Synthesis and biological evaluation of some novel 1-alkyl-3-methylimidazolium carboxylate ionic liquids as potential antifungal agents

Article abstract of DOI:10.1002/jccs.201900366

In an effort to discover lead compounds that may be of importance as potent antifungal agents, a series of novel 1-alkyl-3-methylimidazolium carboxylate ionic liquids were efficiently synthesized through a solvent-free ultrasound-assisted quaternization reaction of 1-methylimidazole and alkyl bromides RBr (R = hexyl, octyl) followed by an anion exchange process with selected carboxylate anions (cinnamate, salicylate, crotonate, and oxalate). Quantitative yields obtained were in the range of 86–94%. Structure characterization was done using FT-IR, ¹H-NMR, and ¹³C-NMR spectroscopic techniques. All the synthesized compounds showed in vitro antifungal activity against the fungus Candida albicans with the minimum inhibitory concentrations found to be less than or equal to 1%. Preliminary cytotoxicity assays (trypan blue exclusion and MTT) were performed on all ionic liquids and findings revealed higher lymphocyte viability in 1-hexyl-3-methylimidazolium carboxylate ionic liquids than in 1-octyl-3-methylimidazolium counterparts. No extensive toxicity effect was observed with the carboxylate anion variation. Among the tested compounds, 1-hexyl-3-methylimidazolium crotonate and 1-hexyl-3-methylimidazolium oxalate exhibited the lowest cytotoxicity in the trypan blue exclusion and MTT assays, respectively. Together, our results highlight the potential of carboxylate-based ionic liquids in the development of next-generation antifungal drugs.

Full text of DOI:10.1002/jccs.201900366

Received: 29 October 2019
DOI: 10.1002/jccs.201900366
Revised: 12 January 2020
Accepted: 18 January 2020
A R T I C L E
Synthesis and biological evaluation of some novel 1-alkyl-
3-methylimidazolium carboxylate ionic liquids as potential
antifungal agents
Ser John Lynon P. Perez | Eduardo C. Atayde Jr. | Susan D. Arco
Synthetic Organic Chemistry Laboratory,
Abstract
Institute of Chemistry, University of the
Philippines Diliman, Quezon City,
Philippines
In an effort to discover lead compounds that may be of importance as potent
antifungal agents, a series of novel 1-alkyl-3-methylimidazolium carboxylate
ionic liquids were efficiently synthesized through a solvent-free ultrasound-
assisted quaternization reaction of 1-methylimidazole and alkyl bromides RBr
(R = hexyl, octyl) followed by an anion exchange process with selected carbox-
ylate anions (cinnamate, salicylate, crotonate, and oxalate). Quantitative yields
obtained were in the range of 86–94%. Structure characterization was done
Correspondence
Ser John Lynon P. Perez, Synthetic
Organic Chemistry Laboratory, Institute
of Chemistry, University of the
Philippines Diliman, Quezon City 1101,
Philippines.
Email: spperez@up.edu.ph
1
using FT-IR, H-NMR, and 13C-NMR spectroscopic techniques. All the synthe-
sized compounds showed in vitro antifungal activity against the fungus
Candida albicans with the minimum inhibitory concentrations found to be less
than or equal to 1%. Preliminary cytotoxicity assays (trypan blue exclusion and
MTT) were performed on all ionic liquids and findings revealed higher lympho-
cyte viability in 1-hexyl-3-methylimidazolium carboxylate ionic liquids than in
1-octyl-3-methylimidazolium counterparts. No extensive toxicity effect was
observed with the carboxylate anion variation. Among the tested compounds,
1-hexyl-3-methylimidazolium crotonate and 1-hexyl-3-methylimidazolium oxa-
late exhibited the lowest cytotoxicity in the trypan blue exclusion and MTT
assays, respectively. Together, our results highlight the potential of carboxylate-
based ionic liquids in the development of next-generation antifungal drugs.
Funding information
Department of Science and Technology -
Philippine Council for Health Research
and Development
K E Y W O R D S
antifungal agents, C. albicans, carboxylate-based ionic liquids, cytotoxicity
1 | INTRODUCTION
afflicted with oral candidiasis alone[2] and vulvovaginal
candidiasis (thrush) continues to affect 70–75% of women
Globally, fungal infections remain a leading cause of
human morbidity and mortality, predominantly on
immunocompromised patients. These fungal infections
invade about 300 million individuals annually with a
global mortality rate of over 1.60 million people per
annum.[1] Around 9.5 million people each year become
at least once during their lifetime.[3] With the increase in
the frequency and severity of these opportunistic infec-
tions and the ever-evolving resistance of microorganisms
to the existing drugs, there remains a never-ending need
for the discovery and development of potential antifungal
agents.
