = 4.0 Hz, C30-H), 0.86 (d, 3H, J = 6.0 Hz, C29-H);
13
C NMR (100 MHz, CDCl3): d ppm 217.8 (C3), 183.8
4637
(C28), 138.0 (C13), 125.5 (C12), 55.2 (C5), 52.5 (C18), 47.9
(C17), 47.3 (C8), 46.7 (C9), 42.0 (C14), 39.4 (C4), 39.2 (C1),
39.0 (C20), 38.7 (C19), 36.6 (C22), 36.6 (C10), 34.1 (C7), 32.4
(C21), 30.5 (C15), 27.9 (C12), 26.5 (C23), 24.0 (C16), 23.5
(C27), 23.3 (C11), 21.4 (C30), 31.1 (C29), 19.5 (C6), 16.9
(C25), 16.9 (C26), 15 (C24); MS m/z (%): 454 (M?, 2.86), 248
(100.00), 203 (90.00); Anal. Calc. for C30H46O3 (%): C
79.25, H 10.20, O 10.56. Found: C 79.28, H 9.88, O 10.29.
3-Oxo-olean-12-en-28-oic acid (O1)
OA was reacted by general procedure to obtain compound O1
as colorless crystals. Yield 96.3 %; mp 169–171 °C (Shirane
et al., 1996, mp: 170–173 °C); IR (KBr) tmax: 2960 (CH3),
2923 (CH2), 1735 (C=O, C28), 1700 (C=O, C3), 1443 (CH),
738 (C=CH) cm-1; 1H NMR (400 MHz, CDCl3): d ppm 5.30
(s, 1H, C12-H), 2.81 (m, 1H, C18-H), 2.56 (m, 1H, C2a-H), 2.38
(m, 1H, C2b-H), 1.14, 1.07, 1.04, 1.03, 0.98, 0.93, 0.86 (each s,
21H, 7 9 CH3); 13C NMR (100 MHz, CDCl3): d ppm 217.8
(C3), 184.4 (C28), 143.6 (C13), 122.3 (C12), 55.2 (C5), 47.4
(C17), 46.8 (C19), 46.5 (C14), 45.7 (C8), 41.6 (C9), 40.9 (C18),
39.2 (C4), 39.0 (C10), 36.7 (C1), 34.1 (C22), 33.7 (C21), 33.0
(C29), 32.3 (C7), 32.1 (C23), 30.6 (C20), 27.6 (C15), 26.4 (C2),
25.8 (C27), 23.5 (C30), 23.4 (C16), 22.8 (C11), 21.4 (C26), 19.5
(C6), 16.9 (C25), 15.0 (C24); MS m/z (%): 454 (M?, 14.28), 203
(100.00), 248 (99.09); Anal. Calc. for C30H46O3 (%): C 79.25,
H 10.20, O 10.56. Found: C 78.13, H 9.56, O 9.33.
3, 11-Dioxo-18b-olean-12-en-30-oic acid (G1)
GA was reacted by general procedure to obtain compound G1
as colorless crystals. Yield 93.6 %; mp 306–308 °C (Rao
et al., 2008, mp: 308–311 °C); IR (KBr) tmax: 2964 (CH3),
1727 (C=O, C30), 1682 (C=O, C3), 1645 (C=O, C11),
1455(CH), 780 (C=CH) cm-1; 1H NMR (400 MHz, CDCl3):
