Triterpenoids from the stem bark of Vitellaria paradoxa (Sapotaceae) and derived esters exhibit cytotoxicity against a breast cancer cell line

Article abstract of DOI:10.1007/s00044-017-2059-7

A study of the chemical constituents of the stem bark of Vitellaria paradoxa (Sapotaceae) has resulted in the isolation and characterization of a new ursane-type triterpenoid, 2β,3β,19α-trihydroxyurs-12-en-28-oic acid (1), together with seven known compounds: betulinic acid (2), 1α,2β,3β,19α-tretrahydroxyurs-12-en-28-oic acid (3), β-sitosterol (7), sigmasterol (8), (-)-epicatechin (9), (+)-catechin (10) and quercetin (11). The structure of the novel, ursane-type acid 1 was elucidated on the basis of detailed spectroscopic analysis including IR, HRMS (ESI), 1D and 2D NMR and a comparison to previously described, related natural products. Preliminary cytotoxicity assays against the MDA-MB-231 breast cancer cell line indicated that betulinic acid 2 and its corresponding methyl ester 5 were the most active compounds tested with IC₅₀ values of 19.9 μM (17.2–23.1 μM, 95% CI) and 32.9 μM (24.9–43.4 μM, 95% CI), respectively. Esterification of acids 1–3 afforded the corresponding methyl esters 4–6 for additional structure-activity relationship (SAR) analysis. In general, the activity against the MDA-MB-231 breast cancer cell line increased upon esterification of the triterpenoids screened.

Full text of DOI:10.1007/s00044-017-2059-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2059-7
ORIGINAL RESEARCH
Triterpenoids from the stem bark of Vitellaria paradoxa
(Sapotaceae) and derived esters exhibit cytotoxicity against a
breast cancer cell line
Kenneth O. Eyong1,2 Guerisson Bairy2 Anna A. Eno3 Joseph Taube4
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Kenneth G. Hull1 Gabriel N. Folefoc2 Harquin S. Foyet5 Daniel Romo1
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Received: 13 February 2017 / Accepted: 30 August 2017
© Springer Science+Business Media, LLC 2017
Abstract A study of the chemical constituents of the stem
bark of Vitellaria paradoxa (Sapotaceae) has resulted in the
isolation and characterization of a new ursane-type tri-
terpenoid, 2β,3β,19α-trihydroxyurs-12-en-28-oic acid (1),
together with seven known compounds: betulinic acid
(2), 1α,2β,3β,19α-tretrahydroxyurs-12-en-28-oic acid (3),
β-sitosterol (7), sigmasterol (8), (-)-epicatechin (9),
(+)-catechin (10) and quercetin (11). The structure of the
novel, ursane-type acid 1 was elucidated on the basis of
detailed spectroscopic analysis including IR, HRMS (ESI),
1D and 2D NMR and a comparison to previously described,
related natural products. Preliminary cytotoxicity assays
against the MDA-MB-231 breast cancer cell line indicated
that betulinic acid 2 and its corresponding methyl ester
5 were the most active compounds tested with IC50 values
of 19.9 μM (17.2–23.1 μM, 95% CI) and 32.9 μM
(24.9–43.4 μM, 95% CI), respectively. Esterification of
acids 1–3 afforded the corresponding methyl esters 4–6 for
additional structure-activity relationship (SAR) analysis. In
general, the activity against the MDA-MB-231 breast can-
cer cell line increased upon esterification of the triterpenoids
screened.
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Keywords plant-derived natural products
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ursane family isolation structure elucidation
Apoptosis/necrosis assay
Introduction
Cancer refers to any of a group of more than 100 distinct
diseases that are characterized by the uncontrolled pro-
liferation of abnormal cells in the body. The genotypic
and phenotypic diversity between tumors originating
from distinct organs and through distinct mutations
necessitate the continued development of novel cytotoxic
molecules as well as the characterization of natural pro-
ducts with cytotoxic properties. Despite a diminished
focus on natural products recently, compounds originally
isolated from natural sources make a significant con-
tribution to the landscape of pharmaceutical agents
(Harvey et al. 2015, Gennari et al. 2007) approved by the
US Food and Drug Administration (FDA). Furthermore,
the World Health Organization estimates that 25% of
pharmaceutical drugs are derived from plants known for
efficacy among practitioners of traditional medicine
(WHO 2003).
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-017-2059-7) contains supplementary
material, which is available to authorized users.
* Kenneth O. Eyong
eyongkk@yahoo.com
1
Department of Chemistry and Biochemistry and the CPRIT
Synthesis and Drug-Lead Discovery Laboratory, Baylor
University, Waco, Texas 76798, USA
2
Department of Organic Chemistry, Faculty of Science, University
of Yaounde I, PO Box 812, Yaounde, Cameroon
3
Department of Microbiology, Faculty of Science, University of
Yaounde I, PO Box 812, Yaounde, Cameroon
4
Department of Biology and Institute for Biomedical Studies,
Baylor University, Waco, Texas 76798, USA
5
In recent years, the use of natural products for drug
discovery has declined in favor of combinational methods
Department of Biological Sciences, Faculty of Science, University
of Maroua, PO Box 46, Maroua, Cameroon

Med Chem Res
Fig. 1 Triterpenoid acids from V. paradoxa and their methyl ester derivatives
which can rapidly generate large libraries of potential lead
compounds. In their perspective, Paterson and Anderson
(Paterson and Anderson 2005) suggest that it may be time to
revisit the prevailing dogma and consider ways in which
natural products may continue to inspire the development of
new drugs. One approach that we have applied in this report
is to use bioactive natural products as a starting point for the
design and synthesis of targeted therapeutic agents with
specific structural modifications.
Results and discussion
Isolation and characterization
A total of 3.2 kg of powdered stem bark of V. paradoxa was
extracted by sonication, successively with hexanes, ethyl
acetate, methanol and water. The MeOH extract was sub-
jected to column chromatography eluting successively with
hexanes, hexanes-EtOAc, EtOAc, EtOAc-MeOH and
MeOH. Three triterpenoids (1–3, Fig. 1) were obtained
together with five non-triterpenoids (7–11, Fig. 3). Ester-
ification of acids 1–3 afforded their corresponding methyl
esters 4–6 (Fig. 1).
