Biological activity of intervenolin analogs with a phenyl substituent

Article abstract of DOI:10.1038/ja.2017.123

Intervenolin analogs with a phenyl substituent at the 2- or 3-position were synthesized. The compounds (3-11) showed weak or no inhibitory activity toward the growth of MKN-74 gastric adenocarcinoma cells, even in the presence or absence of the corresponding Hs738 stromal cells, whereas 2-substituted analogs exhibited selective anti-Helicobacter pylori activity. Introduction of a pendant side chain on the nitrogen alleviated their acute toxicity in mice. The 2-phenyl-substituted analogs are reasonable structural templates for structure-activity relationship studies toward the development of anti-H. pylori agents that do not affect human cells.

Full text of DOI:10.1038/ja.2017.123

The Journal of Antibiotics (2017), 1–5
2017 Japan Antibiotics Research Association All rights reserved 0021-8820/17
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&
ORIGINAL ARTICLE
Biological activity of intervenolin analogs with a phenyl
substituent
Hikaru Abe1, Manabu Kawada1,2, Masayuki Igarashi1, Shun-ichi Ohba2, Chigusa Hayashi1, Chiharu Sakashita1,
Takumi Watanabe1 and Masakatsu Shibasaki1
Intervenolin analogs with a phenyl substituent at the 2- or 3-position were synthesized. The compounds (3–11) showed weak or
no inhibitory activity toward the growth of MKN-74 gastric adenocarcinoma cells, even in the presence or absence of the
corresponding Hs738 stromal cells, whereas 2-substituted analogs exhibited selective anti-Helicobacter pylori activity.
Introduction of a pendant side chain on the nitrogen alleviated their acute toxicity in mice. The 2-phenyl-substituted analogs are
reasonable structural templates for structure–activity relationship studies toward the development of anti-H. pylori agents that do
not affect human cells.
The Journal of Antibiotics advance online publication, 11 October 2017; doi:10.1038/ja.2017.123
INTRODUCTION
first SAR study revealed that (1) substituents at the 2-position have a
large impact on the biological activity, (2) a substituent on the
nitrogen is beneficial for reducing acute toxicity and (3) many of the
synthetic intervenolin analogs exhibit highly selective anti-Helicobacter
pylori activity over a variety of pathogenic bacteria: inspired by
the reported work on natural quinolones,10 the antibacterial activity
was investigated. On the other hand, SAR information on the
3-position has not been collected. More importantly, separation of
the antiproliferative activity and anti-H. pylori activity for lead-
generation purposes would help to avoid undesirable side effects.
In the above-mentioned SAR study, an intervenolin analog with a
4-fluorophenyl group at the 2-position (2, Figure 1) exhibited no
growth-inhibitory activity (IC50: 4100 μg ml− 1) toward MKN-74
gastric adenocarcinoma cells, irrespective of the presence or absence
of stromal cells (Hs738), whereas it exhibited moderate anti-H. pylori
activity (MIC: 0.2 μg ml− 1). These results prompted us to carry out
further SAR studies of intervenolin analogs with aromatic substituents
(Scheme 1), and the results are disclosed herein. Initially, compounds
with a phenyl group at the 2-position were evaluated, followed by
investigation of compounds with a phenyl group at the 3-position
instead of the original methyl group. For the latter analogs, the side
chain at the 2-position was fixed to C8 that was demonstrated to be
the preferred length in a previous SAR study.9
Tumor tissue comprises not only tumor cells, but also stromal cells,
including fibroblasts and endothelial cells, that are normal cells.1,2 In
fact, extensive evidence indicates that stromal cells contribute largely
to the growth of adjacent tumor cells,3,4 as well as to tumor
progression5 and malignancy6 (tumor–stromal interactions). Targeted
cancer therapy using small-molecular-weight molecules that act
on cancer-relevant proteinous proliferation regulators is nowadays
frequently included in the cancer treatment regimen: these therapeutic
agents almost exclusively influence the fate of the tumor cells without
seriously damaging normal cells, and are thus less likely to produce
side effects. Although this relatively new class of therapeutics has
enjoyed great clinical success, the lability of cancer genes often leads to
mutations of the molecular target, thereby increasing the risk of
relapse of the treated cancer. Consequently, exploratory studies of
chemotherapeutic agents with molecular targets of normal cell origin
are in high demand.
