Phenanthrenes from Juncus inflexus with Antimicrobial Activity against Methicillin-Resistant Staphylococcus aureus

Article abstract of DOI:10.1021/acs.jnatprod.6b00581

The present study has focused on an investigation of the antibacterial effects of Juncus inflexus and the isolation and identification of its active compounds. Eleven phenanthrenes were isolated from a methanolic extract of the roots. Four compounds (jinflexins A-D, 1-4) are new natural products, while seven phenanthrenes [juncuenins A (5), B (6), and D (8), juncusol (7), dehydrojuncuenins A (9) and B (11), and dehydrojuncusol (10)] were isolated for the first time from the plant. Jinflexin D (4) is a dimer with an unprecedented heptacyclic ring system. The absolute configurations of the new compounds were determined by TDDFT-ECD calculations, and their enantiomeric purity was checked by chiral HPLC analysis. Extracts of different polarity (n-hexane, dichloromethane, and ethyl acetate) were evaluated for their antimicrobial effects against methicillin-resistant Staphylococcus aureus, extended-spectrum β-lactamase (ESBL)-producing Citrobacter freundii, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, multiresistant Acinetobacter baumannii, and Pseudomonas aeruginosa. The MIC values of the isolated compounds were determined by a microdilution method. Jinflexin B (2), juncusol (7), juncuenin D (8), and dehydrojuncuenin B (11) showed significant activity (MIC value range 12.5-100 μg/mL) against MRSA strains.

Full text of DOI:10.1021/acs.jnatprod.6b00581

Article
pubs.acs.org/jnp
Phenanthrenes from Juncus inflexus with Antimicrobial Activity
against Methicillin-Resistant Staphylococcus aureus
†
†
†
‡
‡
‡
́
Barbara Toth, Erika Liktor-Busa, Norbert Kusz, Adam Szappanos, Attila Mandi, Tibor Kurtan,
́
́
́
́
́
Edit Urban,§ Judit Hohmann,†,⊥ Fang-Rong Chang,*,∥,#,¶ and Andrea Vasas*,†
́
†Department of Pharmacognosy and Interdisciplinary Centre of Natural Products, University of Szeged, 6720 Szeged, Hungary
⊥
‡Department of Organic Chemistry, University of Debrecen, 4032 Debrecen, Hungary
§Institute of Clinical Microbiology, University of Szeged, 6725 Szeged, Hungary
∥Graduate Institute of Natural Products, College of Pharmacy, and #Research Center for Environmental Medicine, Kaohsiung Medical
University, 807 Kaohsiung, Taiwan
¶Department of Marine Biotechnology and Resources, National Sun Yat-sen University, 804 Kaohsiung, Taiwan
S
* Supporting Information
ABSTRACT: The present study has focused on an inves-
tigation of the antibacterial effects of Juncus inflexus and the
isolation and identification of its active compounds. Eleven
phenanthrenes were isolated from a methanolic extract of the
roots. Four compounds (jinflexins A−D, 1−4) are new natural
products, while seven phenanthrenes [juncuenins A (5), B (6),
and D (8), juncusol (7), dehydrojuncuenins A (9) and B (11),
and dehydrojuncusol (10)] were isolated for the first time from
the plant. Jinflexin D (4) is a dimer with an unprecedented
heptacyclic ring system. The absolute configurations of the new
compounds were determined by TDDFT-ECD calculations,
and their enantiomeric purity was checked by chiral HPLC
analysis. Extracts of different polarity (n-hexane, dichloro-
methane, and ethyl acetate) were evaluated for their
antimicrobial effects against methicillin-resistant Staphylococcus aureus, extended-spectrum β-lactamase (ESBL)-producing
Citrobacter freundii, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, multiresistant Acinetobacter baumannii, and
Pseudomonas aeruginosa. The MIC values of the isolated compounds were determined by a microdilution method. Jinflexin B (2),
juncusol (7), juncuenin D (8), and dehydrojuncuenin B (11) showed significant activity (MIC value range 12.5−100 μg/mL)
against MRSA strains.
interesting alternatives in antibiotic therapy, because plants
icrobial infections and diseases are one of the leading
Mcauses of deaths worldwide after cardiovascular diseases. produce these compounds in part in order to defend
themselves from microbes.
Staphylococcus aureus is a major pathogen since it possesses
extreme genetic plasticity. Hence, owing to bacterial evolution
and drug abuse, the resistance of S. aureus has increased
dramatically. Methicillin was introduced in therapy in 1959 in
order to treat penicillinase-producing S. aureus infections,1 but
this bacterium rapidly acquired resistance. The first methicillin-
resistant S. aureus (MRSA) strains were identified in 1961 in
the United Kingdom.2 MRSA is becoming resistant to the most
recently developed antibiotics, with the options for the
successful treatment of MRSA being very limited.3 Moreover,
MRSA variants pose a real threat to become untreatable
multidrug-resistant bacteria,4 so there is an urgent need for
novel anti-MRSA agents with new modes of action.
The family Juncaceae comprises approximately 500 species
worldwide, and these are distributed into seven genera.7,8 The
largest genera are Juncus (n = 347) and Luzula (n = 140). There
are 15 Juncus and six Luzula species native to Hungary. Most of
the Juncus species grow exclusively in wetland habitats. In
traditional oriental medicine, the medulla of J. effusus has been
used for the treatment of colds, insomnia, and anxiety.9,10 An
infusion of J. acutus fruits mixed with grains was applied for the
treatment of cold and fever.11
The most characteristic compounds identified in the species
of the family Juncaceae are phenanthrenes and 9,10-
dihydrophenanthrenes. These compounds are derived from a
specific biosynthetic pathway, presumably formed by oxidative
During their evolution, plants developed different defense
mechanisms and systems in order to protect themselves against
abiotic (e.g., UV radiation)5 and biotic stresses (e.g., micro-
organisms).6 Natural products from plants could therefore be
Received: June 24, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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coupling of the aromatic rings of stilbene precursors or by
coupling of two phenylethane units. Thus, the hydroxylation/
methylation patterns of at least one of the two benzene rings in
most phenanthrenes correspond to the 3,5-disubstitution
pattern of stilbenes.12 Phenanthrenes may occur in both
monomeric and dimeric forms and can be substituted with
hydroxy, methoxy, alkyl, formyl, and carboxyl groups.13
Glycosides have been reported only from J. effusus (effusides
I−V).14 Dimeric phenanthrenes are very rare in this genus, with
only seven such compounds having been reported from J.
acutus and four dimers obtained from J. effusus.15−18
Carotenoids, cycloartane triterpenes, diterpenoids, benzocou-
marins, flavonoids, and phenolic compounds also occur in
genera of the Juncaceae.19−23Phenanthrenoids, the major
bioactive compounds of Juncaceae species, show cytotoxic,
antimicrobial, antiviral, antiallergic, anti-inflammatory, spasmo-
lytic, and antialgal activities.13 It has been found that isolated
dihydrophenanthrenes from the marsh plant J. roemerianus have
potential antimicrobial activity against Staphylococcus aureus
(ATCC 12600).6
The purpose of the present study was to investigate the
antibacterial effects of Juncus inflexus L. and to isolate the
bioactive compounds.