The diversity of flora in West Africa represents a pro-
mising source for novel anti-cancer agents. Vitellaria
paradoxa C. F. Gaertn (Sheanut tree) has long been used in
traditional medicine for the treatment of various ailments,
including inflammation, fever, skin irritations, dermatitis,
sunburn, rheumatism, diarrhea, stomach ache and ulcers
(Orwa et al. 2009, Moore 2008, Foyet et al. 2015). More-
over, according to an ethno-botanical survey, the bark of the
trunk and stems of V. paradoxa has been used historically
for the treatment of cancer (Jiofack et al. 2010). Following
this survey, a preliminary antiproliferation screen of the
methanol extract from V. paradoxa produced very low GI50
values (concentration of the extract causing 50% growth
inhibition of the cells) compared with four cancer cell lines
(NCI-H460, MCF7, PC3 and HeLa) using a sulforhodamine
B (SRB) assay (Tagne et al. 2014). The results from these
preliminary studies compelled us to run more comprehen-
sive experiments to isolate and characterize the bioactive
compounds responsible for the observed cytotoxic effects.
Herein, we report the isolation and structure elucidation
of a new ursane-type triterpenoid, 2β,3β,19α-trihydroxyurs-
12-en-28-oic acid (1) from the air-dried stem bark of V.
paradoxa along with seven known compounds. Triterpe-
noids 1–3 together with their corresponding derived methyl
esters 4–6, were evaluated for cytotoxic activity against the
MDA-MB-231 breast cancer cell line.
Acid 1 was obtained as a white powder from silica gel
column chromatography eluting with 55% EtOAc/hexanes.
It is soluble in MeOH, stains on TLC with iodine or H2SO4
and has a melting point of 260–262 °C. The molecular
formula was determined to be C30H48O5 on the basis of
HRMS (ESI), (m/z 489.1668 [M + H]+) indicating seven
degrees of unsaturation. The IR spectrum of acid 1 con-
firmed the presence of a carboxylic acid group (carbonyl:
1687 cm−1; broad O–H stretch; 2900–3400). Acid 1 dis-
played an optical rotation of ½αŠ26 + 25.5o (c 0.22, THF).
D
1
The H NMR spectrum (600 MHz, MeOH-d4) of com-
pound 1 exhibited signals indicative of seven methyl groups
at δH 1.37 (3H, s), 1.22 (3H, s), 1.04 (3H, s), 1.01 (3H, s),
0.96 (3H, d, 12 Hz), 0.89 (3H, s), and 0.81(3H, s), twenty-
four methylene and methine groups, two of which are
attached to oxygen atoms at δH 3.97 (1H, dt, 6.0, 12.0 Hz)
and 3.35(1H, d, 6.0 Hz), and one olefinic proton at δH 5.32
(1H, t, 6.0 Hz) (Table 1). The 13C NMR spectrum (150
MHz, MeOH-d4) exhibited 30 carbon signals that were
assigned by a DEPT experiment as seven methyl (δC 27.6,
25.7, 23.5, 21.1, 16.1, 15.5, 15.2), eight methylene (δC 44.1,
37.6, 32.7, 28.2, 26.0, 25.2, 23.3, 17.9), seven methine,

Med Chem Res
Table 1 1H and 13C NMR Data (600 and 150 MHz) of compounds 1–6 (δH, CDCl3, MeOH-d4, J in Hz)
C# 1
1H
2
3
4
5
6
13C 1H
13C 1H
13C 1H
13C 1H
13C 1H
13C
1
1.55(1H, m)
1.35(1H, m)
41.1 0.88(1H, m)
1.55(1H, m)
38.7 3.45(1H, d,
12.0)
81.4 1.55(1H, m)
1.32(1H, m)
41.1 0.88(1H, m)
1.65(1H, m)
38.7 3.48 (1H, d, 8.0)
79.8
2
3
3.95 (1H, td, 6.0,
12.0Hz)
65.8 1.75 (2H, m)
27.6 3.63(1H, dd, 3.5,
12.0)
71.7 3.97 (1H, dt, 6.0,
12.0)
65.7 1.75 (2H, m)
27.4 3.68 (1H, dd, 4.0, 70.4
12)
3.35(1H, d, 6.0Hz)
78.7 2.98(1H, m)
77.2 3.39(1H, d, 3.5)
80.9 3.35(1H, d, 6.0)
78.7 3.13(1H, dd, 4.0,
12.0)
79.0 3.42(1H, d, 4.0)
79.3
4
5
6
38.1
39.0
42.5
38.1
38.7
41.1
48.2
18.0
1.23(1H, m)
1.45(1H, m)
1.40 (1H, m)
1.65(1H, m)
1.35(1H,m)
48.7 0.65(1H, m)
17.9 1.45(1H, m)
1.40 (1H, m)
55.4 1.34(1H, br, d)
18.4 1.78(1H, m)
0.94(1H, br)
49.1 1.20(1H, m)
19.6 1.53(1H, m)
1.48 (1H, m)
34.3 1.62(1H, m)
1.32(1H,m)
48.1 0.65(1H, m)
17.9 1.45(1H, m)
1.50 (1H, m)
55.4 1.35(1H, m)
18.3 1.78 (1H, m)
0.94 (1H, m)
7
32.7 1.40(2H, m)
34.4 1.58(1H, m)
1.29(1H, m)
32.6 1.40(2H, m)
34.3 1.58(1H, m)
1.29(1H, m)
32.6
8
38.9
40.7
41.8
41.3
40.7
40.4
48.4
42.9
26.9
9
1.85(1H, m)
46.8 1.33(1H, m)
38.0
50.4 2.00(1H, m)
37.2
49.4 1.89(1H, m)
44.4
46.8 1.25(1H, m)
39.9
50.6 2.05(1H, m)
37.2
10
11 2.00(2H, m)
12 5.32(1H, t, 6.0Hz)
13