ppm 5.76 (s, 1H, C12-H), 2.96 (m, 1H, C2a-H), 2.63 (m, 1H,
C2b-H), 2.45 (s, 1H, C9-H), 2.37 (brd, 1H, J = 15.6 Hz, C18-
H), 1.38, 1.27, 1.23, 1.17, 1.10, 1.07, 0.86 (each s, 21H,
7 9 CH3); 13C NMR (100 MHz, CDCl3): d ppm 217.4 (C3),
199.7 (C11), 181.9 (C30), 170.0 (C13), 128.3 (C12), 60.9 (C9),
55.3 (C5), 48.1 (C18), 47.7 (C4), 45.2 (C14), 43.7 (C20), 43.2
(C8), 40.8 (C19), 39.6 (C1), 37.6 (C22), 36.6 (C10), 34.1 (C7),
32.0 (C21), 31.8 (C17), 30.9 (C29), 30.8 (C2), 28.5 (C28), 28.4
(C23), 26.4 (C16), 26.3 (C15), 23.2 (C27), 21.3 (C26), 18.7 (C6),
18.4 (C25), 15.6 (C24); MS m/z (%): 468 (M?, 15.71), 135
(100.00); Anal. Calc. for C30H44O4 (%): C 76.88, H 9.46, O
13.66. Found: C 76.98, H 9.23, O 12.93.
3-Oxo-lup-20(29)-en-28-oic acid (B1)
BA was reacted by general procedure to obtain compound
B1 as colorless crystals. Yield 95.3 %; mp 252–254 °C
(Urban et al., 2005, mp: 250–254 °C); IR (KBr) tmax: 2943
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(CH3), 1725 (C=O, C30), 1689 (C=O, C3), 884
(C=CH2) cm-1; 1H NMR (400 MHz, CDCl3): d ppm 4.74 (s,
1H, C29a-H), 4.62 (s, 1H, C29b-H), 3.02 (m, 1H, C19-H), 2.45
(m, 2H, C2-H), 1.69 (s, 3H, methyl, C30), 1.07, 1.02, 0.99,
0.93 (each s, 15H, 5 9 CH3); 13C NMR (100 MHz, CDCl3):
d ppm 218.0 (C3), 181.2 (C28), 149.6 (C20), 102.2 (C29), 59.3
(C17), 54.9 (C5), 49.8 (C9), 48.0 (C18), 47.5 (C19), 47.3 (C4),
42.6 (C14), 40.8 (C8), 39.6 (C1), 38.7 (C13), 36.9 (C10), 34.1
(C2), 33.6 (C7), 33.1 (C16), 29.8 (C15), 29.1 (C21), 28.8 (C22),
26.6 (C23), 25.5 (C12), 21.3 (C11), 21.0 (C24), 19.6 (C6), 19.0
(C29), 15.9 (C26), 15.7 (C25), 14.2 (C27); MS m/z (%): 454
(M?, 52.85), 189 (100.00), 248 (97.14); Anal. Calc. for
C30H46O3 (%): C 79.25, H 10.20, O 10.56. Found: C 79.34, H
10.48, O 10.47.
General procedure for production of 2-methylene
of four acids (method A) (Roman, 2013)
3-Keto compound (1.00 mmol) was dissolved in 100 mL of
anhydrous DMF, to which dimethylamine hydrochloride
(88.8 mg, 1.10 mmol), paraformaldehyde (39.39 mg,
1.3 mmol), and acetic acid (0.3 mL) were added. The reaction
mixture was stirred at 100 °C for 1.5 h. When the mixture
became cool, saturated aqueous NaHCO3 was added to it to
adjust the pH to 8, and then the mixture was stirred at 100 °C
for 1 h until the starting material was not observed by TLC.
Mixture was cooled and poured into brine and extracted with
EtOAc (3 9 200 mL). The organic phase was washed with
saturated aqueous NaHCO3 and brine (3 9 100 mL), dried
over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure. The crude products were then chromato-
graphed on silica (petroleum ether-acetone) to obtain the pure
compound.
General procedure for production of 2-methylene
of four acids (method B) (Limberakis et al., 2012)
3-Keto compound (1.00 mmol) was dissolved in 100 mL
methanol, to which KOH (280 mg, 5.00 mmol) and
formaldehyde (1 mL, 10.00 mmol) were added. The reac-
tion solution was refluxed for 3 h to complete reaction by
TLC. The cooled mixture was acidified with 2 M HCl to
pH-7 and extracted with EtOAc (3 9 200 mL). The
organic phase was washed with saturated aqueous NaHCO3
and brine (3 9 100 mL), dried over anhydrous Na2SO4,
filtered, and concentrated. The crude products were then
chromatographed on silica (petroleum ether-acetone) to
obtain pure compound.
