Alkaloids from the Chinese vine gnetum montanum

Article abstract of DOI:10.1021/np200700f

During a high-throughput screening campaign of a prefractionated natural product library, fractions from the Chinese vine Gnetum montanum showed in vitro activity against Pseudomonas aeruginosa wild-type strain, PAO1. UV-directed isolation of the organic extract from the vine leaves resulted in the purification of the new natural products N-methyllaudanosolinium trifluoroacetate (1), 3′-hydroxy-N,N-dimethylcoclaurinium trifluoroacetate (2), 1,9,10-trihydroxy-2-methoxy-6-methylaporphinium trifluoroacetate (3), and 6a,7-didehydro-1,9,10-trihydroxy-2-methoxy-6-methylaporphinium trifluoroacetate (4). Compound 4 is described here for the first time, and this is the first report of compounds 1-3 as natural products. Compounds 1-3 were found to racemize over time. Starting from commercially available (+)-boldine, through a series of semisynthetic reactions, a mechanism for the racemization of the isolated compounds is proposed. The known natural products (-)-latifolian A (5) and magnocurarine (6) were also isolated during these studies. The antibacterial activity was explained by the presence of 5, which displayed an IC₅₀ value of 9.8 μM (MIC = 35 μM).

Full text of DOI:10.1021/np200700f

Article
pubs.acs.org/jnp
Alkaloids from the Chinese Vine Gnetum montanum
Frederic Martin, Tanja Grkovic, Melissa L. Sykes, Todd Shelper, Vicky M. Avery, David Camp,
́
́
Ronald J. Quinn,* and Rohan A. Davis
Eskitis Institute, Griffith University, Brisbane, QLD 4111, Australia
S
* Supporting Information
ABSTRACT: During a high-throughput screening campaign
of a prefractionated natural product library, fractions from the
Chinese vine Gnetum montanum showed in vitro activity
against Pseudomonas aeruginosa wild-type strain, PAO1. UV-
directed isolation of the organic extract from the vine leaves
resulted in the purification of the new natural products N-
methyllaudanosolinium trifluoroacetate (1), 3′-hydroxy-N,N-
dimethylcoclaurinium trifluoroacetate (2), 1,9,10-trihydroxy-2-
methoxy-6-methylaporphinium trifluoroacetate (3), and 6a,7-
didehydro-1,9,10-trihydroxy-2-methoxy-6-methylaporphinium
trifluoroacetate (4). Compound 4 is described here for the first
time, and this is the first report of compounds 1−3 as natural products. Compounds 1−3 were found to racemize over time.
Starting from commercially available (+)-boldine, through a series of semisynthetic reactions, a mechanism for the racemization
of the isolated compounds is proposed. The known natural products (−)-latifolian A (5) and magnocurarine (6) were also
isolated during these studies. The antibacterial activity was explained by the presence of 5, which displayed an IC50 value of 9.8
μM (MIC = 35 μM).
pecies that comprise the group of nonfermenting Gram-
large-scale organic extract from the leaves of G. montanum
Snegative bacteria pose a major risk for health care, as many afforded the new benzylisoquinoline alkaloids N-methyllauda-
possess multidrug resistance.1 Such species include Pseudomo-
nas aeruginosa, Straphylococcus maltophilia, and Acinetobacter
spp., which are opportunistic pathogens and prominent in
critically ill, hospitalized patients.2 Multidrug resistance is
genetically acquired by these pathogens and is primarily due to
the active transport of drugs out of the cell by efflux pump
systems.3,4 In addition, intrinsic resistance further decreases the
efficacy of clinically used treatments, such as ampicillin,
cephalosporins, and macrolide antibiotics, mainly due to
impermeability.1,5 Bacterial resistance, particularly among
Gram-negative organisms, has increased our reliance upon
drugs that have poorer safety profiles.6 These facts highlight the
urgent need to discover new, selective antibiotics with low
toxicity and new mechanisms of action. Natural products
constitute a promising source of new lead compounds or drugs
for antibacterial treatments since most existing antibiotics are
based on natural chemotypes.7
A high-throughput screening (HTS) campaign was under-
taken on the P. aeruginosa efflux pump knockout strain,
PAO200 (MexAB-OprM deficient mutant), in an effort to
increase initial hits by decreasing efflux clearance. HTS hits
were retested against the wild-type strain PAO1 and a Gram-
positive methicillin-resistant bacterium, Staph. aureus (MRSA
strain 01A1095),8 for estimation of Gram selectivity. Three
fractions derived from the Chinese vine Gnetum montanum
showed in vitro activity against P. aeruginosa PAO200. These
fractions eluted early from an analytical C18 HPLC column and
displayed prominent UV profiles. UV-directed isolation on the
nosolinium trifluoroacetate (1) and 3′-hydroxy-N,N-dimethyl-
coclaurinium trifluoroacetate (2), the new aporphine alkaloids
1,9,10-trihydroxy-2-methoxy-6-methylaporphinium trifluoroa-
cetate (3) and 6a,7-didehydro-1,9,10-trihydroxy-2-methoxy-6-
methylaporphinium trifluoroacetate (4), and the known
alkaloids (−)-latifolian A (5) and magnocurarine (6). The
antibacterial activities of compounds 1−6 against P. aeruginosa
PAO1 and Staph. aureus 01A1095 are also reported.
