Novel 3-methyl-2-alkylthio benzothiazolyl-based ionic liquids: Synthesis, characterization, and antibiotic activity

Article abstract of DOI:10.3390/molecules23082011

Three series of novel 3-methyl-2-alkylthio benzothiazolyl ionic liquids (ILs) were synthesized for the first time. After structural identification, their melting point, solubility, and thermostability together with antibiotic activity were determined successively. As a result, 3-methyl-2-alkylthio benzothiazolyl p-toluene sulfonate was found to have the highest antibacterial activity among the three series of ILs. Meanwhile, it has a good solubility in water as well. On the basis of comprehensive comparison with similar compounds, the effect of cations and anions of these benzothiazolium ILs on typical physical properties together with antibiotic performance was explored and discussed, which is very beneficial to take the greatest advantage of their structural designability for various purposes. Furthermore, the experiment data preliminarily discovered the relationships of the structure-properties/activities of the above three kinds ILs to a certain extent, which can provide useful references for future research and for the potential application of these new ILs as surfactant antiseptics or agricultural chemicals.

Full text of DOI:10.3390/molecules23082011

molecules
Article
Novel 3-Methyl-2-alkylthio Benzothiazolyl-Based
Ionic Liquids: Synthesis, Characterization,
and Antibiotic Activity
Teng He Zhang 1,†, Hao Xi He 2,†, Jun Liang Du 3, Zhi Jian He 3,* and Shun Yao 1,
*
ID
1
School of Chemical Engineering, Sichuan University, Chengdu 610065, China; zthrainbow@foxmail.com
Mianyang Blood Center, Mianyang 621000, China; pharmposter2012@163.com
College of Chemistry and Chemical Engineering, Mianyang Normal University, Mianyang 621000, China;
15928065204@139.com
2
3
*
†
Correspondence: youngsun9802@163.com (Z.J.H); cusack@scu.edu.cn (S.Y)
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 23 July 2018; Accepted: 6 August 2018; Published: 12 August 2018
Abstract: Three series of novel 3-methyl-2-alkylthio benzothiazolyl ionic liquids (ILs) were
synthesized for the first time. After structural identification, their melting point, solubility,
and thermostability together with antibiotic activity were determined successively. As a result,
3-methyl-2-alkylthio benzothiazolyl p-toluene sulfonate was found to have the highest antibacterial
activity among the three series of ILs. Meanwhile, it has a good solubility in water as well. On the
basis of comprehensive comparison with similar compounds, the effect of cations and anions of
these benzothiazolium ILs on typical physical properties together with antibiotic performance was
explored and discussed, which is very beneficial to take the greatest advantage of their structural
designability for various purposes. Furthermore, the experiment data preliminarily discovered the
relationships of the structure-properties/activities of the above three kinds ILs to a certain extent,
which can provide useful references for future research and for the potential application of these new
ILs as surfactant antiseptics or agricultural chemicals.
Keywords: 3-methyl-2-alkylthio benzothiazolyl-based ionic liquids; solubility; thermal stability;
antibiotic activity
1. Introduction
Benzothiazole derivatives are an important kind of heterocycles which are popularly applied
as a variety of pharmaceutical agents and bioactive natural products. They have received more and
more attention because of their various pharmacological activities such as their antimicrobial [
1
6
],
],
anticancer [ ], anti-inflammatory [ ], analgesic [ ], antitubercular [ ], and antidepressant activities [
2
3
4
5
etc. Moreover, some of them have efficient catalytic activity and are used in chemical reactions and so
forth [ ]. With a common thermally stable electron-withdrawing nucleus, benzothiazole derivatives
7
are a class of molecules displaying a variety of chemical and biological functions.
At present, ionic liquids (ILs) are exhibiting a series of unique properties, such as a low
melting point, non-flammability, good solvation, high thermal stability and so on [8]. Meanwhile,
ILs possess an ideal designability for their structures, which are promoting people to create
more new ILs based on various nuclei and substituent groups, and the innovation of their
structures always results in new bioactivity. For instance, the biocidal behaviors of larger
organic cations are commonly used because of their inhibition of bacterial, fungal or insect
growth [
9
–
12]. Among them, 1-[(1R,2S,5R)-(
−
)-menthoxymethyl]-3-alkylimidazolium chlorides
Molecules 2018, 23, 2011; doi:10.3390/molecules23082011
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2011
2 of 13
demonstrated an extremely high activity against microbes with a wide spectrum of activity and
alkoxymethyl(2-hydroxyethyl)dimethylammonium acesulfamates have been shown to be potential
insect feeding deterrents. Especially, the series of ILs with imidazolium nucleus have been
widely investigated by a great number of researchers and related work has made a lot of
progress [13]. The mechanism behind their antimicrobial action is also considered similar to that
of cationic surfactants. Moreover, a good correlation of the alkyl chain length in the cations of
1,3-dialkylimidazolium ILs with their cytotoxicity in two different cell cultures and the marine bacteria
V. fischeri has been found [14]. Moreover, some researchers reported and employed a series of important
classes of ionic liquids for their toxicity towards both prokaryotic and eukaryotic microorganisms [15].
By comparison, it was found the imidazolium ion shows higher toxicity towards freshwater algae than
the pyridinium ion [16,17]. However, no research work has been published these bioactivities of the
ionic liquids with benzothiazolium cation together with related sulfhydryl substituents, and a little
study only focused on their synthesis, physical properties and catalytic application (e.g., [18]). In recent
years, some reviews have revealed that many effective antimicrobial agents have a heterocyclic segment
within their structure [19]. Generally, the preceding mercaptobenzothiazole (MBT) derivatives have
good biological activities [20]. However, most of them have a very poor solubility in water, so their
application is limited. Whether the assembled ILs can generate different antimicrobial activity or
other new biological activities needs to be explored. For the above considerations, it was planned
in this study to introduce the substituted benzothiazole group into ionic liquids for the synthesis of
3-methyl-2-alkylthiobenzothiazolyl ILs with three kinds of anions. According to online search with
SciFinder Scholar (Chemical Abstract Service), 13 compounds (A1–A5, B1–B5 and C1–C3) have not
been reported.
2. Results and Discussion
2.1. Synthesis Method and Conditions
In a previous study, the antimicrobial activity of 23 newly synthesized pyridinium ILs was
tested against a panel of bacteria and fungi [21]. The results proved that all tested ILs were effective
−
antibacterial and antifungal agents and that the inhibition zone of these ILs with PF6 is 13.4–20.9 mm
(MIC = 3.9–125 µg/mL). More recently, the antimicrobial activity of 24 ILs paired with different anions
(hydrophilic anions: Cl, Br, SCN, DCA, and BF4; hydrophobic anion: NTf2; amino acid anions: Gly, Ala,
Ser, Pro, and Asn) have been investigated and the results clearly demonstrated that introduction of
the hydrophobic anion of bis((trifluoromethyl)sulfonyl)amide (NTf2−) and elongation of the cations
substitutions could result in higher bioactivity (EC50 = 0.31–3.86 Mm) [8]−. Therefore, the combination
of benzothiazolium cations with three anions of OTs−, PF6−, and NTf2 was finally selected in this
study. The synthetic route of 13 kinds of ILs is shown in Scheme 1, and these ionic liquids were
obtained successfully through the three-step approach as follows:
(1) 2-alkylthio benzothiazole (compound
reaction between MBT and alkyl bromide;
b) was firstly obtained via a nucleophilic substitution
(2) 3-methyl-2-alkylthio benzothiazolyltosilate (compound
A
) was obtained by the reaction
between 2-alkylthiobenzothiazole and the 4-methyl benzene−sulfonic a−cid methyl ester;
(3) 3-methyl-2-alkylthiobenzothiazolyl salts with NTf2 and PF6 (compounds
synthesized lastly by anion exchange reaction.
B and C) were
There are two reaction sites in MBT, which include sulfydryl and amino. Li et al. [22] found
that lower temperature were more beneficial for the sulfydryl substituent, and higher temperature
were more advantageous to the amino substitution reaction for MBT when other conditions were the
same. In summary, the temperature of the reaction plays an important role in the thiol alkylation
reaction of MBT. In our experiment, the synthesis reaction of compound
b was conducted for nearly
10 h at 343 K or 18 h at 333 K. If the temperature was lowered than 313 K, the obvious reaction would
not have been observed within 24 h. Through comparison of various reaction conditions, the range

