Antimicrobial activity of quinoline-based hydroxyimidazolium hybrids

Article abstract of DOI:10.3390/antibiotics8040239

Eight quinoline-based hydroxyimidazolium hybrids 7a-h were prepared and evaluated in vitro against a panel of clinically important fungal and bacterial pathogens, including mycobacteria. Hybrid compounds 7c-d showed remarkable antifungal activity against Cryptococcus neoformans with a minimum inhibitory concentration (MIC) value of 15.6 μg/mL. Against other opportunistic fungi such as Candida spp. and Aspergillus spp., these hybrids showed MIC values of 62.5 μg/mL. Regarding their antibacterial activity, all the synthetic hybrids demonstrated little inhibition of Gram-negative bacteria (MIC ≥50 μg/mL), however, hybrid 7b displayed >50% inhibition against Klebsiella pneumoniae at 20 μg/mL and full inhibition at 50 μg/mL. Moreover, this hybrid was shown to be a potent anti-staphylococcal molecule, with a MIC value of 2 μg/mL (5 μM). In addition, hybrid 7h also demonstrated inhibition of Staphylococcus aureus at 20 μg/mL (47 μM). Hybrids 7a and 7b were the most potent against Mycobacterium tuberculosis H37Rv with MIC values of 20 and 10 μg/mL (46 and 24 μM), respectively. The 7b hybrid demonstrated high selectivity in killing S. aureus and M. tuberculosis H37Rv in comparison with mammalian cells (SI >20), and thus it can be considered a hit molecule for mechanism of action studies and the exploration of related chemical space.

Full text of DOI:10.3390/antibiotics8040239

antibiotics
Article
Antimicrobial Activity of Quinoline-Based
Hydroxyimidazolium Hybrids
Daniel Insuasty 1,
* ,
, Oscar Vidal 1, Anthony Bernal 1, Edgar Marquez 1 , Juan Guzman 2
Braulio Insuasty 3 , Jairo Quiroga 3 , Laura Svetaz 4, Susana Zacchino 4 , Gloria Puerto 5 and
Rodrigo Abonia 3,
*
1
Departamento de Química y Biología, Universidad del Norte, Km 5 vía Puerto Colombia,
Barranquilla 081007, Colombia; oorjuela@uninorte.edu.co (O.V.); awbernal@uninorte.edu.co (A.B.);
ebrazon@uninorte.edu.co (E.M.)
Institute for Insect Biotechnology, Justus-Liebig-University of Giessen, 35392 Giessen, Germany;
Juan.D.Guzman-Vasquez@agrar.uni-giessen.de
Research Group of Heterocyclic Compounds, Department of Chemistry, Universidad del Valle,
A. A. Cali 25360, Colombia; braulio.insuasty@correounivalle.edu.co (B.I.);
jairo.quiroga@correounivalle.edu.co (J.Q.)
2
3
4
Área Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario,
Suipacha 531, Rosario 2000, Argentina; laurasvetaz@hotmail.com (L.S.); szaabgil@citynet.net.ar (S.Z.)
Laboratorio de Micobacterias, Instituto Nacional de Salud, Bogotá 111321, Colombia; gpuerto@ins.gov.co
Correspondence: insuastyd@uninorte.edu.co (D.I.); rodrigo.abonia@correounivalle.edu.co (R.A.)
5
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 25 October 2019; Accepted: 19 November 2019; Published: 28 November 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Eight quinoline-based hydroxyimidazolium hybrids 7a–h were prepared and evaluated
in vitro against a panel of clinically important fungal and bacterial pathogens, including mycobacteria.
Hybrid compounds 7c–d showed remarkable antifungal activity against Cryptococcus neoformans
with a minimum inhibitory concentration (MIC) value of 15.6
fungi such as Candida spp. and Aspergillus spp., these hybrids showed MIC values of 62.5
Regarding their antibacterial activity, all the synthetic hybrids demonstrated little inhibition of
Gram-negative bacteria (MIC 50 g/mL), however, hybrid 7b displayed >50% inhibition against
Klebsiella pneumoniae at 20 g/mL and full inhibition at 50 g/mL. Moreover, this hybrid was shown to
be a potent anti-staphylococcal molecule, with a MIC value of 2
7h also demonstrated inhibition of Staphylococcus aureus at 20
µg/mL. Against other opportunistic
µg/mL.
≥
µ
µ
µ
µg/mL (5
µM). In addition, hybrid
µg/mL (47
µM). Hybrids 7a and 7b
were the most potent against Mycobacterium tuberculosis H37Rv with MIC values of 20 and 10
µg/mL
(46 and 24 M), respectively. The 7b hybrid demonstrated high selectivity in killing S. aureus and
µ
M. tuberculosis H37Rv in comparison with mammalian cells (SI >20), and thus it can be considered a
hit molecule for mechanism of action studies and the exploration of related chemical space.
Keywords: quinoline-based hydroxyimidazolium hybrids; antimicrobial activity; antifungal activity;
tuberculosis; cytotoxicity
1. Introduction
The intensive use of antibacterial and antifungal drugs has led to an increase in difficult-to-eradicate
infections [1]. In recent years, the trend in reducing infectious disease mortality has been threatened
by the emergence of strains of bacteria that are no longer susceptible to the currently available
antimicrobial agents such as the Gram-negative Acinetobacter baumannii, Pseudomonas aeruginosa, or
the Gram-positive Enterococcus faecium, or Staphylococcus aureus added to the multi- and extensively-
drug-resistant Mycobacterium tuberculosis. Regarding fungi, they are also a source of concern in
antifungal chemotherapy because many fungi can be opportunistic pathogens seriously affecting
Antibiotics 2019, 8, 239; doi:10.3390/antibiotics8040239
www.mdpi.com/journal/antibiotics