RESULTS AND DISCUSSION
■
The dried roots of J. inflexus (3.5 kg) were powdered and
extracted with methanol at room temperature. After concen-
tration, the extract was dissolved in 50% aqueous MeOH, and
solvent−solvent partitioning was performed initially with n-
hexane, then with CH2Cl2, and finally with EtOAc, with the
residue providing a remaining H2O extract. From the most
active CH2Cl2 fraction 11 phenanthrene derivatives (1−11)
were isolated with a combination of different chromatographic
techniques. The structure determinations of the new
compounds (1−4) were carried out by extensive spectroscopic
analysis, using 1D and 2D NMR (1H−1H COSY, HSQC,
HMBC) spectroscopy, HRESIMS experiments, and compar-
ison of the spectroscopic data with literature values.
Compound 1 was obtained as an amorphous solid with [α]D26
0 (c 0.1, MeOH). Its HRESIMS provided the molecular
formula C19H22O3 through the presence of a peak at m/z
321.1532 [M + Na]+ (calcd for C19H22O3Na, 321.1461). The
1H NMR spectrum (Table 1) displayed signals of two ortho-
coupled aromatic protons (δH 6.70 and 6.84 d), one aromatic
Table 1. NMR Spectroscopic Data for Jinflexin A (1) (500 MHz, DMSO-d6) and Jinflexin B (2) (400 MHz, CD3OD)
1
2
position
δH (J in Hz)
δC, type
HMBC (H→C)
δH (J in Hz)
δC, type
125.9, C
1
120.2, C
138.8, C
153.7, C
111.6, CH
126.0, CH
125.3, C
137.5, C
126.8, C
110.5, CH
153.8, C
119.6, C
138.1, C
26.3, CH2
25.3, CH2
11.6, CH3
74.5, CH
23.2, CH3
11.7, CH3
1a
141.7, C
2
157.4, C
3
6.70, d (8.0)
6.84, d (8.0)
1, 4a
6.78, d (9.2)
7.65, d (8.7)
108.2, CH
131.8, CH
129.4, C
4
1a, 2, 4a
4a
5
137.9, C
5a
127.0, C
6
6.86, s
5a, 8
122.2, C
7
155.1, C
8
6.64, s
113.7, CH
138.6, C
8a
9
2.32, 2H, m
2.75, 2H, m
2.13, s
2.59, 2H, m
2.80, 2H, m
4.76, 2H, s
31.4, CH2
26.6, CH2
55.9, CH2
139.2, CH
119.5, CH2
13.6, CH3
10
11
1, 1a, 2
12
4.69, d (4.9)
1.47, d (4.9)
2.10, s
5a, 6, OMe-12
5, 12
6.83 dd (18.6, 11.6)
13
5.43 dd (11.4, 1.1), 5.13 dd (18.0, 1.0)
2.21, s
14
7, 8, 8a
OH
OH
2-OMe
12-OMe
9.23, s
9.17, s
3.84, s
56.1, OCH3
2.85, s
55.0, CH3
12
B
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proton as a singlet (δH 6.86), three methyls, two methylenes,
one sp3 methine, and signals of protons belonging to one
methoxy and two hydroxy groups. In the JMOD (J-modulated
spin−echo experiment) spectrum, the presence of 19 carbon
1
signals was detected (Table 1). In the H NMR spectrum, two
methylene signals at δH 2.32 and 2.75 (2 × 2H) indicated this
compound to be a 9,10-dihydrophenanthrene derivative. In the
1H−1H-COSY spectrum, correlations were observed between
the H-9 and H-10 methylenes, δH 6.70 and 6.84 d (H-3/H-4),
and δH 4.69 and 1.47 d (3H) (H-12/H-13). The methyl
doublet at δH 1.47 and a methine proton at δH 4.69 provided
evidence for the presence of an isolated CH3CH structural unit
1
(C-12, C-13) in the molecule. According to the H and 13C
NMR signals at δH 2.85 and δC 55.0, a methoxy group could be
identified, which was connected to C-12 as confirmed by an
HMBC correlation between OCH3 (δH 2.85) and C-12 (δC
74.5) (Table 1). Moreover, on the basis of HMBC correlations
between C-5/13-Me, C-6/H-12, and C-5a/H-12, this side chain
was placed at C-5. One of the methyl groups (δH 2.13, s) was
placed at C-1 on the basis of its HMBC correlation with the
quaternary carbons at δC 138.8 (C-1a), 120.2 (C-1), and 153.7
(C-2), and the other methyl group was assigned at C-8 on the
basis of the correlations of H3-14/C-8. The hydroxy groups
linked to C-7 and C-2 were confirmed by the chemical shift of
the quaternary carbons (δC‑7 153.8 and δC‑2 153.7). NOESY
correlations confirmed the substitution pattern of compound 1.
Nuclear Overhauser effects were detected between H-3/H-4,
H-4/H-12, H-4/H-13, H3-OMe/H-12, H3-13/H-12, H-6/H3-
13, H-9/H3-14, and H-9/H-10. All of the above evidence
confirmed the planar structure of 1 as 2,7-dihydroxy-1,8-
dimethyl-5-(1-methoxyethyl)-9,10-dihydrophenanthrene,
which has been named jinflexin A.
Figure 1. Structures and populations of the low-energy B3LYP/6-
31G(d) in vacuo conformers (≥2%) of (S)-1.
ECD spectra of this conformer reproduced well the
experimental HPLC-ECD curve of the first-eluting enantiomer,
and conformers B (12.0%) and D (5.3%), differing only in the
orientation of the OH-2 and OH-5 protons, showed near
congruent computed ECDs. In contrast, conformers C (8.6%)
and E (3.3%), having M-helicity with −32.78° and −31.20°
ωC1a,C4a,C5a,C8a torsional angles, respectively, showed mirror-
image computed ECD curves of the experimental ECD (Figure
2, Table 2). Thus, the proper estimation of the population for
Jinflexin A (1) showed a zero specific rotation, and the
baseline ECD spectrum suggested it to be a racemic mixture,
which was also confirmed by the separation of its enantiomers
on a Chiralpak IA column using hexane−propan-2-ol (90:10)
as eluent. The HPLC-ECD spectra of the separated
enantiomers were recorded, which enabled the determination
of the absolute configuration by exploiting the solution
TDDFT-ECD protocol.24,25 In a preliminary Merck Molecular
Force Field (MMFF) conformational search of (S)-1, 31
conformers were generated, which were then reoptimized at
both the B3LYP/6-31G(d) in vacuo and B97D/TZVP26,27
PCM/CHCl3 levels, resulting in five and six low-energy
conformers above a 2% Boltzmann population, respectively.