Molecules 2018, 23, 2011
3 of 13
of 338–343 K was finally selected as the appropriate temperature range for the synthesis of b1
–b5,
and the yield of reached 90.35–96.48%. After the reaction, excessive inorganic salts were removed
b
by filtration. Then the filtrate was washed with a saturated Na2CO3 aqueous solution and water
successively. The residual solvent and water were removed under a vacuum to obtain a crude product,
which was further purified with silica gel column chromatography using 5 to 10% (v/v) EtOAc in
petroleum ether (boiling range: 30–60 ◦C) as an eluent to afford pure 2-methylthio benzothiazole.
Scheme 1. The synthetic routes of 3-methyl-2-alkylthio benzothiazolyl-based ILs (R = CH3-, C2H5-,
n-C4H9-, n-C6H13-, n-C8H17-, benzyl groups).
In the synthesis process of compound
was doubled. When it was higher than 408 K, the byproducts of the reaction would increase and
then the purity of would decrease. The optimal temperature of the synthesis reaction of was
determined to be 403 K after repeated experiments. As a result, a 0.02 mol product reacted with
A, if the temperature was below 353 K, the reaction time
A
A
b
equimolar p-methyltoluene sulfonate at 403 K for 6 h, and the viscous fluid product was obtained when
the system was cooled to room temperature. The viscous fluid product was washed with deionized
water four times at 393 K. Then the aqueous solution was merged and centrifuged, and the upper
light yellow water layer was isolated after demulsification. Finally, the product was dehydrated under
vacuum and further dried with magnesium sulfate to give product
A
. As shown in Table 1, the yield
A5 reduced with an increase
of A1 A5 was in the range of 97–68%. It was found that the yield of A1
–
–
of the carbon chains of thiol alkyl at the same reaction temperature, which resulted from the increase
of the steric hindrance of the carbon chain of thiolalkyl.
In the last step, compound
homogeneous solution was obtained. A total of 25 mL of LiNTf2 aqueous solution (0.0105 mol) was
added dropwise to the solution of compound and the mixture was stirred for 12 h. After the
A (0.01 mol) was dissolved in 20 mL of water and a transparent
A
reaction, a light yellow solid product at the bottom of the flask was separated out from the reaction
mixture, and then it was washed with deionized water for four times and recrystallized in ethyl acetate,
successively. The product was dried and obtained as compound B.

Molecules 2018, 23, 2011
4 of 13
Table 1. The structures, yields, and other information of the prepared ILs.
Entry
A1
ILs Structure
ILs abbr.
M.W. (HR-MS)
Yield/%
Reaction Time/h
[3-Me-2-S-C1-MBT][OTs]
367.5064
97
6
6
6
A2
A3
[3-Me-2-S-C2-MBT][OTs]
[3-Me-2-S-C4-MBT][OTs]
381.5328
409.5856
95
85
A4
A5
[3-Me-2-S-C6-MBT][OTs]
[3-Me-2-S-C8-MBT][OTs]
437.6389
465.6920
72
68
6.5
7
B1
B2
B3
[3-Me-2-S-C1-MBT][NTf2]
[3-Me-2-S-C2-MBT][NTf2]
[3-Me-2-S-C4-MBT][NTf2]
476.4586
490.4852
518.5380
95
93
95
12
12
13
B4
[3-Me-2-S-C6-MBT][NTf2]
546.5915
99
13
B5
C1
C2
C3
[3-Me-2-S-C8-MBT][NTf2]
[3-Me-2-S-C2-MBT][PF6]
[3-Me-2-S-C6-MBT][PF6]
[3-Me-2-S-Bn-MBT][PF6]
574.6445
355.3031
411.4093
417.3726
96
90
94
93
13
11
11
11
2.2. Spectral Data and Analysis
The data of IR, 1H NMR, 13C NMR, 19F NMR, and ESI-MS of all the synthesized ILs are shown in
the Supplementary Materials, which can be used to identify their structures and related substituted

