Antimicrobial agent isolated from Coptidis rhizome extract incubated with Rhodococcus sp. strain BD7100

Article abstract of DOI:10.1038/s41429-018-0114-3

Coptidis rhizome (CR) is a widely used herbal medicine that contains protoberberine-type alkaloids. CR extract exhibits various pharmacologic activities. A previous study reported the isolation of Rhodococcus sp. strain BD7100 as a berberine (BBR)-utilizing bacterium, and the BBR-degradation pathway has been investigated. When we incubated strain BD7100 cells with CR extract, the number of viable cells declined with the degradation of components in the CR extract, and the culture broth exhibited antibacterial activity against strain BD7100. These results suggest that CR extract cultured in the presence of strain BD7100 contains one or more antibacterial agents. In this study, we isolated coptirhoquinone A (1) from CR extract incubated with strain BD7100 in Luria–Bertani (LB) medium, and the structure was elucidated using NMR and MS analysis. We also report the total synthesis and antimicrobial activities of 1 against bacteria, fungi, and Pythium sp.

Full text of DOI:10.1038/s41429-018-0114-3

The Journal of Antibiotics
https://doi.org/10.1038/s41429-018-0114-3
ARTICLE
Antimicrobial agent isolated from Coptidis rhizome extract
incubated with Rhodococcus sp. strain BD7100
2
Kazuki Ishikawa1 Kazunori Takahashi
Syun Hosoi1 Hisashi Takeda1 Hinaka Yoshida1 Daigo Wakana1
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Masayoshi Tsubuki2 Fumihiko Sato3 Motoaki Tojo4 Tomoo Hosoe1
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Received: 1 August 2018 / Revised: 1 October 2018 / Accepted: 7 October 2018
© The Author(s) under exclusive licence to the Japan Antibiotics Research Association 2018
Abstract
Coptidis rhizome (CR) is a widely used herbal medicine that contains protoberberine-type alkaloids. CR extract exhibits
various pharmacologic activities. A previous study reported the isolation of Rhodococcus sp. strain BD7100 as a berberine
(BBR)-utilizing bacterium, and the BBR-degradation pathway has been investigated. When we incubated strain BD7100
cells with CR extract, the number of viable cells declined with the degradation of components in the CR extract, and the
culture broth exhibited antibacterial activity against strain BD7100. These results suggest that CR extract cultured in the
presence of strain BD7100 contains one or more antibacterial agents. In this study, we isolated coptirhoquinone A (1) from
CR extract incubated with strain BD7100 in Luria–Bertani (LB) medium, and the structure was elucidated using NMR and
MS analysis. We also report the total synthesis and antimicrobial activities of 1 against bacteria, fungi, and Pythium sp.
Introduction
CR extract and traditional medicines containing CR exhibit
antimicrobial, antimalarial, anti-inflammatory, and analge-
sic activities [3–6]. CR contains protoberberine-type alka-
loids such as berberine (BBR), palmatine (PAL), coptisine
(COP), jatrorrhizine (JAT), epi-berberine (E-BBR), and
columbamine (COL) [7].
Coptidis rhizome (CR) is the dried rhizome of plants of the
genus Coptis, such as C. japonica Makino and C. chinensis
Franch. CR is widely used as a herbal medicine to relieve
abdominal pain and diarrhea in traditional Chinese medicine
and Japanese Kampo [1, 2]. Previous studies reported that
We have been investigating bacterial degradation of
alkaloids. Rhodococcus sp. strain BD7100 is a bacterium
isolated from soil that can utilize BBR as a sole carbon
source [8]. A recent report indicated that strain BD7100
degrades BBR into 2-hydroxy-3,4-dimethoxybenzeneacetic
acid (HDBA) via demethylenation to D-BBR [9]. In this
work, we investigated the degradation of protoberberine-
type alkaloids by strain BD7100 by incubating the bacter-
ium with CR extract, which contains numerous
protoberberine-type alkaloids. Unexpectedly, we found that
the number of viable cells of strain BD7100 declined, and
the culture medium exhibited antimicrobial activity against
strain BD7100 that increased in potency as the level of
protoberberine-type alkaloids decreased. These results
suggested that an antibacterial agent is produced in CR
extract incubated with strain BD7100. In this study, we
report a novel antimicrobial agent was being produced from
the CR extract when incubated strain BD7100 in
Luria–Bertani (LB) medium. We also elucidated the struc-
ture of the antimicrobial agent using NMR and MS analysis
and report its total synthesis and spectrum of activity.