Jinflexin A (1) has an inherently chiral substituted biphenyl
chromophore, of which the conformational freedom is
restricted by an ethylidene linker. Similar to atropisomeric
biphenyls, the populations of the preferred P- or M-helicity
conformers determine the features of the ECD spectra, which,
in turn, are governed by the absolute configuration of the
benzylic chirality center. According to literature data of
substituted 9,10-dihydrophenanthrene derivatives with an
ortho-tetrasubstituted biphenyl moiety,28,29 P- and M-helicity
conformers can be interconverted at ambient temperature by
flipping the C-9 and C-10. The conformational analysis
identified conformers with both P- and M-helicity of the
biphenyl moiety, which showed near mirror-image computed
ECD curves. The lowest energy B3LYP/6-31G(d) in vacuo
conformer (conformer A, 63.5%) of (S)-1 had P-helicity with a
30.78° ωC1a,C4a,C5a,C8a torsional angle (Figure 1), and the C-12−
H-12 bond of the benzylic chirality center was near coplanar
with the benzene ring (ω12H,C12,C5,C5a = 12.18°). The computed
Figure 2. Experimental HPLC-ECD spectrum of the first-eluting
enantiomer of 1 in hexane−propan-2-ol (9:1) compared with the
Boltzmann-weighted BH&HLYP/TZVP ECD spectrum of (S)-1
computed for the B3LYP/6-31G(d) in vacuo conformers. Bars
represent the rotational strength of conformers A and C.
the P- and M-helicity conformers is fundamental for the
unambiguous assignment of the absolute configuration.30 It was
Table 2. Selected Geometrical Parameters of the Computed
B3LYP/6-31G(d) in Vacuo Conformers of (S)-1
helicity of
distance of 4-H
biphenyl ωC1a,C4a,C5a,C8a ω12H,C12,C5,C5a and 13-H (Å)
conformer A
conformer B
conformer C
conformer D
conformer E
P
30.78°
31.48°
−32.78°
31.69°
12.18°
11.85°
27.27°
11.22°
−12.86°
2.13
2.13
4.18
2.13
4.39
P
M
P
M
−31.20°
C
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found that the distance of the H-4 and H-13 protons is 2.13 Å
in the three P-helicity conformers (A, B, and D), while this
distance is larger than 4.10 Å for the M-helicity conformers (C
and E). Since NOE correlation could be observed between H-4
and H-13 protons and there was no correlation between H-4
and H-15, which would be expected from conformers C and E,
the P-helicity conformers were also confirmed experimentally as
the dominant ones in solution. The B97D/TZVP (PCM/
CHCl3) method showed an even smaller contribution of the M-
helicity forms with a total population of 4.5% from two
conformers (Figure S28, Supporting Information). The ECD
spectra of (S)-1 computed at the B3LYP/TZVP, BH&HLYP/
TZVP, and PBE0/TZVP levels reproduced the experimental
HPLC-ECD spectrum of the first-eluting enantiomer, and thus
the S absolute configuration could be assigned to the
enantiomer with a negative Cotton effect (CE) at 275 nm.
Compound 2 was obtained as an amorphous solid; it gave a
molecular formula of C19H20O3 as determined by HRESIMS,
through the presence of a peak at m/z 297.1392 [M + H]+
(calcd for C19H21O3, 297.1380). The 1H and 13C NMR spectra
suggested a 9,10-dihydrophenanthrene skeleton with vinyl
substitution, similar to that of juncusol (7),31 isolated
previously from other Juncus species (J. acutus, J. effusus, and
J. roemerianus) (Table 1).13 The differences found were that a
hydroxymethyl group instead of the methyl group is linked to
the skeleton at position C-1, and a methoxy group (δH 3.84, δC
56.1) could be placed at C-2 according to its HMBC
correlation with C-2 (δC 157.4). The position of the
hydroxymethyl group was determined by the HMBC cross-
peaks between methylene protons (H-11) and C-1, C-1a, and
C-2. Nuclear Overhauser effects were observed between OMe-
2/H-3, H-3/H-4, H-12/H-14, H-13/H-14, H-8/H-9, and H-
10/H2-11. All this evidence confirmed the structure of 2
(jinflexin B) as 7-hydroxy-6-methyl-1-hydroxymethyl-2-me-
thoxy-5-vinyl-9,10-dihydrophenanthrene.
Table 3. NMR Spectroscopic Data (400 MHz, CD3OD) for
Jinflexin C (3)
position
δH (J in Hz)
δC, type
1
123.1, C
1a
2
140.1, C
159.4, C
3
6.71, d (8.4)
113.6, CH
125.2, CH
126.4, C
4
7.23, d (8.4)
4a
5
2.98, m; 2.72, m
3.88, dd (9.6, 4.5)
33.6, CH2
149.4, C
5a
6
74.4, CH
55.5, C
7
8
201.9, C
8a
9
128.8, C
2.65, m; 2.24, m
2.84, m; 2.57, m
2.15, s
21.1, CH2
25.1, CH2
11.4, CH3
138.1, CH
116.5, CH2
20.2, CH3
10
11
12
13
14
6.10, dd (17.7, 10.8)
5.13, d (10.8), 4.90, d (17.7)
1.32, s
(6S*,7S*) relative configuration of 3 was determined on the
basis of NOE correlations observed between α-oriented H-5
(δH 2.98, m) and H-6 and the β-oriented H-5 (δH 2.72 m) and
H-12.
In contrast to 1, 3 was found to be optically active with a
negative specific rotation, and a distinct ECD spectrum could
be recorded as well. In order to check on the possibility of
partial racemization, chiral HPLC analysis of 3 was carried out
under the same conditions used for 1 (Chiralpack IA, hexane−
propan-2-ol, 90:10), which showed an 80% enantiomeric
excess. HPLC-ECD measurements and HPLC-ECD traces
confirmed the enantiomeric relationship of the separated
components. For the determination of the absolute config-
uration, the same ECD calculation protocol was carried out as
for 1. The initial MMFF conformational search of the arbitrarily
chosen (6S,7S) enantiomer yielded 96 conformers, which were
reoptimized at the B3LYP/6-31G(d) level in vacuo (Figure
S32, Supporting Information) and the B97D/TZVP PCM/
CH3CN level, resulting in 10 and 12 conformers above 2%,
respectively. In the lowest energy B3LYP/6-31G(d) conformer,
the C-7 vinyl group adopted an axial orientation, while OH-6
has an equatorial orientation. This conformation of ring C was
represented by seven other computed conformers totaling
71.1% of the population, and an equatorial C-7 vinyl group was
found in conformers C and J with a total population of 10.1%.
