Synthesis, characterization, crystal structure, and antimicrobial studies of 2-morpholinoethyl-substituted benzimidazolium salts and their silver(I)-N-heterocyclic carbene complexes

Article abstract of DOI:10.1007/s11164-017-2995-3

This article describes synthesis of N-morpholinoethylbenzimidazole (1), 2-morpholinoethyl-substituted benzimidazolium salts (NHC precursors, 2a–c), and their Ag(I) N-heterocyclic carbene (NHC) complexes (3a–c). All compounds were characterized by ¹H and ¹³C Nuclear Magnetic Resonance (NMR), Fourier Transform Infrared Spectroscopy (FTIR), and elemental analysis, and their antimicrobial effects examined. The molecular structure of the NHC precursor 2a¹ was established by single-crystal X-ray diffraction analysis. Minimum Inhibition Concentration (MIC) values were determined to evaluate their antimicrobial activity against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus bacterial strains and Candida albicans fungal species. All tested samples were compared with silver nitrate. All the compounds exhibited strong antimicrobial activity.

Full text of DOI:10.1007/s11164-017-2995-3

Res Chem Intermed
DOI 10.1007/s11164-017-2995-3
Synthesis, characterization, crystal structure,
and antimicrobial studies of 2-morpholinoethyl-
substituted benzimidazolium salts and their
silver(I)-N-heterocyclic carbene complexes
1
1
2
3
•
I s¸ ıl Yıldırım
•
Aydın Aktas¸
•
•
Duygu Barut Celepci
2
Muhittin Ayg u¨ n
•
Sevda Kırbag˘
1
1
T u¨ rkan Kutlu
Yetkin G o¨ k
•
Received: 14 April 2017 / Accepted: 28 May 2017
Ó Springer Science+Business Media Dordrecht 2017
Abstract This article describes synthesis of N-morpholinoethylbenzimidazole (1),
-morpholinoethyl-substituted benzimidazolium salts (NHC precursors, 2a–c), and
their Ag(I) N-heterocyclic carbene (NHC) complexes (3a–c). All compounds were
2
1
13
characterized by H and C Nuclear Magnetic Resonance (NMR), Fourier Trans-
form Infrared Spectroscopy (FTIR), and elemental analysis, and their antimicrobial
1
effects examined. The molecular structure of the NHC precursor 2a was estab-
lished by single-crystal X-ray diffraction analysis. Minimum Inhibition Concen-
tration (MIC) values were determined to evaluate their antimicrobial activity against
Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus bacterial
strains and Candida albicans fungal species. All tested samples were compared with
silver nitrate. All the compounds exhibited strong antimicrobial activity.
Keywords N-Heterocyclic carbene Á Ag–NHC complexes Á Morpholine Á
Antimicrobial activity Á Antibiotic
Electronic supplementary material The online version of this article (doi:10.1007/s11164-017-2995-3)
contains supplementary material, which is available to authorized users.
&
I s¸ ıl Yıldırım
isilyld@hotmail.com
1
2
3
Department of Chemistry, Faculty of Science, Inonu University, 44280 Malatya, Turkey
˙
Department of Physics, Faculty of Science, Dokuz Eyl u¨ l University, 35160 Izmir, Turkey
Department of Biology, Faculty of Science, Fırat University, 23200 Elazı g˘ , Turkey
123

I. Yıldırım et al.
Introduction
Many silver(I) complexes containing nitrogen, oxygen, phosphorus, sulfur, and N-
heterocyclic carbene (NHC) donor ligands have been evaluated as potential
antifungal and antibacterial agents against different Gram-positive and Gram-
negative bacteria. Many medicinal applications of Ag–NHC complexes are known,
especially showing activity as antimicrobials [1–3]. Aromatic heterocyclic ligands
(
NHC) containing nitrogen atoms have attracted attention for synthesis of
antimicrobial silver complexes [1–7]. NHC precursors are very important ligands
in bioorganic and coordination chemistry and have found application in the rapidly
developing field of metallosupramolecular chemistry [8, 9].
Although the antimicrobial activity of Ag compounds has been known since
ancient times, the antimicrobial effects of 2-morpholinoethyl-substituted Ag(I)NHC
compounds remain unknown. Therefore, we aimed to synthesize, characterize, and
determine the crystal structure of a new series of 2-morpholinoethyl-substituted
benzimidazole (1), benzimidazolium salts (2a–c), and their respective Ag(I)–NHC
(
3a–c) complexes. We also investigated their in vitro antimicrobial activity against
Gram-positive bacterium Staphylococcus aureus, Gram-negative bacterium Escher-
ichia coli, and Candida albicans fungal species.
Method
Reagents and instruments
The chemicals used in this work including 2-morpholinoethyl chloride, potassium
hydroxide, 1-bromopropane, 2-bromopropane, 3-bromo-1-propanol, diethyl ether,
dimethyl sulfoxide (DMSO), chloroform (CHCl ), dichloromethane (DCM),
3
dimethylformamide (DMF), silver oxide (Ag O), and argon gas as well as Schlenk
2
flask etc. were purchased from Sigma Aldrich, abcr, and Merck. All other reagents
were obtained commercially from Aldrich and used without further purification.
Melting points were determined in glass capillaries under air using an Electrother-
mal-9200 melting point apparatus. FTIR spectra were recorded from KBr pellets in
-
1
1
the range of 400–4000 cm on an AT, UNICAM 1000 spectrometer. Proton ( H)
1
and carbon ( C) NMR spectra were recorded using a Varian AS 300 Merkur
13
spectrometer operating at 300 MHz ( H) or 75 MHz ( C) in CDCl and DMSO-d
3
1
3
6
with tetramethylsilane as internal reference. The Inonu University Scientific and
Technology Center performed elemental analyses. Gram-positive bacterium
Staphylococcus aureus Cowan 1, Gram-negative bacterium Escherichia coli ATCC
2
5922, and Candida albicans FMC 17 fungal strains were used to evaluate
antimicrobial activity. The strains were obtained from Fırat University Faculty of
Science Department of Biology Microbiology Laboratory culture collections. As
medium, Merck mark, Mueller–Hinton broth medium was used. Studies were
performed in 96-well plates. The study was performed in Fırat University
Department of Biology Microbiology Laboratory.
1
23

