Note

pubs.acs.org/jnp

Identification of Pyridinium with Three Indole Moieties as an

Antimicrobial Agent
Masahiro Okada,*,† Tomotoshi Sugita,† Chin Piow Wong, Toshiyuki Wakimoto,‡ and Ikuro Abe*
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
*
S Supporting Information

ABSTRACT: A novel pyridinium with three indole moieties,

tricepyridinium, was obtained from the culture of an Escherichia coli
clone incorporating metagenomic libraries from the marine sponge
Discodermia calyx. For the important structural elements of
tricepyridinium to be investigated for antibacterial activity, tricepyr-
idinium and its analogues were chemically synthesized. Tricepyr-
idinium had antimicrobial activity, but not against E. coli, and
cytotoxicity against P388 cells. Additional bioassays with its synthetic
analogues revealed that the intriguing combination of the indole
moieties, most likely derived from three tryptamines, as well as the
pyridinium moiety were chiefly responsible for its potent biological
activities.

M any natural products, especially secondary metabolites,

are attractive as drugs and drug leads due to their broad
biological activities. The indole ring, derived from tryptophan,
or its derivatives such as tryptamine and indole itself, is one of
the most common moieties in many biologically active
alkaloids.1−4 Hence, numerous biologically active natural
products containing an indole ring have been isolated from
various natural sources including marine sponges using
bioassay-guided screening.
Marine sponges including their symbiotic microorganisms
are attractive sources for the discovery of biologically active
secondary metabolites. For instance, the potent protein
phosphatase inhibitor calyculin A and its derivatives were
isolated from the marine sponge Discodermia calyx collected in
relatively shallow waters from the ocean around Japan.5 We also
previously reported several cytotoxic secondary metabolites,
calyxamides6 and sulfolipodiscamides,7 from D. calyx and D.
kiiensis, respectively.
The metagenome mining approach is a powerful tool not
only to identify biosynthetic gene clusters of secondary
metabolites but also to directly isolate bioactive compounds
likely derived from genes of symbiotic microorganisms.8,9
Indeed, the biosynthetic gene cluster of calyculin A was recently
identified by using metagenomic fosmid libraries of the sponge-
microbe consortium of D. calyx.10 The huge NRPS-PKS hybrid
genes corresponding to calyculin A synthase are encoded by an
uncultured symbiotic bacterium, Candidatus “Entotheonella”.11
On the other hand, the clones transformed with metagenomic
libraries serve as a rich source of various biologically active
compounds. Therefore, we screened the Escherichia coli EPI300
clones transformed with metagenomic libraries of D. calyx to
search for new bioactive molecules and identified several
antibacterial compounds.12−15 In particular, several antibacterial
indole metabolites have been isolated, such as indole-porphyrin
© 2017 American Chemical Society and
American Society of Pharmacognosy
hybrid molecules,14 2,2-di(3-indolyl)-3-indolone,15 and turbo-
mycins.15,16 In this study, a novel pyridinium with three indole
moieties, tricepyridinium (1), originally detected from the
culture extracts of the E. coli clone incorporating metagenomic
libraries of D. calyx, and its analogues were chemically
synthesized. The biological activities of 1 and its synthetic
analogues are also reported herein.
In our previous research, 3-hydroxypalmitic acid was isolated
as a moderate antibacterial compound against Bacillus cereus
from the culture extracts of the E. coli EPI300 clone (pDC113)
incorporating metagenomic libraries of D. calyx.12 After
purification of the culture extracts by column chromatography
with Diaion HP-20, a fraction containing compound 1
exhibited significant antibacterial activity. Compound 1 was
separated by HPLC with a MeCN-aqueous acetic acid solvent
system to give 1 probably as an acetate salt (1a). NMR analysis
indicated the presence of two symmetrical 3-substituted indole
rings and one 3-ethylindole moiety. In addition, two equivalent
aromatic protons and one additional aromatic proton were
Received: December 15, 2016
Published: March 14, 2017
1205 DOI: 10.1021/acs.jnatprod.6b01152
J. Nat. Prod. 2017, 80, 1205−1209
Journal of Natural Products Note

