Synthesis and investigation of antimicrobial activity of some bisbenzimidazole-derived chelating agents

Article abstract of DOI:10.1016/S0223-5234(03)00146-6

The 1,2-bis(2-benzimidazyl)-1,2-ethanediol (1), 1,4-bis(2-benzimidazyl)-1, 2,3,4-butanetetraol (2), 1,3-bis(2-benzimidazyl)-2-thiapropane (3), 1,3-bis(2-benzimidazyl)-2-thia-propane-dihydrochloride (4), 1,5-bis(2- benzimidazyl)-3-thiapentane (5), and 1,5-bis(2-benzimidazyl)-3-thiapentane dihydrochloride (6) chelating ligands are synthesised and characterised by using analytical data and modem spectroscopic methods such as FT-Raman, FT-IR, ¹H- and ¹³C-NMR spectrometers. Their antimicrobial activities are reported by comparing the in vitro activities, with those of ofloxacin, ciprofloxacin, piperacillin, ampicillin and cefazolin antibacterial agents against fresh clinical isolates. Antifungal activities are reported on Candida albicans, Candida utilis, Cryptococcus neoformans fungi, and the results are referenced with amphotericin-B, fluconazole and flucytosine antifungal agents. It has been found that all the compounds have broad spectra activity and was either more active or equipotent to those compared antibiotic and antifungal agents.

Full text of DOI:10.1016/S0223-5234(03)00146-6

European Journal of Medicinal Chemistry 38 (2003) 875ꢀ881
/
www.elsevier.com/locate/ejmech
Laboratory note
Synthesis and investigation of antimicrobial activity of some
bisbenzimidazole-derived chelating agents
a,
Naz M. Agh-Atabay *, Basaran Dulger , Fahrettin Gucin
b
a
a
Faculty of Arts and Science, Fatih University, 34900 Hadimkoy, Istanbul, Turkey
b
Faculty of Arts and Science Department of Biology, Canakkale Onsekizmart University, Canakkale, Turkey
Received 6 December 2002; received in revised form 9 May 2003; accepted 10 July 2003
Abstract
The 1,2-bis(2-benzimidazyl)-1,2-ethanediol (1), 1,4-bis(2-benzimidazyl)-1,2,3,4-butanetetraol (2), 1,3-bis(2-benzimidazyl)-2-thia-
propane (3), 1,3-bis(2-benzimidazyl)-2-thia-propane-dihydrochloride (4), 1,5-bis(2-benzimidazyl)-3-thiapentane (5), and 1,5-bis(2-
benzimidazyl)-3-thiapentane dihydrochloride (6) chelating ligands are synthesised and characterised by using analytical data and
1
13
modem spectroscopic methods such as FT-Raman, FT-IR, H- and C-NMR spectrometers. Their antimicrobial activities are
reported by comparing the in vitro activities, with those of ofloxacin, ciprofloxacin, piperacillin, ampicillin and cefazolin
antibacterial agents against fresh clinical isolates. Antifungal activities are reported on Candida albicans, Candida utilis,
Cryptococcus neoformans fungi, and the results are referenced with amphotericin-B, fluconazole and flucytosine antifungal agents.
It has been found that all the compounds have broad spectra activity and was either more active or equipotent to those compared
antibiotic and antifungal agents.
´
#
2003 Editions scientifiques et m e´ dicales Elsevier SAS. All rights reserved.
Keywords: Ampicillin; Bisbenzimidazole; Chelating ligands; Equipotent; Ofloxacin
1
. Introduction
viruses [7,8]. Other examples include 5-aminoimidazole
ribonucleotide, a key intermediate on the de novo purine
biosynthetic pathway [9] and 5-(formylamino)imidazole
ribonucleoside, a competitive inhibitor of adenosine
deaminase.
Benzimidazole and bis-benzimidazole derivatives are
key components in a great many bioactive compounds
of both natural and synthetic origin. These ligands and
their derivatives display a wide range of pharmacologi-
cal activity, and their inhibitory properties as regards
the replication of polio viruses, adenosine deaminase,
and casein kinase have been fully demonstrated [1,2].