Synthesis, characterization, crystal structure,…
Synthesis of N-2-morpholinoethylbenzimidazole (1) This known compound was
1
13
synthesized and characterized based on analysis of m.p., IR, H and C NMR, and
micro analyses, showing results consistent with literature [10].
Synthesis
of
2a) Compound 2a was synthesized from N-morpholinoethylbenzimidazole
1.16 g, 5 mmol) with 2-bromopropane (0.62 g, 5 mmol) in dry DMF (4 ml) using
1-isopropyl-3-(2-morpholinoethyl)benzimidazolium
bromide
(
(
the Schlenk technique. Then, it was refluxed for 24 h at 80 °C in oil bath. Upon
cooling to room temperature, dry ether was added to the reaction mixture. Then, the
solvents were evaporated under vacuum to afford the product as white solid. The
crude product was recrystallized from ethyl alcohol/diethyl ether (1:3) at room
-
1
temperature. Yield: 1.38 g (78%). M.p.: 276–278 °C. m(CN): 1558.5 cm . Anal.
Cald. for C H BrN O: C: 54.24; H: 6.78; N: 11.86. Found C: 54.21; H: 6.74; N:
1
6
24
3
1
1
2
4
1.90. H NMR (300 MHz, D O) in d ppm: 1.61 (d, 6H, –CH(CH ) , J: 9.0 Hz);
2
3 2
.89 (s, 4H, –NCH CH O–); 3.25 (t, 2H, –NCH CH –NC H O, J: 6.0 Hz); 3.75 (t,
H, –NCH CH O–, J: 4.5 Hz); 4.72 (t, 2H, –NCH CH –NC H O, J: 7.5 Hz); 4.96
2
2
2
2
4 8
2
2
2
2
4 8
1
3
(
(
(
(
m, 1H, –NCH(CH ) ); 7.62–7.91 (m, 4H, Ar–H), 9.40 (s, 1H, 2-CH). C NMR
3 2
300 MHz, D O) in d ppm: 21.0 (–CH(CH ) ); 42.3 (–NCH CH O–); 51.1
–NCH CH –NC H O); 52.3 (–NCH CH –NC H O); 55.1 (–NCH(CH ) ); 65.1
–NCH CH O–); 112.8, 113.8, 126.8, 127.1, 130.9, 131.2 (Ar–C); 139.4 (2-C).
2
3 2
2
2
2
2
4
8
2
2
4
8
3 2
2
2
Synthesis of 1-isopropyl-3-(2-morpholinoethyl)benzimidazolium
1
dibromide
2a ) 2-Bromopropane for synthesis of 2a compound was not commercially
(
available and was synthesized by reaction of hydrogen bromide with isopropyl
1
alcohol. 2a compound was synthesized by reaction of 2-bromopropane synthesized
by this method with N-(2-morpholinoethyl)benzimidazole. The spectroscopic
1
results for the synthesized compound 2a indicated presence of hydrogen bromide
in the structure together with the salt structure. Finally, when the single crystal was
obtained, the salt structure was completely uncovered. Compound 2a was then
resynthesized by reaction of 2-bromopropane with N-(2-morpholinoethyl)benzim-
idazole when 2-bromopropane was commercially available. Unfortunately, we
could not obtain appropriate single crystals of compound 2a for X-ray diffraction
analysis.
1
1
The H NMR spectrum of the resulting single crystals of 2a was found to be
1
different from that of the first compound: H NMR (300 MHz, CDCl -d ) in d ppm:
3
6
2
NC H O); 3.65 (s, 4H, –NCH CH O–); 4.41 (s, 2H, –NCH CH –NC H O); 4.84 (s,
.55 (m, 6H, –CH(CH ) ); 2.89 (s, 4H, –NCH CH O–); 3.15 (s, 2H, –NCH CH –
3 2 2 2 2 2
4
8
2
2
2
2
4 8
1
–
H, –NCH(CH ) ); 7.23–8.51 (m, 4H, Ar–H); 9.18 (s, 1H, 2-CH); 13.30 (s, 1H,
3 2
N–H–Br).
Synthesis of 1-(2-morpholinoethyl)-3-propylbenzimidazolium bromide (2b) Com-
pound 2b was synthesized in the same way as described for 2a, but using
1
-bromopropane (0.62 g, 5 mmol) instead of 2-bromopropane. Yield: 1.66 g
-
1
(
94 %). M.p.: 158–160 °C. m(CN): 1557.5 cm . Anal. Cald. for C H BrN O: C:
16
24
3
1
4.24; H: 6.78; N: 11.86. Found: C: 54.38; H: 6.91; N: 11.94. H NMR (300 MHz,
5
CDCl ) in d ppm: 0.99 (t, 3H, –CH CH CH , J: 7.5 Hz); 2.06 (m, 2H,
3
2
2
3
–
CH CH CH ); 2.57 (s, 4H, –NCH CH O–); 2.91 (t, 2H, –NCH CH –NC H O,
2 2 3 2 2 2 2 4 8
123

