Biocontrol

Science,

2004,

Vol. 9, No. 4, 95-103

Original

Synthesis
and Antimicrobial
Characteristics
of a Novel
Biocide, 4,4'- (1,6-Dioxyhexamethylene)bis(1-alkylpyridinium
halide)
TADAO

YABUHARA, TAKUYA MAEDA, HIDEAKI NAGAMUNE,

AND HIROKI KOURAI*

Department of Biological Science and Technology, Faculty of Engineering,

The University of Tokushima, Minamijosanjima-cho, Tokushima 770-8506, Japan
Received 10 June 2004/Accepted 6 August 2004
We synthesized
a new bis-quaternary
ammonium
compound
(bis-QAC), 4,4'- (1,6dioxyhexamethylene)
bis (1-alkylpyridinium halide) s (4DOBP-6-n-X) (alkyl chain length, n = 6,
8, 10, 12, 14, 16 and 18, X = Br or I) that has a symmetrical dimeric structure, and is composed
of two alkylpyridinium halides connected with a dioxyhexamethylene.
We examined the relationship of the hydrophobicity and antimicrobial activity using 4DOBP-6-n-X. The influences of
pH, temperature,
and hemolytic activity were then measured using 4,4'- (1,6-dioxyhexamethylene)bis (octylpyridinium bromide) (4DOBP-6-8-Br) which showed a superior antimicrobial
activity among the 4DOBP-6-n-X series. The following three chemical compounds were used
as a comparison: 4,4'- (1 ,6-dithiohexamethylene)bis(octylpyridinium
bromide) (4DTBP-6-8-Br)
which is considered
as an excellent antimicrobial
agent based on a previous report,
alkylbenzyldimethyl
ammonium chloride (BAC) and 2- (4'-thiazolyl)benzimida-zole
(TBZ). In
the results of the experiment, 4DOBP-6-8-Br exhibited a wide antimicrobial spectrum, and had
a strong activity against bacteria and fungi comparable to 4DTBP-6-8-Br. The hemolytic activities of 4DOBP-6-8-Br and 4DTBP-6-8-Br were almost same, and lower than that of BAC.
4DTBP-6-8-Br had some defects in that it colored metals or generated a bad smell and a hazardous gas; however, in 4DOBP-6-n-X, these disadvantages
were not present. 4DOBP-6-n-X
seems to be an environmentally friendly antibacterial agent.
Key words : Quaternary ammonium compound/Novel biocide/Antimicrobial activity/4,4'-(1,6dioxyhexamethylene)bis(1-alkylpyridinium
halide) .
INTRODUCTION
Quaternary ammonium compounds (QACs) have
been commonly used for paints, water treatment, textiles, in the food industry and the hospital, and for
medical and domestic use because QACs have a
relatively low toxicity and wide-ranging antimicrobial
spectrum.
For the first time, Domagk disclosed the antimicro* Corresponding
81-88-656-9148.

author

. Tel : +81-88-656-7408,

Fax

: +

bial activity of the long-chain QACs such as

alkylbenzyl dimethyl ammonium chloride (BAC)
(Domagk, 1935), a so-called mono-QAC which consists of only one quaternary ammonium group.
Numerous studies on their synthesis and antimicrobial characteristics are currently underway. In order to improve the antimicrobial activity of the monoQACs, bis-QACs, which consist of two symmetric
quaternary ammonium groups, were synthesized. For
example, N,N,N',N'-tetraalkyl-N,N'-bis (alkylbenzyl) alkylbenzyl)
-N,
N'-2-butynylene-1,4-bis (ammonium halides) [Benneville, P. L. and Bock L.H. (1950) U. S. Patent, No.

96

T. YABUHARA

ET AL.