While others are important potent antiviral agents [3,4],
many are active components of biocides such as
fungicides, and insecticides [5,6]. For instance it is
known that 1,2-bis(2-benzimidazyl)-1,2-ethanediol (1)
shows antifungal and anti-polio viruses character, and
that ligand such as 1,4-bis(2-benzimidazyl)-1,2,3,4-bu-
tanetetraol (2) have a negative effect on intracellular
In addition to their biological importance, these
ligands are strongly coordinating agents and form stable
complexes with various metals [10ꢀ12]. Metal ions play
/
a vital role in vast number of different biological
processes through co-enzymatic system. The interaction
of these ions with biological active ligands, for instance
in drugs, is a subject of great interest. Some of the
biological active compounds do act via chelation, but
for most of them little are known about how metal
coordination influences their activity. It is believed that
they react selectively towards certain biological systems
[
13,14].
We are interested in the microbiological activity of
these ligands towards many bacterial and yeast isolates
* Correspondence and reprints.
E-mail address: natabay@fatih.edu.tr (N.M. Agh-Atabay).
both representing several species.
´
0
223-5234/03/$ - see front matter # 2003 Editions scientifiques et m e´ dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(03)00146-6

876
N.M. Agh-Atabay et al. / European Journal of Medicinal Chemistry 38 (2003) 875ꢀ881
/
2
. Chemistry
respectively. The results of these assays are summarised
in Tables I and II. The data for ofloxacin, ciprofloxacin,
piperacillin, ampicillin and cefazolin antibacterial agents
and amphotericin, fluconazole and flucytosine antifun-
gal agents are used for comparison. Apart from few
exceptions, all the compounds are more active as
compared to antibacterial and antifungal agents against
all the tested micro-organisms. These compounds activ-
ity toward tested micro-organisms are equipotent to that
of ofloxacin and requires over 64 mg ml doses for the
inhibition of Enterococcus faecalis and Proteus species.
It could be seen from the data that if the result of only
few micro-organisms, namely Escherichia coli, Salmo-
nella thyphi, Bacillus subtilis and Stapylococcus aureus
are considered for the basis of this study, this would
have resulted in the failure of achieving, respectable and
valuable information. In this respect, we have used a
reasonable number of micro-organisms, 10 (Gram-
positive and Gram-negative) total, 115 bacteria and 30
yeast isolates, to obtain broad antimicrobial effect
spectrum.
The compounds are prepared either by modified
literature procedures Philips [15] or fused [16] method.
Full general procedures for synthesis of these com-
pounds are given in the experimental and the reactions
sequence are shown in Figs. 1ꢀ4. The spectroscopic data
/
1
13
of H- and C-NMR, FT-Raman and FT-IR do
provide useful information for their formation and
structural identification. The results of the spectroscopic
data are summarized in the end of each experimental.
Apart from few exceptions, the analysis of these spectra
is straightforward. Exception arise that for all the
ligands (1, 2) in H-NMR amino proton resonances
are observed as broad signals at dH 12.24 and 12.07
ppm, respectively. Both of these disappear due deuter-
ꢁ
1
1
ium exchange. For the ligands (3ꢀ6) the amino proton
resonance was not detected due to the fast tautomeric
equilibrium of the proton atom in the imidazole ring.
/
3
. Pharmacological
In classifying the antibacterial activity as Gram-
positive or Gram-negative, it would generally be ex-
pected that a much greater number would be active
against Gram-positive than Gram-negative bacteria [17].
However, in this study, the compounds are active
against both Gram-positive and Gram-negative bacteria
and highly active against yeasts. The activity against
both types of bacteria and yeasts may be indicative of
the presence of broad spectrum.
The results of our study indicate that the compounds
have the potential to generate novel metabolites. The
compounds demonstrating especially anticandidal activ-
ity could result in the discovery of novel anticandidal
agents, showing demonstrating broad spectrum activ-
ities, this may help to discover new chemical classes of
antibiotics that could serve as selective agents against
infectious diseases.
Fresh clinical isolates are obtained from Faculty of
Medicine, Uludag University (Turkey), and are supple-
mented by stock cultures of recent clinical isolates of
some species to achieve target numbers. The total
numbers of isolates are 115 bacteria and 30 yeasts
representing 10 and 3 species, respectively.