Antibiotics 2019, 8, 239
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immunocompromised patients, and some of them, such as Candida albicans, Cryptococcus neoformans,
and Aspergillus spp. can cause complicated systemic infections that are associated with high mortality
rates [2].
With the rise of the difficult-to-eradicate infectious diseases, the need for new antimicrobial agents
is urgently needed. A promising strategy for the development of new antimicrobial drugs is the
synthesis of molecular hybrids containing two or more covalently joined antimicrobial pharmacophores
within a single molecule [3–5].
Quinoline moiety is historically important because it is present in the Cinchona alkaloids quinine
and quinidine, which were the first useful treatment for malaria. Based on the activity of these
natural products, several quinoline-based molecules have shown to be effective inhibitors of essential
proteins from microbial pathogens [
some of them, such as amodiaquine, chloroquine (
piperaquine are still used clinically today, as they are recommended by the WHO [
6
]. For that, synthetic antimalarials have been developed, and
in Figure 1), mefloquine (II in Figure 1), and
]. One of the
I
7
by-products of the synthesis of chloroquine was identified as an active antibacterial principle in 1960,
and further research led to the discovery of nalidixic acid in 1962 and later to the fluoroquinolone
class of antibacterials [8]. Their clinical importance is evident, as these were highly active against
most Enterobacteriaceae, which includes common pathogens such as Escherichia coli, Salmonella spp.,
Yersinia pestis, and others, and in addition these molecules are unique to targeting bacterial DNA
topoisomerases [9]. Moreover, other quinoline-containing compounds have been developed as
antibacterial drugs, and one remarkable example is bedaquiline (III in Figure 1). This diarylquinoline
derivative is a first-in-class anti-tuberculosis drug, acting by inhibition of mycobacterial ATP synthase
and approved to treat multiple-drug resistant tuberculosis [10].
Figure 1. Some examples of biologically important quinoline- and imidazolium-derivatives.
Although quaternary ammonium salts such as benzalkonium and cetylpyridinium chlorides salts
have been used for a long time as antiseptics in a variety of pharmaceutical and cosmetic products [11,12],
the imidazolium salts are not used as antiseptics, nor are they constitutive fragment of any drug thus
far. In contrast, the imidazole ring is a common moiety in antifungal chemotherapeutic agents such as
clotrimazole, ketoconazole, and miconazole [13
in the lepidilines (IV in Figure 1), which are in fact antitumoral natural products from the Neotropical
plant Lepidium meyenii [16]. Synthetic natural products analogs showed potent antimicrobial [17
and antitumoral activities [18 19]. The interesting biological properties of imidazolium salts may be
–15]. Interestingly, the imidazolium moiety is present
]
,
explained by the possibility of not only engaging in ion-dipole and hydrogen bond interactions but
also to participate in acid-base reactions and to coordinate metal atoms. This versatility opens the
door to tuning their selectivity by covalent linking with appropriate steric and electrostatic scaffolds
directed to particular biochemical targets.
In this work, eight quinoline-based hydroxyimidazolium hybrids 7a–h were prepared and
evaluated against a panel of clinically important fungal and bacterial pathogens, including
Mycobacterium tuberculosis H37Rv.