23.3 1.42(1H, m)
1.21(1H, m)
128.0 1.00(1H, m)
1.89(1H, m)
20.9 2.55(1H, m)
2.17(1H, dd, 4, 12)
25.6 5.29(1H, m)
28.4 2.00(2H, m)
23.4 1.45(1H, m)
1.25(1H, m)
20.9 2.67(1H, m)
2.22(1H, dd, 4, 12)
25.5 5.32 (1H, t, 4)
130.8 5.32(1H, m)
138.9
128.1 1.00(1H, m)
1.75(1H, m)
129.4
137.4
138.7 2.26(1H, td, 4.0,
12.0)
38.1
138.6 2.13(1H, m)
38.3
14
41.3
42.5
38.9
41.7
42.4
37.4
28.2
15 1.00(1H, m)
1.77(1H, m)
16 1.52 (1H, m)
28.2 1.26(1H, m)
1.85(1H, m)
30.6 1.00 (1H, m)
1.77(1H, m)
32.2 1.51(1H, m)
2.5(1H, m)
29.8 1.00(1H, m)
1.77(1H, m)
28.1 1.35(1H, m)
1.81(1H, m)
30.6 1.00 (1H, m)
1.77(1H, m)
32.2 1.51(1H, m)
2.61(1H, m)
25.9 1.55 (1H, m)
2.19 (1H, m)
26.8 1.52 (1H, m)
2.67 (1H, td, 6.0, 12)
25.8 1.45 (1H, m)
2.18 (1H, m)
25.2
2.62 (1H, td, 6.0,
12 Hz)
17
47.9
55.9
49.0
48.1
56.6
48.1
53.6
72.1
41.6
25.8
18 2.52(1H, s)
19
53.7 1.55(1H, m)
72.2 3.00 (1H, m)
41.7
49.0 2.48(1H, s)
47.1
55.1 2.54(1H, s)
73.6
53.7 1.55(1H, m)
72.1 2.95 (1H, m)
41.7
49.5 2.53(1H, s)
47.0
20 1.35(1H, m)
21 1.35(2H, m)
150.8 1.34(1H, m)
29.7 1.51(1H, m)
1.48(1H, m)
43.2 1.32(1H, m)
27.5 1.65(1H, m)
1.25(1H, s)
150.1 1.34(1H, m)
29.7 1.51(1H, m)
1.48(1H, m)
25.2 1.22(1H, m)
1.45(1H, m)
25.2 1.20(1H, m)
1.40(1H, m)
22 1.65(1H, m)
1.75(1H, m)
37.6 1.45(1H, m)
1.82(1H, m)
36.8 1.72(1H, m)
1.62 (1H, m)
39.2 1.65(1H, m)
1.75(1H, m)
37.4 1.45(1H, m)
1.86(1HH, m)
37.0 1.72(1H, m)
1.62 (1H, m)
37.5
23 1.40(3H, s)
24 0.89 (3H, s)
25 1.02(3H, s)
26 0.81(3H, s)
27 1.21(3H, s)
28
23.5 0.94(3H, s)
21.1 0.77 (3H, s)
15.5 0.65(3H, s)
16.1 0.87(3H, s)
25.7 0.88(3H, s)
180.8
28.6 1.17(3H, s)
16.2 0.86(3H, s)
16.3 1.00 (3H, s)
16.4 0.77(3H, s)
14.9 1.33(3H, s)
177.7
29.3 1.37(3H, s)
22.5 0.89 (3H, s)
13.1 1.01(3H, s)
17.8 0.71(3H, s)
25.1 1.21(3H, s)
182.4
23.5 0.89(3H, s)
21.0 0.75 (3H, s)
15.4 0.68(3H, s)
16.0 0.89(3H, s)
25.6 0.84(3H, s)
179.1
28.0 1.22(3H, s)
15.4 0.90(3H, s)
16.0 1.03 (3H, s)
16.1 0.72(3H, s)
14.7 1.37(3H, s)
176.7
27.7
21.0
11.5
16.2
23.6
179.1
25.6
29 1.01(3H, s)
27.6 4.70(1H, d, 4.0Hz) 110.1 0.97(3H, s)
4.57(1H, d, 4.0 Hz)
27.2 1.01(3H, s)
27.8 4.69(1H, d, 4.0)
4.53(1H, d, 4.0)
15.1 1.61 (3H, s)
50.7 3.60(3H, s)
109.4 0.99(3H, s)
30 0.96 (3H, d, 12)
15.2 1.65 (3H, s)
19.4 0.92(3H, d, 8.0)
16.8 0.96 (3H, d, 12.0)
3.60(3H, s)
19.4 0.95(3H, d, 8.0)
51.3 3.60(3H, s)
15.1
50.7
31

Med Chem Res
Fig. 2 Main HMBC and NOESY correlations of compound 1
including two –CH(OH) (δC 78.7, 65.8), four –CH (δC 53.7,
48.0, 46.8, 41.7) and three sp2-hybridized carbons (δC 180.8
(C=O), 138.7 (C=C), 127.9 (=CH), one tertiary alcohol
(72.2 (C–OH)) and five quaternary carbons (δC 47.9, 41.3,
38.9, 38.1, 38.0) (Table 1). The above NMR data are
characteristic of the triterpenoid class of natural products
(Peng et al. 1998, Liu et al. 2016).
with C-15(δ 28.2), C-17(47.91), C-18(53.7) and C-28
(180.8) support the ring D connectivity. Moreover, the
NOESY correlations between H-18 (δ 2.52) with H-27
(1.21), H-2 (3.95) with H-3 (3.35) and H-5 (1.23) with H-9
(1.85) confirmed the relative configuration of C-2, C-3, C-5,
C-9 and C-18 in Fig. 2. The coupling constant between H-2,
H-3 (3JH-2,H-3 = 6.0 Hz) indicated a syn configuration of the
two hydrogens and thus a syn diol as previously observed in
similar triterpenoids (Eyong et al. 2015). Therefore, com-
pound 1 was determined to be 2β,3β,19α-trihydroxyurs-12-
en-28-oic acid.
Compound 2 was obtained as a white powder from a
silica gel column eluting with 25 → 27.5% EtOAc/hexanes.
It is soluble in methanol, stains on TLC in iodine or H2SO4,
has a melting point of 282–285 °C, and gave LRMS
(APCI+) Calcd. for C30H47O3 (M–H)+ 455.4, found m/z:
455.3. These properties along with the IR and NMR data
(supplemental information) matched those previously
reported for betulinic acid (Peng et al. 1998) (Fig. 1).
Compound 3 was obtained as a white powder from a
silica gel column eluting with 70 → 80% EtOAc/hexanes. It
is soluble in methanol, stains on TLC in iodine or H2SO4,
has a melting point of 262–264 °C and gave LRMS(APCI+)
Calcd. for C30H47O6 (M–H)+ 503.4, found m/z: 503.3.