Electronic supplementary material The online version of this article
(https://doi.org/10.1038/s41429-018-0114-3) contains supplementary
material, which is available to authorized users.
* Kazunori Takahashi
takakazu@hoshi.ac.jp
* Tomoo Hosoe
hosoe@hoshi.ac.jp
1
Department of Organic Chemistry, Hoshi University, 2-4-41
Ebara, Shinagawa-ku, Tokyo 142-8501, Japan
2
Institute of Medical Chemistry, Hoshi University, 2-4-41 Ebara,
Shinagawa-ku, Tokyo 142-8501, Japan
3
Department of Plant Gene and Totipotency, Graduate School of
Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto
606-8502, Japan
4
Graduate School of Life and Environmental Sciences, Osaka
Prefecture University, 1-1, Gakuen-cho, Nakaku, Sakai, Osaka
599-8531, Japan

K. Ishikawa et al.
A
B
BBR
0 h
130
60
PAL
COP
JAT
E-BBR
COL
-10
BBR
COP
PAL
JAT
COL
18 h
130
D-BBR
60
-10
130
28 h
48 h
D-COP
60
-10
130
E-BBR
60
-10
C
0
6
12
18
24
60
40
20
0
Retention time (min)
LB
LB + CR extract
10
20
30
40
50
Incubation time (h)
Fig. 1 Incubation of CR extract with Rhodococcus sp. strain BD7100
in LB medium. a Chemical structures of protoberberine-type alkaloids
identified in CR extract. b HPLC chromatograms (λ = 280 nm) of CR
extract incubated with cells of strain BD7100. c Change in the number
of viable bacteria. Solid line indicates the number of viable bacteria in
LB medium, and dotted line indicates the number of viable bacteria in
LB medium containing CR extract. Each value represents the mean
SD of four independent experiments
Table 1 Time course of antibacterial effect of culture medium against
strain BD7100, as determined using the paper-disk method
degraded during incubation, and demethyleneberberine and
demethylenecoptisine were produced. BBR, COP, and JAT
were completely degraded by 48 h, whereas PAL, E-BBR,
and COL were still detected after 48 h (Figure S1).
The number of viable BD7100 cells in culture broth with
and without CR extract was determined using the dilution-
plate method (Fig. 1c). The viable cell counts exhibited
similar trends until 36 h. In culture broth containing CR
extract, the number of viable cells decreased markedly by
48 h. Antibacterial activity in culture broth containing CR
extract was tested against strain BD7100 using the paper-
disk method. At 36 and 48 h, culture broth exhibited anti-
bacterial activity against BD7100 cells; however, there
was no activity observed at other time points (Table 1).
These results suggested that one or more antimicrobial
compounds active against strain BD7100 is produced in
CR extract incubated with BD7100 cells. Then we tried
to bioassay-guided isolation to identify bioactive
compounds.
Incubation time (h)
Bacterium
BD7100
0
12
24
32
36
15
48
n.d.
n.d.
n.d.
n.d.
12.5
Amount of sample: 50 μL/disc (8 mm), unit: mm
Results and Discussion
Production of antimicrobial agent from CR extract
by Rhodococcus sp. strain BD7100 in LB medium
CR extract was added to LB medium and incubated with
cells of strain BD7100. After 48 h, the culture broth was
analyzed using HPLC. BBR, PAL, COP, JAT, E-BBR, and
COL were detected in samples analyzed by HPLC at the
beginning of the incubation period (Fig. 1a, b). HPLC
chromatograms indicated that BBR, COP, and JAT were

Antimicrobial agent isolated from Coptidis rhizome extract incubated with Rhodococcus sp. strain. . .
