18β-Glycyrrhetinic Acid Derivatives Possessing a Trihydroxylated A Ring Are Potent Gram-Positive Antibacterial Agents

Article abstract of DOI:10.1021/acs.jnatprod.5b00641

The oleanane-type triterpene 18β-glycyrrhetinic acid (1) was modified chemically through the introduction of a trihydroxylated A ring and an ester moiety at C-20 to enhance its antibacterial activity. Compounds 22, 23, 25, 28, 29, 31, and 32 showed more potent inhibitory activity against Streptomyces scabies than the positive control, streptomycin. Additionally, the inhibitory activity of the most potent compound, 29, against Bacillus subtilis, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus was greater than that of the positive controls. The antibacterial mode of action of the active derivatives involved the regulation of the expression of genes associated with peptidoglycans, the respiratory metabolism, and the inherent virulence factors found in bacteria, as determined through a quantitative real-time reverse transcriptase PCR assay.

Full text of DOI:10.1021/acs.jnatprod.5b00641

Article
pubs.acs.org/jnp
18β-Glycyrrhetinic Acid Derivatives Possessing a Trihydroxylated A
Ring Are Potent Gram-Positive Antibacterial Agents
Li-Rong Huang,†,‡ Xiao-Jiang Hao,†,‡ Qi-Ji Li,‡ Dao-Ping Wang,‡ Jian-Xin Zhang,‡ Heng Luo,*,‡
and Xiao-Sheng 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, People’s Republic of China
‡The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences, Guiyang 550002,
People’s Republic of China
S
* Supporting Information
ABSTRACT: The oleanane-type triterpene 18β-glycyrrhetinic acid (1) was modified chemically through the introduction of a
trihydroxylated A ring and an ester moiety at C-20 to enhance its antibacterial activity. Compounds 22, 23, 25, 28, 29, 31, and 32
showed more potent inhibitory activity against Streptomyces scabies than the positive control, streptomycin. Additionally, the
inhibitory activity of the most potent compound, 29, against Bacillus subtilis, Staphylococcus aureus, and methicillin-resistant
Staphylococcus aureus was greater than that of the positive controls. The antibacterial mode of action of the active derivatives
involved the regulation of the expression of genes associated with peptidoglycans, the respiratory metabolism, and the inherent
virulence factors found in bacteria, as determined through a quantitative real-time reverse transcriptase PCR assay.
18β-Glycyrrhetinic acid (1), an oleanane-type triterpene, is
found as a minor component of licorice (the roots of the
leguminous plants Glycyrrhiza glabra L., Glycyrrhiza uralensis
Fisch., and Glycyrrhiza inflata Batalin).1 However, it can also be
obtained from the hydrolysis of glycyrrhizic acid, the major
active component of licorice.1,2 18β-Glycyrrhetinic acid (1) and
its analogues have demonstrated a variety of biological
activities, such as cytotoxic, hepatoprotective, antiviral, and
anti-inflammatory effects.3 Furthermore, these compounds
exhibit broad-spectrum antibacterial activity by inhibiting the
growth of Bacillus subtilis,4 Mycobacterium tuberculosis,5 Staph-
ylococcus aureus,6 and Actinobacillus actinomycetemcomitans.6
Structure−activity relationship studies of 1 have indicated that
its A ring and the carboxylic acid group at C-20 not only are
involved in various biological activities but also act as important
sites for structural modification.7 Other researchers have also
reported that the introduction of oxygen functionalities into
ring A of 1 and the esterification of the carboxylic acid group
confer improved biological activities.8
and integrity of human and plant life. A significant proportion
of the currently approved drugs that have been designed as new
antibacterial agents are natural products or are derived from a
natural product scaffold.9 Natural products and their derivatives
with unique and diverse chemical structures usually exhibit
broad-spectrum bactericidal activity, particularly potent inhib-
itory activity against antibiotic-resistant bacterial strains, which
provides a platform for the rational design and development of
novel antimicrobial therapeutics.9
1α-Hydroxy-12-olean-30-oic acid shows weak inhibitory
activity against Escherichia coli, with a minimum inhibitory
The increasing morbidity and mortality caused by infections
with pathogenic bacteria, particularly multi-drug-resistant
pathogens, have highlighted an urgent requirement for
developing novel antibacterial agents to protect the health
Received: July 22, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Scheme 1. Synthesis of Derivatives of 18β-Glycyrrhetinic Acid
concentration (MIC) greater than 250 μg/mL.10 However,
1α,3β-dihydroxy-12-oleanen-30-oic acid, which possesses one
more hydroxy group in ring A than the above-mentioned
compound, displays better antibacterial activity against
Escherichia coli, with a MIC of 16 μg/mL.10 Similarly, oleanolic
acid exhibits MIC values of 1.9 and 7.8 μg/mL against Bacillus
thuringiensis and Escherichia coli, respectively, whereas its
analogue, maslinic acid, which bears an additional hydroxy
group at position C-2 in ring A, exhibits MIC values of 0.9 and
3.9 μg/mL, respectively, against these bacterial strains.11 It thus
appears that the addition of a hydroxy group to ring A of
oleanane-type triterpenes increases their antibacterial proper-
B
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ties. On the basis of these observations, modifying 18β-
glycyrrhetinic acid (1) through the addition of two hydroxy
groups at positions C-1 and C-2 may be expected to enhance
the resultant antibacterial activity. To evaluate this hypothesis, a
series of trihydroxylated derivatives was synthesized in the
present study using 18β-glycyrrhetinic acid (1) as the starting
scaffold. The antibacterial properties of the parent compound
and the synthesized derivatives were assayed against three
Gram-negative bacteria, namely, Escherichia coli (E. coli),
Pseudomonas aeruginosa (P. aeruginosa), and multi-drug-
resistant Pseudomonas aeruginosa (MDRPA), and four Gram-
positive bacteria, namely, Streptomyces scabies (S. scabies),
Bacillus subtilis (B. subtilis), Staphylococcus aureus (S. aureus),
and methicillin-resistant Staphylococcus aureus (MRSA), to
identify the structure−activity relationships of the derivatives. It
was found that some of the trihydroxylated compounds
obtained showed markedly enhanced inhibitory activity against
Gram-positive bacterial strains when compared with the parent
compound 1 and were even more potent than the positive
controls. Furthermore, the most potent derivatives were
selected and assayed to investigate the potential mechanisms
underlying their activity. These findings may provide new
insights for the design of new pentacyclic triterpene derivatives
as promising antibiotics.
group at the C-10 position. Similar signals were also observed
for compound 10.
Epoxy ketones 9 and 10 were reacted with NaBH4 to obtain
the 1α,2α-epoxy-3β-ols 11 and 12 as the major products (yields
more than 60%) and the 1α,2α-epoxy-3α-ols 13 and 14 as the
minor products (yields less than 25%), after purification by
silica gel column chromatography.16 Based on 1D NMR
analysis, the protons at C-1, C-2, and C-3 in compounds 11 and
13 were assigned according to their HSQC, HMBC, and COSY
spectra. The OH-3 relative configuration was deduced from the
ROESY spectrum. Strong NOE correlations for compound 11
were observed between H-3 and H-5α and between H-1 and
H-25β, and no NOE correlation was observed between H-3
and H-25α, implying that H-3 is in the β-orientation and that
the hydroxy group is α-oriented. However, the distinct NOE
correlations between H-25β, H-1, H-2, and H-3 for compound
13 indicated β-orientations for these protons, thereby
suggesting an α-orientation for OH-3. Similarly, the OH-3
configuration of compounds 12 and 14 was confirmed in each
case from their ROESY data.
The treatment of the major product 11 with perchloric acid
in acetone produced the trihydroxylated compounds 15 and 17
with yields of 66% and 16%, respectively, after purification by
silica gel column chromatography.13 These compounds were
crystallized from EtOH, and the structures were confirmed by
X-ray crystallography (Figures 1 and 2). The same reaction
RESULTS AND DISCUSSION
■
Chemistry. It was hypothesized that the antibacterial
activities of 18β-glycyrrhetinic acid (1) and its derivatives can
be changed significantly by the reduction of the carbonyl group
at position C-11 to a methylene group. Thus, to determine the
influence of the carbonyl group on the antibacterial activity, 11-
deoxo-18β-glycyrrhetinic acid (2) was prepared from 1.12
Similar modifications were then made to compounds 1 and 2.