RESULTS AND DISCUSSION
■
The air-dried and ground leaves of G. montanum (Gnetaceae)
were sequentially extracted with n-hexane, CH2Cl2, and MeOH.
The n-hexane extract was discarded, and the remaining organic
extracts were combined and fractionated on polyamide gel
using MeOH as eluent. The resulting MeOH wash was
subjected to several steps of C18 semipreparative HPLC
[MeOH/H2O (0.1% TFA)] to yield 1 (6.1 mg, 0.24% dry
wt), 2 (7.2 mg, 0.17% dry wt), 3 (6.3 mg, 0.15% dry wt), 4 (1.3
mg, 0.03% dry wt), 5 (3.6 mg, 0.09% dry wt), and 6 (6.4 mg,
0.15% dry wt). All alkaloids were isolated as their
trifluoroacetate salts. Compounds 5 and 6 were assigned to
the known compounds (−)-latifolian A and racemic
magnocurarine, respectively, following comparison of spectro-
scopic data.9,10 (−)-Latifolian A was previously isolated from G.
Received: August 26, 2011
Published: October 31, 2011
© 2011 American Chemical Society and
American Society of Pharmacognosy
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Chart 1
a
latifolium and exhibited inhibition in an assay against the JNK3
kinase.10 Magnocurarine has been isolated from a variety of
plants and has been reported to display weak antinociceptive
activity.11
Table 1. NMR Data for Compounds 1 and 2
1
2
δH mult (J in
δH mult (J in
position
δC mult
Hz)
δC mult
Hz)
Compound 1 was isolated as a brown gum, and the
molecular formula of the quaternary ammonium cation of 1 was
1
71.3 CH
4.55 dd (9.4,
2.9)
71.2 CH
4.59 dd (9.3,
2.8)
determined to be C18H22NO4 by HR-ESIMS ([M − TFA]+:
+
3
4
54.3 CH2
22.4 CH2
α 3.54 m
β 3.68 m
α 2.97 m
β 2.97 m
54.3 CH2
23.8 CH2
α 3.58 m
β 3.71 m
α 3.05 m
β 3.05 m
1
m/z 316.1544). Analysis of the H NMR and gCOSY spectra
(Table 1) revealed the presence of two isolated aromatic
singlets at δH 6.58 and 5.94, one aromatic ABX system (δ H
6.65, d, J = 8.0 Hz; δH 6.53, d, J = 1.9 Hz; and δH 6.35, dd, J =
8.0, 1.9 Hz), two N+Me groups (δH 3.23 and 3.03), two
contiguous methylene groups (δH 3.68/3.54 and 2.97), and
one methine-methylene system (δH 4.55/3.44, 2.68). Fur-
thermore, correlations observed in the gHSQC and gHMBC
spectra showed the presence of two different substituted
aromatic rings (rings A and C), both of which possessed a
catechol substructure [δC 143.6 and 145.5 (ring A), 145.2 and
144.1 (ring C)]. The two isolated aromatic singlets (δ H 6.58/
δC 114.7 and δH 5.94/δC 114.5) were positioned para to each
other on ring A on the basis of their overlapping HMBC
correlations. The aromatic protons in the ABX system could be
assigned to carbons that belonged to ring C (δ C 115.2, 116.6,
and 119.9) following HSQC analysis. The two last carbons
belonging to ring A were quaternary (δ C 118.9 and 121.6). The
carbon at δC 118.9 showed HMBC correlations with the two
contiguous methylenes (δC 54.3 and 22.4), while the carbon at
δC 121.6 displayed HMBC correlations with the methine-
methylene system (δC 71.3 and 36.5). Moreover the protons in
both N+Me groups showed HMBC correlations with the
carbons at δC 71.3 and 54.3, to generate a closed ring and
deliver an isoquinolinium moiety. Furthermore, correlations
between the carbon at δC 36.5, present in the methine-
methylene system, with protons belonging to ring C confirmed
that 1 was a benzylisoquinoline derivative. These two systems
were connected through a quaternary carbon at δC 127.0.
Compound 1 was thus assigned to N-methyllaudanosolinium
trifluoroacetate. The synthetic preparation of the iodide salt of
1 has previously been reported but was only partially
charaterized.12 We report here the full spectroscopic character-
ization of 1.
