Cytotoxic and NF-κB and mitochondrial transmembrane potential inhibitory pentacyclic triterpenoids from Syzygium corticosum and their semi-synthetic derivatives

Article abstract of DOI:10.1016/j.bmc.2018.07.025

Syzygium is a large genus of flowering plants, with several species, including the clove tree, used as important resources in the food and pharmaceutical industries. In our continuing search for anticancer agents from higher plants, a chloroform extract of the leaves and twigs of Syzygium corticosum collected in Vietnam was found to be active toward the HT-29 human colon cancer cell line. Separation of this extract guided by HT-29 cells and nuclear factor-kappa B (NF-κB) inhibition yielded 19 known natural products, including seven triterpenoids, three ellagic acid derivatives, two methylated flavonoids, a cyclohexanone, four megastigmanes, a small lactone, and an aromatic aldehyde. The full stereochemistry of (+)-fouquierol (2) was defined for the first time. Biological investigations showed that (+)-ursolic acid (1) is the major cytotoxic component of S. corticosum, which exhibited also potent activities in the NF-κB and mitochondrial transmembrane potential (MTP) inhibition assays conducted, with IC₅₀ values of 31 nM and 3.5 μM, respectively. Several analogues of (+)-ursolic acid (1) were synthesized, and a preliminary structure-activity relationship (SAR) study indicated that the C-3 hydroxy and C-28 carboxylic acid groups and 19,20-dimethyl substitution are all essential in the mediation of the bioactivities observed for this triterpenoid.

Full text of DOI:10.1016/j.bmc.2018.07.025

Accepted Manuscript
Cytotoxic and NF-κB and mitochondrial transmembrane potential inhibitory
pentacyclic triterpenoids from Syzygium corticosum and their semi-synthetic
derivatives
Yulin Ren, Gerardo D. Anaya-Eugenio, Austin A. Czarnecki, Tran Ngoc Ninh,
Chunhua Yuan, Hee-Byung Chai, Djaja D. Soejarto, Joanna E. Burdette,
Esperanza J. Carcache de Blanco, A. Douglas Kinghorn
PII:
S0968-0896(18)31038-1
https://doi.org/10.1016/j.bmc.2018.07.025
BMC 14462
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 June 2018
7 July 2018
14 July 2018
Please cite this article as: Ren, Y., Anaya-Eugenio, G.D., Czarnecki, A.A., Ngoc Ninh, T., Yuan, C., Chai, H-B.,
Soejarto, D.D., Burdette, J.E., de Blanco, J.C., Kinghorn, A.D., Cytotoxic and NF-κB and mitochondrial
transmembrane potential inhibitory pentacyclic triterpenoids from Syzygium corticosum and their semi-synthetic
derivatives, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.07.025
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Cytotoxic and NF-κB and mitochondrial transmembrane potential
inhibitory pentacyclic triterpenoids from Syzygium corticosum and
their semi-synthetic derivatives
a
a
b
c
Yulin Ren , Gerardo D. Anaya-Eugenio , Austin A. Czarnecki , Tran Ngoc Ninh , Chunhua
b,e
b
Yuan d, Hee-Byung Chai a, Djaja D. Soejarto , Joanna E. Burdette , Esperanza J. Carcache de
Blanco a, A. Douglas Kinghorn a,*
a Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus,
OH 43210, United States.
b Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago,
Chicago, IL 60612, United States.
c Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, Hoang Quoc Viet,
Cau Giay, Hanoi, Vietnam.
d Campus Chemical Instrument Center, The Ohio State University, Columbus, OH 43210, United States.
e Science and Education, Field Museum of Natural History, Chicago, IL 60605, United States.
* Corresponding author.
E-mail address: kinghorn.4@osu.edu (A. D. Kinghorn).
1

ABSTRACT: Syzygium is a large genus of flowering plants, with several species, including the
clove tree, used as important resources in the food and pharmaceutical industries. In our
continuing search for anticancer agents from higher plants, a chloroform extract of the leaves and
twigs of Syzygium corticosum collected in Vietnam was found to be active toward the HT-29
human colon cancer cell line. Separation of this extract guided by HT-29 cells and nuclear
factor-kappa B (NF-κB) inhibition yielded 19 known natural products, including seven
triterpenoids, three ellagic acid derivatives, two methylated flavonoids, a cyclohexanone, four
megastigmanes, a small lactone, and an aromatic aldehyde. The full stereochemistry of (+)-
fouquierol (2) was defined for the first time. Biological investigations showed that (+)-ursolic
acid (1) is the major cytotoxic component of S. corticosum, which exhibited also potent activities
in the NF-κB and mitochondrial transmembrane potential (MTP) inhibition assays conducted,
with IC50 values of 31 nM and 3.5 µM, respectively. Several analogues of (+)-ursolic acid (1)
were synthesized, and a preliminary structure-activity relationship (SAR) study indicated that the
C-3 hydroxy and C-28 carboxylic acid groups and 19,20-dimethyl substitution are all essential in
the mediation of the bioactivities observed for this triterpenoid.
Keywords:
Syzygium corticosum
Pentacyclic triterpenoids
Cytotoxicity
NF-κB inhibition
Mitochondrial transmembrane potential inhibition
2