On the basis of a previous study, triterpenoid acids bearing a
trihydroxylated A ring were prepared as detailed in Scheme 1.13
Enones 7 and 8 were obtained through a two-step process with
moderate yields (approximately 70%). Ketones 5 and 6 were
first treated with phenylselenenyl chloride (PhSeCl) in CH2Cl2
at −78 °C for 15 min, and the mixture was further oxidized
with 30% hydrogen peroxide in the presence of pyridine.14
Additionally, the enones (7 and 8) could be synthesized
directly by the treatment of the ketones (5 and 6) with
selenium dioxide in acetic anhydride.13 However, this method is
undesirable because it generates many byproducts in the
process, lowering the yield (<50%). The epoxy ketone
intermediates (9 and 10) were prepared with high selectivity
through the epoxidation of the enones (7 and 8) using
hydrogen peroxide, and yields higher than 90% were obtained
after purification by silica gel column chromatography.15 The
relative configuration of the epoxy ketones was proposed based
on the results of 1D NMR (1H, 13C, and DEPT NMR) and 2D
NMR (HSQC, HMBC, and ROESY) analysis. For example, for
compound 10, oxygenated methines δH 3.51 (d, J = 4.4 Hz)
and 3.38 (d, J = 4.4 Hz) were assigned to H-1 and H-2 based
on the HSQC and HMBC spectra, respectively. The ROESY
spectra were used to confirm the relative configuration. NOE
correlations were observed between H3-25β, H-1, and H-2,
indicating that these protons are in the β-orientation and
therefore suggesting that the epoxy ring is in the α-orientation.
Additionally, the formation of the epoxy ring in the α-
orientation is in agreement with the reaction mechanism, which
involves the nucleophilic attack of the hydrogen peroxide anion
at the less-hindered C-1 from the α-side opposite the β-methyl
Figure 1. X-ray crystallographic structure of compound 15. Displace-
ment ellipsoids are shown at the 30% probability level.
with compound 12 generated compound 16 with a yield of
78%. The relative configuration of the hydroxy groups in
compound 16 was determined by comparison of its NMR
spectra with the data for compounds 15 and 17.
The protected benzyl ester groups of compounds 15−17
were then hydrogenated with 10% palladium over carbon in the
presence of hydrogen gas in tetrahydrofuran (THF) to produce
the target compounds 18−20 in high yields (>85%).16
The acid-catalyzed ring-opening reaction of the epoxy
compound 11 produced compound 15 and its 3α-hydroxy
epimer 17. In general, this reaction follows mechanism a
(Scheme 2): the weak nucleophile H2O attacks the C-2
position from the β-side in the presence of acid to obtain a
trans-hydroxy group at positions C-1 and C-2 and thereby
C
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inhibited bacterial growth. After identification of the active
compounds, the MIC values of these compounds, determined
through broth microdilution assays and representing the lowest
concentration of the antibacterial agent at which bacterial
growth was not apparent when compared with the negative
control, which consisted of vehicle without microorganisms,
were determined to assess the potency of the compounds.
The results indicated that none of the derivatives showed
antibacterial activity against the Gram-negative bacterial strains,
namely, E. coli, P. aeruginosa, and MDRPA. In contrast, some of
the compounds exhibited an improvement in potency against
the Gram-positive bacterial strains, namely, S. scabies, B. subtilis,
S. aureus, and MRSA, as presented in Table 1. Similar to the
findings obtained in previous studies, these results confirmed
that the oleanane-type triterpenes preferentially inhibit the
growth of Gram-positive bacterial growth, although the
mechanism of action has not been fully elucidated.20
The intermediates (7−14) possessing an α,β-unsaturated
ketone or an α-epoxy group in ring A showed negligible
inhibitory activity against all of the bacterial strains tested.
Notably, the derivatives with the highest activity against Gram-
positive bacteria were compounds 22, 23, 25, 28, 29, 31, and
32, which possess three hydroxy groups in ring A and short-
chain alkyl (C1−C4) ester functionalities at C-20 (Table 1).
However, both the trihydroxylated derivatives (26, 27, 33−42),
which contain long-train alkyl ester groups or substituted
benzyl ester groups, and compounds 18, 19, and 20, which
have a carboxylic acid group at C-20, were inactive or exhibited
weak activity, suggesting that the short-chain alkyl ester moiety
is essential for the antibacterial activity of the trihydroxylated
compounds. Among the compounds tested, compound 29,
which has an ethyl ester group at C-20, showed the highest
activity against B. subtilis, S. aureus, and MRSA, with MIC
values lower than those of the positive controls. Additionally,
compounds 22, 23, 25, 28, 29, 31, and 32 exhibited potent
inhibitory activity against S. scabies, with MIC values ranging
from 1.0 to 4.4 μg/mL. Of these compounds, 28, which
possesses a methyl ester group, was the most active, with a MIC
value that was 5-fold lower than that of the positive control.
Csuk et al. found no direct relationship between the C-11
ketone group and the apoptotic activity of compound 1
derivatives.21 However, in the present study, a significant
difference in bacterial inhibition activity was observed between
the 11-oxo and 11-deoxo compounds. Of these two compound
types, the 11-oxo compounds, such as 13 and 23, showed
higher activity levels than their corresponding 11-deoxo
Figure 2. X-ray crystallographic structure of compound 17. Displace-
ment ellipsoids are shown at the 30% probability level.
generate compound 15.17 The formation of compound 17 is
explained by mechanism b (Scheme 2): the position of the
oxirane ring moves in the presence of the neighboring hydroxy
group, and the nucleophile H2O then attacks C-3 from the α-
side to produce compound 17.18 However, the same reaction
with 12 did not yield the 3α-hydroxy epimer of 16, perhaps due
to high stereospecificity or its removal during the purification
process.
Additional modifications were focused on the esterification of
the carboxylic acid group at C-20 of compounds 19 and 20
(Scheme 3). Due to the insufficient quantities obtained,
compound 18 could not be further modified. Compounds 19
and 20 were reacted with various alkyl halides to obtain ester
derivatives (compounds 21−42),16 including alkyl esters
(linear-chain alkyl ester and branched-chain alkyl ester
derivatives) and benzyl esters (nitro-substituted and chloro-
substituted benzyl ester derivatives), in high yields (>95%).
In Vitro Antibacterial Activity. The parent compound 1
and all of its prepared derivatives were subjected to
turbidimetric assays to assess their in vitro inhibitory activities
against the bacterial growth of seven bacterial strains, namely, E.
coli, P. aeruginosa, MDRPA, S. scabies, B. subtilis, S. aureus, and
MRSA.19 Ampicillin, streptomycin, and vancomycin were used
as positive controls. The compounds were tested initially using
a single dose of 100 μM to determine whether they completely
Scheme 2. Mechanism for the Acid-Catalyzed Epoxide Ring-Opening Reaction
D
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Scheme 3. General Synthesis of Compounds 21−42
Table 1. In Vitro Antibacterial Activity of Selected
Derivatives
that trihydroxylated derivatives (compounds 23, 29, and 31)
exhibited potent inhibitory activity against the antibiotic-
resistant strain MRSA, implying that this type of compound
exhibits the potential to be developed as agents against
antibiotic-resistant bacteria.
a
MIC (μg/mL)
compound
B. subtilis
S. scabies
S. aureus
MRSA
8
>34.6
>36.7
>37.8
>36.9
>37.8
>31.2
16.9
34.6
36.7
>37.8
>36.0
>37.8
31.2
2.1
>34.6
>36.7
>37.8
>36.9
>37.8
15.6
4.2
>32.0
>32.0
4.0
Time-Kill Assays. The inhibitory activities of derivatives 28
and 29, which had the lowest MIC values, were studied using
time-kill assays.19 As shown in Figure 3, compounds 28 and 29
displayed dose-dependent inhibitory activity against the growth
of three Gram-positive bacterial strains. At concentrations equal
to 0.5 times their MIC values, the compounds showed a limited
inhibitory effect before regrowth, whereas at the other
concentrations tested (1× MIC, 2× MIC, and 4× MIC), the
survival initially decreased at a rapid rate and then gradually
slowed. Similar growth inhibition patterns were observed for
both B. subtilis and S. aureus using different concentrations of
the compounds. The killing time of compound 28 was longer
than 24 h for S. scabies due to the slow growth of this
bacterium.
Effects of the Compounds on the Expression of
Bacterial Genes Related to Metabolism and Virulence.