In 2013, Kawada et al.7 reported a quinolone-based natural product,
intervenolin (1, Figure 1), produced by Nocardia sp. ML96-86F2 as a
modulator of tumor–stroma interactions. Intervenolin inhibits the
growth of gastric and colon tumor cell lines more potently in the
presence of the corresponding stromal cells than in their absence. Our
laboratory established a synthetic route to this compound by taking
advantage of Suzuki–Miyaura coupling and thiocyanate–isothiocyanate
rearrangement as key transformations to introduce a geranyl side
chain and pendant iminodithiocarbonate moiety, respectively.8 Both
side chains are rare chemical motifs as substituents of quinolone-
related compounds; the latter is particularly uncommon in natural
products as a whole. The successful chemical synthesis of intervenolin
enabled detailed biological investigations, including in vivo antitumor
RESULTS AND DISCUSSION
Synthesis
The synthesis was accomplished uneventfully according to the reported
protocol9 (Scheme 1) starting from aniline derivatives equipped with a
ketone functionality that was converted to the corresponding amide
tests and structure–activity relationship (SAR) studies.9 Indeed, the with installation of the structural element for the 2-substituent.
1Institute of Microbial Chemistry (BIKAKEN), Tokyo, Japan and 2Institute of Microbial Chemistry (BIKAKEN), Shizuoka, Japan
Correspondence: Dr T Watanabe or Dr Professor M Shibasaki, Institute of Microbial Chemistry (BIKAKEN), Tokyo, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan.
E-mail: mshibasa@bikaken.or.jp or twatanabe@bikaken.or.jp
Received 5 June 2017; revised 16 August 2017; accepted 24 August 2017

Biological activity of intervenolin analogs
H Abe et al
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Subsequent annulation constructed the requisite quinoline core, monocultured conditions, consistent with the results of the
followed by functionalization of the nitrogen, if needed.
2-(4’-flurophenyl)-substituted compound (2). The tendency for
substituents at the 1-position to reduce acute toxicity (i.v., single
dose), which was observed in the previous study, was also observed in
Antiproliferative activity toward gastric cancer cells (MKN-74)
under cocultured conditions with gastric stromal cells (Hs738) and this case (compare 2 with 3 and 5). Introduction of a phenyl group
monocultured conditions
instead of a methyl group at the 3-position (6–11) had detrimental
We first examined the activity of each synthesized intervenolin analog effects regardless of the substituent on the nitrogen. Further SAR
(3–11) as a modulator of tumor–stromal interactions (Table 1), as the studies are needed to clarify whether the results were due to the
growth-inhibitory activity toward MKN-74 gastric tumor cells in bulkiness of the benzene ring.
the presence or absence of Hs738 stromal cells was evaluated as in
the previous SAR study. For 2-phenyl-substituted analogs 3–5, no Anti-H. pylori activity
antiproliferative activity was observed under either cocultured or In contrast to the data regarding their properties as modulators of
tumor–stromal interactions shown in Table 1, the intervenolin analogs
with the phenyl functionality at the 2-position (3–5) displayed anti-H.
pylori activity (Table 2). In particular, the potency of 3 containing a
methoxycarbonylmethyl moiety at the 1-position exhibited the best
anti-H. pylori activity among the compounds synthesized herein;
fourfold less potent toward JCM 12093 strain (MIC: 0.25 μg ml− 1
)
than the positive control used in this study, clarithromycin (MIC:
0.0625 μg ml− 1). The potency of 2 is cited from the previous study in
Table 2 that was twofold less potent than clarithromycin (MIC:
0.0078 μg ml− 1).9 It is noteworthy that this class of compounds (3–5)
exhibited limited antibacterial activity toward other species, such as
Figure 1 Intervenolin and an analog.
Scheme 1 Intervenolin analogs in this study, and outline of the synthesis.
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Campylobacter coli, Staphylococcus aureus, Enterococcus faecalis and br: broad), coupling constant (Hz) and integration. HR-MS (electrospray
ionization (ESI)− time-of-flight (+)) were measured on Thermo Fisher
Scientific (Yokohama, Japan) LTQ Orbitrap XL. Unless otherwise noted,
materials were purchased from commercial suppliers and were used without
purification.