1. Introduction
Syzygium (Myrtaceae) is a large genus of flowering plants, containing about 1200‒1800
species that are distributed mainly in tropical and subtropical regions.1 Of these, the clove tree
(Syzygium aromaticum Merr. & Perry) is used as an important economic plant in the food and
pharmaceutical industries with particular use as a spice and a flavoring agent that has
antioxidant, antimicrobial, antinociceptive, antiviral, and cytotoxic activities.2 Syzygium cumini
(L.) Skeels, known as “jamun” or “jambolan” (black plum) in Hindi, is another important species
used widely in several traditional medicine systems for the treatment of diabetes, allergy,
arthritis, and other diseases.3 Chemical purification has shown that benzoic acid analogues and
flavonoids are the main bioactive components of clove,2 while chalcones and phloroglucinol
derivatives have been identified as the major cytotoxic components of S. samarangense Merr. &
Perry and S. jambos (L.) Alston, respectively.4‒6 Interestingly, the flowers of S. aromaticum were
found to contain large quantities of (+)-oleanolic acid [1.6% (w/w)] and eugenol [89% (w/w) of
the clove essential oil], and thus this species is regarded as an important natural source of these
both compounds.2,7
Pentacyclic triterpenes are common secondary plant metabolites, as represented by ursane-,
oleanane-, and lupane-type triterpenes, with (+)-ursolic acid, (+)-oleanolic acid, and (+)-betulinic
acid being the representative members. These three natural products are attracting increasing
interest for their promising bioactivities, including their anticancer potential as mediated by
induction of cancer cell apoptosis and suppression of angiogenesis.7‒9 Among these, (+)-ursolic
acid was found to induce doxorubicin-resistant HepG2 cell apoptosis through both caspase-
3

dependent and independent pathways.10 Tumor growth was suppressed significantly when 4-
week-old athymic BALB/c nude male mice inoculated with DU145 human prostate cancer cells
were treated with (+)-ursolic acid (i.p., 200 mg/kg, twice a week) for six weeks,11 and such an
antitumor efficacy was found to be mediated through inhibition of NF-κB activation.11,12 Several
clinical studies suggested that ursolic acid has potential for the development of an anticancer
drug.13 Two other well-known plant triterpenoids, (+)-betulinic acid and (+)-oleanolic acid, have
been reported to inhibit proliferation of melanoma and human gallbladder cancer cells,
respectively, and in turn, they suppressed tumor growth in representative tumor xenograft
models.14,15 Mechanistically, both compounds mediated their anticancer potential by induction of
apoptosis through activation of the mitochondrial pathway.15,16
In our continuing search for anticancer agents from higher plants,17 a chloroform partition of
the leaves and twigs of Syzygium corticosum (Lour.) Merr. & Perry collected in Vietnam was
found to be cytotoxic toward the HT-29 human colon cancer cell line, but the chemical
constituents of this plant have not been reported previously. Separation of this extract guided by
cytotoxicity for HT-29 cells and NF-κB inhibition yielded 19 known natural products. All
compounds isolated were tested for their cytotoxicity against HT-29 cells, and those isolated in
sufficient quantities were evaluated further for their cytotoxicity toward other human cancer cell
lines and for their NF-κB and mitochondrial transmembrane potential (MTP) inhibitory effects.
The functional groups of the major active compound, (+)-ursolic acid (1), have been determined,
and a preliminary structure-activity relationship (SAR) study for pentacyclic triterpenes and their
cancer cell line cytotoxicity and NF-κB and MTP inhibitory activities are reported.
2. Results and discussion
4

2.1. Purification and identification of the bioactive compounds from Syzygium corticosum
The MeOH extract of the leaves and twigs of S. corticosum was partitioned with n-hexane
and CHCl3, and all the dried partitions were evaluated for their cytotoxicity toward HT-29 cells.
A major known compound, (+)-ursolic acid (1),18 was purified from this extraction process, and
it was found to exhibit cytotoxic and NF-κB inhibitory activities. Following this result, the
CHCl3 extract was subjected to chromatographic separation guided by HT-29 cells and NF-κB
inhibition, and 18 known compounds, (+)-fouquierol (2),19 (+)-melaleucic acid 28-methyl ester
(3),20 (+)-melaleucic acid (4),21 (+)-alphitolic acid (5),22 (+)-cylicodiscic acid (6),23 (+)-3-O-β-
trans-p-coumaroylalphitolic acid (7),24 3,4-methylenedioxy-3'-O-methylellagic acid (S1),25 3,4-
methylenedioxy-3',4',5'-tri-O-methylellagic acid (S2),26 3,3',4'-tri-O-methylellagic acid (S3),27
sideroxylin (S4),28 (+)-2,3-dihydrosideroxylin (S5),29 (+)-3-hydroxy-6,10-seco-muurol-1-ene-
6,10-dione (S6),30 (+)-dehydrovomifoliol (S7),31 (+)-3-oxo-α-ionol (S8),32 (+)-annuionone D
(S9),33 (+)-vomifoliol (S10),34 (‒)-loliolide (S11),35 and syringaldehyde (S12),36 were isolated
from S. corticosum.
(+)-Fouquierol (2) was isolated originally from Fouquiera splendens Engelm
(Fouquieraceae),37 with its 13C NMR data presented in the literature.19 However, detailed
information about its configuration and complete assignments of its NMR spectroscopic data
have not been reported.38 A C30H52O3 molecular formula deduced from the HRESIMS and NMR
spectroscopic data, along with seven tertiary methyl groups at δH 0.77, 0.84, 0.88, 0.96, 0.97,
1.15, and 1.73 (singlets, 3H each) exhibited in the 1H NMR spectrum, indicated a tetracyclic
5

triterpene for 2, which contains three hydroxyl groups and a double bond, as supported by its UV
(λmax 201 nm) and IR (νmax 3418 and 1667 cm-1) spectra.39
Fig. 1. Structures of triterpenoids (1‒7) isolated from Syzygium corticosum.
A dammarane skeleton could be indicated for 2 by comparison of its 1H and 13C NMR data
(Table 1)40 with those of lupeol,41 which showed that both compounds possess closely similar
NMR spectroscopic data for their A-, B-, and C-ring units. In addition, three hydroxy groups
connected to the C-3, C-20, and C-24 positions could be deduced from its two oxygenated
6