I. Yıldırım et al.
J: 6.0 Hz); 3.58 (s, 4H, –NCH CH O–); 4.53 (t, 2H, –CH CH CH , J: 7.5 Hz); 4.78
2
2
2
2
3
(
t, 2H, –NCH CH –NC H O, J: 4.5 Hz); 7.59–7.80 (m, 4H, Ar–H); 11.10 (s, 1H,
2 2 4 8
1
3
2
-CH). C NMR (300 MHz, CDCl ) in d ppm: 11.0 (–CH CH CH ); 22.7
3
2
2
3
(
NC H O); 56.2 (–NCH CH –NC H O); 66.8 (–NCH CH O–); 113.1, 113.2, 127.1,
–CH CH CH ); 44.1 (–NCH CH O–); 48.9 (–CH CH CH ); 53.4 (–NCH CH –
2 2 3 2 2 2 2 3 2 2
4
8
2
2
4
8
2
2
1
27.2, 131.1, 131.3 (Ar–C); 143.3 (2-C).
Synthesis of 1-(3-hydroxypropyl)-3-(2-morpholinoethyl)benzimidazolium bromide
(
2c) Compound 2c was synthesized in the same way as described for 2a, but using
-bromo-1-propanol (0.7 g, 5 mmol) instead of 2-bromopropane. Yield: 1.40 g
3
-
1
(
75 %). M.p.: 238–240 °C. m(CN): 1562.8 cm . Anal. Cald. for C H BrN O : C:
16
24
3 2
1
1.90; H: 6.53; N: 11.35. Found: C: 51.94; H: 6.45; N: 11.40. H NMR (300 MHz,
5
CDCl ) in d ppm: 2.13 (s, 2H, –CH CH CH OH); 2.58 (s, 4H, –NCH CH O–);
3
2
2
2
2
2
2
2
–
.94 (s, 2H, –NCH CH –NC H O); 3.61 (s, 2H, –NCH CH –NC H O); 3.61 (s,
H, –NCH CH CH OH); 3.61 (s, 2H, –NCH CH CH OH); 4.74 (s, 4H,
2 2 4 8 2 2 4 8
2
2
2
2
2
2
NCH CH O–); 5.20 (s, 1H, –CH CH CH OH); 7.58–7.76 (m, 4H, Ar–H); 10.94
2
2
2
2
2
1
3
(
s, 1H, 2-CH). C NMR (300 MHz, CDCl ) in d ppm: 31.0 (–CH CH CH OH);
3
2
2
2
4
5
1
4.1 (–NCH CH –NC H O); 44.5 (–NCH CH O–); 53.4 (–NCH CH –NC H O);
6.3 (–NCH CH CH OH); 56.9 (–CH CH CH OH); 66.6 (–NCH CH O–); 113.1,
13.3, 127.1, 131.0, 131.4 (Ar–C); 144.0 (2-C).
2 2 4 8 2 2 2 2 4 8
2
2
2
2
2
2
2
2
Synthesis of bromo[1-isopropyl-3-(2-morpholinoethyl)benzimidazol-2-ylidene]sil-
ver(I) (3a) Solution of 1-isopropyl-3-(2-morpholinoethyl)benzimidazolium bro-
˚
mide (0.5 g, 1.4 mmol), Ag O (0.16 g, 0.7 mmol), and activated 4-A molecular
2
sieves in DCM (20 mL) was stirred at room temperature for 24 h in the dark. The
reaction mixture was filtered through Celite, and solvent removed under vacuum.
The crude product was recrystallized from dichloromethane/hexane at room
-
1
temperature. Yield: 0.59 g (91 %). M.p.: 188–190 °C. m(CN): 1478.1 cm . Anal.
Cald. for C H AgBrN O: C: 41.67; H: 5.03; N: 9.11. Found C: 41.75; H: 5.08; N:
1
6
23
3
1
9
9
6
6
.18. H NMR (300 MHz, DMSO-d ) in d ppm: 1.67 (d, 6H, –CH(CH ) , J:
6
3 2
.0 Hz); 2.43 (m, 4H, –NCH CH O–); 2.73 (t, 2H, –NCH CH –NC H O, J:
.0 Hz); 3.55 (m, 4H, –NCH CH O–); 4.55 (t, 2H, –NCH CH –NC H O, J:
2
2
2
2
4 8
2
2
2
2
4 8
1
.0 Hz); 5.09 (m, 1H, –NCH(CH ) ); 7.41–7.93 (m, 4H, Ar–H). C NMR
3
3
2
(
(
(
300 MHz, DMSO-d ) in d ppm: 22.5 (–CH(CH ) ); 46.5 (–NCH CH O–); 53.5
6
3 2
2
2
–NCH CH –NC H O); 53.6 (–NCH(CH ) ); 57.7 (–NCH CH –NC H O); 66.1
–NCH CH O–); 112.2, 112.6, 123.7, 124.0, 132.2, 133.5 (Ar–C); 188.5 (2-C).
2
2
4
8
3 2
2
2
4 8
2
2
Synthesis of bromo-[1-(2-morpholinoethyl)-3-propylbenzimidazol-2-ylidene] sil-
ver(I) (3b) Compound 3b was synthesized in the same way as described for 3a,
but using 1-(2-morpholinoethyl)-3-propylbenzimidazolium bromide (0.5 g,
1
.4 mmol) instead of 1-isopropyl-3-(2-morpholinoethyl)benzimidazolium bromide.
Yield: 0.49 g (75 %). M.p.: 259–261 °C. Anal. Cald. for C H AgBrN O: C:
1
6
23
3
-
1 1
4
1.67; H: 5.03; N: 9.11. Found: C: 41.73; H: 5.06; N: 9.16. m(CN): 1452.1 cm . H
NMR (300 MHz, DMSO-d ) in d ppm: 0.89 (t, 3H, –CH CH CH , J: 6.0 Hz); 1.89
6
2
2
3
(
NC H O, J: 6.0 Hz); 3.53 (t, 4H, –NCH CH O–, J: 4.5 Hz); 4.46 (t, 2H,
m, 2H, –CH CH CH ); 2.44 (s, 4H, –NCH CH O–); 2.75 (t, 2H, –NCH CH –
2 2 3 2 2 2 2
4
8
2
2
–
CH CH CH , J: 6.0 Hz); 4.58 (t, 2H, –NCH CH –NC H O, J: 6.0 Hz);
2 2 3 2 2 4 8
1
23