2,525, 778], the salts of decamethylene-bis-4aminoquinaldinium (Babbs et al., 1956) and bisQACs derived
from bis-(2-dimethylaminoethyl)
glutarate (Pavlikova-Moricka et al., 1994) have been
prepared. Subsequently, a series of modified bisQACs as potential antimicrobial agents, 4,4'-( a, copolymethylenedithio) bis (1-alkylpyridinium
halide) s
(4DTBP-m-n-X) (Okazaki et al., 1997), 4,4'-(1,6-hexamethylene-dioxydicarbonyl) bis (1-alkylpyridinium
iodide)s (4DOCBP-6-n) (Maeda et al., 1999a), 5,5'[2,2'-(tetramethylenedicarbonyldioxy)
diethyl]bis (3al kyl-4-methylthiazolyium
iodide) s
(5DEBT-4-n)
(Maeda et al., 1999b), and N,N'-hexamethylenebis(4-carbamoyl-1-decylpyridinium bromide) (D-38)
(Yoshida et al., 2000) were synthesized and studied.
Previous studies revealed that QACs act on the cell
wall and have a direct or indirect lethal effect on the
cell. In addition, it was proved that the factors which
control their antimicrobial activity are molecular
hydrophobicity (Kourai et al., 1983b and 1995),
adsorbability (Kourai et al., 1983a), and the electron
density of the ammonium nitrogen atom (Maeda et
al., 1996; Okazaki et al., 1996) or bacterioclastic activity (Kourai et al., 1994).
We considered that the 4DTBP-m-n-X was the best
QAC as described below. In previous studies, it was
demonstrated that it had wide antimicrobial effects
against both bacteria and fungi, and these activities
were stronger than those of the typical bactericide
BAC or the popularly used fungicide TBZ. Moreover,
the bactericidal activities of 4DTBP-m-n-X were not
affected by the length of the hydrophobic alkyl chain.
They were comparatively uninfluenced by environmental conditions, such as pH or temperature.

However, at the same time, the 4DTBP-m-n-X has

some defects as follows. First, in an aqueous solution
it colors metal surfaces. Also, hazardous gases, such
as hydrogen sulfide, sulfur dioxide, etc., are generated from 4DTBP-m-n-X, not only during its manufacturing process but also during its decomposition.
These phenomena would cause the contamination of
the environment. In order to modify these disadvantages in this study, we presented 4DOBP-6-n-X as a
new compound, in which the sulfur atom of 4DTBPm-n-X is substituted by oxygen. Therefore, if the
antimicrobial activities of 4DOBP-6-n-X are better
than those of 4DTBP-m-n-X, it will be a novel
antimicrobial agent that is environmentally friendly.
MATERIALS

AND METHODS

Chemicals
The procedure to synthesize 4DOBP-6-n-X is
shown in Fig.1. The abbreviations, n and X, indicate
the carbon number of the alkyl chain and the halogen
atom,
respectively.
4,4'-(1,6-Dithiohexamethylene)bis(1-octylpyridinium bromide) (4DTBP-6-8-Br)
was chosen as a representative of 4DTBP-m-n-X. All
chemicals used to synthesize the compounds were
reagent grade commercial materials and used without
further purification. The synthesis of 4DOBP-6-n-X
was more difficult than that of 4DTBP-6-n-X since it is
dependent on its chemical reactivity, i.e., the less reactive 4-hydroxypyridine does react with the
alkylhalide directly. Therefore, it was synthesized by
a method different (Fig.1) from that described in the
previous report (Okazaki, 1997). The reaction of 4hydroxypyridine with sodium in N,N-dimethylformal-

FIG. 1. Procedure to synthesize 4,4'-(1,6-dioxyhexamethylene)bis(1-alkylpyridinium

halide)s (4DOBP-6n-X) .

NOVEL ANTIMICROBIAL
dehyde

provided

verted

into

the

alcoholate.

4DODP-6

It was

by

the

hexamethylenedibromide.

The

4DOBP-6-n-X's,

were

4DODP-6

and

anhydrous

the

at

were

ether

to

The

give

60

by

white
to

F254 plates

ucts
CHN

Corder,

EX

MT-5,

points

were

(Mitamura
sition

Kogyo

Rigaku,

in

from

was
a

(alkyl

San-ai

previous

Oil,

Tokyo).
a

The

solubility

agitation

report

Inc.

Ltd.

(Tokyo).

by

-18)

was

(Tokyo).

24
a

decompoapparatus

was

was

The

judged
25.

rithm
mate

of

the

the

(Franke,

was

de-

coefficient,

molecular

can

be

hydrophobicities

from

the

used
of

tography

study,

thin

(DC-Fertigplatten,

TABLE

1. 1H-NMR

a1H -NMR
the

spectra

layer

to

the

partition

RP-18,

:10)

rates

with

as

4DOBP-6-n-X.

preparation

12713

et

were

also

2h

agar

these

The
by

al.,

and

1991)
Nutrient

previously
kept

potato

dextrose

and

Co.