Table 2 NMR chemical shifts of coptirhoquinone A (1) and synthetic
compound in CDCl3
Elucidation of the structure of coptirhoquinone A (1)
1
Synthetic
compound
Compound 1 was isolated as a red, amorphous solid, and
the molecular formula was determined as C12H14O6S (six
degrees of unsaturation) by high-resolution electrospray
ionization mass spectrometry (HRESIMS). IR spectra
(1740, 1716, and 1653 cm−1) and 13C NMR signals indi-
cated the presence of two α, β-unsaturated ketones
(δC 181.1 and 180.2) and an ester bond (δC 169.7) in the
a
b
a
b
Position
δC
δH
HMBCc
δC
δH
1
137.9
181.1
145.4
144.9
180.2
–
–
–
–
–
–
138.0
181.0
145.4
144.9
180.1
–
–
–
–
–
–
2
3
1
4
structure of 1. H NMR spectra of 1 exhibited signals for
5
three methyl groups with an oxygen atom (δH 4.01, 4.00,
and 3.70), a methyl group with a sulfur atom (δH 2.56), and
a methylene group (δH 3.77). A heteronuclear multiple bond
coherence (HMBC) correlation of 8-OCH3 [δH 3.70 (s)]
with C-8 (δC 169.7) indicated the presence of a methyl ester
residue. HMBC correlations of 3-OCH3 (δH 4.01 [s]) with
C-3 (δC 145.4) and 4-OCH3 (δH 4.00 [s]) with C-4 (δC
144.9) indicated linkages between two methoxy groups at
the C-3 and C-4 positions. An HMBC correlation of 6-
SCH3 (δH 2.56 [s]) with C-6 (δC 145.6) indicated a linkage
between a sulfurated methyl group and the C-6 position.
From these results, the structure of 1 was determined as
shown in Fig. 2 and Table 2.
6
145.6
7
33.7
3.77 s
–
1, 2, 6, 8
33.7
3.76 s
–
8
169.7
61.5 *
61.4 *
17.9
169.7
61.5*
61.4*
17.9
3-OCH3
4-OCH3
6-SCH3
7-OCH3
8-COOCH3
4.01 s
4.00 s
2.56 s
3
4
6
4.01 s
4.00 s
2.56 s
52.5
3.70 s
8
52.5
3.70 s
a100 MHz for 13C-NMR
b400 MHz for H-NMR
cHMBC correlations are from the proton(s) stated to the indicated
1
carbon
*Exchangeable
Total chemical synthesis of coptirhoquinone A (1)
Total synthesis of compound 1 was carried out to confirm
its structure. Benzylation of known phenol 4 [10, 11]
(readily derived from 2,3,4-trimethoxylbenzaldehyde in
three steps) with benzyl bromide in the presence of K2CO3
afforded benzyl ether 5 at 94% yield (Scheme 1). Rear-
rangement of 5 with HMBI (7) (1H-1-hydroxy-5-methyl-
1,2,3-benziodoxanthiole 3,3-dioxide) [12] and trimethyl
orthoformate was carried out at room temperature for 2 h to
furnish the methyl acetate 6 at 66% yield [13]. Bromination
of 6 with NH4Br-oxone [14] brought about the expected
bromination to give the desired product 8 at 67% yield.
Hydrogenation of 8 with Pd/C, followed by oxidation of the
resulting phenol 9 with nBu4NBr-oxone [15], gave quinone
10 at 76% yield over two steps. Finally, substitution of 10
with sodium thiomethoxide [16] gave the desired product 1
at 76% yield. The spectroscopic data of synthesized 1 were
superimposable with those of the natural product. Thus, we
accomplished the total synthesis of 1 in six steps from 4 at
16.3% overall yield (Scheme 1).
1
Fig. 2 Structure of 1
ampicillin and kanamycin (25 and 24.7 mm, respectively).
Compound 1 also inhibited the growth of Staphylococcus
aureus (32.7 mm) and Proteus mirabillis (16.3 mm) at 0.25
mg per disk. No inhibition of the growth of Escherichia
coli, Klebsiella neumoniae, or Pseudomonus aerginosa was
observed.
Compound 1 was also tested for activity against patho-
gens associated with deep mycosis, such as Aspergillus
fumigatus, A. niger, Candida albicans, and Cryptococcus
neoformans. Compound 1 inhibited the growth of A. fumi-
gatus (12.3 mm), C. albicans (10.3 mm), and C. neofor-
mans (12.7 mm).