Standardised powders of floxacin (an effective broad
spectrum bactericidal, with inhibition of DNA gyrase
activity), ciprofloxacin (antibaterial), piperacillin (b-
lactamase a sensitive penicillin antibiotic), ampicillin
(
broad spectrum antibiotic), cefazolin (for intravenous
and intramuscular administration), were purchased
together with NAD from Sigma Chemicals. Amphoter-
icin-B (antifungal and antiprotozoal), fluconazole
(
broad-spectrum bis-triazole antifungal) and flucytosine
are obtained from Fluka. MuellerꢀHinton media was
/
purchased from Difco and the yeast extracts were
obtained from Oxoid.
5. Experimental protocols
5.1. Chemistry
4
. Results and discussion
All chemicals and solvents were reagent grade and
Bisbenzimidazole derived compounds are evaluated
used without further purification, unless otherwise
indicated. Purity of the compounds was tested on thin
layer chromatography (TLC) plates (silica gel 60 F254
Merck) in the solvent system ethanol:water (5:3). Spots
by broth macro dilution procedure for their in vitro
antibacterial activity against 115 pathogenic bacteria
and 30 yeast-isolates representing 10 and 3 species,
Fig. 1. Syntheses of ligands (1ꢀ2), (a) refluxed in 5 M and neutralised with dilute base, (b) fused at high temperature.
/

N.M. Agh-Atabay et al. / European Journal of Medicinal Chemistry 38 (2003) 875ꢀ
/881
877
Fig. 2. Syntheses of ligands nꢂ
/
1 (4) and nꢂ2 (6), refluxed in 5 M HCl.
/
were located under UV light or by exposure to iodine
vapours. Melting points were determined with electro-
thermal 9100 melting point apparatus. Analytical data
for ligands 5 and 6 were obtained with a Carlo Erba
in warm ethanol. The solution mixture was decolourised
with charcoal at reflux temperature for about 10 min,
filtered and crystallised by gradual addition of water.
Fine colourless crystals are deposited when the solution
was left standing overnight. These crystals are isolated
1
106 analyser. Infrared (IR) spectra were recorded (as
KBr pellets or as mulls in Nujol) on a Mattson Satellite
FT-JR spectrometer. FT-Raman spectra were obtained
from powdered samples placed in a Pyrex tube using the
Bruker RFS 100/S spectrometer (Nd: YAG-Laser, 1064
nm, 200 mW). Routine solution H- and C-nuclear
magnetic resonance (NMR) spectra were recorded at
ambient temperature (20 8C), on a Bruker DPX 400
by filtration and dried in vacuo (4.2 g, 70%), m.p.
1
2218 C. H-NMR (400 MHz): d 3.85 (4H, s, CH ), 6.96
2
1
(s, 4H), 7.32 (s, 4H), (2H, NH not detected); C{ H}-
3
1
NMR (100 MHz): 6 29.04 (2C, CH ), 115.17 (4C),
2
1
13
122.19 (4C), 138.93 (4C), 152.10 (2C); JR (KBr pellets):
ꢁ
1
nmax/cm
C); Raman (solid): nmax/cm
2916 (CꢀH), 1590 (CÄC), 770 (CÄ
includes 1670 (CÄ
/
N), 1620 (CÄ
/
C), 742 (CÄ
includes 3055 (CꢀH),
C).
/
ꢁ
1
/
ultra-shield NMR spectrometer in DMSO-d . Chemical
/
/
/
6
shifts (d) were expressed in units of ppm relative to
TMS. The residual DMSO-d signal was also used as an
6
5.1.1.2. Ligand 4 is also synthesized using the direct
internal reference. The NMR peaks are expressed as:
singlets (s), doublet (d), triplet (t), quartet (q), multiplet
reaction route, commonly applied for solid-state reactions
according to the Fused method [16]. In a mullite mortar,
a freshly sublimed o-phenylenediamine (2.00 g, 16.66
mmol) was ground together with 2,2?-thiodiethanoic
acid (1.4 g, 9.0 mmol). To obtain dense pellets, about a
quarter of the finely ground powder was introduced into
(
m) and broad (br).
