Synthesis of 2,4-dihalogenofluorobenzenes and their antimicrobial and antifungal activity studies

Article abstract of DOI:10.1007/s00044-007-9024-9

The aim of this study was to synthesize and identify 2,4- dihalogenofluorobenzene (or trihalogenobenzene) derivatives by spectroscopic means, ¹H-NMR and ¹⁹F-NMR. The 2,4-dihalofluorobenzene derivatives considered were 2,4-dichlorofluorobenzene (1), 2-bromo-4- chlorofluorobenzene (2), 2-iodo-4-chlorofluorobenzene(3), 2,5- dichlorofluorobenzene (4), 2-bromo-5-chlorofluorobenzene (5), 2-iodo-5-chlorofluorobenzene (6). The in vitro antibacterial and antifungal activities of trihalogenobenzene derivatives were studied against the bacteria Staphylococcus aureus ATCC 25923 Gram(+), Bacillus cereus ATCC 6633 Gram(+), Micrococcus flavus (clinical isolate) Gram(+), Morgenella morganii (clinical isolate) Gram(-), Escherichia coli ATCC 27853 Gram(-) and fungus Candida albicans (clinical isolate), obtained from the biology departments of the Pamukkale and Gazi Universities. The antibacterial and antifungal activities were measured by minimum inhibition concentration (MIC) method and the disc-diffusion method used to measure zone diameter against Gram-positive and Gram-negative bacteria and fungi. All these bacteria and fungi were studied against antibiotics to compare the zone diameters with the results from our treatments. Birkhaeuser 2007.

Full text of DOI:10.1007/s00044-007-9024-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2007) 16:205–212
DOI 10.1007/s00044-007-9024-9
ORIGINAL RESEARCH
Synthesis of 2,4-dihalogenofluorobenzenes and their
antimicrobial and antifungal activity studies
Hikmet Katırcıo g˘ lu Æ Elif Lo g˘ o g˘ lu Æ Tahir Tilki Æ
¨
Atilla Oktemer
Received: 12 February 2007 / Accepted: 29 June 2007 / Published online: 19 September 2007
Ó Birkh a¨ user Boston 2007
Abstract The aim of this study was to synthesize and identify 2,4-dihalogeno-
1
fluorobenzene (or trihalogenobenzene) derivatives by spectroscopic means, H-
1
9
NMR and F-NMR. The 2,4-dihalofluorobenzene derivatives considered were 2,4-
dichlorofluorobenzene (1), 2-bromo-4-chlorofluorobenzene (2), 2-iodo-4-chloroflu-
orobenzene(3), 2,5-dichlorofluorobenzene (4), 2-bromo-5-chlorofluorobenzene (5),
2
-iodo-5-chlorofluorobenzene (6).
The in vitro antibacterial and antifungal activities of trihalogenobenzene deriv-
atives were studied against the bacteria Staphylococcus aureus ATCC 25923
Gram(+), Bacillus cereus ATCC 6633 Gram(+), Micrococcus flavus (clinical iso-
late) Gram(+), Morgenella morganii (clinical isolate) Gram(-), Escherichia coli
ATCC 27853 Gram(-) and fungus Candida albicans (clinical isolate), obtained from
the biology departments of the Pamukkale and Gazi Universities. The antibacterial
and antifungal activities were measured by minimum inhibition concentration
(MIC) method and the disc-diffusion method used to measure zone diameter against
Gram-positive and Gram-negative bacteria and fungi. All these bacteria and fungi
H. Katırcıo g˘ lu (&)
Faculty of Education, Department of Biology Education, Gazi University, Teknikokullar, Ankara,
Turkey
e-mail: hturk@gazi.edu.tr
E. Lo g˘ o g˘ lu
Faculty of Arts and Sciences, Chemistry Department, Gazi University, Teknikokullar, Ankara,
Turkey
T. Tilki
Faculty of Arts and Sciences , Department of Chemistry, S u¨ leyman Demirel University, Isparta,
Turkey
¨
A. Oktemer
Science Faculty Department of Chemistry, Ankara University, Tandogan, 06100 Ankara, Turkey