Antimicrobial activity of coptirhoquinone A (1)
The antibacterial activity of 1 against strain BD7100 and
various intestinal bacteria was tested using the paper-disk
method (Table 3). In the presence of disks permeated with
1, growth of strain BD7100 was inhibited at 0.25 mg per
disk (25.7 mm), similar to the same concentrations of
Compounds consisting of a benzoquinone skeleton have
been reported to exhibit antibacterial activity against a
variety of microorganisms [17, 18], and synthesis of various
derivatives has been reported [19]. In particular, benzo-
quinone derivatives containing
a
methylthio group

K. Ishikawa et al.
Scheme 1 Total synthesis of 1
via rearrangement reaction
7
reportedly exhibit enhanced antibacterial activity. However,
the isolation of natural benzoquinone derivatives containing
methylthio groups is rare; examples include T1801B, iso-
lated from the bacterium Pseudomonas [20], and thio-
graphisquinone, isolated from Graphis [21], both of which
Ltd., Tokyo, Japan) and a FT/IR-4100 instrument (JASCO
1
Co., Ltd., Tokyo, Japan), respectively. H and 13C NMR
spectra were recorded using a Bruker AVANCE-400
spectrometer (400.13 MHz for 1H, 100.61 MHz for 13C;
Bruker Biospin K.K., Kanagawa, Japan). Chemical shifts
(δ) were measured in ppm using tetramethylsilane as an
internal standard. BioShakers BR43-FH (TAITEC Co.,
Ltd., Saitama, Japan) and FTW-502M (Hirayama Manu-
facturing Corp., Saitama, Japan) were used for incubation of
bacteria. The turbidity of cultures was monitored using an
OD-MonitorA&S (TAITEC Co.). Samples were analyzed
and quantified by PDA-HPLC using PU-980 and PU-1580
pumps (JASCO Co.) equipped with an Inertsil ODS-3
column (5 µm, 4.6 mm × 250 mm; GL Science Inc., Tokyo,
Japan; maintained at a temperature of 30 °C using a CO-
2065 Plus column oven [JASCO Co.]) and an MD-2010
Plus photodiode array detector (JASCO Co.). Compounds
were isolated using a reverse-phase MPLC system equipped
with an SSC-3160 pump (Senshu Scientific Co., Ltd.,
Tokyo, Japan), ULTRA PACK ODS-SM-50A column (300
mm × 11 mm [50 µm], Yamazen Corp., Osaka, Japan), and
YRU-8883 RI-UV detector (Shimamuratech, Tokyo,
Japan). Reverse-phase HPLC was also carried out using an
LC-20AT prominence pump (Shimadzu Corp., Kyoto,
Japan) equipped with an Inertsil ODS-3 column (250 mm ×
4.6 mm [5 µm], GL Science Inc.), CO-965 column oven
consist of
a 2-methylthio-3,4-benzoquinone skeleton.
T1801B reportedly exhibits antibacterial activity against S.
aureus and C. neoformans.
In this study, a novel antimicrobial substance was pro-
duced by incubating CR extract with a BBR-utilizing bac-
terium. These results suggest that it would be possible to
more effectively utilize resources by preparing CR extract
from waste generated during the manufacture of herbal
medicine preparations and using it as a raw material. In
addition, this study serves as a model for the preparation of
novel compounds by the biotransformation of herbal
extracts, and we hypothesize that the results could be
applied to other herbal extracts and bacteria.
Materials and methods
Experimental instruments
UV and IR spectra were recorded on an Ultrospec 2100 pro
UV-visible spectrophotometer (Amersham Biosciences,

Antimicrobial agent isolated from Coptidis rhizome extract incubated with Rhodococcus sp. strain. . .
Table 3 Antimicrobial activity
Microorganisms
1
Amp
Kan
AmB
of 1 and various antibiotics, as
determined using the paper-disk
method
Bacterial sp.
Rhodococcus sp. BD7100
Staphylococcus aureus
Escherichia coli
25.7 0.6
32.7 3.2
0.0 0.0
8.0 7.0
16.3 0.6
0.0 0.0
25.0 1.0
47.7 11.0
29.0 1.0
14.7 0.6
34.3 2.1
0.0 0.0
24.7 2.5
40.7 2.9
27.3 0.6
18.3 9.0
26.7 1.5
14.7 0.6
–
–
–
–
–
–
Klebsiella neumoniae
Proteum mirabillis
Pseudomonas aeruginosa
Fungal sp.