The structures of the low-energy conformers were in agreement
with the observed NOE correlations of 3. The ECD spectra of
(6S,7S)-3 computed for the gas-phase or solvent model
conformers gave mirror-image agreement of the experimental
ECD recorded in acetonitrile (Figure 3), allowing for the
assignment of the absolute configuration for the major
enantiomer (second-eluting enantiomer in the chiral HPLC
analysis) of 3 as (6R,7R).
Compound 3 was isolated as an amorphous, yellow powder,
with [α]2D6 −19 (c 0.2, MeOH). Its HRESIMS provided a
molecular formula of C18H20O3 through the presence of a peak
at m/z 285.1498 [M + H]+ (calcd for C18H21O3, 285.1485).
1
The H and 13C NMR chemical shifts at δH 2.24, 2.65, 2.57,
and 2.84 and δC 21.1 and 25.1 (C-9, C-10) confirmed a
1
dihydrophenanthrene structure of 3. The H NMR spectrum
exhibited two methyl singlets at δH 1.32 and 2.15, two ortho-
coupled aromatic protons at δH 7.23 (J = 8.6 Hz) and 6.71 (J =
8.5 Hz), and a vinylic system at δH 5.13, 4.90, and 6.10 (C-12,
C-13) (Table 3). The 1H−1H-COSY correlations of the
protons at δH 2.72, 2.98, and 3.88 and the chemical shifts of
their carbons (δC 33.6 and 74.4) indicated a −CH2−
CH(OH)− unit (−C-5−C-6−) in the molecule. This structural
part was found to form a keto-substituted six-membered ring
together with quaternary carbons at δC 149.4, 128.8, and 55.5,
according to the long-range correlations between H-5 and C-7,
C-4a, C-8a, C-5a; H-6 and C-12, C-14; and H-12/C-8, as
shown in structural formula 3. The methyl and vinyl
substitution at C-7 was indicated by the HMBC cross-peaks
between H2-13/C-12 and C-7 as well as H3-14 and C-6, C-7, C-
8, and C-12, respectively. In the HSQC spectrum, the protons
of the methyl group at δH 2.15 (H-11) were correlated to the
carbon at δC 11.4 (C-11) and had HMBC correlations with C-
1, C-1a, and C-2. There was a hydroxy group present at
position C-2, which was suggested by the HMBC correlations
between C-2 (δC 159.4)/H-3 and H-4. All the above evidence
obtained confirmed the planar structure of 3 (jinflexin C). The
Compound 4 was isolated as an amorphous solid, with [α]D26
0 (c 0.1, MeOH). The HRESIMS (m/z 513.2448 [M + H]+)
established a molecular formula of C36H32O3 (calcd for
C36H33O3 513.2424), suggesting the dimeric nature of
compound 4. In the JMOD spectrum, the presence of 36
signals was detected (Table 4). The 1H NMR spectrum showed
sharp singlets for the H-11, H-11′, H-14, and H-14′ methyls,
D
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doublets for H-3, H-4, H-9, H-10, H-3′, and H-4′, aromatic
singlets for the H-5 and H-8 protons, and signals for H-12′ and
H-13′ (Table 4). According to the NMR data, one of the
monomers is a tetrasubstituted phenanthrene containing two
methyls and one hydroxy group and a trisubstituted carbon,
while the other part of the molecule is a dihydrophenanthrene
substituted with two methyls and two hydroxy groups. The
linkage between these units was determined on the basis of
1
1H,1H-COSY and HMBC experiments (Figure 4). The H,1H-
Figure 3. Experimental ECD spectrum of 3 in CH3CN compared with
the Boltzmann-weighted PBE0/TZVP ECD spectrum of (6S,7S)-3
computed for the B3LYP/6-31G(d) in vacuo conformers. Bars
represent the rotational strength of the lowest energy conformer.
Figure 4. Diagnostic COSY (bold lines) and HMBC correlations (→)
for 4.
Table 4. NMR Spectroscopic Data (500 MHz, DMSO-d6) for
Jinflexin D (4)
position
δH (J in Hz)
δC, type
HMBC (H→C)
COSY spectrum defined the structural fragment −CHCH2−
CHCH− with the relevant correlated protons (H-12−H-
13−H-13′−H-12′). The H-12 proton was correlated in the
HMBC spectrum with C-5, C-5′, C-6, C-6′, C-7, C-7′, and C-
13′. The long-range correlations of the H-13 protons with the
C-6, C-6′, HC-12, C-12′, and C-13′ carbons also indicated
linkages to occur between C-12/C-6′ and C-13/C-13′ of the
units, forming a new ring system containing a tri- and a
tetracyclic unit. Most probably, during the biosynthesis process,
two vinyl-substituted phenanthrene monomers were connected
through their vinyl groups, producing a new ring. The HMBC
spectroscopic long-range correlations observed between H3-11/
C-1, C-1a, and C-2; H3-14/C-8, C-8a, and C-7; H3-11′/C-1′,
C-1a′, and C-2′; and H3-14′/C-8′, C-8a′, and C-7′ indicated
the C-1, C7, C-1′, and C-7′ positions of methyl groups and the
C-2, C-2′, and C-7′ positions of hydroxy groups, respectively.
The NOESY correlations between H-3/H-4, H-4/H-5, H-8/
H3-14, H-9/H-10, H-10/H3-11, H3-14/H-12 and H-13, H-13/
H-13′, H-12′/H-13′, H-4′/H-12′, H3-11′/H-10′, H9′/H-10′,
and H-9′/H3-14′ confirmed the proposed structure of the
molecule. On the basis of the above findings, the structure of
this compound (jinflexin D) was established as depicted in
structural formula 4.