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Med Chem Res (2007) 16:205–212
were studied against antibiotics to compare the zone diameters with the results from
our treatments.
Keywords Antimicrobial activity Á MIC Á 2,4-dihalofluorobenzenes
Introduction
In the last three decades the environmental impact of halogenated chemicals has
become increasingly apparent. Similarly many other haloagens are important
environmental pollutants (Lu et al., 2000). Halogenated organic compounds are
frequently employed as solvents, pesticides, and refrigerants and for the study of
electron transfer reactions in radiation and electrochemical investigations (Alfassi
et al., 1989; Emmi et al., 1989; Lal et al., 1986, Grodkowski and Neta, 1984). Many
of these compounds are toxic in nature if metabolized in living species (M o¨ nig
et al., 1983; Reynolds et al., 1980; Slater et al., 1982). Halogenated organic
compounds are also known to play an important role in depletion of tropospheric
ozone (Recknegel et al., 1977). The widespread use of halogenated organic
compounds has toxicological and environmental implications (Kime et al., 1987).
The effects on hexachlorobenzene (HCB) dechlorination of several factors,
including electron donors, electron acceptors, and microbial inhibitors [including
bromoethane sulfonic acid (BESA), vancomycin, and molybdate] have been
recognized as selective inhibitors of metlianogen, eubactefia, and sulfate-reducing
bacteria, respectively (Naik and Mohan, 2005). Polychlorinated benzenes (PcBzs)
are used as solvents and starting materials or intermediates in the synthesis of many
other substances, e.g., phenols, dyestuffs in the chemical industry and as pesticides
and fungicides in agriculture. Hexachloro benzene (HcBz) has many uses in
industry, e.g., as a plasticizer for polyvinylchloride (PVC) and as a fungicide in
agriculture (Chang et al., 1998).
The biological activities of tetrasubstituted benzene derivatives against S. aureus,
B. subtilis, Y. enterocolitica, E. coli and C. albicans have been reported
(Nowakowska and Sczcubialka, 1999). The antimicrobial activity of piperidine
and pyrrolidine substituted halogenobenzene derivatives against some bacteria and
¨
fungi have also been studied (Lo g˘ o g˘ lu and Oktemer, 2005).
Among the polybromobenzenes, hexabromobenzene has been used most widely.
The products that result from the hexabromobenzene debromination (penta-, tetra-,
and tribromobenzenes) are formed by means of environmental degradation or
through metabolisms of various organisms (Kashimoto and Tatsukawa, 1986; de
Roode and Klomp, 2002; Colacci et al., 1990). They are more volatile and water
soluble than the parent compound. Dibromobenzenes found in natural environments
mainly originate from their use as fumigants, additives to cleaning agents, or as
intermediates in the production of pharmaceutical preparations (Gustafson et al.,
1
998). For the determination of hepatocarcinogenicity, some scientist have focused
on bioassays to assess the concentration of 1,2,4,5-tetrachlorobenzene and 1,4-
dichlorobenzene using a medium-term liver (GLFC, 1996; Tilki, 2005).