methines and one oxygenated quaternary carbon shown in the 13C NMR spectrum and the
HMBC correlations between H-28 and H-29 and C-3, H-21 and C-17, C-20, and C-22, and H-26
and H-27 and C-24, respectively (Fig. S29, supplementary data).
Table 1
1H and 13C NMR spectroscopic data of 2 and 3 a
position
1
2 b
2 c
3 d
3 e
39.19 CH2
α 0.97 m
β 1.68 m
1.63 m
1.59 m
α 3.21 m
39.62 CH2
α 0.91 m
β 1.74 m
1.54 m
1.57 m
α 3.06 m
2
27.57 CH2
28.25 CH2
3
4
5
79.11 CH
39.14 C
56.14 CH
78.54 CH
39.58 C
56.55 CH
α 0.74 m
α 0.65 dd
(11.6, 1.6)
α 1.49 m
β 1.37 m
α 1.52m
6
7
18.42 CH2
35.37 CH2
α 1.53 m
β 1.44 m
α 1.28 m
β 1.51 m
19.04 CH2
38.44 CH2
β 1.61 m
8
40.52 C
41.07 C
9
10
11
50.77 CH
37.28 C
21.67 CH2
α 1.33 m
52.04 CH
38.20 C
21.42 CH2
α 1.51 m
β 1.23 m
α 1.49 m
α 1.47 m
β 1.70 m
β 1.62 m
1.48 m
1.32 m
α 2.14 m
β 1.67 m
β 2.35 m
12
24.99 CH2
26.91 CH2
13
14
15
42.55 CH
50.48 C
31.33 CH2
40.49 CH
59.95 C
28.36 CH2
α 1.07 m
β 1.46 m
1.63 m
α 1.51 m
β 1.27 m
α 2.32 m
β 1.45 m
16
27.57 CH2
34.99 CH2
1.59 m
17
18
19
20
21
50.27 CH
15.63 CH3
16.37 CH3
75.33 C
α 1.76 m
β 0.96 s
β 0.84 s
56.96 C
52.64 CH
47.91 CH
150.95 C
31.06 CH2
α 1.70 m
β 3.09 m
25.54 CH3
β 1.15 s
β 1.87 m
α 1.39 m
β 1.41 m
α 1.91 m
α 0.93 s
β 0.74 s
β 0.88 s
β 1.02 s
22
36.19 CH2
1.46 m
1.27 m
1.26 m
4.08 t (5.6)
37.56 CH2
23
24
25
26
29.52 CH2
76.13 CH
147.91 C
110.93 CH
28.62 CH3
16.19 CH3
17.09 CH3
17.42 CH3
4.85 br s
4.97 br s
1.73 s
α 0.97 s
β 0.77 s
27
28
29
18.17 CH3
28.15 CH3
15.51 CH3
177.05 C
176.84 C
110.50 CH2
4.59 br s
4.73 br s
1.69 s
3.35 brs
3.67 s
30
OH-3
OCH3-28
16.63 CH3
α 0.88 s
β 3.67 brs
19.11 CH3
51.67 CH3
7

aAssignments of chemical shifts are based on the analysis of 1D- and 2D-NMR
spectra. The overlapped signals were assigned from 1H−1H COSY, HSQC, and
HMBC spectra without designating multiplicity.
b 13C NMR spectroscopic data (δ) measured in CDCl3 at 100.61 MHz and
referenced to the solvent residual peak at δ 77.16.40
c 1H NMR spectroscopic data (δ) measured in CDCl3 at 400.13 MHz and referenced
to the solvent residual peak at δ 7.26.40
d 13C NMR spectroscopic data (δ) measured in acetone-d6 (different from that used
in literature20) at 100.61 MHz and referenced to the solvent residual peak at δ
29.84.40
e 1H NMR spectroscopic data (δ) measured in acetone-d6 (different from that used in
literature20) at 400.13 MHz and referenced to the solvent residual peak at δ 2.05.40
The same relative configuration for the A-, B-, and C-ring unit as that of (+)-betulinic acid
(9) was assigned for 2 from the consistent 2D NOESY correlations observed for both compounds
(Fig. S31, supplementary data). Also, a 3β-hydroxy group of 2 was evident from a large
difference observed for the 13C NMR spectroscopic chemical shifts of C-28 and C-2939 and the
NOESY correlations between H-3 and H-5 and H-28. A 20β,24β-dihydroxy functionality group
could be assigned from the NOESY correlations between H-17/H-21, H-17/H-30, H-21/H-24, H-
21/H-27, and H-24/H-27 (Fig. S31, supplementary data). This determination has also been
supported by a comparison of the 1H NMR spectrum of 2 with reported values. The coupling
constant of the 1H NMR signal for H-24 of 2 is similar to that assigned for a 24R-
hydroxydammarane at δH 3.33 (dd, J = 7.6 and 4.9 Hz) but different from that for a 24S-
hydroxydammarane that appeared at δH 3.28 (dd, J = 11.0, 1.8 Hz).42 Thus, compound 2 could be
defined as 3β,20β,24β-dammar-25-ene-3,20,24-triol, with the full
8