observed as a singlet in the 1H NMR spectrum (Table 1, palladium(II) dichloride [PdCl2(dppf)] and potassium acetate
Figures S1 and S2). These findings together with MS analysis resulted in the synthesis of diboronate 3.17 Successive Suzuki−
Miyaura cross-coupling of 1-Boc-3-bromoindole with catalytic
Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data of PdCl2(dppf) and potassium carbonate gave bisindolyl com-
Tricepyridinium in CD3OD pound 4.18 After deprotection of the Boc group, the resulting 5
was treated with 3-(2-bromoethyl)indole at 90 °C for 42 h to
tricepyridinium (1)
give the desired 1-[2-(3-indolyl)ethyl]-3,5-di(3-indolyl)-pyridi-
position δC, type δH (J in Hz) HMBC nium (1) as a bromide salt (1b). As expected, all the
1 spectroscopic data for synthetic 1 were identical to those of
2, 6 136.2, CH 8.49, 2H, s 2 (6), 3′, 9′ the bacterial product. Thus, the chemical structure of 1 was
3, 5 137.0, C confirmed to be 1-[2-(3-indolyl)ethyl]-3,5-di(3-indolyl)-pyridi-
4 135.7, CH 8.78, s 2 (6), 3′ nium, which we named tricepyridinium. In addition, we
1′ (NH) synthesized pyridinium derivatives with two indole moieties
2′ 126.0, CH 7.70, 2H, s 3 (5), 3′, 3a′, 7a′ (6 and 7), a monoindolylpyridine derivative (8), and
3′ 109.3, C pyridinium derivatives with one indole moiety (9 and 10) to
3a′ 124.1, C investigate structure−activity relationships (Figure 1 and
4′ 118.0, CH 7.39, 2H, d (7.6) 3a′, 6′, 7a′ Supporting Information).
5′ 121.1, CH 7.14, 2H, dd (7.4, 7.6) 3a′, 7′
6′ 122.5, CH 7.22, 2H, dd (7.4, 7.9) 4′, 7a′
7′ 112.0, CH 7.47, 2H, d (7.9) 3a′, 5′
7a′ 137.6, C
1″ (NH)
2″ 124.0, CH 7.01, s 3″, 3a″, 7a″
3″ 108.4, C
3a″ 126.7, C
4″ 117.4, CH 7.53, d (7.6) 3a″, 7a″
5″ 119.3, CH 7.06, dd (7.4, 7.6) 3a″, 7″
6″ 121.9, CH 7.18, dd (7.4, 7.9) 4″, 7a″
7″ 111.7, CH 7.34, d (7.9) 3a″, 5″
7a″ 136.9, C Figure 1. Chemical structures of synthetic tricepyridinium analogues.
8″ 27.4, CH2 3.58, 2H, t (4.8) 2″, 3″, 3a″
9″ 62.5, CH2 5.00, 2H, t, (4.8) 2 (6), 3″
The antimicrobial activity of 1 was investigated by measuring
indicated that 1 was 1-[2-(3-indolyl)ethyl]-3,5-di(3-indolyl)- the minimum inhibitory concentration (MIC) according to the
pyridinium, which has never been isolated previously. Clinical & Laboratory Standards Institute (CLSI) protocol.19
To confirm the structure of 1 and evaluate its biologically Tricepyridinium bromide (1b) displays significant antibacterial
activities, we chemically synthesized 1. Three indole moieties activity against Bacillus cereus with an MIC value of 0.78 μg/mL
were introduced in two steps via Suzuki−Miyaura cross- and methicillin-sensitive Staphylococcus aureus (MSSA) with an
coupling and an SN2 reaction of an alkyl bromide, as shown in MIC value of 1.56 μg/mL as well as moderate antifungal
Scheme 1. Using 3,5-dibromopyridine 2 as a starting material, a activity against Candida albicans with an MIC value of 12.5 μg/
diborylation reaction by cross-coupling of bis(pinacolato)- mL with no activity against E. coli at 100 μg/mL, as shown in
diboron with catalytic [1,1′-bis(diphenylphosphino)ferrocene]- Table 2.