Escherichia coli, clearly demonstrating that these analogs maintain
high selectivity toward H. pylori, a characteristic property of some
intervenolin analogs synthesized in the previous study. As expected,
the antibacterial activity was independent of the growth-inhibitory
activity. In fact, 3-phenyl-substituted analogs (6–11) again exhibited
limited anti-H. pylori potency (that is, MIC: 4–64 μg ml− 1); this mode
of substitution is not suitable for molecular design aimed at pursuing
both biological activities.
Synthesis
Intervenolin analogs were synthesized according to the reported procedure.
Characterization data for 3–11 (novel compounds) are listed below. A copy of
NMR charts is in Supplementary Material.
Methyl 2-(3-methyl-4-oxo-2-phenylquinolin-1(4H)-yl)acetate (3). 1H NMR
(500 MHz, CDCl3) δ 8.56 (1H, dd, J = 8.2, 1.6 Hz), 7.64 (1H, m), 7.55–7.51
(3H, m), 7.40 (1H, m), 7.31–7.28 (2H, m), 7.19 (1H, d, J = 8.5 Hz), 4.61
(2H, s), 3.71 (3H, s), 3.62–3.51 (2H, m), 1.84 (3H, s); 13C NMR (125 MHz,
CDCl3) δ 177.8, 168.6, 150.6, 140.5, 135.0, 132.3, 129.5, 129.4, 128.3, 127.4,
125.1, 123.4, 118.6, 114.6, 52.8, 50.3, 13.4; HR-MS (ESI) Anal calcd for
C19H18NO3 m/z 308.1287 [M+H]+, found 308.1279.
EXPERIMENTAL PROCEDURES
General
NMR was recorded on JEOL (Akishima, Japan) ECS-400 and ECA-600
spectrometers. Chemical shifts of protons are reported in ppm downfield from
TMS or are referenced to residual protium in the NMR solvent (CDCl3:
δ 7.26 ppm, CD3OD: δ 3.30 ppm, DMSO-d6: 2.49). For 13C NMR, chemical
shifts were reported in ppm downfield from TMS or are relative to the NMR
solvent (CDCl3: 77.0 ppm, CD3OD: δ 49.0 ppm, DMSO-d6: 39.7 ppm) as an
internal reference. NMR data are reported as follows: chemical shifts, multi-
plicity (s: singlet, d: doublet, dd: doublet of doublets, t: triplet, m: multiplet,
2-(3-Methyl-4-oxo-2-phenylquinolin-1(4H)-yl)acetic acid (4). 1H NMR
(500 MHz, DMSO-d6) δ 8.28 (1H, dd, J = 7.9, 1.6 Hz), 7.72 (1H, m),
7.62–7.55 (4H, m), 7.41 (1H, m), 7.38–7.22 (2H, br), 1.66 (3H, s); 13C
NMR (125 MHz, DMSO-d6) δ 176.0, 169.5, 150.9, 140.4, 134.7, 132.1, 129.4,
129.3, 128.0, 125.7, 124.2, 123.1, 116.6, 116.5, 50.1, 13.1; HR-MS (ESI) Anal
calcd for C18H16NO3 m/z 294.1130 [M+H]+, found 294.1126.
Table 1 Effects of the synthetic intervenolin derivatives on cocultured
MKN-74 cells and Hs738 cells
S-Methyl((3-methyl-4-oxo-2-phenylquinolin-1(4H)-yl)methyl)carbamothioate
(5). 1H NMR (500 MHz, CDCl3) δ 8.66 (1H, br), 8.12–8.08 (1H, br), 7.59–
7.50 (5H, m), 7.40 (1H, m), 7.23 (1H, d, J = 8.5 Hz), 7.17 (1H, br), 4.61 (2H,
s), 2.47 (3H, s), 1.47 (3H, s); 13C NMR (125 MHz, CDCl3) δ 177.8, 169.9,
151.1, 140.2, 134.6, 132.4, 129.8, 129.5, 128.2, 126.7, 125.0, 124.0, 118.7, 115.2,
53.6, 22.6, 13.2; HR-MS (ESI) Anal calcd for C19H19NO2S m/z 339.1167
[M+H]+, found 339.1155.
