synthesis and antimicrobial characteristics of a novel biocide

8/20/2019 Synthesis and Antimicrobial Characteristics of a Novel Biocide

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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 - (1,6-

dioxyhexamethylene) 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 rela-

tionship 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-dioxyhexame-

thylene)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 ac-

tivities 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 haz-

ardous 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 -(1,6-

dioxyhexamethylene)bis(1-alkylpyridinium halide) .

INTRODUCTION

Quaternary ammonium compounds (QACs) have

been commonly used for paints, water treatment, tex-

tiles, 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-

bial activity of the long-chain QACs such as

alkylbenzyl dimethyl ammonium chloride (BAC)

(Domagk, 1935), a so-called mono-QAC which con-

sists of only one quaternary ammonium group.

Numerous studies on their synthesis and antimi-

crobial characteristics are currently underway. In or-

der to improve the antimicrobial activity of the mono-

QACs, bis-QACs, which consist of two symmetric

quaternary ammonium groups, were synthesized. For

example, N,N,N ,N -tetraalkyl-N,N -bis (alkylbenzyl) alkylbenzyN,

N -2-butynylene-1,4-bis (ammonium halides) [Benne-

ville, P. L. and Bock L.H. (1950) U. S. Patent, No.

* Corresponding author

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

81-88-656-9148.


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96 T. YABUHARA ET AL.

2,525, 778], the salts of decamethylene-bis-4-

aminoquinaldinium (Babbs et al., 1956) and bis-

QACs derived from bis-(2-dimethylaminoethyl)

glutarate (Pavlikova-Moricka et al., 1994) have been

prepared. Subsequently, a series of modified bis-

QACs as potential antimicrobial agents, 4,4 -( a, co

polymethylenedithio) bis (1-alkylpyridinium halide) s

(4DTBP-m-n-X) (Okazaki et al., 1997), 4,4 -(1,6-he-

xamethylene-dioxydicarbonyl) bis (1-alkylpyridinium

iodide)s (4DOCBP-6-n) (Maeda et al., 1999a), 5,5 -

[2,2 -(tetramethylenedicarbonyldioxy) diethyl]bis (3-

al kyl-4-methylthiazolyium iodide) s (5DEBT-4-n)

(Maeda et al., 1999b), and N,N -hexamethylene-

bis(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 ac-

tivity (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 environ-

mental 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 gener-

ated from 4DTBP-m-n-X, not only during its manufac-

turing process but also during its decomposition.

These phenomena would cause the contamination of

the environment. In order to modify these disadvan-

tages in this study, we presented 4DOBP-6-n-X as a

new compound, in which the sulfur atom of 4DTBP-

m-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-Dithiohexamethy-

lene)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 re-

active 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 4-

hydroxypyridine with sodium in N,N-dimethylformal-

FIG. 1. Procedure to synthesize 4,4 -(1,6-dioxyhexamethylene)bis(1-alkylpyridinium halide)s (4DOBP-6n-X)

.


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NOVEL ANTIMICROBIAL AGENT 97

dehyde provided the alcoholate. It was further con-

verted into 4DODP-6 by the addition of 1,6-

hexamethylenedibromide. The final products,

4DOBP-6-n-X s, were obtained in the reaction of

4DODP-6 and the corresponding n-alkyl halides, in

anhydrous ethanol at 80•Ž, and 80MPa for 72 h. The

products were recrystallized from ethanol-diethyl

ether to give a white powder and dried in a vacuum.

The procedures to synthesize 4DTBP-6-n-X were

traced by thin-layer chromatography (Merck silica gel

60 F254 plates and Merck RP-18 F254S plates, thickness

0.25mm, Merck Japan, Ltd., Tokyo). The final prod-

ucts were identified by elemental analyses (Yanaco

CHN Corder, MT-5, Yanagimoto, Kyoto) and proton

nuclear magnetic resonance (NMR, 400MHz, JEM-

EX 400, JEOL, Tokyo) spectra data. The melting

points were measured with a melting point apparatus

(Mitamura Riken Kogyo Inc., Tokyo). The decompo-

sition points were recorded on a TG-DTA apparatus

(PTC-10A, Rigaku, Tokyo). The solubility was judged

by visual observation after agitation for 24 h at 25•Ž.

4DTBP-6-8-Br was synthesized by a method de-

scribed in a previous report (Okazaki et al., 1997).

BAC (alkyl =CnH2n+1 n= 8 -18) was purchased from

Kanto Chemical Co., Inc. (Tokyo). TBZ was obtained

from San-ai Oil, Ltd. (Tokyo).

Molecular hydrophobicity

The chromatographic RM value, related to the loga-

rithm of the partition coefficient, can be used to esti-

mate the molecular hydrophobicities of the QACs

(Franke, 1984).