Although a distinct ECD spectrum for 4 could be recorded in
acetonitrile, chiral HPLC analysis showed only 9% enantio-
meric excess, and the HPLC-ECD spectra of the separated
enantiomers were also recorded. DFT reoptimization of the
initial 40 MMFF conformers generated for the arbitrarily
chosen (R)-4 yielded nine and nine low-energy conformers
(≥2%) at B3LYP/6-31G(d) in vacuo and B97D/TZVP PCM/
CHCl3 levels, respectively. The B97D/TZVP PCM/CHCl3
conformers differed only in the orientation of the OH protons,
while the relative arrangement of the fused tricyclic and
tetracyclic units was near identical (Figure S36, Supporting
Information). The ECD spectra of (R)-4 computed for the
B97D/TZVP PCM/CHCl3 conformers and the Boltzmann-
weighted ECD curve reproduced well the HPLC-ECD
spectrum of the second-eluting enantiomer of 4 (Figure 5),
and it was a mirror image of the experimental solution ECD
spectrum. Thus, the absolute configuration of the first-eluting
enantiomer of 4 was determined unambiguously as R, with the
1
117.1, C
131.6, C
152.9, C
1a
2
3
7.02, d (8.9)
116.4, CH 1, 2, 4a
119.9, CH 1a, 2, 5a
122.7, C
4
7.59, d (9.0)
4a
5
7.69, s
120.3, CH 4a, 7, 12
128.0, C
5a
6
141.6, C
7
133.2, C
8
7.67, s
128.8, CH 5a, 6, 9, 14
128.1, C
8a
9
7.64, d (9.2)
7.74, d (9.2)
2.41, s
126.1, CH 1a, 5a
121.7, CH 1, 1a, 4a
10.9, CH3 1, 1a, 2
10
11
12
13
14
1′
1a′
2′
3′
4′
4a′
5′
5a′
6′
7′
8′
8a′
9′
4.90, d (8.6)
2.85, m; 2.40, m
2.68, s
32.0, CH
5, 5′, 6, 6′, 7, 7′, 13, 13′
28.7, CH2 6, 6′, 12, 12′, 13′
19.3, CH3 6, 7, 8
120.3, C
138.5, C
153.9, C
6.77, d (8.3)
7.13, d (8.3)
111.6, CH 1′, 2′, 4a′
127.1, CH 1a′, 2′, 5a′
125.4, C
128.4, C
125.7, C
124.5, C
149.8, C
121.4, C
136.5, C
2.91, dt (4.3)
2.36, m
26.1, CH2 1a′, 5a′, 8′, 8a′, 10′
10′
2.98, dt (4.1)
2.50, m
25.3, CH2 1′, 1a′, 4a′, 8a′, 9′
11′
12′
13′
14′
2.26, s
11.7, CH3 1′, 1a′, 2′
128.0, CH 6′, 13
6.96, dd (9.7, 2.7)
5.71, ddd (9.7, 7.1, 2.1) 122.8, CH 5′, 12, 13
2.09, s 12.6, CH3 7′, 8′, 8a′
E
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Figure 6. Structures and populations of the low-energy B3LYP/6-
31G(d) in vacuo conformers (≥2%) of (S)-8.
well the HPLC-ECD spectrum of the second-eluting
enantiomer of 8, allowing the determination of absolute
configuration for the separated enantiomers.
Figure 5. Experimental HPLC-ECD spectrum (first-eluting enan-
tiomer) of 4 in hexane−propan-2-ol (9:1) compared with the
Boltzmann-weighted BH&HLYP/TZVP PCM/CHCl3 ECD spectrum
of (R)-4 computed for the B97D/TZVP PCM/CHCl3 conformers.
Bars represent the rotational strength of the lowest energy conformer.
enantiomeric excess derived from the S enantiomer (second-
eluting enantiomer).
Compound 4 may be considered as being derived by the
coupling of 2-hydroxy-1,7-dimethyl-6-vinylphenanthrene (de-
hydrojuncuenin A, 9) with 2,7-dihydroxy-1,8-dimethyl-5-vinyl-
9,10-dihydrophenanthrene through their vinyl groups, forming
an unprecedented heptacyclic structure. Previously, phenan-
threne dimers were published from J. acutus and J. effusus, but
in those compounds either no or only one vinyl group was
found to be involved in the dimerization.15−18
Besides the new compounds (1−4), four dihydrophenan-
threnes [juncuenin A (5), juncuenin B (6), juncusol (7),
juncuenin D (8)], three phenanthrenes [dehydrojuncuenin A
(9), dehydrojuncusol (10), dehydrojuncuenin B (11)], and the
flavonoid chrysoeriol were isolated from the roots of J. inflexus.
Their structures were determined by analysis of their MS and
1D and 2D NMR data and by comparison with literature
values.32−35 In the case of jucuenin B (6), juncusol (7),
juncuenin D (8), dehydrojuncusol (10), and dehydrojuncuenin
B (11) the previously published NMR data were completed
with 1H and 13C NMR data determined in methanol or
dimethyl sulfoxide (see Supporting Information).
Although juncuenin D (8) is a chiral derivative, it had been
isolated previously as a racemic mixture, and thus character-
ization of its enantiomers was not reported.32 The present
chiral HPLC analysis of 8 showed 4% enantiomeric excess,
which was not sufficient to record an acceptable solution ECD
spectrum. Thus, the HPLC-ECD spectra of the separated
enantiomers were recorded and ECD calculations were
performed to assign the absolute configuration. The 13 initial
MMFF conformers of the arbitrarily chosen (S)-8 were
reoptimized at B3LYP/6-31G(d) in vacuo and B97D/TZVP
PCM/CHCl3 levels, resulting in six and nine low-energy
conformers above 2%, respectively. Two groups of the low-
energy B3LYP/6-31G(d) in vacuo conformers (Figure 6) could
be distinguished based on the relative orientation of the vinyl
group. Conformers of the two groups had substantially different
computed ECD curves, as represented by conformers A and B.
The Boltzmann-weighted ECD spectra of the B3LYP/6-
31G(d) in vacuo conformers of (S)-8 (Figure 7) reproduced
Figure 7. Experimental HPLC-ECD spectrum of the second-eluting
enantiomer of 8 in hexane−propan-2-ol (9:1) compared with the
Boltzmann-weighted BH&HLYP/TZVP ECD spectrum of (S)-8
computed for the B3LYP/6-31G(d) in vacuo conformers. Bars
represent the rotational strength of conformers A and B.
All compounds obtained in the present investigation were
isolated for the first time from J. inflexus. The methyl group at
C-1, a hydroxy group at C-2, and the vinyl, methyl, and hydroxy
group substitution on ring B are characteristic features of the
isolated phenanthrenes. In compounds 3 and 8, a carbonyl
group can be found in each molecule. In the case of J. inflexus,
the main compound is juncuenin B (6), occurring at up to
0.024% w/w at the roots. For the new compounds (1, 3, 4) and
8 the absolute configuration was determined by HPLC-ECD
measurements and TDDFT-ECD calculations.
The wetland Juncus plants (e.g., J. acutus, J. roemerianus, and J.
effusus) are considered rich sources of nitrogen-free alkylated
phenanthrenoids; the first example of this class was juncusol
(7) isolated from J. roemerianus.36 To date, more than 90
phenanthrenes were isolated from five Juncus species (J. acutus,
J. effusus, J. roemerianus, J. setchuensis, and J. subulatus).13,37,38
Among them, J. acutus and J. effusus are the most abundant
sources of phenanthrenes.
The n-hexane, dichloromethane, and ethyl acetate fractions
of a methanol extract prepared from the roots of the plant were
evaluated for their antimicrobial effects against methicillin-
F
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Journal of Natural Products
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Bruker Avance DRX 500 NMR spectrometer at 500 MHz (1H) and
125 MHz (13C). The signals of the deuterated solvents were taken as
reference. Two-dimensional (2D) experiments were performed with
standard JEOL or standard Bruker software. In the COSY, HSQC, and
HMBC experiments, gradient-enhanced versions were applied. Two-
dimensional data were acquired and processed with standard JEOL
software. High-resolution MS data were recorded on a Waters-
Micromass Q-TOF Premier mass spectrometer equipped with an
electrospray source. The resolution was over 1 ppm. The data were
acquired and processed with MassLynx software.