4a
118.9 C
114.7 CH
143.6 C
119.2 C
113.3 CH
147.7 C
5
6.58 s
9.11 s
6.79 s
6
6-OH
7
145.5 C
145.5 C
7-OH
8.84 s
5.94 s
8.89 s
5.95 s
8
114.5 CH
121.6 C
114.5 CH
123.3 C
8a
α
36.5 CH2
3.44 dd (13.9,
2.9)
36.4 CH2
3.46 dd (13.9,
2.8)
2.68 dd (13.9,
9.4)
2.68 dd (13.9,
9.3)
1′
127.0 C
116.6 CH
145.2 C
127.0 C
116.5 CH
145.7 C
2′
6.53 d (1.9)
8.77 s
6.52 d (1.5)
8.75 s
3′
3′-OH
4′
144.1 C
143.9 C
4′-OH
5′
8.92 s
8.91 s
115.2 CH
119.9 CH
6.65 d (8.0)
115.2 CH
119.9 CH
6.65 d (8.0)
6′
6.35 dd (8.0,
1.9)
6.35 dd (8.0,
1.5)
−N+Me2
50.2 CH3
50.0 CH3
3.23 s
3.03 s
50.3 CH3
50.0 CH3
55.1 CH3
3.25 s
3.04 s
3.75 s
−OMe
a
Spectra were recorded in DMSO-d6 at 30 °C.
Comparison of the NMR and UV data of the major
metabolite 2 with 1 clearly identified that 2 also belonged to
the benzylisoquinoline structure class. The major difference in
the NMR spectrum of 1 was that one of the hydroxy moieties
had been replaced by a methoxy group in 2 (δH 3.75/δC 55.1).
This was confirmed by HR-ESIMS ([M − TFA]+: m/z
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330.1691), which indicated that the molecular formula of the
quaternary ammonium cation of 2 was C19H24NO4 . The
correlations were observed from this signal to two oxygenated
carbons (δC 148.1 and 142.1) and a quaternary aromatic
carbon at δC 119.5. The carbon at δC 119.5 showed HMBC
correlations to both contiguous methylene protons (with
carbons at δC 23.1 and 60.4) as well as the methine-methylene
system with carbons at δC 68.3 and 28.1. The carbon at δC 28.1
also displayed correlations with the two other aromatic proton
singlets belonging to ring D. The four remaining carbons of 3
were determined to be two ortho-substituted (δC 144.6 and
143.7) and quaternary (δ C 123.4 and 122.5) carbons following
HMBC data analysis. Finally the aromatic proton at δH 7.85
displayed a correlation with a quaternary carbon at δC 120.4,
which belongs to ring A, validating the aporphine skeleton. The
methoxy group of 3 was positioned at C-2 following 2D NMR
data analysis. The methoxy protons (δ H 3.85) showed a strong
HMBC correlation with the C-2 phenolic carbon (δ C 148.1)
and showed a ROESY correlation to the aromatic proton H-3
(δ H 6.78). Compound 3 was thus assigned as the
trifluoroacetate salt of 1,9,10-trihydroxy-2-methoxy-6-methyl-
aporphinium. This is the first report of 3 as a natural product.
The synthesis of 3 has been reported by the same group that
+
methoxy position was determined following gHMBC analysis.
A strong correlation was observed between the methoxy
protons and the carbon C-6 (δC 147.7). Moreover, the ROESY
spectrum showed a strong correlation between the methoxy
protons and the aromatic proton H-5 (δ H 6.79). The structure
of compound 2 was therefore assigned as the trifluoroacetate
salt of 3′-hydroxy-N,N-dimethylcoclaurinium. This is the first
report of 2 as a natural product. As with compound 1, the
synthesis of 2 has been previously reported.13 The complete
NMR assignment of 2 is presented in Table 1.
Compound 3 exhibited a markedly different UV spectrum
from that of 1 and 2. Moreover, the molecular formula of the
+
quaternary ammonium cation of 3 (C19H22NO4 ) obtained
from the HR-ESIMS ([M − TFA]+: m/z 328.1532) indicated
that 3 was a didehydro analogue of 2. Analysis of the 1H NMR
spectrum (Table 2) showed three aromatic singlets at δH 7.85,
a
Table 2. NMR Data for Compounds 3 and 4
synthesized 2.13 Our data were consistent with the H NMR
3
4
1
δH mult (J
in Hz)
and UV spectra from the literature.
position
δC mult
δH mult (J in Hz)
δC mult
In order to determine the absolute configuration of
compounds 1−3, we measured their optical rotation and
ECD spectra with the intention to compare against the
reported spectra of natural products of known absolute
configuration, such as (+)-roefractine,16 (+)-laudanosine,17
and (+)-reticuline.18 We also obtained commercially available
(+)-boldine for comparison. The recorded ECD spectra of
compounds 1−3 all showed an absence of Cotton effects,
indicating that the isolated natural products were racemic.