Med Chem Res (2007) 16:205–212
207
Nowadays, because of pathogenic bacterial resistance against all known
antibiotics, there is a need to identify many new chemicals that could be used as
antibiotics. Therefore, scientists must study new chemicals to see whether medicinal
raw material may have antibiotic action or not. In this work, we report on the
synthesis of trihalogenobenzene derivatives and the biological activities of these
compounds against Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC
6633, Morgenella morganii (clinical isolate), Escherichia coli ATCC 27853,
Micrococcus flavus (clinical isolate), and Candida albicans (clinical isolate).
Results and Discussions
Results of chemical part
Trihalobenzene derivatives were synthesized and identified by spectroscopic means
1
19
H-NMR and F-NMR. The structures of the trisubstituted benzene derivatives are
given in Table 1.
1
H-NMR results
1
,4-dichlorofluorobenzene: H-NMR : JHa-Hb:Meta 2,54 Hz , JHa-F: meta 6,37 ,
2
Ha:7,4 ppm , JHb-Ha:2,57Hz, JHb-Hc; meta8,81,JHb-F:4,12 , Hb: multiplet7,2 ppm
1
9
,
JHc-Hb: ortoho 8,65 Hz , JHc-F:8,68 Hz , Hc:7,1 F-NMR: F: -117,6 ppm.
-Bromo-4-chlorofluorobenzene: JHa-Hb: ortoho 2,55 Hz , JHa-F: meta 5,95
2
Hz , Ha:7,6 ppm , JHb-Ha:meta 2,57 Hz, JHb-Hc;meta 8,81 Hz ,JHb-F:para 4,48 ,
Hb: multiplet 7,3 ppm , JHc-Hb: meta 8,11 Hz , JHc-F:8,66 Hz , Hc:7,1 ppm.
2
-Iodo-4-chlorofluorobenzene: JHa-Hb: meta 2,16 Hz , JHa-F: meta 6,84, Ha:
,6 ppm , JHb-Ha: ortho 2,17Hz, JHb-Hc; ortho 8,65 Hz , JHb-F: 4.43 Hz, Hb:
multiplet 7,4 ppm , JHc-Hb: meta 8,78 Hz , JHc-F: 8,89 Hz , Hc: 6,8 ppm.
,5-Dichlorofluorobenzene: JHa-Hb:ortoho 8,22 Hz , JHa-F: meta 8,11 ,
Ha:7,35 ppm , JHb-Ha: ortho 8,59Hz, JHb-Hc;meta 2,28 Hz ,JHb-F:para 1,29 , Hb:
7
2
1
9
multiplet 7,1 ppm , JHc-Hb: meta 2,32 Hz , JHc-F:8,79 Hz , Hc:7,2 ppm. F-NMR:
F: -112,2 ppm.
2-Bromo-5-chlorofluorobenzene: JHa-Hb:ortoho 8,43 Hz , JHa-F: meta 7,55 ,
Ha:7,5 ppm , JHb-Ha: ortoho 8,58Hz, JHb-Hc;meta 2,23 Hz ,JHb-F:para 0,98 ,
Hb: multiplet 7,05 ppm , JHc-Hb: meta 2,34 Hz , JHc-F:8,34 Hz , Hc:7,15 ppm.
2-Iodo-5chlorofluorobenzene: JHa-Hb: ortoho 8,43 Hz , JHa-F: meta 6,85 ,
Ha:7,65 ppm , JHb-Ha: ortoho 8,44 Hz, JHb-Hc;meta 2,22 Hz , Hb: multiplet 6,95
ppm , JHc-Hb: meta 2,28 Hz , JHc-F: ortoho 7,76 Hz , Hc:7,1 ppm.
Biological results
In the present study, the antimicrobial activities of compounds were determined and
are given in Tables 2 and 3. Table 2 shows activity estimated in terms of the

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Med Chem Res (2007) 16:205–212
Table 1 The structures of the trisubstituted benzene derivatives
Compound
no.
Structures
Compound
no.
Structures
1
2
3
4
5
6
F
F
Cl
Cl
Cl
Cl
2
,5-dichlorofluorobenzene
2
,4-dichlorofluorobenzene
F
F
Br
Br
Cl
Cl
2
-bromo-5-chlorofluorbenzene
2
-bromo-4-chlorofluorobenzene
F
F
I
I
Cl
Cl
2-iodo-5-chlorofluorobenzene
2-iodo-4-chlorofluorobenzene
minimum concentration (MIC) inhibiting microbial growth. Table 3 shows the
antibacterial activity of the investigated compounds against Staphylococcus aureus
ATCC 25923, Bacillus cereus ATCC 6633, Morgenella morganii (clinical isolate),
Escherichia coli ATCC 27853, Micrococcus flavus (clinical isolate), and Candida
albicans (clinical isolate). All compounds were found to be more effective against
the tested Gram-positive bacteria. Although E. coli and M. morganii were the most
resistant Gram-negative bacteria, we determined that these bacteria were sensitive
to most of these compounds. All compounds also showed antifungal activity against
Candida albicans, but compound II was twice as sensitive to the reference antibiotic
(
ketoconazole).
According to a statistical test, there was no significant diversity between the
activities of the compounds against the tested microorganisms (F = 0.151, df = 5,
p [ 0.05).
From these results, we may state that differences between the activities of the
compounds were due to different substituent such as Br, Cl and I over the benzene
ring. Compared to the reference discs, compound II showed approximately twofold
more activity against M. flavus, C. albicans, and B. cereus. Compounds III, IV, and
VI were found to be active against all test microorganisms.