Aspegillus fumigatus
Aspegillus niger
12.3 0.6
0.0 0.0
10.3 0.6
12.7 1.2
–
–
–
–
–
–
–
–
13.3 0.6
14.7 1.5
17.3 6.4
19.3 0.6
Candida albicans
Cryptococcus neoformans
Amp ampcillin, Kan kanamycin, AmB amphotericin B, n.d. not detected, – not tested
Each value represents the mean SD of three independent experiments (n = 3)
(JASCO Co.), and SPD-20A UV detector (Shimadzu
Corp.). Molecular weight and molecular formula were
determined by LC-ESI-TOF-MS using a JMS-T100LP
spectrometer (JEOL, Ltd., Tokyo, Japan) and 1200 series
system (Agilent Technologies, Inc., Tokyo, Japan).
at 180 rpm. Next, 0.5-mL samples of the culture were col-
lected at various time points and mixed with an equal
amount of methanol (MeOH) containing 0.4% (v/v)
hydrochloric acid. This mixture was centrifuged (18,800 ×
g, 4 °C, 5 min) to remove cells and debris.
Strains and culture conditions
PDA-HPLC analysis
The isolation and identification of Rhodococcus sp. strain
BD7100 has been described previously [9]. For the present
work, strain BD7100 was incubated in LB medium (0.5 g
Bacto yeast extract, 1.0 g Bacto tryptone, and 0.5 g NaCl in
100 mL of water) or on 0.1 × LB agar medium (containing
Bacto yeast extract and Bacto tryptone at 10-fold lower
levels and 1.5% [w/v] agar). CR extract was added to these
media as indicated.
The flow rate was 1 mL/min, and the column oven tem-
perature was 30 °C. Analysis was performed using a mobile
phase gradient as follows. Initial conditions were 15% for
5 min, followed by a linear gradient to 50% B from 5 to
15 min and 100% B at 15.1 min, which was maintained
until 18 min. The mobile phase was returned to initial
conditions at 18.1 min and maintained until the end of the
run at 25 min. Mobile phase solvents A and B were H2O
containing 0.1% trifluoroacetic acid and acetonitrile,
respectively.
Preparation of CR extract
Dried Coptidis rhizome (100 g, Uchida Wakan-yaku,
Tokyo, Japan) was boiled in water (590 mL) at 100 °C for
30 min, and the extract was then filtered directly. The filtrate
was centrifuged (2610 × g, r.t., 15 min), and the supernatant
was filtered using a sterilized 0.2-µm filter. The resulting
filtrate was used as the CR extract.
Determination of the number of viable bacteria
Rhodococcus sp. strain BD7100 was incubated in flasks
(500 mL) containing 100 mL of LB medium with or without
5 mL of CR extract at 30 °C with rotation (180 rpm). Sam-
ples (100 µL) of the cultures were collected at 12, 24, 36, and
48 h of incubation and diluted 107 fold using sterilized water.
Aliquots (100 µL) of the diluted cultures were inoculated
onto LB agar plates, which were incubated at 30 °C for 48 h.
After incubation, colonies on the plates were counted.
Incubation of CR extract with strain BD7100 in LB
medium
Rhodococcus sp. strain BD7100 was grown at 30 °C on
0.1 × LB agar plates containing 1.0 mM BBR. Individual
colonies of strain BD7100 were used to inoculate 10 mL of
LB medium in a test tube, which was incubated for 24 h at
30 °C on a rotary shaker (180 rpm). After incubation, the
culture was inoculated into LB medium (100 mL) contain-
ing CR extract (5 mL) in a 500-mL Erlenmeyer flask rotated
Investigation of antibacterial activity in cultures
containing CR extract
Strain BD7100 was incubated in LB medium containing CR
extract. The incubation conditions were the same as those
described above. After incubation, 20 mL of each culture

K. Ishikawa et al.
was removed, frozen, and lyophilized. Next, 1 mL of
MeOH containing 0.4% (v/v) hydrochloric acid was added
to each sample and centrifuged (2610 × g, r.t., 10 min). The
resulting supernatant was used to test antibacterial activity
using the paper-disk method.
1H and 13C NMR spectra were obtained on a Bruker AV III
400 (1H-NMR: 400 MHz, 13C NMR: 100 MHz) instrument
for solutions in CDCl3, and chemical shifts are reported on
the δ scale using TMS as an internal standard of 0.00 for 1H
NMR spectra and CDCl3 as an internal standard of δ 77.00
for 13C NMR spectra. MS spectra were obtained on a JEOL-
600 spectrometer. Elemental analyses were performed on a
Yanaco-MT5.
Testing antibacterial activity using the paper-disk
method
A 50-µL aliquot of sample was applied to an 8-mm disk.
The test samples were dissolved in MeOH containing 0.4%
(v/v) hydrochloric acid. The disks containing sample were
placed on an LB agar plate inoculated with bacteria, and the
plate was incubated at 30 °C for 12 or 24 h. After incuba-
tion, the diameter of inhibitory zone around each disk was
measured.
