Synthesis, Characterization and Antimicrobial properties of Methylbenzyl and Nitrobenzyl containing Imidazolium-based Silver N-Heterocyclic Carbenes

Article abstract of DOI:10.1016/j.molliq.2017.03.021

A series of imidazolium-based novel ionic liquids (ILs) containing methylbenzyl and nitrobenzyl substituents were prepared and further converted to neutral as well as ionic silver complexes. The ILs and their Ag-NHCs (N-Heterocyclic Carbenes) were success

Full text of DOI:10.1016/j.molliq.2017.03.021

Accepted Manuscript
Synthesis, characterization and antimicrobial properties of
methylbenzyl and nitrobenzyl containing imidazolium-based
silver N-heterocyclic carbenes
Sainath Aher, Anamika Das, Prashant Muskawar, Jabez Osborne,
Pundlik Bhagat
PII:
DOI:
Reference:
S0167-7322(17)30406-3
doi: 10.1016/j.molliq.2017.03.021
MOLLIQ 7056
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
29 January 2017
4 March 2017
6 March 2017
Please cite this article as: Sainath Aher, Anamika Das, Prashant Muskawar, Jabez Osborne,
Pundlik Bhagat , Synthesis, characterization and antimicrobial properties of methylbenzyl
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ACCEPTED MANUSCRIPT
Synthesis, Characterization and Antimicrobial Properties of Methylbenzyl and
Nitrobenzyl Containing Imidazolium-Based Silver N-Heterocyclic Carbenes
Sainath Aher a, Anamika Das b, Prashant Muskawar c, Jabez Osborne b, Pundlik Bhagat a,*
a Department of Chemistry, School of Advanced Sciences, VIT University,
Vellore, Tamilnadu, India- 632014
E-mail: drprbhagat111@gmail.com
b Department of Biosciences, School of Biosciences and Biotechnology,
VIT University, Vellore, Tamilnadu, India- 632014
c Department of Chemistry, Amlokchand Mahavidyalaya, Yavatmal,
Sant Gadge Baba Amravati University, Amravati- 444602, India
Abstract
A series of imidazolium-based novel ionic liquids (ILs) containing methylbenzyl and nitrobenzyl
substituents were prepared and further converted to neutral as well as ionic silver complexes.
The ILs and their Ag-NHCs (N-Heterocyclic Carbenes) were successfully characterized by
NMR, FT-IR, and EI-MS studies. The ILs and Ag-NHCs were screened for antimicrobial
activity based on the zone of inhibition, Minimum Inhibitory Concentration (MIC)
determination, and stability studies against Salmonella enterica and Staphylococcus aureus. The
neutral complex NS-32 ((1-((2'-cyano-[1,1'-biphenyl]-4-yl)methyl)-3-(4-nitrobenzyl)-2,3-
dihydroimidazol-2-yl)silver bromide) was the most active with MIC of 25 µM and showed
efficient growth inhibition of bacterial pathogens even up to 21 d. The complex NS-32 was
further monitored for cell wall disruption by scanning electron microscopy (SEM). The
photomicrographs of bacterial cells obtained by SEM after treatment of NS-32 with S. enterica
and S. aureus showed rupture of a cell wall, thus proving it to be the site of action and ultimately
having inhibitory effects on the microbial cell growth.
Keywords
Antimicrobial, Cell wall distortion, Minimum inhibitory concentration, N-heterocyclic carbenes,
Silver
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1. Introduction
Silver based antimicrobials are of much focus in recent years due to the appearance of
resistant bacteria where these complexes have exhibited sufficient activity against a wide variety
of bacterial strains [1,2]. The Silver Sulfadiazine (SSD) is widely used for the treatment of third-
degree burns against various pathogens. SSD releases Ag+ ions when applied as Silvadene (1%
cream) at the wound site, wherein sulfonamide dye and silver ions act synergistically by
possessing bactericidal properties [3,4]. Ag+ ions have a high potency of interactions with cell
membrane functionalities such as amino acids, -SH groups, and can interact with enzymes
essential for cell wall synthesis with additional effects on nuclear materials, cell respiration, and
metabolism [5,6].