Synthesis, characterization, crystal structure,…
1
3
7
.42–7.83 (m, 4H, Ar–H). C NMR (300 MHz, DMSO-d ) in d ppm: 11.1
6
(
NC H O); 53.6 (–NCH CH O–); 57.8 (–NCH CH –NC H O); 66.1 (–NCH CH O–
–CH CH CH ); 23.2 (–CH CH CH ); 45.9 (–CH CH CH ); 49.9 (–NCH CH –
2 2 3 2 2 3 2 2 3 2 2
4
8
2
2
2
2
4
8
2
2
)
; 112.0, 112.1, 123.8, 133.1, 133.3 (Ar–C); 189.4 (2-C).
Synthesis of bromo-[1-(3-hydroxypropyl)-3-(2-morpholinoethyl)benzimidazol-2-yli-
dene] silver(I) (3c) Compound 3c was synthesized in the same way as described
for 3a, but using 1-(3-hydroxypropyl)-3-(2-morpholinoethyl)benzimidazolium bro-
mide (0.52 g, 1.4 mmol) instead of 1-isopropyl-3-(2-morpholinoethyl)benzimida-
zolium bromide. Yield: 0.52 g (78 %). M.p.: 214–216 °C. Anal. Cald. for
C H AgBrN O : C: 40.28; H: 4.86; N: 8.81. Found: C: 40.32; H: 4.89; N: 8.75.
1
6
23
3 2
-
1
1
m(CN): 1443.6 cm . H NMR (300 MHz, DMSO-d ) in d ppm: 2.00 (m, 2H,
6
–
J: 6.0 Hz); 3.42 (t, 2H, –NCH CH CH OH, J: 6.0 Hz); 3.53 (t, 4H, –NCH CH O–
CH CH CH OH); 2.44 (s, 4H, –NCH CH O–); 2.75 (t, 2H, –NCH CH –NC H O,
2 2 2 2 2 2 2 4 8
2
2
2
2
2
J: 6.0 Hz); 4.55 (m, 2H, –NCH CH CH OH); 4.75 (s, 1H, –NCH CH CH OH);
2
2
2
2
2
2
1
3
7
.43–7.84 (m, 4H, Ar–H). C NMR (300 MHz, DMSO-d ) in d ppm: 32.9
6
(
–CH CH CH OH); 44.9 (–NCH CH –NC H O); 45.7 (–NCH CH O–); 53.5, 57.1
2 2 2 2 2 4 8 2 2
(
–NCH CH –NC H O); 57.4 (–NCH CH CH OH); 57.8 (–NCH CH CH OH);
2 2 4 8 2 2 2 2 2 2
66.1 (–NCH CH O–); 112.0, 112.1, 113.6, 113.7, 123.8, 126.6, 133.1, 133.4 (Ar–
2 2
C); 188.5 (2-C).
Crystal structure determination
1
Single-crystal X-ray diffraction data for 2a were collected at room temperature on
a Rigaku-Oxford Xcalibur diffractometer with an Eos Charge Coupled Device
˚
(
CCD) detector using graphite-monochromated Mo K radiation (k = 0.71073 A)
a
Pro
with CrysAlis software [11]. Data reduction and analytical absorption correction
Pro
were performed by CrysAlis program [12]. Utilizing OLEX2 [13], the structure
was solved by direct methods with SHELXT [14] and refined by full-matrix least
2
squares on F in SHELXL [15]. Anisotropic thermal parameters were applied to all
˚
nonhydrogen atoms. Methyl H atoms (C–H = 0.96 A), methylene H atoms (C–
˚
˚
H = 0.97 A), and aromatic H atoms (C–H = 0.93 A) were positioned geometri-
cally. Hydrogen atoms attached to carbon of benzimidazole ring and nitrogen were
found in the difference map and refined freely.
1
Crystallographic details for 2a
3
C H Br N O, M = 435.21, colorless prismatic, 0:48 Â 0:26 Â 0:19 mm , orthorhom-
16
25
2 3
˚
bic, space group Pbca, a = 13.0956(9) A, b = 12.1176(5) A, c = 22.8729(13) A,
˚
˚
3
V = 3629.6(4) A , Z = 8, D
3
= 1.593 g/cm , F(000) = 1760, T = 294(2) K,
˚
calc
7
272 reflections collected, 3425 unique (Rint = 0.034). Final goodness-of-
fit = 1.001 R = 0.040, wR = 0.069, R indices based on 2346 reflections with
1
2
2
-1
I C 2r(I) (refinement on F ). 216 parameters, 0 restrains, l = 4.472 mm
.
123