© 2020 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
J Chin Chem Soc. 2020;1–8.
http://www.jccs.wiley-vch.de
1

2
PEREZ ET AL.
Ionic liquids (ILs) are salts that possess melting points
presented in Scheme 1.[24] Initially, the reaction mix-
ture was biphasic due to the poor miscibility of the reac-
tants. Upon sonication, the mixture then turned opaque
until a clear, yellowish viscous liquid was obtained (1a,
1b). As shown in Table 1, the product yields for
[RMIM]Br ILs were both greater than 90%, rendering
the one-pot, solvent-free, sonochemically mediated
method, an efficient and facile direct access to halide-
based ILs.
The prepared [RMIM]Br ILs were used to synthesize
the novel set of [RMIM]RCOO ILs using a halide-to-
anion exchange process. By passing the [RMIM]Br ILs
through a loaded anion exchange resin, the [RMIM]
RCOO ILs can be obtained (2a–2h) in high purity and
quantitative yields (86–94%) (Table 1).
below 100ꢀC due to the poor coordination of the constitu-
ent ions, which prevents the formation of a stable crystal
lattice.[4] Given the possibility of tuning their attractive
features (negligible vapor pressure, high thermal and
chemical stability, good conductivity, and wide electro-
chemical range), ILs have been labeled as “designer sol-
vents.”[5] Most of the earliest interest in ILs was as
alternative green solvents for chemical synthesis and
catalysis.[6–12] Since then, ILs have found applications in
electrochemistry[13–15] and biomass conversion.[16–19]
These diverse applications prove the tunable nature of
ILs which eventually led to the transition from “designer
solvents” to “designer drugs.” Through the incorporation
of active pharmaceutical ingredients (API), a generation
of novel ILs with biological activity (API-ILs) can be
synthesized.[5,20–23]
In our previous work[24] on pharmaceutically active
ILs, the antifungal activity of a series of 1-alkyl-
3-methylimidazolium [RMIM]Br ILs against Candida
albicans has been demonstrated. It was also shown that an
increase in the alkyl chain length on the imidazolium cat-
ion corresponds to an increase in the bioactivity of the syn-
thesized ILs.[24] With research studies establishing that
organic carboxylic acids (cinnamic acid,[25–28] oxalic
acid,[29,30] salicylic acid,[31,32] and crotonic acid[33]) inhibit
fungal growth, in this recently published patented
study,[34] we now report the efficient synthesis of a set of
novel imidazolium-based API-ILs through the anion
exchange reaction of 1-alkyl-3-imidazolium bromide
[RMIM]Br (R = hexyl, octyl) with organic carboxylates
(cinnamate, crotonate, oxalate, and salicylate) as confirmed
2.2 | Characterization of the ILs
All synthesized ILs were characterized based on NMR
and FT-IR spectroscopic data (found in Data S1) to con-
firm the purity and structure of the products. With the
ILs bearing a similar cation [RMIM]+ and differing only
in their corresponding anions, a general spectral interpre-
tation was provided.
The observed upfield 1H-NMR signals (δ = 0.79–
4.4 ppm) are attributed to the long alkyl chain of the ILs.
The downfield peaks (δ = 7.5–10 ppm) are due to the pro-
tons attached to the sp2-hybridized carbon of the
imidazolium ring. The absence of more downfield signals
(δ > 11–12 ppm) implies that there are no carboxylic acid
1
by FTIR, H-NMR, and 13C-NMR spectroscopy. The mini-
TABLE 1 Percent yields of synthesized ILs
mum inhibitory concentrations (MICs) of these potential
antifungal agents against C. albicans have been tested using
the broth dilution method. Cytotoxicity of the synthesized
API-ILs toward human lymphocytes were evaluated
through the trypan blue exclusion and MTT assays.
Ionic liquid
Yield (%)
[HMIM]Br
90
93
87
92
92
87
94
88
88
86
[OMIM]Br
[HMIM]crotonate
[OMIM]crotonate
[HMIM]cinnamate
[OMIM]cinnamate
[HMIM]salicylate
[OMIM]salicylate
[HMIM]oxalate
[OMIM]oxalate
2 | RESULTS AND DISCUSSION
2.1 | Synthesis of [RMIM]Br and
[RMIM]RCOO ILs
The ultrasound-assisted preparation of 1-alkyl-3-methyl-
imidazolium bromide ILs followed the general scheme
SCHEME 1 Synthetic route to
[RMIM]RCOO ILs

PEREZ ET AL.
