Structure-activity relationship study of intervenolin derivatives: Synthesis, antitumor, and anti-Helicobacter pylori activities

Article abstract of DOI:10.1016/j.tet.2013.05.033

Intervenolin is a natural antitumor quinolone that inhibits the growth of MKN-74 gastric tumor cells cocultured with Hs738 gastric stromal cells, and exhibits selective anti-Helicobacter pylori activities. In this report, a structure-activity relationship study of intervenolin is presented. Suitable substituents on the nitrogen, including iminodithiocarbonate moiety, alleviated acute toxicity, and four derivatives displayed specific anti-H. pylori activity. One intervenolin analog with quinoline scaffold also exhibited selective growth inhibition toward cocultured MKN-74, and in vivo antitumor effect.

Full text of DOI:10.1016/j.tet.2013.05.033

Tetrahedron 69 (2013) 7608e7617
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Structureeactivity relationship study of intervenolin derivatives:
synthesis, antitumor, and anti-Helicobacter pylori activities
,
b
b
b
Hikaru Abe a, Manabu Kawada b , Hiroyuki Inoue , Shun-ichi Ohba , Tohru Masuda ,
*
Chigusa Hayashi a, Masayuki Igarashi a, Akio Nomoto a, Takumi Watanabe a
,
,
*
,
Masakatsu Shibasaki a
*
a Institute of Microbial Chemistry (BIKAKEN), Tokyo, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
b Institute of Microbial Chemistry (BIKAKEN), Numazu, 18-14 Miyamoto, Numazu, Shizuoka 410-0301, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Intervenolin is a natural antitumor quinolone that inhibits the growth of MKN-74 gastric tumor cells
cocultured with Hs738 gastric stromal cells, and exhibits selective anti-Helicobacter pylori activities. In
this report, a structureeactivity relationship study of intervenolin is presented. Suitable substituents on
the nitrogen, including iminodithiocarbonate moiety, alleviated acute toxicity, and four derivatives
displayed specific anti-H. pylori activity. One intervenolin analog with quinoline scaffold also exhibited
selective growth inhibition toward cocultured MKN-74, and in vivo antitumor effect.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 21 March 2013
Received in revised form 9 May 2013
Accepted 10 May 2013
Available online 16 May 2013
Keywords:
Antitumor agent
Tumorestroma interaction
Anti-Helicobacter pylori agent
Quinolone
Iminodithiocarbonate
1. Introduction
Tumor tissues comprise not only tumor cells but also the sur-
rounding stroma.11 The stroma comprises normal cells, including
The pros and cons of conventional cancer chemotherapy are
a matter of great debate: a patient’s life may be prolonged at the
expense of severe adverse side effects because the majority of
clinically used antitumor agents are cytotoxic substances that act
not only on tumor cells but also normal cells. To address this issue,
investigators have searched for modulators of signaling systems
unique to tumor cells. This relatively novel paradigm of tumor
chemotherapy using molecules of this class is referred to as targeted
cancer therapy. Since the first approval in 2001 of imatinib,1 a tyro-
sine kinase inhibitor, targeted cancer drug with low molecular
weight, fewsimilardrugs were reported in the last decade, including
a Raf kinase inhibitor2 and a proteasome inhibitor,3 in addition to
other tyrosine kinase inhibitors.4e10 Despite the triumph of targeted
cancer drugs against specific classes of tumors, not all subtypes of
treated cancers are universally susceptible because of their hetero-
geneous genetic backgrounds. In an attempt to overcome this ob-
stacle, we designed a paradoxical strategy, i.e., the development of
chemotherapeutic agents that selectively affect normal cells.
endothelial cells, fibroblast-like cells (termed stromal cells), and
extracellular matrix.12 Recent studies revealed that stromal cells
regulate the growth of adjacent tumor cells either positively or
negatively via direct or indirect communication influenced by cell
adhesion or secreted factors.13 Signals transmitted from stromal
cells are particularly interesting because the machineries come
from normal cells wherein the participating molecules are believed
to be barely mutated. To search for modulators of tumorestroma
interactions, we constructed an assay system to pick molecules that
inhibit the growth of tumor cells cocultured with stromal cells
more potently compared to monocultured tumor cells.14
Some natural products exhibit the desired selectivity, including
phthoxazolin A,15 NBRI23477
leucinostatins.18
A
and B,16 NMRI16716s,17 and
* Corresponding authors. Tel.: þ81 55 924 0601; fax: þ81 55 922 6888 (M.K.);
tel.: þ81 3 3441 4173; fax: þ81 3 3441 7589 (T.W.) tel.: þ81 3 3447 7779; fax: þ81 3
3441 7589 (M.S.); e-mail addresses: kawadam@bikaken.or.jp (M. Kawada), mshi-
basa@mol.f.u-tokyo.ac.jp, mshibasa@bikaken.or.jp (M. Shibasaki).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.05.033

H. Abe et al. / Tetrahedron 69 (2013) 7608e7617
7609
We recently discovered an unprecedented second metabolite of
microorganisms, intervenolin (1),19 which also has selective in-
hibitory activity on the proliferation of human gastric and colon
cancer cell lines. Intervenolin belongs to the 4-quinolone family
whose scaffold is considered as a privileged structure, especially for
anti-infective medicines. Despite its familiar core structure, inter-
venolin has two characteristic substituents that are unusual for
quinolone: an iminodithiocarbonate moiety on the nitrogen and
a geranyl side chain at C2.
Interestingly, intervenolin exhibits antitumor effects in a xeno-
graft modelof human coloncancercells invivo.19 To furtherelucidate
the details of its biological activity, especially its in vivo antitumor
effectsagainstavarietyof tumorsubtypes requires a large quantityof
intervenolin. The production of this compound by fermentation,
however, was as low as 3.9 mg/10 L, which was not sufficient to meet
the demand. To resolve this supply issue, we developed a practical
synthetic route to intervenolin using a thiocyanateeisothiocyanate
rearrangement and SuzukieMiyaura coupling as key steps to install
the two substructures rare for quinolones.20
In addition, intervenolin is structurally closely related to CJ-
13,136 (2)21 of microbial origin discovered by the Pfizer group as
an anti-Helicobacter pylori agent, which led us to examine the an-
tibacterial activity of intervenolin. Our findings indicated that
intervenolin has profoundly highly selective anti-H. pylori activity
over other bacteria.19
Herein we disclose a structureeactivity relationship (SAR) study
of intervenolin-related compounds. The practical synthetic pro-
cedure enabled elucidation of antitumor effects in detail,20 and
anti-H. pylori activity of intervenolin analogs. To access more de-
tailed insight into the SAR, biological activity of the side products
obtained in the course of model synthetic studies to establish
a route to intervenolin,20 and structurally related quinolones re-
ported previously,21e24 were also evaluated.
Fig. 1. Structure of intervenolin derivatives.
2. Results and discussion
2426 was converted to amides 25e29, which were cyclized to af-
ford the desired products by treatment with NaOH in 1,4-dioxane
under reflux conditions. One of the quinolone derivatives, 7, was
used for further model studies of the synthesis of intervenolin. Ini-
tially, a cyano group was installed on the nitrogen of 7 to give 14
using BrCN27 after deprotonation of NH by LiOt-Bu. Although the
product 14 was not useful for the synthesis of intervenolin because
the cyano group of this product could not be reduced to provide the
aminomethylated intermediate,20 the biological activity of this
compound should be examined as an intervenolin derivative. The
next approach aimed at intervenolin synthesis was to introduce an
acetate unit on the nitrogen followed by conversion into an ami-
nomethyl functional group, taking advantage of Curtius rearrange-
ment as a key transformation. Toward this end, 7 was again treated
with LiOt-Bu, and methyl bromoacetate was added successively to
afford 15, which has an acetate unit at the 1-position. The more
common bases, such as K2CO3, resulted almost exclusively in O-al-
kylation product 22.20 Saponification of 15 proceeded uneventfully
to give 17, which was subsequently subjected to the Curtius rear-
rangement conditions developed by Yamada and Shioiri28,29 using
DPPA as the azide source. The transient isocyanate functionality was
trapped in situ by methylthiolate to provide S-thiocarbamate 18. A
thioester 16 was obtained when the reactionwas initiated byadding
DPPA and NaSMe at almost the same time, which is not surprising,
because DPPA is known to be a coupling reagent for peptide syn-
thesis.28,30 Unfortunately, S-thiocarbamate 18 could not be used as
an intermediate for the synthesis of intervenolin: we envisioned
that triflation of 18 followed by the addition of a methylthio group
and instantaneous elimination of the OTf leaving group would form
the iminodithiocarbonate structure, but the first triflation did not
occur at all.20 Ester hydrolysis of O-alkylated compound 22 also
proceeded smoothly to afford 23.