Reports of naturally occurring racemates of benzylisoquinoline
and aporphine-type alkaloids are not commonly found in the
literature. Racemic isococlaurine trifluoroacetate has been
reported from Cimmampelos mucronata,19 and the racemic
perchlorate salt of xylopinidine has been identified as a
constituent of Xylopia parviflora.20 Interestingly, in both
publications the authors had also isolated a number of other
chiral benzylisoquinoline alkaloids.
1
142.1 C
142.9 C
b
1-OH
1a
1b
2
9.58 s
120.4 C
119.5 C
148.1 C
109.0 CH
119.1 C
23.1 CH2
119.3 C
115.3 C
147.6 C
111.3 CH
118.8 C
23.7 CH2
3
6.78 s
7.40 s
3a
4
α 2.93 m
β 3.18 m
3.47 t (5.9)
4.02 t (5.9)
5
60.4 CH2
α 3.65 ddd (12.9,
60.9 CH2
12.9, 4.5)
β 3.74 dd (12.9,
5.8)
6a
7
68.3 CH
28.1 CH2
4.56 dd (13.7, 2.3)
135.7 C
α 3.16 dd (13.7,
116.3 CH
8.10 s
7.30 s
2.3)
β 2.73 dd (13.7,
13.7)
With the intent to examine whether the racemic quaternary
benzylisoquinoline and aporphine alkaloids identified in this
study were artifacts of the isolation procedure, we first reacted
commercially available (+)-boldine with an excess of MeI
according to a known literature procedure.21 The crude
reaction mixture was chromatographed on a semipreparative
7a
123.4 C
114.9 CH
143.7 C
124.3 C
112.7 CH
143.8 C
8
6.71 s
9
b
c
9-OH
10
9.73
144.6 C
146.5 C
b
c
10-OH
11
9.74
C
18 HPLC column using a gradient from H2O to MeOH (0.1%
116.3 CH
122.5 C
7.85 s
113.6 CH
124.1 C
9.22 s
TFA) to give N-methylboldine trifluoroacetate (7) as well as a
small amount of N-methylsecoboldine trifluoroacetate (8) and
N,N-dimethylsecoboldine trifluoroacetate (9). Compound 7
initially showed an ECD spectrum comparable to that of
(+)-boldine; however, racemization was observed in a MeOH
(0.1% TFA) solution over a period of one week, as shown by a
complete loss of the ECD Cotton effects (see Supporting
Information).
Lee et al. have reported solvolysis of 2-hydroxyaporphines to
phenanthrene-type alkaloids via reflux in a 80% solution of
propionic acid.22 The authors proposed a mechanism where the
dissociated acid abstracts a proton from the acidic 2-
hydroxyaporphine and opens the B ring, followed by
deprotonation at C-9 to give a stable phenenthrene product.
We propose that the quaternary trifluoroacetates of benzyliso-
quinoline and aporphine alkaloids isolated in this study can
11a
−N+Me2
52.9 CH3
42.8 CH3
55.9 CH3
3.34 s
2.93 s
3.85 s
54.5 CH3
54.5 CH3
3.64 s
3.64 s
3.99 s
−OMe
56.4 CH3
b
a
Spectra were recorded in DMSO-d6 at 30 °C. Signals not observed.
c
Interchangeable signals.
6.78, and 6.71. Signals were still apparent for the two
contiguous methylenes (δH 3.74/3.65 and 3.18/2.93), the
methine-methylene system (δH 4.56/3.16, 2.73), the two
N+Me groups (δH 3.34 and 2.93), and the methoxy group (δ H
3.85). These signals are typical for aporphine derivatives14 and
aporphinium salts.15 The correlations observed in the gHSQC
and gHMBC spectra confirmed the structure. The isolated
singlet at δH 6.78 (δC 109.0) was attached to ring A. HMBC
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Figure 1. Proposed mechanism for the racemization of 7 and 3.
rearrange via a similar mechanism as shown in Figure 1.
Nucleophilic attack of the nitrogen at C-10 is favored over
deprotonation at C-9 in the intermediate and accounts for the
racemization of the aporphine alkaloids isolated in this work.
The minor plant metabolite 4 was isolated as an optically
inactive gum. The molecular formula of the quaternary
xylem, a feature more commonly found in the angiosperms.
Plants of this genus include trees, shrubs, and vines and are
found in Africa, Asia, and Central and South America. The
Gnetum genus is a well-known source of polyphenols, which are
predominantly stilbenoid derivatives.24 To date, only 10
alkaloids have been reported.25 G. montanum is a vine that
can reach more than 10 m in length. It can be found in the
forests of southern China, as well as in other South-Asian
countries (Bhutan, India, Laos, Myanmar, Thailand, and
Vietnam). Fibers from the stem bark have been used for
making bags, fishing nets, and ropes. The seeds can be eaten
fried and produce an edible oil.26 This plant has been used as a
traditional medicine in China to treat arthritis and bronchitis.