5
.1.1. General procedure for the preparation of the
compounds 1ꢀ
/
6
a stainless steel die (Øꢂ13 mm) which was then
/
evacuated shortly to remove the air between the
particles. The powder was then pressed into a disc
5
.1.1.1. The
1,3-bis(2-benzimidazyl)-2-thiapropane-
dihydrochloride (4) and 1,3-bis(2-benzimida-zyl)-2-
thiapropane (3) ligands are synthesized according to
Philip’s method [15]. Aqueous HC1 (5 M, 50 mL) was
added to a mixture of 2,2?-thiodiethanoic acid (3.0 g,
between stainless steel anvils at a pressure of 10ꢀ12 tons.
/
The pellets were transferred into a flame dried Schlenk
tube, which was connected to a vacuum line and heated
up to about 180 8C or until it melted, with occasional
opening the vacuum line to remove condensed water.
2
(
0.0 mmol) and freshly sublimed o-phenylenediamine
4.32 g, 40.0 mmol). The solution mixture was refluxed
The completion of the reactions took 2ꢀ3 h. The bluish-
/
for 24 h, followed by immediate filtration. As the filtrate
cooled using ice-bath, large light-green crystals formed.
These are collected by vacuum filtration, washed several
times with cold water, dried in vacuo, and identified as
the dihydrochloride (4), decompose at 2468 C.
purple solid product was formed, which was finely
ground, and either sublimed at 160 8C or crystallized
as above (yield 60%).
The free base ligand (3) was obtained by treatment of
the 4 with excess aqueous 10% ammonia solution. The
dark brown oily residue, which separated are dissolved
5.1.1.3. 1,5-bis(2-Benzimidazyl)-3-thiapentane (5). A
mixture of 2,2?-thiodiethanoic acid (3.0 g, 20.0 mmol)
and freshly sublimed o-phenylenediamine (3.64 g, 40.0
Fig. 3. Syntheses of ligands nꢂ
/
1 (3) and nꢂ2 (5), neutralised with dilute base.
/
Fig. 4. Syntheses of ligands nꢂ
/
1 (3) and nꢂ2 (5), fused at high temperature.
/

Table I
MICs of compounds 1ꢀ
/3 for 115 bacteria and 30 yeasts and their rates of susceptibility to five compared antibiotics
ꢁ
1
a
ꢁ1
b
A
B
MIC (mg ml
)
Isolates % for MICsB
/
64 mg ml
% of isolates susceptible to
No Compound 1
Compound 2
Compound 3
1
2
3
AMP CIP CFZ OFL PIP AM FLU FL
5
0%
90%
Range
32ꢀ256
50%
90%
Range
32ꢀ128
50%
90%
Range
A
B
C
D
E
F
G
H
I