These properties along with the IR and NMR data (SI)
matched those previously reported to the known
1α,2β,3β,19α-tretrahydroxyurs-12-en-28-oic acid (Eyong
et al. 2015) (Fig. 1).
The presence of a methyl doublet assigned to proton H-
20 (δ 0.96, d, 3H, J = 12 Hz) and a quaternary carbon atom
at δ 72.2, attributed to C-19, are signals typical of an ursane-
type triterpenoid (Eyong et al. 2015, Liu et al. 2016). The
1H and 13C NMR data (Table 1) were assigned using 1H–1H
COZY, HSQC, HSQC–TOCSY, HMBC and NOESY
spectra (Supporting Information). In the 1H–1H COZY,
correlations were observed between H-2 (δ 3.95, td, J = 6.0,
12.0 Hz) and H-3 (δ 3.35, d, J = 6.0 Hz) with a coupling
3
constant JH-2,H-3 = 6.0 Hz suggesting a cis coupling
(Minch 1994). Also observed were correlations between H-
2, H-3, H-1a (δ 1.55, m) and H-1b (δ 1.35, m) from the ring
A nucleus. The ring A correlations were observed in the
HSQC–TOCSY and TOCSY experiments. Additional
1H–1H-COZY correlations were identified between H-5 (δ
1.23, m), H–6a (1.45, m), H-6b (1.40, m), H-7a (1.65, m)
and H-7b (1.35, m) in ring B, H-12 (δ 5.32, t, 6.0 Hz), H-11
(δ 2.00, m, 2H) and H-9 (δ 1.85, m) for ring C, H-16a (δ
2.62 td, 6.0, 12.0 Hz), H-16b (δ 1.52, m), H-15a (δ 1.77, m)
and H-15b (δ1.00, m) for ring D, and correlations between
Me-30 (δ 0.96, d, 12), H-20 (1.35, m), H-21 (1.35, m), H-
22a (1.75, m) and H-22b (1.65, m) for ring E. The
HSQC–TOCSY correlations of H-12 (δ 5.32) with C-11 (δ
23.3), C-9 (46.8) and C-12 (128.0), H-2 (δ 3.97) with C-1(δ
41.1), C-2(65.8) and C-3(78.7), H-16β(δ 2.62) with C-16(δ
25.9) and C-15(δ 28.2) support our assignment. Typical in
the HMBC spectrum are correlations of H-12(δ 5.32) with
C-11(δ 23.3), C-14(41.3), C-9(46.8), and C-18 (53.7) sup-
To obtain derivatives for structure activity relationship
(SAR) studies, esterification was carried out on compounds
1–3 using diazomethane generated in situ from 1-methyl-3-
nitro-1-nitrosoguanidine (MNG) (McKay 1948) to afford
methyl esters 4–6 (Fig. 1).
After esterification of compound 1 with diazomethane,
compound 4 was purified by column chromatography on
silica gel using 50% EtOAc/hexanes (75% yield) and iso-
lated as a white powder. It is soluble in MeOH, stains on
2
porting H-12 connectivity in J or 3J to these carbon atoms
(Fig. 2). The HMBC correlations between H-16β(δ 2.62)

Med Chem Res
Fig. 3 Non-triterpenoids from the methanol extract of V. paradoxa
Fig. 4 Triterpenoids 2, 4 and 5
display cytotoxic activity against
MDA-MB-231 breast cancer
cells. MDA-MB-231 cells were
plated at a density of 2000 cells
per well and treated with the
indicated compounds for 3 days
followed by cell viability assay.
Error bars indicate the standard
deviation of three independent
measurements. The curves
indicate the best fit of a non-
linear curve fitting the equation,
Y = 100/(1 + 10X−log IC50
)
1
TLC in iodine or H2SO4 and had a melting point of
103–105 °C. The IR spectrum showed λmax: 3438, 2928,
2873, 1708, 1456, 1378 cm−1 and LRMS(APCI+) Calcd.
for C31H51O5 (M + H)+ 503.4, found m/z: 503.18. The H
NMR (600 MHz) and 13C NMR spectrum of compound 4 in
MeOH-d4 was similar to compound 1 except for the

Med Chem Res
Table 2 IC50 values calculated
from Fig. 4 for the indicated
compounds upon a 3 day
exposure to MDA-MB-231
breast cancer cells
Compounds
1
2
3
4
5
6
IC50 (μM)
230
19.9
63.5
46.7
32.9
430
95% Confidence intervals (μM) 179–295 17.2–23.1 51.5–78.3 39.8–54.9 24.9–43.4 328–564
appearance of a new methyl singlet at δ 3.60 (3H, s) in the
1H NMR and at δ 50.7 in the 13C NMR (Table 1).
After esterification of acid 2 with diazomethane, methyl
ester 5 was purified by column chromatography on silica gel
using 20% EtOAc/hexanes (95% yield) and isolated as a
white powder. It is soluble in CH2Cl2, stains on TLC with
iodine or H2SO4 and melts at 205–207 °C. An IR spectrum
showed signals at 3437, 2927, 1698, 1452, 1378 cm−1 and a
low resolution LRMS(APCI+) was obtained: Calcd. for
C31H51O3 (M + H)+ 471.4, found m/z: 471.20. The 1H
NMR (600 MHz) and 13C NMR spectra of methyl ester 5 in
CDCl3 were similar to those of (3β)-3-hydroxy-lup-20(29)-
en-28-oic acid (2) except for the appearance of a new
1
methyl singlet at δ 3.60 (3H, s) for the H NMR and at δ
51.3 for the 13C NMR (Table 1).
After esterification of compound 3 with diazomethane,
compound 6 was purified by column chromatography on
silica gel using 65% EtOAc/hexanes (75% yield) and iso-
lated as a white powder. It is soluble in MeOH, stains on
TLC with iodine or H2SO4 and exhibits a m.p. 155–157 °C.
The IR spectrum showed signals at 3435, 2929, 2871, 1700,
1454, 1378 cm−1 and a low resolution MS(APCI+) was
obtained: Calcd. for C31H51O6 (M + H)+ 519.4, found m/z:
Fig. 5 Induction of apoptosis by triterpenoids 2, and 5. Cells treated
with IC50 doses of the indicated compounds for 24 h were stained with
annexin V and propidium iodide (PI). Flow cytometry was performed
and the proportion of cells positive for annexin V is indicated. Note
that the y-axis is shortened to better indicate the differences in the
annexin V-positive populations. The proportion of cells positive for
annexin V is indicated in the legend
1
519.16. The H NMR (600 MHz) and 13C NMR spectra of
relative to 20% in vehicle-treated cells) indicating induction
of apoptosis (Fig. 5).
ester 6 in MeOH-d4 were similar to those of acid 3 except
for the appearance of the new methyl peak at δ 3.60 (3H, s)
1
in the H NMR and at δ 50.7 in the 13C NMR (Table 1).
