Bactericidal Action of b-Lactam Ionic Liquids
products, 1-hexadecyl-3-methylimidazolium ampicillin
[C16M1Im][Amp], 1-hexadecyl-2,3-dimethylimidazolium ampicillin
[C16M2Im][Amp], and cetylpyridinium ampicillin [CP][Amp], were iso-
lated as solids at room temperature and purified by washing with
anhydrous diethyl ether. These salts have limited solubility in water
but are soluble in ethanol, isopropanol, dimethylsulfoxide, and chlo-
roform.
20H), 0.86 (s, 2H). Anal. Cacld for C37H56N4O4S: C, 68.06; H, 8.64;
N, 8.58; S,4.91. Found: C,67.97; H, 8.57; N, 8.55S, 4.84.
ing
1-hexadecyl-3-methylimidazolium ampicillin ([C16M1Im][Amp]), off-
white solid, yield, 91%. Mp = 55 ꢀC [C16M1Im][Amp].CMC = 24 lM
[C16M1Im][Amp]; 430 lM [C16M1Im][Br]. Water solubility: 379 lg ⁄ mL.
1H NMR (250 MHz, DMSO) d 9.11 (s, 1H), 8.31 (s, 1H), 7.72 (d, 2H),
7.59 – 7.12 (m, 5H), 5.36 (s, 2H), 4.14 (t, 2H), 3.85 (s, 2H), 2.09 (s,
3H), 1.77 (t, 2H), 1.47 (dd, 10.0 Hz, 6H), 1.24 (s, 28H), 1.09 (s, 3H),
0.85 (d, 2H).Anal. Cald for C36H57N5O4S: C, 65.92; H, 8.76; N, 10.68;
S, 4.89. Found: C, 65.79; H, 8.81; N, 10.42; S, 4.87.
The solubility of Amp-ILs in water was characterized using UV ⁄ vis
spectroscopy. We observed that the solubilities of Amp-ILs were
475, 379, and 348 lg ⁄ mL for [C16M2Im][Amp], [C16M1Im][Amp], and
[CP][Amp], respectively. A 100- to 150-fold reduction (R2 = 0.99) in
the aqueous solubility of ampicillin was observed once the sodium
was replaced with a quaternary ammonium group. A positive con-
trol consisting of Amp-ILs in isopropanol confirmed that the anionic
ampicillin was an intact part of the IL structure as evidenced by
absorption bands at 257, 262, and 268 nm.
1-hexadecyl-2,3-dimethylimidazolium ampicillin ([C16M2Im][Amp]), off-
white solid, yield, 94%. Mp = 65 ꢀC [C16M2Im][Amp]. CMC = 92 lM
[C16M2Im][Amp]; 450 lM [C16M2Im][Br]. Water solubility: 475 lg ⁄ mL.
1H NMR (250 MHz, DMSO) d 8.27 (s, 1H), 7.60 (dd, 2H), 7.53 –
6.95 (m,5H), 5.70 (s, 2H), 5.30 (d, 4H), 4.07 (t, 2H), 3.73 (s, 3H), 2.09
(s, 1H), 1.68 (s, 6H), 1.22 (s, 30H), 0.83 (d, 3H).Anal. Cald for
C37H59N5O4S: C, 66.33; H, 8.88; N, 10.45; S, 4.79. Found: C, 66.21;
H, 8.84; N, 10.44; S, 4.72.
1
Amp-ILs were also characterized using H-NMR and elemental anal-
ysis. All Amp-ILs contained the chemical shifts of the ampicillin
anion and the respective cations. In the case of [C16M1Im][Amp], a
singlet peak was observed with a chemical shift at 9.11 ppm, which
was attributed to the acidic proton in the C2 position. However, this
acidic peak is absent in the spectra for [C16M2Im][Amp] because of
the methyl group substituted on C2. In addition, the chemical shifts
between the hydrogen on the C4 and C5 positions of the imidazoli-
um rings decreased upon successful metathesis. A secondary set of
Antibacterial activity
Minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC)
The MIC values were determined in triplicate by the broth dilution
method in a 96-well microtiter plate using Mueller-Hinton Broth
(38). The test organisms were grown individually on BHI agar for
24 h at 37 ꢀC prior to each antibacterial test. The growth was
adjusted using colony plate counts. Bacteria of 105 CFU ⁄ mL concen-
trations were exposed to an Amp-IL concentration range of
0.8–0.2 mM. The MIC for each Amp-IL was recorded as the lowest
concentration that showed no turbidity after 24 h of incubation at
37 ꢀC. Turbidity is an indication of microbial growth and if present,
the corresponding concentration of antibacterial agent is considered
ineffective. To determine whether the Amp-ILs inhibited growth or
killed the bacteria, twenty microliters of 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyl tetrazolium bromide or MTT (1 mg ⁄ mL) was added to
the non-turbid wells of the MIC assay plate and incubated for 2 h
at 37 ꢀC for the bacteriostatic ⁄ bacteriocidal status determination
(39,40). In the case of viable cells with inhibited growth, the tetra-
zolium dye (i.e. yellow solution) would be metabolically reduced to
aqueous soluble formazan crystals (i.e. purple solution); however, a
solution containing dead bacterial cells would remain yellow (40).
1
multiplets ranging from 8.27 to 9.19 ppm present in the H-NMR of
[CP][Amp] are attributed to the more electron-withdrawing nitrogen
in the pyridinium ring. The anion exchange from bromide to ampicil-
lin anion was confirmed by examining the multiplet ranging from
7.11 to 7.52 ppm that was directly contributed by the benzyl group
in the ampicillin structure. Lastly, a strong singlet at approximately
1.22 ppm validated the existence of the long alkyl chain in the cat-
ion moiety for each Amp-IL.
Evaluation of antibacterial activity
MIC results
The trend of increasing toxicity with long alkyl chain lengths paired
with ampicillin as an anion was observed in both ampicillin-type,
pyridinium- and imidazolium-based ILs. To quantify their antibacte-
rial activities, the MICs and MBC were determined. As controls, the
components of the ILs were evaluated for antimicrobial activity to
determine the difference in activity between the molecular and IL
forms of the compounds. The MIC results demonstrate that each
Amp-IL exhibited similar or improved activity in the Amp-IL form
against both Gram-positive and Gram-negative bacteria (Table 2).
Overall increases in activity ranged from 2 to 43 times compared
with [Na][Amp].
Results and Discussion
Synthesis and characterization
The synthesis of 1-alkyl-3-methylimidazolium ILs involved the quat-
ernization of 1-methylimidazole or 1,2-dimethylimidazole with 1-
bromohexadecane followed by anion exchange. Quaternization was
carried out for 48 h under reflux in anhydrous ethanol under argon
atmosphere. Amp-ILs (Table 1) were synthesized by anion-exchange
reactions between the synthesized imidazolium bromides [Im][Br] or
commercially available QAC, CPB [CP][Br], and excess sodium ampi-
cillin [Na][Amp] in a chloroform ⁄ water (4:1 v ⁄ v) mixture. The result-
Imidazolium-based ampicillin ionic liquids
The imidazolium-type ampicillin salts were tested against E. coli
O157:H7, K. pneumoniae, S. aureus, and E. faecium to investigate
structure and activity relationships. These compounds are composed
of three parts: the head imidazole, the C16-alkyl chain, and the
Chem Biol Drug Des 2011; 78: 33–41
35