G. montanum has been the source of stilbenes, dimeric
stilbenes, flavonoids, and triterpenes.27
Antibacterial screening of compounds 1−6 against P.
aeruginosa PAO1 showed that compound 5 displayed moderate
inhibitory activity against P. aeruginosa (PAO1), with an IC50
value of 9.8 μM (MIC 35 μM). Compounds 3 and 4 displayed
weak activities (59% inhibition at 175 μM and 59% inhibition
at 87.5 μM, respectively), while 1, 2, and 6 were not active at
350 μM. It is worth noting that both aporphine derivatives were
slightly active, while the three benzylisoquinolines were
inactive. In order to further evaluate the potential antibacterial
activity of 1−6, all compounds were tested against the Gram-
positive methicillin-resistant bacterium Staph. aureus (MRSA
strain 01A1095).8 Compound 5 displayed 55% growth
inhibition at 350 μM, while 1−4 and 6 showed no activity at
350 μM. These data identified that 5 showed a ∼36-fold
+
ammonium cation of 4 (C19H20NO4 ) was obtained following
HR-ESIMS analysis ([M − TFA]+: m/z 326.1382). The
molecular formula difference between 4 and 3 was two
hydrogen atoms, which indicated that 4 was an oxidized
analogue of 3. Inspection of the 1H NMR spectrum of 4 (Table
2) clearly showed the disappearance of the signals belonging to
the methine-methylene system. The aromatic portion of the
spectrum also showed major modifications. Four aromatic
singlets were detected (δ H 9.22, 8.10, 7.40, and 7.30), with
downfield chemical shifts in comparison with the aromatic
proton signals of 3. These data suggested that 4 belonged to the
6a,7-didehydro-aporphine structure class. The other proton
signals of 4 included a N+Me singlet (δH 3.64, 6H), a methoxy
singlet (δH 3.99, 3H), and a methylene-methylene system (δ H
4.02 and 3.47). The correlations observed in the gHSQC and
gHMBC spectra were in accordance with this structure class.23
The methoxy group was positioned at C-2 (δ C 147.6) on the
basis of HMBC and ROESY correlations. With the position of
the methoxy group established, the structure of 4 was assigned
as the trifluoroacetate of 6a,7-didehydro-1,9,10-trihydroxy-2-
methoxy-6-methylaporphinium.
Gnetum is currently the only genus belonging to the family
Gnetaceae. These gymnosperms have vessel elements in their
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MI, USA), from concentrated frozen stocks of culture. The final
bacterial concentration in the assay was 1500 cfu/well. Diluted bacteria
(45 μL) were added to a 384-well lidded, sterile clear plate (Becton
Dickinson, Franklin Lakes, NJ, USA) containing controls/fractions by
a Multidrop liquid handler (Thermo Scientific, Barrington, IL, USA).
Plates were incubated at 37 °C in a humidified incubator for 18 h or
until the wells reached an optical density (OD620) of between 0.7 and
0.8, then allowed to cool for 30 min. Clear plate seals (Perkin-Elmer,
Meriden, CT, USA) were placed over the plate surface before reading
on a Multiskan Ascent reader (Thermo Scientific) at 620 nm.
Test fractions, compounds, or control samples (5 μL) were added
to the assay plate prior to the addition of bacteria. Samples were
prepared by dilution of stock fractions/compounds/controls in DMSO
into the assay plate with addition of 0.875 μL of stock and 4.125 μL of
autoclaved Milli-Q filtered H2O with a Minitrak (Perkin-Elmer,
Meriden, CT, USA) liquid handler. The final concentration of DMSO
in the assay was 1.75%. Each assay plate contained both positive and
negative controls in columns 23 and 24, respectively. The positive
control, for uninhibited growth, consisted of 5 μL of DMSO/Milli-Q
H2O to a final concentration of 1.75%, and the negative control, or
100% cell death, was comprised of 5 μL of the broad-spectrum
antibiotic ciprofloxacin at a final concentration of 5 μg/mL. Whole
control plates were included for each assay run, which consisted of
duplicate dose−response curves of ciprofloxacin, each in triplicate.
Ciprofloxacin was shown to have an IC50 value of 0.038 μM against P.
aeruginosa PAO1.
selectivity for P. aeruginosa (PAO1) compared to Staph. aureus
(01A1095).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a Jasco P-1020 polarimeter. Circular dichroism spectra
were recorded on a Jasco J-715 spectropolarimeter. UV spectra were
recorded on a Jasco V650 UV/vis spectrophotometer. NMR spectra
were recorded at 30 °C on either a Varian 500 or 600 MHz Unity
INOVA spectrometer. The latter spectrometer was equipped with a
1
triple resonance cold probe. The H and 13C NMR chemical shifts
were referenced to the solvent peaks for DMSO-d6 at δH 2.49 and δC
39.5, respectively. LR-ESIMS were recorded on a Mariner time-of-
flight spectrometer equipped with a Gilson 215 eight-probe injector.