2.1. Structurally related compounds of intervenolin
Fig. 1 lists the structurally related compounds of intervenolin
that were prepared for examination of their biological activities.
Because both intervenolin and CJ-13,136 selectively inhibit gastric
cancer cell growth at almost the same potency,19 the initial SAR
study was performed using 4-quinolones without the iminodi-
thiocarbonate moiety whose synthesis is not trivial. We first syn-
thesized analogs with saturated side chains that varied in their
length and branching (3e7), instead of the geranyl group. Next we
attempted to introduce unsaturation and aromatic moieties to ac-
cess analogs 8e11. A structurally simplified analog 12 in which the
methyl group at C3 was removed demonstrated the significance of
this substituent. Compounds 13e20 were obtained during model
studies of the synthesis of intervenolin, which would be used to
probe the SAR regarding functionalities at the 1-position. Analog 21
was synthesized, and using this substance, the effects of different
degrees of saturation of the aliphatic side chain on biological ac-
tivities could be determined. The quinoline derivatives 22 and 23
were dead-end byproducts obtained in the model synthetic study
of intervenolin, but nevertheless displayed serendipitous and in-
triguing biological activities (vide infra).
2.2. Preparation of intervenolin analogs
The preparative methods of intervenolin analogs are summa-
rized in Scheme 1. Reported compounds, 3, 5, 8, 9, 12 and 13 were
synthesized according to the previously described procedures.22e24
Other 1-unsubstituted-2-aliphatic derivatives, 4, 6, 7, 10, and 11,
were prepared by Camps quinolone synthesis:25 a 2-ketoaniline

7610
H. Abe et al. / Tetrahedron 69 (2013) 7608e7617
Scheme 1. Synthesis of intervenolin derivatives.
Another model study starting from quinolone 6 led us to elaborate
intervenolin 1 alleviated the acute toxicity observed for the 1-
unsubstituted congener 2 (CJ-13,136). Except for 8 and 10, com-
pounds with no substituent on the nitrogen (2e7, 9, 11, and 12)
substantially inhibited the growth of MKN-74 cells. It is noteworthy
the successful synthesis of intervenolin.20 In fact, an intermediate 19
was obtained by the thiocyanomethylation of the quinolone skeleton
at the 1-position and simultaneous rearrangement to isothiocyanate.
The low isolated yield (26%) resulted from the instability of this
compound: a longer reaction time caused extensive decomposition
of the product, and the crude mixture after a 2-h reaction required
purification by quick silica gel chromatography. Subsequently,19 was
transformed into 21 with the same appended substructure found in
intervenolin by the addition of methylthiolate and subsequent
methylation of the carbonimidodithioate monoanion. When the re-
action was quenched after the first addition of NaSMe, a di-
thiocarbamate 20 was readily obtained.
Table 1
Effects of the synthetic intervenolin derivatives on cocultured MKN-74 cells and
Hs738 cells
Compound
In vitro IC50
Cocultured
(
m
g/mL)
Monocultured
In vitro MTD (mg/kg)
1a
2a
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
0.17
0.010
14.13
1.32
0.37
0.71
0.011
>100
4.30
>100
0.012
0.21
0.21
0.007
0.53
1.40
4.15
0.27
0.12
2.91
0.78
0.35
0.37
3.0
0.25
>50
2.5
NT
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
2.36
>100
>100
5.84
71.0
5.84
25.0
12.5
6.25
12.5
6.25
12.5
12.5
1.56
6.25
>50
6.25
6.25
>50
>50
>50
6.25
>50
>50
>50
>50
2.3. Inhibition of gastric cancer cell line growth
With the intervenolin analogs synthesized as described above,
we examined their inhibitory effects on the growth of a gastric
cancer cell line, MKN-74, cultured in the presence or absence of
a stromal cell line, Hs738 (Table 1). The growth of tumor cells was
determined by measuring the fluorescence intensity of green fluo-
rescence protein (GFP), which enabled us to determine the tumor
growth selectively even when cocultured with stromal cells. First
the stromal cells were cultured with the test compounds for 2 days,
and then the tumor cells were inoculated onto the pre-cultured
stromal cells and further cultured for 3 days. To evaluate an acute
toxicity, we designated a maximum tolerated dose (MTD) as half of
a dose that caused death or severe toxic effect during 2-week ob-
servation after the injection of test compounds to mice. As reported
in our previous paper,19 the iminodithiocarbonate moiety of
90.0
40.0
1.36
28.1
2.95
a
Data from Ref. 19.

H. Abe et al. / Tetrahedron 69 (2013) 7608e7617
7611
that all of the active compounds exhibited significantly more potent
inhibitory activities when cocultured with Hs738 cells compared to
monoculture conditions. Among the linear aliphatic substituents
thus examined (3 and 5e7), the longest C8 chain was most prefer-
iminodithiocarbonate moiety and saturated C8 linear aliphatic side
chain at the C2 position, and 23 with a quinoline scaffold were
chosen as the test compounds and compared with intervenolin 1.
The upper two panels of Fig. 2 show the antitumor effects when
the gastric tumor cell line MKN-74 alone was inoculated into
model mice, whereas the lower panels show the effects of si-
multaneous inoculation of both MKN-74 cells and gastric stromal
Hs738 cells. Each test sample was administered on the days in-
dicated in the diagrams of the two left panels at a dose of 12.5 mg/
kg. When MKN-74 cells alone were inoculated, intervenolin
exhibited substantial antitumor effects as previously reported.19 As
expected based on the higher IC50 value from in vitro studies,
analog 21 did not affect the tumor volume. The quinoline de-
rivative 23 exerted antitumor activity comparable to that of
intervenolin. The results indicate that 23 with a different struc-
tural platform from that of intervenolin is another promising lead
for further SAR studies. None of these substances led to a decrease
in the body weight of the model mice, indicating their low acute
toxicity as predicted by the high MTD values (Supplementary data,
Fig. S1).
able (7, IC50 [mg/mL]: 0.011 for cocultured vs >100 for monocultured)
with respect to potency and selectivity. The presence of steric bulk
near the quinolone core had detrimental effects on the potency,
based on the substantially lowered IC50 value of the isobutyl and
homobenzyl analogs 4 and 9, as well as aryl and styryl derivatives 8
and 10 (IC50 [mg/mL]: 14.13 for 4, >100 for 8, 4.30 for 9, and >100 for
10 under monoculture conditions). Analog 11 having a long aliphatic
chain with an unsaturation and a branch near the periphery of this
alkyl functionality exhibited one of the lowest IC50s for cocultured
cells (0.012
mg/mL), and no growth inhibitory effects under mono-
culture conditions (>100
mg/mL). Nevertheless, this compound had
relatively high acute toxicity (maximum tolerated dose [MTD]:
1.56 mg/kg). The profile of the biological activities of this compound
resembled those observed for 2 and 7 (excellent potency and se-
lectivity, but unfavorable toxicity). Compound 12 with the same
structure as 7 except for a missing methyl group at the 3-position
displayed almost 20-fold less potent growth inhibitory activity un-
der cocultured conditions (IC50 [mg/mL]: 0.21 for 12), suggesting that
this functionality should remain for further SAR studies.
Comparison of the results obtained from 7 with 13e18, and 6
with 19e21 revealed the SAR concerning functionality at the 1-
position. While substitution by a cyano group at this site (14,
IC50 [mg/mL]: 0.007 for cocultured, >100 for monocultured, MTD
6.25 mg/kg) preserved the biological activity profile of the parent
compound 7, the analogs 13 and 16e18 had reduced potency and
selectivity for growth inhibition, albeit nontoxic properties (MTD:
>50 mg/kg). It is noticeable that intervenolin derivatives 13 and 18
had practically the same sets of biological activities as that of
intervenolin itself (for 13 and 18, IC50 [mg/mL]: 0.21 and 0.27 for
cocultured, 2.36 and 5.84 for monocultured, MTD: >50 mg/kg for
both). Introduction of an isothiocyano group (19) also maintained
the growth inhibitory properties (IC50 [mg/mL]: 0.12 for cocultured,
90 for monocultured, MTD 6.25 mg/kg) exerted by its parent
compound 6. Although derivative 20 exhibited less potency and
selectivity, mitigated acute toxicity was observed for 13 and
16e18. At this stage, the following tendency was detected: com-
pounds with higher selectivity had higher toxicity and vice versa.