In addition to the triterpenoids, five non-triterpenes were
obtained and include β-sitosterol (7), sigmasterol (8),
(−)-epicatechin (9), (+)-catechin (10), and quercetin (11)
(See Fig. 3) (Zhang et al. 2014).
Discussion
In the present investigation, the methanolic extract of the
stem bark of V. paradoxa afforded eight compounds
belonging to three main classes of natural products: tri-
terpenoids (1–3), steroids (7–8) and flavonoids (9–11). Of
the triterpenoids isolated, 2β,3β,19α-trihydroxyurs-12-en-
28-oic acid (1) is reported for the first time. Esterification of
the triterpenoids was carried out for SAR studies and a
general trend was determined. The methyl esters 4–6 were
more active than the isolated carboxylic acids 1–3 against
the MDA-MB-231 breast cancer cell line. This trend could
be due to greater cell permeability of the more lipophilic
ester derivatives. The carboxylic acid natural products carry
a negative charge at physiological pH and this could hinder
passive diffusion across cell membranes. Observed IC50
values of these compounds are near the range of clinically
Cytotoxic activity
We next sought to determine the cytotoxic activity of each
of these compounds in the context of breast cancer. For this,
we chose the triple-negative MDA-MB-231 breast cancer
cell line, commonly used for in vitro assays (Lacroix and
Leclercq 2004). The compounds screened exhibited differ-
ential activity, with compounds 2, 4 and 5 showing the
highest cytotoxic activity against MDA-MB-231 cells (Fig.
4 and Table 2). To determine whether the compounds
induced programmed cell death or necrosis, we next per-
formed staining with annexin V and propidium iodide (PI).
Necrosis was not observed with compounds 2 and 5 at the
approximate IC50 doses, however, the proportion of cells
positive for annexin V increased (38 and 45%, respectively,
useful chemotherapeutic agents such as cisplatin (IC50
6.7 μM for MDA MB 231 cells, data not shown). Clinically,
breast cancer can be divided into distinct subtypes that have
=

Med Chem Res
prognostic and therapeutic implications. Most breast cancer
patients have a subtype defined by the expression of the
estrogen receptor (ER), progesterone receptor (PR), or
amplification of HER-2/Neu (Chave et al. 2010, Foulkes
et al. 2010). These markers allow classification of breast
cancer tumors as hormone receptor positive or HER-2/Neu
amplified. However, a fourth type of breast cancer referred
to as triple-negative breast cancer (TNBC) does not express
ER, PR, or have HER-2/Neu amplification. The name of
this cancer type, triple-negative is based on the lack of these
three molecular biomarkers. Given the lack of validated
molecular targets and the poor outcome of patients with
TNBC, there is a clear need for a greater understanding of
TNBC and for the development of novel therapies to treat
this specific cancer type.
investigations into their putative cellular receptors and a
molecular level understanding of their mode of action.
Experimental
Plant material
The stem bark of V. paradoxa was harvested in Garoua-
Cameroon (July, 2012) and identified by Mr. Bamba Jean
Paul (plant taxonomist) of the National School of Fauna,
Garoua, where voucher specimens were deposited (HEFGN
6276). The stem bark was collected, cut into small pieces,
dried at ambient temperature (~25 °C) and powdered.
The transport of drugs across membranes involves one or
more of the following processes: (1) passive diffusion, (2)
filtration, (3) bulk flow, (4) active transport, (5) facilitated
transport, (6) ion-pair transport, (7) endocytosis, and (8)
exocytosis. Passive drug absorption depends on a number of
physicochemical factors, the three most important of which
are lipophilicity, solubility and charge (Alavijeh et al.
2005). The formation of methylesters from the corre-
sponding carboxylic acids has a considerable impact on the
net charge of these compounds since esters are neutral and
acids are negatively charged at physiological pH. We also
observed a considerable decrease in the melting points of
the esters which corresponds with decreased polarity and
intramolecular interactions (e.g. hydrogen bonding). These
results suggest that esterification of the natural products
may have improved their passive cell membrane perme-
ability. Lipophilic substances that do not possess a charge
tend to more readily diffuse passively across the hydro-
phobic cellular membranes. These substances are more
hydrophobic with a tendency to be driven into cellular
membranes from an aqueous environment.
Extraction and isolation
A total of 3.2 kg of powdered stem bark was extracted by
sonication, successively with hexane, ethyl acetate, metha-
nol and water. A portion (400 g) of the MeOH extract was
then subjected to silica gel F60, 40–63 μm column chro-
matography using a hexanes, hexanes-ethyl acetate gra-
dient, ethyl acetate, ethyl acetate-methanol gradient and
finally methanol as eluent. Eight natural products:
2β,3β,19α-trihydroxyurs-12-en-28-oic acid (1, 60 mg),
betulinic acid (2, 700 mg), 1α,2β,3β,19α-tretrahydroxyurs-
12-en-28-oic acid (3, 200 mg), β-sitosterol (7, 100 mg),
sigmasterol (8, 80 mg), (-)-epicatechin (9, 45 mg),
(+)-catechin (10, 75 mg), and quercetin (11, 20 mg) were
isolated.