HR-ESIMS were recorded on a Bruker Daltronics Apex III 4.7e
Fourier-transform mass spectrometer. A BIOLINE orbital shaker was
used for the large-scale extraction of plant material. Machery Nagel
Polyamide CC6 (0.05−0.016 mm) was used for tannin/polyphenolic
removal. Alltech Davisil 40−60 μm 60 Å C18 bonded silica was used
for preadsorption work. A Waters 600 pump equipped with a Waters
996 PDA detector and a Waters 717 autosampler were used for HPLC.
A Thermo-Electron C18 Betasil 143 Å column (5 μm, 21.2 × 150 mm)
or a Phenomenex Luna C18 column (5 μm, 21.2 × 250 mm) was used
for semipreparative HPLC separations. All solvents used for
chromatography, UV, and MS were Lab-Scan HPLC grade (RCI
Lab-Scan, Bangkok, Thailand), and the H2O was Millipore Milli-Q PF
filtered. (+)-Boldine (cat. # B3916) and ciprofloxacin (cat. # 17850)
were purchased from Sigma-Aldrich.
Staph. aureus Optical Density Viability Assay. The S. aureus
assay was carried out as per the P. aeruginosa assay, with the following
modifications: the final bacterial concentration used was 1980 cfu/
well, and ciprofloxacin for the internal assay control wells was at 500
μg/mL. Incubation was for 19 h, or until the OD620 reached 0.45.
Ciprofloxacin was shown to have an IC50 value of 125 μM against
methicillin-resistant Staph. aureus 01A1095.
Plant Material. The leaves of Gnetum montanum Markgr.
(Gnetaceae) were collected by the Zi Yuan Medicine Company in
Guangxi Province, China, in January 2000. A voucher specimen (no.
03101699C) has been lodged at the Zi Yuan Medicine Company,
China.
N-Methyllaudanosolinium trifluoroacetate (1): brown gum;
Extraction and Isolation. The dried and ground leaves of G.
montanum (10 g) were sequentially extracted with n-hexane (250 mL),
CH2Cl2 (250 mL), and MeOH (2 × 250 mL). All CH2Cl2 and MeOH
extracts were combined and dried under reduced pressure to yield a
dark green-brown solid (2.88 g). This material was resuspended in
MeOH (150 mL), loaded onto MeOH-conditioned polyamide gel (30
g) in a sintered glass column, and washed with MeOH (300 mL) to
yield 2.45 g of a tannin-free extract. A portion of this semipurified
extract (1 g) was preadsorbed to C18-bonded silica and then packed
into a stainless steel HPLC guard cartridge (10 × 30 mm) that was
subsequently attached to a C18 Betasil HPLC column. Isocratic HPLC
conditions of 90% H2O (0.1% TFA)/10% MeOH (0.1% TFA) were
initially employed for 10 min; then a linear gradient to MeOH (0.1%
TFA) was run over 40 min, followed by isocratic conditions of MeOH
(0.1% TFA) for a further 10 min, all at a flow rate of 9 mL/min. Sixty
fractions (60 × 1 min) were collected from time = 0 min, then
analyzed by (+)-LR-ESIMS. Fraction 18 afforded 9.8 mg of 1 (0.24%
dry wt). Fraction 20 was purified by HPLC using the same Betasil
column as before and a gradient of H2O (0.1% TFA)/MeOH (0.1%
TFA) (80:20 to 50:50 in 60 min) to yield 7.2 mg of 2 (0.17% dry wt).
Compound 5 (3.6 mg, 0.09% dry wt) was obtained by separating
fraction 21 by HPLC with a Phenomenex Luna C18 column using a
gradient of H2O (0.1% TFA)/MeOH (0.1% TFA) (80:20 to 50:50 in
30 min). Fraction 26 was purified by HPLC using a Phenomenex Luna
C18 column and a gradient of H2O (0.1% TFA)/MeOH (0.1% TFA)
(70:30 to 50:50 in 30 min) to yield 1.3 mg of 4 (0.03% dry wt).
Finally, fraction 22 was separated with the Phenomenex Luna C18
column using a gradient of H2O (0.1% TFA)/MeOH (0.1% TFA)
(70:30 to 50:50 in 30 min). Thirty fractions (30 × 1 min) were
collected from time = 0 min, then analyzed by (+)−LR-ESIMS.
Fraction 21 afforded 6.3 mg of 3 (0.15% dry wt), while fraction 23
yielded 6.4 mg of 6 (0.15% dry wt).
ECD (MeOH) λmax (Δε) 0 (0) nm; UV (MeOH) λmax (log ε) 233 sh
1
(3.19), 287 (2.99) nm; H and 13C NMR data, see Table 1; (+)-HR-
ESIMS m/z 316.1544 (C18H22NO4: [M − TFA]+, requires 316.1543).