Therefore, in this study, thin layer partition chroma-

tography (DC-Fertigplatten, RP-18, F254S Merck

Japan Ltd.) was used to determine the RM values, and

then these were employed to estimate their molecular

hydrophobicities. The thin layer chromatography was

performed using an acetonitrile-ethyl alcohol (10 :10)

solvent system at 30•Ž for 30min. The flow rates of

the samples were determined under irradiation with

UV light. The RM value is defined as

RM = log ((1/Rf) -1)

where a is the flow ra te of a specific 4DOBP-6-n-X.

Microbes, cultivation and preparation

Escherichia coli IFO 12713 was used in these ex-

periments, unless otherwise noted. Bacteria and fungi

were basically prepared by a method described in

previous report (Maeda et al., 1999b). The bacterial

cell suspensions were also prepared by a previously

reported method (Kourai et al., 1991) and kept in ice-

cold water for 2h before use. Nutrient broth medium

was purchased from Becton, Dickinson and Co.

Nutrient agar medium was obtained from OXOID Ltd.

Sabouraud agar and potato dextrose agar medium

was bought from Nissui Pharmaceutical Co., Ltd.

Antibacterial activity

The minimum bactericidal concentration (MBC)

and minimum inhibitory concentration (MIC) of the

antimicrobials were measured by the method previ-

ously described (Okazaki et al., 1997). Bacteriostatic

activity and bactericidal activity were defined as log

MIC-1 and log MBC-1.

Erythrocyte preparation and hemolytic activity

These procedures were carried out using a modi-

fied method of Fogt et al. (1995). Human blood

TABLE 1. 1H-NMR spectra of 4DOBP-6-n-X.

a1H

-NMR spectra were measured in Materials and Methods using tetramethylsilane as an internal standard as described.

b See the text for the full name of this compound

.


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drawn from the first author was washed three times in

a phosphate-buffered saline (PBS) by centrifugation

at 1500 •~g for 5 min at 4•Ž to obtain pure erythro-

cytes. PBS (pH7.4) consisted of 6.78g NaCI, 1.42g

Na2HPO4, and 0.4g KH2PO4 in 1 liter of distilled water.

The erythrocytes were then suspended at a concen-

tration of 2 •~ 109cells/ml in the PBS by determining

the number of erythrocytes with a hemocytometer

(Thoma Blutkorperzahlapparat, Tiefe 0.1mm,

1/400qmm). The antimicrobial agents like QACs

were stepwise diluted by PBS to a final volume of 1

ml. Ten ƒÊ l of the erythrocyte suspension (2 •~ 109

cells/ml) was then added to the above solution and

the final erythrocyte concentration was 2 •~ 107cells/-

ml. The erythrocyte solutions were next incubated at

37•Ž for 30min. Following the incubation, the solu-

tions were centrifuged at 600 •~g for 5 min at 4•Ž, and

the percent hemolysis was determined by comparing

the absorbance at 540 nm of the supernatants with

that of a control sample totally hemolyzed with dis-

tilled water :

hemolysis ( ) = C (AQAC - A PBS / (A control - A PBS) ) •~

100

where APBss the absorbance of the supernatant with

PBS. The hemolytic activity, HC50, s the concentra-

tion which induces a 50 release of hemoglobin from

the erythrocytes, which was determined from the plot

of the percentage of hemolysis versus the concentra-

tions of the QACs.

RESULTS AND DISCUSSION

Chemical structures and properties of 4DOBP-6-

n-X

In Table' 1, the chemical structure assignments for

the compounds are based on their 1H-NMR. For ex-

ample, 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

TABLE 2. Elemental analyses of 4DOBP-6-n-X.

a Numbers indicate

.

TABLE 3. Yields and physical properties of 4DOBP-6-n-X.

a The yields of 4DOBP

-6-n-X were calculated as 4DOBP-6-n-X versus 4

-hydroxypyridine

.

b Decomposition was observed at two points by TG/DTA

, heating rate 10•Ž/min, under a nitrogen flow of 200ml/min, ref-

erence, Al2O3.

che solubilities of 4DOBP-6-n-X were measured in water at 25•Ž and indicated as weight

.

d Values are mean•}S

.D., obtained from three independent experiments.


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nitrogen, the chemical shifts were seen at 4.36ppm

(t,=6.5Hz, 4H) or 4.46ppm (t,J=7.6Hz, 4H). These

chemical shifts were confirmed by a two dimensional

proton-carbon NMR correlation spectroscopy (C-H

cosy) experiment (data not shown). The signals at

7.51ppm (d, J=7.6Hz, 4H) and 8.75ppm (d, J=7.6Hz,

4H) were assigned to the protons of the pyridine ring.