2,3,4-Trimethoxyphenol
To a solution of 2,3,4-trimethoxybenzaldehyde (1.0 g, 5.1
mmol) in MeOH (7.3 mL), 31% H2O2 (0.75 mg, 6.6 mmol)
and H2SO4 (0.07 mL) were added at room temperature.
After stirring for 12 h at the same temperature, the volatile
solvent was removed in vacuo to leave a residue. The
residue was treated with H2O (30 mL) and extracted twice
with CHCl3. The extract was washed with H2O, saturated
NaHCO3 aqueous solution, and brine. The organic layer
was dried over Na2SO4 and concentrated in vacuo to give
crude 2,3,4-trimethoxyphenol (1.1 g), which was used for
the next step without further purification.
Isolation of compound coptirhoquinone A (1)
Rhodococcus sp. strain BD7100 was incubated in 36 flasks
containing LB medium (100 mL) with CR extract (5 mL) at
30 °C for 48 h. The culture was then centrifuged (2610 × g,
r.t., 15 min) to remove cells, and the resulting supernatant
was extracted using two volumes of ethyl acetate, two
volumes of ethyl acetate containing 0.08% (v/v) hydro-
chloric acid, and an equal volume of ethyl acetate. The ethyl
acetate was evaporated, and the residue (2.08 g) was sus-
pended in water. The suspension was extracted twice with
chloroform, and then the chloroform was evaporated. The
chloroform extract (571.8 mg) was dissolved in MeOH, and
the solution was fractionated using a reverse-phase MPLC
system at a flow rate of 5 mL/min and the following gra-
dient: initial conditions, 0% B (0–30 min); 15% (30–75
min), 30% B (75–105 min), 50% (105–125 min), 70% B
(125–160 min), 100% (160–180 min) (A: H2O, B: acetoni-
trile). The sixth fraction was chromatographed to isolate and
purify compound 1 (3.0 mg) using reverse-phase HPLC
under the following conditions: flow rate, 1.0 mL/min;
column oven temperature, 30 °C; detector, UV at 210 nm;
mobile phase, 65% H2O containing 0.1% (v/v) tri-
fluoroacetic acid and 35% acetonitrile.
1H NMR (CDCl3; 400 MHz) δ 6.63 (1H, d, J = 9.0 Hz),
6.56 (1H, d, J = 9.0 Hz), 5.39 (1H, s), 3.96 (3H, s), 3.90
(3H, s), 3.81 (3H, s).
2,3,4-Trimethoxyphenyl acetate
To the crude 2,3,4-trimethoxyphenol (1.1 g) and pyridine
(2.0 g, 25.5 mmol) in CHCl3 (20 mL), Ac2O (1.9 mL, 20.4
mmol) and pyridine (12 mL, 95.4 mmol) were added at
room temperature under Ar gas. After stirring for 6 h at the
same temperature, the reaction mixture was treated with
H2O and extracted with ethyl acetate (EtOAc). The extract
was washed with saturated KHSO4 aqueous solution, brine,
saturated NaHCO3 aqueous solution, and brine. The organic
layer was dried over Na2SO4 and concentrated in vacuo to
give 2,3,4-trimethoxyphenylacetate (1.2 g), which was used
for the next step without further purification. 1H NMR
(CDCl3; 400 MHz) δ 6.71 (1H, d, J = 9.0 Hz), 6.59 (1H, d,
J = 9.0 Hz), 3.86 (3H, s), 3.85 (3H, s), 3.82 (3H, s), 2.28
(3H, s).
Compound 1: Red, amorphous solid; HRESIMS obsd.
287.0601 calcd. for C12H15O6S1 [M+H]+ 287.0589; UV
λmax (MeOH) nm (log ε): 206 (4.0), 270 (3.5), 342 (3.4); IR
(KBr) cm−1: 1740, 1716, 1653. The NMR data see Table 2.
1-(2-Hydroxy-3,4,5-trimethoxyphenyl)ethanone (4)
To the crude 2,3,4-trimethoxyphenylacetate (1.2 g),
BF3·OEt2 (4 mL, 31.8 mmol) was added at room tempera-
ture under Ar gas. After stirring for 30 min at 100 °C, the
reaction mixture was poured into H2O, and the resulting
mixture was extracted with EtOAc. The organic layer was
washed with saturated NaHCO3 aqueous solution, brine,
and then dried over Na2SO4. Removal of the solvent gave a
General methods
Melting points were determined using a Yanagimoto MP
apparatus, and the values are uncorrected. Optical rotations
were measured using a JASCO DIP-360. IR spectra were
obtained using a JASCO FT/IR-4100 spectrophotometer.