3
TABLE 2 MIC of [RMIM]RCOO ILs against C. albicans
groups present, establishing the efficiency of the anion
exchange method. For the [RMIM]RCOO ILs, the appear-
ance of distinct peaks of their anions further confirms
the synthesis success. Signals at 5.6–7.0 ppm attributed
to vinylic protons were observed for [RMIM]crotonate
and [RMIM]cinnamate ILs while aromatic protons
(δ > 7 ppm) were detected for [RMIM]cinnamate and
[RMIM]salicylate ILs.
Sample concentration, %
Sample
0a
+
+
+
+
+
+
+
+
1.0
−
−
−
−
−
−
−
−
2.5
−
−
−
−
−
−
−
−
5.0
−
−
−
−
−
−
−
−
7.5
−
−
−
−
−
−
−
−
10
−
−
−
−
−
−
−
−
[HMIM]crotonate
[OMIM]crotonate
[HMIM]cinnamate
[OMIM]cinnamate
[HMIM]salicylate
[OMIM]salicylate
[HMIM]oxalate
[OMIM]oxalate
The 13C-NMR spectra of all products showed three
downfield signals (δ
=
121–122, 123–124, and
134–138 ppm) for the carbons of the imidazolium ring.
For the [RMIM]RCOO ILs, the more downfield signals
(δ = 165–172 ppm) confirmed the presence of the car-
bonyl carbon of the carboxylate anions. On the other
hand, the presence of upfield signals (δ = 22.0–28.0 ppm)
and (δ = 14.0 ppm) are due to the CH2 and CH3
groups of the alkyl chain substituent. The number of
signals also corresponded to the number of carbon
atoms expected in the molecular structure of the
synthesized ILs.
aSabouraud dextrose broth only.
Note: +, growth on agar plate after subculture from tube; −, no growth on
agar plate after subculture from tube.
Results suggest that the MICs of the samples against
C. albicans must be less than or equal to 1%. Given that
most over-the-counter antifungal topical creams being
marketed contain 1% (w/w) active ingredient, our find-
ings highlight the efficacy of these novel API [RMIM]
RCOO ILs as growth inhibitors of C. albicans and their
promising application as next-generation antifungal
agents.
Functional group analyses using FT-IR spectroscopy
further confirmed the structure of the desired ILs. The
prominent peaks observed for the [RMIM]+ cation corre-
spond to C=N stretch (1,600–1,640 cm−1, m), C=C sym-
metric stretch (1,460–1,600 cm−1, m), C-N stretch (1,100–
1,300 cm−1, s), aromatic C-H stretch (3,059–3,144 cm−1),
and aliphatic C H stretch (2,850–2,942 cm−1). The broad
peak (3,340–3,400 cm−1) can be attributed to the hydra-
tion of the IL samples because of their hygroscopic
nature.[24] The FT-IR spectra of the [RMIM]RCOO ILs
showed peaks at 1,550–1,700 cm−1 (C=O stretch) and
1,020–1,180 cm−1 (C O stretch) affirming the presence
of a carboxylate functional group.
2.4 | Evaluation of cell viability in
[RMIM]RCOO ILs
In the aim of further assessing the potential of [RMIM]
RCOO as antifungal agents, a preliminary evaluation of
its effect on cell viability was performed using trypan
blue exclusion assay. The assay involved normal human
lymphocytes with supplemented RPMI (culture medium)
and phenol as the negative and positive controls,
respectively.
Table 3 summarizes the results obtained from the cell
viability test. For [HMIM]RCOO ILs, the percent cell via-
bilities of lymphocytes ranged from 65–82%, which are
significantly higher than phenol (2.1%). Among them,
[HMIM]crotonate provided the highest cell survival per-
centage at 81.8%, considerably close to the percent cell
survival in the medium (90.8%).
For the [OMIM]RCOO ILs, the determination of the
percent cell viability via the trypan blue exclusion test
was not possible due to chemical-aided cellular aggrega-
tion. ILs with long hydrocarbon chains are known to have
aggregation behaviors in aqueous solutions and
are observed to behave similar to conventional
surfactants.[38–40] The molecular structures of long-chain
ILs resemble classical surface-active agents due to the
2.3 | MIC determination
Azole-based antimycotic agents act by inhibiting the
enzyme cytochrome p450 lanosterol 14α-demethylase by
binding in the active site of the enzyme, which then
blocks ergosterol synthesis.[35–37] Ergosterol regulates the
fluidity, asymmetry, and integrity of membranes in fun-
gal cells. Depletion of ergosterol and accumulation of ste-
rol precursors lead to the formation of an altered
membrane with impaired functionality, and ultimately to
the prevention of fungal growth.[37]
The antifungal activities were evaluated by determin-
ing the MICs of the synthesized agents against
C. albicans via broth microdilution method. The biologi-
cal assay results are presented in Table 2. Since the
growth of C. albicans was inhibited at the lowest concen-
tration tested (1%), the MICs of test samples were not
exactly determined in the chosen concentration range.