Ag-NHCs resembles with SSD’s mechanism of Ag+ ions release, which holds enormous
potential for Ag-NHC based antimicrobials preventing the infection and promotes wound healing
by a slow and sustained release of Ag+ ions at the wound site. The main objective is to design
potent Ag-NHCs to accomplish slow dissociation of the complex with sustained release of Ag+
ions at the wound site which is influenced by the strength of Ag-NHC bond affected by
functionalities present on NHCs [7–10]. This is well supported by the in vitro as well as in vivo
antimicrobial studies of NHC-Ag-OAc (NHC-silver acetate) complex SBC3 against MRSA
(Methicillin Resistant S. aureus) where the complex SBC3 displayed MIC of 12.5 µg/mL [11–
13].
We report the imidazolium-based novel ILs and their silver complexes in neutral as well
as ionic form and further evaluated for antibacterial activity against S. aureus, S. enterica with
MIC determination. Additionally, the stability of these compounds was studied up to 21 d with
effects of NS-32 on the bacterial cell wall by SEM analysis.
2. Experimental Section
2.1. Chemistry
2.1.1. Synthesis of Ionic Liquids
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Scheme 1 Synthesis of ligands i.e. Ionic Liquids (ILs)
IL-31: Imidazole (680 mg, 10 mmol), K2CO3 (1.38 gm, 10 mmol) and 4-methyl benzyl bromide
(5.5 gm, 30 mmol) were refluxed in ACN for 8-10 h. Acetonitrile was removed under vacuum
and the residue was suspended in DCM followed by stirring at RT, filtered over whatman paper.
The solid sticky product obtained after removal of DCM was resuspended in DEE (3×10 mL),
followed by removing the solvent under high vacuum to obtain a white powder (Scheme 1) [14].
IL-32-35: IL-32 was prepared from (4'-((1H-imidazol-1-yl)methyl)-[1,1'-biphenyl]-2-
carbonitrile, IL-33 from 1-benzlimidazole, IL-34 from 1-butylimidazole, and IL-35 from 1-
methylimidazole. The 4-nitrobenzyl bromide and N-functionalized imidazole were taken in
toluene and charged to 100 ml RBF. The reaction mixture was refluxed at 100-110 °C for 48 h.
The heating was stopped and allowed to attain RT. The sticky solid settled at the bottom was
separated by decanting the toluene. To this solid, ethyl acetate was added and washed (2×20
mL). The white powder was filtered over whatman filter paper, washed twice during filtration
followed by drying at 60 °C (Scheme 1)[15].
2.1.2. Synthesis of Ag-NHCs
The formation of neutral or ionic silver complexes is greatly influenced by solvent
polarity. Non-polar solvent favors neutral species (NHC-Ag-X) while polar solvent stabilizes
ionic species ([NHC]2Ag+X-) [16,17]. Neutral Ag-NHCs were prepared by stirring ILs and Ag2O
(1:0.6 mole equivalents) in DCM for 3-4 h in the dark (Scheme 2). The reaction mixture filtered
over celite bed and the further procedure is mentioned below.
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NS-31 and NS-32: The DCM was reduced to 4-5 ml under vacuum and n-pentane was added to
form a fine powder. The suspension was stirred at RT in the dark and solvent decanted. The
residual solvent was removed under vacuum to get light brown powder of NS-31 and light pink
color powder of NS-32 (Scheme 2).
NS-33, NS-34: DCM was reduced to 4-5 ml and n-pentane was added to it. Unfortunately, we
were not able to obtain the precipitate. Alternatively, DCM was removed under vacuum to get
sticky to get sticky solid which was not able to precipitate upon addition of precipitating solvent.
NS-35: The silver complex was insoluble in the reaction mixture, so the DCM was removed at
40 °C to get grayish solid. Acetone was added to dissolve the precipitate, filtered over Whatman
paper. The filtrate was reduced to 5 ml at 50 °C and allowed to attain RT. The slurry was further
cooled to precipitate the dull white powder and filtered over Whatman. The complex was washed
twice with cold acetone to get dull white powder of NS-35.
2.1.3. Synthesis of Ionic Ag-NHCs
IL and Ag2O (1:0.6 mole equivalents) were stirred in MeOH for 24 h in the dark and filtered
over celite. The solvent was removed under vacuum to get sticky solid which was then dissolved
in n-hexane (1 part) and DEE (3-4 parts) was added to get a fine powder (Scheme 1). IS-33 and
IS-34 did not form powder after work up. Despite trying all possible solvent systems we were
not able to get the product for IS-33 and IS-34.