Molecules 2018, 23, 2011
5 of 13
groups through comparison with the spectral information from the previous study of benzothiazolium
ILs [23]. As an example, the four signals of 8.15 (1H), 7.93 (1H), 7.72–7.68 (1H), 7.59–7.56 (1H)
in 1H NMR (600 MHz, CDCl3) of compound A1 ((3-Me-2-S-C1-MBT)(OTs)) can be assigned to the
four protons in the benzene ring in the benzothiazolium nucleus, and the two peaks at 7.65 (2H)
and 7.06 (2H) can be assigned to the protons of the AA0BB0 system in the benzene ring of the anion.
The signals at 4.17 (s, 3H), 3.06 (s, 3H), and 2.30 (s, 3H) are attributable to the three -CH3 groups
δ
δ
δ
connected with N, S, and O atoms, respectively. The corresponding carbon signals can also prove the
existence of above nucleus and groups. In its IR spectra, an absorbance at 3092.6 and 3023.6 cm−1 can
be assigned to the stretching vibration of aromatic hydrogens in the benzene rings of the cation and
anion, which also cause the signals below 1000 cm−1 because of bending vibrations. The stretching and
−1
bending vibrations of the various methyl groups result in several peaks in the range of 3000–2900 cm
and 1500–1400 cm−1, respectively; the stretching vibration region of the benzenesulfonyl group in
the anion exists in the range of 1260–1000 cm−1, and the wavenumber of the asymmetric stretching
vibrations is higher than that of the symmetric one. The absorbance in the range of 1600–1400 cm−1
belongs to the skeleton vibration of the benzene rings and the C=N signal usually appears around
1600 cm−1. Furthermore, the Finnigan TSQ Quantum Ultra LC/MS/MS system (Thermo Fisher,
San Jose, CA, USA) in a dual ion pattern and full-scan mode (100–500 m/z) was operated to determine
the cations and anions (see Figure S1 in Supplementa◦ry Materials) under the following conditio◦ns:
N2dryinggas with a flow rate of 700 L
·
h−1 and 400 C, a 3.0 kV capillary voltage−, and a 100 C
−
capillary temperature. The MS bars of the three various anions appear at m/z 171 (TsO ), 280 (NTf2 ),
−
and 145 (PF6 ), as calculated, successively. Moreover, it was found that the mass to charge ratio of the
cations increases with the growth of their alkyl length. As a conclusion, MS can be used to discriminate
them rapidly.
2.3. Physical State and Melting Points of Synthesized ILs
As known to all, as a kind of organic molten salts displaying ionic-covalent crystalline structures,
ILs can remain in a liquid status over a very wide temperature range (e.g., 233.15–423.15 K) and has
a low melting point ( 423.15 K). Generally, a working definition is that an ionic liquid is a salt with
≤
a melting temperature below the boiling point of water. Seddon et al. [24] also hold the opinion that
there is nothing sacred about a temperature of 100 ◦C and that it is merely a convenient and arbitrary
marker. The melting point is one of the important properties of ILs, which depends on the different
compositions of cations and anions. The total lattice energy of the ionic compounds is higher and their
melting point is higher. Various combinations of cation and anion will have significant effects on the
melting point of the ILs on the basis of their volume, structural symmetry, planarity, H-bond, induction
effect, and so on [25]. As shown in Table 2, it can be observed that the ILs of A1
–A5 have lower melting
points than the other two series of ILs when their cations are the same. The melting points of B1
–B4
decrease with the increasing length of the alkyl chain of the cation, which have the same anion of
(NTf2−). However, if the length of the alkyl chain of the cation was much longer, the melting point
◦
would rise up (such as B5, 78.9–79.5 C). This U-type trend is the same as that stated in previous
reports on the melting points of immidazolium ILs with different alkyl side chains [26]. The results
show that the symmetry of the cation of the ILs will become lower with the increasing length of the
alkyl chain of the cation. If the length of the alkyl chain of the cation is further increased, it will result
in the increase of the intermolecular van der Waals force and the aggregation among the lipophilic
−
groups in the structures of different ILs. When the cation is the same, PF6 has the smallest volume
among the three kinds of anions, which leads to a highe−r crystal stacking density and a higher lattice
−
−
energy. The order of the anion volume is as follows: OTs > NTf2 > PF6 , so their melting points are
significantly different.