4
PEREZ ET AL.
TABLE 3 Cell viability in [HMIM]RCOO ILs using trypan
blue assay
lymphocytes had survival rates ranging from 20–29%, all
lower than the measured viability in phenol. From the
results of the MTT assay, the least cytotoxic IL was found
to be [HMIM]oxalate (87% cell viability). Inconsistencies
among different cytotoxicity assays are reported in sev-
eral literature,[41–46] mainly attributed to their mecha-
nisms and principles. The exceedingly high cell
proliferation rate in [HMIM]oxalate may be due to possi-
ble overestimation of MTT assay over trypan blue test[45]
and the fact that the oxalate anion is a minor metabolite
involved in cellular metabolism.
Results from both assays suggest that varying the
anion in the ILs did not contribute significantly to the
toxicity. Moreover, [OMIM]RCOO ILs were found to be
more cytotoxic than [HMIM]RCOO ILs. This is attributed
to the tendency of ILs with longer alkyl chains to be
incorporated in cellular membranes, as a consequence of
their higher lipophilicity, resulting in an alteration in the
membrane permeability.[24]
Average
number of
Treatment viable cellsa
Average total
number of
cellsa
Percent cell
viability, %
Supplemented 195.00
RPMI
214.78
90.8
Phenol
4.11
193.00
214.22
2.1
[HMIM]
175.33
81.8
crotonate
[HMIM]
cinnamate
165.66
106.78
85.44
216.56
163.44
143.33
76.4
65.3
59.6
[HMIM]
salicylate
[HMIM]
oxalate
aCell density is expressed as n × 104 cells/ml. where n is the number of cells
reported above.
3 | EXPERIMENTAL
3.1 | General remarks
All the reagents used in this study were of analytical
grade and were used without further purification. Oxalic
acid was purchased from J.T. Baker Chemical Company.
Salicylic acid was purchased from Fluka. Methanol was
purchased from Scharlau Chemie. 1-bromohexane,
1-bromooctane, 1-methylimidazole, ethyl acetate,
cinnamic acid, crotonic acid, and ion exchange resin
Amberlyst A-26 (OH form) were purchased from Sigma-
Aldrich.
The ILs were synthesized through irradiation at
40 kHz in an MRC ultrasonic cleaner, washing with ethyl
FIGURE 1 Cell survival rates of lymphocytes in [RMIM]
RCOO ILs using MTT assay, showing higher cell viability for all
[HMIM]RCOO ILs than for their [OMIM] counterparts. [HMIM]
oxalate was found to be the least cytotoxic
acetate, oven-drying in
a vacuum oven (Yamato
ADP310C), and lyophilization using Yamato DC801
freeze dryer. Halide-to-carboxylate exchange reaction
was performed using Amberlyst A-26 resin (OH− form).
Structural spectral data were obtained and recorded using
Agilent Varian 500 MHz NMR spectrometer and
Shimadzu IR Prestige-21 spectrometer (with attenuated
total reflectance accessory).
presence of charged hydrophilic head groups and hydro-
phobic alkyl chain tails. It has been shown that true
aggregates are formed when the imidazolium-based IL
has a general formula of [Cnmim]X (where n = number
of carbon atoms in alkyl branch, mim = methyl-
3.2 | Solventless sonochemical
preparation and characterization of 1-alkyl-
3-methylimidazolium bromide [RMIM]
Br ILs
imidazolium, and X = Cl−, Br−, I−, BF4 , etc.) is ≥8.[38]
−
Measured cell viabilities using MTT assay are illus-
trated in Figure 1. In agreement with the trypan blue
assay, all [HMIM]RCOO ILs provided cell survival per-
centages (43–87%) higher than phenol (35%). On the
other hand, upon exposure to [OMIM]RCOO ILs, the
In a 100-ml round-bottom flask, a reaction mixture of 1:1
bromoalkane RBr (R = C6, C8) and 1-methylimidazole

PEREZ ET AL.
5
was sonicated in an ultrasonic bath for 8 hr until a vis-
cous yellow liquid is obtained. The resulting liquid was
cooled to room temperature before it was washed thrice
with ethyl acetate to remove any unreacted materials.
The mixture was vacuum-dried for 3–4 hr at 80ꢀC and
lyophilized overnight to obtain the pure IL as a viscous
yellow liquid. Both [HMIM]Br and [OMIM]Br ILs were
characterized via FT-IR, 1H-NMR, and 13C-NMR
spectroscopy.