HPLC-ECD analysis: Chiral HPLC separations were performed
with a JASCO HPLC system using a Chiralpak IA column with 250
mm × 4.6 mm i.d., 5 μm (Daicel, Chemical Industries, Ltd.), and n-
hexane−propan-2-ol as eluent at a flow rate of 1.0 mL/min. HPLC-UV
and OR chromatograms were recorded with a JASCO MD-910
multiwavelength and OR-2090Plus chiral detector, respectively. The
online ECD and UV spectra were measured simultaneously by
stopping the flow at the UV absorption maximum of each peak. The
values of the ECD ellipticity (ϕ) were not corrected for the
concentration. Three consecutive scans were recorded and averaged
for an HPLC-ECD spectrum with standard sensitivity, 2 nm
bandwidth, and 1 s response. The background HPLC-ECD spectrum
of the eluent was recorded in the same way.
resistant Staphylococcus aureus, extended-spectrum β-lactamase
(ESBL)-producing Citrobacter freundii, Escherichia coli, Enter-
obacter cloacae, Klebsiella pneumoniae, multidrug-resistant
Acinetobacter baumannii, and Pseudomonas aeruginosa. The
dichloromethane fraction possessed high antibacterial activity
(diameter of inhibition zone: 14.6
1.2 mm) against only
methicillin-resistant Staphylococcus aureus, while n-hexane and
ethyl acetate fractions proved to be inactive. The compounds
isolated from this fraction were tested for their anti-MRSA
activity (Table 5). Among the isolated phenanthrenes,
noteworthy inhibitory activities were recorded for jinflexin B
(2), juncusol (7), juncuenin D (8), and dehydrojuncuenin B
(11). Juncuenin D (8, inhibition zone 12.0 0.3 mm, MIC
12.5 μg/mL) and juncusol (7, inhibiton zone 12.0 0.6 mm,
MIC 25 μg/mL) were the most potent in inhibition of MRSA
(ATCC43300) growth, in accordance with previously reported
studies.5,6 Moreover, dehydrojuncuenin B (11, inhibiton zone
10.0 0.2 mm, MIC 25 μg/mL) and jinflexin B (2, inhibiton
zone 7.0
0.1 mm, MIC 100 μg/mL) possessed marked
activity. The other compounds proved to be inactive against
MRSA. In a previous study, the antibacterial activity of
dehydroeffusol and juncusol (7) isolated from J. effusus was
tested against methicillin-resistant and -sensitive S. aureus. It
was observed that the activity of the compounds increased 16-
and 2-fold, respectively, by irradiation with UVA.39
Plant Material. The roots and aerial parts of J. inflexus were
collected in June 2014 from the Southern Great Plain (46°24′14.42″
N 20°20′53.08″ E), Hungary, and identified by Gusztav
́
Jakab
University, Szarvas,
(Institute of Environmental Sciences, Szent Istvan
́
Hungary). A voucher specimen (No. 863) has been deposited at the
Department of Pharmacognosy, University of Szeged.
Extraction and Isolation. The air-dried and powdered roots of J.
inflexus (3.5 kg) were extracted with MeOH (60 L) at room
temperature in a percolator. The crude extract was evaporated under
reduced pressure (393 g) and subjected to solvent−solvent
partitioning first with 3.5 L of n-hexane, then with 5 L of CH2Cl2,
and finally with EtOAc (5 L). After concentration, the CH2Cl2 fraction
(52.81 g) was chromatographed on a polyamide column (150 mm ×
350 mm) with mixtures of MeOH and H2O [2:3, 1:1, 3:2, 4:1, 1:0
(2.5, 5, 10, 40, and 5 L, respectively); each eluent was collected as a
fraction]. The fractions were concentrated and monitored by thin-layer
chromatography using cyclohexane−EtOAc−EtOH (20:10:1) as the
solvent system. According to their TLC chromatograms, the fractions
obtained from the polyamide column with methanol−water (3:2 and
4:1) were similar, so these fractions were combined (J3). The four
fractions (J1−4) obtained from the polyamide column were subjected
to antibacterial screening, and, among them, J3 showed the most
potent inhibitory activity against two MRSA strains (ATCC43300,
Table 5. Anti-MRSA Activity of Isolated Compounds
MRSA (ATCC43300) inhibitory activity
inhibition (diameter of inhibition zone in
mm)
MIC (μg/
mL)
compound
1
inactive
2
7.0 0.1
inactive
100
3
4
inactive
5
inactive
6
inactive
7
12.0 0.6
12.0 0.3
inactive
25
8
12.5
9
10
inactive
11
10.0 0.2
inactive
25
2
-64326) (inhibition zones = 15.3
0.6 and 14.3
0.6 mm,
chrysoeriol
vancomycin
respectively), while J2 (inhibition zones = 7.3
0.6 mm for both
a
15.5 0.6
strains) and J4 (inhibition zones = 7.6 0.6 mm for both strains)
possessed moderate activity, and J1 proved to be inactive. Fraction J3
a
Positive control: vancomycin (5 μg/disk).
(32.9 g) was further chromatographed by VLC (Kieselgel GF254
,
Merck) on silica gel with a gradient system of cyclohexane−EtOAc−
EtOH [from 95:5:0 to 5:5:1 (1000 mL/eluent) and finally with EtOH
(1500 mL); volume of collected fractions 100 mL] to yield the major
fractions (J3/1−14) according to their TLC patterns (detection at 254
and 366 nm and at daylight after staining with vanillin−sulfuric acid
reagent and heating at 120 °C for 5 min).
From fraction J3/3 (35.9 mg), compound 5 (16.2 mg) was
crystallized (from methanol). Fraction J3/4 (71.8 mg) was purified on
a Sephadex LH 20 column using MeOH as eluent (120 mL; volume of
collected fractions 3 mL) to afford compound 9 (21.6 mg).
Compounds 10 (35.5 mg) and 7 (12.2 mg) were purified from
fraction J3/6 (93.7 mg) by preparative TLC on silica gel 60 F254 plates
using cyclohexane−EtOAc (7:3) as solvent system.
Fraction J3/7 (3.6 g) was separated by VLC, by elution with a
gradient system of n-hexane−CH2Cl2−MeOH [from 3:7:0 to 0:97:3
(500 mL/eluent) and finally MeOH (500 mL); volume of collected
fractions 100 mL] to yield six subfractions. Subfraction J3/7/3 (668
mg) was separated by VLC using a gradient solvent system of CHCl3−
acetone [from 99:1 to 4:1 (200 mL/eluent) and finally acetone (200
EXPERIMENTAL SECTION
■
General Experimental Procedures. Vacuum liquid chromatog-
raphy (VLC) was carried out on silica gel G (15 μm, Merck), and
LiChroprep RP-18 (40−63 μm, Merck) was used for reversed-phase
VLC. Column chromatography (CC) was performed on polyamide
(MP Biomedicals) and Sephadex LH-20 (25−100 μm, Pharmacia Fine
Chemicals); preparative thin-layer chromatography (preparative
TLC), on silica gel 60 F254 plates (Merck). Medium-pressure liquid
chromatography (MPLC) was performed by a Biotage SP1
purification system using a KP-C18HS 40+M column. HPLC analyses
were performed with a Shimadzu LC-10AT pump interface equipped
with a Shimadzu SPD-20A UV−vis detector using a Luna phenyl-hexyl
column.