Scheme 2 Synthesis of neutral and ionic Ag-NHCs
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Table 1 The physical properties of synthesized compounds
S. No.
Test Compd.
mp (°C)
(Uncorrected)
113-115
Appearance
Yield %
1
2
IL-31
IL-32
IL-33
IL-34
IL-35
NS-31
NS-32
NS-35
IS-31
IS-32
IS-35
White amorphous powder
White amorphous powder
White amorphous powder
White amorphous powder
White amorphous powder
Light brown color powder
Light pink color powder
Dull white powder
60.8
65
62
68
72
75
79
65
71
72
70
146-148
131-133
108-109
110-112
132-134
149-151
137-139
154-156
147-149
141-143
3
4
5
6
7
8
9
Light brown powder
10
11
Dull white powder
Dark brown powder
Table 2 Characterization data for synthesized compounds
Test
Compd.
FT-IR
(cm-1)
NMR (DMSO-d6, 25 °C, ppm)
IL-31
3064, 3005, 1H NMR (400 MHz): δ 9.37 (s, 1H, NHC-H), 7.79 (s, 2H, Im-H), 7.32 (d, J=7.5 Hz, 4H, Ar-H), 7.24
1556, 1514, (d, J=7.5 Hz, 4H, Ar-H), 5.37 (s, 4H, CH2), 2.3 (s, 6H, CH3); 13C NMR (100 MHz): δ 143.52,
1340, 1143
136.95, 134.79, 133.65, 128.01, 57.12, 25.95.
IL-32
3088, 3014, 1H NMR (400 MHz): δ 9.55 (s, 1H, NHC-H), 8.30 (s, 1H, Im-H), 8.28 (s, 1H, Im-H), 7.97 (t, J= 3.4
2220, 1600, Hz, 1H, Ar-H), 7.95 (s, 1H, Ar-H), 7.81 (t, J= 15.5 Hz, 1H, Ar-H), 7.72 (d, J= 8.5 Hz, 2H, Ar-H),
1516, 1342, 7.67 (d, J= 8.5 Hz, 2H, Ar-H), 7.63 (d, J= 3.4 Hz, 1H, Ar-H), 7.61 (m, 2H, Ar-H), 7.58 (s, 1H, Ar-
1342, 1149
H), 5.65 (s, 2H, CH2), 5.56 (s, 2H, CH2); 13C NMR (100 MHz): 147.62, 143.72, 141.95, 138.16,
136.97, 135.28, 133.91, 133.62, 130.12, 133.62, 130.12, 129.63, 129.33, 128.59, 128.49, 124.04,
123.21, 123.17, 118.46, 110.14, 51.69, 51.19.
IL-33
IL-34
3076, 3008, 1H NMR (400 MHz): δ 9.56 (s, 1H, NHC-H), 8.29 (s, 1H, Im-H), 8.26 (s, 1H, Im-H), 7.91 (d, J= 1.5
1604, 1512, Hz, 2H), 7.71 (d, J= 8.5 Hz, 2H, Ar-H), 7.44 (m, 4H), 7.40 (m, 1H), 5.65 (s, 2H, CH2), 5.48 (s, 2H,
1346, 1165
CH2); 13C NMR (100 MHz): δ 147.59, 142.00, 136.74, 134.65, 129.63, 129.01, 128.79, 128.42,
124.01, 123.07, 123.03, 52.08, 51.07.
3074, 2964, 1H NMR (400 MHz): δ 9.46 (s, 1H, NHC-H), 8.28 (d, J= 8.2 Hz, 2H, Ar-H), 7.89 (s, 2H, Im-H),
1558, 1516, 7.71 (d, J= 8.2 Hz, 2H, Ar-H), 5.65 (s, 2H, CH2), 4.21 (t, J= 14.5 Hz, 2H, CH2), 1.79 (m, J= 32 Hz,
1344, 1151
2H, CH2), 1.28 (m, J= 39.3 Hz, 2H, CH2), 0.90 (t, J= 12.9 Hz, 3H, CH3); 13C NMR (100 MHz): δ
125.86, 147.32, 142.05, 141.63, 134.80, 129.24, 128.24, 127.94, 56.24, 54.06, 36.45, 24.04, 18.50.