1a: 1-hexyl-3-methylimidazolium bromide [HMIM]
Br: 90% yield. IR (cm−1, neat): 3,369, 3,071, 2,924, 1,569,
1,454, 1,169, 1,042. 1H-NMR (500 MHz, DMSO-D6):
δ(ppm) = 10.11 (s, 1H), 7.52 (d, J = 80.2 Hz, 2H), 4.26 (t,
J = 7.4 Hz, 2H), 4.06 (s, 3H), 1.90–1.76 (m, 2H), 1.38–1.76
(m, 6H), 0.80 (t, J = 6.7 Hz, 3H). 13C-NMR (500 MHz,
CDCl3): δ(ppm) = 137.08, 123.75, 122.01, 50.05, 36.71,
31.02, 30.20, 25.82, 22.33, 13.9.
1b: 1-octyl-3-methylimidazolium bromide [OMIM]Br:
93% yield. IR (cm−1, neat) νmax = 3,415, 3,066, 2,924,
2,854, 1,570, 1,462, 1,377, 1,336, 1,166. 1H-NMR
(500 MHz, DMSO-D6): δ(ppm) = 9.16 (s, 1H), 7.76 (dd,
J = 19.4, 18.0 Hz, 2H), 4.15 (t, J = 7.2 Hz, 2H), 3.84 (s,
3H), 1.80–1.72 (m, 2H), 1.36–1.13 (m, 10H), 0.84 (t,
J = 6.9 Hz, 3H). 13C-NMR (500 MHz, DMSO-D6):
δ(ppm) = 138.9,124.02, 122.69, 49.2, 36.21, 31.62, 29.84,
28.94, 28.79, 25.94, 22.52, 14.41.
fractions containing the desired product were collected,
subjected to rotary evaporation at 40ꢀC, and further lyophi-
lized for 24 hr to obtain the [RMIM]RCOO IL (2a–2h). All
carboxylate-based ILs were then characterized via FT-IR,
1H-NMR, and 13C-NMR spectroscopy.
2a: 1-hexyl-3-methylimidazolium crotonate [HMIM]
crotonate: 87% yield. IR (cm−1, neat) vmax = 3,334, 3,038,
2,920, 2,847, 1,550, 1,349, 1,164 cm−1
.
1H-NMR
(500 MHz, DMSO-D6): δ(ppm) = 9.820 (s, 1H), 7.840 (m,
2H), 6.212 (m, 1H), 5.639 (dq, J = 15.2, 1.6 Hz, 1H), 4.154
(t, J = 7.3 Hz, 2H), 3.850 (s, 3H), 1.729 (m, 2H), 1.618 (m,
3H), 1.207 (m, 6H), 0.807 (t, J = 6.8 Hz, 3H). 13C-NMR
(500 MHz, DMSO-D6): δ(ppm) = 170.477, 137.883,
134.140, 132.573, 123.955, 122.644, 49.021, 35.963, 31.031,
29.928, 25.608, 22.347, 17.492, 14.245.
2b: 1-octyl-3-methylimidazolium crotonate [OMIM]
crotonate: 92% yield. IR (cm−1, neat) νmax = 3,340, 3,140,
1
3,030, 2,900, 2,850, 1,650, 1,550, 1,350, 1,180 cm−1. H-
NMR (500 MHz, DMSO-D6): δ(ppm) = 9.884 (s, 1H),
7.779 (dt, J = 38.3, 1.6 Hz, 2H), 6.149 (m, 1H), 5.617 (m,
1H), 4.139 (t, J = 7.2 Hz, 2H), 3.834 (s, 3H), 1.731 (m,
2H), 1.611 (dd, J = 6.8, 1.7 Hz, 3H), 1.203 (m, 10H), 0.797
(t, J = 6.9 Hz, 3H). 13C-NMR (500 MHz, DMSO-D6):
δ(ppm) = 170.204, 137.993, 134.807, 131.810, 123.941,
122.642, 49.017, 35.969, 31.628, 29.956, 28.970, 25.968,
22.512, 17.490, 14.362.
2c: 1-hexyl-3-methylimidazolium cinnamate [HMIM]
cinnamate: 92% yield. IR (cm−1, neat) vmax = 3,345,
3.3 | Synthesis and characterization of
1-alkyl-3-methylimidazolium carboxylate
[RMIM]RCOO ILs
3,066, 2,931, 2,853, 1,550, 1,354, 1,181 cm−1 1H-NMR
.