ECD spectra were recorded on a JASCO J-810 spectropolarimeter.
NMR spectra were recorded in CD3OD and DMSO-d6 on a JEOL
ECS 400 MHz FT-NMR spectrometer at 400 MHz (1H) and 100
MHz (13C), with tetramethylsilane as internal standard, and on a
G
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Journal of Natural Products
Article
mL); volume of collected fractions 20 mL] to yield six subfractions.
Furthermore, subfraction J3/7/3/3 (84 mg) was purified by reversed-
phase VLC using a MeOH−H2O gradient elution procedure [from 3:2
to 85:15 (150 mL/eluent) and finally MeOH (150 mL); volume of the
collected fractions 12 mL] to yield six subfractions. Subfraction J3/7/
3/3/4 (12 mg) was purified on a Sephadex LH-20 column, using
MeOH−CH2Cl2 (1:1) as eluent (90 mL; volume of collected fractions
2 mL), to afford compound 4 (4.1 mg). Furthermore, from J3/7/4
(1.1 g) and J3/7/6 (1.5 g) compound 6 was crystallized (from
methanol, 849 mg). Subfraction J3/7/5 (324 mg) was separated on a
Sephadex LH-20 column, using MeOH as eluent (80 mL; volume of
collected fractions 2 mL) to afford compound 1 (52.1 mg).
Fraction J3/11 (370 mg) was subjected to RP-MPLC on a Biotage
SNAP KP-C18HS 40+M column using a gradient system of MeOH−
H2O [from 1:1, 3:2, 4:1 and finally with MeOH (300 mL each), at a
flow rate of 6 mL/min; volume of collected fractions 9 mL] to yield 12
subfractions. Compound 3 (7.5 mg) was obtained from fraction J3/
11/4 (11.0 mg) using preparative TLC on silica gel 60 F254 plates with
CH2Cl2−MeOH (95:5) as solvent system. Subfraction J3/11/8 (23.3
mg) was chromatographed on a Sephadex LH-20 column using the
mobile phase MeOH (100 mL; volume of collected fractions 3 mL) to
afford compounds 2 (6.7 mg) and 8. Final purification of compound 8
(5.2 mg) was performed by RP-HPLC on a Shimadzu LC-10AT
instrument with an SPD-20A UV−vis detector using a Luna phenyl-
hexyl column (250 × 10 mm, 5 μm), eluted with MeOH−H2O (7:3)
at 2 mL/min (tR 21.5 min). Subfraction J3/11/10 (25.2 mg) was
further chromatographed on a Sephadex LH-20 column using the
mobile phase MeOH (200 mL; volume of collected fractions 4 mL) to
obtain compound 11 (3.8 mg).
2-ol, 80:10, λmax (ϕ)} 331 (−7.71), 304 (−7.38), 293sh (6.78), 275
(109.51), 259 (−148.97), 241 (77.25), 210 (−29.04) nm.
Juncuenin B (6): white powder; NMR data were in good agreement
with data published for 6 in CDCl3.32 NMR assignments for CD3OD
solution, made on the basis of 2D measurements, were not published
previously (Supporting Information).
Juncusol (7): amorphous solid; 1H NMR (CD3OD, 500 MHz)
(data measured for the first time in this solvent, Supporting
Information).
Juncuenin D (8): yellow powder; UV (CH3CN) λmax (log ε) 347
(3.88), 275sh (3.85), 251 (4.04), 213 (4.39) nm; 1H NMR (CD3OD,
400 MHz) and 13C NMR (CD3OD, 100 MHz) (data measured for the
first time in this solvent, Supporting Information).
(R)-8: tR 4.72 min on Chiralpak IA column (hexane−propan-2-ol,
80:10); HPLC-ECD {hexane−propan-2-ol, 90:10, λmax (ϕ)} 376
(−24.82), 275.5 (22.63), 258 (−20.83), 238 (9.32) nm; (S)-8: tR 5.20
min on Chiralpak IA column (hexane−propan-2-ol, 80:10); HPLC-
ECD {hexane−propan-2-ol, 90:10, λmax (ϕ)} 376 (29.86), 275.5
(−22.40), 258 (15.23), 238 (−9.46) nm. 4% ee as determined by
chiral HPLC.
Dehydrojuncusol (10): amorphous solid; 1H NMR (DMSO-d6, 400
MHz) and 13C NMR (DMSO-d6, 100 MHz) (data measured for the
first time in this solvent, Supporting Information).
1
Dehydrojuncuenin B (11): brown, amorphous powder; H NMR
(DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) (data
measured for the first time in this solvent, Supporting Information).
In the case of juncuenin A (5) and dehydrojuncuenin A (9), the
spectroscopic data obtained were identical with those previously
published.32
Test Microorganisms. The test microorganisms were one
standard and nine clinical isolates with different antibiotic resistance
profiles, which originated from various departments of Albert Szent-
Fraction J3/12 (405 mg) was further separated by reversed-phase
VLC with a gradient system of MeOH−H2O [from 3:7 to 9:1 (100
mL/eluent) and finally MeOH (200 mL); volume of collected
fractions 15 mL] to yield 10 subfractions. From subfraction 4,
chrysoeriol (6.1 mg) was isolated by the use of preparative TLC, using
CH2Cl2−MeOH (95:5) as solvent system.
Gyorgyi Health Center and were identified at the Institute of Clinical
̈
Microbiology at University of Szeged by conventional methods. The
standard strain was methicillin-resistant Staphylococcus aureus
(ATCC43300). The clinical strains were multiresistant (MR)
Acinetobacter baumannii (64060/2 and 61748/2), ESBL-positive
Citrobacter freundii (63458), ESBL-positive Enterobacter cloacae
(63033), ESBL-positive Escherichia coli (64663), ESBL-positive
Klebsiella pneumoniae (63735), MR Pseudomonas aeruginosa (61485/
1 and 64658), and methicillin-resistant S. aureus (64326). Microbial
cultures were grown on standard Mueller-Hinton agar plates and
maintained at 4 °C throughout the study to use as stock cultures.