2β,3β,19α-trihydroxyurs-12-en-28-oic acid (1)
White powder (MeOH) obtained from Hexane-EtOAc 55%;
mp 260–262 °C; [α]25D + 25.5 (c 0.22, THF); IR νmax 2953,
1702, 1449 cm-1. 1H NMR (MeOH-d4, 600 MHz,): δ = 5.32
(1H, t, 6.0 Hz, H-12), 3.95 (1H, td, 6.0, 12.0 Hz, H-2), 3.35
(1H, d, 6.0 Hz, H-3), 2.62 (1H, td, 6.0, J = 12 Hz, H-16a),
2.52(1H, s, H-18), 2.00(2H, m, H-11), 1.85(1H, m, H-9),
1.77(1H, m, H-15a), 1.75(1H, m, H-22a), 1.65(1H, m, H-
22b), 1.65(1H, m, H-7a), 1.55(1H, m, H-1b), 1.52 (1H, m,
H-16b), 1.45(1H, m, H-6a), 1.40 (1H, m, H-6b), 1.40(3H, s,
H-23), 1.35(1H, m, H-1a), 1.35(1H, m, H-7b), 1.35(1H, m,
H-20), 1.35(2H, m, H-21), 1.23(1H, m, H-5), 1.21(3H, s, H-
27), 1.02(3H, s, H-25), 1.01(3H, s, H-29), 1.00(1H, m, H-
15b), 0.96 (3H, d, J = 12.0 Hz, H-30), 0.89 (3H, s, H-24),
0.81(3H, s, H-26) and 13C NMR (MeOH-d4, 150 MHz,): δ
= 180.8(C-28), 138.7(C-13), 128.0 (C-12), 78.7(C-3), 72.2
(C-19), 65.8(C-2), 53.7 (C-18), 48.7(C-5), 47.9 (C-17), 46.8
(C-9), 41.7 (C-20), 41.1(C-1), 41.3 (C-14), 38.9 (C-8), 38.1
(C-4), 38.0 (C-10), 37.6 (C-22), 32.7(C-7), 28.2 (C-15),
27.6 (C-29), 25.9(C-16), 25.7(C-27), 25.2 (C-21), 23.5(C-
23), 23.3 (C-11), 21.1(C-24), 17.9(C-6), 16.1(C-26), 15.5
Conclusion
Phytochemical studies on the methanol extract of the stem
bark of V. paradoxa afforded a new ursane triterpenoid 1,
together with two known ursane (3) and lupeol-type (2)
triterpenoids. Methylation of compounds 1–3 afforded their
methyl esters 4–6. In a general trend, the methyl esters were
more active than the isolated natural triterpenoids 1–3
against the MDA-MB-231 breast cancer cell line. Further
derivatization of these triterpenoids could lead to a better
understanding of structure-activity relationships for this
class of natural products. Additional screening against a
broader range of cancer cell lines may reveal additional
utility of these compounds as drug leads and may fuel

Med Chem Res
(C-25), 15.2(C-30), LRMS(APCI+) m/z: Calcd. for
C30H47O5 (M–H)+; 487.4. Found 487.3.
2β,3β,19α-trihydroxyurs-12-en-28-oic methyl ester (4)
White powder (MeOH) obtained from hexane-AcOEt 50%.
mp 103–105 °C. 1H NMR (MeOH-d4, 600 MHz,): δ = 5.32
(1H, m, H-12), 3.97 (1H, dt, J = 6.0, 12.0 Hz, H-2), 3.60
(3H, s, H-31), 3.35(1H, d, J = 6.0 Hz, H-3), 2.67 (1H, td, J
= 6.0, 12 Hz, H-16a), 2.54(1H, s, H-18), 2.00(2H, m, H-
11), 1.89(1H, m, H-9), 1.77(1H, m, H-15a), 1.75(1H, m, H-
22a), 1.65(1H, m, H-22b), 1.65(1H, m, H-21a), 1.62(1H, m,
H-7a), 1.55(1H, m, H-1a), 1.53(1H, m, H-6a), 1.52 (1H, m,
H-16a), 1.48 (1H, m, H-6b), 1.37(3H, s, H-23), 1.32(1H, m,
H-20), 1.32(1H, m, H-7b), 1.32(1H, m, H-1b), 1.25(1H, s,
H21b), 1.21(3H, s, H-27), 1.20(1H, m, H-5), 1.01(3H, s, H-
29), 1.01(3H, s, H-25), 1.00(1H, m, H-15b), 0.96 (3H, d, J
= 12.0 Hz, H-30), 0.89 (3H, s, H-24), 0.71(3H, s, H-26),
and 13C NMR (MeOH-d4, 150 MHz,): δ = 179.1(C-28),
138.6(C-13), 128.1(C-12), 78.7(C-3), 72.1(C-19), 65.7(C-
2), 53.7(C-18), 50.7(C-31), 48.1(C-17), 48.1(C-5), 46.8(C-
9), 41.7(C-20), 41.7(C-14), 41.3(C-8), 41.1(C-1), 39.9(C-
10), 38.1(C-4), 37.4(C-22), 32.6(C-7), 28.1(C-15), 27.8(C-
29), 23.5(C-23), 25.8(C-16), 25.6(C-27), 25.2(C-21), 23.4
(C-11), 21.0(C-24), 17.9(C-6), 16.0(C-26), 15.4(C-25), 15.1
(C-30); LRMS(APCI+) m/z: Calcd. for C31H51O5 (M +
H)+; 503.4. Found 503.18.