Med Chem Res (2007) 16:205–212
209
Table 2 The MICs of the tested compounds and reference antibiotics
Compound
MIC, lg/ml
E. coli
ATCC
M. morgani
(clinical
isolate)
M. flavus
(clinical
isolate)
St. aureus
ATCC
25923
B. cereus
ATCC
6633
C. albicans
(clinical
isolate)
27853
I
1200
2500
3000
3000
3000
2500
1500
2500
1500
2500
2500
2500
1500
2500
1500
2500
2500
2500
1200
2500
2000
2500
2500
2500
1500
2500
2000
2000
2000
2000
1
II
2500
2500
2000
2500
2500
III
IV
V
VI
Table 3 Inhibition zone diameters of compounds and reference antibiotic discs against the test
microorganisms
Compound
Inhibition zone (mm)
E. coli
ATCC27853
M. morgani
(clinical
isolate)
M. flavus
(clinical
isolate)
St. aureus
ATCC
25923
B. cereus
ATCC
6633
C. albicans
(clinical
isolate)
I
12
-
-
11
32
20
14
13
11
-
15
-
19
15
16
16
17
13
-
12
30
14
17
11
14
-
II
-
III
10
11
13
11
19
-
10
12
-
13
15
14
12
-
IV
V
VI
13
-
Penicillin
Chloramphenicol
Tetracycline
Ampicillin
Oxacillin
Ketoconazole
15
-
-
-
-
-
-
20
-
-
-
-
-
-
9
-
-
-
-
-
-
9
-
-
-
-
-
-
16
Experimental Procedures
Synthesis of 2,4-dihalogenoanilines
The trisubstituted 2,4-dihalogenanilines were 2,4-dichlorofluorobenzene (1), 2-
bromo-4-chlorofluoro benzene (2) (yield: 35%; bp: 187°C), 2-iodo-4-chlorofluoro
benzene (3) (yield: 27%; bp: 214°C], 2,5-dichlorofluorbenzene (4) (yield: 38%, bp:
1
68°C), 2-bromo-5-chlorofluoro benzene (5) (yield: 29%, bp: 181°C), and 2-iodo-5-
chlorofluoro benzene (6) (yield: 31%, bp: 206°C).