Compound 1 represses the synthesis of DNA, RNA, and
protein in B. subtilis and reduces the expression of virulence
genes in MRSA.22 To investigate the antibacterial mechanism
of the active derivatives at the molecular level, the parent
compound 1 and selected derivatives (28 and 29) were
subjected to a Q-RT-PCR assay to determine whether these
compounds (at a concentration equal to 0.5× MIC) could
regulate the expression of bacterial genes related to metabolism
and virulence.22b,23 The genes selected for this experiment are
associated with peptidoglycans (ykuD and lytF in B. subtilis,
lytM and fmhB in S. aureus and MRSA, and murX in S. scabies),
respiratory metabolism (mmgD in B. subtilis, narG in S. aureus
and MRSA, and p450 in S. scabies), and virulence regulation
(yhdT and yqhB in B. subtilis, seaR and hla in S. aureus, seaR, hla,
and mecA in MRSA, and txtA, txtC, nec1, and tomA in S.
scabies).
13
15
16
8.0
17
4.0
20
>32.0
4.0
22
23
>34.8
>34.8
>35.7
4.0
4.3
4.3
2.0
24
>34.8
4.4
>34.8
>35.7
2.0
8.0
25
4.0
28
1.0
>32.0
1.0
29
2.0
4.1
1.0
30
>33.9
>33.9
>34.8
>33.9
4.2
8.4
8.0
31
8.4
2.0
32
4.3
>34.8
1.4
>32.0
nt
b
ampicillin
streptomycin
vancomycin
nt
nt
2.9
nt
5.8
nt
nt
nt
nt
2.0
a
The derivatives not listed in this table were inactive (MIC > 50 μg/
b
mL). Not tested.
compounds, 14 and 30, respectively (Table 1). Conversely, the
11-deoxo compounds, such as 8, 20, and 28, displayed higher
activity levels than their corresponding 11-oxo compounds, 7,
19, and 21, respectively (Table 1). Thus, these results support
the initial hypothesis that the antibacterial activity of compound
1 and its derivatives can be altered significantly by the reduction
of the carbonyl group at position C-11 to a methylene group,
although the structure−activity relationships are not entirely
clear.
The results indicated that the introduction of two hydroxy
groups at the C-1 and C-2 positions of compound 1 can
improve its antibacterial activity against Gram-positive bacteria.
Moreover, although no studies conducted to date have
concentrated on the role of the hydroxy groups of pentacyclic
triterpenes as a possible contributor to the inhibitory activity
against antibiotic-resistant bacterial strains, this study showed
Peptidoglycans, which are essential cell-wall components in
bacteria, play an important role in adhesion, morphological
control, and signal transduction and have been the most
important target for therapeutic intervention in bacterial
infections, particularly antibiotic treatment.24 The ykuD gene
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Figure 3. Time-kill analysis of selected compounds 28 and 29 against
three Gram-positive bacterial strains. The curves represent the survival
rates of the bacteria treated with different concentrations of the
compounds. Each data point represents the results from three
experiments, and the error bars show the standard errors of the
means (SEM).
encodes a peptidoglycan transglycosylase that participates in
peptidoglycan degradation during bacterial autolysis,25 and lytF
encodes a peptidoglycan endopeptidase that plays essential
roles in peptidoglycan turnover, cell separation, and bacterial
autolysis.26 These two genes were significantly up-regulated by
compound 29 in B. subtilis (Figure 4A), suggesting that the
inhibitory activity of this compound may induce bacterial
autolysis. Another autolytic gene, lytM, was not altered in S.
aureus in response to treatment with compounds 1 and 29
(Figure 4B).23 However, compound 29 inhibited the expression
of this gene in MRSA (Figure 4C). The fmhB and murX genes
encode the peptidoglycan synthases FmhB and phospho-N-
acetylmuramoyl-pentapeptide-transferase, respectively.27 Nota-
bly, the FmhB protein, which catalyzes the synthesis of the
Figure 4. Relative gene expression in three bacterial strains treated
with the test compounds in vitro. The data were analyzed by Q-RT-
PCR and are expressed as the means SEM (n = 3). The asterisks
indicate statistically significant differences between the derivative-
treated group and the control group (*p < 0.05), and the pound signs
indicate statistically significant differences between the derivative-
treated group and the compound 1-treated group (#p < 0.05).
pentaglycine interpeptide in S. aureus peptidoglycans, is
important for cell-wall cross-linking and stability and acts as
an anchor for cell-wall-associated proteins and pathogenicity
determinants.27 The expression of fmhB in S. aureus and MRSA
was increased after treatment with compound 29 compared
F
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with the control and the parent compound 1, whereas murX
expression in S. scabies was reduced by treatment with
compound 28 as compared with the control (Figure 4B, C,
and D). These results provide the first evidence that the active
derivatives of compound 1 may affect the enzymes participating
in peptidoglycan metabolism, resulting in cell-wall instability or
disruption. A similar finding was also observed in previous
studies of oleanolic acid, which belongs to the family of
oleanane-type triterpenes, similar to compound 1.28 After this
alteration in the peptidoglycan structure, the Gram-positive
bacteria are perhaps more susceptible to the compounds, which
can now enter the cell more easily.
The levels of the mmgD and narG genes, which are related to
respiratory metabolism, were reduced in the bacterial strains
when treated with the test compounds when compared with
the control (Figure 4A, B, and C). However, these genes were
not significantly reduced in compound 1-treated bacteria,
indicating that compound 1 and its derivatives have different
antibacterial mechanisms. Interestingly, p450 expression was
decreased in S. scabies after treatment with compounds 1 and
28 (Figure 4D). The mmgD gene contributes to the
methylcitric acid cycle by converting propionyl-CoA to
pyruvate, which is a key intersection in the metabolic
network.29 The inhibition of mmgD expression would block
the methylcitric acid cycle and affect propionyl-CoA metabo-
lism and bacterial growth.29 The narG gene in S. aureus encodes
a membrane-bound nitrate reductase that participates in the
nitrogen metabolic pathway.30 In turn, p450 monooxygenase
gene encodes the key functional enzyme in the respiratory
chain, which transforms a wide variety of secondary metabolite
and xenobiotic substrates and thus plays an important role in
detoxification.31 These critical respiratory metabolism genes
were down-regulated by the selected compounds, which may
indicate that the derivatives lower metabolic efficiency and
detoxification and inhibit bacterial energy mechanisms,
resulting in the observed antibacterial activity.
The virulence genes yhdT and yqhB in B. subtilis and seaR
and hla in S. aureus, which are related to hemolytic activity,
were up-regulated by compounds 1 and 29 (Figure 4A and B).
However, the expression of seaR and hla in MRSA was
inhibited by these two compounds (Figure 4C), suggesting that
the test compounds may decrease the pathogenicity of MRSA.
The mecA gene is the main determinant for methicillin
resistance and encodes a specific penicillin-binding protein
(PBP), PBP2a, in MRSA to confer resistance to β-lactam
antibiotics.22b This gene was down-regulated by compound 29
when compared with the control and compound 1 (Figure 4C).
This observation indicates that compound 29 has the potential
to restore the inhibitory activity of β-lactam antibiotics against
MRSA when administered together with these antibiotics.The
txtA, txtC, nec1, and tomA genes in S. scabies, which are
responsible for potato scab disease, were significantly down-
regulated by the test compounds, with the exception of txtA
(Figure 4D).32 The altered expression of the virulence genes
demonstrates the influence of the derivatives produced an in
vitro investigation on bacterial pathogenicity.