Antimicrobial agent isolated from Coptidis rhizome extract incubated with Rhodococcus sp. strain. . .
residue, which was purified by column chromatography
using hexane-EtOAc (8:2, v/v) as the eluent to give 1-(2-
hydroxy-3,4,5-trimethoxyphenyl)ethanone (4) (690.1 mg,
Methyl 2-(2-[benzyloxy]-6-bromo-3,4,5-trimethoxyphenyl)
acetate (8)
1
68%) as a yellow solid. H-NMR (CDCl3; 400 MHz) δ
To a solution of methyl phenylacetate 6 (77.4 mg, 0.33
mmol) in MeOH-H2O (1:1, 6 mL), NH4Br (33.9 mg, 0.35
mmol) and oxone (151.3 mg, 0.25 mmol) were added at
0 °C. After stirring for 30 min at the same temperature, the
reaction was quenched with H2O, and the whole sample was
extracted with EtOAc. The extract was washed with satu-
rated Na2S2O3 aqueous solution, saturated NaHCO3 aqu-
eous solution, and brine, and then dried over Na2SO4. The
solvent was removed in vacuo to leave a residue, which was
purified by column chromatography on silica gel using
hexane-EtOAc (85:15, v/v) as the eluent to give bromo-
benzene 8 (64.1 mg, 67%) as a colorless oil. IR ν max 1743,
12.44 (1H, s), 6.93 (1H, s), 4.04 (3H, s), 3.92 (3H, s), 3.86
(3H, s), 2.59 (3H, s); 13C-NMR (CDCl3; 100 MHz) δ 203.1,
153.1, 149.9, 145.0, 141.4, 114.2, 107.4, 61.3, 61.0, 59.7,
26.7.
1-(2-[Benzyloxy]-3,4,5-trimethoxyphenyl)ethanone (5)
To a solution of 1-(2-hydroxy-3,4,5-trimethoxyphenyl)
ethanone (4) (94.6 mg, 0.42 mmol) in acetone, K2CO3
(122.0 mg, 0.88 mmol) and BnBr (74.6 μL, 0.63 mmol)
were added at room temperature under Ar gas. After stirring
for 49.5 h at the same temperature, the reaction mixture was
diluted with EtOAc and then filtered through a pad of
Celite. The filtrate was removed in vacuo. The residue was
purified by column chromatography on silica gel using
hexane-EtOAc (8:2, v/v) as the eluent to give benzyl ether 5
1
1415, 1083, 1042 cm−1; H NMR (CDCl3; 400 MHz) δ
7.43–7.32 (5H, m), 5.00 (2H, s), 3.97 (3H, s), 3.93 (3H, s),
3.87 (3H, s), 3.82 (2H, s), 3.66 (3H, s); 13C NMR (CDCl3;
100 MHz) δ 171.3, 147.8, 147.4, 147.1, 146.5, 137.2,
128.5, 128.1, 128.1, 124.2, 114.8, 75.6, 61.4, 61.2, 61.0,
52.0, 35.7; HRMS (ESI+) calcd for C19H2179BrNaO6 [M
+Na]: 447.0419; found: 447.0406.
(124.9 mg, 94%) as a yellowish oil. IR ν
1672, 1117,
max
1065 cm−1 1H NMR (CDCl3; 400 MHz) δ 7.45–7.33
;
(5H, m), 5.06 (1H, s), 3.99 (3H, s), 3.94 (3H, s), 3.87 (3H,
s), 2.56 (3H, s); 13C NMR (CDCl3; 100 MHz) δ 198.5,
149.3, 147.1, 147.1, 147.0, 136.8, 128.6, 128.3, 128.3,
127.5, 106.5, 76.4, 61.4, 61.2, 56.1, 31.4; HRMS (ESI+)
calcd for C18H21O5 [M+H]: 317.1389; found: 317.1392.