IL-35
3055, 2941, 1H NMR (400 MHz): δ 9.32 (s, 1H, NHC-H), 8.28 (d, J= 8.8 Hz, 2H, Ar-H), 7.85 (s, 1H, Im-H),
1560, 1512, 7.78 (s, 1H, Im-H), 7.69 (d, J= 8.8 Hz, 2H, Ar-H), 5.63 (s, 2H, CH2), 3.87 (s, 3H, CH3); 13C NMR
1342,1143
(100 MHz): δ 147.55, 142.18, 137.12, 129.49, 124.18, 123.97, 122.46, 50.84, 35.97.
NS-31
3020, 2939, 1H NMR (400 MHz): δ 7.51 (s, 2H, Im-H), 7.17 (d, J= 7.8 Hz, 4H, Ar-H), 7.13 (d, J= 7.8 Hz, 4H,
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1514, 1444, Ar-H), 5.23 (s, 4H, CH2), 2.24 (s, 6H, CH3); 13C NMR (100 MHz): δ 137.29, 134.16, 129.28,
1217
127.55, 122.38, 54.01, 20.64.
NS-32
NS-35
3165, 2935, 1H NMR (400 MHz): δ 8.19 (d, J= 8.8 Hz, 2H, CH, Ar-H), 7.95 (d, J= 7.8 Hz, 1H, CH, Ar-H), 7.76
2229, 1600, (t, J= 15.1 Hz, 1H, CH, Ar-H), 7.69 (d, J= 1.8 Hz, 1H, Im-H), 7.64 (d, J= 1.8 Hz, 1H, Im-H), 7.56
1517, 1340, (d, J= 8.4 Hz, 4H, Ar-H), 7.48 (d, J=8.5 Hz, 2H, Ar-H), 7.43 (d, J= 8.5 Hz, 2H, Ar-H), 5.53 (s, 2H,
1230
CH2), 5.46 (s, 2H, CH2); 13C NMR (100 MHz): δ 147.04, 144.69, 143.81, 137.65, 137.43, 133.84,
133.49, 130.01, 129.12, 128.46, 128.3, 27.71, 123.87, 123.03, 122.97, 118.46, 110.07, 53.87, 53.32.
3055, 2941, 1H NMR (400 MHz): δ 8.21 (d, J= 8.4 Hz, 2H, Ar-H), 7.56 (d, J= 1.7 Hz, 1H, Im-H), 7.51 (d, J= 1.7
1579, 1514, Hz, 1H, Im-H), 7.49 (d, J= 8.4 Hz, 2H, Ar-H), 5.50 (s, 2H, CH2), 3.8 (s, 3H, CH3); 13C NMR (100
1340, 1222
3022, 2918, 1H NMR (400 MHz): δ 7.53 (s, 2H, Im-H), 7.06 (s, 8H, Ar-H), 5.20 (, 4H, CH2), 2.21 (s, 6H, CH3);
1571, 1118
13C NMR (100 MHz: δ 137.21, 134.20, 129.24, 127.33, 122.57, 53.91, 20.60.
3165, 2935, 1H NMR (400 MHz):δ 8.18 (d, J= 8.8 Hz, 2H, CH, Ar-H), 7.94 (d, J= 8.6 Hz, 1H, CH, Ar-H), 7.75
2229, 1600, (t, J= 15.3 Hz, 1H, CH, Ar-H), 7.68 (d, J= 1.8 Hz, 1H, Im-H), 7.62 (d, J= 1.8 Hz, 1H, Im-H), 7.55
1517, 1340, (d, J= 8.1 Hz, 4H, Ar-H), 7.47 (d, J= 8.6 Hz, 2H, Ar-H), 7.42 (d, J= 8.5 Hz, 2H, Ar-H), 5.52 (s, 2H,
MHz): δ 147.06, 144.85, 128.55, 123.85, 123.50, 122.41, 53.07, 38.23.