(3S,5R,8R,9R,10R,13R,14R,17S,20S,24R) configuration characterized for the first time. Based on
our best knowledge, (+)-fouquierol (2) is the first member of the dammarane-type triterpenes
isolated from the genus Syzygium.
All other known compounds, including six pentacyclic triterpenoids, three ellagic acid
derivatives, two flavonoids, four megastigmanes, a cyclohexanone, a small lactone, and an
aromatic aldehyde isolated from the leaves and twigs of S. corticosum, were identified by
comparison of their spectroscopic data with the literature values (Figs. S1‒S7 and S14‒S25 and
Tables S1‒S6, supplementary data). The NMR spectroscopic data for (+)-melaleucic acid 28-
methyl ester (3) were measured in a deuterated solvent different from that used previously,20
owing to its limited solubility in CDCl3, and these data were assigned completely in the present
study (Table 1).
2.2. Evaluation of the cytotoxicity and NF-κB and MTP inhibitory effects for compounds isolated
from S. corticosum
All chromatographically and spectroscopically pure compounds (Figs. S1‒S7 and S14‒S25,
supplementary data) isolated from S. corticosum were evaluated for their cytotoxicity against the
HT-29 human colon cancer cell line, using paclitaxel as the positive control.43,44 The compounds
isolated in sufficient quantity were also tested for their cytotoxicity toward the MDA-MB-231
breast, the MDA-MB-435 melanoma, and the OVCAR3 ovarian human cancer cell lines.43,44
Only compounds 1 and (+)-2,3-dihydrosideroxylin (S5) were found to show activity, with IC50
values of 5.9 µM (toward MDA-MB-231 cells) and 6.0 µM (against MDA-MB-435 cells),
respectively, and all other compounds tested were inactive, with IC50 values being larger than 10
9

µM (Table 2 and Table S9, supplementary data), indicating that (+)-ursolic acid (1) is the major
cytotoxic component of S. corticosum.
To further test for their bioactivities, the compounds isolated in a sufficient amount from S.
corticosum were also evaluated for their inhibitory effects in standard in vitro NF-κB and MTP
bioassays, with rocaglamide and staurosporine used as the positive control, respectively.43,45 Five
of these compounds [1, 3, 5, 7, and 3,3',4'-tri-O-methylellagic acid (S3)] tested were found to be
active, among which compound 1 was the most potent in the NF-κB inhibition assay, showing an
IC50 value of 31 nM. It also exhibited MTP inhibitory activity, with an IC50 value of 3.5 µM
(Table 2), indicating that this compound mediates its cytotoxicity at least in part through
mechanisms involving inhibition of NF-κB activation and mitochondrial transmembrane
potential (MTP).
Table 2
Cytotoxicity and NF-κB and MTP inhibition of triterpenoids isolated
from S. aromaticum *
compound
A a
B b
C c
D d
E e
F f
>10*1 5.9
>10*2 >10*3 0.031 3.5
1
>10
>10
>10
>10
>10
>10
9
>10
>10
>10
>10
>10
>10
17
>10
NT
>10
>10
>10
>10
13
>10
NT
>10
>10
>10
>10
13
NT
1.5
>10
8.7
>10
4.7
NT
2
3
4
5
6
7
>10
>10
>10
>10
>10
Paclitaxel g
Rocaglamide h
Staurosporine i
70
20
* IC50 values are the concentration (µM) required for 50% inhibition of
cell viability for a given test compound and were calculated using
nonlinear regression analysis with measurements performed in triplicate
and representative of three independent experiments, where the values
generally agreed within 10%.
a IC50 value (µM) toward the HT-29 human colon cancer cell line with 72
h treatment.
10

b IC50 value (µM) toward the MDA-MB-231 human breast cancer cell line
with 72 h treatment.
c IC50 value (µM) toward the MDA-MB-435 human melanoma cell line
with 72 h treatment.
d IC50 value (µM) toward the OVCAR3 human ovarian cancer cell line
with 72 h treatment.
e IC50 value (µM) for inhibition of nuclear factor kappa B (NF-κB p65) in
HeLa cells with 3 h treatment.
f IC50 value (µM) for inhibition of mitochondrial transmembrane potential
(MTP) in HT-29 cells with 3 h treatment.
NT: Not tested yet.
g Positive control for the cytotoxicity assay (IC50 values presented in nM).
h Positive control for the NF-κB p65 inhibition assay (IC50 values
presented in nM).
i Positive control for the mitochondrial potential assay (IC50 values
presented in nM).
*1‒3 IC50 values of 11.1, 13.1, and 12.9 µg/mL, respectively.
2.3. Structure-activity relationships (SARs) of pentacyclic triterpenoids and their cytotoxicity and
NF-κB and MTP inhibition
To identify the main functional groups responsible for the activities of (+)-ursolic acid (1),
two of its analogues, (+)-oleanolic acid (8, a commercially available sample) and (+)-betulinic
acid (9),39 were evaluated for their cytotoxic and NF-κB and MTP inhibitory activities. Neither 8
nor 9 showed activity in any of the cytotoxicity assays. In the NF-κB inhibition assay,
compounds 1 and 8 exhibited activity, with the potency of these compounds found to be
11

increased following the sequence 9 (IC50 14.3 µM), 8 (IC50 2.4 µM), and 1 (IC50 0.031 µM). In
the MTP assay, compound 9 showed the same potency as 1, but compound 8 was inactive (Chart
1 and Table S9, Supplementary data). These results indicate that the 19,20-dimethyl substitution
is required structurally for (+)-ursolic acid (1) to mediate its cytotoxicity and NF-κB inhibitory
effect, but replacement of the C-19 methyl group with a prop-1-en-2-yl substituent and removal
of C-20 and its methyl group did not change its potency of MTP inhibition. In addition,
compound 1 showed activity in all the cytotoxicity against MDA-MB-231 cells and NF-κB and
MTP inhibition assays, while, 8 was active only in the NF-κB inhibition assay, and 9 exhibited
potency only in the MTP inhibition assay, suggesting that (+)-ursolic acid (1), (+)-oleanolic acid
(8), and (+)-betulinic acid (9) mediate their bioactivities probably through a different mechanism
of action.
To test the importance of the C-28 carboxylic acid substituent, three pentacyclic triterpene
derivatives, (+)-uvaol (10),46 (+)-erythrodiol (11),47 and (+)-betulin (12) (Figs. S10‒S12 and
Tables S7 and S8, supplementary data),48 were prepared by reduction of 1, 8 or 9 with lithium
aluminium hydride (LAH) (Scheme 1),47 with their cytotoxic and NF-κB and MTP inhibitory
activities evaluated. Except for compound 12, which exhibited activity in the NF-κB inhibition
assay, all these products (10, 11, and 12) did not show any activities in any of the assays utilized
(Chart 1 and Table S9, supplementary data). Thus, reduction of the C-28 carboxylic acid group
to a primary alcohol led to the loss of the cytotoxic and NF-κB and MTP inhibitory activities of 1
and the NF-κB inhibitory activity of 8, decreased the MTP inhibitory activity of 9, and a slightly
increased cytotoxicity of 8 and 9, indicating that this carboxylic acid group is important for 1, 8,
and 9 to mediate their bioactivities evaluated. Also, it implies that introduction of an electron-
12