Antibacterial Screening. Antibacterial activities of the fractions
and the pure compounds were first screened for their inhibitory zones
by a disk-diffusion method. The plant extracts were prepared at 50
mg/mL, while pure compounds were diluted to 10 mg/mL using
DMSO. The sterile filter paper disks (6 mm diameter) impregnated
with the extracts (50 μL) were placed on the agar plate seeded with
the respective bacterial suspension (inoculums 0.5 McFarland, (1−2)
× 108 CFU/mL). The solvent served as a negative control. The plates
were then incubated at 37 °C for 24 h under aerobic conditions. The
diameters of inhibition zones produced by the plant extracts (including
the disk) were measured and recorded. All experiments were carried
out in triplicate.
( )-Jinflexin A (1): amorphous solid; [α]2D6 0 (c 0.1, MeOH); UV
1
(CH3CN) λmax (log ε) 276 (4.01), 211 (4.50) nm; H and 13C NMR
data, see Table 1; (+)-HRESIMS m/z 321.1532 [M + Na]+ (calcd for
C19H22O3Na, 321.1461).
(S)-1: tR 5.51 min on a Chiralpak IA column (hexane−propan-2-ol,
90:10); HPLC-ECD {hexane−propan-2-ol, 90:10, λmax (ϕ)} 308
(1.51), 292sh (−8.37), 275 (−15.91), 233.5 (63.95), 210 (−31.40)
nm; (R)-1: tR 5.93 min on a Chiralpak IA column (hexane−propan-2-
ol, 90:10); HPLC-ECD {hexane−propan-2-ol, 90:10, λmax (ϕ)} 308
(−1.32), 292sh (6.80), 275 (13.72), 233.5 (−52.72), 210 (19.89) nm.
Jinflexin B (2): amorphous solid; UV (MeOH) λmax (log ε) 280
(3.81), 219 (3.99) nm; 1H and 13C NMR data, see Table 1;
+
(+)-HRESIMS m/z 297.1392 [M + H]+ (calcd for C19H21O3 ,
297.1380).
Jinflexin C (3): amorphous, yellow powder; [α]2D6 −19 (c 0.2,
MeOH, 80% ee); UV (CH3CN) λmax (log ε) 329 (3.75), 242 (3.52),
217sh (3.63), 195sh (4.06) nm; ECD {CH3CN, λmax (Δε)} 360
(1.36), 351sh (1.02), 319 (−1.35), 255 (−1.22), 228 (1.13), 209
(−0.77) nm; 1H and 13C NMR data, see Table 3; (+)-HRESIMS m/z
+
285.1498 [M + H]+ (calcd for C18H20O3 , 285.1485).
(6R,7R)-3 tR = 15.48 min on a Chiralpak IA column (hexane−
propan-2-ol, 90:10); (6S,7S)-3: tR = 8.44 on a Chiralpak IA column
(hexane−propan-2-ol, 90:10).
The active test compounds with diameters of inhibition zone of ≥10
mm were further investigated to determine their minimal inhibitory
concentration (MICs) by a microdilution method. Briefly, in 96-well
plates, the stock solutions of the compounds (50 mg/mL in DMSO)
were serially diluted with Mueller-Hinton broth to arrive at final
concentrations between 2.5 mg/mL and 4.9 μg/mL. Then, 100 μL of
inoculum (0.5 McFarland, (1−2) × 108 CFU/mL) was then added to
the wells. A sterility check (medium and DMSO in amounts
corresponding to the highest concentration), negative control
(medium, DMSO, and inoculum), and positive control (medium,
DMSO, inoculum, and vancomycin) were included for each
experiment. The plates were then incubated at 37 °C for 24 h
under an aerobic environment. The MIC of each compound was the
lowest concentration that completely inhibited the visible bacterial
growth. All experiments were performed twice in triplicate.
Jinflexin D (4): amorphous solid; [α]2D6 0 (c 0.1, MeOH, 9% ee); UV
(CH3CN) λmax (log ε) 361 (3.30), 321sh (3.64), 306sh (4.08), 281sh
(4.33), 271sh (4.55), 263 (4.64), 226sh (4.53) nm; ECD {CH3CN,
λmax (Δε) 331 (−0.12), 301 (−0.24), 288sh (0.10), 275 (1.94), 260
1
(−2.86), 241 (1.10), 202 (−1.51) nm; H and 13C NMR data, see
+
Table 4; (+)-HRESIMS m/z 512.2299 [M + H]+ (calcd for C36H32O3
512.2346).
(R)-4: tR 4.38 min on Chiralpak IA column (hexane−propan-2-ol,
80:10); HPLC-ECD {hexane−propan-2-ol, 80:10, λmax (ϕ)} 331
(6.44), 304 (6.28), 293sh (−6.50), 275 (−103.43), 259 (151.40), 241
(−69.62), 210 (27.05) nm; (S)-4: tR 4.77 min on a Chiralpak IA
column (hexane−propan-2-ol, 80:10); HPLC-ECD {hexane−propan-
H
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Computational Section. Mixed torsional/low-frequency mode
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implicit solvent model for CHCl3.40 Geometry reoptimizations were
carried out at the B3LYP/6-31G(d) level in vacuo and the B97D/
TZVP level26,27 with the PCM solvent model for CH3CN or CHCl3.
TDDFT ECD calculations were run with various functionals (B3LYP,
BH&HLYP, PBE0) and the TZVP basis set as implemented in the
Gaussian 09 package with the same or no solvent model as in the
preceding DFT optimization step.41 ECD spectra were generated as
sums of Gaussians with 3000 cm−1 widths at half-height (correspond-
ing to ca. 24 at 280 nm), using dipole-velocity-computed rotational
strength values.42 Boltzmann distributions were estimated from the
ZPVE-corrected B3LYP/6-31G(d) energies in the gas-phase calcu-
lations and from the B97D/TZVP energies in the solvated ones. The
MOLEKEL software package was used for visualization of the
results.43
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AUTHOR INFORMATION
Corresponding Authors
■
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*Tel (F.-R. Chang): +886-7-3121101 (ext. 2162). Fax: +886-7-
3114773. E-mail: aaronfrc@kmu.edu.tw.
*Tel (A. Vasas): +36-62-546451. Fax: +36-62-545704. E-mail:
vasasa@pharmacognosy.hu.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
Financial support from the Hungarian Scientific Research Fund
(OTKA K109846) is gratefully acknowledged. A.V. acknowl-
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edges the award of a Janos Bolyai scholarship of the Hungarian
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Academy of Sciences. T.K. thanks the Hungarian National
Research Foundation (OTKA K105871) for financial support
and the National Information Infrastructure Development
Institute (NIIFI 10038) for CPU time. This work was
supported by grants from the Ministry of Science and
Technology of Taiwan (NSC 102-2628-B-037-003-MY3,
MOST 103-2320-B-037-005-MY2, awarded to F.-R.C.). This
study is also supported partially by Kaohsiung Medical
University (Aim for the Top Universities Grant, nos. KMU-
TP104E39, KMU-TP104A26), Ministry of Health and Welfare
of Taiwan (MOHW105-TDU-B-212-134007), and Health and
Welfare Surcharge of Tobacco Products. The authors thank G.
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