displayed the most potent activity, with MIC values that were
lower than those of the positive controls. Structure−activity
relationship studies showed that the substitution of positions C-
1 and C-2 of 18β-glycyrrhetinic acid (1) with α- and β-hydroxy
groups, respectively, and the replacement of the carboxylic acid
group at C-20 with a short-chain alkyl ester group led to
increased antibacterial activity against antibiotic-sensitive and
-resistant Gram-positive bacteria. Furthermore, Q-RT-PCR
studies revealed that the antibacterial activity of the active
derivatives involved the regulation of several genes associated
with peptidoglycans, respiratory metabolism, and virulence
factors. Therefore, the modifications, structure−activity rela-
tionships, and mechanistic studies reported herein provide
insights that should enable the further development of 18β-
glycyrrhetinic acid (1) or its analogues as viable and highly
effective antibacterial agents, particularly as inhibitors of
antibiotic-resistant bacteria.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Ampicillin, streptomycin,
and vancomycin were purchased from Sigma Chemical Co. (St. Louis,
MO, USA), and the commercial reagents used were of analytical
reagent grade. The melting points are uncorrected (Leica hot-stage
microscope). The optical rotations were measured using a
PerkinElmer 241 polarimeter, and the [α]D values are given in 10−1
deg cm2 g−1. The IR spectra were recorded using KBr disks on a
PerkinElmer FT-IR spectrometer. The 1D NMR and 2D NMR
spectroscopic data were collected on a Varian INOVA 400 MHz or a
Varian Nova 500 MHz spectrometer using tetramethylsilane as the
internal standard. EIMS was performed using an HP1100-MSD
spectrometer, and ESIMS was performed using an Agilent 1100/MS-
G1946 spectrometer. TLC was performed on silica gel 60 F254 plates
purchased from Qingdao Haiyang Chemical Co., Ltd. (Qingdao,
People’s Republic of China) and visualized by exposure to UV light
and the use of an iodine chamber or by spraying with a 10% solution
of sulfuric acid in ethanol followed by heating. All of the compounds
produced were subjected to HPLC analysis on a Waters Sunfire CH,
C18 (5 μm, 250 × 4.6 mm) column, with two solvent systems
(acetonitrile−water and methanol−water). The X-ray crystal structure
data were obtained using a Bruker APEX-II CCD diffractometer at
100(2) K with Cu Kα radiation (λ = 1.541 78 Å).
Microorganisms and Culture Media. The E. coli ATCC 25922,
P. aeruginosa ATCC 27853, B. subtilis ATCC 6051, S. aureus ATCC
25923, and S. scabies CGMCC4.7610246 bacterial strains were
obtained from the National Institute for the Control of Pharmaceutical
and Biological Products (Beijing, People’s Republic of China).
MDRPA and MRSA were isolated from clinical specimens obtained
from Kunming General Hospital of Chengdu Military Area Command,
People’s Republic of China, from 2009 to 2010 and were stored frozen
at −80 °C. Specimen collection procedures were approved by the
Institutional Review Board (human studies review committee), and
the protocol number was 96−-049. E. coli, S. aureus, and B. subtilis were
cultured in Luria−Bertani medium (LB, Oxoid, Basingstoke, UK) on a
shaker at 37 °C, MDRPA and MRSA were cultured in Mueller-Hinton
broth (Oxoid, Basingstoke, UK) supplemented with 25.0 mg/L Ca2+
and 12.5 mg/L Mg2+ at 35 °C, and S. scabies was cultured in oat bran
broth prepared according to the instructions provided by Goyer et al.
at 28 °C.33 The microorganisms were collected at the log phase of
growth by centrifugation, washed once, and suspended in the broth for
turbidimetry assays.
Plant Material. The roots of Glycyrrhiza uralensis were produced
in the People’s Republic of China and were purchased from the
Sanqiao Crude Drug Market, Guiyang, People’s Republic of China, in
October 2011. The plants were identified by Prof. Lu-Tai Pan at the
School of Pharmacy, Guiyang College of Traditional Chinese
Medicine. A voucher specimen (No. 2010-0916) has been deposited
at the Herbarium of the Guiyang College of Traditional Chinese
Medicine.
In summary, 28 new compounds possessing a trihydroxylated
A ring were synthesized from the natural product 18β-
glycyrrhetinic acid (1), and the absolute structure of this
novel compound type was confirmed by X-ray crystallography
as compounds 15 and 17. The derivatives exhibited preferential
growth-inhibiting activity against Gram-positive bacteria in
vitro. Among the compounds prepared, compounds 28 and 29
G
DOI: 10.1021/acs.jnatprod.5b00641
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Extraction and Isolation of 18β-Glycyrrhetinic Acid (1).2 Air-
dried and powdered roots of G. uralensis (20 kg) were extracted three
times with 75% ethanol for 2 h under reflux. The extract was
concentrated under a vacuum to yield a residue, which was partitioned
successively three times between dichloromethane, ethyl acetate, n-
butanol, and water. After evaporation, the n-butanol fraction (530 g)
was chromatographed over a silica gel column to obtain eight fractions,
which were eluted with a gradient of MeOH in CHCl3 (0 to 50%).
Fraction 5 was subjected to silica gel H (average particle size of 10
μM) column chromatography, with an eluent of CHCl3−MeOH
(8:2). This was purified further using Sephadex LH-20 (MeOH) to
afford a glycoside (5.6 g), which was identified as glycyrrhizic acid
based on a comparison of its NMR spectroscopic data with literature
values.2 Glycyrrhizic acid (650 mg) was then dissolved in 200 mL of
MeOH containing 10% H2SO4 for hydrolysis under reflux for 4 h. The
reaction mixture was diluted with water, neutralized with a 10% NaOH
solution, and extracted three times with CHCl3, and the organic layer
was evaporated under vacuum. The crude aglycone was purified by
silica gel H column chromatography and then recrystallized from
acetic acid to yield compound 1 as white needle crystals (purity >95%
by HPLC; mp 293−295 °C, lit.34 mp 295 °C); its spectroscopic data
were consistent with the published literature data.34
Synthesis of α,β-Unsaturated Ketones (Compounds 7 and
8). The 3-ketones 5 and 6 were prepared with good yields (>90%) as
described in the literature.12,16 PhSeCl (1.84 g, 9.6 mmol, 1.2 equiv)
was added to a solution of the 3-ketone compound (5 or 6, 8.0 mmol,
1.0 equiv) in dry dichloromethane (200 mL). The mixture was stirred
at −78 °C for 15 min, and 30% hydrogen peroxide (0.29 mL, 9.6
mmol, 1.2 equiv) and pyridine (20 mL) were then added to the
mixture. The reaction mixture was stirred at room temperature for 1.5
h, and most of the solvent was then distilled off under vacuum. The
residue was extracted with EtOAc (3 × 100 mL), and the organic
phase was washed with saturated aqueous NaHCO3 and brine, dried
with anhydrous MgSO4, filtered, and concentrated under vacuum.
Each crude product was purified by silica gel column chromatography
with petroleum ether−ethyl acetate as the eluent to yield the enone
products.
distilled off under vacuum. The residue was extracted with EtOAc (3 ×
100 mL). The organic phase was washed with saturated aqueous
NaHCO3 and brine, dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. Each crude product was then
chromatographed on a silica gel column with a petroleum ether−
acetone solution as the eluent to obtain the pure compound.
Benzyl 1α,2α-epoxy-3,11-dioxo-18β-olean-12-en-30-oate (9): 2.9
g, 93% yield; white, amorphous powder; [α]30 +243.8 (c 0.26,
D
CHCl3); IR (KBr) νmax 2955, 2866, 1731, 1698, 1649, 1466, 1389,
1216, 1160, 1085, 865, 750, 696 cm−1; 1H NMR (400 MHz, CDCl3) δ
7.41−7.33 (5H, m, H-Ar), 5.64 (1H, s, H-12), 5.21 (1H, d, J = 12.4
Hz, Bn-CH2a), 5.10 (1H, d, J = 12.4 Hz, Bn-CH2b), 4.49 (1H, d, J =
4.4 Hz, H-2), 3.38 (1H, d, J = 4.4 Hz, H-1), 2.90 (1H, s, H-9), 1.41,
1.21, 1.17, 1.13, 1.11, 1.02, 0.75 (each 3H, s, CH3); 13C NMR (100
MHz, CDCl3) δ 212.4, 198.9, 176.1, 170.7, 136.0, 128.5, 128.5, 128.3,
128.2, 128.2, 127.9, 66.2, 64.6, 57.2, 54.5, 48.2, 45.5, 45.1, 44.8, 43.9,
43.4, 41.0, 38.3, 37.5, 31.7, 31.6, 31.0, 28.4, 28.2, 27.9, 26.5, 26.2, 23.2,
20.9, 18.6, 18.1, 15.8; EIMS m/z 572 [M]+ (40), 557 (6), 481 (35),
463 (22), 91 (100).