Scheme 1. Synthesis of Tricepyridinium Bromide (1b)

1206 DOI: 10.1021/acs.jnatprod.6b01152

J. Nat. Prod. 2017, 80, 1205−1209
Journal of Natural Products
Table 2. Antimicrobial Activities of Tricepyridinium
Bromide and Its Analogues
MIC [μg/mL]
compound B. cereus S. aureus (MSSA) C. albicans E. coli (W3110)
1b 0.78 1.56 12.5 >100
5 50.0 50.0 50.0 >100
6b 1.56 1.56 25.0 100
7b 25.0 50.0 100 >100
8 >100 100 100 >100
9b >100 >100 100 >100
10b >100 >100 100 >100
ampicillin 0.063 0.50 100 4.00
The synthetic analogues with two indole moieties (5, 6b, and
7b) exhibited antimicrobial activity against B. cereus, MSSA, and
C. albicans, but the intensities of 5 and 7b were much weaker
than that of 1b. Intriguingly, the 1-ethylpyridinium analogue
with two indole moieties (6b) exhibited antimicrobial activity
not only superior to all synthetic analogues, comparable to that
of 1b, but also weak activity against E. coli with an MIC value of
100 μg/mL. In contrast, the synthetic analogues with one
indole moiety (8, 9b, and 10b) exhibited no or only weak
antimicrobial activity with the MIC value of 100 μg/mL.
Subsequently, we tested the toxicity of these compounds
against the murine leukemic cell line P388. Compound 1b had
cytotoxicity to P388 cells with an IC50 value of 0.53 ± 0.07 μg/
mL (1.0 ± 0.1 μM). Interestingly, the 1-ethylpyridinium
analogue with two indole moieties (6b) exhibited the most
potent cytotoxicity to P388 cells with the IC50 value of 0.093 ±
0.029 μg/mL (0.22 ± 0.07 μM), whereas the other analogues
showed weaker cytotoxicity than that of 1b (Table S1).
These results indicated that the three indole moieties of 1
and the charged pyridinium were chiefly responsible for the
high cytotoxicity toward Gram-positive bacteria and fungi. In
particular, the indole group at the 3- or 5-position was more
crucial than an indole group at the 8″-position. Previous
structure−activity relationship studies on pyridinium com-
pounds demonstrated that 1-alkylpyridinium compounds
generally exhibited antimicrobial activity.20−23 However,
previous studies also revealed that the hydrophobic alkyl
chain could improve antimicrobial activity in most cases up to
an alkyl chain having 18 carbon atoms, and the 1-
alkylpyridinium compounds exhibited broad-spectrum anti-
microbial activity including against E. coli. Actually, the 1-
ethylpyridinium analogue (6b) exhibited weaker antimicrobial
activity against B. cereus and C. albicans but stronger activity
against E. coli compared to that of 1b. Therefore, tricepyr-
idinium (1) is likely to be a unique antimicrobial agent with a
different mechanism of action from typical 1-alkylpyridinium
compounds. In contrast to antimicrobial activity, the presence
of the indole group at the 8″-position does not enhance
cytotoxicity against P388 cells. This structure−activity relation-
ship assessment indicated that the two indole moieties played a
concerted role with the 1-ethylpyridinium moiety to induce
cytotoxicity of P388 cells. It is remarkable that a molecule with
three simple indole moieties displays high bioactivities, and the
synthetic route we established is highly applicable to
preparation of its derivatives.
The biosynthesis of 1 is predicted to involve tryptamine and
formaldehyde precursors (Scheme S1), and the three indole
rings are likely derived from three tryptamines. Because an
ORF involved in the biosynthesis of tryptamine is not present
1207
Note
in the fosmid containing the metagenomics gene cluster
(pDC113) based on our previous research,12 the genes
responsible for the biosynthesis of 1 remain elusive.
Unfortunately, it is difficult to completely eliminate the
possibility at this time that tricepyridinium is nonenzymatically
synthesized.24 We propose that the introduction of the fosmid