3′-Hydroxy-N,N-dimethylcoclaurinium trifluoroacetate (2):
brown gum; ECD (MeOH) λmax (Δε) 0 (0) nm; UV (MeOH) λmax
1
(log ε) 231 sh (3.52), 286 (3.29) nm; H and 13C NMR data, see
Table 1; (+)-HR-ESIMS m/z 330.1691 (C19H24NO4: [M − TFA]+,
requires 330.1699).
1,9,10-Trihydroxy-2-methoxy-6-methylaporphinium tri-
fluoroacetate (3): brown gum; ECD (MeOH) λmax (Δε) 0 (0)
nm; UV (MeOH) λmax (log ε) 229 (3.80), 270 sh (3.28), 281 (3.33),
1
308 (3.42), 317 sh (3.39) nm; H and 13C NMR data, see Table 2;
(+)-HR-ESIMS m/z 328.1532 (C19H22NO4: [M − TFA]+, requires
328.1543).
6a,7-Didehydro-1,9,10-trihydroxy-2-methoxy-6-methyla-
porphinium trifluoroacetate (4): brown gum; UV (MeOH) λmax
(log ε) 222 sh (3.74), 270 (3.88), 288 sh (3.67), 335 (3.35), 353
1
(3.33), 371 (3.32) nm; H and 13C NMR data, see Table 2; (+)-HR-
ESIMS m/z 326.1382 (C19H20NO4: [M − TFA]+, requires 326.1387).
Methylation of (+)-Boldine. Boldine (45 mg, 137 μmol) was
dissolved in a mixture of MeCN/MeOH (1:1) to which six molar
equivalents of MeI (50 μL, 863 μmol) was added. The reaction
mixture was stirred at room temperature for 6 h, after which the
solvent was removed under a stream of N2. The crude reaction mixture
was chromatographed on a C18 Betasil HPLC column. Isocratic HPLC
conditions of 90% H2O (0.1% TFA)/10% MeOH (0.1% TFA) were
initially employed for 10 min; then a linear gradient to MeOH (0.1%
TFA) was run over 40 min, followed by isocratic conditions of MeOH
(0.1% TFA) for a further 10 min, all at a flow rate of 9 mL/min. Sixty
fractions (60 × 1 min) were collected from time = 0 min, then
analyzed by (+)-LR-ESIMS. Fractions 24−26 afforded compound 7 as
a trifluoroacetate salt (15.8 mg, 51% yield), fractions 30−32 afforded 8
as a trifluoroacetate salt (2.1 mg, 7% yield), and fraction 29 afforded 9
as a trifluoroacetate salt (1.1 mg, 3% yield).
P. aeruginosa Optical Density (OD620) Viability Assay. P.
aeruginosa PAO1 or PAO200 strain (supplied by Pfizer Global
Research and Development) cultures were prepared at 3.5 × 104 cfu/
mL in cation-adjusted Mueller Hinton (caMH) broth (Difco, Detroit,
N-Methylboldine trifluoroacetate (7): brown gum; [α]D +39 (c
1
0.1, MeOH); ECD (MeOH) λmax (Δε) 242 (+28.1) nm; H and 13C
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Journal of Natural Products
Article
NMR data were in good agreement with those reported for the iodide
salt;21 (+)-LR-ESIMS m/z 342.1, C20H24NO4 [M − TFA]+.
N-Methylsecoboldine trifluoroacetate (8): brown gum; 1H
and 13C NMR data were in good agreement with those reported for
the iodide salt;21 (+)-LR-ESIMS m/z 342.1, C21H26NO4 [M − TFA]+.
N,N-Dimethylsecoboldine trifluoroacetate (9): brown gum;
1H and 13C NMR data were in good agreement with those reported
for the iodide salt;21 (+)-LR-ESIMS m/z 356.1, C20H24NO4 [M −
TFA]+.
(17) Meyers, A. I.; Dickman, D. A.; Boes, M. Tetrahedron 1987, 43,
5095−5108.
(18) Lee, S.-S.; Doskotch, R. W. J. Nat. Prod. 1999, 62, 803−810.
(19) Tsibangu, J. N.; Wright, A. D.; Konig, G. M. Phytochem. Anal.
̈
2003, 14, 13−22.
(20) Nishiyama, Y.; Moriyasu, M.; Ichimaru, M.; Iwasa, K.; Kato, A.;
Mathenge, S. G.; Chalo Mutiso, P., B.; Juma, F., D. Phytochemistry
2004, 65, 939−944.
(21) Milian
́ ́
, L.; Estelles, R.; Abarca, B.; Ballesteros, R.; Sanz, M., J.;
Blaz
́
quez, M., A. Chem. Pharm. Bull. 2004, 52, 696−699.