In vitro IC50 (μg ml− 1
)
Compound
Cocultured
Monocultured
In vivo MTD (mg kg− 1
)
1
0.52
410
410
410
410
410
5.9
3.9
410
410
410
410
410
410
410
9.3
450
6.25a
450
NT
2
3
2-Octyl-3-phenylquinolin-4(1H)-one (6). 1H NMR (400 MHz, CDCl3) δ 8.34
(1H, d, J = 8.3 Hz), 7.41 (1H, m), 7.29–7.19 (6H, m), 7.15–7.10 (1H, m), 2.30
(2H, m), 1.42 (2H, m), 1.19–1.03 (10H, m), 0.87 (3H, t, J = 7.1 Hz); 13C NMR
(100 MHz, CDCl3) δ 176.9, 152.4, 139.9, 136.4, 131.1, 131.0, 128.1, 126.8,
125.4, 124.6, 123.0, 121.6, 118.7, 32.3, 31.7, 29.2, 29.1, 28.9, 22.5, 14.0; HR-MS
(ESI) Anal calcd for C23H18NO m/z 334.2171 [M+H]+, found 334.2167.
4
5b
450
NT
6
7
450
450
450
NT
8
410
4.3
9
Methyl 2-(2-octyl-4-oxo-3-phenylquinolin-1(4H)-yl)acetate (7). 1H NMR
(400 MHz, CDCl3) δ 8.48 (1H, dd, J =8.1, 1.5 Hz), 7.63 (1H, m), 7.44–7.32
(4H, m), 7.28–7.25 (3H, m), 4.95 (2H, s), 3.83 (3H, s), 2.54 (2H, br), 1.50 (2H,
m), 1.28–1.08 (10H, m), 0.86 (3H, t, J =7.1 Hz); 13C NMR (100 MHz, CDCl3)
δ 176.7, 168.6, 151.8, 141.1, 136.6, 132.3, 130.6, 128.4, 127.6, 127.2, 126.2, 124.7,
10
11
410
5.9
410
~ 10
NT
Abbreviations: MTD, maximum tolerated dose; NT, not tested.
aData from Abe et al.9
bContains a small amount of impurity.
Table 2 Antibacterial activities of the synthetic intervenolin derivatives (MIC: μg ml− 1
)
Helicobacter pylori JCM
12093
H. pylori JCM
12095
Campylobacter coli JCM
2529
Staphylococcus aureus
FDA209P
Enterococcus faecalis JCM
5803
Escherichia coli
K-12
Compound
1
2a
0.125
0.0156
0.25
1
0.0625
32
NT
128
128
4128
4128
4128
4128
4128
128
4128
128
0.0156
3
0.5
2
64
128
128
4
5b
64
128
128
0.5
1
64
128
128
6
32
64
8
4128
4128
128
64
128
4128
128
7
4
4
4128
4128
4128
4128
4128
0.25
8
32
64
16
32
16
0.0312
4128
4128
128
128
9
8
4128
4128
4128
16
10
11
CAM
32
64
16
64
4128
o0.125
0.0625
4
Abbreviations: CAM, clarithromycin; NT, not tested.
aData from Abe et al.9
bContains a small amount of impurity.
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Biological activity of intervenolin analogs
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123.5, 114.4, 53.0, 48.5, 31.6, 31.5, 29.4, 28.88, 28.86, 28.7, 22.5, 14.0; HR-MS
further for 3 days. For monoculture of MKN-74 cells, only assay medium with
test samples was first incubated for 2 days, and then MKN-74 cells were
inoculated as described above and further cultured for 3 days. The growth of
MKN-74 cells was determined by measuring GFP fluorescence intensity
(excitation at 485 nm and emission at 538 nm) by lysing the cells in 10 mM
Tris-HCl, 150 mM NaCl, 0.9 mM CaCl2 and 1% Triton X-100.
(ESI) Anal calcd for C26H32NO3 m/z 406.2382 [M+H]+, found 406.2374.
2-(2-Octyl-4-oxo-3-phenylquinolin-1(4H)-yl)aceticacid (8). 1H NMR (400 MHz,
CD3OD) δ 8.35 (1H, dd, J=8.1, 1.5 Hz), 7.72 (1H, m), 7.66 (1H, d, J=8.6 Hz),
7.47–7.35 (4H, m), 7.26–7.24 (2H, m), 5.04 (2H, br), 2.62 (2H, br), 1.56 (2H,
m), 1.29–1.06 (10H, m), 0.87 (3H, t, J=6.9 Hz); 13C NMR (100 MHz, CD3OD)
δ 178.1, 156.3, 142.9, 138.2, 133.6, 132.0, 129.6, 128.4, 127.3, 126.9, 125.2, 124.9,
117.7, 51.6, 32.9, 32.7, 30.5, 30.0, 29.6, 29.4, 23.6, 14.4; HR-MS (ESI) Anal calcd
for C25H30O3 m/z 392.2226 [M+H]+, found 392.2215.