IS-31
IS-32
1230
CH2), 5.44 (s, 2H, CH2); 13C NMR (100 MHz):δ 138.13, 137.92, 134.33, 133.98, 130.50, 129.61,
128.94, 128.81, 128.20, 124.36, 123.52, 118.95. 110.56, 54.36, 53.82;
IS-35
3105, 2941, 1H NMR (400 MHz):δ 8.22 (d, J= 8.6 Hz, 2H, Ar-H), 7.57 (d, J= 1.8 Hz, 1H, Im-H), 7.51 (d, J= 1.8
1608, 1517, Hz, 1H, Im-H), 7.50 (d, J= 8.6 Hz, 2H, Ar-H), 5.50 (s, 2H, CH2), 3.80 (s, 3H, CH3); 13C NMR (100
1344, 1222
MHz): δ 144.84, 128.55, 123.85, 123.50, 122.40, 53.07, 38.23;
Table 3 EI-MS data of synthesized compounds
Test
Compd.
IL-31
Molecular
Formula
C %
H %
N %
EI-MS (70 eV) m/z,
Major Fragments
Calcd. Found
Calcd. Found Calcd. Found
C19H21N2Br
63.87
60.84
64.22
60.53
5.92
4.03
5.98
4.15
7.84
7.51
451.53,
274.93,
261.06,
194.26, 167.33, 97.99
IL-32
IL-33
IL-34
IL-35
C24H19N4O2Br
C17H16N3O2Br
C14H18N3O2Br
C11H12N3O2Br
11.79
12.05 350.43,
272.38,
258.45,
191.65, 169, 157.75, 90.92
54.56
49.42
44.31
54.34
49.21
44.79
4.31
5.33
4.06
4.25
5.48
4.11
11.23
12.35
14.09
11.18 316.41,
272.81,
258.95,
192.13, 165.19, 158.23
12.70 274.33,
253.30,
166.71, 153.74, 97.38
193.59,
256.87,
14.37 638.30,
268.77,
190.09, 163.14, 89.35
NS-31
C19H20N2AgBr
49.06
49.46
4.55
3.28
5.67
3.34
6.02
5.92
538.53,
191.43, 157.53, 90.70
272.17,
251.13,
314.45,
NS-32
IS-31
C24H18N4O2AgBr
C38H40N4AgBr
49.43
55.33
49.85
55.68
9.61
9.82
508.18,
273.52, 257.38, 192.42
424.38,
4.43
4.86
10.75
10.58
461.10, 377.48, 273.14,
207.03, 68.61
IS-32
C48H36N8O4AgBr 58.91
59.05
3.91
4.08
11.45
11.23
1008.93, 803.91, 461.70,
249.38, 199.10, 183.04
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2.2. Biological Screening
2.2.1. Antibacterial Evaluation
Inhibition of bacterial growth by ILs and Ag-NHCs was assayed by the agar well
diffusion method. The human pathogenic S. enterica and S. aureus bacterial cultures were
clinically isolated and used for antibacterial evaluation. The bacteria were incubated in Luria-
Bertani (LB) medium at 37 °C. After incubation, 100 μL of the bacterial suspension
(106 CFU/mL) was spread evenly on Mueller-Hinton agar plates with a sterile cotton swab.
25 μL of the test compound solution with varying concentrations of 25, 50, 75 and 100 µL was
added to the wells on the agar plate. DMSO was used as a control and Kanamycin solution as a
reference standard. All plates were incubated overnight at room temperature. Experiments were
performed in triplicate. The zones of inhibition were measured and reported as an average [18].
2.2.2. MIC Determination
MIC test was performed to obtain the most effective compounds with the ability to inhibit
the growth of pathogenic bacteria at its minimum concentration. Bacterial strains S. enterica and
S. aureus were grown overnight in LB with vigorous shaking at 37 °C. The cell density was
adjusted to 106-107 CFU/mL before a series of the concentration of the prepared test compounds
(100, 50, 25, 12.5 and 6.25 µM) with equal volumes were added to the solution. Bacterial growth
was determined by observing the bacterial growth after 24 h of incubation at 28 °C. Bacterial
growth rates were measured by monitoring the optical density at 600 nm (OD600) with different
time intervals using a UV–vis spectrophotometer (Shimadzu UV-2550). The LB medium without
any supplement was used as a control. The lowest concentration of the antibacterial agent at
which no bacterial growth is observed in the culture suspensions is defined as the MIC. The
experiment was performed with triplicates [19].