Molecules 2018, 23, 2011
6 of 13
Table 2. The state and melting points of the synthesized ILs.
◦
◦
Entry
State under Room Temperature
Entry
Melting Point/ C
Entry
Melting Point/ C
A1
A2
A3
A4
A5
Brown viscous liquid
Brown viscous liquid
Brown viscous liquid
Brown viscous liquid
Brown semi-solid
B1
B2
B3
B4
B5
78.9–79.9
58.1–58.8
55.1–56.0
49.9–50.1
78.9–79.5
C1
C2
C3
146.4–147.3
206.6–207.0
171.3–171.9
2.4. Solubility Investigation
The solubility behavior of ILs in common solvents always play key roles in their usage.
Based on previous study [16], many hydrophilic acesulfamate-based ILs were antimicrobially active.
In this section, the solubility values of all obtained ILs were evaluated in eight common solvents at
298.0 ± 0.5 K, which was investigated using Al-mohammed0s method [27]. The results were defined
as miscible (marked with +), partially miscible (marked with
respectively [28]. Generally, ionic compounds have an ideal solubility in polar solvents on the basis of
the “like dissolves like” theory, and there is a certain relationship between their dissolution behavior
±
), or immiscible (marked with
−
),
and the dielectric constant (ε) of the related solvents. According to the results of the solubility test
shown in Table 3, it was found that some ILs can be dissolved in the solvents with moderate or low
polarity, except in n-hexane; and the individual cations and anions can be tunable to produce ILs with
the desired solubility. For instance, the typ−e of anion plays a strong impact on the solubility of ILs
−
in water [29], those ILs with NTf2 or PF6 are immiscible with water, which can hardly provide
H-bond interaction with hydrone and always appear as hydrophobic anions by researchers in the
previous study on immidazolium ILs. All of the ILs with OTs− are immiscible with ethyl acetate,
but the ILs combined with NTf2– are miscible with ethyl acetate. All the studied ILs are moderately
miscible or miscible with acetone, which has an unsaturated and polarized carbon–oxygen bond and
can provide more intermolecular interaction than protic solvents. When their polarity and dielectric
constant further declines, the solvents only have the selective dissolving capacity for a part of the
tested ILs. Finally, it can be concluded that the dispersion force between these ILs and alkane is weaker
than that between alkane molecules. Therefore, they are not miscible with n-hexane.
Table 3. The solubility of the 13 ionic liquids in various common solvents.
No. Water Alcohol Acetone Tetrahydrofuran Ethyl Acetate Chloroform Toluene n-Hexane
ε
80.1
+
+
+
+
25.8
+
±
±
+
20.7
±
±
±
+
7.58
-
-
7.3
-
-
-
-
−
+
+
+
+
+
−
+
−
5.1
-
2.4
-
-
1.9
-
-
A1
A2
A3
A4
A5
B1
B2
B3
B4
B5
C1
C2
C3
+
+
+
+
+
+
+
+
+
−
+
−
+
-
-
−
−
−
−
±
−
±
+
−
±
−
−
−
−
−
−
−
−
−
−
−
+
+
±
+
−
−
−
−
−
−
−
−
±
+
+
+
+
+
+
+
+
+
+
+
+
+
+
±
-
+
−
−
−
−
+
−
Note: Al-mohammed’s method [27] was employed for the determination of solubility. For liquid samples, (+) means
miscible: a drop of the compound dissolved in a few drops (1–5) of solvent; (
of the compound dissolved in 10 drops (<2 mL) of solvent; ( ) means immiscible: a drop of the compound did
not dissolve in 1–2 mL of solvent. For solid samples, (+) means miscible: 50 mg of the compound dissolved
in a few drops (1–5) of solvent; ( ) means moderately miscible: 50 mg of the compound dissolved in 10 drops
(2 mL) of solvent; ( ) means immiscible: 50 mg of the compound undissolved in 1–2 mL of solvent. Measurement
temperature: 298.0 ± 0.5 K.
±
) means moderately miscible: a drop
−
±
−

Molecules 2018, 23, 2011
7 of 13
2.5. Thermal Stability
Considering the potential conditions in their application, the thermal stability of these ILs was
evaluated in terms of the decomposition temperature, which is determined with a TGA/SDTA851
◦
thermogravimetric analyzer (Metrohm, Switzerland) with a heating rate of 10 C/min from 30 to
450 ◦C under a nitrogen atmosphere. Their thermal stability is influenced by the interactions between
the carbon atom and the heteroatom or the heteroatom and the H-bond [30]. The structures, together
with properties of the cation and anion, are closely related with the decomposition temperature of the
ILs [31]. As a result, the TGA thermograms of A1–A5 IL (see Figure S2 in the Supplementary Materials)
show two common weight loss regions, successively. The first region is within the temperature range
of 50–145 ◦C, and the weight loss of the ILs in this range is 7–17 wt %. This weight loss is ascribed
to the evaporation of residual water, and compounds A1
temperature, which is in accordance with the fact that they have a higher solubility in water. The second
stage is from 145 to 350 ◦C, the onset decomposition temperature of A1 A5 is 145 ◦C and they were
decomposed completely around 350 ◦C. The degradation of A1
A5 with a 20–80% total weight loss
–A5 have a strong hygroscopicity at room
–
–
within the above range of decomposition temperatures is due to the breakage of the nuclear structure
of these ILs. Figure S2c–f display the thermogravimetric analysis results of the ILs and the two series of
−
B
and
C
(e.g., B1
–
B5 containing NTf2 ). Generally, the coordination, affinity, and hydrophilicity of the
anions are closely related with the thermostability of ILs. As a result, it can be concluded that the order
−
−
of thermal stability of anions is NTf2 > OTs− > PF6−. ILs containing NTf2 are the most promising
candidates for higher−temperature application, while the thermal stability of ILs with OTs− is weaker
than those with NTf2 because of the electron-donating effect of methyl on the benzene ring reduces
the force constant of the C-S bond of the sulfonic acid group. On the other hand, the relationship curve
of thermal stability of the cations and the total carbon number of the side chain on the cation zigzags.
The cation of 3-methyl-2-alkylthio benzothiazolyl has a worse thermostability than the immidazolium
cation for its more unsaturated structure, and the latter usually begins to degrade at temperatures
above 350 ◦C. Of course, the maximum applicable temperature of these ionic liquids depends on the
duration of the applications and tolerance to changes in quantity and quality.
2.6. Antibiotic Activity
Finally, the antibiotic effect of ILs was also investigated by testing their activity against some
disease-causing crop bacteria with different antimicrobial resistance profiles. The related data of this
study were the average values of the three parallel experiments unless there is special explanation.
The results of the inhibitory zone diameter (mm) are summarized in Table 4. Through careful
observation and comparison, it can be found that the two series of
effects compared against the blank control groups. Furthermore, the inhibitory zone diameters (mm)
of the ILs of are obviously wider than those of the ILs of , so the antibiotic function of the ILs of
A1 A5 is more ideal than the series of . Compared with the other study on the antimicrobial activity
of benzoazole ionic liquids (an 8 mm inhibition zone against Gram-positive B. subtillis for [C6mim][TBT]
A and B all have obvious antibiotic
A
B
–
B
and a 9 mm inhibition zone against P. aeruginosa for both [C4mim][TBO] and [C2mim][TBI]) [32], the
A
series has more ideal antibiotic activity.
Table 4. The inhibitory zone diameter s (mm) of the ILs in the A and B series.
Staphylococcus Aureus
Escherichia coli
Chinese Cabbage Soft Rot Disease
Sample
Mean
SD
Mean
SD
Mean
SD
A1
A2
A3
A4
A5
0.0
0.0
2.0
1.2
3.6
2.6
6.0
0.0
1.0
2.6
1.5
7.0
0.0
6.7
16.7
16.7
15.7
0.0
1.2
3.1
0.6
0.6
14.0
19.3
24.0
23.0
15.0
21.0
19.7
13.3

