2266
N. IWAI et al.
4
medium agar plates (1.5% agar) containing 10 cells/ml
of SH3210 and 40 ꢄg/ml of 5-bromo-4-chloro-3-indolyl
ꢀ
Determination of minimum inhibitory concentration
(MIC). MICs were determined by using a standard 2-
fold serial dilution format on Lennox agar plates (1.5%
agar).
-D-galactopyranoside. The plates were incubated at
ꢀ
4
2 C for 24 h, and the development of a blue color and
the growth inhibitory zone around the paper disks were
evaluated. The development of a blue color in this assay
suggests the production of anucleate cells.
Results
In order to define the structural element(s) required
for the activity to induce spherical cells in E. coli, the
biological activity of two S-benzylisothiourea deriva-
tives and seven related compounds, together with A22
(1), was examined in this study (Table 1).
Microscopic observation. E. coli K-12 strain MG1655
ꢁ ꢁ
7)
(
F
ꢂ ) was grown in Lennox broth containing 1%
Polypepton, 0.5% yeast extract, 0.5% NaCl, and 0.1%
glucose (pH 7.2). Cells spread on a slide glass were
treated with methanol for 5 min and covered with poly-
L-lysine. The fixed cells were observed by the differ-
ential interference contrast system through an Axioskop
Since 1 had originally been found through screening
1
,2)
by the anucleate cell blue assay,
the effect of each
compound was first examined by this assay. The
development of a blue color in the anucleate cell blue
assay suggests the production of anucleate cells, and the
formation of a growth inhibitory zone represents anti-
bacterial activity.
2
Germany). To observe the anucleate cells, cells were
microscope (Carl Zeiss Co., Ltd., Oberkochen,
0
stained with a 4 ,6-diamidino-2-phenylindole (DAPI)
solution (5 ꢄg/ml in saline) and then observed by the
fluorescence phase-contrast combined method.6
)
S-(4-Chlorobenzyl)isothiourea (2) induced a deep
blue zone around a growth inhibitory zone almost
comparable to those of 1. S-benzylisothiourea (3) also
developed a blue zone just around the paper disk, but
only a marginal growth inhibitory zone was detected.
These indicate that 3- and/or 4-chloro-substitution of
the S-benzyl group was not absolutely required for the
activity to induce a blue zone in this assay, but did
enhance the activity. S-Cyclohexylmethylisothiourea (4)
induced neither a growth inhibitory zone nor blue zone
in this assay. Interestingly, S-heptylisothiourea (5)
induced a growth inhibitory zone as well as a blue
zone, but, as mentioned later, did not induce spherical
cells. S-Ethylisothiourea (6) induced neither a growth
inhibitory zone nor blue zone in this assay. These results
indicate that the S-benzyl group was necessary to show
activity for inducing blue zones in this assay (Table 1).
Benzylthiocyanate (7) and benzylisocyanate (9) in-
duced neither a growth inhibitory zone nor blue zone.
Benzylisothiocyanate (8) induced a growth inhibitory
zone, but no blue zone developed around the growth
inhibitory zone in this assay. N-Phenylthiourea (10)
induced neither a growth inhibition nor blue zone. These
results suggest that the isothiourea group was also
necessary for the activity to induce blue zones. Judging
from all these results, the S-benzylisothiourea structure
was necessary and sufficient for the activity to develop a
blue color in this assay (Table 1).
Chemical compounds. S-(3,4-Dichlorobenzyl)iso-
thiourea, S-(4-chlorobenzyl)isothiourea, S-cyclohexyl-
methylisothiourea and S-heptylisothiourea were synthe-
sized as described later. S-Benzylisothiourea, ben-
zylthiocyanate and benzylisocyanate were purchased
from Wako Pure Chemical Industries Ltd. (Osaka,
Japan), benzylisothiocyanate from Avocado Research
Chemicals Ltd. (Lancs, UK), thiourea, N-phenylthiourea
and S-ethylisothiourea from Sigma-Aldrich Co. (St.
Louis, Missouri, USA). 3,4-Dichlorobenzyl chloride, 4-
chlorobenzyl chloride, bromomethylcyclohexane and 1-
chloroheptane were purchased from Tokyo Kasei Kogyo
Co., Ltd. (Tokyo, Japan).
Synthesis of the compounds. Thiourea was suspended
in dehydrated ethanol, and 3,4-dichlorobenzyl chloride
was added to the suspension. The mixture was heated at
ꢀ
1
30 C to reflux for several hours and then cooled to
room temperature. The reaction mixture was concen-
trated under vacuum, and the resulting residue was
diluted with methanol. The product was purified by
recrystallization from diethyl ether. The structure of the
compound was confirmed by 1H-NMR spectrometry.
S-(4-Chlorobenzyl)isothiourea, S-cyclohexylmethyliso-
thiourea and S-heptylisothiourea were similarly synthe-
sized by using 4-chlorobenzyl chloride, bromomethyl-
cyclohexane and 1-chloroheptane, respectively, instead
of 3,4-dichlorobenzyl chloride.
The MIC of each compound for E. coli and other
several bacteria was determined. 1 showed antibacterial
activity toward E. coli and S. typhimurium at a rela-
tively low concentration (3.13 ꢄg/ml), but was less
effective against P. putida (100 ꢄg/ml) and Gram-posi-
tive bacteria (100 ꢄg/ml for B. subtilis and more than
Other bacterial strains used. Bacillus subtilis 168
was from laboratory stock. Pseudomonas putida
NBRC14164 (the same as ATCC12633), Salmonella
typhimurium NBRC13245 (the same as LT2), and
Staphylococcus aureus subsp. aureus NBRC15035 (the
same as ATCC29213) were obtained from the National
Institute of Technology and Evaluation (Kisarazu,
Chiba, Japan).
2
)
100 ꢄg/ml for S. aureus). 2 showed similar antibacte-
rial activity to that of 1 against these bacteria (see
Table 1). MIC of 3 was 100 ꢄg/ml for E. coli and more
than 100 ꢄg/ml for the other bacteria tested. These
results concur with those of the anucleate cell blue