2.2.3. Longevity experiment
The stability or longevity of the ILs and Ag-NHCs against S. enterica and S. aureus was
evaluated for a period of 21 d with the working concentration of 100 µM. 2% inoculum of the
overnight grown S. aureus and S. enterica bacterial cultures was inoculated in the compound
solution and the long-term stability was determined by visual inspection of the bacterial growth
followed by an absorbance reading at 600 nm by UV–vis spectroscopy [20].
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2.2.4. SEM analysis
The morphology of S. enterica and S. aureus was observed by SEM after treatment with
the NS-32 for 48 h. A thin smear of the culture was made on the cover slip followed by fixation
using 2.5% glutaraldehyde, a series of acetone wash and observed for cell morphology [21].
3. Results and Discussion
3.1. Chemistry
3.1.1. NMR Studies
1
All synthesized compounds were amorphous and stable towards moisture. In H NMR
spectra, the NHC proton appeared downfield between δ 9-10 ppm attributed to high acidity and
electronic properties responsible for stabilization of NHCs [22,23]. The ionic liquids i.e. IL-31-
35 are well stabilized by the presence of phenyl rings. NHC H in IL-31 appeared at δ 9.37 ppm,
while it appears at δ 9.56 ppm in IL-32, δ 9.56 in IL-33, δ 9.46 in IL-34, and 9.32 ppm in IL-35.
1
The main characteristic feature of silver complexation is the absence of H signal for
NHC proton which validates the successful Ag-NHC formation. The signal responsible for NHC
H was completely absent in all Ag-NHCs. The silver complexation causes significant alteration
1
in electron density on imidazolium ring and causes considerable peak shifting of H and 13C
signals in silver complexes [24,25]. As it was observed in the case of NS-31, IS-31, NS-32, and
IS-32. The 13C signal for carbene C appeared at δ 138.19 ppm which had shown a drastic change
in position on NMR scale. It was appeared at δ 137.29 ppm in NS-31 and at δ 137.21 ppm in IS-
21, which was considerable downfield shifting. While in the case of IL-32, it had appeared at δ
135.28 ppm which was surprisingly missing upon complexation as in NS-32 and IS-32 (Fig. 1).
f
e
d
c
b
a
Fig. 1 13C NMR spectra overlay of a)IL-31, b)NS-31, c)IS-31, d)IL-32, e)NS-32, and f)IS-32 in DMSO-d6
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3.1.2. FT-IR characterization
Ionic liquids and their silver complexes were characterized by FT-IR in the range of
4000-400 cm-1. The characteristic IR peaks for functional groups showed a significant change in
appearance upon complexation. The characteristic peaks for aromatic C-H stretch appear in the
range of 3100-3000 cm-1 in IL-32 which showed a reduction in intensity upon complexation as of
in NS-32 and IS-32. The peaks are completely absent in NS-32 and very weak signals in IS-32.
The same observation was repeated for aliphatic C-H stretching vibrations. The signal for CN at
~2222 cm-1 and –NO2 group in the range of 1560-1515 & 1385-1345 cm-1 remained unaltered
upon complexation without any significant changes in peak intensity. The intensity of peaks for
aromatic C=C stretching in the range of 1600-1400 cm-1 had shown a considerable reduction in
NS-32 and IS-32, which are the consequences of metallation. There are considerable changes in
electron density on imidazolium ring upon complexation resulting in reduced intensity of FT-IR
peaks for C-N of imidazolium moiety in the range of 1360-1080 cm-1. This is called a "four
fingers (f.fs)" which showed variations upon complexation (Fig. 2). The peaks for aromatic C-H
bending vibrations also displayed a sufficient reduction in intensity peaks because of silver
metallation [26–28].