Molecules 2018, 23, 2011
8 of 13
Table 4. Cont.
Escherichia coli
Staphylococcus Aureus
Chinese Cabbage Soft Rot Disease
Sample
Mean
6.7
10.3
9.3
6.7
9.3
SD
1.2
4.5
3.1
1.2
1.2
Mean
6.0
10.7
10.0
8.7
SD
0.0
4.2
2.0
3.1
2.3
Mean
10.7
11.3
8.3
11.3
6.7
SD
1.2
2.3
4.0
2.3
1.2
B1
B2
B3
B4
B5
7.3
5% Methanol (c)
10% Acetone (d)
0.0
1.0
0.0
2.4
0.0
3.0
0.0
0.0
0.0
0.0
0.0
0.0
Rice Bacterial Blight
Xanthomonas Axonopodispv. Citri
Ginger Bacterial Wilt
Sample
Mean
SD
Mean
SD
Mean
SD
A1
A2
A3
A4
A5
B1
B2
B3
B4
B5
6.0
6.0
6.0
12.3
17.7
6.0
7.3
6.0
7.3
6.0
0.0
0.0
0.0
5.5
3.2
0.0
2.3
0.0
2.3
0.0
0.0
11.7
14.0
16.3
16.7
6.0
8.0
8.0
9.3
6.0
0.0
1.5
2.6
1.2
3.1
0.0
3.5
3.5
3.1
0.0
2.0
6.0
12.0
14.0
16.3
6.0
7.3
7.3
10.0
6.0
1.2
0.0
1.0
0.0
0.6
0.0
2.3
2.3
3.5
0.0
5% Methanol (c)
10% Acetone (d)
2.0
3.0
4.2
4.2
0.0
1.0
0.0
0.0
1.0
2.0
0.0
0.0
Note: The concentration of A1
–
A5 and B1–B5 was 1 mg/mL. Methanol and acetone were used as blank controls.
The activity of C1–C3 against some disease-causing crop bacteria was not investigated due to their poor solubility.
Then the minimum inhibitory concentrations (MIC) of the ILs were determined, which were
used to compare both the antibacterial activities of the and series of ILs against representative
A
B
standard strains of Gram-positive and Gram-negative bacteria by the tube double dilution method [33].
For comparison, a common aminoglycoside antibiotic (neomycin) and an important natural
antibacterial compound (emodin) were selected as positive controls, which are the representatives
of water-soluble and lipid-soluble agents with conjugated unsaturated structures, respectively.
Three parallel experiments were carried out in each group and the relevant data were processed
to obtain Table 5. The various combinations of different cations and anions could significantly affect
the biological activities of the target ILs [34]. Considering that the structure of ILs is similar to
surfactants and cationic surfactants possessing interfacial properties, these ILs can also injure the
membrane of common cellular structures by attacking the lipid structure of the lipo-polysaccharide
membrane of microorganisms with the alkyl groups on their cations [35]. On the one hand, the effective
concentration of hydrophobic ionic liquids in water is not enough to produce a significant impact on
the cell membrane, so this could result in lower activity. On the other hand, the ionic liquids depend
on hydrophobic groups to combine with the phospholipid bimolecular layer of the microbial cell
membrane to change its structure and permeability. The hydrogen on the sulfydryl of A1
ILs was replaced by the alkyl groups with various lengths, which could result in considerable difference
in the antibacterial functions of the two series of ILs. For example, the antibacterial action of A1 A5
–A5/B1–B5
–
increased successively against Staphylococcus aureus, Xanthomon asaxonopodispv. citri, and the Ginger
bacterial wilt strains with the increase of the total number of carbons in the side chain (see Table 4).
All the previous studies have also proved that each ionic liquid has the most appropriate alkyl chain
length for a specific microorganism. It cannot be simply concluded that the longer the alkyl chain is,
the higher their activity is. When the cation is the same, the anion in the ILs will show a significant
influence on their biological activity (see Table 5). The MIC values of typical cationic surface-active
fungicides and antimicrobials usually range from 8 to 500
realized by attacking the cell membrane and making it lose permeability. Here, the MIC values of
µg
mL−1, and their inhibitory effects are
·