15 32
15
15 16
10
64
16
16
8
/
32
8
128
16
/
64
16
8
256
32
16
8
64ꢀ/256
85
96
88
100
1000
12
7
67
60
97
6
70
93
50
100
88
60 Nd Nd
67 Nd Nd
Nd Nd Nd
Nd Nd Nd
100 Nd Nd
96 Nd Nd
100 Nd Nd
100 Nd Nd
100 Nd Nd
100 Nd Nd
100 100 100
Nd 100 100
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
100
100
92
8
8ꢀ
8ꢀ
4ꢀ
/
16
16
8
8ꢀ
8ꢀ
4ꢀ
/
32
32
8
16ꢀ/32
100
100
100
100
96
100
100
100
100
96
/
8
16
/
8ꢀ/16
100
100
100
16
100
4
/
8
8
64
/
8
4ꢀ/8
96 100
88
80
10 16
10 32
10 64
32
128
128
16ꢀ
32ꢀ
64ꢀ
/
32
16
64
64
16ꢀ
64ꢀ
64ꢀ
/
64
32
16
64
64
64
128
32ꢀ
16ꢀ
64ꢀ
/
64
100
100
96
80
100
0
4
/128
128
128
/128
/64
92
97
/128
/256
/128
96
0
96
0
100
100
100
100
Nd
Nd
Nd
100 100
10
10
10
10
10
10
ꢀ
/
256
256
256
ꢀ
/
256 256ꢀ
256 256ꢀ
256 256ꢀ
/
ꢀ
/
256
256
256
ꢀ
/
256
256
256
ꢀ
/
256 256ꢀ
256 256ꢀ
256 256ꢀ
/
ꢀ
/
256
256
256
ꢀ
/
256
256
256
ꢀ
/
256 256ꢀ
256 256ꢀ
256 256ꢀ
/
ꢀ
/
256
256
256
0
96
96
87
85
85
0
92
94
ꢀ
/
ꢀ
/
/
ꢀ
/
ꢀ
/
ꢀ
/
/
ꢀ
/
ꢀ
/
ꢀ
/
/
ꢀ
/
0
0
0
J
ꢀ
/
ꢀ
/
/
ꢀ/
ꢀ
/
ꢀ
/
/
ꢀ/
ꢀ
/
ꢀ
/
/
ꢀ/
0
0
0
83
K
L
M
4
4
8
8
8
8
4ꢀ
4ꢀ
4ꢀ
/
8
8
8
4
8
4
8
8
8
4ꢀ
4ꢀ
4ꢀ
/
8
8
8
4
4
2
8
8
4
4ꢀ/8
100
100
100
Nd
100
100
Nd
100
100
Nd Nd
Nd Nd
Nd Nd
100
Nd
Nd
/
/
4ꢀ/8
/
/
0.125ꢀ
/
4
Nd
98 100
A: microorganisms; B: number of isolates. (A) Staphylococcus aureus, (B) Escherichia coli, (C) Enterobacter aerogenes, (D) Klebsiella pneumoniae, (E) Alcaligenes faecalis, (F) Citrobacter freundii,
G) Streptococcus pyogenes, (H) Proteus vulgaris, (I) Proteus mirabilis, (J) Enterococcusfaecalis, (K) Candida albicans, (L) Candida utilis, (M) Cryptococcus neoformans.
(
a
5
0% and 90%, MICs at which 50 and 90% of the isolates are inhibited, respectively.
ꢁ1
b
ꢁ1
1.0 mg ml ); CFZ, cefazolin (B
8.0 mg mlꢁ1); PIP,
ꢁ1
Susceptibility breakpoints are in parentheses. OFL, ofloxacin (B
/
2.0 mg ml ); AMP, ampicillin (B
/
8.0 mg ml ); CIP, ciprofloxacin (B
/
/
ꢁ
1
ꢁ1
ꢁ1
ꢁ1
piperacillin (B
/
16 mg ml ); AM, amphotericin-B (B
/
16 mg ml ); FLU, fluconazoie (B
/
8.0 mg ml ); FL, flucytosine (B16 mg ml ).
/

Table II
MICs of compounds 4ꢀ
/6 for 115 bacteria and 30 yeasts and their rates of susceptibility to compared antibiotics and fungi
ꢁ
1
)
a
ꢁ1
b
A
B
MIC (mg ml
Isolates % for MICsB
/
64 mg ml