Fig. 2 FT-IR overlay of IL-32, NS-32 and IS-32
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3.1.2. EI-MS studies
In addition to the NMR and FT-IR, the ILs and Ag-NHCs were further confirmed with
EI-MS studies and the fragmentation pattern of IL-32 was studied in detail (Fig. 3). IL-32 had
formed several major fragments such as peak at m/z 68.52 corresponds for imidazole, where 1-
methylimidazole appeared at m/z 84.41. The peak for benzonitrile fragment appeared at m/z
104.34 while the peak at m/z 171.06 corresponds to 1-benzyl-3-methyl-1H-imidazol-3-ium
cation, whereas the same fragment with additional –NO2 appeared at m/z 217.97. The next major
fragment formed was of 1-((2'-cyano-[1,1'-biphenyl]-4-yl)methyl)-3-methyl-1H-imidazol-3-ium
cation at m/z 275.78 while other cation i.e. 1-([1,1'-biphenyl]-4-ylmethyl)-3-(4-nitrobenzyl)-1H-
imidazol-3-ium appeared at m/z 371.62 with molecular cation peak at m/z 395.15. The
appearance of corresponding fragments validates the successful formation of IL-32.
Fig. 3 EI-MS spectra of IL-32
The M+ peak appeared at m/z 580.97 for NS-32 and Im-Ag appeared at m/z 171.81.
Benzonitrile fragment appeared at m/z/ 105.03 along with 1-methylimidazole at m/z 85.12. The
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fragment appearing at m/z 276.52 corresponds to 1-((2'-cyano-[1,1'-biphenyl]-4-yl)methyl)-3-
methyl-1H-imidazol-3-ium cation. This fragmentation pattern has been given in Fig. 4.
Fig. 4 EI-MS spectra of NS-32
3.2. Antimicrobial Screening
It is necessary to understand the mechanism of action of Ag-NHCs when subjected to
activity evaluation. The silver complexes when comes in vicinity with water molecules, releases
Ag+ ions into the solution which then induces bactericidal effects. This release of silver ions
depends upon the substitutions present in NHC moiety which provides extra stability and assures
release of Ag+ ions over a period of several days to achieve faster healing and prevent the
spreading of infection.
3.2.1. Antibacterial activity
The efficacy of prepared ILs and silver complexes was investigated against S. aureus and
S. enterica. Antibacterial activity was measured in terms of zone of inhibition for the test
compounds with varying concentrations (25-100 ppm) using DMSO as a control and the
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Kanamycin solution as a reference standard. All compounds, including ILs as well as silver
complexes showed a zone of inhibition ranging from 7 to 15 mm (Fig. 5) (Table S1 & S2
supporting information). There is no significant impact of increasing the test compound’s
concentration on an antibacterial activity which indicates the absence of dose-dependent activity.
25
Against S. aureus
Against S. enterica
16
14
12
10
8
20
15
10
5
DMSO
DMSO
Kanamycin
25 ppm
50 ppm
75 ppm
100 ppm
Kanamycin
25ppm
6
50ppm
4
75ppm
2
100ppm
0
0
Test Compounds
Test Compounds
Fig. 5 Antibacterial activity of test compounds in the terms of zone of inhibition (mm)
3.2.2. Determination of MIC
MIC is the lowest concentration of an antimicrobial agent that inhibits visible growth of
microbes. For precise determination of MIC, OD600 was measured after 5 d to assign relative
potency amongst the test compounds (Table 1, see Table S3 Supporting information).
Table 2. MIC (expressed in µM) values of test compounds
Entry
1
2
3
4
5
6
7
8
9
10
11
Test Compd.
IL-31
NS-31
IS-31
IL-32
NS-32
IS-32
IL-33
IL-34
IL-35
NS-35
IS-35
100
*
50
50
100
50
*
50
25
100
50
*
*
*
100
100
100
100
100
100
S. enterica
S. aureus
100
100
* - OD>0.5 at 100 µM
The silver complexes NS-31, IS-31, NS-32, and IS-32 exhibited MIC of 50, 50, 25, and
50 µM respectively against S. aureus. Remaining all test compounds displayed higher MIC
values. The ionic derivative of NS-32 i.e. IS-32 showed MIC of 50 µM against S. aureus but
failed to show similar MIC against S. enterica. NS-31, IS-31, NS-32, and IS-32 had displayed
OD600 value of 0.49, 0.50, 0.44 and 0.51 against S. aureus at 50, 50, 25, and 50 µM respectively.
While these complexes showed OD600 as 0.52, 0.50, 0.46, and 0.55 at 50, 100, 50, and 100 µM
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concentrations respectively against S. enterica. From this overall data, NS-32 was finalized as
most potent and selected for further studies.