Methyl 2-(2-bromo-6-hydroxy-3,4,5-trimethoxyphenyl)
acetate (9)
To a solution of bromobenzene 8 (52.5 mg, 0.12 mmol) in
MeOH (4 mL), 10% Pd/C (4.2 mg) was added under an
atmosphere of H2 at room temperature. After stirring for
1.5 h at the same temperature, the reaction mixture was
filtered through a pad of celite. The filtrate was concentrated
to leave a residue, which was used for the next steps without
further purification.
Methyl 2-(2-[benzyloxy]-3,4,5-trimethoxyphenyl)acetate (6)
To a solution of the benzyl ether 5 (54.1 mg, 0.17 mmol) in
MeOH (2 mL), H2SO4 (8.4 mg, 0.09 mmol) and trimethyl
orthoformate (0.6 mL) were added at room temperature.
After stirring for 10 min at the same temperature, HMBI (7,
1H-1-hydroxy-5-methyl-1,2,3-benziodoxanthiole 3,3-diox-
ide) (54 mg, 0.17 mmol) was added to the reaction mixture.
After stirring for 15 min, HMBI (60 mg, 0.19 mmol) was
added to the above mixture. After stirring for 2 h, the
reaction mixture was poured into H2O, and the resulting
mixture was extracted with EtOAc. The extract was washed
with saturated NaHCO3 aqueous solution, brine, and dried
over Na2SO4. Removal of the solvent gave a residue, which
was purified by column chromatography on silica gel using
hexane-EtOAc (8:2, v/v) as the eluent to give phenylacetate
Methyl 2-(2-bromo-4,5-dimethoxy-3,6-dioxocyclohexa-1,4-
dienyl)acetate (10)
To a solution of the corresponding phenol 9 (45.4 mg) in
CH3CN-H2O (2:1, 6 mL), nBu4NBr (4.7 mg, 0.01 mmol)
and oxone (64.7 mg, 0.11 mmol) were added at 0 °C.
After stirring for 2.5 h at the same temperature, the
reaction mixture was filtered through a pad of SiO2.
The filtrate was concentrated in vacuo to leave a residue,
which was purified by column chromatography on silica gel
using hexane-EtOAc (30%, v/v) as the eluent to give qui-
6 (38.8 mg, 66%) as a yellowish oil. IR ν
1739, 1494,
max
1464, 1128, 1087 cm−1; H NMR (CDCl3; 400 MHz) δ
7.49–7.30 (5H, m), 6.52 (1H, s), 4.99 (2H, s), 3.94 (3H, s),
3.91 (3H, s), 3.83 (3H, s), 3.64 (3H, s), 3.56 (2H, s); 13C
NMR (CDCl3; 100 MHz) δ 172.2, 149.4, 147.1, 144.3,
142.3, 137.6, 128.3, 128.1, 127.9, 122.4, 108.2, 75.1, 61.1,
56.1, 51.9, 35.4; HRMS (ESI+) calcd for C19H22NaO6 [M
+Na]: 369.1314; found: 369.1322.
none 10 (30.1 mg, 76%) as a reddish oil. IR ν
1743,
max
1
1669, 1604, 1262 cm−1; H NMR (CDCl3; 400 MHz) δ
4.05 (3H, s), 4.03 (3H, s), 3.76 (2H, s), 3.73 (3H, s); 13C
NMR (CDCl3; 100 MHz) δ 180.1, 176.3, 168.4, 145.1,
144.3, 139.9, 136.0, 61.6, 61.3, 52.5, 35.8; HRMS (ESI+)
calcd for C11H1279BrO6 [M+H]: 318.9817; found:
318.9808.
1

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CHCl3-H2O (6:1, 3.5 mL), nBu4NBr (3 mg, 9.3 μmol) and
NaSMe (16.9 mg, 0.24 mmol) were added at room tem-
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1
mg, 76%) as a reddish oil. H NMR (CDCl3; 400 MHz) δ
4.02 (3H, s), 4.01 (3H, s), 3.78 (2H, s), 3.71 (3H, s), 2.57
(3H, s); 13C NMR (CDCl3; 100 MHz) δ 180.9, 180.0,
169.5, 145.4, 145.2, 144.7, 137.7, 61.3, 61.2, 52.3, 33.5,
17.7.
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Acknowledgements This work was supported by Hoshi University
Otani Research Grants and a JSPS Grant-in-Aid for Young Scientists
(B) (grant number 972018).
Compliance with ethical standards
compounds with ammonium bromide and oxone. Synth
2013;45:1497–504.
.
Conflict of interest The authors declare that they have no conflict of
interest.
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