% of isolates susceptible to
No Compound 4
Compound 5
Compound 6
1
2
3
AMP CIP CFZ OFL PIP AM FLU FL
5
0%
90%
Range
32ꢀ64
50%
90%
Range
64ꢀ128
50%
90%
Range
64ꢀ256
A
B
C
D
E
F
G
H
I
15 32
64
16
16
8
/
64
8
128
16
/
64
8
256
16
/
85
96
88
100
100
100
100
100
96
7
67
60
97
70
93 100
100
96 100
50
60 Nd Nd
67 Nd Nd
88 Nd Nd
88 Nd Nd
100 Nd Nd
96 Nd Nd
100 Nd Nd
100 Nd Nd
100 Nd Nd
100 Nd Nd
100 100 100
Nd 100 100
Nd 98 100
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
100
100
92
15
15
10
8
8
4
8ꢀ
8ꢀ
4ꢀ
/
16
16
8
8ꢀ
8ꢀ
4ꢀ
/
16
16
8
8ꢀ
8ꢀ
4ꢀ
/
32
16
8
100
100
100
100
96
100
100
100
100
96
/
8
16
8
/
8
16
8
/
0
12
100
100
80
6
/
8
/
8
/
10 32
10 32
10 64
54
64
128
32ꢀ
32ꢀ
32ꢀ
/
64
32
32
64
32
32ꢀ
32ꢀ
64ꢀ
/
64
32
64
64
64
32ꢀ
64ꢀ
32ꢀ
/
64
100
16
0
4
80
92
97
92
94
83
/64
128
128
/128
128
128
/128
100
/128
/256
/128
96
0
96
0
100
100
100
100
Nd
Nd
Nd
100 100
10
10
10
10
10
10
ꢀ
ꢀ
ꢀ
2
2
4
/
256
256
256
ꢀ
/
256 256ꢀ
256 256ꢀ
256 256ꢀ
/
ꢀ
/
256
256
256
ꢀ
ꢀ
ꢀ
4
8
8
/
256
256
256
ꢀ
ꢀ
ꢀ
8
8
8
/
256 256ꢀ
256 256ꢀ
256 256ꢀ
/
ꢀ
/
256
256
256
ꢀ
ꢀ
ꢀ
2
2
4
/
256
256
256
ꢀ
ꢀ
ꢀ
4
4
8
/
256 256ꢀ
256 256ꢀ
256 256ꢀ
/
ꢀ
/
256
256
256
0
96
96
87
85
85
0
/
ꢀ
/
/
ꢀ
/
/
/
/
ꢀ
/
/
/
/
ꢀ
/
0
0
0
J
/
ꢀ/
/
ꢀ
/
/
/
/
ꢀ
/
/
/
/
ꢀ
/
0
0
0
K
L
M
4
4
8
5/
0.125ꢀ
/4
4ꢀ
4ꢀ
4ꢀ
/
8
8
8
5/0.125ꢀ
/4
100
100
100
100
100
100
100
100
100
Nd Nd 100
2ꢀ/4
/
5/0.125ꢀ
/44ꢀ
/8
Nd Nd
Nd Nd
Nd
Nd
4ꢀ/8
/
4ꢀ/8
A: microorganisms; B: number of isolates. (A) Staphylococcus aureus, (B) Escherichia coli, (C) Enterobacter aerogenes, (D) Klebsiella pneumoniae, (E) Alcaligenes faecalis, (F) Citrobacter freundii,
G) Streptococcus pyogenes, (H) Proteus vulgaris, (I) Proteus mirabilis, (J) Enterococcusfaecalis, (K) Candida albicans, (L) Candida utilis, (M) Cryptococcus neoformans.
(
a
5
0% and 90%, MICs at which 50 and 90% of the isolates are inhibited, respectively.
ꢁ1
b
ꢁ1
1.0 mg ml ); CFZ, cefazolin (B
8.0 mg mlꢁ1); PIP,
ꢁ1
Susceptibility breakpoints are in parentheses. OFL, ofloxacin (B
/
2.0 mg ml ); AMP, ampicillin (B
/
8.0 mg ml ); CIP, ciprofloxacin (B
/
/
ꢁ
1
ꢁ1
ꢁ1
ꢁ1
piperacillin (B
/
16 mg ml ); AM, amphotericin-B (B
/
16 mg ml ); FLU, fluconazoie (B
/
8.0 mg ml ); FL, flucytosine (B16 mg ml ).