stimulated an endogenous biosynthetic pathway to tryptamine
in the host E. coli cells. Notably, tryptamine and formaldehyde,
which are toxic to the E. coli host, are consumed to produce
tricepyridinium (1) that can be exported as a possible
detoxification mechanism. Intriguingly, the released tricepyr-
idinium (1) exhibits significant toxicity to other micro-
organisms but not to the E. coli producer. The discovery of
novel compound 1 demonstrates that transformation of an
environmentally derived DNA library is a useful approach to
induce serendipitous synthesis of small molecules in a host
organism.
In this study, we isolated a novel pyridinium with three
indole moieties, tricepyridinium (1), from E. coli transformed
with metagenomic libraries of D. calyx, based on functional
screening. Tricepyridinium (1) possessed three unsubstituted
indole moieties and exhibited antibacterial activity, but not
against E. coli, antifungal activity, and cytotoxicity against P388
cells. All three indoles contributed to these activities. In
contrast to antimicrobial activity, elimination of the indole
group at the 8″-position (6b) resulted in more potent
cytotoxicity against P388 cells than that of tricepyridinium
bromide (1b). It was demonstrated in this study that the
synthesis of tricepyridinium (1) is feasible and that three indole
moieties are easily substituted with similar derivatives using our
synthetic scheme. The biological activity of 1 can be expected
to significantly improve through future structure−activity
relationship studies.
■
EXPERIMENTAL SECTION
General Experimental Procedures. The UV spectrum was
recorded on a Shimadzu UV-1280 spectrophotometer. The IR
spectrum was recorded on a JASCO FT/IR-4100 spectrometer with
CaF2 plates. NMR spectra were recorded on a JEOL ECX-500 or
ECA-500 spectrometer (1H NMR: 500 MHz; 13C NMR: 125 MHz)
with either TMS (at 0.0 ppm) or residual solvent peaks with specific
shift values as an internal standard (CDCl3: δH 7.26, δC 77.1; CD3OD:
δH 3.31, δC 49.0). ESI-HRMS analysis was performed with a Bruker
Compact QqTOF mass spectrometer. HPLC was performed on a
Shimadzu LC-20A system using an ODS column (4.6 × 250 mm,
COSMOSIL 5C18-PAQ, NACALAI TESQUE, Inc.). Solvents and
chemicals were purchased from Wako Pure Chemical Industry, Kanto
Chemical Co., Inc., or Sigma-Aldrich JAPAN unless otherwise noted.
Preparation of E. coli EPI300 Clone pDC113. The preparation
of the metagenomic DNA library from the marine sponge was
previously reported.13,14 Briefly, metagenomic DNA was prepared
from the marine sponge D. calyx collected at Shikine-jima Island in
Japan, and then the DNA fragments larger than 35 kbp were separated
from the total DNA by agarose gel electrophoresis. The DNA was
blunt-ended, ligated into the pCC1FOS fosmid vector, and packaged
to transfect E. coli according to the manufacturer’s instructions
(Epicenter). The packaged vector was transformed into E. coli EPI300-
T1R, and the cells were plated on LB agar containing chloramphenicol
(12.5 mg/L) as a selection marker according to the manufacturer’s
instructions (Epicenter).
Purification of Tricepyridinium Acetate (1a). After incubation
of the clone pDC113 in LB medium supplemented with
chloramphenicol (12.5 mg/L) at 30 °C and 120 rpm for 3 days, the
broth was subjected to solid phase extraction with Diaion HP-20 resin
(Mitsubishi Chemical Corp.) and then eluted with MeOH. The eluent
was purified by HPLC on an ODS column (Cosmosil COSMOSIL
DOI: 10.1021/acs.jnatprod.6b01152
J. Nat. Prod. 2017, 80, 1205−1209
Journal of Natural Products
5C18-PAQ, Nacalai Tesque), eluting with a gradient system from H2O
to MeOH containing 0.1% acetic acid to give compound 1a as a white
powder (0.76 mg/L broth). UV (MeOH) λmax (log ε) 224 (5.47), 261