Molecules 2018, 23, 2011
9 of 13
related ILs against Staphylococcus aureus can prove the antibacterial activity of OTs− is stronger than
that of NTf2−. Compounds A1
A5 have higher biological activities against Staphylococcus aureus than
–
against Escherichia coli. It can be noticed that neomycin shows the lowest MIC value for the two
bacteria, which is usually used to treat related microorganisms in crops. However, the bacteria can
develop resistance to it very easily after exposure to neomycin, which is gradually being restricted
from being used in the field. More importantly, its stability is very unsatisfactory; e.g., solutions of
neomycin are significantly unstable to heat, which will change its color above 30 ◦C. Thus, it is very
unsuitable for outdoor anti-microorganism use. On the contrary, related benzothiazolium ILs are
relatively stable, and the antibacterial activity of A3
–A5 is obviously higher than that of emodin and
A1/A2, which should be ascribed to the contribution of the alkyl side chain their cations.
Table 5. The minimal inhibitory concentration (MIC/
µg
·
mL−1) of the tested ILs in the
A and B series.
Escherichia coli
Staphylococcus Aureus
Samples
Mean
SD
Mean
SD
A1
A2
A3
A4
A5
B1
B2
B3
B4
B5
>250
> 250
>250
>250
>250
250
250
250
250
250
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
125
62.5
15.6
15.6
15.6
>250
>250
>250
250
5.3
3.1
2.3
2.3
2.3
0.0
0.0
0.0
0.0
0.0
250
Neomycin
Emodin
3.1
16
1.3
2.0
2.9
20.0
1.1
3.5
Note: The MIC was determined by the concentration of a sample with 100% inhibitory, and neomycin and emodin
were used as positive controls. The standard stock solution of the bacterial strain was equivalent to 1
108
×
colony-forming units per milliliter. The activity of C1–C3 was not investigated because of their poor solubility.
3. Materials and Methods
3.1. Reagents and Methods
The compounds 2-mercaptobenzothiazole (MBT, purity: 98%), methyl iodide (purity: 99%),
bromine ethane (purity: 98%), 1-bromobutane (purity: 99%), 1-bromohexane (99%), 1-bromooctane
(purity: 98%), benzylbromide (purity: 99%), 4-methyl-benzenesulfonicacimethylester (purity: 99%),
bis (trifluoromethane) sulfonamidelithium salt (purity: 99%), and sodium hexafluorophosphate (purity:
98%) were purchased from the Borhett chemical technology company (Chengdu, China). All the solvent
and reagents were used without further purification.
Column chromatography was implemented using HG/T2354-92 silica gel (Haiyang Chemical Co.
Ltd., Qingdao, China) with various specified eluents. Thin layer chromatography was carried out
on 0.15–0.2 mm layer silica coated HSGF 254 plates (Haiyang Chemical Co. Ltd., Qingdao, China).
The IR spectra were recorded on JASCO, Bomem MB, and Shimadzu-8400S FTTR spectrophotometers.
The Finnigan TSQ Quantum Ultra LC/MS/MS system (Thermo Fisher, San Jose, USA) in a dual ion pattern
and full-scan mode (100–500 m/z) was operated to determine the cations and anions under the following
conditions: N2 drying gas with a flow rate of 700 L h
−1 and a temperature of 400 ◦C, a 3.0 kV capillary
·
voltage, and a 100 ◦C capillary temperature.1H NMR and 13C NMR spectra were recorded on a Bruker
600 spectrometer in CDCl3. The thermal stability of ILs was evaluated in terms of their decomposition
temperature by using a TGA/SDTA851 thermogravimetric analyzer (Metrohm, Switzerland).

Molecules 2018, 23, 2011
10 of 13
3.2. Synthesis of 3-Methyl-2-alkylthio Benzothiazolyl-Based Ionic Liquids
MBT (5.017 g, 0.03 mol), bromomethane (2.848 g, 0.03 mol) and anhydrous potassium carbonate
◦
(4.140 g, 0.03 mol) were mixed and stirred in ethyl acetate at 65 C. The reaction was monitored
by thin layer chromatography (TLC). After the completion of the reaction, the inorganic salts that
had precipitated from the reaction mixture were removed by filtration, the filtrate was washed with
aqueous saturated Na2CO3 and water successively, and then it was dried by MgSO4. Finally, MgSO4
was removed by filtration; the filtrate was concentrated by reduced pressure distillation and then
purified by silica gel column chromatography using 5 to 10% EtOAc in petroleum ether (v/v) as an
eluent to obtain pure 2-methylthio benzothiazole (b1). The above general method was also used for
the synthesis of different compounds of b2–b5.
Product b1 (3.625 g, 0.02 mol) and 4-methyl benzene sulfonic acid methyl ester (3.724 g, 0.02 mol)
◦
were reacted at 130 C for 6 h, and then a very sticky fluid was obtained after the system was cooled to
room temperature. It was washed four times with water at 20 ◦C and then the aqueous solution was
merged and centrifuged to eliminate emulsification. The upper layer was collected and concentrated
by reduced pressure distillation and then the product A1 was obtained. The preparation method of
A2–A5 was analogous to that of A1.
A1 (3.665 g, 0.01 mol) was dissolved completely in water and a LiNTf2 (3.014 g, 0.0105 mol)
aqueous solution was added dropwise to above solution. After stirring for 1 h, oil droplets began to
appear at the bottom of the flask and a light yellow solid was separated out from the reaction mixture
after stirring for 12 h. After filtration, the filter cake was washed with water for four times and dried
under a vacuum. The crude product was recrystallized in ethyl acetate and then the product B1 was
obtained. The preparation method of B2–B5 was analogous to that of B1. The preparation method
of C1 C2 was analogous to that of C1 A1 (3.665 g, 0.01 mol) was dissolved completely in water and
–
.
asodium hexafluorophosphate (1.7635 g, 0.0105 mol) aqueous solution was added dropwise into the
solution of A1. After stirring for 13 h, a yellow solid was finally separated from the reaction mixture.
After filtration, the filter cake was washed by water four times and dried under vacuum. The crude
product was recrystallized in acetone and then the product C1 was obtained. The preparation method
of C2–C3 was analogous to that of C1.
3.3. Antibacterial Activity
The antibacterial activity of ILs (A1–A5 and B1–B5) against 6 kinds of bacteria was evaluated by
the modified Oxford cup method [36] and the inhibition zone diameters were measured. Beef extract
peptone agar media was used for the bacterial culture. A total of 1 mL of an 18-hour-old culture was
added to 60 mL of the medium, and 20 mL of this culture was shaken and placed into sterile Petri dishes.
After the solidification of the agar medium, the sterile stainless steel cylinders (6 × 10 mm) were placed
on the surface of the seeded agar and filled with 100 mL of the tested samples at 1 mg·mL−1. The plates
were incubated at 37 ◦C for 24 h.
Commercial antibiotics—neomycin and emodin—were used as positive controls with the same
range of concentrations. Distilled water was used as a negative control to dissolve all the tested ILs
in a concentration range of 0.0165–0.250 mg
mL−1. An overnight culture grown in the beef extract
·
peptone broth was diluted to approximately 108 CFUmL−1 in sterile water. The diluted bacterial
suspension was inoculated onto agar plates containing serial two-fold dilutions of neomycin and
tested ILs at a final concentration ranging from 250 to 15.6
lowest concentration of antibiotic that was able to prevent visible organism growth after incubation for
16 to 18 h at 37 ◦C.
µg [37]. The MIC was defined as the
mL−1
·
4. Conclusions
In summary, a total of 13 novel 3-methyl-2-alkylthiobenzothiazolyl ionic liquids (ILs) in three
series were synthesized and first reported. Their melting point, solubility, thermostability, and typical