Acute toxicity in vivo
Female ICR mice, 4-week old, were purchased from Charles River
Breeding Laboratories (Yokohama, Japan) and maintained in a specific
pathogen-free barrier facility according to our institutional guidelines. Samples
were injected i.v. into mice. Over 2 weeks of observation, half of the dose that
caused death or severe toxic effect was designated as the maximum tolerated
dose in this study.
S-Methyl((2-octyl-4-oxo-3-phenylquinolin-1(4H)-yl)methyl)carbamothioate
(9). 1H NMR (400 MHz, CDCl3) δ 8.37 (1H, m), 7.63 (1H, m), 7.54 (1H, d,
J = 8.5 Hz), 7.36–7.28 (4H, m), 7.13 (2H, d, J = 6.9 Hz), 6.92 (1H, br), 5.71
(2H, s), 2.69 (2H, m), 2.37 (3H, s), 1.48 (2H, m), 1.27–1.08 (10H, m), 0.86
(3H, t, J =7.3 Hz); 13C NMR (100 MHz, CDCl3) δ 176.7, 168.7, 152.3, 140.1,
132.6, 130.5, 128.4, 127.6, 126.0, 124.5, 123.7, 115.0, 52.2, 31.7, 31.1, 29.8, 29.4,
28.9, 28.8, 22.6, 14.1, 12.4; HR-MS (ESI) Anal calcd for C26H33N2O2S m/z
437.2263 [M+H]+, found 437.2253.
Antimicrobial effect
H. pylori JCM12093, H. pylori JCM12095, C. coli JCM2529, and E. faecalis
JCM5803 were purchased from Japan Collection of Microorganisms (Tsukuba,
Japan). S. aureus FDA209P and E. coli K-12 were from the in-house collection
of the Institute of Microbial Chemistry. The MICs were determined by using
the microdilution broth method in 96-well plates. Twofold serial dilutions of
the test samples were prepared with DMSO. The dilution series were initially
prepared at 200-fold final concentrations from this stock to provide a final test
range of 128–0.001 μg ml− 1. Then, 50 μl of each dilution and 50 μl of test
organisms (2–9 × 104 CFUs per ml) were dispensed into each well. The MIC
was defined as the lowest concentration of compound at which no visible
growth could be detected. The test organisms, medium and culture conditions
were as follows: H. pylori JCM12093 and H. pylori JCM12095 and C. coli
S-Methyl 2-(2-octyl-4-oxo-3-phenylquinolin-1(4H)-yl)ethanethioate (10). 1H
NMR (400 MHz, CDCl3) δ 8.48 (1H, m), 7.63 (1H, m), 7.45–7.32 (4H, m),
7.29–7.25 (3H, m), 5.07 (2H, br), 2.54 (2H, m), 2.36 (3H, s), 1.50 (2H, m),
1.27–1.06 (10H, m), 0.86 (3H, t, J =7.1 Hz); 13C NMR (100 MHz, CDCl3)
δ
196.5, 176.7, 151.9, 141.1, 136.5, 132.3, 130.6, 128.4, 127.6, 127.2,
126.2, 125.0, 123.7, 114.8, 56.0, 31.6, 31.5, 29.3, 29.0, 28.8, 28.6, 22.5, 14.0,
11.4; HR-MS (ESI) Anal calcd for C26H32NO2S m/z 422.2154 [M+H]+, found
422.2145.
Methyl((2-octyl-4-oxo-3-phenylquinolin-1(4H)-yl)methyl)carbamodithioate
(11). 1H NMR (400 MHz, CDCl3) δ 8.30 (1H, m), 8.03 (1H, br), 7.66 (1H,
m), 7.47 (1H, d, J = 8.6 Hz), 7.40–7.26 (4H, m), 7.10 (1H, m), 6.13 (2H, br),
2.66 (3H, s), 2.60 (2H, m), 1.52 (2H, m), 1.27–1.06 (10H, m), 0.86 (3H, t,
J = 7.1 Hz); 13C NMR (100 MHz, CDCl3) δ 200.1, 176.9, 152.2, 140.2, 136.1,
132.9, 130.4, 128.4, 127.4, 127.3, 125.7, 124.6, 124.0, 115.2, 58.1, 31.7, 31.5,
29.5, 29.4, 28.9, 28.7, 22.6, 18.2, 14.1; HR-MS (ESI) Anal calcd for
C26H33N2OS2 m/z 453.2034 [M+H]+, found 453.2024.