donating hydroxy functional group at the C-28 position results in the bioactivities of these
pentacyclic triterpenes changed.
Chart 1. Structures and bioactivities of (+)-ursolic acid (1), (+)-oleanolic acid (8), (+)-betulinic acid (9) and their
semi-synthetic derivatives. a IC50 value (µM) toward the MDA-MB-231 human breast cancer cell line. b IC50 value
(µM) for inhibition of NF-κB (p65) in HeLa cells. c IC50 value (µM) for inhibition of mitochondrial transmembrane
potential (MTP) in HT-29 cells.
To investigate the functionality of the C-3 hydroxy group of (+)-ursolic acid (1), a new ester,
(+)-3-O-(4-chlorobenzoyl)ursolic acid (13), was prepared (Scheme 1) 43 and evaluated
13

biologically. This product was found to be inactive in all the assays tested (Chart 1), indicating
that the free C-3 hydroxy group is important for (+)-ursolic acid (1) to mediate its cytotoxic and
NF-κB and MTP inhibitory activities, and also the introduction of a chlorine in the C-3 ester unit
did not improve these activities of 1. This means that introduction of a COC6H5Cl or –Cl
electron-withdrawing functional group at the C-3 hydroxy group results in the cytotoxic and
MTP inhibitory activities of 1 abolished and the NF-κB inhibitory activity decreased (Chart 1).
Scheme 1. Synthesis of compounds 10‒13. Reagents and conditions: (a) LAH/THF (dried), reflux, 5 h. (b) pyridine,
reflux, 3 h.
Previously, it has been reported that (+)-betulinic acid (9) exhibited potent and selective
antitumor activity targeted at the mitochondria.16 In the present study, compound 9 and several of
its analogues [3‒7, 12, and (+)-3β-O-trans-feruloylbetulinic acid (S13)] have been evaluated
biologically. All these derivatives did not exhibit any activities in the cytotoxicity assays (IC50
>10 µM), and 3‒7 and 12 were also inactive in the MTP inhibition assay, in which compounds 9
and S13 were active (Chart 2 and Table 2 and Table S9, supplementary data). In the NF-κB
inhibition assay, analogues 3, 5, 7, 12, and S13 exhibited activity, but 4 and 6 were inactive.
14

These results indicate that changing the C-27 methyl group of 9 to an electron-withdrawing
carboxylic acid unit in 4 or to an electron-donating hydroxy group of 6 leads to a loss of NF-κB
and MTP inhibition potency, but methyl esterification of C-28 carboxylic acid group of 4 or
introducing a hydroxy group at the C-2 position in 5 results in an increased NF-κB inhibition and
abolished MTP inhibition potency of 9. In addition, reduction of the C-28 carboxylic acid group
of 9 to a primary alcohol group of 12 or esterification of the C-3 hydroxy group of 5 with 4-p-
coumaric acid (7) increased the cytotoxic and NF-κB inhibitory activity but decreased the MTP
inhibitory potency of 9. However, esterification of the C-3 hydroxy group of 9 with a trans-
feruloyl unit (S13) increased both NF-κB and MTP inhibitory activities of 9. Thus, the C-2
methylene protons, the C-3 hydroxy, the C-27 methyl, and the C-28 carboxylic acid substituents
all are important for mediating the NF-κB and MTP inhibitory effect of (+)-betulinic acid (9).
Inhibition of NF-κB activation can contribute to both induction of cancer cell apoptosis and
limit tumor cell resistance to the chemotherapeutic agents.49 Similar to these mechanisms,
disruption of mitochondrial function results subsequently in apoptosis through interfering in the
release of cytochrome c and several antiapoptotic proteins located in the inner or outer
mitochondrial membranes.50 Thus, these molecular targets have been used for discovery of new
anticancer agents to overcome the serious reverse effects and the mechanisms of resistance of
cancer cells to the cytotoxic drugs.49,50 Several pentacyclic triterpenoids have been investigated
in some detail for their potent and promising anticancer activity mediated through the NF-κB and
or mitochondrial pathways.11‒16 Among these, (+)-ursolic acid (1) was reported to induce DU145
prostate cancer cell apoptosis partially through NF-κB inhibition, and it was found to suppress
prostate tumor growth, without significant decrease of mouse body weight.11 (+)-Betulinic acid
(9) was demonstrated to inhibit completely melanoma tumor growth in vivo, without any obvious
15

toxicity observed in mice,14 and its molecular target was identified as the mitochondrial
apoptosis.16
Chart 2. Structures and bioactivities of (+)-betulinic acid (9) and its analogues. a IC50 value (µM) toward the MDA-
MB-231 human breast cancer cell line. b IC50 value (µM) for inhibition of NF-κB (p65) in HeLa cells. c IC50 value
(µM) for inhibition of mitochondrial transmembrane potential (MTP) in HT-29 cells.
16