(3β)-3-Hydroxy-lup-20(29)-en-28-oic acid or Betulinic acid
(2)
White powder (MeOH) obtained from Hexane-AcOEt
25–27.5%. mp 282–285 °C. 1H NMR (MeOH-d4, 600
MHz,): δ = 4.70(1H, d, 4.0 Hz, H-29a), 4.57(1H, d, 4.0 Hz,
H-29b), 3.00 (1H, m, H-19), 2.98(1H, m, H-3), 2.26(1H, td,
J = 4.0 12.0 Hz, H-13), 2.19 (1H, m, H-16a), 1.89(1H, m,
H-12a), 1.85(1H, m, H-15a), 1.82(1H, m, H-22a), 1.75 (2H,
m, H-2), 1.65 (3H, s, H-30), 1.55(1H, m, H-18), 1.55 (1H,
m, H-16b), 1.55(1H, m, H-1a), 1.45(1H, m, H-6a), 1.45(1H,
m, H-22b), 1.45(1H, m, H-21a), 1.42(1H, m, H-11a), 1.40
(1H, m, H-6b), 1.40(2H, m, H-7), 1.33(1H, m, H-9), 1.22
(1H, m, H-21b), 1.21(1H, m, H-11b), 1.00(1H, m, H-12b),
0.94(3H, s, H-23), 0.88(1H, m, H-1b), 0.88(3H, s, H-27),
0.87(3H, s, H-26), 0.65(3H, s, H-25), 0.65(1H, m, H-5) and
13C NMR (MeOH-d4, 150 MHz,): δ = 177.7(C-28), 150.8
(C-20), 110.1(C-29), 77.2(C-3), 55.9(C-17), 55.4(C5), 50.4
(C-9), 49.0(C-18), 47.1(C-19), 42.5(C-14), 40.7(C-8), 39.0
(C-4), 38.7 (C-1), 38.1(C-13), 37.2(C-10), 36.8(C-22), 34.4
(C-7), 32.2(C-16), 30.6(C-15), 29.7(C-21), 28.6(C-23), 27.6
(C-2), 25.6(C-12), 20.9(C-11), 19.4(C-30), 18.4(C-6), 16.4
(C-26), 16.3(C-25), 16.2(C-24), 14.9(C-27) LRMS(APCI+)
m/z: Calcd. for C30H47O3 (M–H)+; 455.4. Found 455.33
(3β)-3-Hydroxy-lup-20(29)-en-28-oic acid methyl ester or
Betulinic acid methyl ester (5)
White powder (CH2Cl2) obtained from Hexane-AcOEt
1
1α,2β,3β,19α-tretrahydroxyurs-12-en-28-oic acid (3)
20%, mp 205–207 °C. H NMR (CDCl3, 600 MHz,): δ =
4.69(1H, d, J = 4.0 Hz, H-29a), 4.53(1H, d, J = 4.0 Hz, H-
29b), 3.60(3H, s, H-31), 3.13(1H, dd, J = 4.0, 12.0 Hz, H-
3), 2.95 (1H, m, H-19), 2.18 (1H, m, H-16a), 2.13(1H, m,
H-13), 1.86(1H, m, H-22a), 1.81(1H, m, H-15b), 1.75 (2H,
m, H-2), 1.75(1H, m, H-12a), 1.65(1H, m, H-1a), 1.61 (3H,
s, H-30), 1.55(1H, m, H-18), 1.50 (1H, m, H-6a), 1.45 (1H,
m, H-16b), 1.45(1H, m, H-6b), 1.45(1H, m, H-11a), 1.45
(1H, m, H-22b), 1.40(1H, m, H-21a), 1.40(2H, m, H-7),
1.35(1H, m, H-15a), 1.25(1H, m, H-11b),1.25(1H, m, H-9),
1.20(1H, m, H-21b), 1.00(1H, m, H-12b), 0.89(3H, s, H-
26), 0.89(3H, s, H-23), 0.88(1H, m, H-1b), 0.84(3H, s, H-
27), 0.75 (3H, s, H-24), 0.68(3H, s, H-25), 0.65(1H, m, H-
5) and 13C NMR (MeOH-d4, 150 MHz,): δ = 176.7(28),
150.1(20), 109.4(C-29), 79.0(C-3), 56.6(C-17), 55.4(C-5),
51.3(C-31), 50.6(C-9), 49.5(C-18), 47.0(C[19), 42.4(C-14),
40.7(C-8), 38.7(C-4), 38.7 (C-1), 38.3(C-13), 37.2(C-10),
37.0(C-22), 34.3(C-7), 32.2(C-16), 30.6(C-15), 29.7(C-21),
28.0(C-23), 27.4(C-2), 25.5(C-12), 20.9(C-11), 19.4(C-30),
18.3(C-6), 16.1(C-26), 16.0(C-25), 15.4(C-24), 14.7(C-27);
LRMS(APCI+) m/z: Calcd. for C31H51O3 (M + H)+; 471.4.
Found 471.20.
White powder (MeOH) obtained from Hexane-AcOEt 70%.
mp 262 °C. H NMR (MeOH-d4, 600 MHz,): δ = 5.29(1H,
1
m, H-12), 3.63(1H, dd, J = 3.5, 12.0 Hz, H-2), 3.45(1H, d,
J = 12.0 Hz, H-1), 3.39(1H, d, J = 3.5 Hz, H-3), 2.55(1H,
m, H-11a), 2.50(1H, m, H-16a), 2.48(1H, s, H-18), 2.17
(1H, dd, J = 4, 12 Hz, H-11b), 2.00(1H, m, H-9), 1.78(1H,
m, H-6a), 1.77(1H, m, H-15a), 1.72(1H, m, H-22b), 1.62
(1H, m, H-22a), 1.58(1H, m, H-7a), 1.51(1H, m, H-21a),
1.51(1H, m, H-16b), 1.48(1H, m, H-21b), 1.34(1H, m, H-
20), 1.34(1H, br, H-5), 1.33(3H, s, H-27), 1.29(1H, m, H-
7b), 1.17(3H, s, H-23), 1.00 (1H, m, H-15b), 1.00 (3H, s, H-
25), 0.97(3H, s, H-29), 0.94(1H, br, H-6b), 0.92(3H, d, 8.0,
H-30), 0.86(3H, s, H-24), 0.77(3H, s, H-26) and 13C NMR
(MeOH-d4, 150 MHz,): δ = 182.4(C-28), 138.9(C-13),
130.8(C-12), 81.4(C-1), 80.9(C-3), 73.6(C-19), 71.7(C-2),
55.1(C-18), 49.4(C-9), 49.1(C-5), 49.0(C-17), 44.4(C-10),
43.2(C-20), 42.5(C-4), 41.8(C-8), 39.2(C-22), 38.9(C-14),
34.3(C-7), 29.8(C-15), 29.3(C-23), 28.4(C-11), 27.5(C-21),
27.2(C-29), 26.8(C-16), 25.1(C-27), 22.5(C-24), 19.6(C-6),
17.8(C-26), 16.8(C-30), 13.1(C-25); LRMS(APCI+) m/z:
Calcd. for C30H47O6 (M–H)+; 503.4. Found 503.30

Med Chem Res
1α,2β,3β,19α-tretrahydroxyurs-12-en-28-oic acid methyl
ester (6)
MHZ spectrometer equipped with a cryoprobe. The che-
mical shifts are quoted relative to TMS, and the coupling
constants are provided in Hz. The chromatographic silica
gel (450–550 mesh) purchased from SiliCycle, F60, 40–63
μm 60 Å was used.