(500 MHz, DMSO-D6): δ(ppm) = 9.717 (s, 1H), 7.780 (dt,
J = 37.1, 1.7 Hz, 2H), 7.413 (dd, J = 8.1, 0.9 Hz, 2H),
7.293 (m, 2H), 7.053 (m, 1H), 7.021 (d, J = 15.9 Hz, 1H),
6.370 (d, J = 15.9 Hz, 1H), 4.150 (t, J = 7.3 Hz, 2H), 3.857
(s, 3H), 1.733 (m, 2H), 1.197 (m, 6H), 0.793 (t, J = 5.0 Hz,
3H). 13C-NMR (500 MHz, DMSO-D6): δ(ppm) = 169.882,
137.742, 137.375, 135.004, 131.628, 128.992, 128.121,
127.193, 123.976, 122.656, 49.076, 36.042, 31.019, 29.892,
25.626, 22.341, 14.272.
A glass column (1-cm diameter) was packed with 5.0 g of
wet anion exchange resin Amberlyst A-26 (OH− form).
The column was then washed with deionized water,
followed by equilibration using increasing concentrations
(25, 50, 75, and 100%) of methanol (eluent). To load the
resin with the carboxylate anion, a 1% carboxylic acid
(crotonic, cinnamic, salicylic, or oxalic acid) in methanol
solution was allowed to pass slowly through the resin
until the pH of the eluate matched the pH of the original
acid solution. Subsequently, the column was washed with
methanol until the pH is constant.
A 0.05 M methanolic solution of [RMIM]Br (1a, 1b)
was then slowly passed through the column and the eluates
were obtained in portions. The fractions were subjected to a
silver nitrate test (3 drops of 1% AgNO3) to detect the pres-
ence of bromide ions. The presence of the desired IL
([RMIM]RCOO) is signified by the formation of a white
precipitate (silver carboxylate salt) while a yellow precipi-
tate (AgBr) indicates the presence of bromide ions. Further
addition of a few drops of 1% HNO3 solution dissolves the
silver carboxylate salts but not the AgBr precipitate. All
2d: 1-octyl-3-methylimidazolium cinnamate [OMIM]
cinnamate: 87% yield. IR (cm−1, neat) νmax = 3,340,
3,140, 3,070, 2,900, 2,850, 1,600, 1,550, 1,300, 1,250, 1,170,
1
1,020 cm−1. H-NMR (500 MHz, DMSO-D6): δ(ppm) =
9.70 (s, 1H), 7.78 (dt, J = 38.7, 1.7 Hz, 2H), 7.41 (m, 2H),
7.289 (dd, J = 10.4, 4.7 Hz, 2H), 7.209 (ddt, J = 12.1, 10.9,
5.5 Hz, 1H), 7.02 (d, J = 15.9 Hz, 1H), 6.369 (d,
J = 15.9 Hz, 1H), 4.149 (t, J = 7.2 Hz, 2H), 3.856 (s, 3H),
1.734 (m, 2H), 1.181 (m, 10H), 0.797 (t, J = 7.0 Hz, 3H).
13C-NMR (500 MHz, DMSO-D6): δ(ppm) = 169.880,
137.763, 137.375, 135.004, 131.615, 128.975, 128.107,
127.187, 123.963, 122.662, 49.085, 36.048, 31.614, 29.945,
28.963, 28.829, 25.982, 22.503, 14.372.
2e: 1-hexyl-3-methylimidazolium salicylate [HMIM]
salicylate: 94% yield. IR (cm−1, neat) vmax = 3,071, 2,953,

6
PEREZ ET AL.
2,926, 1,583, 1,450, 1,371, 1,287, 1,169 cm−1
.
1H-NMR
medium consisting of Sabouraud Dextrose Broth (Difco).
For each dilution, duplicate 2-ml volumes were prepared.
The inoculum was prepared from 24-hr old culture of the
test organism on Glucose Yeast Peptone agar (GYP) using
0.1% peptone water as suspending medium. The inocu-
lum used contained approximately 106 colony-forming
units (CFU) per ml. Forty microliters of the inoculum
was added to each tube containing the test dilution to
reach an inoculum level of approximately 104 CFU per
ml. The tubes were then incubated at 35ꢀC for 24 hr and
culture broth from each tube was streaked on GYP plates.
The plates were incubated at 35ꢀC and observed for
growth after 24 hr.
(500 MHz, DMSO-D6): δ(ppm) = 9.29 (s, 1H), 7.74 (dt,
J = 38.2, 1.5 Hz, 2H), 7.663 (m, 1H), 7.097 (m, 1H), 6.608
(m, 1H), 6.570 (m, 1H), 4.124 (t, J = 7.3 Hz, 2H), 3.836 (s,
3H), 1.726 (m, 2H), 1.200 (m, 6H), 0.803 (t, J = 7.2 Hz,
3H). 13C-NMR (500 MHz, DMSO-D6): δ(ppm) = 171.999,
163.243, 137.114, 131.848, 130.335, 123.989, 122.671,
120.740, 116.225, 110.045, 49.165, 36.103, 30.978, 29.819,
25.589, 22.317, 14.250.