Recently, a synthetic oleanane-type triterpenoid, bardoxolone methyl (CDDO-Me), which
suppressed the activity of NF-κB, has been developed as a promising agent that has been tested
in phase I/II trials for the treatment of leukemias and solid tumors.51 In a previous preliminary
SAR study, the C-28 carbonyl group was proposed as the critical functional group for (+)-
betulinic acid (9) to induce melanoma cell apoptosis.52 Consistent with these previous studies,
the present study showed that (+)-ursolic acid (1) exhibits cytotoxic and NF-κB and MTP
inhibitory activities, with its C-28 carboxylic acid unit functioned critically. Thus, this
pentacyclic triterpene [(+)-ursolic acid (1)] might be a promising lead for design and discovery
of new anticancer drugs with decreased multidrug resistance, based on a previous proposal that
inhibition of NF-κB activation indicates a potential candidate for new anticancer drug
development.53
3. Conclusion
Several triterpenoids, ellagic acid analogues, flavonoids, and megastigmanes have been
isolated and identified for the first time from the leaves and twigs of Syzygium corticosum
collected in Vietnam, of which (+)-ursolic acid (1) was isolated in a large quantity, with a yield
of up to 0.2% (w/w). Thus, S. corticosum can be regarded as a new natural source of (+)-ursolic
acid (1), which exhibited cytotoxicity against the MDA-MB-231 human breast cancer cells and
potent NF-κB and MTP inhibitory activities, with the 19,20-dimethyl substitution, the C-28
carboxylic acid unit, and the C-3 hydroxy group being important for these biological activities
observed.
17

4. Materials and methods
4.1. General experimental procedures
Optical rotations were measured at room temperature on an Anton Paar MCP 150
polarimeter. UV spectra were recorded on a Hitachi U2910 ultraviolet spectrophotometer. ECD
measurements were performed using a JASCO J-810 spectropolarimeter. IR spectra were
recorded on a Nicolet 6700 FT-IR spectrometer. 1H and 13C, DEPT 90, DEPT 135, HSQC,
HMBC, NOESY, and COSY NMR spectra were recorded at room temperature on a Bruker
Avance DRX 400, a Bruker Avance II 400, a Bruker Avance III HD 700, or a Bruker Avance III
HD 800 MHz NMR spectrometer. ESIMS and HRESIMS data were collected on a Bruker Maxis
4G Q-TOF mass spectrometer in the positive-ion mode. Column chromatography was conducted
using silica gel (65 × 250 or 230 × 400 mesh, Sorbent Technologies, Atlanta, GA, USA).
Analytical thin-layer chromatography (TLC) was performed on precoated silica gel 60 F254
plates (Sorbent Technologies). Sephadex LH-20 was purchased from Amersham Biosciences,
Uppsala, Sweden. For visualization of TLC plates, H2SO4 was used as spray reagent. All
procedures were carried out using solvents purchased from commercial sources and employed
without further purification. Paclitaxel and compounds used for chemical synthesis were
purchased from Sigma-Aldrich (St. Louis, MO, USA) (purity ≥ 98%), if not otherwise noted.
4.2. Plant material
18

A sample of the leaves and twigs (acquisition number AA06822) of Syzygium corticosum
was collected in July 2011 by D.D.S. and T.N.N. (voucher specimen: DDS 14648) from a tree
(30-meters tall) at Cotuy forest at Vung Olim campsite (11o 43.469' N; 109o 08.255' E), Nui
Chua National Park, Ninh Hai District, Ninh Thuan Province, Vietnam. A voucher herbarium
specimen has been deposited at the John G. Searle Herbarium of the Field Museum of Natural
History, Chicago, IL, USA, under the accession number FM2294588.
4.3. Extraction and isolation
The milled air-dried leaves and twigs of S. corticosum (sample AA06822, 6100 g) was
extracted with MeOH (6 L × 7) at room temperature. The solvent was evaporated in vacuo, and
the dried MeOH extract (299.8 g, 4.9%) was resuspended in 10% H2O in MeOH (2000 mL) and
partitioned with n-hexane (1000, 1000, 600, and 600 mL) to yield a n-hexane-soluble residue
(35.4 g, 0.58%). Then, 200 mL of H2O were added to the aqueous MeOH layer, and this was
partitioned with CHCl3 (1000, 500, and 500 mL). The interfacial layer was filtered, and 12.0 g of
precipitate were obtained, which showed cytotoxicity toward HT-29 cells (IC50 < 20 μg/mL). Re-
crystallization of 60 mg of this filtrate in MeOH yielded 30 mg of (+)-ursolic acid (1), and the
material left is the impure form of 1.
The CHCl3 partition was washed with a 1% aqueous solution of NaCl, to partially remove
plant polyphenols, and the solvent was evaporated to afford a CHCl3-soluble extract (66.1 g,
1.08%). All the CHCl3-, n-hexane- and water-soluble extracts were inactive (IC50 > 20.0 μg/mL)
in the cytotoxicity bioassay used. However, when (+)-ursolic acid (1) was evaluated, it was
found to be both cytotoxic against the MDA-MB-231 human breast cancer cell line (IC50 5.9 µM)
19