/

880
N.M. Agh-Atabay et al. / European Journal of Medicinal Chemistry 38 (2003) 875ꢀ881
/
mmol) in 5 M aqueous HCl (50 mL) was refluxed
overnight. After cooling to room temperature a light
grey product was collected by filtration and dried in
vacuo (4.6 g, 70%), m.p. 145 8C. Anal. Found: C, 63.6;
H, 5.4; N, 13.8; S, 7.9. Calc. for C H N C1 S: C, 54.7;
5.2. Pharmacology
.2.1. The experiments were carried out by preparing a
5
broth micro dilution, following the procedure outlined by
the National Committee for Clinical Laboratory
1
8
20
4
2
H, 5.1; N, 14.2; S, 8.1%. The free base ligand was
crystallised (3.2 g, 85%), m.p. 183ꢀ184 8C. Anal. Found:
C, 66.7; H, 5.9 N, 17.0; S, 9.4. Calc. for C H N S: C,
Standards*
A cation adjusted Muellerꢀ
for testing the susceptibility of microorganisms to
antimicrobial agents. For streptocci and enterococci,
the medium was supplemented with 5% lysed horse
/
NCCLS [18]
/
/
Hinton Broth was used
1
8
18
4
1
6
3
7.1; H, 5.6; N, 17.4; S, 9.9%. H-NMR (400 MHz): d
.03 (4H, t, CH ), 3.12 (4H, t, CH ), 7.12 (in, 4H), 7.48
2
2
1
3
1
(
in, 4H), (2H, NIH not resolved); C{ H}-NMR (400
ꢁ1
ꢁ1
blood, 5 mg ml
of yeast extract, and 15 mg ml
NAD. The final inoculation (inoculums) approximately
of
MHz): d 29.96 (2C, Cꢀ
/
CH ), d 30.05 (2C, Sꢀ
/
CH2),
2
1
15.34 (4C), 122.12 (4C), 139.55 (4C), 154.29 (2C); JR
5
ꢁ1
was 10 cfu ml . For 50 of the isolates, bactericidal
endpoints are determined, following the method recom-
mended by the NCCLS [19]. Susceptibilities of yeasts are
performed by the broth macro dilution testing method
using RPMI 1640 broth [20]. Tested concentrations of
the compounds are serial twofold dilutions ranging from
1
(
(
(
KBr pellets): nmax/cmꢁ includes 3055 (Cꢀ
/H), 2916
CH ), 1620 (CÄN), 1540 (CÄ
/
/
C), 1450, 1270 and 735
2
ꢁ
1
CÄ
/
C); Raman (solid): nmax/cm includes 3055 (Cꢀ
916 (CꢀH), 1590 (CÄC), 1540 (CÄC), 1450, 1270 and
70 (CÄC).
/H),
2
7
/
/
/
/
ꢁ
1
256 to 0.125 mg ml
.
5
.1.1.4. 1,2-bis(2-Benzimidazyl)-1,2-ethanediol (1). A
mixture of DL-tartaric acid (1.50 g, 10 mmol) and freshly
sublimed o-phenylenediamine (2.16 g, 20.0 mmol) in 5
M aqueous HC1 (30 mL) was refluxed overnight. After
cooling to room temperature a light grey product was
collected by filtration and dried in vacuo. After neu-
tralisation with dilute alkali, the free base ligand was
References
[
1] M.P. Groziak, Z.-W. Huan, H. Ding, Z. Meng, W.C. Stevens,
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/
/
1
crystallised (1.95 g, 70%), m.p. 268 8C. H-NMR (400
(
/
MHz): aliphatic; d 5.33 (1H, d), 5.85 (1H, d), aromatic;
H
[
7
NH); deuteriated with D O, aliphatic; d 5.32 (1H, s),
.10ꢀ
/
7.15 (2H, m), 7.49ꢀ7.52 (2H, m); 12.24 (1H, br,
/
3
/
[5] W.B. Pratt, Chemotherapy of Infection, Oxford University Press,
New York, 1977.
[6] A.S. Demir, H. Hamamci, O. Sesenoglu, R. Neslihanoglu, B.
2
aromatic; 7.10ꢀ
/
7.15 (2H, m), 7.49ꢀ
C{ H}-NMR (100 MHz): dC 70.80 (CHOH), 114.83
2C), 138.75 (2C), 155.45 (1C); IR (KBr pellets): nmax/
/
7.52 (2H, m);
1
3
1
Asikoglu, D. Capanoglu, Tetrahedron Lett. 43 (2002) 6447ꢀ
/
6449.
C¸ akir, E. B u¨ y u¨ kbig o¨ l, U. U c¸ ucu, U. Abbaso oˆ lu, N. Noya-
nalpan, J. Fac. Pharm. Gazi. 71 (1988) 71ꢀ77.
[8] S. Akihama, K. Takahashi and N. Miyajama, Yakogaku Zasshi,
4, 247 (1974). CA 81: 9648.