This led us to hypothesize that 21 would have high selectivity and
low acute toxicity, because the IC50s for monocultured cells of the
analogs with a saturated side chain (3e7) were greater than
100 mg/mL, and the presence of the iminodithiocarbamate moiety
in the structure of intervenolin seemed responsible for its low
toxicity. Disappointingly, the selectivity of this analog was not as
high as 3e7, but the growth inhibitory activity of 21 toward
cocultured cells remained potent (IC50 [mg/mL]: 0.78 for cocul-
tured, 1.36 for monocultured) and a high MTD was realized
(>50 mg/kg).
Surprising biological activities were observed for quinoline-
based compounds 22 and 23 with an acetate unit at the oxygen
substituted at the 4-position. Despite their distinct tautomeric
skeletons, the inhibition of cocultured cell growth demonstrated by
Fig. 2. Effect of intervenolin on tumor growth of MKN-74 cells in vivo. MKN-74 human
gastric cancer cells were inoculated subcutaneously with (lower panels) or without
(upper panels) Hs738 gastric stromal cells in female nude mice. The test compounds
(control: open circle, intervenolin 1: filled circle, 21: open square, 23: filled square)
were administered intravenously at 12.5 mg/kg on the days indicated by arrows (left
panels). The mice were sacrificed at day 21 after the tumor inoculation, and the tumors
were excised (right panels, control: open bar, intervenolin 1: black-filled bar, 21: gray-
filled bar, 23: shaded bar). Values are meansꢀSD of five mice. *P <0.05 and **P <0.01
versus the values obtained with vehicle.
these compounds was more potent than expected (IC50 [mg/mL]:
0.35 for 22, 0.37 for 23). Notably, neither analog exhibited acute
toxicity, suggesting that the structure could be another reasonable
starting point of further SAR studies to back up intervenolin de-
rivatives of the quinolone family.
Unexpectedly, however, the in vivo antitumor activity of inter-
venolin and 23 toward MKN-74 in the presence of Hs738 was al-
most the same as that in its absence. At present, the reasons for this
finding are unclear. Our preliminary experiments have revealed
that stromal cells derived from a host mouse apparently intruded in
the xenograft tumor tissues. Thus, intervenolin and 23 could act on
the interactions between the tumor cells and the mouse stromal
cells, which must be studied further.
2.4. In vivo inhibition of tumor growth
Based on the SAR study of intervenolin described above, we
examined the in vivo antitumor effects of the synthetic
analogs. Among the nontoxic compounds, 21 having the

7612
H. Abe et al. / Tetrahedron 69 (2013) 7608e7617
2.5. Inhibitory activity against other tumor cell lines
profound, similar to the case of MKN-74 and Hs738. This combi-
nation should be a target of the future in vivo studies of antitumor
activities. In addition, the quinoline analog 23 exhibited selective
inhibitory activity against colon cancer HCT-15 cells with CCD-18Co
colon stromal cells, albeit with less potency than intervenolin. To
examine the SAR for the development of clinical medicines, it is
preferable to have several leads with the similar biological profiles
but different structural frameworks.
Encouraged by this observation, we then investigated the
in vitro growth-inhibitory activities against a diverse array of cell
lines originating from a variety of tumor tissues cultured with or
without stromal cells using intervenolin, 21, and 23 as test com-
pounds. Fig. 3 presents representative data for pancreatic (BxPC-
3ꢀPS), prostate (DU-145ꢀPrSC), breast (BSY-1ꢀHs371), colon (HCT-
15ꢀCCD18Co), and lung (A549ꢀNHLF) tumor cells. Intervenolin
inhibited the growth of all type of cells most effectively, and tended
to be the same against the other cell lines examined in this study
(Supplementary data, Figs. S2 and S3). In particular, the pro-
liferation of colon cancer HCT-15 cells cocultured with colon stro-
mal CCD-18Co cells was efficiently inhibited, and the selectivity
between the cocultured and monocultured conditions was
2.6. Anti-H. pylori activity
All 14 compounds tested in this study also showed highly se-
lective anti-H. pylori activities similar to intervenolin (Table 2).19
Intriguingly, 6, 7, 11, and 20 exhibited antibacterial activity only
toward H. pylori strains with efficiency comparable to that of clar-
ithromycin, the current first-choice clinical medicine used to
eliminate the bacteria from the stomach.31 This outstanding se-
lectivity is a favorable characteristic for a lead of anti-Helicobacter
agents less prone to the emergence of resistance of nontarget mi-
croorganisms. The mechanistic background of this selectivity
should be a subject of further study. The quinoline derivative 23
exhibited moderate activity.
3. Summary
Utilizing our practical synthetic route, we conducted a SAR
study of intervenolin. Suitable substituents on the nitrogen, such as
the iminodithiocarbonate substructure decreased the acute toxic-
ity, whereas higher selectivity against tumor cells cocultured with
stromal cells was observed for many 1-unsubstituted intervenolin
analogs. Moreover, four derivatives synthesized in this study dis-
played specific anti-H. pylori activities over other bacteria. One of
the side products (23) of the model synthetic study of intervenolin
with a distinct scaffold, a quinoline core, also exhibited selective
growth inhibition of MKN-74 cells cocultured with Hs738 cells with
no acute toxicity. Further SAR studies of antitumor and anti-H.
pylori activities employing the promising leads identified in the
present study are underway and will be reported in due course.
4. Experimental
4.1. General
All the reactions were performed in flame-dried glass pear
shaped flasks or test tubes with a TeflonÒ-coated magnetic stirring
bar. The flasks or test tubes were fitted with a 3-way glass stopcock
and reactions were run under argon atmosphere. Air- and
moisture-sensitive liquids were transferred via a gas-tight syringe
and a stainless-steel needle. All work-up and purification pro-
cedures were carried out with reagent-grade solvents under am-
bient atmosphere. Column chromatography was performed using
silica gel 60N (40e100 mm) purchased from KANTO chemical co.,
inc. Infrared (IR) spectra were recorded on a JASCO FT/IR 4100
Fourier transform infrared spectrophotometer. NMR was recorded
on JEOL ECS-400 and ECA-600 spectrometers. Chemical shifts for
proton are reported in parts per million downfield from tetrame-
thylsilane and are referenced to residual protium in the NMR sol-
Fig. 3. Effect of intervenolin on coculture of tumor cells and stromal cells. The growth
of tumor cells cocultured with stromal cells (filled) or that of tumor cells alone (open)
in the presence of the indicated concentrations of the test compounds (intervenolin 1:
red circle, 21: green triangle, 23: blue square) was determined by measuring fluo-
rescence intensity of green fluorescent protein (GFP). MKN-74 human gastric cancer
cell lines were cocultured with Hs738 gastric stromal cells. BxPC-3 pancreatic cancer
cell lines were cocultured with PS pancreatic stromal cells. DU-145 human prostate
cancer cell lines were cocultured with PrSC prostate stromal cells. BSY-1 human breast
cancer cell lines were cocultured with Hs371 breast stromal cells. HCT-15 human co-
lorectal cancer cells were cocultured with CCD-18Co colorectal stromal cells. A549
human lung cancer cell lines were cocultured with NHLF lung stromal cells.
vent (CDCl3:
13C NMR, chemical shifts were reported in the scale relative to NMR
solvent (CDCl3: 77.0 ppm, CD3OD: 49.0 ppm, DMSO-d6: 39.7 ppm)
d 7.26 ppm, CD3OD: d 3.30 ppm, DMSO-d6: 2.49). For
d
as an internal reference. NMR data are reported as follows: chem-
ical shifts, multiplicity (s: singlet, d: doublet, dd: doublet of dou-
blets, ddd: doublet of doublet of doublets, t: triplet, m: multiplet,
br: broad), coupling constant (Hertz), and integration. High-
resolution mass spectra (ESI Orbitrap) were measured on Thermo
Fisher Scientific LTQ Orbitrap XL. Unless otherwise noted, materials

H. Abe et al. / Tetrahedron 69 (2013) 7608e7617
7613
Table 2
Antibacterial activities of the synthetic intervenolin derivatives
Test organisms
compound
Helicobacter pylori
JCM12093
H. pylori
JCM12095
Staphylococcus
aureus FDA209P
Enterococcus
faecalis JCM5803
Escherichia
coli K-12
Haemophilus
influenzae T-196
H. influenzae
ARD476
1a
4
6
7
8
9
10
11
12
15
17
19
20
21
23
CAMb
ABPCb
0.0156
0.5
0.0156
0.0078
0.0156
2
0.0078
0.25
0.0156
0.0078
0.0156
1
0.5
0.0156
0.5
2
0.25
0.0156
0.0625
2
64
128
>128
>128
>128
128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
0.5
>128
128
>128
>128
128
>128
>128
>128
>128
>128
128
>128
>128
>128
128
64
64
>128
>128
128
64
128
>128
>128
128
>128
64
>128
>128
>128
128
>128
>128
>128
64
4
64
128
64
1
0.0078
0.25
2
>128
>128
>128
128
>128
>128
>128
64
1
0.0156
0.0312
2
4
>128
4
>128
<0.125
<0.125
1
0.5
0.0078
0.125
0.0078
0.25
16
4
8
0.5
0.5
a
Data from Ref. 19.