The described 1H-NMR data are consistent with the

proposed chemical structure of 4DOBP-6-8-Br. Data

for the other compounds are shown in Table 1. It is

worth noting that there was a remarkable difference

between the chemical shifts of the methylene proton

which is bonded to the oxygen or sulfur. That is, in

4DOBP-6-8-Br and 4DTBP-6-8-Br, they were respec-

tively 4.36 ppm (t, J=6.5Hz, 4H) and 3.24ppm (t, J

=7.4Hz

, 4H), while the other data did not show a sig-

nificant difference between the two compounds. It

can be presumed that the differences are attributable

to the gap between the electronegativity of the oxy-

gen and sulfur atoms.

In Table 2, the data for the elemental analyses were

all within •}0.3 of the calculated values. In Table 3,

the yields, the melting points, the decomposition

points and the solubilities are listed. As a result, all of

the synthesized 4DOBP-6-n-X compounds had the

FIG. 2. Relationship between molecular hydrophobicity

(RM) and the bacteriostatic activity (log MIC-1) of 4DOBP-6-

n-Br. The unit of MIC is molarity(M). Symbols : •œ,

Escherichia cob' IFO 12713 ; •, Staphylococcus aureus

IFO 12732. The number under the symbol in the figure ex-

presses n. Solid lines represent the equations : E. coil IFO

12713, log MIC-1 =4.96-0.756 RM-1.86(RM)2 ; (coefficient

of correlation r=0.791); St. aureus IFO 12732, log MIC-1

=6

.05+0.992 RM-4.80 (RM)2 ; (r= 0.932) .

FIG. 3. Relationship between molecular hydrophobicity

(RM) and the bactericidal activity (log MBC-1) of 4DOBP-6-

n-Br. The unit of MIC is molarity(M). Symbols : •œ,

Escherichia coil IFO 12713 ; •, Staphylococcus aureus

IFO 12732. The number under the symbol in the figure ex-

presses n. Solid lines represent the equations : E. cob' IFO

12713, log MBC-1=5.10+0.952 RM -.03(RM)2 ; (coefficient

of correlation r=0.506); St. aureus IFO 12732, log MBC-1

=5.90+3.20 RM-3.30 (RM)2 ;r= 0.860) .

proposed chemical structures, and the analytical data

indicated sufficient purity for the following investiga-

tion of their antimicrobial characteristics.

Molecular hydrophobicity of 4DOBP-6-n-X

In Table 3, an approximately linear relationship ex-

ists between the alkyl chain length of 4DOBP-6-n-Br

and its molecular hydrophobicity. Therefore, the

change in its molecular hydrophobicity will be de-

pendent on the length of the alkyl group of the pyri-

dine.

Effect of molecular hydrophobicity on the

antimicrobial activity

As described above, the attachments of the differ-

ent lengths of alkyl groups changed the molecular

hydrophobicity of the QACs, and then the effects of

the molecular hydrophobicities (RM) on their

antimicrobial activities were evaluated. The RM data of

4DOBP-6-n-Br are shown in Table 1, and plotted ver-

sus their antimicrobial activities. The resulting curves

were parabolic against E. coil IFO 12713 and

Staphylococcus aureus IFO 12732, but the coeffi-

cients of the second-order equation of the

bacteriostatic activities (footnote in Fig. 2) were

greater than for their bactericidal activities (footnote


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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 in-

creased when n=6 increased to n=8, and slightly de-

creased when n=8 increased to n=18. On the other

hand, in Fig. 3, their bactericidal activities were simi-

larly increased with the range from n=6 to n=8, but

FIG. 4. Effect of temperature A) and pH B) on the bacte-

ricidal activity log MBC-1) of 4DOBP-6-8-Br and BAC

against Escherichia coil IFO 12713. The unit of MIC is

molarity M). Symbols:•œ4DOBP-6-8-Br;•, BAC.

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) resem-

bled 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 ma-

terials, while the bacteriostatic activity reflects the in-

teraction in the nutrient medium containing

hydrophobic materials such as peptone. Therefore, it

is implied that the hydrophobic materials in the me-

dium interact with the hydrophobic group of the

antimicrobial molecule such as the alkyl chain, inhibit

the contact with the bacterial cell surface, and even-

tually 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 4DTBP-

6-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-

TABLE 4. Minimum inhibitory concentrations MICs) of 4DOBP-6-8-X, 4DTBP-6-8-Br and BAC against bacteria.

a Alkylbenzyldimethyl ammonium chloride

.

b Values are mean •}S



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TABLE 5. Minimum Inhibitory concentrations (MICs) of 4DOBP-6-8-Br, 4DTBP-6-8-Br and TBZ against fungi

.

a Values are mean•}S


b Obtained from National Institute of Health Sciences

.