OXOID

Ltd.

agar

Pharmaceutical

in icemedium

Dickinson
from

in

bacterial

broth

obtained

fungi

described

1999b).

Becton,

ex-

and

prepared

use.

Nissui

in

method

al.,

was

and

from

used
Bacteria

et

before

medium

bought

was
noted.

medium

Co.,

Ltd.

Antibacterial
The

activity
minimum bactericidal

minimum

loga-

activity

esti-

MIC-1

were

described
and
and

concentration

inhibitory

concentration
measured

(Okazaki

log

by
et

bactericidal

al.,

the

1997).

activity

(MBC)
(MIC)

were

of

method
Bacteriostatic
defined

as

log

MBC-1.

QACs

chroma-

F254S

in Materials
and
of this compound

the

previ-

These
fied

Merck

preparation
procedures

method

of

and
were

Fogt

et

hemolytic

carried
al.

out

activity
using

modi-

(1995).

Human

blood

standard

as described.

of 4DOBP-6-n-X.

spectra
were measured
text for the full name

of

irradiation

specific

by

from

agar

Erythrocyte
in this

(10

flow

1997).

1984).

Therefore,

b See

partition

of

(Kourai

for

Sabouraud

obtained

to

rate

(Maeda

method
water

ously
related

is defined

prepared

purchased

and

RM value,

was

alcohol
The

under

otherwise

report

Nutrient

hydrophobicity
chromatographic

and

molecular

chromatography

30min.

and

unless

antimicrobials
Molecular

for

IFO

suspensions

cold

purchased
TBZ

RM values,
their

-1)

coli

previous
cell

reported

al.,

layer

determined

flow

basically

apparatus

method
et

the

estimate

acetonitrile-ethyl

cultivation

were

melting

h at

thin

RM value

is the

periments,

JEM-

was

for

(Okazaki

Co.,

The

TG-DTA

synthesized

=CnH2n+1 n=

Chemical

point

to

30

((1/Rf)

Escherichia

proton

The

melting

on

after

and

The
log

Microbes,

prod-

400MHz,

data.

Inc.,

Tokyo).

observation

scribed

recorded

4DTBP-6-8-Br

Kanto

with

were

(PTC-10A,

BAC

spectra

where

(Yanaco

Kyoto)
(NMR,

an
at

were

light.

determine

97

gel

thickness
final

using

RM =

to

employed
The

samples

UV

were

The

used

were

system

the

The

silica

analyses

Yanagimoto,

solvent

a vacuum.

F254S plates,
Tokyo).

elemental

Tokyo)

Riken
points

visual

RP-18

measured

in

(Merck

resonance

JEOL,

h.

4DTBP-6-n-X

Ltd.,
by

magnetic

400,

by

Merck
Japan,

performed

in

ethanol-diethyl

dried

synthesize

identified

nuclear

and

72

was

these

of

halides,
for

Ltd.)

then

hydrophobicities.

reaction

80MPa
from

chromatography

and

Merck
were

and

powder

thin-layer

0.25mm,

80,

Japan

1,6-

products,

the
n-alkyl

recrystallized

procedures

traced

in

conof

final

obtained

corresponding

ethanol

products

further

addition

AGENT

Methods
.

using

tetramethylsilane

as an internal

98

T. YABUHARA

drawn
a

from

the

first

ET AL.
author

phosphate-buffered

at

1500 ~g

cytes.

for

PBS

The

min

(pH7.4)

Na2HPO4,

and

the

of

0.4g

of

then

were

the

stepwise

was

by

the

erythrocyte

PBS

added

to

final

erythrocyte

concentration

The

erythrocyte

solutions

30min.

were
percent

the

absorbance
of

water

(%)

a The

nm

volume

2 ~

of

107cells/-

at

by

the

and

at

the

5 min

solu-

4,

and

comparing

supernatants

with

hemolyzed

with

dis-

indicate%

3. Yields

yields

of

b Decomposition

was

of 4DOBP-6-n-X.

and

4DOBP

- A PBS) / (A control - A PBS) ) ~

analyses

physical

-6-n-X

properties

were

observed

at

of 4DOBP-6-n-X.