Benzyl 1α,2α-epoxy-3-oxo-18β-olean-12-en-30-oate (10): 2.8 g,
92% yield; white, amorphous powder; [α]30D +160.0 (c 0.40, CHCl3);
IR (KBr) νmax 2956, 1727, 1699, 1455, 1383, 1212, 1155, 1084, 732,
697 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.37−7.26 (5H, m, H-Ar),
5.22 (1H, br s, H-12), 5.21 (1H, d, J = 12.4 Hz, Bn-CH2a), 5.08 (1H,
d, J = 12.4 Hz, Bn-CH2b), 3.51 (1H, d, J = 4.4 Hz, H-1), 3.38 (1H, d, J
= 4.4 Hz, H-2), 1.20, 1.16, 1.11, 1.01, 1.01, 0.98, 0.75 (each 3H, s,
CH3); 13C NMR (100 MHz, CDCl3) δ 212.9, 198.9, 176.9, 144.8,
136.3, 128.4, 128.4, 128.0, 128.0, 128.0, 121.7, 65.9, 63.9, 56.9, 48.2,
45.9, 44.8, 44.2, 42.6, 41.7, 40.5, 39.9, 38.4, 38.2, 32.0, 31.9, 31.2, 28.5,
28.1, 27.9, 26.8, 26.1, 25.7, 23.9, 20.9, 18.8, 17.1, 15.1; EIMS m/z 558
[M]+ (3), 467 (38), 421 (24), 338 (16), 247 (63), 91 (100).
General Procedure for the Synthesis of Compounds 11−14.
The epoxy ketone (9 or 10, 4.3 mmol, 1.0 equiv) was dissolved in
MeOH (100 mL), and NaBH4 (817.0 mg, 21.5 mmol, 5.0 equiv) was
added slowly to the solution while stirring in an ice bath for 2 h. After
quenching with water (200 mL), the mixture was extracted with
EtOAc (3 × 200 mL), and the organic phase was washed with
saturated aqueous NaHCO3 and brine. The combined organic phases
were dried over anhydrous MgSO4, filtered, and concentrated. Each
residue was then isolated by silica gel column chromatography using
petroleum ether−acetone as the eluent to yield a pure compound.
Benzyl 1α,2α-epoxy-3β-hydroxy-11-oxo-18β-olean-12-en-30-
Benzyl 3,11-dioxo-18β-olean-1,12-dien-30-oate (7): 3.14 g, 71%
yield; yellow, amorphous powder; [α]30 +190.6 (c 0.67, CHCl3); IR
D
(KBr) νmax 3062, 3033, 2968, 2867, 1727, 1652, 1615, 1462, 1386,
1212, 1162, 766, 750, 716 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.73
(1H, d, J = 10.0 Hz, H-1), 7.38−7.34 (5H, m, H-Ar), 5.79 (1H, d, J =
10.0 Hz, H-2), 5.64 (1H, s, H-12), 5.22 (1H, d, J = 12.4 Hz, Bn-CH2a),
5.09 (1H, d, J = 12.4 Hz, Bn-CH2b), 2.65 (1H, s, H-9), 1.41, 1.37, 1.17,
1.16, 1.16, 1.11, 0.75 (each 3H, s, CH3); 13C NMR (100 MHz,
CDCl3) δ 204.6, 198.9, 176.1, 170.5, 161.6, 136.0, 128.5, 128.5, 128.3,
128.2, 128.2, 128.0, 124.5, 66.2, 52.5, 52.7, 48.2, 45.4, 44.7, 43.9, 43.4,
41.0, 38.7, 37.5, 31.9, 31.7, 31.0, 28.4, 28.2, 27.5, 26.4, 26.2, 23.3, 21.5,
20.0, 18.8, 18.1; EIMS m/z 556 [M]+ (50), 541 (10), 468 (24), 91
(100).
Benzyl 3-oxo-18β-olean-1,12-dien-30-oate (8): 3.13 g, 72% yield;
yellow, amorphous powder; [α]30D +115.2 (c 0.58, CHCl3); IR (KBr)
νmax 3442, 2929, 1728, 1668, 1455, 1382, 1214, 1155, 1083, 824, 733,
697 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.38−7.34 (5H, m, H-Ar),
7.04 (1H, d, J = 10.4 Hz, H-1), 5.81 (1H, d, J = 10.4 Hz, H-2), 5.22
(1H, br s, H-12), 5.20 (1H, d, J = 12.4 Hz, Bn-CH2a), 5.09 (1H, d, J =
12.4 Hz, Bn-CH2b), 1.18, 1.15, 1.14, 1.13, 1.10, 1.04, 0.75 (each 3H, s,
CH3); 13C NMR (100 MHz, CDCl3) δ 205.3, 176.8, 159.2, 144.8,
136.3, 128.4, 128.4, 128.0, 128.0, 128.0, 124.9, 121.7, 65.9, 53.3, 48.2,
44.5, 44.2, 42.5, 41.8, 41.6, 40.5, 39.3, 38.1, 32.3, 31.9, 31.2, 28.5, 28.1,
27.8, 26.8, 26.0, 25.8, 23.4, 21.6, 18.9, 18.8, 17.3; EIMS m/z 542 [M]+
(6), 451 (64), 405 (35), 247 (62), 91 (100).
oate (11): 1.49 g, 61% yield; white, amorphous powder; [α]30
D
+173.9 (c 0.29, CHCl3); IR (KBr) νmax 3446, 2957, 2868, 1728,
1
1658, 1455, 1386, 1215, 1160, 826, 751, 697 cm−1; H NMR (400
MHz, CDCl3) δ 7.40−7.33 (5H, m, H-Ar), 5.59 (1H, s, H-12), 5.21
(1H, d, J = 12.4 Hz, Bn-CH2a), 5.09 (1H, d, J = 12.4 Hz, Bn-CH2b),
4.10 (1H, d, J = 2.8 Hz, H-1), 3.55 (1H, d, J = 5.6 Hz, H-3), 3.05 (1H,
d, J = 3.6 Hz, H-2), 2.79 (1H, s, H-9), 1.36, 1.30, 1.16, 1.12, 0.94, 0.81,
0.73 (each 3H, s, CH3); 13C NMR (100 MHz, CDCl3) δ 199.9, 176.2,
169.8, 136.0, 128.6, 128. 6, 128.2, 128.1, 128.1, 128.0, 75.3, 66.2, 61.2,
57.9, 56.1, 48.2, 45.2, 44.5, 43.9, 43.3, 40.9, 37.6, 36.9, 36.3, 32.4, 31.7,
31.1, 28.3, 28.2, 28.2, 26.4, 26.3, 23.2, 18.9, 17.9, 17.8, 16.0; EIMS m/z
574 [M]+ (12), 559 (13), 491 (27), 91 (100).
Benzyl 1α,2α-epoxy-3β-hydroxy-18β-olean-12-en-30-oate (12):
1.67 g, 69% yield; white, amorphous powder; [α]30 +109.1 (c 0.33,
D
CHCl3); IR (KBr) νmax 3442, 2955, 1729, 1658, 1455, 1381, 1214,
1148, 1082, 1049, 1027, 752, 697 cm−1; 1H NMR (400 MHz, CDCl3)
δ 7.37−7.32 (5H, m, H-Ar), 5.20 (1H, d, J = 12.0 Hz, Bn-CH2a), 5.18
(1H, br s, H-12), 5.08 (1H, d, J = 12.8 Hz, Bn-CH2b), 3.57 (1H, s, H-
3), 3.07 (1H, d, J = 4.0 Hz, H-2), 2.99 (1H, d, J = 4.0 Hz, H-1), 1.15,
1.14, 1.13, 0.97, 0.95, 0.79, 0.74 (each 3H, s, CH3); 13C NMR (100
MHz, CDCl3) δ 176.9, 144.4, 136.4, 128.4, 128.4, 128.0, 127.9, 127.9,
121.9, 75.8, 65.9, 60.3, 57.3, 48.1, 45.5, 44.2, 42.7, 41.5, 41.1, 39.7,
38.2, 36.9, 36.4, 32.3, 31.8, 31.3, 28.5, 28.4, 28.1, 26.8, 26.1, 25.8, 23.6,
18.0, 16.9, 16.8, 16.6; EIMS m/z 558 [M − 2H]+ (6), 451 (22), 405
(22), 338 (82), 247 (100), 91 (65).