(22) Lee, S.-S.; Chiou, C.-M.; Lin, H.-Y.; Chen, C.-H. Tetrahedron
Lett. 1995, 36, 1531−1532.
ASSOCIATED CONTENT
■
S
* Supporting Information
(23) Daskalova, E.; Iskrenova, E.; Kiryakov, H. G.; Evstatieva, L.
Phytochemistry 1988, 27, 953−955.
1H and 13C NMR spectra of compounds 1−3, H and 2D
1
(24) Ali, Z.; Tanaka, T.; Iliya, I.; Iinuma, M.; Furusawa, M.; Ito, T.;
Nakaya, K.; Murata, J.; Darnaedi, D. J. Nat. Prod. 2003, 66, 558−560.
(25) Dictionary of Natural Products on DVD version 20.1. Taylor and
Francis Group/CRC Publishing: London, UK, 2011.
(26) Fu, L.; Yu, Y.-F.; Gilbert, M. G. In Flora of China; Science Press
(Beijing) and Missouri Botanical Garden (St. Louis), 1999; Vol. 4, pp
102−105.
NMR spectra of compound 4, and the ECD spectra of boldine
and compound 7. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
(27) Xiang, W.; Jiang, B.; Li, X.-M.; Zhang, H.-J.; Zhao, Q.-S.; Li, S.-
H.; Sun, H.-D. Fitoterapia 2002, 73, 40−42.
*Tel: +61-7-3735-6000. Fax: +61-7-3735-6001. E-mail: r.
quinn@griffith.edu.au.
ACKNOWLEDGMENTS
■
The authors would like to acknowledge Pfizer Global Research
and Development for financial support. We thank A. Miller, D.
Fisher, and J. Montgomery at Pfizer Global Research and
Development for advice during the screening and isolation
campaign. We thank the Australian Research Council (ARC)
for support toward NMR and MS equipment (LE0668477 and
LE0237908). The authors thank C. Lewis and K. Watts from
the Molecular Libraries group (Eskitis Institute), for their
assistance in the preparation of the screening library. We also
thank H. Vu from Griffith University for acquiring the HR-
ESIMS measurements.
REFERENCES
■
(1) McGowan, J. E. Jr. Am. J. Infect. Control 2006, 34, S29−37, ;
S64−73.
(2) Quinn, J. P. Clin. Infect. Dis. 1998, 27 (Suppl 1), S117−124.
(3) Higgins, C. F. Nature 2007, 446, 749−57.
(4) Zgurskaya, H. I.; Nikaido, H. Mol. Microbiol. 2000, 37, 219−225.
(5) Hancock, R. E. Clin. Infect. Dis. 1998, 27 (Suppl 1), S93−99.
(6) Leibovici, L.; Vidal, L.; Paul, M. J. Antimicrob. Chemother. 2009,
63, 246−251.
(7) Pelaez, F. Biochem. Pharmacol. 2006, 71, 981−990.
(8) Starr, J. T.; Sciotti, R. J.; Hanna, D. L.; Huband, M. D.; Mullins,
L. M.; Cai, H.; Gage, J. W.; Lockard, M.; Rauckhorst, M. R.; Owen, R.
M.; Lall, M. S.; Tomilo, M.; Chen, H.; McCurdy, S. P.; Barbachyn, M.
R. Bioorg. Med. Chem. Lett. 2009, 19, 5302−5306.
(9) Lee, S.-S.; Lin, Y.-J.; Chen, C.-K.; Liu, K. C. S.; Chen, C.-H. J.
Nat. Prod. 1993, 56, 1971−1976.
(10) Rochfort, S. J.; Towerzey, L.; Carroll, A.; King, G.; Michael, A.;
Pierens, G.; Rali, T.; Redburn, J.; Whitmore, J.; Quinn, R. J. J. Nat.
Prod. 2005, 68, 1080−1082.
(11) Zhao, Q.; Zhao, Y.; Wang, K. J. Ethnopharmacol. 2006, 106,
408−413.
(12) Brossi, A.; Ramel, A.; O’Brien, J.; Teitel, S. Chem. Pharm. Bull.
1973, 21, 1839−1840.
(13) Kametani, T.; Noguchi, I. J. Chem. Soc. C 1967, 1440−1443.
(14) Lee, S.-S.; Lai, Y.-C.; Chen, C.-K.; Tseng, L.-H.; Wang, C.-Y. J.
Nat. Prod. 2007, 70, 637−642.
́ ́
́
(15) Iturriaga-Vasquez, P.; Perez, E. G.; Slater, E. Y.; Bermudez, I.;
Cassels, B. K. Bioorg. Med. Chem. 2007, 15, 3368−3372.
(16) Gozler, B.; Kivca
1990, 53, 666−668.
̧
k, B.; Gozler, T.; Shamma, M. J. Nat. Prod.
̈
̈
2430
dx.doi.org/10.1021/np200700f|J. Nat. Prod. 2011, 74, 2425−2430