(5.13), 279 (5.13), 324 (5.19), 394 (sh) (4.60) nm; IR (CaF2) νmax
3181, 1589, 1537, 1455, 1430, 1338, 1245 cm−1; 1H NMR (500 MHz,
CD3OD) δH 8.77 (1H, s), 8.47 (2H, s), 7.68 (2H, s), 7.51 (1H, d, J =
7.6 Hz), 7.46 (2H, d, J = 7.9 Hz), 7.40 (2H, d, J = 7.9 Hz), 7.37 (1H,
d, J = 7.9 Hz), 7.22 (2H, dd, J = 7.4, 7.9 Hz), 7.13 (2H, dd, J = 7.4, 7.9
Hz), 7.05 (1H, dd, J = 7.4, 7.6 Hz), 4.99 (2H, t, J = 4.8 Hz), 3.57 (2H,
t, J = 4.8 Hz), 1.87 (3H, s); 13C NMR (125 MHz, CD3OD) δC 174.7,
137.9, 137.54, 137.49, 136.8, 136.5, 127.1, 126.3, 124.6, 124.4, 122.9,
122.3, 121.4, 119.7, 118.3, 117.8, 112.4, 112.4, 109.8, 108.8, 62.9, 27.8,
23.1; HRESIMS m/z 453.2072 [M]+ (calcd for C31H25N4, 453.2074).
Synthetic Protocols for Tricepyridinium (1). Synthesis of 3,5-
Bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (3). To a
solution of 3,5-dibromopyridine (2) (1.39 g, 5.87 mmol) in 1,4-
dioxane (50 mL) were added bis(pinacolato)diboron (3.42 g, 13.5
mmol), KOAc (3.45 g, 35.2 mmol), and [1,1′-bis-
(diphenylphosphino)ferrocene]dichloropalladium(II) [PdCl2(dppf)2]
(341 mg, 0.418 mmol). After the reaction mixture was stirred at 85 °C
for 19 h, it was concentrated on a rotary evaporator. After the residue
was suspended in CH2Cl2, the suspension was filtered. The filtrate was
concentrated on the rotary evaporator to give a black solid. After the
solid residue was dissolved in H2O containing formic acid (pH 1), the
aqueous solution was washed with EtOAc. The aqueous solution was
evaporated to give 3 (1.38 g, 4.17 mmol, 71%) as a brown powder,
which was used without any further purification. 1H NMR (500 MHz,
CDCl3) δH 8.87 (3H, s), 1.20 (24H, s); 13C NMR (125 MHz, CDCl3)
δC 156.2, 149.5, 128.2, 84.3, 24.7. Unfortunately, HRESIMS could not
be obtained.
Synthesis of 3,5-Bis(1-Boc-indol-3-yl)pyridine (4). To a solution of
3 (231 mg, 0.698 mmol) in DMSO (7.0 mL) were added 1-Boc-3-
bromoindole (496 mg, 1.67 mmol), K2CO3 (964 mg, 6.98 mmol), and
PdCl2(dppf)2 (57.0 mg, 69.8 μmol). After the mixture was stirred at 90
°C for 2 h, it was quenched with H2O and filtered through Celite to
remove the palladium catalyst. The filtrate was extracted with EtOAc,
washed with saturated aqueous NaCl, dried over Na2SO4, and
evaporated. The residue was purified by silica gel column
chromatography (hexane/acetone = 15:1 to 10:1) to give a mixture
of dindolyl/monoindolyl compounds in a 5:1 molar ratio as a white
powder. The two compounds were completely separated by HPLC to
give 4 (261 mg, 0.512 mmol, 73%) as a white powder. 1H NMR (500
MHz, CDCl3) δH 8.89 (2H, brs), 8.25 (3H, d, J = 1.7 Hz), 7.91−7.76
(4H, m), 7.49−7.29 (4H, m), 1.72 (18H, s); 13C NMR (125 MHz,
CDCl3) δC 149.6, 146.4, 136.0, 134.8, 130.6, 128.5, 125.2, 123.9, 123.5,
119.5, 118.2, 115.8, 84.5, 28.3; HRESIMS m/z 510.2389 [M + H]+
(calcd for C31H32N3O4, 510.2387).
Synthesis of 3,5-Di(indol-3-yl)pyridine (5). To a solution of 4 (171
mg, 0.336 mmol) in CH2Cl2 (1.0 mL) was slowly added TFA (3.0
mL) in CH2Cl2 (1.0 mL) at 0 °C, and the mixture was stirred at 0 °C
for 1 h. The reaction mixture was quenched and basified with 1 M
aqueous KOH. The mixture was extracted with EtOAc, washed with
saturated aqueous NaCl, dried over Na2SO4, and evaporated to give 5
(101 mg, 0.326 mmol, 97%) as a brown powder, which was used
without any further purification. 1H NMR (500 MHz, CDCl3) δH 8.93
(1H, s), 8.53 (2H, s), 7.94 (2H, m), 7.50−7.35 (4H, m), 7.30−7.20
(4H, m); 13C NMR (125 MHz, CDCl3) δC 148.1, 136.8, 134.8, 131.9,
130.4, 125.5, 122.9, 122.4, 120.9, 119.4, 111.7; HRESIMS m/z
310.1332 [M + H]+ (calcd for C21H16N3, 310.1339).
Synthesis of Tricepyridinium Bromide (1b). To a solution of 5
(90.0 mg, 0.291 mmol) in 1,4-dioxane (0.60 mL) was added 3-(2-
bromoethyl)indole (98.0 mg, 0.437 mmol), and the mixture was
stirred at 90 °C for 42 h. After the mixture was quenched with H2O,