I. Yıldırım et al.
Antimicrobial activity
Microorganism culture preparation and inoculation
Bacterial strains Staphylococcus aureus Cowan 1 and Escherichia coli ATCC
5922 were inoculated in nutrient broth and incubated at 35 ± 1 °C for 24 h. Yeast
Candida albicans FMC 17 was inoculated in yeast malt extract broth and incubated
2
at 25 ± 1 °C for 48 h. After density adjustment according to McFarland standard
(
0.5), growing cultures in media were inoculated at 1 % in culture broth then kept in
6
refrigerator at ?4 °C. The number of microorganisms in 1 ml was calculated as 10 /
ml for bacteria and 10 /ml for yeast.
4
Preparation of medium
Mueller–Hinton broth (5.5 g) was weighed and diluted to 250 ml, then the density
was adjusted according to McFarland standard (0.5).
Minimal inhibitory concentration (MIC) method
Each synthesized compound or standard drug was diluted to 2000 lg/ml concen-
tration as stock solution. DMSO solvent was used as diluent. Three separate 96-well
plates were used (one for each type of microorganism), to which 100 ll medium
was added. Then, 200 ll (i.e., 400 mg/ml) of stock solution was taken and added to
the medium in the first well by pipette. From the first well, 200 ll was extracted and
added to the second well, and so on to obtain dilution. Then, 100 ll culture was
added to each plate. The final concentrations obtained were therefore 66.66, 44.4,
29.6, 19.75, 13.15. 8.75, and 5.85 lg/ml. Plates were incubated at 37 °C for 24 h,
after which the microbial growth was observed for each each sample. The
concentration with greatest turbidity on each plate was taken as an indication of the
minimum inhibitory concentration. As positive control, AgNO was used.
3
Results and discussion
Synthesis
Reaction of benzimidazole with 2-morpholinoethyl chloride resulted in formation
1
of N-morpholinoethylbenzimidazole (1) (Scheme 1). The H NMR spectrum of 1
N
+
O
H
N
N K+
N
N
N
1
KOH
EtOH
EtOH
+
N
O
KCl
Cl
N
0
12 h.
reflux
1
h., 25 C
Scheme 1 Synthesis of N-morpholinoethylbenzimidazole (1)
1
23

Synthesis, characterization, crystal structure,…
N
O
N
O
N
N
N
DMF
Br-
+
R-Br
0
80 C, 24 h.
N
R
1
2
R:
,
OH
,
a
b
c
Scheme 2 Synthesis of 2-morpholinoethyl-substituted NHC precursors (2a–c)
in CDCl displayed the 2-CH proton at 7.97 ppm. The benzimidazolium salts 2a–
3
c were prepared by reaction of alkyl bromide (alkyl: iso-propyl, n-propyl, and
1
-hydroxypropyl)
with
N-morpholinoethylbenzimidazole,
respectively
Scheme 2). The reaction mixture was stirred in dimethylformamide overnight
(
at 80 °C. After evaporation of solvent under vacuum, the oily product was washed
with diethyl ether. The crude product was recrystallized from dichloromethane:di-
ethyl ether (1:2) at room temperature. The compounds were obtained as white
1
solid in very good yield (75–94 %). The H NMR spectra of 2a–c in CDCl
3
1
3
displayed the 2-CH protons at 9.40, 11.10, and 10.94 ppm, respectively. In the
C
NMR spectra, the 2-C signals appeared at 139.4, 143.3, and 143.9 ppm for 2a, b,
and c, respectively.
Ag(I)–NHCs 3a–c were prepared by reaction of Ag O with the benzimidazolium
2
salts 2a–c under stirring in dichloromethane for 24 h at room temperature in the
dark (Scheme 3). The reaction mixture was then filtered through Celite, and the
solvents were evaporated under vacuum to afford the product as white solid. The
crude product was recrystallized from dichloromethane:diethyl ether (1:3) at room
temperature. Absence of a downfield benzimidazolium proton (NCHN) signal and
an upfield shift for all the signals were observed with respect to the ligand
1
precursors in the H NMR spectra of complexes 3a–c in DMSO-d , indicating
6
1
successful complex formation. In the C NMR spectra of the silver complexes,
3
resonances due to carbene carbon (NCN) atoms appeared as singlet peaks at 185.9,
1
89.4, and 188.5 ppm, respectively. These signals were shifted more toward the
downfield region compared with the NHC precursors 2a–c. This finding further
demonstrates formation of the expected complexes. This signal shift is in
accordance with literature reports [16–19].
123