CAM: clarithromycin, ABPC: ampicillin.
b
were purchased from commercial suppliers and were used without
purification. THF, DMF, and CH2Cl2 were purified by passing
through a solvent purification system (Glass Contour). Anhydrous
1,4-dioxane was purchased from Wako Pure Chemical Co. Ltd.
(Osaka, Japan).
172.8, 141.1, 134.8, 130.6, 122.1, 121.3, 120.8, 38.8, 33.1, 31.7, 29.2,
29.0, 25.5, 22.6, 14.1, 8.4.
4.2.3. N-(2-Propionylphenyl)nonanamide (27). To a solution of 24
(2.0 g, 13.0 mmol) in CH2Cl2 (25 mL) was added Et3N (2.17 mL,
15.6 mmol). The mixture was cooled to 0 ꢁC followed by the ad-
dition of nonanoyl chloride (2.60 mL, 14.3 mmol) and stirred for 2 h
at room temperature. 0.1 N HCl (10 mL) was added to the reaction
mixture and extracted with CH2Cl2 (3ꢂ20 mL). The combined or-
ganic layers were washed with saturated aqueous NaHCO3 (30 mL),
and brine (30 mL). The organic layer was dried over Na2SO4, fil-
tered, and concentrated in vacuo. The resultant crude residue was
purified using column chromatography (EtOAc/n-hexane¼1/10) to
4.2. Synthesis
4.2.1. 3-Methyl-N-(2-propionylphenyl)butanamide (25). To a solu-
tion of 2426 (200 mg, 1.34 mmol) in CH2Cl2 (5 mL) was added Et3N
(0.22 mL, 1.61 mmol). The mixture was cooled to 0 ꢁC followed by
the addition of isovaleryl chloride (0.18 mL, 1.47 mmol) and stirred
for 3 h at room temperature. 0.1 N HCl (5 mL) was added to the
reaction mixture and extracted with CH2Cl2 (3ꢂ10 mL). The com-
bined organic layers were washed with saturated aqueous NaHCO3
(10 mL), and brine (10 mL). The organic layer was dried over
Na2SO4, filtered, and concentrated in vacuo. The resultant crude
residue was purified using column chromatography (EtOAc/n-
afford 27 (3.09 g, 82%) as a colorless oil; IR (KBr) n 3255, 2953, 2928,
1698, 754 cmꢃ1; HRMS (ESI) Anal. Calcd for C18H27NNaO2 m/z
312.1934 [MþNa]þ, found 312.1933; 1H NMR (400 MHz, CDCl3)
d
11.76 (br s, 1H), 8.77 (dd, J¼8.5, 1.1 Hz, 1H), 7.93 (dd, J¼8.0, 1.6 Hz,
1H), 7.53 (ddd, J¼8.5, 7.3, 1.6 Hz, 1H), 7.09 (ddd, J¼8.0, 7.3, 1.1 Hz,
1H), 3.07 (q, J¼7.3 Hz, 2H), 2.43 (t, J¼7.6 Hz, 2H), 1.74 (m, 2H),
1.20e1.41 (m, 13H), 0.87 (t, J¼6.6 Hz, 3H); 13C NMR (100 MHz,
hexane¼1/7) to afford 25 (303 mg, 97%) as a colorless oil; IR (KBr)
n
3254, 2959, 1697, 1376, 754 cmꢃ1; HRMS (ESI) Anal. Calcd for
C14H19NNaO2 m/z 256.1308 [MþNa]þ, found 256.1306; 1H NMR
CDCl3) d 205.4, 172.8, 141.1, 134.8, 130.6, 122.1, 121.4, 120.9, 38.8,
(400 MHz, CDCl3)
d
11.75 (br s, 1H), 8.78 (d, J¼8.5 Hz, 1H), 7.93 (dd,
33.2, 31.9, 29.3, 29.2, 29.1, 25.6, 22.6, 14.1, 8.5.
J¼8.0, 1.4 Hz, 1H), 7.53 (ddd, J¼8.5, 6.8, 1.4 Hz, 1H), 7.10 (ddd, J¼8.4,
6.8, 1.1 Hz,1H), 3.07 (q, J¼7.1 Hz, 2H), 2.31 (d, J¼6.4 Hz, 2H), 2.24 (m,
1H), 1.22 (t, J¼7.1 Hz, 3H), 1.01 (d, J¼6.4 Hz, 6H); 13C NMR (150 MHz,
4.2.4. N-(2-Propionylphenyl)cinnamamide (28). To a solution of 24
(200 mg, 1.34 mmol) in CH2Cl2 (5 mL) was added Et3N (0.22 mL,
1.61 mmol). The mixture was cooled to 0 ꢁC followed by the addi-
tion of cinnamoyl chloride (246 mg, 1.47 mmol) and stirred for 2 h
at room temperature. 0.1 N HCl (5 mL) was added to the reaction
mixture and extracted with CH2Cl2 (3ꢂ10 mL). The combined or-
ganic layers were washed with saturated aqueous NaHCO3 (10 mL),
and brine (10 mL). The organic layer was dried over Na2SO4, filtered,
and concentrated in vacuo. The resultant crude residue was puri-
fied using column chromatography (EtOAc/n-hexane¼1/5) to afford
CDCl3)
d 205.4, 172.1, 141.0, 134.9, 130.6, 122.2, 121.4, 120.8, 48.1,
33.2, 26.2, 22.5, 8.45.
4.2.2. N-(2-Propionylphenyl)octanamide (26). To a solution of 24
(4.00 g, 26.8 mmol) in CH2Cl2 (100 mL) was added Et3N (11.2 mL,
80.4 mmol). The mixture was cooled to 0 ꢁC followed by the ad-
dition of octanoyl chloride (10.1 mL, 59.0 mmol) and stirred for 2 h
at room temperature. 0.1 N HCl (20 mL) was added to the reaction
mixture and extracted with CH2Cl2 (3ꢂ20 mL). The combined or-
ganic layers were washed with saturated aqueous NaHCO3 (30 mL),
and brine (30 mL). The organic layer was dried over Na2SO4, fil-
tered, and concentrated in vacuo. The resultant crude residue was
purified using column chromatography (EtOAc/n-hexane¼1/9) to
28 (234 mg, 62%) as a white powder (mp 109e111 ꢁC); IR (KBr)
n
3223, 3023, 1677, 1653, 751, 727 cmꢃ1; HRMS (ESI) Anal. Calcd for
C18H17NNaO2 m/z 302.1152 [MþNa]þ, found 302.1151; 1H NMR
(400 MHz, CDCl3)
d
12.10 (br s, 1H), 8.91 (dd, J¼8.5, 1.4 Hz, 1H), 7.96
(dd, J¼8.0, 1.4 Hz, 1H), 7.76 (d, J¼15.5 Hz, 1H), 7.56e7.61 (m, 3H),
7.36e7.44 (m, 3H), 7.13 (ddd, J¼8.0, 7.1, 1.1 Hz, 1H), 6.65 (d,
J¼15.5 Hz, 1H), 3.11 (q, J¼7.1 Hz, 2H), 1.25 (t, J¼7.1 Hz, 3H); 13C NMR
afford 26 (7.31 g, 98%) as a colorless oil; IR (KBr) n 3255, 2954, 2928,
1698, 754 cmꢃ1; HRMS (ESI) Anal. Calcd for C17H25NNaO2 m/z
298.1778 [MþNa]þ, found 298.1777; 1H NMR (400 MHz, CDCl3)
(100 MHz, CDCl3)
d 205.6, 164.9, 142.3, 141.3, 134.9, 134.7, 130.7,
d
11.65 (br s, 1H), 8.77 (dd, J¼8.4, 1.4 Hz, 1H), 7.92 (dd, J¼8.2, 1.4 Hz,
130.0, 128.8, 128.1, 122.4, 122.1, 121.5, 121.1, 33.2, 8.4.