9] F.J. Schendel, Y.S. Cheng, J.D. Otvos, S. Wehrli, J. Stubbe,
Biochemistry 27 (1988) 2614ꢀ2623.
10] S.E. Castilo-Blum, N. Barba-Behrens, Coordination Chem. Rev.
96 (2000) 3ꢀ30.
[11] P. Sing, S.P. Sing, Plant Physiol. 83 (1987) 12.
(
cm
[7] B. .
¨
ꢁ
1
includes 3431 (Nꢀ
C), 1270 and 735 (CÄ
includes 3060 (CꢀH), 2930 (Cꢀ
C), 1532 and 1271.
/
H), 3184 (CH), 1623 (CÄ
C); Raman (solid): nmax/
H), 1623 (CÄN),
/
N),
/
1
cm
540 (CÄ
/
/
ꢁ
1
9
/
/
/
[
1
594 (CÄ
/
/
[
1
/
5
(
.1.1.5. 1,4-bis(2-Benzimidazyl)-1,2,3,4-butanetetraol
2). A mixture of mucic acid (1.80 g, 10 mmnol) and
¨
[12] A. Tavman, B.
.
Ulk u¨ seven, N.M. Agh-Atabay, Transition Metal
328.
Chem. 25 (2000) 324ꢀ
/
freshly sublimed o-phenylenediamine (2.16 g, 20.0
mmol) in 5 M aqueous HC1 (30 mL) was refluxed
overnight. After neutralisation with dilute alkali, the
[
[
13] J. Sunberg, R.B. Martin, Chem. Rev. 74 (1974) 471.
14] M.A. Ali, A.H. Mirza, M. Nazimuddin, P.K. Dhar, R.J. Butcher,
Transition Metal Chem. 27 (2002) 27ꢀ
/
33.
free base ligand was crystallised (2.50 g, 75%), m.p.
[15] M.A. Philips, J. Chem. Soc. (1931) 1143.
1
[
16] A.W. Addison, P.J. Burke, Heterocyclic Chem. 18 (1981) 803ꢀ
05.
17] A.R. Mc Cutcheon, S.M. Ellis, R.E.W. Hancock, G.H.N.
Towers, J. Ethnopharmacol. 37 (1992) 213ꢀ223.
/
2
4
7
82 8C. H-NMR (400 MHz): aliphatic; d 4.11 (1H, s),
.755.85 (1H, s), 5.16 (1H, d), 5.44 (1H, d) aromatic;
H
8
[
.10ꢀ
/
7.13 (2H, m), 7.49ꢀ7.51 (2H, m); 12.07(1H, br,
/
/
NH); deuteriated with D O, aliphatic; dH 4.11 (1H, s),
2
[18] National Committee for Clinical Laboratory Standards, Methods
for dilution antimicrobial susceptibility tests for bacteria that
grow aerobically, 4th ed. Approved standard (M7-A4). National
Committee for Clinical Laboratory Standards, Wayne, PA, 1997.
5
.16 (1H, s), aromatic; 7.11ꢀ
/
7.13 (2H, m), 7.49ꢀ
/
7.51
2H, m); C{ H} (not soluble enough); IR (KBr
1
3
1
(
pellets): nmax/cm
ꢁ
1
includes 3438 (NꢀH), 3200br, 1615
/
[
19] National Committee for Clinical Laboratory Standards, Methods
for determining bactericidal activity of antimicrobial agents.
Proposed guideline (M26-P). National Committee for Clinical
Laboratory Standards, Villonova, PA, 1987.
(
CÄ
nmax/cm includes 3065 (Cꢀ
N), 1594 (CÄC) and 1278.
/
N), 1592 (CrC), 1276 and 745 (CrC); Raman (solid):
ꢁ
1
/
H), 2938 (Cꢀ
/
H), 1620 (CÄ
/
/

N.M. Agh-Atabay et al. / European Journal of Medicinal Chemistry 38 (2003) 875ꢀ
/881
881
[
20] National Committee for Clinical Laboratory Standards, Refer-
ence method for broth dilution antifungal susceptibility testing of
yeasts. Tentative standard (M27-T). National Committee for
Clinical Laboratory Standards, Wayne, PA, 1995.