I. Yıldırım et al.
N
O
+
N
O
N
N
DCM
Br-
2
Ag O
2
2
AgBr
+
H O
2
0
N
R
N
R
2
5 C, 24 h.
2
3
R:
,
OH
,
a
b
c
Scheme 3 Synthesis of 2-morpholinoethyl-substituted Ag(I)NHC complexes (3a–c)
FTIR spectral studies
-
1
The IR spectrum for complex 2a shows peaks at 500–600 cm
-
for C–Br
for C–N bond, peak at around
1
stretching vibration, peak at 1200 cm
-
1
-1
1
400–1600 cm for aromatic structure, and peak at 1558.5 cm for benzimi-
dazole C–N. Cross peaks for C=C and C=N groups are observed at
1
600–1690 cm . The peak at 2800 cm corresponds to C–H, while the peak
-
-1
1
at around 3500–3700 cm
-
1
corresponds to NH stretching vibration. The IR
-
spectrum for 2b shows a peak at 600 cm for C–Br stretching vibration, and peak
1
-
at 1200 cm for C–N bond. The peak observed at around 1300 cm corresponds
1
-1
-
1
to C–H bending vibration, while the aromatic structure peak is seen at 1600 cm
-
,
and the benzimidazole C–N peak at 1557.5 cm . –N=C=N stretching vibration is
1
-
seen at 2800 cm . The peak at 2800 cm corresponds to C–H. The peak at
1
-1
-
1
around 3500–3700 cm
spectrum for 2c shows a peak for C–Br stretching vibration at 500–600 cm
corresponds to N–H stretching vibration. The IR
-
1
,
peak at 1200 cm for C–N bond, peak at around 1400–1600 cm for aromatic
-
1
-1
-
structure, and benzimidazole C–N peaks at around 1562.8 cm . It also shows
1
-
peaks between 1600 and 1690 cm for C=C and C=N groups. The peak at
1
-
800 cm corresponds to C–H bonds, and the peak at 3500 cm corresponds to
1
-1
2
-
1
N–H stretching vibration. The O–H stretching vibration peak at 3600 cm
corresponds to propanol group.
1
23

Synthesis, characterization, crystal structure,…
The IR spectrum for complex 3a shows C–Br stretching vibration peak between
-
00 and 600 cm , signals in the silver region of 720–850 cm , peak at
1
-1
5
-
200 cm for C–N bond, peak at around 1400–1600 cm for aromatic structure,
1
-1
1
and benzimidazole C–N peaks at 1478.1 cm . The peak at 2800 cm corresponds
-
1
-1
-
to C–H, while the peak at around 3500–3700 cm corresponds to N–H stretching
1
-
vibration. The IR spectrum for complex 3b shows a peak at 500–600 cm for C–Br
1
-
stretching vibration, signals at 720–850 cm in the Ag region, and a peak at
1
-
200 cm for C–N bond. The peak at around 1300 cm corresponds to C–H
1
-1
1
bending vibration, that at 1600 cm to aromatic structure, and that at 1452.1 cm
-
1
-1
-
to benzimidazole C–N. The peaks at around 2800 cm correspond to C–H bond.
1
-
The peaks in the vicinity of 3500 cm correspond to N–H stretching vibration. The
1
-
IR spectrum for complex 3c shows peak at 500–600 cm for C–Br stretching
1
-
1
-1
vibration, signals in the Ag region at 720–850 cm , and a peak at 1200 cm for
-
1
C–N bond, while the peaks at 1400–1600 cm
structure. Benzimidazole C–N peaks are seen at 1443.6 cm . The peaks observed
1
at 2800 cm correspond to C–H bond. The peaks in the vicinity of 3500 cm
correspond to the aromatic
-
1
-
-1
-
1
correspond to N–H stretching vibration. The peak at 3600 cm corresponds to O–
H stretching vibration.
Structural studies
1
Crystals suitable for determination of the molecular structure of 2a by X-ray
crystallography were obtained by slow diffusion of diethyl ether into saturated
1
dichloromethane solution. The ORTEP [20] diagram for 2a is depicted in Fig. 1a,
and selected geometric parameters are presented in Table 1. The benzimidazole
˚
ring is almost planar with maximum deviation of 0.010(2) A for atom C2, and links
to the morpholine ring via –CH –CH – bridge, in which the N2–C11–C12–N1 unit
2
2
adopts a gauche conformation with torsion angle of 78.2(4)°. Also, the morpholine
˚
ring has chair conformation with puckering amplitude q = 0.019(2) A,
2
u = 356.7(2)° [21]. Bond distances in the morpholine ring are comparable to
2
1
literature [22–24]. The crystal structure of 2a is stabilized by intermolecular N–
HÁÁÁBr and C–HÁÁÁBr hydrogen bonds (Table 2, Fig. 1b). A strong hydrogen bond is
observed between the acidic proton of the imidazolium cation and the bromide
anion (Br1): The N–HÁÁÁBr ionic bond angle is 170° with N–H and HÁÁÁBr distance
˚
of 0.97 [25, 26] and 2.29 A, respectively. Br2 connects the molecules as an
acceptor to form an infinite One-Dimensional (1D) zigzag chain structure along a-
axis.
123