1H), 7.53 (ddd, J¼8.4, 7.1,1.4 Hz,1H), 7.09 (ddd, J¼8.2, 7.1,1.4 Hz,1H),
3.07 (q, J¼7.1 Hz, 2H), 2.43 (t, J¼7.6 Hz, 2H), 1.74 (m, 2H), 1.20e1.41
4.2.5. (E)-8-Methyl-N-(2-propionylphenyl)non-6-enamide (29). To
a solution of 24 (100 mg, 0.67 mmol) in CH2Cl2 (5 mL) was added
(m, 11H), 0.87 (t, J¼6.6 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d 205.4,

7614
H. Abe et al. / Tetrahedron 69 (2013) 7608e7617
Et3N (0.19 mL, 1.34 mmol). The mixture was cooled to 0 ꢁC followed
by the addition of trans-8-methyl-6-nonenoyl chloride (0.15 mL,
0.74 mmol) and stirred for 4 h at room temperature. 0.1 N HCl (5 mL)
was added to the reaction mixture and extracted with CH2Cl2
(3ꢂ10 mL). The combined organic layers were washed with satu-
rated aqueous NaHCO3 (10 mL), and brine (10 mL). The organic layer
was dried over Na2SO4, filtered, and concentrated in vacuo. The re-
sultant crude residue was purified using column chromatography
(EtOAc/n-hexane¼1/10) to afford 29 (172 mg, 85%) as a colorless oil;
NMR (400 MHz, DMSO-d6)
d
11.23 (br s, 1H), 8.06 (dd, J¼8.2, 1.4 Hz,
1H), 7.70e7.74 (m, 3H), 7.58e7.62 (m, 1H), 7.35e7.53 (m, 5H), 7.24
(ddd, J¼8.5, 6.8, 1.1 Hz, 1H), 2.16 (s, 3H); 13C NMR (100 MHz, DMSO-
d6)
d 176.8, 143.1, 139.8, 135.9, 135.1, 131.6, 129.3, 129.2, 127.6, 125.1,
123.1, 122.6, 121.1, 118.2, 115.7, 10.7.
4.2.6.5. (E)-2-(7-Methyloct-5-en-1-yl)-3-methylquinolin-4(1H)-
one (11). A white powder (mp 178e181 ꢁC) (49% yield); IR (KBr)
3064, 2957, 2933, 1670, 1638, 1614, 1555, 1500, 1371, 1358, 1152,
1028, 998, 967, 756, 691 cmꢃ1; HRMS (ESI) Anal. Calcd for C19H26NO
m/z 284.2009 [MþH]þ, found 284.2011; 1H NMR (600 MHz, CDCl3)
n
IR (KBr) n
3255, 2956, 2937, 1655, 969, 754 cmꢃ1; HRMS (ESI) Anal.
Calcd for C19H27NNaO2 m/z 324.1934 [MþNa]þ, found 324.1932; 1H
NMR (400 MHz, CDCl3)
d
11.77 (br s, 1H), 8.77 (dd, J¼8.5, 1.1 Hz, 1H),
d
8.65 (br, 1H), 8.36 (dd, J¼8.2, 1.4 Hz, 1H), 7.52 (ddd, J¼8.2, 5.5,
7.92 (dd, J¼8.0,1.4 Hz,1H), 7.53 (ddd, J¼8.5, 7.3,1.4 Hz,1H), 7.09 (ddd,
J¼8.0, 7.3, 1.1 Hz, 1H), 5.37 (m, 2H), 3.07 (q, J¼7.1 Hz, 2H), 2.44 (t,
J¼7.3 Hz, 2H), 2.22 (m, 1H), 2.02 (m, 2H), 1.75 (m, 2H), 1.44 (m, 2H),
1.22 (t, J¼7.1 Hz, 3H), 0.95 (d, J¼6.8 Hz, 6H); 13C NMR (100 MHz,
1.4 Hz, 1H), 7.32 (br d, J¼8.2 Hz, 1H), 7.28 (ddd, J¼8.2, 5.8, 1.0 Hz,
1H), 5.36e5.39 (m, 1H), 5.27e5.32 (m, 1H), 2.70 (m, 2H), 2.22 (m,
1H), 2.15 (s, 3H), 2.01 (q, J¼6.8 Hz, 2H), 1.69 (m, 2H), 1.44 (m, 2H),
0.95 (d, J¼6.5 Hz, 6H); 13C NMR (150 MHz, CDCl3)
d 178.2, 148.5,
CDCl3)
d
205.4, 172.7, 141.1, 138.0, 134.8, 130.7, 126.5, 122.1, 121.4,
138.8, 138.5, 131.3, 126.3, 126.1, 123.7, 123.0, 116.7, 115.7, 33.0, 32.1,
31.0, 29.2, 27.8, 22.6, 10.7.
120.8, 38.7, 33.1, 32.2, 31.0, 29.2, 25.0, 22.6, 8.5.
4.2.6. General procedure for synthesis of 4(1H)-quinolones. To
4.2.7. 2-Octyl-3-methyl-4-oxoquinoline-1(4H)-carbonitrile (14). To
a solution of 7 (50.0 mg, 0.18 mmol) in THF (1 mL) was added LiOt-
Bu (29.4 mg, 0.37 mmol) and stirred for 20 min at room tempera-
ture. The mixture was cooled to 0 ꢁC followed by the addition of
a large amount of cyanogen bromide (0.61 mL, 1.84 mmol) and
stirred for 2 h at room temperature. H2O (5 mL) was added to the
reaction mixture and extracted with EtOAc (3ꢂ5 mL). The com-
bined organic layers were dried over Na2SO4, filtered, and con-
centrated in vacuo. The resultant crude residue was purified using
column chromatography (EtOAc/n-hexane¼1/2) to afford 14
a
mixture of amide (25e29, 1.0 equiv) and crushed NaOH
(3.0 equiv) was added 1,4-dioxane, the concentration was adjusted
to 0.1 M. The reaction mixture was stirred for several hours at
110 ꢁC and allowed to cool to room temperature. H2O was added to
the reaction mixture and neutralized using 1 N HCl and then n-
hexane was added to the flask. The flask was sonicated for a few
minutes until the solid precipitate. The solid precipitated was fil-
tered and washed with H2O, n-hexane and mixed solvent (EtOAc/n-
hexane¼1/1) and then dried in vacuo.
(34.0 mg, 62%) as a colorless oil; IR (KBr)
n 2961, 2926, 2853, 2237,
4.2.6.1. 2-Isobutyl-3-methylquinolin-4(1H)-one (4). A white
1628, 1576, 1470, 1292, 1191, 761, 693 cmꢃ1, HRMS (ESI) Anal.
powder (mp 240e244 ꢁC) (44% yield); IR (KBr)
n
3059, 2956, 1636,
Calcd for C19H25N2O m/z 297.1961 [MþH]þ, found 297.1961; 1H
1609, 1554, 1505, 1369, 1359, 1189, 998, 762, 695 cmꢃ1; HRMS (ESI)
(400 MHz, CDCl3)
d
8.33 (ddd, J¼8.0, 0.9, 1.1 Hz, 1H), 7.73 (m, 2H),
Anal. Calcd for C14H18NO m/z 216.1383 [MþH]þ, found 216.1385; 1H
7.47 (m, 1H), 2.93 (m, 2H), 2.15 (s, 3H), 1.71 (m, 2H), 1.21e1.50 (m,
NMR (400 MHz, DMSO-d6)
d
11.26 (br s, 1H), 8.04 (dd, J¼8.2, 1.1 Hz,
10H), 0.87 (t, J¼6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d 177.3,
1H), 7.55 (ddd, J¼8.2, 6.6, 1.4 Hz, 1H), 7.49 (d, J¼8.2 Hz, 1H), 7.21
(ddd, J¼8.2, 6.6, 1.4 Hz, 1H), 2.56 (d, J¼7.5 Hz, 2H), 2.02 (m, 1H), 1.97
(s, 3H), 0.92 (d, J¼6.6 Hz, 6H); 13C NMR (100 MHz, DMSO-d6)
146.1, 137.3, 133.4, 127.1, 126.2, 123.4, 120.3, 116.3, 106.4, 31.8, 31.7,
29.4, 29.1, 29.0, 28.0, 22.6, 14.1, 11.2.
d
176.4, 148.8, 139.3, 131.1, 125.2, 123.0, 122.4, 117.7, 114.7, 28.3, 22.3,
4.2.8. Methyl 2-(3-methyl-2-octyl-4-oxoquinolin-1(4H)-yl)acetate
(15). To a solution of 7 (800 mg, 2.95 mmol) in THF (20 mL) was
added LiOt-Bu in THF (4.42 mL, 4.42 mmol) and stirred for 20 min at
room temperature followed by the addition of methyl bromoace-
tate (1.40 mL, 14.7 mmol) and stirred for 12 h at 80 ꢁC. H2O (20 mL)
was added to the reaction mixture and extracted with EtOAc
(3ꢂ20 mL). The combined organic layers were dried over Na2SO4,
filtered, and concentrated in vacuo. The resultant crude residue was
purified using column chromatography (EtOAc/n-hexane¼1/ 2) to
11.0.