calculated
two

as

points

by

4DOBP-6-n-X

versus

4 -hydroxypyridine

TG/DTA
, heating

erence,

AND DISCUSSION

Chemical structures and properties of 4DOBP-6n-X

In Table' 1, the chemical structure assignments for
the compounds are based on their 1H-NMR. For example, in 4DOBP-6-8-Br, the signal at 0.90ppm (t, J
=7.0Hz , 6H) indicated the terminal methyl protons of
the alkyl chain which is connected to the ammonium
nitrogen. The signals of 1.30-1.40ppm (m, 20H),
1.41-1.54 (m, 4H), 1.88ppm (m, 4H) and 1.99ppm
(m, 4H) were assigned to the methylene protons of
the alkyl and alkylene chains. As for the methylene
protons bonded to the ether oxygen or the ammonium

1
109

incubated

incubation,
for

of
(2 ~

next

RESULTS

QACs

solution

was

totally

C (AQAC

2. Elemental

a Numbers

TABLE

final

determined

sample

0.1mm,
like

hemolysis

TABLE

540

concen-

hemocytometer

above

600 ~g

was

at

control

the

the

at

hemolysis

to

were

Following

centrifuged

the

suspension

ml.

for

water.

determining

agents

the

tilled

at
by

antimicrobial

then

1.42g

distilled

Tiefe

diluted
of

cells/ml)

that

of

PBS

with

100
where APBsis the absorbance of the supernatant with
PBS. The hemolytic activity, HC50,is the concentration which induces a 50% release of hemoglobin from
the erythrocytes, which was determined from the plot
of the percentage of hemolysis versus the concentrations of the QACs.

in

erythro-

NaCI,

suspended
in

erythrocytes

The

Ten l

tions

pure

6.78g

1 liter

times

centrifugation

obtain
of

Blutkorperzahlapparat,

1/400qmm).

37

three
by

to

in

109cells/ml

number

ml.

KH2PO4
were

2 ~

(Thoma

at

washed
(PBS)

consisted

erythrocytes

tration

was

saline

rate

10/min,

under

nitrogen

Al2O3.

The
solubilities
c
d Values
are

of
mean}S

4DOBP-6-n-X
.D.,

obtained

were
from

measured
three

in

water

independent

at

25

experiments.

and

indicated

as

weight

% .

flow

of

200ml/min,

ref-

NOVEL ANTIMICROBIAL
nitrogen,

the

chemical

J=6.5Hz,

4H)

chemical

shifts

(d,

4H)

were

assigned

The

described

proposed
for
worth
between
is

to

ppm

, 4H),

nificant

the

gap
and

shifts

of

the

between
sulfur

methylene

the

That
were

and

data

did

the

two

It is

proton

sulfur.

4H)

between

1.

difference

they

other

the
Data

Table

remarkable

or

J=6.5Hz,

ring.
with

in

the

at

J=7.6Hz,

pyridine

4DOBP-6-8-Br.

oxygen

that

the

shown
a

the

presumed

In Table
all

(t,

while

(d,

of

was

the

of

signals

consistent

4DTBP-6-8-Br,

difference
be

gen

and

(C-H

The

are

are

there

bonded

4.36

=7.4Hz

protons
data

chemical

4DOBP-6-8-Br
tively

the

4.36ppm

dimensional

8.75ppm

structure

that

the

which

to

to

a two

shown).

and

compounds

noting

can

4H)

1H-NMR

other

at

spectroscopy

not

J=7.6Hz,

chemical

the

by

correlation
(data

7.51ppm

seen

is,

3.24ppm
not

in

respec(t,

show

a sig-

compounds.

differences

are

electronegativity

It

attributable
of

the

oxyFIG.

atoms.

2, the

data

within }0.3%

of

for

the

the

elemental

calculated

analyses
values.

In Table

3.

(RM)

were
3,

Relationship

and

n-Br.

the

The

yields,

points
the

the

and

the

melting

points,

solubilities

synthesized

are

the
listed.

4DOBP-6-n-X

decomposition
As

IFO

a result,

compounds

all

of

had

the

12732.

of

of

coil

presses
12713,

between

bactericidal
unit

Escherichia
the

99

4H). (t, These

confirmed

NMR

experiment

were

4.46ppm (t,J=7.6Hz,

were

proton-carbon
cosy)

shifts

or

AGENT

MIC

IFO

The

molecular

activity
is

;,

under

correlation

r=0.506);

St.