A prominent observation was made that the silver complexes have higher MIC i.e. less
activity in S. eneterica (gram-negative) compared to S. aureus (gram-positive). This could be the
result of resistance for silver ions against gram-negative pathogens. This resistance is the effect
of plasmid-mediated gene expression responsible for efflux of Ag+ from a bacterial cell caused
by a well determined periplasmic silver binding protein called SilE and thereby making gram-
negative bacteria resistant to silver [29–31].
3.2.3. Stability studies
The stability or longevity of silver complexes with respect to time has been studied by
inoculating 100 ppm solutions of each compound with 0.5 OD bacterial culture in LB broth. The
growth was observed visually on 7, 14 and 21 d (Table 2, Table S4-Supporting information).
Table 3. Study of test compounds for bacterial growth inhibition
S. aureus
S. enterica
d
d
Entry Test Compounds
7
‒
14
+
21
+
7
‒
14
‒
21
+
1
IL-31
NS-31
IS-31
IL-32
NS-32
IS-32
IL-33
IL-34
IL-35
NS-35
IS-35
2
‒
‒
‒
‒
‒
‒
‒
‒
‒
‒
‒
‒
‒
‒
‒
‒
+
‒
‒
‒
+
+
+
‒
+
+
+
+
+
+
‒
‒
‒
‒
‒
‒
‒
‒
‒
‒
‒
‒
+
‒
‒
+
+
‒
‒
‒
+
+
+
‒
+
+
+
+
+
+
3
4
5
6
7
8
9
10
11
˗ (Growth Not Observed), + (Growth Observed)
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The test compounds had shown bacterial growth inhibition up to 7 d in the case of both
pathogens. After 7 d, IL-31, IL-32, IL-33, and IL-34 had lost their activity. The Ag-NHCs had
shown growth inhibition up to 14 d, but only NS-32 was able to retain the activity up to 21 d.
This pattern of growth inhibition was similar in the case of S. aureus and S. enterica.
3.2.4. SEM Analysis
The antimicrobial action of Ag NHCs was confirmed by inspecting the disruption of cell
wall morphology. The highly reactive Ag+ released from Ag-NHCs interacts very strongly with
cell wall components and shows subsequent inhibition of bacterial growth. The complex NS-32
was studied for cell wall disruption in S. aureus and S. enterica. The SEM images of a bacterium
without treatment showed no disruption of the cell wall and presents intact rod and cocci shape
(Fig. 6 a & c). The treatment of bacteria with NS-32 led to an alteration in cell morphology
where cell wall distortion is clearly visible (Fig. 6 b & d).
a
b
c
d
Fig. 6 SEM images of S. enterica a) without treatment b) treated with NS-32; S. aureus c) without treatment
d) treated with NS-32
4. Conclusions
The prepared imidazolium-based novel ionic liquids (ILs) containing methylbenzyl and
nitrobenzyl substituents and their neutral as well as ionic silver complexes were obtained as a
fine powder and stable towards moisture. The formation of neutral or ionic silver complex was
influenced by solvent polarity. The widely used method of coprecipitation was successfully
implemented throughout the experiment. The nitrobenzyl and biphenyl carbonitrile based neutral
silver complex NS-32 was the most active amongst all showing MIC of 25 µM in S. enterica as
well as S. aureus and inhibiting the bacterial cell growth up to 21 d. The silver complexes had
higher MIC in gram-negative (S. enterica) because of the resistance for silver ions. The other
factors such as hydrophobic substitution, steric bulk affected the release of Ag+ ions in an
aqueous environment.
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Acknowledgements
We gratefully acknowledge SIF DST-VIT-FIST Vellore for providing NMR, FT-IR data,
TT-310 for providing excellent research facilities and SEM facility at SBST, and RGEMS, VIT
University, Vellore for funding the research work. Authors are highly thankful to SAIF, IIT
Madras for EI-MS analysis.
Supporting Information Available
Supporting data associated with this article can be found in the online version.
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Highlights
 Imidazolium based novel ILs, their neutral as well as ionic complexes are prepared.
 The IL-32 and NS-32 was studied for fragmentation pattern in EI-MS.
 The test compounds had shown dose independent activity.
 The complex NS-32 inhibited bacterial growth up to 21 days with MIC of 25 µM.
 Ag-NHCs induce ruptures in bacterial cell wall as a site of action.
18