4.2.6.2. 2-Heptyl-3-methylquinolin-4(1H)-one
(6). A
white
powder (mp 228e231 ꢁC) (47% yield); IR (KBr)
n 3061, 2954, 2925,
1637, 1608, 1556, 1504, 1188, 998, 754, 692 cmꢃ1; HRMS (ESI) Anal.
Calcd for C17H24NO m/z 258.1852 [MþH]þ, found 258.1853; 1H NMR
(400 MHz, CD3OD)
d
8.21 (d, J¼8.2 Hz, 1H), 7.61 (m, 1H), 7.52 (m,
1H), 7.32 (t, J¼8.1 Hz, 1H), 2.78 (t, J¼7.7 Hz, 2H), 2.14 (s, 3H),
1.65e1.73 (m, 2H), 1.30e1.46 (m, 8H), 0.88 (d, J¼6.6 Hz, 3H); 13C
afford 15 (780 mg, 76%) as a colorless oil; IR (KBr)
n 2953, 2922,
NMR (100 MHz, CD3OD)
d
179.5, 153.4, 140.6, 132.7, 126.2, 124.5,
1743, 1635, 1617, 1558, 1507, 1214, 994, 760, 688 cmꢃ1, HRMS (ESI)
124.4, 118.7, 116.2, 33.5, 32.9, 30.5, 30.2, 30.0, 23.7, 14.4, 10.8.
Anal. Calcd for C21H30NO3 m/z 344.2220 [MþH]þ, found 344.2222;
1H (400 MHz, CDCl3)
d
8.47 (dd, J¼8.0, 1.6 Hz, 1H), 7.58 (ddd, J¼8.7,
4.2.6.3. 2-Octyl-3-methylquinolin-4(1H)-one (7). A white pow-
7.1, 1.6 Hz, 1H), 7.33 (ddd, J¼8.0, 6.8, 0.7 Hz, 1H), 7.20 (d, J¼8.7 Hz,
1H), 4.90 (s, 2H), 3.80 (s, 3H), 2.74 (br, 2H), 2.22 (s, 3H), 1.60 (m, 2H),
1.21e1.51 (m, 10H), 0.89 (t, J¼6.6 Hz, 3H); 13C NMR (100 MHz,
der (mp 228e231 ꢁC) (66% yield); IR (KBr)
n 3059, 2952, 2923, 1638,
1607, 1555, 1500, 1190, 998, 754, 693 cmꢃ1; HRMS (ESI) Anal.
Calcd for C18H26NO m/z 272.2009 [MþH]þ, found 272.2010; 1H
CDCl3) d 177.4, 168.7, 150.7, 140.7, 131.9, 127.3, 124.8, 123.1, 117.6,
NMR (400 MHz, DMSO-d6)
d
11.33 (br s, 1H), 8.03 (dd, J¼8.0, 1.4 Hz,
114.2, 53.0, 48.5, 31.8, 30.9, 29.8, 29.2, 29.1, 28.1, 22.6, 14.1, 11.7.
1H), 7.55 (ddd, J¼8.2, 6.7, 1.4 Hz, 1H), 7.47 (d, J¼8.0 Hz, 1H), 7.21 (dd,
J¼8.2, 6.6 Hz, 1H), 2.65 (t, J¼7.5 Hz, 2H), 1.97 (s, 3H), 1.60 (m, 2H),
1.17e1.39 (m, 10H), 0.83 (d, J¼6.6 Hz, 3H); 13C NMR (100 MHz,
4.2.9. 2-(3-Methyl-2-octyl-4-oxoquinolin-1(4H)-yl)acetic acid (17).
To a stirred solution of 15 (890 mg, 2.59 mmol) in THF (5 mL) and
EtOH (5 mL) was added 2 M aqueous NaOH (2 mL) and stirred for
2 h at room temperature. The reaction mixture was cooled to 0 ꢁC
and acidified with 1 N HCl and then extracted with EtOAc
(3ꢂ20 mL). The combined organic layers were dried over Na2SO4,
filtered, and concentrated in vacuo. The resultant crude residue was
treated with Et2O, and a precipitate was collected by filtration to
afford 17 (530 mg, 62%) as a white powder (mp 161e163 ꢁC); IR
DMSO-d6) d 176.4, 149.8, 139.4, 131.1, 125.2, 123.1, 122.4, 117.7, 113.9,
31.8, 31.4, 29.0, 28.9, 28.8, 28.5, 22.3, 14.2, 10.5.
4.2.6.4. (E)-2-Styryl-3-methylquinolin-4(1H)-one (10). A yellow
powder (mp >260 ꢁC) (62% yield); IR (KBr)
n 3064, 2938,1628,1570,
1507, 1387, 1359, 1187, 965, 755, 690 cmꢃ1; HRMS (ESI) Anal.
Calcd for C18H15NNaO m/z 284.1046 [MþNa]þ, found 284.1046; 1H

H. Abe et al. / Tetrahedron 69 (2013) 7608e7617
7615
(KBr)
n
2955, 2925, 2853, 1725, 1635, 1593, 1506, 1191, 976, 760,
139.9, 132.4, 127.3, 124.6, 123.8, 118.2, 114.4, 56.3, 31.7, 30.6, 29.8,
28.9, 28.6, 22.6, 14.1, 11.6.
689 cmꢃ1, HRMS (ESI) Anal. Calcd for C20H28NO3 m/z 330.2064
[MþH]þ, found 330.2063; 1H (400 MHz, CDCl3)
d
8.39 (dd, J¼8.0,
1.1 Hz, 1H), 7.54 (ddd, J¼8.7, 6.8, 1.1 Hz, 1H), 7.42 (d, J¼8.7 Hz, 1H),
7.26 (t, J¼8.0 Hz, 1H), 4.98 (br, 2H), 2.80 (br, 2H), 2.19 (s, 3H),
1.20e1.65 (m, 12H), 0.87 (t, J¼6.4 Hz, 3H); 13C NMR (100 MHz,
4.2.13. Methyl ((2-heptyl-3-methyl-4-oxoquinolin-1(4H)-yl)methyl)
carbamodithioate (20). A mixture of 19 (100 mg, 0.30 mmol) and
sodium thiomethoxide (23.4 mg, 0.33 mmol) in CH3CN (1.5 mL) was
stirred for 20 min at room temperature. Saturated aqueous NaHCO3
(5 mL) was added to the reaction mixture and extracted with EtOAc
(3ꢂ5 mL). The combined organic layers were dried over Na2SO4,
filtered, and concentrated in vacuo. The resultant crude residue was
purified using column chromatography (EtOAc/n-hexane¼1/2) to
afford 20 (31.7 mg, 27%) as a yellow powder (mp 167e170 ꢁC); IR
CDCl3)
d 176.6, 169.4, 154.2, 140.5, 132.5, 126.7, 123.9, 123.8, 117.2,
115.5, 49.7, 31.7, 31.3, 29.8, 29.1, 27.9, 22.7, 14.1, 11.9.
4.2.10. S-Methyl 2-(3-methyl-2-octyl-4-oxoquinolin-1(4H)-yl)etha-
nethioate (16). To a suspension of 17 (100 mg, 0.30 mmol) in THF
(5 mL) were added Et3N (50.8
mL, 0.36 mmol), DPPA (72.0 mL,
0.33 mmol), and sodium thiomethoxide (23.0 mg, 0.33 mmol). The
reaction mixture was stirred for 2 h at 80 ꢁC. Saturated aqueous
NH4Cl (5 mL) was added to the reaction mixture and extracted with
EtOAc (3ꢂ5 mL). The combined organic layers were dried over
Na2SO4, filtered, and concentrated in vacuo. The resultant crude
residue was purified using column chromatography (EtOAc/n-
(KBr) n 3119, 2958, 2918, 2850, 1619, 1598, 1538, 1282, 1199, 1105,
938, 764, 688 cmꢃ1; HRMS (ESI) Anal. Calcd for C20H28N2NaOS2 m/z
399.1535 [MþNa]þ, found 399.1534; 1H NMR (400 MHz, CDCl3)
d
10.93 (br, 1H), 8.15 (d, J¼7.8 Hz, 1H), 7.59 (ddd, J¼8.7, 7.8, 1.1 Hz,
1H), 7.44 (d, J¼8.7 Hz, 1H), 7.22 (t, J¼7.8 Hz, 1H), 5.68e6.41 (br, 2H),
2.76 (s, 3H), 2.22e2.58 (br, 2H), 1.20e1.50 (m, 13H), 0.88 (t,
hexane¼1/3) to afford 16 (39.3 mg, 36%) as a colorless oil; IR (KBr)
n
J¼6.6 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d 199.8, 177.1, 152.2, 139.4,
2924, 2852, 1687, 1614, 1594, 1542, 1193, 1028, 757, 558 cmꢃ1, HRMS
132.7, 126.4, 123.9, 123.5, 116.8, 115.9, 58.2, 31.6, 30.9, 29.6, 28.8,
28.3, 22.5, 18.0, 14.0, 10.7.