RM-3.30

(RM)2

chemical

structures,

sufficient

purity

tion

of

their

ists

3,

an

between

and

pendent

its

on

of

alkyl

molecular
in

12732,

log

the

MBC-1

analytical

following

data

investiga-

4DOBP-6-n-X
linear

chain

length

relationship
of

Therefore,

hydrophobicity

length

of

the

ex-

4DOBP-6-n-Br

hydrophobicity.
molecular

the

ex-

characteristics.

approximately

the

its

change

the

figure

: E. cob' IFO
; (coefficient

and

hydrophobicity

In Table

in the

IFO
0.860)

for

antimicrobial

Molecular

symbol

aureus

indicated

: ,
aureus

the equations
RM -.03(RM)2

;r=

proposed

of 4DOBP-6-

Symbols

Staphylococcus

the

n. Solid
lines
represent
log MBC-1=5.10+0.952

=5.90+3.20

hydrophobicity

MBC-1)

molarity(M).

12713

number

(log

the

will

alkyl

group

be

of

de-

the

pyri-

on

the

dine.

Effect

of

molecular

antimicrobial
As
ent

described
lengths

above,
of

hydrophobicity
the
FIG.

2.

(RM)

and

n-Br.

Relationship
the

The

unit

Escherichia
IFO

of

cob'

12732.

between

bacteriostatic

The

IFO

molecular

activity
MIC

is

hydrophobicity
MIC-1)

molarity(M).

12713

number

(log

;,

under

of

Symbols

the

symbol

in

molecular

sus

are

their

n.

12713,

log

of

correlation

=6

.05+0.992

Solid

lines

MIC-1

=4.96-0.756

r=0.791);
RM-4.80

represent

the

equations

St.
(RM)2

aureus

the

figure

ex-

(r=

0.932)

coil

12732,
.

changed

QACs,

and

the
then

evaluated.

antimicrobial

in

effects

activities.

1, and
The

of

on

The

Table

differ-

molecular

the

(RM)

were
shown

the

their

RM data
plotted

resulting

of
ver-

were

parabolic

against

log

aureus

E.
IFO

coil

IFO

curves

12732,

12713
but

and

the

coeffi-

IFO

cients

; (coefficient

IFO

of

aureus

: E.

RM-1.86(RM)2

attachments

groups
the

activities

Staphylococcus
presses

the

hydrophobicities

4DOBP-6-n-Br

: ,

Staphylococcus

alkyl
of

antimicrobial

4DOBP-6-

hydrophobicity

activity

MIC-1

of

the

bacteriostatic
greater

second-order
activities

than

for

their

equation
(footnote

bactericidal

in
activities

of
Fig.

2)

the
were

(footnote

100

T. YABUHARA

ET AL.

in Fig.3). Though there were differences in the values

of the antimicrobial activity against the two bacteria,
similar behavior was seen. As indicated in Fig. 2, the
bacteriostatic activities of 4DOBP-6-n-Br were increased when n=6 increased to n=8, and slightly decreased when n=8 increased to n=18. On the other
hand, in Fig. 3, their bactericidal activities were similarly increased with the range from n=6 to n=8, but

FIG.

4. Effect

ricidal
against
molarity

of temperature

activity

(log

Escherichia
(M).

(A)

MBC-1)
coil

of

IFO

and

pH

(B)

on

4DOBP-6-8-Br

12713.

The

and
unit

Symbols:4DOBP-6-8-Br; ,

b Values

are

ammonium
mean }S

.D.,

obtained

of

bacteBAC
MIC

BAC.