2f: 1-octyl-3-methylimidazolium salicylate [OMIM]
salicylate: 88% yield. IR (cm−1, neat) νmax = 3,400, 3,140,
3,070, 2,850, 1,580, 1,480, 1,450, 1,370, 1,320, 1,290,
1,160, 1,000 cm−1
.
1H-NMR (500 MHz, DMSO-D6):
δ(ppm) = 9.329 (s, 1H), 7.750 (dt, J = 38.3, 1.6 Hz, 2H),
7.659 (dd, J = 7.6, 1.6 Hz, 1H), 7.095 (m, 1H), 6.597 (m,
1H), 6.556 (m, 2H), 4.127 (t, J = 7.3 Hz, 2H), 3.842 (s,
3H), 1.730 (m, 2H), 1.187 (m, 10H), 0.814 (t, J = 7.0 Hz,
3H). 13C-NMR (500 MHz, DMSO-D6): δ(ppm) = 171.953,
163.354, 137.169, 131.665, 130.312, 123.984, 122.681,
121.010, 116.248, 116.178, 49.169, 36.091, 31.612, 29.887,
28.937, 28.794, 25.953, 22.512, 14.359.
2g: 1-hexyl-3-methylimidazolium oxalate [HMIM]2-
oxalate: 88% yield. IR (cm−1, neat) vmax = 3,088, 2,931,
2,859, 1,712, 1,628, 1,567, 1,460, 1,371, 1,169 cm−1. 1H-
NMR (500 MHz, DMSO-D6): δ(ppm) = 9.333 (s, 1H), 7.740
(d, J = 36.9 Hz, 2H), 4.139 (t, J = 7.2 Hz, 2H), 3.834 (s, 3H),
1.731 (m, 2H), 1.203 (m, 6H), 0.797 (t, J = 7.0 Hz, 3H). 13C-
NMR (500 MHz, DMSO-D6): δ(ppm) = 165.395, 137.207,
123.990, 122.661, 49.123, 36.040, 30.971, 29.824, 25.552,
22.301, 14.226.
2h: 1-octyl-3-methylimidazolium oxalate [OMIM]2-
oxalate: 86% yield. IR (cm−1, neat) νmax = 3,440, 3,150,
3,000, 2,900, 2,850, 1,700, 1,630, 1,460, 1,370, 1,170 cm−1.
1H-NMR (500 MHz, DMSO-D6): δ(ppm) = 9.340 (s, 1H),
7.780 (dd, J = 19.8, 18.3 Hz, 1H), 4.147 (t, J = 7.2 Hz, 2H),
3.841 (s, 3H), 1.739 (m, 2H), 1.193 (m, 10H), 0.807 (t,
J = 7.0 Hz, 3H). 13C-NMR (500 MHz, DMSO-D6):
δ(ppm) = 165.214, 137.152, 123.988, 122.673, 49.127, 36.108,
31.606, 29.886, 28.933, 28.795, 26.927, 22.496, 14.356.
3.5 | Evaluation of the cytotoxicity of
the ILs
3.5.1 | Lymphocyte culture
The cell viability tests (trypan blue exclusion and MTT
assays) were performed by the Biological Research and
Services Laboratory of UP NSRI. Blood sample from a
healthy individual was collected in
a green-top
vacutainer tube. About 3.5 ml of whole blood was care-
fully overlaid onto an equal volume of Histopaque® in
two centrifuge tubes, followed by centrifugation at
2,000 rpm for 30 min at room temperature. Approxi-
mately, 1.5 ml of clear plasma top layer was discarded
from each tube and the cloudy buffy coat layer was
transferred to a clean centrifuge tube. Ten milliliters of
serum-free RPMI medium were added and the tube was
inverted several times to wash the lymphocytes. The
tube was centrifuged at 1,000 rpm for 15 min at room
temperature. Afterward, the supernatant was discarded,
leaving about 0.5 ml of supernatant with the pellet.
Another 10 ml of serum-free RPMI medium was added
to the resuspended pellet. The tube was inverted several
times before centrifugation at 1,000 rpm for 10 min at
room temperature. The supernatant was removed, leav-
ing about 0.5 ml, and supplemented RPMI medium
(RPMI with fetal bovine serum, Penicillin–Streptomy-
cin, Amphotericin B) was added such that the final cell
density was about 2 × 106 cells/ml. The pellet was
resuspended by inverting the tube several times and the
culture was incubated at 37ꢀC for an hour with occa-
sional mixing.