(ESI) Anal. Calcd for C21H30NO2S m/z 360.1992 [MþH]þ, found
360.1994; 1H (400 MHz, CDCl3)
d
8.47 (dd, J¼8.0, 1.4 Hz, 1H), 7.58
(ddd, J¼8.7, 6.6, 1.4 Hz, 1H), 7.34 (dd, J¼8.0, 6.6 Hz, 1H), 7.21 (d,
J¼8.7 Hz, 1H), 5.01 (br, 2H), 2.51e2.99 (br, 2H), 2.33 (s, 3H), 2.23 (s,
3H), 1.60 (br, 2H), 1.20e1.50 (m, 10H), 0.88 (t, J¼6.4 Hz, 3H); 13C
4.2.14. Dimethyl ((2-heptyl-3-methyl-4-oxoquinolin-1(4H)-yl)methyl)
carbonimidodithioate (21). A mixture of 19 (50.0 mg, 0.15 mmol) and
NaSMe (10.6 mg, 0.15 mmol) in CH3CN (1.5 mL) was stirred for
NMR (100 MHz, CDCl3)
d
196.8, 177.5,150.7,140.7,132.0,131.9,127.3,
20 min at room temperature. MeI (8.5 mL, 0.17 mmol) was then
123.3, 117.9, 114.6, 56.0, 31.7, 31.0, 29.8, 29.2, 29.1, 28.2, 22.6, 14.1,
11.7, 11.4.
added, the reaction mixture was stirred for a further 30 min at room
temperature. Saturated aqueous NaHCO3 (2 mL) was added to the
reaction mixture and extracted with EtOAc (3ꢂ5 mL). The combined
organic layers were dried over Na2SO4, filtered, and concentrated in
vacuo. The resultant crude residue was purified using column
chromatography (EtOAc/n-hexane¼1/2) to afford 21 (30.6 mg, 52%)
4.2.11. S-Methyl ((3-methyl-2-octyl-4-oxoquinolin-1(4H)-yl)methyl)
carbamothioate (18). To a suspension of 17 (100 mg, 0.30 mmol) in
THF (5 mL) were added Et3N (50.8 mL, 0.36 mmol), DPPA (72.0 mL,
0.33 mmol) and stirred at 80 ꢁC. After 1 h, sodium thiomethoxide
(23.0 mg, 0.33 mmol) was added to the reaction mixture and ad-
ditionally stirred for 1 h at 80 ꢁC. Saturated aqueous NH4Cl (5 mL)
was added to the reaction mixture and extracted with EtOAc
(3ꢂ5 mL). The combined organic layers were dried over Na2SO4,
filtered, and concentrated in vacuo. The resultant crude residue was
purified using column chromatography (EtOAc/n-hexane¼1/5) to
as a white powder (mp 92e94 ꢁC); IR (KBr)
n 2958, 2922, 2852, 1618,
1595, 1566, 1492, 1370, 1277, 1192, 1004, 769, 700 cmꢃ1; HRMS (ESI)
Anal. Calcd for C21H30N2NaOS2 m/z 413.1692 [MþNa]þ, found
413.1689; 1H NMR (400 MHz, CDCl3)
d
8.46 (dd, J¼8.0, 1.6 Hz, 1H),
7.55 (ddd, J¼8.4, 7.1, 1.6 Hz, 1H), 7.31 (m, 2H), 5.60 (s, 2H), 2.77 (m,
2H), 2.71 (s, 3H), 2.28 (s, 3H), 2.22 (s, 3H), 1.64 (m, 2H), 1.24e1.50 (m,
8H), 0.89 (t, J¼6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d 177.5, 161.4,
afford 18 (48.6 mg, 43%) as a colorless oil; IR (KBr)
n
3169, 2955,
151.4, 141.1,131.3,126.8,124.8,122.8,117.0, 115.8, 63.8, 31.7, 30.7, 29.8,
28.9, 28.3, 22.6, 15.0, 14.8, 14.1, 11.5.
2927, 1671, 1615, 1595, 1556, 1492, 1195, 1084, 760, 651 cmꢃ1, HRMS
(ESI) Anal. Calcd for C21H30N2NaO2S m/z 397.1920 [MþNa]þ, found
397.1921; 1H (600 MHz, CDCl3)
d
8.77 (br,1H), 8.26 (dd, J¼7.9,1.4 Hz,
4.2.15. Methyl 2-((3-methyl-2-octylquinolin-4-yl)oxy)acetate (22).
To a solution of 7 (100 mg, 0.37 mmol) in DMF (5 mL) was added
1H), 7.59 (ddd, J¼8.6, 6.8, 1.4 Hz, 1H), 7.49 (d, J¼8.6 Hz, 1H), 7.25
(ddd, J¼7.9, 6.8, 1.0 Hz, 1H), 5.67 (br, 2H), 2.46 (s, 3H), 2.45 (br, 2H),
1.22e1.45 (m, 15H), 0.89 (t, J¼6.7 Hz, 3H); 13C NMR (150 MHz,
K2CO3 (332 mg, 2.40 mmol) and methyl bromoacetate (53.0
mL,
0.56 mmol). The reaction mixture was stirred for 12 h at 80 ꢁC. H2O
(20 mL) was added to the reaction mixture and extracted with EtOAc
(3ꢂ10 mL). The combined organic layers were dried over Na2SO4,
filtered, and concentrated in vacuo. The resultant crude residue was
purified using column chromatography (EtOAc/n-hexane¼1/2) to
CDCl3)
d 177.3, 168.6, 151.6, 139.6, 132.3, 126.9, 124.4, 123.2, 117.0,
115.5, 52.6, 31.8, 30.7, 29.7, 29.2, 29.1, 28.5, 22.6, 14.0, 12.2, 11.1.
4.2.12. 2-Heptyl-1-(isothiocyanatomethyl)-3-methylquinolin-4(1H)-
one (19). To a solution of 6 (1.0 g, 3.89 mmol) in THF (10 mL) was
added LiOt-Bu in THF (5.80 mL, 5.80 mmol, 1 M) and stirred for
20 min at room temperature. The mixture was cooled to 0 ꢁC fol-
lowed by the addition of chloromethyl thiocyanate (6.7 mL,
38.9 mmol) and stirred for 2 h at room temperature. Brine (10 mL)
was added to the reaction mixture and extracted with EtOAc
(3ꢂ10 mL). The combined organic layers were dried over Na2SO4,
filtered, and concentrated in vacuo. The resultant crude residue was
purified using column chromatography (EtOAc/n-hexane¼1/2) to
afford 22 (83.0 mg, 66%) as a colorless oil; IR (KBr) n 2953, 2925, 2854,
1765, 1618, 1596, 1437, 1123, 968, 768, 680 cmꢃ1, HRMS (ESI) Anal.