EtOAc was added, and the mixture was extracted with H2O. The
aqueous layer was evaporated to give 1b (97.0 mg, 0.182 mmol, 63%)
as an orange solid. For further analysis, the compound was purified by
HPLC to give analytically pure 1b as a yellow powder. 1H NMR (500
MHz, CD3OD) δH 8.78 (1H, s), 8.49 (2H, s), 7.70 (2H, s), 7.53 (1H,
d, J = 7.6 Hz), 7.47 (2H, d, J = 7.9 Hz), 7.39 (2H, d, J = 7.6 Hz), 7.34
(1H, d, J = 7.9 Hz), 7.22 (2H, dd, J = 7.4, 7.9 Hz), 7.14 (2H, dd, J =
1208
Note
7.4, 7.6 Hz), 7.06 (1H, dd, J = 7.4, 7.6 Hz), 5.00 (2H, t, J = 4.8 Hz),
3.58 (2H, t, J = 4.8 Hz); 13C NMR (125 MHz, CD3OD) δC 137.6,
137.0, 136.9, 136.2, 135.7, 126.7, 126.0, 124.1, 124.0, 122.5, 121.9,
121.1, 119.3, 118.0, 117.4, 112.0, 111.7, 109.3, 108.4, 62.5, 27.4;
HRESIMS m/z 453.2075 [M]+ (calcd for C31H25N4, 453.2074).
Antimicrobial Activity. According to the CLSI protocol,19 the
turbidity of suspensions of E. coli W3110, MSSA1, B. cereus, and C.
albicans was adjusted to 0.5 McFarland, respectively, in PBS(−) and
then diluted 300-fold with growth medium (Mueller Hinton Broth,
Sigma-Aldrich) for E. coli, MSSA, and B. cereus, Roswell Park Memorial
Institute 1640 medium (RPMI 1640, Sigma-Aldrich) with glutamine,
without sodium bicarbonate, with 2% (w/v) glucose, and with phenol
red, buffered to pH 7.0 with MOPS at 165 mM for C. albicans) in 96-
well plates. Each well contained serially diluted solutions of the
compounds. Final culture volume for each well was adjusted to 100
μL, and after 20 h (24 h for C. albicans) incubation at 37 °C, MICs
were scored visually. Ampicillin was used as a positive control.
Cell Culture and Cytotoxic Activity. The murine leukemic cell
line P388 was maintained in Dulbecco’s modified Eagle medium
supplemented with 10% fetal bovine serum (MP Biomedicals) and
1.0% penicillin (10,000 U/mL)-streptomycin (10 mg/L). Cytotoxicity
of the compounds was evaluated via MTT assay based on
mitochondrial succinate dehydrogenase activity and confirmed via
microscopic observation. At the end of the incubation, 15 μL of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 5.0
mg/mL was added to each of the wells. The cultures were incubated
for another 3 h before the cell supernatants were removed. After the
removal of the cell supernatants, 100 μL of dimethyl sulfoxide was
added to each well to dissolve the formed formazan. The optical
density was measured using a microplate reader (Bio-Rad) at 550 nm
with reference wavelength at 700 nm. Cisplatin and doxorubicin were
used as positive controls.
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b01152.
Synthetic protocols for analogues, NMR spectra, table of
cytotoxicity, and plausible biosynthetic pathway (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail: okadam@mol.f.u-tokyo.ac.jp.
*E-mail: abei@mol.f.u-tokyo.ac.jp.
ORCID
Toshiyuki Wakimoto: 0000-0003-2917-1797
Ikuro Abe: 0000-0002-3640-888X
Present Address
‡
T.W.: Faculty of Pharmaceutical Sciences, Hokkaido Uni-
versity, Kita 12, Nishi 6, Kita-ku, Sapporo 060−0812, Japan
Author Contributions
†
M.O. and T.S. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports,
Science and Technology, Japan (JSPS KAKENHI Grant
Number JP15H01836, JP16H06443, and JP24688011). Sup-
port was also provided by Takeda Science Foundation,
Kobayashi International Scholarship Foundation, and a Grant-
in-Aid for the Cooperative Research Project from Institute of
DOI: 10.1021/acs.jnatprod.6b01152
J. Nat. Prod. 2017, 80, 1205−1209
Journal of Natural Products Note

Natural Medicine, University of Toyama in 2016 (to M.O.).

We thank Dr. Rui He and Dr. Yuya Takeshige for their
technical assistance.

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1209 DOI: 10.1021/acs.jnatprod.6b01152

J. Nat. Prod. 2017, 80, 1205−1209