TABLE 6. Hemolytic concentrations (NC50)a of 4DOBP-6-8-Br, 4DTBP-6-8-Br and BAC against human red blood cells.

a HC

50 (ƒÊM) was measured by a dilution method using a phosphate-buffered saline at 37•Ž for 30 min and was the con-

centration of QACs inducing 50 hemolysis.

b Values are mean•}S


tericidal activity of BAC decreased under acidic con-

ditions. It is known that the antimicrobial activity of a

mono-QAC, such as BAC, is lower in acid conditions

than in alkali (Kourai et al., 1994) which is one of its

disadvantages. To the contrary, the bactericidal activ-

ity of 4DOBP-6-8-Br was not dependent on pH, which

is due to its dimeric structure.

ubsequently, the effects of temperature on the an-

tibacterial activities of 4DOBP-6-8-Br and BAC were

examined at various temperatures (10, 20, 30, and

40•Ž) against E coil IFO 12713. The other conditions

of this antibacterial activity test were the same as in

the method described above in Materials and

Methods. There were no effects of the bacterial

growth on this result, since the MBCs were measured

in a suspension without nutrient substances for 30

min. As can be seen in Fig. 4, the bactericidal activity

of 4DOBP-6-8-Br was almost equal, which was higher

than that of BAC, at temperatures from 10•Ž to 40•Ž.

However, the activity of BAC was slightly influenced

by the temperature, and decreased by lowering the

temperature. In a previous study, the antimicrobial ac-

tivity of bis-QACs was hardly influenced by tempera-

ture, but that of mono-QACs was. The compounds

with other alkyl groups showed a similar tendency

(Maeda et al., 1999b).

In this experiment, the activities of 4DOBP-6-8-Br

and BAC had a tendency to resemble those in a pre-

vious study. Generally, an increase in the temperature

tends to enhance the bactericidal activities of the

QACs, because the temperature is closely related to

the fluidity of the bacterial cell membrane and most

QACs interact with the membrane to produce a bac-

tericidal 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.

Antimicrobial spectra of 4DOBP-6-8-Br, 4DTBP-6-

8-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 bac-

teria (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 nutri-

ent broth composed of organic materials. The anti-

bacterial 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 experi-

ment.

In this experiment, 4DOBP-6-8-Br and 4DTBP-6-8-


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Br had higher activities against gram-positive bacteria

than against the gram-negative, as in the case of the

general QACs. Moreover, remarkable differences in

their activities were seen between bis-QACs

(4DOBP-6-8-Br, 4DTBP-6-8-Br) and a mono-QAC

(BAC) against gram-negative bacteria, though not so

against the gram-positive bacteria. This would be due

to the fact that the cell surface of the gram-positive

bacteria is more hydrophobic than that of gram-

negative bacteria (Kourai et al.,1989). In conclusion,

it was implied that the excellent antimicrobial activi-

ties of 4DOBP-6-n-X are due to its unique dimeric

structure.

Hemolytic activity of 4DOBP-6-8-Br, 4DTBP-6-8-

Br and BAC

The hemolytic activity of 4DOBP-6-8-Br was evalu-

ated and compared to that of 4DTBP-6-8-Br and a

commercial BAC on human red cells (Table 6). Tac

was assumed as one of the indications of acute

cytotoxicity using the data of HC50 and MIC. It is

thought that the safety of an antibacterial agent also

becomes high as the value of Tac becomes large. The

Tao is defined as

Tac=Hac/BMIC

where Hacand BMIC are the aforedescribed HC50 value

ƒÊ) and MIC(ƒÊM).

From the above formula using MIC of Table 4 and

HC50 of Table 6, Tac of 4DOBP-6-8-Br was 138 to

1.44,and that of BAC was 72.7 to 0.48. Therefore, it is

presumed that the safety of 4DOBP-6-8-Br is higher

than BAC. On the other hand, the hemolytic activity

HC50 value of 4DTBP-6-8-Br was almost same as that

of 4DOBP-6-8-Br.

It is possible that the differences between the

HC50 values of 4DOBP-6-8-Br and BAC could be at-

tributed to their chemical structures. Further work is

now in progress to confirm this mechanism.

In conclusion, in this study, it was proved that

4DOBP-6-n-X had excellent antimicrobial activities

and safety regarding hemolysis. Moreover, we cannot

expect 4DOBP-6-n-X to generate a foul odor and

harmful gases due to its chemical structure nor to

have the property of coloring metals. Hence 4DOBP-

6-n-X would be a new excellent antimicrobial agent

that is environmentally friendly. Further investigations

to characterize 4DOBP-6-n-X are now in progress.

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