TABLE 4. Minimum inhibitory concentrations

a Alkylbenzyldimethyl

the

chloride
from

is

they were almost constant over n=8. These results

suggest that the hydrophobicities of 4DOBP-6-n-Br
have a smaller influence on the bactericidal activities
than on the bacteriostatic activities. Except for n=6,
the tendency of 4DOBP-6-n-Br (n=8 to n=18) resembled the results previously reported for 4DTBP-6-n-X
(n=8 to n=18) (Maeda et al., 1998). The reason why
the bacteriostatic activities of 4DOBP-6-n-Br (n=8 to
18) declined in Fig. 2 compared with the bactericidal
activities is thought to be due to the difference in the
measurement system. The bactericidal activity is
based on the interaction between the antimicrobial
agent and bacterial cell in the water without other materials, while the bacteriostatic activity reflects the interaction
in the nutrient medium containing
hydrophobic materials such as peptone. Therefore, it
is implied that the hydrophobic materials in the medium interact with the hydrophobic group of the
antimicrobial molecule such as the alkyl chain, inhibit
the contact with the bacterial cell surface, and eventually reduce the antibacterial activity. The result of
our experiment clearly showed that the antimicrobial
activities of 4DOBP-6-n-X were a little affected by the
molecular hydrophobicities like in the case of 4DTBP6-n-X.
Effects of pH and temperature
on bactericidal
activity
The effects of pH on the bactericidal activity of
4DOBP-6-8-Br and BAC were measured using 0.05M
phosphate buffer (pH=5, 6, 7, 8 and 8.7) against E.
coil IFO 12713.
In Fig. 4, the bactericidal activity of 4DOBP-6-8-Br
was constant and higher than that of BAC in the
range from pH5 to pH8.7. On the other hand, the bac-

(MICs) of 4DOBP-6-8-X, 4DTBP-6-8-Br and BAC against bacteria.

.
three

independent

experiments.

NOVEL ANTIMICROBIAL

TABLE 5. Minimum Inhibitory concentrations

a Values

are

mean}S

b Obtained

from

TABLE

6.

obtained
Institute

Hemolytic

of

(MICs) of 4DOBP-6-8-Br, 4DTBP-6-8-Br and TBZ against fungi .

from
three
independent
Health
Sciences

concentrations

(NC50)a

101

of

experiments.
.

4DOBP-6-8-Br,

4DTBP-6-8-Br

and

BAC

against

human

red

blood

cells.

HC
50 (M)

centration
b Values

was

measured

of QACs
mean}S

are

tericidal

activity

ditions.

It is

than

of

as

its

this

on

min.
of

As

this

that

be

of

However,

BAC,

tivity

but

with

other

(Maeda
In

et
this

almost

of

study.

tends

to

had

the

which

of

30,

as

bacterial
measured
for

was
10

slightly
by
the

was.
a

40.

influenced
lowering

by
The

showed

higher
to

the

antimicrobial

influenced

30

activity

which
from

mono-QACs

the

activities

Generally,
enhance

an
the

the
the

to

actempera-

compounds

similar

of

tendency

4DOBP-6-8-Br

resemble

increase

in a

closely

membrane

pre-

temperature

activities
is

cell

those
in the

bactericidal

temperature
bacterial

saline

at

37

for

30

min

and

was

the

con-

of

the

related
and

QACs interact with the membrane to produce a bactericidal action. In this study, the bactericidal activity
of BAC was influenced by the fluidity of the bacterial
cell membrane but that of 4DOBP-6-8-Br was not. It
could be suggested that this was caused by the
dimeric structure of 4DOBP-6-n-X.
As described above, the bactericidal activities of
4DOBP-6-8-Br were not affected by conditions of pH
or temperature. These are considered the merits of
4DOBP-6-n-X when it is used as an antimicrobial
agent.

in
and

the

were

equal,

study,

and

same

bactericidal

was

anwere

conditions

substances
the

hardly

the

BAC

Materials

decreased

a tendency

of

its

1999b).

because
fluidity

were
in

BAC

groups

experiment,

BAC

vious

QACs,

al.,

pH,

20,

other

MBCs

4,

temperatures

was
of

(10,

the

at

and

alkyl

of
activ-

on

The

nutrient

activity

that

conditions
is one

on

effects

was

bis-QACs

ture,

and

no

In a previous

of

of

and

test

in Fig.

temperature,

temperature.

con-

temperature

12713.

since

seen

the

the

of

without

can

acidic

acid

dependent

above

result,

4DOBP-6-8-Br

than

by

IFO

were

suspension

phosphate-buffered

experiments.

activity

bactericidal

temperatures

coil

There

growth

in

4DOBP-6-8-Br

described

Methods.

independent

which
the

not

activity

method

lower
1994)

effects
of

antibacterial

the

three

under

contrary,

various

against

is

using

structure.
the

at

40)

from

antimicrobial

al.,

was

activities

examined

in

the

dimeric

Subsequently,
tibacterial

of

et

To

to

the

BAC,

(Kourai

method

hemolysis.