Synthesis of Epoxy Ketones (Compounds 9 and 10). A 10%
NaOH aqueous solution (7.7 mmol, 1.4 equiv) was added dropwise to
a solution of ketone (7 or 8, 5.5 mmol, 1.0 equiv) in MeOH (200 mL)
with continuous stirring in an ice bath for 30 min. After a 30% H2O2
solution (1.7 mL, 55.0 mmol, 10.0 equiv) was added to the mixture,
the reaction mixture was stirred at room temperature until the starting
material was not observed by TLC. The resulting reaction mixture was
quenched with a 1 M HCl solution, and most of the solvent was then
Benzyl 1α,2α-epoxy-3α-hydroxy-11-oxo-18β-olean-12-en-30-
oate (13): 575.1 mg, 23% yield; white, amorphous powder; [α]30
+122.4 (c 0.49, CHCl3); H NMR (400 MHz, CDCl3) δ 7.40−7.33
D
1
H
DOI: 10.1021/acs.jnatprod.5b00641
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(5H, m, H-Ar), 5.59 (1H, s, H-12), 5.21 (1H, d, J = 12.4 Hz, Bn-
CH2a), 5.09 (1H, d, J = 12.0 Hz, Bn-CH2b), 4.29 (1H, d, J = 4.4 Hz, H-
1), 3.54 (1H, d, J = 4.8 Hz, H-3), 3.42 (1H, dd, J = 4.4, 4.8 Hz, H-2),
2.80 (1H, s, H-9), 1.39, 1.28, 1.17, 1.11, 0.89, 0.82, 0.74 (each 3H, s,
CH3); 13C NMR (100 MHz, CDCl3) δ 199.8, 176.2, 169.9, 136.1,
128.6, 128. 6, 128.2, 128.1, 128.1, 128.0, 72.5, 66.2, 63.1, 56.2, 55.9,
48.3, 45.3, 43.9, 43.3, 41.7, 40.9, 37.6, 37.3, 36.4, 32.4, 31.7, 31.1, 28.4,
28.2, 26.4, 26.3, 25.8, 23.7, 23.3, 18.9, 17.8, 16.6; EIMS m/z 575 [M +
H]+ (10), 560 (27), 491 (17), 393 (31), 352 (21), 135 (53), 91 (100).
Benzyl 1α,2α-epoxy-3α-hydroxy-18β-olean-12-en-30-oate (14):
484.0 mg, 20% yield; white, amorphous powder; [α]30D +120.6 (c 0.49,
(SHELXS-97) and refined with full-matrix least-squares on F2. All of
the non-hydrogen atoms were refined anisotropically, and all of the
hydrogen atoms were placed in idealized positions and refined as
riding atoms with relative isotropic parameters. The absolute
structures were determined by refining the Flack parameter and
computing the Hooft parameter. The absorption correction was
performed by SADABS using multiscan parameters. The crystallo-
graphic data for compounds 15 (deposition number CCDC 1042471)
and 17 (deposition number CCDC 1042473) have been deposited in
the Cambridge Crystallographic Data Centre. Copies of the data can
be obtained free of charge by applying to the Director of the
Cambridge Crystallographic Data Centre (12 Union Road, Cambridge
CB2 1EZ, UK; fax: + 44-(0)1223-336033 or e-mail: deposit@ccdc.
cam.ac.uk).
1
CHCl3); H NMR (400 MHz, CDCl3) δ 7.35−7.29 (5H, m, H-Ar),
5.18 (1H, d, J = 12.4 Hz, Bn-CH2a), 5.16 (1H, br s, H-12), 5.06 (1H,
d, J = 12.4 Hz, Bn-CH2b), 3.51 (1H, d, J = 4.0 Hz, H-3), 3.42 (1H, t, J
= 4.4 Hz, H-2), 3.16 (1H, d, J = 4.0 Hz, H-1), 1.14, 1.13, 1.07, 0.94,
0.87, 0.78, 0.72 (each 3H, s, CH3); 13C NMR (100 MHz, CDCl3) δ
176.9, 144.6, 136.4, 128.4, 128.4, 127.9, 127.9, 127.9, 121.8, 72.7, 65.9,
62.3, 55.4, 48.1, 44.2, 42.7, 42.6, 41.5, 41.4, 39.8, 38.2, 37.2, 36.4, 32.3,
31.8, 31.3, 28.5, 28.1, 26.8, 26.0, 25.9, 25.8, 23.8, 23.6, 17.3, 16.9, 16.4;
EIMS m/z 558 [M − 2H]+ (9), 405 (6), 338 (41), 247 (82), 201 (12),
91 (100).
Crystal Data for Compound 15: C37H52O6·2(H2O), M = 628.82,
colorless prism, size 1.21 × 0.37 × 0.27 mm3, monoclinic, a =
34.1981(12) Å, b = 6.6186(2) Å, c = 15.6744(6) Å, α = 90.00°, β =
108.2830(10)°, γ = 90.00°, V = 3368.7(2) Å3, space group C2, Z = 4,
Dc = 1.240 g/cm3, μ(Cu Kα) = 0.688 mm−1, F(000) = 1368 mm−1. A
total of 27 880 reflections and 5872 independent reflections (Rint
=
0.0499) were collected in the θ range from 2.72° to 69.55°, with index
ranges of h(−41/41), k(−8/7), l(−18/18), and completeness θmax
=
General Procedure for the Synthesis of Target Compounds
15−17 Using the Ring-Opening Reaction. The major compound
(11 or 12, 2.5 mmol, 1 equiv) obtained from the above-described
reaction was dissolved in acetone (100 mL), and perchloric acid (5.0
mmol, 2 equiv) was added dropwise while stirring in an ice bath.
Stirring was continued for an additional 24 h. The mixture was
neutralized with 10% NaOH solution and then extracted with EtOAc
(3 × 200 mL). The organic phase was washed with saturated aqueous
NaHCO3 and brine, and the combined organic phases were dried with
anhydrous MgSO4, filtered, and concentrated. The residue was
purified by silica gel column chromatography using petroleum
ether−acetone as the eluent to yield the target product.
97.8%. The final R indices [I > 2σ(I)] were R1 = 0.0548 and wR(F2) =
0.1530. The final R indices (all data) were R1 = 0.0549 and wR(F2) =
0.1532 (all data). The goodness of fit on F2 was 1.083. The largest
difference peak and hole were 0.673 and −0.385 e·Å−3, respectively.
The Flack parameter was 0.0(2), and the Hooft parameter was 0.11(5)
for 2490 Bijvoet pairs.
Crystal Data for Compound 17: C37H52O6·2(H2O), M = 628.82,
colorless prism, size 0.95 × 0.46 × 0.42 mm3, monoclinic, a =
15.7402(6) Å, b = 6.5876(2) Å, c = 17.5417(6) Å, α = 90.00°, β =
93.2380(10)°, γ = 90.00°, V = 1816.00(11) Å3, space group P21, Z = 2,
Dc = 1.150 g/cm3, μ(Cu Kα) = 0.638 mm−1, F(000) = 684 mm−1. A
Benzyl 1α,2β,3β-trihydroxy-11-oxo-18β-olean-12-en-30-oate
(15): 982.7 mg, 66% yield; colorless, monoclinic crystals; mp 89−91
total of 13 439 reflections and 5404 independent reflections (Rint
=
0.0432) were collected in the θ range from 2.52° to 69.31°, with index
°C, [α]30 +178.5 (c 0.29, CHCl3); IR (KBr) νmax 3441, 2948, 2863,
ranges of h(−16/19), k(−7/7), l(−20/21), and completeness θmax
=
D
1
1731, 1655, 1455, 1384, 1214, 1159, 1045, 999, 732, 695 cm−1; H
95.0%. The final R indices [I > 2σ(I)] were R1 = 0.0996 and wR(F2) =
0.2866. The final R indices (all data) were R1 = 0.1011 and wR(F2) =
0.2944 (all data). The goodness of fit on F2 was 1.504. The largest
difference peak and hole were 1.204 and −0.914 e·Å−3, respectively.
The Flack parameter was 0.0(3), and the Hooft parameter was 0.04(7)
for 1976 Bijvoet pairs.