Calcd for C21H30NO3 m/z 344.2220 [MþH]þ, found 344.2222; 1H
(400 MHz, CDCl3)
d
8.05 (d, J¼8.2 Hz,1H), 8.00 (d, J¼8.4 Hz,1H), 7.62
(ddd, J¼8.4, 6.8, 1.3 Hz, 1H), 7.47 (ddd, J¼8.2, 6.8, 1.1 Hz, 1H), 4.62 (s,
2H), 3.86 (s, 3H), 2.95 (m, 2H), 2.43 (s, 3H),1.75 (m, 2H),1.20e1.48 (m,
10H), 0.87 (t, J¼6.6 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d 169.0,164.3,
159.0, 147.8, 128.9, 128.8, 125.7, 121.8, 121.4, 120.9, 120.7, 70.3, 52.3,
37.0, 31.8, 29.9, 29.5, 29.2, 29.0, 22.6, 14.1, 11.9.
afford 19 (330 mg, 26%) as a yellow oil; IR (KBr) n 2961, 2926, 2853,
2237, 1628, 1576, 1470, 1292, 1191, 987, 761, 693 cmꢃ1, HRMS (ESI)
4.2.16. 2-((3-Methyl-2-octylquinolin-4-yl)oxy)acetic acid (23). To
a stirred solution of 22 (80.0 mg, 0.23 mmol) in THF (1 mL) and
EtOH (1 mL) was added 2 M aqueous NaOH (0.5 mL) and stirred for
2 h at room temperature. The reaction mixture was cooled to 0 ꢁC
and acidified with 1 N HCl and then extracted with EtOAc
(3ꢂ20 mL). The combined organic layers were dried over Na2SO4,
Anal. Calcd for C19H24N2OS m/z 329.1682 [MþH]þ, found 329.1682;
1H (400 MHz, CDCl3)
d
8.45 (dd, J¼8.0, 1.6 Hz, 1H), 7.68 (ddd, J¼8.7,
6.8, 1.6 Hz, 1H), 7.46 (d, J¼8.7 Hz, 1H), 7.38 (ddd, J¼8.0, 6.8, 0.9 Hz,
1H), 5.71 (s, 2H), 2.84 (m, 2H), 2.19 (s, 3H), 1.23e1.71 (m, 10H), 0.90
(t, J¼6.6 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d 177.6, 149.6, 141.7,

7616
H. Abe et al. / Tetrahedron 69 (2013) 7608e7617
filtered, and concentrated in vacuo. The resultant crude residue was
treated with Et2O, and a precipitate was collected by filtration to
afford 23 (68.2 mg, 88%) as a white powder (mp 59e62 ꢁC); IR (KBr)
5.4. 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 in-
stitutional guidelines. Samples were injected intravenously or in-
traperitoneally into mice. During 2 weeks observation, half of
a dose that caused death or severe toxic effect is designated as
a maximum tolerated dose (MTD) in this study.
n
2927, 2855, 2713,1736,1642,1589,1227,1181,1078, 764, 724 cmꢃ1
,
HRMS (ESI) Anal. Calcd for C20H28NO3 m/z 330.2064 [MþH]þ, found
330.2064; 1H (600 MHz, CD3OD)
d
8.42 (br d, J¼8.2 Hz, 1H), 8.07 (br
d, J¼8.5 Hz, 1H), 7.92 (ddd, J¼8.5, 6.8, 1.0 Hz, 1H), 7.54 (ddd, J¼8.2,
7.2, 1.0 Hz, 1H), 4.92 (s, 2H), 3.15 (m, 2H), 2.54 (s, 3H), 1.78 (m, 2H),
1.50 (m, 2H), 1.25e1.41 (m, 8H), 0.88 (t, J¼6.8 Hz, 3H); 13C NMR
(150 MHz, CD3OD)
d 171.9, 167.5, 164.3, 142.1, 133.9, 129.2, 124.6,
123.9, 123.7, 122.8, 72.7, 35.1, 33.0, 30.7, 30.3, 30.2, 29.9, 23.7,
14.4, 12.2.
5.5. Antitumor effect in vivo
Female BALB/c nude mice, 5-week old, were purchased from
Charles River Breeding Laboratories (Yokohama, Japan) and main-
tained in a specific pathogen-free barrier facility according to our
institutional guidelines. Cancer cells (8ꢂ106) were trypsinized and
resuspended with or without stromal cells (8ꢂ106) in 0.3 mL of 10%
FBSeDMEM and then combined with 0.5 mL of growth factor-
reduced Matrigel (BD Biosciences). One hundred microliters of
the cell suspension (1ꢂ106 cells) were injected subcutaneously in
the left lateral flank of mice. Five mice were used for each experi-
mental set. Test samples were administered as indicated. Tumor
volume was estimated using the following formula: tumor volume
(mm3)¼(lengthꢂwidth2)/2. After the indicated times, tumors were
surgically dissected.
5. Biological experiments
5.1. Cells and reagents
Human prostate cancer DU-145 cells, human colon cancer DLD-
1 cells, human pancreatic cancer cell lines: MiaPaca2, BxPC-3,
Capan-1, and Panc-1, were obtained from American Type Cultured
Collection (ATCC). Human prostate cancer PC-3 cells were obtained
from DS pharma. LNCaP-CR cell line was established in our labo-
ratory from human prostate cancer LNCaP cells (DS Pharma). Other
cancer cell lines were described elsewhere.32,33 All cancer cell lines
were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM)
(Nissui) supplemented with 10% fetal bovine serum (FBS; Sigma),
100 units/mL penicillin G (Invitrogen), and 100 mg/mL streptomycin
(Invitrogen) at 37 ꢁC with 5% CO2. Hs738 human gastric stromal
cells (CRL-7869), CCD-18Co human colon fibroblasts (CRL-1459),
and Hs371 mammary gland fibroblasts (CRL-7256) were obtained
from ATCC. NHLF normal human lung fibroblasts and PrSC human
prostate stromal cells were obtained from BioWhittaker. PS human
pancreatic stromal cells were obtained from DS pharma. All stromal
cells were maintained in DMEM supplemented with 10% FBS,
5.6. Antimicrobial effect
H. pylori JCM12093, H. pylori JCM12095, and Enterococcus fae-
calis JCM5803 were purchased from Japan Collection of Microor-
ganisms (Tsukuba, Japan). Haemophilus influenzae T-196 and H.
influenzae ARD476 were generously provided by Prof. Kimiko
Ubukata (Kitasato University). Minimum inhibitory concentrations
(MICs) were determined by using microdilution broth method in
96-well plates. Two-fold serial dilutions of the test samples were
prepared with dimethyl sulfoxide. The dilution series were initially
prepared at 200-fold final concentrations from this stock to provide
100 units/mL penicillin G, 100
mg/mL streptomycin, ITH (5
mg/mL
insulin, 5
m
g/mL transferrin, and 1.4 mM hydrocortisone), and 5 ng/
mL basic-FGF (Pepro Tech, NJ, USA) at 37 ꢁC with 5% CO2 as
described.14a
a final test range of 128e0.001 mg/mL. Fifty microliters of each di-
5.2. GFP transfection
lution and 50
m
L of test organisms (2e9ꢂ104 cfu/mL) were then
dispensed into each well. The MIC was defined as the lowest con-
centration of compound at which no visible growth could be seen.
The test organisms, medium, and culture conditions were follow-
ing: H. pylori JCM12093 and H. pylori JCM12095 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; Staphylococcus aureus FDA209P and Escherichia 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 18 h at 37 ꢁC; E. faecalis
JCM5803 was cultured in a Heart Infusion Broth medium (BD Bio-
sciences) at 37 ꢁC for 18 h; H. influenzae T-196 and H. influenzae
ARD476 were cultured in a Muller Hinton medium (BD Biosciences)
with 5% Fildes Enrichment (BD Biosciences) at 37 ꢁC for 18 h.
Cells (2ꢂ105) were cultured in 6-well plates in 10% FBSeDMEM
for 2 days. The medium was replaced by 800 mL of OPTI-MEM (Life
Technologies), and then 200
pEGFP-C1 vector (BD Biosciences), 4
(Life Technologies) and 6 L of PLUS Reagent (I Life Technologies)
mL of OPTI-MEM containing 1
mg of
mL of Lipofectamine Reagent
m
was added to the cells. After 3 h incubation, 1 mL of 20% FBSeOPTI-
MEM was added to each well and the cells were cultured for 24 h.
Stably transfected cells were selected with 400
(Promega, Tokyo, Japan).
mg/mL of G418
5.3. Cell growth and coculture experiment
For coculture experiment, stromal cells were first inoculated in
96-well plates at 5ꢂ103 cells per well in 0.1 mL of DMEM supple-
mented with 1% D-FBS and ITH. Test samples were added into the
Acknowledgements
well and the stromal cells were cultured for 2 days. Then 10 mL of
cancer cell suspension (5ꢂ103) in serum-free DMEM were in-
oculated onto a monolayer of the stromal cells and the cells were
further cultured for 3 days. For monoculture of cancer cells, only
assay medium with test samples was first incubated for 2 days, and
then cancer cells were inoculated as described above and further
cultured for 3 days. The growth of the cancer cells was determined
measuring GFP fluorescence intensity (excitation at 485 nm and
emission at 538 nm) by the lysis of cells in 10 mM TriseHCl,
150 mM NaCl, 0.9 mM CaCl2, and 1% Triton X-100.
The authors thank Dr. Ryuichi Sawa, Ms. Yumiko Kubota, and
Ms. Yuko Takahashi for collection of spectral data. The authors
thank Dr. Daishiro Ikeda for valuable discussions.
Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2013.05.033.

H. Abe et al. / Tetrahedron 69 (2013) 7608e7617
7617
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