3-Oxo-urs-2(31),12-dien-28-oic acid (U2)
U1 was reacted by method A to obtain compound U2 as a
white amorphous powder. Yield 78.4 %; mp 115–118 °C;
123
Med Chem Res (2014) 23:4631–4641
IR (KBr) tmax: 2926 (CH3), 1694 (C=O, C3), 895
(=CH) cm-1; 1H NMR (400 MHz, CDCl3): d ppm 6.00 (s,
1H, C31-H), 5.28 (brs, 1H, C12-H), 5.16 (s, 1H, C31-H),
2.64 (d, 1H, J = 4.0 Hz, C18-H), 1.11, 1.03, 1.00, 0.98,
0.86 (each s, 15H, 5 9 CH3), 0.95 (d, 3H, J = 6.4 Hz, C30-
H), 0.87 (d, 3H, J = 6.8 Hz, C29-H); 13C NMR (100 MHz,
CDCl3): d ppm 207.4 (C3), 183.8 (C28), 141.8 (C2), 137.9
(C13), 125.5 (C12), 123.7 (C31), 54.0 (C5), 52.6 (C18), 48.0
(C4), 46.9 (C1), 46.6 (C17), 45.1 (C9), 42.1 (C14), 39.3 (C8),
39.0 (C19), 38.7 (C20), 36.7 (C10), 36.4 (C22), 33.0 (C7),
30.5 (C21), 29.3 (C23), 28.2 (C15), 25.7 (C27), 24.0 (C16),
23.5 (C6), 23.4 (C36), 22.6 (C29), 21.1 (C30), 20.0 (C11),
16.7 (C25), 15.1 (C24); MS m/z (%): 466 (M?, 5.71), 248
(100.00); Anal. Calc. for C31H46O3 (%): C 79.78, H 9.93, O
10.28. Found: C 79.57, H 9.90, O 10.38.
3-Oxo-olean-2(31),12-dien-28-oic acid (O2)
O1 was reacted by method A to obtain compound O2 as a
white amorphous powder. Yield 80.1 %; mp 117–119 °C;
IR (KBr) tmax: 2926 (CH3), 1694 (C=O, C3), 1461
(CH2) cm-1; 1H NMR (400 MHz, CDCl3): d ppm 5.99 (s,
1H, C31-H), 5.32 (s, 1H, C12-H), 5.16 (s, 1H, C31-H), 1.16,
1.13, 1.04, 0.93, 0.92, 0.91, 0.75 (each s, 21H, 7 9 CH3);
13
C NMR (100 MHz, CDCl3): d ppm 207.4 (C3), 183.7
(C28), 143.6 (C13), 141.8 (C2), 123.7 (C31), 122.2 (C12),
54.0 (C5), 46.6 (C19), 46.5 (C17), 45.8 (C10), 45.7 (C9), 45.3
(C1), 41.7 (C14), 41.0 (C18), 39.1 (C4), 46.7 (C8), 33.7
(C22), 33.0 (C29), 32.3 (C21), 31.9 (C7), 30.6 (C20), 28.1
(C23), 27.6 (C15), 25.6 (C27), 23.5 (C30), 23.3 (C11), 22.8
(C16), 22.6 (C26), 20.0 (C6), 16.7 (C24), 14.8 (C25); MS m/z
(%): 466 (M?, 4.28), 203 (100.00), 248 (97.14); Anal.
Calc. for C31H46O3 (%): C 79.78, H 9.93, O 10.28. Found:
C 79.55, H 10.02, O 10.15.
3,11-Dioxo-olean-2(31),12-dien-30-oic acid (G2)
G1 was reacted by method A to obtain compound G2 as a
white amorphous powder. Yield 85.6 %; mp 323–325 °C;
IR (KBr) tmax: 2927 (CH3), 1703 (C=O, C30), 1657 (C=O,
C3) cm-1; 1H NMR (400 MHz, CDCl3): d ppm 5.76 (s,
1H, C31-H), 5.48 (s, 1H, C12-H), 5.05 (s, 1H, C31-H), 3.12
(s, 1H, C9-H), 1.19, 1.02, 0.98, 0.92, 0.92, 0.87, 0.61 (each
s, 21H, 7 9 CH3); 13C NMR (100 MHz, CDCl3): d ppm
208.5 (C3), 200.8 (C11), 179.9 (C30), 172.2 (C13), 142.4
(C2), 128.4 (C12), 124.7 (C31), 59.6 (C9), 54.6 (C5), 48.8
(C18), 47.4 (C1), 46.5 (C4), 45.5 (C14), 44.1 (C20), 43.8
(C8), 41.6 (C19), 38.1 (C22), 37.0 (C10), 32.2 (C17), 32.1
(C21), 31.3 (C7), 28.8 (C29), 28.6 (C28), 28.4 (C23), 26.8
(C16), 26.6 (C15), 23.5 (C27), 22.9 (C25), 19.7 (C6), 18.4
(C26), 15.3 (C24); MS m/z (%): 480 (M?, 38.57), 135 (100);
Anal. Calc. for C31H44O4 (%): C 77.46, H 9.23, O 13.31.
Found: C 77.83, H 8.97, O 13.32.