210
Med Chem Res (2007) 16:205–212
These compounds were prepared from aniline according to the methods given in
the literature (NCCLS, 1997a). Aniline, concentrated HCl and water were (500 ml)
stirred in a flask until the aniline was dissolved in the solvent. Acetanhydride was
added to the solutions, forming aniline-hiydrochlorure during stirring in the flask,
after which the mixture was stirred and sodium acetate was added. The mixture was
cooled while stirring. Row acetanilide was synthesized and recrystallized. The
halogenogase was added to this cold mixture to prepare 2,4-dihalogenoaniline.
Schieman reactions of 2,4-dihalogeno anilins
2
,4-dihalogeno aniline was reacted with NaNO2 and NaBF4 to prepare 2,4-
dihalogeno benzenediazonyum tetrafluoroborate compounds. 2,4-dihalogenoben-
zenediazonyum tetrafluoroborate compounds react to form trihalogenobenzene
compounds at 150–170°C.
The general reaction scheme for all of 2,4-dihalogenanilines and their Scheiman
reactions are given in Fig. 1. The structures of the trisubstituted benzene derivatives
are given in Table 1.
Test microorganisms
The bacterial subcultures for Staphylococcus aureus ATCC 25923, Bacillus cereus
ATCC 6633, Morganella morganii (clinical isolate), Escherichia coli ATCC 27853,
Micrococcus flavus (clinical isolate), and Candida albicans (clinical isolate) were
obtained from the biology departments of the Pamukkale and Gazi Universities.
Bacterial strains were cultured overnight at 37°C in nutrient broth and the yeast was
cultured overnight at 30°C in yeast extract peptone dextrose (YEPD) broth for
antibacterial and antifungal activity tests. These stock cultures were stored in the
dark at 4°C during the survey.
Determination of MICs
Minimum inhibitory concentrations (MICs) were determined by microdilution broth
method following the procedures recommended by the National Committee for
Clinical Laboratory Standards (NCCLS, 1997b; Kartal et al., 2003). MICs were
defined as the lowest concentrations of the antimicrobial agents that inhibited visible
growth of the microorganism.
All tests were performed in Mueller–Hinton broth (MHB) and YEPD Broth. The
compounds under the test were dissolved in analytically pure dimethylsulfoxide
(DMSO) and geometric dilutions ranging from 300 lg/ml to 3000 lg/ml of the
compounds.

Med Chem Res (2007) 16:205–212
211
NHCOCH3
NHCOCH3
NH2
NH2
HCl
acetanhydride
X2
cooling
X
X
X
X
NH2
+
-
F
N2 BF 4
NaNO2 /H
NaBF4
X
X
X
Sch.reac.
0
1
50-170 C
X
X
X
Fig. 1 The structures of the 2,4-dihalogenanilines and trihalogenobenzene derivatives
Screening of antimicrobial activity
Antimicrobial activity of compounds was determined by the disc diffusion method
Kartal et al., 2003). The antimicrobial screening was performed using Mueller–
(
Hinton agar and YEPD agar for the yeast. The culture suspensions were prepared
and adjusted by comparing against 0.3 McFarland turbidity standard tubes.
Muller–Hinton and YEPD agar (20 ml) were poured into each sterile Petri dish after
injecting cultures (100 ll) of microorganisms and distributing medium homoge-
neously in each Petri dish. Compounds were filtered with a pore size of 0.45 lm. All of
the compounds were dissolved in DMSO at a concentration of 5 mg/ml. 6 mm empty
sterilized discs of (no. 2668, Schleicher and Schuell, Germany) were each
impregnated with 50 ll of compounds. Discs were placed on the agar plates, which
were incubated at 37°C for 24 h for bacteria, and 48 h for C. albicans. Inhibition zones
formed on the medium were evaluated in millimeters. The solvent control (DMSO)
did not show any antimicrobial activity. Studies were performed in duplicate and the
inhibition zones were compared with those of reference discs. Reference discs used
for control were as follows: ketoconazole (50 lg), ampicillin (10 lg), tetracycline
(
30 lg), penicillin (10 U), chloramphenicol (30 lg), and oxacillin (1 lg).
Statistical analysis
Data were analyzed and treatments compared using the one-way analysis of
variance (ANOVA) with 95% confidence limits (p \ 0.05) using SPPS 9.0 for
Windows. Values of p \ 0.05 were considered statistically significant.
Conclusions
In this study, 2,4-dihalofluorobenzene (or trihalogenobenzene) derivatives were
1
synthesized and identified by spectroscopic means, H-NMR, and F-NMR. The
19
in vitro antibacterial and antifungal activities of these trihalogenobenzene derivatives

212
Med Chem Res (2007) 16:205–212
against bacteria and yeast and antimicrobial screening clearly indicate that these
compounds had much more-powerful antimicrobial activity for Gram-positive
bacteria and yeast than Gram-negative bacteria. They proved more effective on
bacteria than antibiotics, and may be suggested for industrial applications.
Acknowledgements We would like to extend our thanks to the Ankara University Research Foundation
(no. 2002-0705071) for financial support.
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