I. Yıldırım et al.
1
Fig. 1 a ORTEP view of 2a with 30 % probability level ellipsoids and b packing view (b-axis
projection) of the compound
1
23

Synthesis, characterization, crystal structure,…
Table 1 Selected bond lengths
1
N1–C12
1.513(5)
1.498(5)
1.400(4)
1.321(4)
1.489(4)
1.413(5)
125.1(3)
126.3(3)
109.1(3)
109.1(3)
N1–C13
N2–C1
1.497(5)
1.340(4)
1.468(4)
1.401(4)
1.411(5)
(
A˚ ) and angles (°) for 2a
N1–C16
N2–C2
N2–C11
N3–C7
N3–C1
N3–C8
O1–C14
O1–C15
C1–N2–C11
C2–N2–C11
C13–N1–C12
C16–N1–C13
C1–N2–C2
C1–N3–C8
C16–N1–C12
108.1(3)
126.9(3)
113.5(3)
Table 2 Hydrogen-bonding
1
D–HÁÁÁA
D–H
HÁÁÁA
DÁÁÁA
D–HÁÁÁA
˚
geometry (A, °) for 2a
N1–H1ÁÁÁBr1
0.97
0.93
0.98
0.97
0.97
2.29
2.66
2.84
2.88
2.92
3.2515(2)
3.4774(2)
3.7324(3)
3.7688(3)
3.8847(3)
170
147
151
153
177
C1–H1AÁÁÁBr2
i
C8–H8ÁÁÁBr2
C11–H11BÁÁÁBr2
C16–H16BÁÁÁBr2
Symmetry code (i): -1/2 ? x,
1/2 - y, -z
Antimicrobial activity
A study published in 2015 described synthesis of benzimidazole-based Ag(I)–NHC
complexes and investigation of their antibacterial activity against Gram-positive
bacterium Staphylococcus aureus (ATCC 12600) and Gram-negative bacterium
Escherichia coli (ATCC 25922). It was concluded that these complexes inhibited
both bacteria noticeably [27].
In their studies, Kalinowska-Lis et al. determined the antimicrobial activity of
derivatives of benzimidazole Ag(I)–NHC complexes against three Gram-negative
pathogens: Pseudomonas aeruginosa ATCC 15442, Escherichia coli ATCC 25922,
and Proteus hauseri ATCC 1331 using the MIC method. They concluded that
complex 5 was the most effective against E. coli with values of 21 lM, while
complex 1 was least effective against E. coli with values of 145 lM. Moreover,
complexes 2, 3, 4, and 5 were very effective when compared with AgNO and Silver
3
Sulfadiazine (AgSD). In addition, complex 2 was most effective against P. hauseri
with values of 41 lM. Complex 5 exhibited the highest antibacterial activity against
P. aeruginosa with values of 21 lM. Complexes 2, 4, and 5 were more active
against P. aeruginosa when compared with AgNO3 and AgSD as reference
compounds [28].
A publication by Sarı and coworker reported the antimicrobial activity of
novel benzimidazolium-based Ag(I)–NHC complexes. The results showed that,
while complex 3 was especially effective against E. coli, P. aeruginosa,
S. aureus, and Enterococcus faecalis with values of 50 lg/ml, it was most
123

I. Yıldırım et al.
effective against C. albicans and C. tropicalis with values of 25 lg/mL. Also, all
complexes showed some influence at 25 lg/mL. Moreover, complex 6 had the
same effect against E. coli, P. aeruginosa, C. albicans, and C. tropicalis with
values of 50 lg/ml. In comparison with antibiotic controls, all their complexes
exhibited antibacterial and antimicrobial activity, but complexes 3 and 6 were
more effective than the others. Compound 3 was more effective against the yeast
strains C. albicans and C. tropicalis than against the bacterial strains, while
complex 5 exhibited same activity across all microorganisms with values of
1
00 lg/ml. Complexes 2 and 4 were the least effective against all microorgan-
isms, with values of 200 lg/ml [29].
A study carried out in 2016 reported the antibacterial activity of derivatives of
benzimidazole Ag(I)–NHC complexes (7–12). The results showed that these
complexes exhibited low antibacterial activity (against Gram-negative and Gram-
positive bacteria) compared with ampicillin antibiotic used as standard. Gram-
negative bacterium (E. coli) showed increased sensitivity at high concentration. In
particular, complexes 10 and 11 showed the highest activity with zone of
inhibition of 11 mm. All the complexes had the same effect on E. coli at 3 ll
concentration (zone of inhibition of 6 mm), while complex 9 showed the highest
antimicrobial activity against Gram-positive bacterium (S. aureus) with zone of
inhibition of 9 mm. Moreover, the MIC results showed that complex 9 exhibited
high activity against E. coli and S. aureus with MIC level of 25 lg/ml. While
complex 7 showed an effect at 25 lg/ml against E. coli, the value against
S. aureus was 12.5 lg/ml. Complexes 10–12 exhibited the same effect (25 lg/ml
MIC) against Gram-positive bacteria as well as against Gram-negative bacterial
species (12.5 lg/ml MIC) [30].
In a previous work, the antimicrobial activity of derivatives of 5-fluoro-2-
substituted phenyl)-6-morpholino-1H-benzimidazole compounds (6–15) was inves-
(
tigated by diffusion method against Bacillus subtilis, S. aureus, E. coli, and
C. albicans. In particular; 5-fluoro-2-(4-fluoro phenyl)-6-morpholino-1H-benzimi-
dazole (compound 7) showed antifungal activity against C. albicans species with
inhibition zone diameter of 17 mm, comparable to the standard fluconazole
(18 mm). Additionally, 5-fluoro-2-(4-chlorophenyl)-6-morpholino-1H-benzimida-
zole (compound 11) and 5-fluoro-2-(3,4-dimethoxyphenyl)-6-morpholino-1H-ben-
zimidazole (12) exhibited antibacterial activity with inhibition zone diameter of
1
0 mm and 12 mm, respectively, against B. subtilis [31].
In our study, the antimicrobial activity of the synthesized compounds (1, 2a–c,
a–c) was estimated against one Gram-positive bacterial strain (S. aureus), one
3
Gram-negative bacterial strain (E. coli), and one yeast (C. albicans) by
evaluating the minimum inhibitory concentration (MIC, lg/ml) using an agar
dilution procedure. All the synthesized compounds (1, 2a–c, 3a–c) exhibited
antimicrobial activity against the fungus and bacteria when tested at concentra-
tions of 66.6–5.85 lg/ml; the results are presented in Table 3. The tested
complexes were found to be effective in inhibiting the growth of bacteria with
1
23