Med Chem Res (2014) 23:4631–4641
3-Oxo-lup-20(29), 2(31)-dien-28-oic acid (B2)
B1 was reacted by method A to obtain compound B2 as a
white amorphous powder. Yield 82.0 %; mp 121–124 °C;
IR (KBr) tmax: 2934 (CH3), 1723 (C=O, C30), 1695 (C=O,
C3) cm-1; 1H NMR (400 MHz, CDCl3): d ppm 7.26 (s,
1H, C31-H), 5.97 (s, 1H, C31-H), 4.74 (s, 1H, C29-H), 4.61
(s, 1H, C31-H), 1.70 (s, 3H, methyl, C30), 1.11, 1.04, 1.00,
0.98, 0.84 (each s, 15 H, 5 9 CH3); 13C NMR (100 MHz,
CDCl3): d ppm 207.7 (C3), 150.2 (C20), 142.2 (C2), 123.7
(C31), 109.7 (C30), 56.3 (C17), 53.9 (C5), 50.8 (C9), 49.0
(C19), 48.3 (C4), 47.0 (C1), 46.8 (C18), 45.8 (C14), 40.8
(C8), 48.4 (C13), 37.0 (C22), 36.8 (C10), 33.3 (C7), 32.0
(C16), 30.4 (C15), 29.6 (C21), 28.0 (C23), 25.4 (C12), 22.3
(C11), 21.3 (C6), 20.0 (C29), 19.3 (C26), 15.6 (C25), 15.4
(C24), 14.5 (C27); MS m/z (%): 466 (M?, 48.57), 55 (100);
Anal. Calc. for C31H46O3 (%): C 79.78, H 9.93, O 10.28.
Found: C 79.87, H 9.68, O 10.38.
Anti-bacterial and anti-tumor activities assay
Reagents, bacterial strains, and cell lines
Luria Broth was purchased from Oxoid, Basingstoke, UK.
Ampicillin, streptomycin, MTT (3-[4,5-dimethylthiazol-
2yl-diphenyl tetrazolium bromide), and Hoechst 33258
were purchased from Sigma, USA. Other reagents were of
analytical reagent quality.
Escherichia coli ATCC 25922, Staphylococcus aureus
ATCC 25923, and Bacillus subtilis ATCC 6051 were
obtained from the National Institute for the Control of
Pharmaceutical and Biological Products (Beijing, China).
The cell lines K562, A549, and MCF-7 were purchased
from the Type Culture Collection (Shanghai, China).
Cell culture
Cell cultures were maintained as monolayer in RPMI 1640
(Hyclone, Germany) supplemented with 10 % heat inacti-
vated research grade fetal bovine serum (Hyclone, Ger-
many) and penicillin/streptomycin (Sigma, USA) at 37 °C
in a humidified atmosphere of 5 % CO2.
Anti-bacterial activity assay
Kirby-Bauer test (Bastian et al., 2012; Djoukeng et al.,
2005)
Paper disks (6 mm diameter) were wet with 30 lL of
solution containing the compounds at concentration of
100 lM, then dried in a desiccator overnight, and used for
the next day. Bacteria were seeded in Luria Broth agar
plates at concentration of 0.5 McFarland with sterile
4639
cotton. The plates were then dried for 30 min. Six paper
disks loaded with compounds were then placed on each
plate, one paper kept in center for control. Plates were
incubated for 24 h at 37 °C and subsequently the diameter
of the inhibition growth zone was measured. Tests were
performed in triplicate.
Turbidimetry test (Kalemba and Kunicka, 2003)
Another method for assay anti-bacterial activities in vitro
was turbidimetry. Tested compounds were prepared at
0.1 M in DMSO and diluted with Luria Broth, and the
concentration of DMSO never exceeded 1 % (v/v). The
bacteria were seeded in 96-well microculture plates
(90 lL/well) at concentration of 1 9 105 CFU/mL of
Luria Broth. The compounds were then added to obtain the
final concentration at 100 lM. Medium containing vehicle
without microorganisms in wells was used as the negative
controls. Medium containing vehicle and microorganisms
in wells was used as positive controls. The microplates
were vigorously shaken on a vibrating platform at 37°Cfor
20 h. The plates were measured in an ELISA plate reader
at 570 nm for the absorbance to determine the growth
inhibitory activity as described (Mageswari and Subrama-
nian, 2012). Results were expressed by the means of three
independent experiments.
ODcontrol  ODsample
Inhibitory activity ¼  100 %;
ODcontrol
where ODcontrol was the optical density of bacteria sus-
pension added the vehicle and ODsample was the optical
density of bacteria suspension treated with tested
compounds.