decreased

that

4DOBP-6-8-Br

is due

a dilution
50%

obtained

BAC

such

disadvantages.
ity

.D.,

of

in alkali

by

inducing

known

mono-QAC,

the

.D.,

National

AGENT

to
most

Antimicrobial spectra of 4DOBP-6-8-Br, 4DTBP-68-Br, BAC and TBZ

The antibacterial
spectra
of 4DOBP-6-8-Br,
4DTBP-6-8-Br and BAC were examined using a MIC
measurement system against the gram-positive bacteria
(6strains)
and
gram-negative
bacteria
(7strains)
of stationary-phase
cells, and fungi
(8strains). The MIC measurement system is an
antimicrobial evaluation which is carried out in a nutrient broth composed of organic materials. The antibacterial and the antifungal spectra
of their
compounds are summarized in Tables 4 and 5. Both
4DOBP-6-8-Br
and
4DTBP-6-8-Br
had
wide
antimicrobial spectra
and exhibited a higher
antimicrobial activity than BAC or TBZ. In Table 4,
the difference in the antimicrobial activity could not
be found between 4DOBP-6-8-Br and 4DOBP-6-8-I.
Therefore, 4DOBP-6-8-Br was used for the experiment.
In this experiment, 4DOBP-6-8-Br and 4DTBP-6-8-

102
Br

T. YABUHARA
had

higher

than

activities

against

general

the

to

fact

that
is

negative

bacteria

ties

cell

surface

that

et

the

of

are

mono-QAC
not

would

the

of

In

to

due

gram-

conclusion,

antimicrobial

due

so

be

gram-positive
that

al.,1989).

excellent

4DOBP-6-n-X

the
in

though

than

(Kourai

implied
of

This

hydrophobic

of

bis-QACs

and

bacteria.

the

more

case

between

bacteria,

gram-positive

bacteria

it was

seen

gram-negative

the

the

differences

4DTBP-6-8-Br)

against

the

in

bacteria

remarkable

were

(4DOBP-6-8-Br,

against

gram-positive
as

Moreover,

activities

(BAC)

against

gram-negative,

QACs.

their

ET AL.

its

activi-

unique

dimeric

structure.

Hemolytic
Br

activity

and

of

4DOBP-6-8-Br,

4DTBP-6-8-

BAC

The

hemolytic

ated

and

commercial
was

activity

of

compared

to

BAC

assumed

on
as

cytotoxicity

using

thought

that

becomes

the

high

of

human
one

of
data
of

the

cells

the

value

evaluand

(Table

HC50

6).

indications

of
an

was

4DTBP-6-8-Br

red

the
safety

as

Tao is defined

4DOBP-6-8-Br

that

and

Tac

of

acute

MIC.

It

is

antibacterial

agent

also

Tac becomes

large.

The

of

as

Tac=Hac/BMIC
where

Hacand
M)

From
HC50

of

above

Table

presumed

HC50

value

of

Tac

using

of

BAC

that

the

safety

On

the

other

value

of

MIC

of

Table

4DOBP-6-8-Br

was

BAC.

72.7

to

of

was

0.48.

4DTBP-6-8-Br

the

was

to

Therefore,

4DOBP-6-8-Br

hand,

and

138

it is

is

higher

hemolytic

almost

activity

same

as

that

4DOBP-6-8-Br.
It

is

HC50

possible

values

tributed
now

to
in

In

that
of

and

their

to

conclusion,

in

gases

have

the

6-n-X
that

due

to
of

be

it

was

proved

is

chemical

we
foul

cannot

odor

structure

metals.

excellent

that

activities

Moreover,

Hence

and
nor

Further
are

to

4DOBP-

antimicrobial

friendly.

4DOBP-6-n-X

at-

work

antimicrobial

coloring

new

be

Further

generate

its

is environmentally
characterize

study,

to

the

could

mechanism.

hemolysis.

property

would

this

this

4DOBP-6-n-X

harmful

BAC

structures.

excellent

regarding

between

and

confirm

had

safety

differences

chemical

progress

expect

the

4DOBP-6-8-Br

4DOBP-6-n-X

to

aforedescribed

formula
6,

that

than
HC50

the

MIC(M).

the

1.44,and

of

BMIC are

and

agent

investigations

now

in

progress.

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