Molecules 2018, 23, 2011
11 of 13
biological activities were determined. The results of this bioactivity research indicated that compounds
A3 A5 had an interesting antibacterial activity with the lowest MIC value of 16.7
mL−1 against
Staphylococcus aureus, which was better than natural antibacterial compounds. Moreover, ILs A3 A5
–
µg
·
–
are easily soluble in water and more stable than common aminoglycoside antibiotics, which make
them have favorable application foreground.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/23/8/2011/
s1, Figure S1 and Figure S2.
Author Contributions: Conceptualization, T.H.Z., H.X.H. and S.Y.; Methodology, J.L.D. and Z.J.H.; Data Curation,
T.H.Z. and H.X.H.; Writing-Original Draft Preparation, T.H.Z. and H.X.H.; Writing-Review & Editing, S.Y. and
Z.J.H. All authors read and approved the final manuscript.
Funding: The work was supported by the Project of Education Department in Sichuan province (14ZB0267) and
Basic Research Project of Mianyang Normal University (2014A12).
Acknowledgments: The authors are grateful to Ling Ma, Liang-Chun Li and Ning Chen for their assistance for
this study.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Park, S.W.; Nile, S.H.; Chikhalia, K.H.; Patel, R.V. Ionic liquid mediated tandem synthesis of bioactive
quinoline based thiophene/thiazole linked multi-heterocomponentugi adducts. Curr. Org. Chem.
2013, 17, 1125–1129. [CrossRef]
2.
3.
4.
5.
Malhotra, S.V.; Kumar, V.; Velez, C.; Zayas, B. Imidazolium-derived ionic salts induce inhibition of cancerous
cell growth through apoptosis. Med. Chem. Commun. 2014, 5, 1404–1409. [CrossRef]
Keri, R.S.; Patil, M.R.; Patil, S.A. A comprehensive review in current developments of benzothiazole-based
molecules in medicinal chemistry. Eur. J. Med. Chem. 2015, 89, 207–251. [CrossRef] [PubMed]
Kaur, H.; Kumar, S.; Singh, I.; Saxena, K.K.; Kumar, A. Synthesis, characterization and biological activity of
various substituted benzothiazole derivatives. Dig. J. Nanomater. Biostruct. 2010, 5, 67–76.
Huang, Q.; Mao, J.; Wan, B.; Wang, Y.; Brun, R.; Franzblau, S.G.; Kozikowski, A.P. Searching for new
cures for tuberculosis: Design, synthesis, and biological evaluation of 2-methylbenzothiazoles. Med. Chem.
2009, 52, 6757–6767. [CrossRef] [PubMed]
6.
7.
8.
9.
Wang, S.; Chen, Y.; Zhao, S.; Xu, X.; Liu, X.; Liu, B.F.; Zhang, G. Synthesis and biological evaluation of
a series of benzoxazole/benzothiazole-containing 2,3-dihydrobenzo-[1,4]dioxine derivatives as potential
antidepressants. Med. Chem. Lett. 2014, 24, 1766–1770. [CrossRef] [PubMed]
Zhang, H.; Liu, D.; Kang, T.; Wang, Y.; Zhang, X.; Zhu, X. Synthesis of novel benzothiazolium ionic liquids
and research on their catalytic esterification for ricinoleic acid. Chin. J. Org. Chem. 2016, 36, 1104–1107.
[CrossRef]
Ghanem, O.B.; Shah, S.N.; Lévêque, J.M.; Mutalib, M.; Elharbawi, M.; Khan, A.S.; Alnarabijia, M.S.;
Al-Absig, H.R.H.; Ullahhi, Z. Study of the antimicrobial activity of cyclic cation-based ionic liquids via
experimental and group contribution QSAR model. Chemosphere 2018, 195, 21–28. [CrossRef] [PubMed]
Seter, M.; Thomson, M.J.; Stoimenovski, J.; Macfarlane, D.R.; Forsyth, M. Dual active ionic liquids and organic
salts for inhibition of microbially influenced corrosion. Chem. Commun. 2012, 48, 5983–5985. [CrossRef]
[PubMed]
10. Deive, F.J.; Rodríguez, A.; Varela, A.; Rodrígues, C.; Leitão, M.C.; Houbraken, J.A.M.P.; Pereiro, A.B.;
Longo, M.A.; Sanroman, M.A.; Samson, R.A.; et al. Impact of ionic liquids on extreme microbial biotypes
from soil. Green Chem. 2011, 13, 687–696. [CrossRef]
11. Pernak, J.; Syguda, A.; Mirska, I.; Pernak, A.; Nawrot, J.; Pradzyn´ska, A.; Griffin, S.T.; Rogers, R.D.
Choline-derivative-based ionic liquids. Chem. Eur. J. 2010, 3, 6817–6827. [CrossRef] [PubMed]
12. Pernak, J.; Feder-Kubis, J.; Cieniecka-Rosonkiewicz, A.; Fischmeister, C.; Griffin, S.T.; Rogers, R.D. Synthesis
and properties of chiral imidazolium ionic liquids with a (1, 2, 5)-( )-menthoxymethyl substituent.
−
New J. Chem. 2007, 31, 879–892. [CrossRef]
13. Hodyna, D.; Bardeau, J.F.; Metelytsia, L.; Riabov, S.; Kobrina, L.; Laptiy, S.; Kalashnikova, L.;
Parkhommenko, V.; Tarasyuk, O.; Rogalsky, S. Efficient antimicrobial activity and reduced toxicity of