Synthesis, characterization, crystal structure,…
MIC values between 44.4 and 5.85 lg/ml. The starting compound N-morpholi-
noethylbenzimidazole (1) showed effective activity with MIC ranging from 13.15
to 19.75 lg/mL against the Gram-negative bacterial strain (E. coli), Gram-
positive bacterial strain (S. aureus), and fungus (C. albicans). The 2-morpholi-
noethyl-substituted benzimidazolium salts (2a–c) showed effective activity with
MIC ranging from 29.6 to 5.85 lg/ml against the Gram-negative bacterial strain
(
E. coli), Gram-positive bacterial strain (S. aureus), and fungus (C. albicans).
Generally, the benzimidazolium salt 2c was more effective than the benzimi-
dazolium salts 2a, b, which can be attributed to presence of hydroxypropyl group
on the N-atom.
The 2-morpholinoethyl-substituted Ag(I)–NHC complexes (3a–c) showed
-
1
effective activity with MIC ranging from 44.4 to 5.85 lg ml against the tested
bacterial strains and fungus. The silver carbene complexes 3a, c showed greater
antibacterial activity than the other complex 3b. Among all the synthesized
compounds, 2c and 3c were most effective against the Gram-positive bacterium
(
(
S. aureus) and Gram-negative bacterium (E. coli). All the synthesized compounds
1, 2a–c, 3a–c) exhibited antifungal activity with MIC ranging from 44.4 and
5
.85 lg/ml. Compounds 1, 2a, b, 3a exhibited the same activity against the
fungus. Also, compounds 2c, 3c exhibited the same activity against the fungus.
Synthesized compounds 1, 2b, 3a, b showed high activity against the fungus
C. albicans. The results of this study suggest that the alkyl groups, which contain
bulky (isopropyl), electron-donating (propyl), and H-bonding (hydroxypropyl)
substituents on the N-atom, may play an important role in the antimicrobial
activity.
We have determined the antimicrobial activity of these new complexes, but the
underlying mechanism and characteristics remain unclear. According to previous
literature, it can be suggested that, as an antibacterial component, silver ions can
inhibit cell division or cause structural abnormalities by damaging the cell
membrane or harming cellular contents of the bacteria such as the cytoplasmic
membrane or cytoplasmic contents. In addition, silver ions can interact especially
with DNA [32, 33].
Table 3 Antimicrobial activity results (lg/ml) for 1, NHC salts (2a–c), and Ag(I)-NHC complexes (3a–
c)
1
2a
2b
5.85
2c
3a
3b
3c
AgNO
3
Escherichia coli ATCC 25922
Staphylococcus aureus Cowan 1 13.15 13.15 13.15
Candida albicans FMC 17 19.75 19.75 19.75 29.6
19.75 19.75
5.85 29.6
44.4
5.85 44.4
5.85 44.4
19.75 44.4 29.6 44.4
5.85 19.75 29.6
123

I. Yıldırım et al.
Conclusions
The compounds synthesized in this work showed antimicrobial activity, having
potential as antibiotics for treatment of bacterial infections. In the next phase of this
work, we will investigate their anticancer effect. Further development of the results
presented herein as well as future work by the authors and other researchers will
provide basic information for scientific research.
Acknowledgements The authors are grateful for financial support provided by Inonu University,
Research Project Unit (project no. I.U.A.F:2011/25), and support for NMR and elemental analyses
provided by Inonu University Central Research Laboratory. The authors acknowledge Dokuz Eyl u¨ l
University for use of the Agilent Xcalibur Eos diffractometer (purchased under university research grant
1
no. 2010.KB.FEN.13). CCDC 1499376 contains the supplementary crystallographic data for 2a . Copies
of the data can be obtained free of charge at http://www.ccdc.cam.ac.uk/conts/retrieving.html or from the
Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge, CB2 1EZ, UK. fax: (?44)
1223-336-033, email: deposit@ccdc.cam.ac.uk.
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