White powder (MeOH) obtained from Hexane-AcOEt 65%,
mp 155–157 °C. 1H NMR (MeOH-d4, 600 MHz,): δ = 5.32
(1H, t, J = 4 Hz, H-12), 3.68 (1H, dd, J = 4.0, 12 Hz, H-2),
3.60 (3H, s, H-31), 3.48 (1H, d, J = 8.0 Hz, H-1), 3.42(1H,
d, J = 4.0 Hz, H-3), 2.67(1H, m, H-11a), 2.61(1H, m, H-
16a), 2.53(1H, s, H-17), 2.22(1H, dd, J = 4, 12 Hz, H-11b),
2.05(1H, m, H-9), 1.78 (1H, m, H-6a), 1.77(1H, m, H-15a),
1.72(1H, m, H-22a), 1.62 (1H, m, H-22b), 1.58(1H, m, H-
7a), 1.51(1H, m, H-21a), 1.51(1H, m, H-16b), 1.48(1H, m,
H-21b), 1.37(3H, s, H-27), 1.35(1H, m, H-5), 1.34(1H, m,
H-20), 1.29(1H, m, H-7b), 1.22(3H, s, H-23), 1.03 (3H, s,
H-25), 1.00 (1H, m, H-15b), 0.99(3H, s, H-29), 0.95(3H, d,
J = 8.0 Hz, H-30), 0.94 (1H, m, H-6b), 0.90(3H, s, H-24),
0.72(3H, s, H-26), and 13C NMR (MeOH-d4, 150 MHz,): δ
= 179.1(C-28), 137.4(C-13), 129.4(C-12), 79.8(C-1), 79.3
(C-3), 72.1(C-19), 70.4(C-2), 50.7(C-31), 53.6(C-18), 48.4
(C-9), 48.2(C-5), 48.1(C-17), 42.9(C-10), 41.6(C-20), 41.1
(C-4), 40.4(C-8), 37.5(C-22), 37.4(C-14), 32.6(C-7), 28.2
(C-15), 27.7(C-23), 26.9(C-11), 25.8(C-21), 25.6(C-29),
25.2(C-16), 23.6(C-27), 21.0(C-24), 18.0(C-6), 16.2(C-26),
15.1(C-30), 11.5(C-25); LRMS(APCI+) m/z: Calcd. for
C31H51O6 (M + H)+; 519.4. Found 519.16.
Chemicals
1-Methyl-3-nitro-1-nitrosoguanidine (wetted with ca. 50%
water) a product of TGI, Lot. JBCSI-TA was used to gen-
erate diazomethane (McKay 1948). Potassium hydroxide
and diethyl ether were purchased from Sigma-Chemical Co.
(St Louis, MO, USA). MTS reagent (CellTiter 96 AQ
Assay) was purchased from TCI (Mfr No: M0527).
Biological assays
MDA-MB-231 cells were cultured in Dulbecco’s modified
Eagle medium (Corning), supplemented with 10% fetal
bovine serum (Gibco) and antibiotics (Lonza). For the
determination of cytotoxicity, cells were plated in 96-well
plates (Nest Scientific) at a density of 2000 cells per well in
100 μl of media per well. On the following day, the media
was aspirated, and 100 μl of media in which a serial dilution
of DMSO-dissolved compound (or DMSO as a control) was
added to wells in triplicate. 3 days later, the relative num-
bers of metabolically active cells was determined by addi-
tion of the CellTiter-Glo reagent (Promega) and
measurement of chemiluminesence according to the man-
ufacturer’s protocol using a Fluoroskan Ascent FL (Thermo
Scientific). To calculate the percent viability, signal from
background wells (media only) was subtracted from each
well and then the remaining chemiluminesence value was
normalized to signal from wells with the equivalent
amounts of DMSO. Apoptosis induction was measured
using the Annexin V / Propidium Iodide staining kit (BD
Biosciences) per manufacturer’s instructions. Assays were
performed in at least triplicate.
Analytical measurements
High resolution mass spectrometry (HRMS) was carried out
using an Accela liquid chromatograph coupled to an Orbi-
trap Q-Exactive Focus mass spectrometer. Compounds
were injected (5 µL) into the LC system consisting of a 15
cm × 2.1 mm (5 µm) Eclipse Plus-C8 column at a constant
flow rate of 350 µL/min). A binary mobile phase gradient
containing 0.1 % (v/v) formic acid in water (A) and acet-
onitrile (B) was applied as follows: 97% A for 5 min, to
98% B in 30 min, held for 5 min, back to 97% A in 1 min,
and equilibrated for 5 min at 97% A. Analytes were intro-
duced into the mass spectrometer using positive electro-
spray ionization (+ESI). Full-scan accurate mass spectra
(m/z range: 50–750) of eluting compounds were obtained at
high resolution (70,000 FWHM) on the Orbitrap mass
analyzer using internal calibration (accuracy of measure-
ments < 1 ppm) and processed using Xcalibur
4.0.27.10 software. Electrospray source conditions were as
follows: sheath and auxiliary gas flow 49 and 12 arbitrary
units (a.u.), respectively; electrospray voltage 3.5 kV;
capillary temperature 300 °C; and S-Lens RF level 50 V.
Optical rotations were recorded on a polarimeter in
MeOH at 26 °C. Fourier Transform Infrared (FTIR) spectra
were recorded as thin films. 1D (1H, 13C and DEPT) and 2D
(1H–1H COZY, HSQC, HSQC-TOCSY, HMBC, TOCSY,
NOESY and ROESY) NMR spectra were recorded at 600
Statistical analysis
Graphing was done using GraphPad Prism 6 (GraphPad
Software). IC50 values were calculated by fitting a non-linear
curve using the “log-inhibitor vs normalized response”
function given by the equation Y = 100/(1 + 10X-log IC50).
Methylation
Methylation of carboxylic acids 1–3 with diazomethane. To
a stirred slurry of 50% aq. KOH (10 mL) in Et2O (5.0 mL)
at 0 °C, 1-methyl-3-nitro-1-nitroso guaidine (11.25 mg,
0.08 mmol) was added slowly. The resulting yellow colored
organic layer containing diazomethane was separated, dried

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Acknowledgements The authors would like to acknowledge the
council for International Exchange of Scholars (CIES) for the J.
William Fulbright Visiting Scholar program to Dr Kenneth Eyong.
The Baylor CPRIT Synthesis and Drug Lead Discovery Laboratory
(supported by the Cancer Prevention and Research Institute of Texas
R1309 and Baylor University, College of Arts and Sciences) and the
Molecular Bioscience Center hosted Dr. Eyong. The Departments of
Chemistry & Biochemistry and Department of Biology at Baylor
University are acknowledged for infrastructure and financial support.
The University of Yaounde I and the Government of Cameroon are
acknowledged for financial support through the Fonds d’Appuis a la
Recherche.
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