The MIC values were measured by broth microdilution
method in 96-well microculture plates (Pereira et al.,
2011). The bacteria were seeded in 96-well microculture
plates at concentration of 1 9 105 CFU/mL in Luria Broth;
a series of different concentrations of tested compounds
were then added and culture incubated at 37 °C for 20 h.
The MIC value was the lowest concentration of compound
whose absorbance by ELISA was parallel to the negative
control in three independent experiments.
Cytotoxicity assay
Cytotoxicity assay was performed by MTT assay (Meng
et al., 2009). K562, A549, and MCF-7 cells were seeded in
96-well microculture plates at a density of 4 9 103 to
8 9 103 cells/well and allowed to adhere for 24 h. Cells
were then exposed to different concentrations of the com-
pounds for 48 h. Cells were detected under inverted
microscope with a CCD camera (Nikon, Japan). Later,
20 lL of MTT solution (5 mg/mL) was added to each well
123
4640
and incubated at 37 °C for an additional 4 h. The medium
was then removed, and 200 lL Tris-DMSO solution was
added. Shaking continued lightly to dissolve the dark blue
formazan crystals, and the absorbance was measured in an
ELISA plate reader at 570 nm.
Apoptosis test-Hoechst 33258 dye
Apoptosis was analyzed by Hoechst 33258 dye (Shi et al.,
2006). A549 cells were seeded on cover slips in a 6-well
plate at 5 9 105 cells/well and incubated for 24 h. The
medium was then removed, and 20 lM of U2 or O2 com-
pounds were added to the medium and incubated for 48 h.
Supernatant was discarded; cells-on cover glasses were
carefully washed with phosphate buffer solution (PBS) and
fixed in 4 % paraformaldehyde solution for 20 min. After
washing twice with PBS, cells were stained with 20 lg/mL
Hoechst 33258 for 10 min, washed again twice with PBS
and analyzed with fluorescence microscope (Nikon, Japan)
using 350 nm excitation and 460 nm emission.
Apoptosis assay-DNA laddering
Another determination of apoptosis was DNA laddering
(Kommera et al., 2011). A549 cells were incubated with
20 lM of U2 or O2 compounds for 72 h, collected by
centrifugation (1,600 rpm, 5 min) and washed twice with
cold PBS. Cells were then resuspended in 0.5 mL of
extraction buffer (100 mM NaCl, 10 mM Tris–HCl pH 8.0,
25 mM EDTA pH 8.0, 0.1 mg/mL Proteinase K) and
incubated at 50 °C overnight by mixing from time to time.
After cell lysate cooled in room temperature, DNA was
extracted with an equal volume of phenol saturated/
chloroform/isoamyl alcohol (25:24:1) and then with a
combination of chloroform/isoamyl alcohol (24:1). After
centrifugation (6,600 rpm, 20 min), 0.1 equal sodium ace-
tate solution (3 M, pH 5.2) and 2 equal cold absolute ethyl
alcohol were added. The solution was then kept at -20 °C
for 30 min and centrifuged at 12,000 rpm for 20 min.
Precipitate was washed with 1 mL ethyl alcohol (70 %) for
two times and dried under room temperature for 15 min.
Precipitate was suspended in 50 lL TE solution (5 mL 1 M
Tris–HCl buffer pH 8.0, 1 mL 0.5 M EDTA pH 8.0, added
dd H2O to 500 mL), and DNA was analyzed on a 1.2 %
agarose gel and imaged using gel imaging system.
Statistical analysis
The IC50 value was calculated from the semi-logarithmic
dose-response curves. The data were analyzed using SPSS
18.0 and reported as mean ± SD of the number of exper-
iments indicated. For all measurements, one-way ANOVA
followed by a Student’s t test was used to assess the
123
Med Chem Res (2014) 23:4631–4641
statistical significance of difference between each group.
LSD method was used to assess the statistical significance
of difference between every two groups. A statistically
significant difference was considered at the level of
P \ 0.05. Data were presented as the mean ± SEM of
three assays.
Acknowledgments The authors are deeply thankful to the staff
members of the analytical center for spectral measurement and
activity test centre for cytotoxicity assay of the laboratory of chem-
istry for natural products of Guizhou Province and Chinese academy
of sciences. The authors also thank Dr. Yaacov Ben-David for con-
structive advice and revision of the manuscript. The work was sup-
ported by the National Basic Research 973 Program of China (No.
2012CB722601) and the Major Special Project in Guizhou Province
(No. 2013-6006).
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