3.4 | Determination of the MIC of
the ILs
The MICs of the synthesized ILs were determined by the
Microbiological Research and Services Laboratory of the
University of the Philippines Natural Sciences Research
Laboratory (UP NSRI) through the broth dilution
method. The test organism C. albicans (UPCC 2168) used
in the assay was obtained from the University of the Phil-
ippines Culture Collection (UPCC).
3.5.2 | Trypan blue exclusion assay
The tube method with 2-ml volume of test dilutions
was used. Each IL sample was diluted with a broth
Fifty microliters of 1% IL sample solution, supplemented
RPMI, sterile distilled water, and 6.5 mg/ml phenol were

PEREZ ET AL.
7
dispensed into three microcentrifuge tubes each. Then,
450 μl aliquots of lymphocyte culture were added to the
tubes containing different treatment solutions. Cultures
were incubated at 37ꢀC for 24 hr. Afterward, treated cul-
tures were obtained for cell counting. Seven μl of incu-
bated culture were added to 7 μl of trypan blue, and 7 μl
of the mixture were placed in a hemocytometer. The
number of live lymphocytes and the number of dead lym-
phocytes were counted in all 25 squares within the 1 mm
center grid. Cell density (number of cells per ml) is com-
puted as follows:
(>90%). Selected 1-alkyl-3-methylimidazolium carboxyl-
ate [RMIM]RCOO (RCOO = cinnamate, crotonate, oxa-
late, salicylate) ILs were also obtained via a facile, direct
halide-to-anion exchange method. The efficiency of the
method was proven by the relatively high % yield of the
[RMIM]RCOO ILs (≥86%). Chemical structures and
purity were confirmed by spectroscopic methods. The
MICs of all [RMIM]RCOO ILs were found to be ≤1%.
Cytotoxicity tests using trypan blue exclusion and MTT
assays showed that [HMIM]RCOO ILs are less cytotoxic
than [OMIM]RCOO ILs and that varying the carboxyl-
ates has no extensive effect on toxicity. [HMIM]crotonate
and [HMIM]oxalate ILs provided the highest cell viability
percentages in trypan blue exclusion and MTT assays,
respectively.
total number of cells
Celldensity =
× 104 × DF
number of 1 mm2 squares
where, DF is dilution factor = 2.
Our findings continue to emphasize the potential of
these novel ILs as antifungal agents. A lower concentra-
tion range is suggested for MIC determination for future
studies. We further suggest ADMET studies on these
compounds to investigate their physicochemical proper-
ties and toxicity.
3.5.3 | MTT assay
Ninety microliters of the cell culture suspension was
placed into the wells of two 96-well flat-bottomed micro-
plate containing 10 μl of the treatment solutions in tripli-
cates (plate 1: supplemented RPMI, phenol, [RMIM]
RCOO; plate 2: [RMIM]RCOO, 0.5% methanol). Then,
100 μL of supplemented RPMI medium without cells was
placed into three wells which serves as the blank. The
plates were then incubated at 37ꢀC with 5% CO2 (using a
CO2 pack) for a duration of 24 hr. After incubation, 10 μl
of MTT (5 mg/ml in PBS) were added to each well. The
plates were incubated again at 37ꢀC with 5% CO2 for
another 4 hr. Afterward, 100 μl of acidic isopropanol
(0.04 N HCl in isopropanol) was added into each well.
The plates were incubated at room temperature in the
dark overnight. The absorbance values were measured at
560 nm using 650 nm as the reference wavelength. The
cell survival rates (%CS) based on the corrected optical
densities (A560–A650) were computed using the following
formula:
ACKNOWLEDGMENTS
The authors are grateful for the funding provided by the
Department of Science and Technology-Philippine Coun-
cil for Health and Research and Development (DOST-
PCHRD). The authors also acknowledge the University
of the Philippines Diliman-Natural Sciences Research
Institute for performing the biological assays. The
authors would like to thank Ms. Kim Marie D. Sisican
and Ms. Zyra Grace C. Zaragosa for their kind assistance
in the manuscript preparation and Mr. Jonathan
N. Patricio for his invaluable help in designing the graph-
ical abstract.
CONFLICT OF INTEREST
The authors declare no conflicts of interest.
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SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
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How to cite this article: Perez SJLP,
Atayde Jr. EC, Arco SD. Synthesis and biological
evaluation of some novel 1-alkyl-3-
methylimidazolium carboxylate ionic liquids as
potential antifungal agents. J Chin Chem Soc. 2020;
1–8. https://doi.org/10.1002/jccs.201900366