and potently active in the NF-κB inhibition assay, with an IC50 value of 31 nM. To isolate more
analogues of this active compound, a portion of the CHCl3-soluble extract (10.0 g) was subjected
to silica gel column chromatography (6.0 × 45 cm), eluted with a gradient of n-hexane-acetone.
Eluates were pooled by TLC analysis to give 21 combined fractions (D2F1−D2F21), of which all
were inactive in the cytotoxicity assay, but fractions D2F8 and D2F9 were found to show activity
in the NF-κB inhibition assay. Thus, fractions D2F7−D2F9 were selected for further
chromatography, based on their TLC analysis and NF-κB inhibitory activity.
Fraction D2F7 was chromatographed over a silica gel column (4.5 × 40 cm), eluted with a
gradient of n-hexane-acetone to yield three pooled subfractions, D2F7F1–D2F7F3. Of these,
fraction D2F7F1 was further chromatographed over a silica gel column (2.5 × 20 cm), eluted
with a gradient of n-hexane-acetone, and purified by separation over a Sephadex LH-20 column,
eluted with CH2Cl2-MeOH (1:1), affording (+)-melaleucic acid 28-methyl ester (3, 3.0 mg), (+)-
3-hydroxy-6,10-seco-muurol-1-ene-6,10-dione (S6, 1.0 mg), and (‒)-loliolide (S11, 1.0 mg).
Fraction D2F7F2 was further chromatographed over a silica gel column (2.5 × 20 cm), eluted
with a gradient of n-hexane-acetone, and purified by separation over a Sephadex LH-20 column,
eluted with CH2Cl2-MeOH (1:1), affording 3,4-methylenedioxy-3'-O-methylellagic acid (S1, 1.5
mg), 3,4-methylenedioxy-3',4',5'-tri-O-methylellagic acid (S2, 5.0 mg), and (+)-annuionone D
(S9, 1.0 mg). Fraction D2F7F3 was further chromatographed over a silica gel column (2.5 × 20
cm), eluted with a gradient of n-hexane-acetone, and purified by separation over a Sephadex LH-
20 column, eluted with CH2Cl2-MeOH (1:1), affording sideroxylin (S4, 3.0 mg), (+)-2,3-
dihydrosideroxylin (S5, 1.0 mg), and (+)-3-oxo-α-ionol (S8, 1.0 mg).
Fraction D2F8 was chromatographed over a silica gel column (2.5 × 20 cm), eluted with a
gradient of n-hexane-acetone to yield two pooled subfractions, D2F8F1 and D2F8F2. Of these,
20

D2F8F1 was further chromatographed over a silica gel column (2.5 × 20 cm), eluted with a
gradient of n-hexane-acetone, and purified by separation over a Sephadex LH-20 column, eluted
with CH2Cl2-MeOH (1:1), affording (+)-melaleucic acid (4, 12.0 mg). Fraction D2F8F2 was
further chromatographed over a silica gel column (2.5 × 20 cm), eluted with a gradient of n-
hexane-acetone, and purified by separation over a Sephadex LH-20 column, eluted with CH2Cl2-
MeOH (1:1), yielding (+)-dehydrovomifoliol (S7, 2.0 mg) and syringaldehyde (S12, 1.0 mg).
Fraction D2F9 was chromatographed over a silica gel column (2.5 × 30 cm), eluted with a
gradient of n-hexane-acetone, to yield three pooled subfractions, D2F9F1‒D2F9F3. Of these,
fraction D2F9F1 was further chromatographed over a silica gel column (2.5 × 20 cm), eluted
with a gradient of n-hexane-acetone and purified by passage over a column containing Sephadex
LH-20, eluted with CH2Cl2-MeOH (1:1), to yield (+)-alphitolic acid (5, 3.0 mg) and (+)-3-O-β-
trans-p-coumaroylalphitolic acid (7, 2.0 mg). Fraction D2F9F2 was further chromatographed
over a silica gel column (2.5 × 20 cm), eluted with a gradient of n-hexane-acetone and purified
by passage over a column containing Sephadex LH-20, eluted with CH2Cl2-MeOH (1:1), to yield
(+)-cylicodiscic acid (6, 1.5 mg) and 3,3',4'-tri-O-methylellagic acid (S3, 15.0 mg). Finally,
fraction D2F9F3 was chromatographed over a silica gel column (2.5 × 20 cm), eluted with a
gradient of n-hexane-acetone and purified by passage over a column containing Sephadex LH-20,
eluted with CH2Cl2-MeOH (1:1), to yield (+)-fouquierol (2, 1.5 mg) and (+)-vomifoliol (S10, 1.0
mg).
3β,20β,24β-Dammar-25-ene-3,20,24-triol [(+)-fouquierol (2)]: Amorphous colorless powder;
[α]25D +3.3 (c 0.6, MeOH) (c: mg/mL); UV (MeOH) λmax (log ε) 201 (3.96) nm; ECD (MeOH,
nm) λmax (Δε) 202.7 (+8.37); IR (dried film) νmax 3417, 1667, 1556, 1463, 1378, 1263, 1088 cm-1;
21

1H and 13C NMR data, see Table 1; positive-ion HRESIMS m/z 483.3815 (calcd for C30H52O3Na,
483.3809).
4.4. Reduction of C-28 carboxylic acid group of 1, 8, and 9
To a dried 25 mL glass vial equipped with a magnetic stirrer, containing 13.7 mg (0.03 mmol)
of (+)-ursolic acid (1), 2 mL of a 0.02 M solution of LiAlH4 in dried THF (0.04 mmol) were
added to the vial under argon. The vial was sealed, and the mixture was stirred with reflux for 5
h, it was cooled to room temperature. Next, the reaction mixture was diluted with aqueous ether,
extracted with CH2Cl2 (DCM), dried over anhydrous Na2SO4, evaporated to dryness and
chromatographed over silica gel to obtain 12.0 mg (0.027 mmol) of the product, (+)-uvaol (10),
with a reaction yield of 90.9%. Following this same procedure, 12.0 mg (0.03 mmol) of the
product (+)-erythrodiol (11, with a yield of 90.9%) were prepared from 13.7 mg (0.03 mmol) of
(+)-oleanolic acid (8, purchased from Sigma), and 7.9 mg (0.018 mmol) of the product (+)-
betulin (12) (with a yield of 90.0%) were prepared from 9.1 mg (0.02 mmol) of (+)-betulinic acid
(9, isolated from our previous study).39
4.5. Esterification of 1
To a dried 25 mL glass vial equipped with a magnetic stirrer, containing 13.7 mg (0.03
mmol) of (+)-ursolic acid (1), 5 µL of 4-chlorobenzoyl chloride and 1 mL of pyridine were
added. The vial was sealed, and the mixture was stirred with reflux for 3 h. The vial was cooled
to room temperature, and CH2Cl2 (DCM, 5 mL) was added, and the solution was extracted with
distilled H2O. The organic layer was washed with distilled H2O, dried over anhydrous Na2SO4,
22