NMR (400 MHz, CDCl3) δ 7.41−7.33 (5H, m, H-Ar), 5.61 (1H, s, H-
12), 5.20 (1H, d, J = 12.4 Hz, Bn-CH2a), 5.09 (1H, d, J = 12.0 Hz, Bn-
CH2b), 4.69 (1H, br s, H-1), 4.00 (1H, br s, H-2), 3.57 (1H, br s, H-3),
3.28 (1H, br s, OH), 3.24 (1H, s, H-9), 3.00 (1H, br s, OH), 2.68 (1H,
br s, OH), 1.39 (3H, s, H-25), 1.36 (3H, s, H-27), 1.16 (3H, s, H-29),
1.15 (3H, s, H-26), 1.02 (3H, s, H-23), 0.99 (3H, s, H-24), 0.74 (3H,
s, H-28); 13C NMR (100 MHz, CDCl3) δ 202.7, 176.3, 171.4, 136.1,
128.6, 128. 6, 128.4, 128.3, 128.2, 128.2, 76.0, 75.2, 74.5, 66.2, 54.7,
48.0, 47.5, 44.9, 43.9, 43.6, 41.2, 41.1, 38.0, 37.7, 31.8, 31.7, 31.1, 29.8,
28.5, 28.2, 26.5, 26.4, 23.5, 19.2, 17.7, 17.7, 16.9; EIMS m/z 592 [M]+
(1), 574 (25), 491 (21), 393 (14), 225 (30), 135 (31), 91 (100).
Benzyl 1α,2β,3α-trihydroxy-11-oxo-18β-olean-12-en-30-oate
(17): 232.4 mg, 16% yield, colorless monoclinic crystal; mp 114−
Benzyl 1α,2β,3β-trihydroxy-18β-olean-12-en-30-oate (16): 1.13 g,
78% yield; white, amorphous powder; [α]30D +121.6 (c 0.33, CHCl3);
IR (KBr) νmax 3425, 2949, 1729, 1455, 1382, 1214, 1155, 1029, 697
1
cm−1; H NMR (400 MHz, CD3OD) δ 7.39−7.32 (5H, m, H-Ar),
5.19 (1H, d, J = 12.0 Hz, Bn-CH2a), 5.13 (1H, br s, H-12), 5.07 (1H,
d, J = 12.0 Hz, Bn-CH2b), 3.93 (1H, t, J = 4.0 Hz, H-2), 3.61 (1H, d, J
= 2.4 Hz, H-1), 3.45 (1H, d, J = 4.0 Hz, H-3), 1.24 (3H, s, H-25), 1.17
(3H, s, H-27), 1.11 (3H, s, H-29), 1.00 (3H, s, H-23), 0.99 (3H, s, H-
26), 0.97 (3H, s, H-24), 0.71 (3H, s, H-28); 13C NMR (100 MHz,
CDCl3) δ 177.1, 144.5, 136.3 128.4, 128.4, 128.0, 128.0, 128.0, 122.2,
75.9, 74.5, 73.9, 65.9, 48.1, 47.9, 44.2, 42.7, 42.0, 40.2, 39.6, 38.5, 38.3,
38.2, 32.0, 31.8, 31.2, 29.5, 28.5, 28.1, 26.9, 26.1, 26.0, 23.4, 17.9, 17.2,
17.0, 14.3; EIMS m/z 578 [M]+ (1), 560 (10), 338 (64), 247 (100),
91 (63).
116 °C, [α]30 +185.6 (c 0.37, CHCl3); IR (KBr) νmax 3424, 2959,
D
2869, 1728, 1650, 1456, 1385, 1214, 1152, 1007, 876, 751, 698 cm−1;
1H NMR (400 MHz, CDCl3) δ 7.41−7.32 (5H, m, H-Ar), 5.70 (1H, s,
H-12), 5.21 (1H, d, J = 12.0 Hz, Bn-CH2a), 5.10 (1H, d, J = 12.0 Hz,
Bn-CH2b), 3.90 (1H, d, J = 8.0 Hz, H-1), 3.72 (1H, d, J = 11.6 Hz, H-
3), 3.54 (1H, dd, J = 8.0, 11.6 Hz, H-2), 3.14 (1H, s, H-9), 2.89 (1H,
br s, OH), 2.77 (1H, br s, OH), 2.48 (1H, br s, OH), 1.35 (3H, s, H-
27), 1.17 (3H, s, H-29), 1.16 (3H, s, H-25), 1.15 (3H, s, H-26), 1.02
(3H, s, H-23), 0.94 (3H, s, H-24), 0.76 (3H, s, H-28); 13C NMR (100
MHz, CDCl3) δ 203.6, 176.1, 172.5, 136.0, 128.5, 128.5, 128.3, 128.2,
128.1, 128.1, 82.6, 74.6, 74.3, 66.2, 54.5, 48.1, 46.8, 44.5, 43.9, 43.7,
42.0, 41.1, 37.6, 37.5, 31.6, 31.0, 30.9, 28.5, 28.1, 26.5, 26.3, 23.4, 23.3,
23.3, 21.3, 19.3, 18.8; EIMS m/z 574 [M − H2O]+ (82), 557 (32), 491
(54), 393 (52), 225 (35), 135 (30), 91 (100).
General Procedure for the Synthesis of Target Compounds
18−20 Using a Hydrogenolysis Reaction. After 10% palladium/
carbon was added to a solution of trihydroxylated compound (15, 16,
or 17) in THF (50 mL), the resulting mixture was stirred at 30 °C
under H2 at atmospheric pressure for 24 h and then filtered through
Celite. The residue was washed with THF (10 mL × 3), and the
filtrate was evaporated to yield a crude product, which was subjected to
silica gel column chromatography using CHCl3−MeOH as the eluent
to produce a pure compound.
Crystallization and X-ray Crystallographic Analysis of
Compounds 15 and 17. Compounds 15 and 17 were crystallized
through the slow evaporation of an EtOH solution. The cell parameter
measurements and data collection were performed at T = 100(2) K
with a Bruker APEX II CCD diffractometer using Cu Kα radiation (λ
= 1.541 78 Å).35 The structure was resolved by direct methods
1α,2β,3α-Trihydroxy-11-oxo-18β-olean-12-en-30-oic acid (18):
72.5 mg, 85% yield, colorless needles; mp 114−116 °C; [α]30
D
+153.0 (c 0.29, CHCl3); IR (KBr) νmax 3424, 2959, 2869, 1728,
1
1650, 1456, 1385, 1214, 1152, 1007, 876, 751, 698 cm−1; H NMR
(400 MHz, CDCl3) δ 5.85 (1H, s, H-12), 3.92 (1H, d, J = 7.2 Hz, H-
I
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J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1), 3.73 (1H, d, J = 11.6 Hz, H-3), 3.55 (1H, dd, J = 7.2, 11.6 Hz, H-
2), 3.16 (1H, s, H-9), 2.89 (1H, br s, OH), 1.35 (3H, s, H-27), 1.23
(3H, s, H-29), 1.17 (3H, s, H-25), 1.16 (3H, s, H-26), 1.03 (3H, s, H-
23), 0.95 (3H, s, H-24), 0.87 (3H, s, H-28); 13C NMR (100 MHz,
CDCl3 and CD3OD) δ 203.9, 179.2, 173.5, 127.9, 81.9, 74.6, 74.5,
54.4, 48.2, 46.5, 44.5, 43.7, 43.5, 41.8, 41.1, 37.6, 37.3, 31.6, 30.9, 30.8,
28.5, 28.2, 26.5, 26.2, 23.4, 23.2, 23.0, 20.7, 19.1, 18.6; EIMS m/z 502
[M]+ (2), 484 (22), 303 (18), 135 (60), 43 (100).
the bacterial suspension was then measured. The MIC values were
determined as the minimum concentration of the test compounds that
caused no apparent bacterial growth compared with the negative
control. The assay was performed in triplicate to verify the MIC values.
Time-kill assays were performed according to previously published
methods.19 Briefly, each bacterial suspension was adjusted to a
concentration of 5 × 105 CFU/mL and divided into three groups:
negative control, positive control (as described above), and the test
compounds at concentrations of 0.5 × , 1 × , 2 ×, and 4 × the MIC.
Under the appropriate culture conditions, the S. aureus and B. subtilis
cultures were incubated for 0, 2, 4, 6, 8, 12, and 24 h, and the S. scabies
cultures were incubated for 0, 4, 8, 16, 24, and 36 h. At the indicated
time points, 1 mL samples of the treated bacteria and controls were
removed, serially diluted, and plated onto agar plates to enumerate the
viable colonies, and the survival was then calculated. The tests were
repeated in triplicate, and the killing kinetics curves were constructed.