JCM2529 were cultured in
a Brain Heart Infusion Broth medium
(BD Biosciences) with 10% FBS at 37 °C for 144 h under microaerobic
conditions of 85% N2, 5% O2 and 10% CO2; S. aureus FDA209P and E. coli
K-12 were cultured in a nutrient broth medium consisting of 1% polypentone
(Nihon Pharmaceutical, Tokyo, Japan), 1% fish extract (Kyokuto, Tokyo,
Japan) and 0.2% NaCl for 18 h at 37 °C. E. faecalis JCM5803 was cultured in a
Heart Infusion Broth medium (BD Biosciences) at 37 °C for 18 h.
Cells and reagents
Human gastric adenocarcinoma MKN-74 cells were obtained from the
RIKEN cell bank (Tsukuba, Japan) and maintained in Dulbecco’s modified
Eagle’s medium (DMEM) (Nissui, Tokyo, Japan) supplemented with 10%
fetal bovine serum (FBS; Sigma, Tokyo, Japan), 100 units per ml penicillin
G (Life Technologies, Carlsbad, CA, USA) and 100 μg ml− 1 streptomycin
(Life Technologies) at 37 °C with 5% CO2. Hs738 human gastric stromal cells
(CRL-7869) were obtained from ATCC (Manassas, VA, USA) and maintained
in DMEM supplemented with 10% FBS, 100 units per ml penicillin G,
100 μg ml− 1 streptomycin, ITH (5 μg ml− 1 insulin, 5 μg ml− 1 transferrin, and
1.4 μM hydrocortisone) and 5 ng ml− 1 basic-fibroblast growth factor (Pepro
Tech, Rocky Hill, NJ, USA) at 37 °C with 5% CO2 as described previously.11
CONCLUSIONS
Intervenolin analogs with a phenyl substituent at the 2- or 3-position
were synthesized. The compounds (3–11) showed weak or no
inhibitory activity toward the growth of MKN-74 gastric cancer cells,
even in the presence or absence of the corresponding Hs738 stromal
cells. On the other hand, 2-substituted compounds had selective anti-
H. pylori activity with reasonable potency. Further SAR studies focused
on 2-aromatic analogs to generate anti-H. pylori lead compounds,
mechanistic study on antiproliferative and anti-H. pylori activities and
investigation on antibacterial activity toward drug-resistant strains of
H. pylori are underway and will be reported in due course.
Green fluorescence protein (GFP) transfection
MKN-74 cells (2× 105) were cultured in 6-well plates in 10% FBS–DMEM for
2 days. The medium was replaced by 800 μl OPTI-MEM (Life Technologies),
and then 200 μl OPTI-MEM containing 1 μg pEGFP-C1 vector (BD
Biosciences, Franklin Lakes, NJ, USA), 4 μl Lipofectamine Reagent (Life
Technologies) and 6 μl PLUS Reagent (Life Technologies) was added to the
cells. After 3 h of incubation, 1 ml of 20% FBS-OPTI-MEM was added to each
well and the cells were cultured for 24 h. Stably transfected cells were selected
with 400 μg ml− 1 G418 (Promega, Tokyo, Japan).
DEDICATION
Dedicated to the late Professor Hamao Umezawa for his great
achievement on development of antibiotics including kanamycin.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
Cell growth and coculture experiment
MK, HA and TW are grateful to the Japan Agency for Medical Research and
development (AMED) (the Project for Cancer Research And Therapeutic
Evolution (P-CREATE)) for financial support. We thank Dr Ryuichi Sawa, Ms
Yumiko Kubota, Dr Kiyoko Iijima and Ms Yuko Takahashi (BIKAKEN) for
For coculture experiments, Hs738 cells were first inoculated in 96-well plates at
5 × 103 cells per well in 0.1 ml DMEM supplemented with 1% D-FBS and ITH.
Test samples were added to the wells and Hs738 cells were cultured for 2 days.
Then, 10 μl of MKN-74 cell suspension (5 × 103 cells) in serum-free DMEM
was inoculated onto a monolayer of Hs738 cells and the cells were cultured spectroscopic analysis.
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Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
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