and evaporated at reduced pressure. The residue was purified by silica gel column
chromatography, using n-hexane-acetone (5:1→1:1), to afford 11.0 mg (0.019 mmol) of 13 (with
a yield of 61.7%).
(+)-3-O-(4-Chlorobenzoyl)ursolic acid (13): Amorphous colorless powder showing a pink
color under UV light at 365 nm; [α]25D +33.8 (c 0.8, MeOH) (c: mg/mL); UV (MeOH) λmax (log
ε) 239 (4.21), 201 (4.54) nm; ECD (MeOH, nm) λmax (Δε) 208.2 (+11.0); IR (dried film) νmax
1721, 1609, 1557, 1455, 1392, 1272, 1116, 1015 cm-1; 1H and 13C NMR data, see Tables S7 and
S8 (supplementary data); positive-ion HRESIMS m/z 617.3382 (calcd for C37H51O4ClNa,
617.3368).
4.6. Cell lines
All the HT-29 colon, MDA-MB-231 breast, MDA-MB-435 melanoma, and OVCAR3
ovarian human cancer cell lines were purchased from the American Type Culture Collection
(ATCC, Manassas, VA, USA), and they all were cultured in RPMI 1640 medium, supplemented
with FBS (10%), penicillin (100 units/mL), and streptomycin (100 μg/mL).
4.7. Cytotoxicity assay
Following a protocol used previously,44 HT-29, MDA-MB-231, MDA-MB-435, or OVCAR3
cells in log phase growth were harvested by trypsinization and seeded in 96-well clear flat-
bottomed plates (Microtest 96, Falcon). Cells were incubated at 37 °C in 5% CO2 overnight and
then treated with the samples or paclitaxel (the positive control) (both dissolved in DMSO and
23

diluted to different concentrations required) or the vehicle (DMSO) for 72 h. Viability of cells
was evaluated by a commercial absorbance assay (CellTiter 96 AQueous One Solution Cell
Proliferation Assay, Promega Corp, Madison, WI, USA), with the IC50 values calculated from
the vehicle control.
4.8. Enzyme-based ELISA assay for NF-κB inhibition
A NF-κB inhibition assay was carried out using a published procedure, with an EZ-Detect
Transcription Factor Assay System ELISA kit (Pierce Biotechnology) used.43 The nuclear
extracts of HeLa cells treated with the controls and the test samples at four different
concentrations for 3 h were used to determine the specific binding ability of the activated p65
subunit of NF-κB to the biotinylated-consensus sequence and was measured by detecting the
chemiluminescent signal in a Fluostar Optima plate reader (BMG Labtech, Inc.). Rocaglamide
was used as a positive control.
4.9. Mitochondrial transmembrane potential (ΔΨm) inhibition assay
The mitochondrial transmembrane potential assay kit from Cayman Chemical Company was
used to assess the inhibition of mitochondrial transmembrane potential (ΔΨm) in HT-29 colon
cancer cells following a previously published protocol.45 Briefly, cells were seeded in 96-well
culture plates at 1 × 106 density. After 24 h incubation at 37 °C in 5% CO2, cells were treated
with samples or the controls for 3 h, and then 10 µL of 5,5′,6,6′-tetrachloro-1,1′,3,3′-
tetraethylbenzimidazoylcarbocyanine iodide (JC-1 staining solution) were added to each well,
24

followed by 15 min incubation. Cells were washed with the assay buffer, of which 100 µL were
added to each well, and the fluorescent intensity was measured using a FLUOstar Optima plate
reader. In healthy cells, JC-1 forms J-aggregates, and ΔΨm displays strong red fluorescence
intensity, which was measured with the excitation and emission at 535 and 595 nm, respectively.
However, in apoptotic cells, JC-1 remains in the monomeric form that exhibits strong green
fluorescence intensity, which was measured with the excitation and emission at 485 and 535 nm,
respectively. Staurosporine was used as a positive control in this assay.
4.10. Statistical analysis
The measurements were performed in triplicate and are representative of two three
independent experiments, where the values generally agreed within 10%. The dose response
curve was calculated for IC50 determinations using non-linear regression analysis (Table
Curve2DV4; AISN Software Inc.). Differences among samples were assessed by one-way
ANOVA followed by Tukey-Kramerʼs test, and the significance level was set at p < 0.05.
Disclosure statement
The authors declare no competing financial interest.
Acknowledgments
This investigation was supported by grant P01 CA125066 funded by the National Cancer
Institute, NIH, Bethesda, MD. The plant sample of Syzygium corticosum was collected under a
25

collaborative agreement between the University of Illinois at Chicago (USA) and the Institute of
Ecology and Biological Resources of the Vietnam Academy of Science and Technology, Hanoi
(Vietnam). We thank Drs. Appaso Jadhav and Nelson Freitas Fernandes, Division of Medicinal
Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, for
assistance for the chemical synthesis and bioassay testing, respectively. Drs. Arpad Somogy and
Nanette M. Kleinholz of the Mass Spectrometry and Proteomics of the Campus Chemical
Instrument Center, The Ohio State University, are thanked for acquisition of the MS data. Drs.
Craig A. McElroy and Deepa Krishnan, College of Pharmacy, The Ohio State University, are
thanked for access to some of the NMR spectroscopic and other instrumentation used in this
investigation.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at
https://doi.org/10.1016/j.bmc.
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