Quantitative Real-Time Reverse-Transcriptase PCR (Q-RT-
PCR) Assay. The test bacterial strains were cultured in broth
supplemented with either the test compounds at a concentration of 0.5
× MIC or vehicle alone. The bacteria were allowed to grow to the
exponential phase (OD620 = 0.4) and were then harvested by
centrifugation.22b,23 The total RNA was isolated according to a
previously described procedure.22b The mRNAs were reverse-tran-
scribed into cDNAs using the PrimeScript reverse transcriptase kit
(Takara, Tokyo, Japan) according to the manufacturer’s instructions.
Q-RT-PCR was performed with an UltraSYBR mixture (with ROX)
kit (CWBIO, Beijing, People’s Republic of China) using a
StepOnePlus real-time PCR system (Applied Biosystems, Grand
Island, NY, USA); the sequences of the primers used are presented in
the Supporting Information. The relative expression levels for each
sample compared with the control were determined by the 2−ΔΔCT
method.36 The yqxC, 16s rRNA, and rpoB genes were used as
housekeeping genes for the B. subtilis, S. aureus, MRSA, and S. scabies
strains, respectively. Each experiment was performed in triplicate.
1α,2β,3β-Trihydroxy-11-oxo-18β-olean-12-en-30-oic acid (19):
520.4 mg, 86% yield, colorless needles; mp 167−169 °C; [α]30
D
+155.3 (c 0.21, CHCl3); IR (KBr) νmax 3431, 2949, 2867, 1704, 1646,
1
1464, 1387, 1219, 1117, 1049, 975, 819 cm−1; H NMR (400 MHz,
C5D5N) δ 6.06 (1H, s, H-12), 5.74 (1H, br s, H-1), 4.81 (1H, t, J = 3.6
Hz, H-2), 4.28 (1H, d, J = 4.0 Hz, H-3), 3.63 (1H, s, H-9), 2.56 (1H,
d, J = 10.4 Hz, H-18), 2.29 (1H, d, J = 11.2 Hz, H-21), 2.02 (3H, s, H-
25), 1.51 (3H, s, H-24), 1.46 (3H, s, H-27), 1.43 (3H, s, H-23), 1.32
(3H, s, H-29), 1.29 (3H, s, H-26), 0.83 (3H, s, H-28); 13C NMR (100
MHz, C5D5N) δ 201.4, 182.9, 169.7, 129.0, 77.4, 74.8, 74.4, 54.9, 48.7,
48.4, 45.4), 44.2, 44.1, 41.9, 41.7, 39.6, 38.5, 32.8, 32.2, 31.6, 30.4, 28.8,
28.7, 26.8, 26.7, 23.7, 19.5, 18.1, 17.8, 16.6; EIMS m/z 503 [M + H]+
(1), 484 (38), 469 (18), 401 (32), 207 (30), 135 (77), 43 (100).
1α,2β,3β-Trihydroxy-18β-olean-12-en-30-oic acid (20): 498.9 mg,
85% yield, colorless needles; mp 255−257 °C; [α]30 +140.6 (c 0.17,
D
CHCl3); IR (KBr) νmax 3428, 2948, 1701, 1455, 1383, 1227, 1177,
1
1029, 969 cm−1; H NMR (400 MHz, C5D5N) δ 5.59 (1H, br s, H-
12), 4.80 (1H, t, J = 3.6 Hz, H-2), 4.29 (1H, br s, H-1), 4.27 (1H, d, J
= 4.0 Hz, H-3), 1.69 (3H, s, H-25), 1.49 (3H, s, H-24), 1.41 (3H, s, H-
27), 1.40 (3H, s, H-23), 1.40 (3H, s, H-29), 1.18 (3H, s, H-26), 0.95
(3H, s, H-28); 13C NMR (100 MHz, C5D5N) δ 179.8, 145.1, 123.5,
77.6, 74.7, 74.3, 49.1, 48.8, 44.4, 43.6, 42.7, 41.2, 40.1, 39.3, 39.2, 38.9,
32.9, 32.5, 31.9, 30.5, 29.2, 28.7, 27.4, 26.7, 26.4, 24.0, 18.7, 18.3, 17.6,
15.0; EIMS m/z 488 [M]+ (2), 470 (8), 248 (100).
General Procedure for the Preparation of Target Com-
pounds (21−42) from Compound 19 or 20. K2CO3 (0.1 mmol,
2.0 equiv) was added to a solution of compound 19 or 20 (0.05 mmol,
1.0 equiv) in dry DMF (20 mL). After 30 min of stirring at room
temperature, an alkyl halide (0.06 mmol, 1.2 equiv) was added, and the
mixture was stirred for an additional 4 h. The mixture was poured into
water (50 mL), neutralized with 1.0 M HCl solution, and then
extracted with EtOAc (3 × 100 mL). The organic phase was washed
with saturated aqueous NaHCO3 and brine, and the combined organic
phases were dried over anhydrous MgSO4, filtered, and concentrated.
The residue was purified by silica gel column chromatography using
petroleum ether−acetone as the eluent to yield a pure compound. The
NMR spectra and MS data for compounds 22−42 are presented in the
Supporting Information.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00641.
Spectroscopic data for the new compounds (compounds
22−42), copies of the NMR spectra of all of the new
compounds (compounds 7−42), and the sequences of
the primers used in the Q-RT-PCR assay (PDF)
Methyl 1α,2β,3β-trihydroxy-11-oxo-18β-olean-12-en-30-oate
(21): 24.6 mg, 95% yield, colorless needles; mp 128−130 °C; [α]30
AUTHOR INFORMATION
Corresponding Authors
*Tel (H. Luo): +86 0851-3876201. Fax: +86 0851-3876210. E-
mail: luohengzz@sina.com.
*Tel (X.-S. Yang): +86 0851-3806465. Fax: +86 0851-3806465.
E-mail: gzcnp@sina.cn.
D
■
+169.4 (c 0.43, CHCl3); IR (KBr) νmax 3438, 2945, 2864, 1733, 1655,
1
1464, 1386, 1216, 1155, 1116, 1044, 998, 866 cm−1; H NMR (400
MHz, CDCl3) δ 5.72 (1H, s, H-12), 4.68 (1H, br s, H-1), 4.00 (1H, br
s, H-2), 3.70 (3H, s, COOCH3), 3.57 (1H, br s, H-3), 3.29 (1H, br s,
OH), 3.24 (s, 1H, H-9), 3.02 (br s, 1H, OH), 2.69 (br s, 1H, OH),
1.39, 1.37, 1.18, 1.15, 1.02, 0.99, 0.83 (each 3H, s, CH3); 13C NMR
(100 MHz, CDCl3) δ 202.9, 177.0, 171.4, 128.4, 75.4, 75.2, 74.3, 54.6,
51.8, 48.2, 47.5, 44.9, 44.0, 43.7, 41.1, 41.1, 38.1, 37.7, 31.8, 31.7, 31.1,
29.7, 28.6, 28.3, 26.5, 26.4, 23.5, 19.2, 17.6, 17.6, 16.7; EIMS m/z 516
[M]+ (3), 498 (100), 483 (35), 317 (21), 276 (50).
Evaluation of the Antibacterial Activity in Vitro. Turbidi-
metric assays were performed to determine the MIC values.19 In brief,
the bacterial cultures were seeded in 96-well microculture plates at a
concentration of 1 × 105 CFU/mL. Twofold serial dilutions of the test
compounds were prepared with broth in microplates using DMSO as
the cosolvent, and the DMSO concentration never exceeded 1% (v/v).
Negative (medium containing vehicle without microorganisms) and
normal controls (medium containing vehicle and microorganisms)
were also designed. After sedimentation and measurement of the first
absorbance at 620 nm using an ELISA plate reader, the bacteria were
incubated for an appropriate time with vigorous shaking on a vibrating
platform at the appropriate temperature. The second optical density of
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for the help provided by the staff at the
Analytical Center for Spectral Measurement of the Laboratory
of Chemistry for Natural Products of Guizhou Province and the
Chinese Academy of Sciences. The authors also thank Dr. Y.
Ben-David for the constructive advice provided and for helping
to revise the manuscript and Prof. G.-Y. Zuo at the Kunming
General Hospital of Chengdu Military Area Command for
providing the resistant bacterial strains. The work was
supported by the National Basic Research 973 Program of
the People’s Republic of China (No. 2012CB722601), the
J
DOI: 10.1021/acs.jnatprod.5b00641
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Journal of Natural Products
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J. Nat. Prod. XXXX, XXX, XXX−XXX