Molecules 2018, 23, 2011
12 of 13
1-dodecyl-3-methylimidazolium tetrafluoroborate ionic liquid/
2016, 284, 1136–1145. [CrossRef]
β-cyclodextrin complex. Chem. Eng. J.
14. Ranke, J.; Bottin-Weber, M.U.; Stock, F.; Stolte, S.; Arning, J.; Störmann, R.; Jastorff, B. Lipophilicity parameters
for ionic liquid cations and their correlation to in vitro cytotoxicity. Ecotox. Environ. Saf. 2007, 67, 430–438.
[CrossRef] [PubMed]
15. Scammells, P.J.; Scott, J.L.; Singer, R.D. Ionic liquids: The neglected issues. Aust. J. Chem. 2005, 58, 155–169.
[CrossRef]
16. Wells, A.S.; Coombe, V.T. On the freshwater ecotoxicity and biodegradation properties of some common
ionic liquids. Org. Process. Res. Dev. 2006, 10, 794–798. [CrossRef]
17. Cho, C.W.; Pham, T.P.T.; Jeon, Y.C.; Vijayaraghavan, K.; Choe, W.S.; Yun, Y.S. Toxicity of imidazolium salt
with anion bromide to a phytoplankton selenastrumcapricornutum: Effect of alkyl-chain length. Green Chem.
2007, 69, 1003–1007. [CrossRef]
18. Peng, Q.; Zhang, Y.W.; Wang, X.M.; Wang, Z.D.; Yao, S.; Song, H. Synthesis of novel ionic liquid with
benzothiazolium and research on their physical chemical property. Chin. J. Synth. Chem. 2012, 1, 28–31.
[CrossRef]
19. Daidone, G.; Maggio, B.; Schillaci, D. Salicylanilide and its heterocyclic analogues. A comparative study of
their antimicrobial activity. Pharmazie 1990, 45, 441–442. [PubMed]
20. Zhang, L; Peng, X.M.; Damu, G.L.V.; Geng, R.X.; Zhou, C.H. Comprehensive Review in Current
Developments of Imidazole-Based Medicinal Chemistry. Med. Res. Rev. 2014, 34, 340–437. [CrossRef]
[PubMed]
21. Messali, M. Eco-friendly synthesis of a new class of pyridinium-based ionic liquids with attractive
antimicrobial activity. Molecules 2015, 20, 14936–14949. [CrossRef] [PubMed]
22. Li, X.K.; Yuan, T.J.; Yang, Y.; Chen, J.M. Novel copper/PEG-400 catalyst systems for chemoselective S- and
N-arylation of 2-mercaptobenzothiazole. Tetrahedron 2014, 70, 9652–9660. [CrossRef]
23. Zhou, X.S.; Liu, J.B.; Luo, W.F.; Zhang, Y.W.; Song, H. Novel Brønsted-acidic ionic liquids based on
benzothiazoliumcations as catalysts for the acetalization reactions. J. Serb. Chem. Soc. 2011, 76, 1607–1615.
[CrossRef]
24. Tawfik, S.M. Simple one step synthesis of gemini cationic surfactant-based ionic liquids: Physicochemical,
surface properties and biological activity. J. Mol. Liq. 2015, 209, 320–326. [CrossRef]
25. Seddon, K.R. Ionic liquids: A taste of the future. Nat. Mater. 2003, 2, 363–365. [CrossRef] [PubMed]
26. Li, H.; Yu, C.; Chen, R.; Li, J.; Li, J. Novel ionic liquid-type gemini surfactants: Synthesis, surface property
and antimicrobial activity. Colloids Surf. A 2012, 395, 116–124. [CrossRef]
27. Al-mohammed, N.N.; Alias, A.; Abdullah, Z. Bis-imidazolium and benzimidazolium based gemini-type
ionic liquids structure: Synthesis and antibacterial evaluation. RSC Adv. 2015, 5, 92602–92617. [CrossRef]
28. Xu, W.; Wang, L.M.; Nieman, R.A.; Angell, C.A. Ionic liquids of chelated orthoborates as model ionic
glassformers. J. Phys. Chem. B. 2003, 107, 11749–11756. [CrossRef]
29. Freire, M.G.; Neves, C.M.S.S.; Carvalho, P.J.; Gardas, R.L.; Fernandes, A.M.; Marrucho, I.M.;
Santos, L.M.N.B.F.; Coutinho, J.A.P. Mutual solubilities of water and hydrophobic ionic liquids. J. Phys.
Chem. B. 2007, 111, 13082–13089. [CrossRef] [PubMed]
30. Shah, S.N.; Lethesh, K.C.; Mutalib, M.I.A.; Pilus, R.B.M. Evaluation of thermophysical properties of
imidazolium-based phenolate ionic liquids. Ind. Eng. Chem. Res. 2015, 54, 3697–3705. [CrossRef]
31. Cao, Y.; Mu, T. Comprehensive investigation on the thermal stability of 66 ionic liquids by thermogravimetric
analysis. Ind. Eng. Chem. Res. 2014, 53, 8651–8664. [CrossRef]
32. Czekan´ski, L.; Almeida, T.S.D.; Mota, J.P.; Rijo, P.; Araújo, M.E.M. Synthesis of benzoazole ionic liquids and
evaluation of their antimicrobial activity. Biomed. Biopharm. Res. 2014, 11, 227–235. [CrossRef]
33. Hiremath, G.S.; Kulkarni, R.D.; Naik, B.D. Evaluation of minimal inhibitory concentration of two new
materials using tube dilution method: An in vitro study. J. Conserv. Dent. 2015, 18, 159–162. [CrossRef]
[PubMed]
34. Pernak, J.; Sobaszkiewicza, K.; Mirska, I. Anti-microbial activities of ionic liquids. Green Chem. 2003, 5, 52–56.
[CrossRef]
35. Ryu, H.; Lee, H.; Iwata, S.; Choi, S.; Kim, M.K.; Kim, Y.R.; Maruta, S.; Kim, S.M.; Jeon, T.J. Investigation of
ion channel activities of gramicidin a in the presence of ionic liquids using model cell membranes. Sci. Rep.
2015, 5, 11935. [CrossRef] [PubMed]

Molecules 2018, 23, 2011
13 of 13
36. Wang, Y.; Lu, Z.X.; Wu, H.; Lv, F. Study on the antibiotic activity of microcapsule curcumin against foodborne
pathogens. Int. J. Food Micobiol. 2009, 136, 71–74. [CrossRef] [PubMed]
37. Dienstag, J.; Nue, H.C. In vitro studies of tobramycin and aminoglycoside antibiotic. Antimicrob. Agents
Chemother. 1972, 1, 41–45. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds A1–A5, B1–B5 and C1–C3 are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).