Antibiotics 2019, 8, 239
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2. Results and Discussion
2.1. Chemistry
The synthesis of the quinoline-based hydroxyimidazolium hybrids
7
was carried out by following
our previously reported methodology [20]. Briefly, the non-commercial 3-formyl-2-oxo-quinoline and
3-formyl-2-alkoxy-quinoline precursors 5a–h were synthesized by a Meth-Cohn reaction [21], followed
by acid hydrolysis and alkylation (Figure 2). Subsequently, the aldehydes 5a–h were subjected to
reaction with 3-butyl-1-methylimidazolium chloride ([Bmim]Cl)
6 in the presence of AcONa and ACN
◦
as a solvent, under ultrasound irradiation at 80 C during 1–7 h. This straightforward procedure
afforded the corresponding quinoline-based hydroxyimidazolium hybrids 7a–h in 60–91% yield.
Structures of hybrids 7 were confirmed by 1H NMR (see Figure S1 in Supplementary Materials).
Figure 2. Synthesis of quinoline-based hydroxyimidazolium hybrids 7a–h.
2.2. Antifungal Activity
Hybrids 7a–h were tested in vitro for antifungal activity against the most common causes of
invasive fungal diseases Candida albicans and Cryptococcus neoformans and the molds Aspergillus niger,
Aspergillus fumigatus, and Aspergillus flavus. The minimum inhibitory concentration (MIC) of all
hybrids was determined with the clinical and laboratory standards institute (CLSI) microbroth dilution
methods M27-A3 and M38-A2 [22
in the range of 250 to 62.5 g/mL, with moderate or low activity, and hybrids with MICs
were considered high activity.
,
23]. Hybrids with MICs >250
µ
g/mL were considered inactive;
µ
≤
31.25 g/mL
µ

Antibiotics 2019, 8, 239
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Results in Table 1 clearly show that C. neoformans were the most susceptible sp., with all hybrids
showing some degree of antifungal activity with MIC values between 15.6 and 250 g/mL. Instead,
MIC values of 7a–h against C. albicans were in the range of 62.5 to (>250 g/mL) with two hybrids
7e and 7h) showing to be inactive (MIC >250 g/mL). Regarding Aspergillus spp., they were the less
µ
µ
(
µ
sensitive spp, since 5 hybrids (7a, 7b, 7e–g) (of the 8 hybrids tested) were inactive.
Table 1. Minimum inhibitory concentration (MIC in µg/mL) of hybrids 7a–h.
Structure
Hybrid R1
R2
R3
R4
Ca
Cn
Afu
Afl
Ani
7a
7b
7c
OMe
Me
H
H
H
H
Cl
H
H
Me
H
-
-
-
-
125
62.5
62.5
62.5
62.5
62.5
15.6
15.6
i
i
i
i
i
i
62.5
62.5
62.5
62.5
62.5
62.5
7d
H
7e
7f
7g
7h
OMe
Me
H
H
H
Cl
H
H
H
H
H
Bu
Bu
Bu
Bn
i
250
125
62.5
250
i
i
i
i
i
i
i
i
i
250
250
i
H
250
250
250
AmpB
0.78
0.25
0.50
0.50
0.50
Antifungal activity was determined with the microbroth dilution assay following the clinical laboratory standards
institute (CLSI) guidelines; inactive (i) = MIC >250 g/mL; Ca: Candida albicans ATCC10231, Cn: Cryptococcus
µ
neoformans ATCC32264, An: Aspergillus niger ATCC9029, Afl: Aspergillus flavus ATCC9170, Afu: Aspergillus fumigatus
ATCC26934. AmpB: Amphotericin B; Bu = Butyl; Bn: benzyl.
In order to have a deeper look at the antifungal behavior of hybrids
the percentages of inhibition of C. neoformans for each hybrid 7a–h at concentrations from 250 to
3.90 g/mL (obtained by two-fold dilutions) were determined. Results are recorded in Table 2 and
7 against C. neoformans,
µ
represented in Figure 3, where the differences in the activity of the 7 hybrids 7 against C. neoformans
can be clearly observed.
Table 2. Percentages of inhibition of C. neoformans ATCC 32264 by hybrids 7a–h.
Concentration in µg/mL
MIC in
µg/mL
Comp.
250
125
62.50
31.25
15.62
7.81
3.90
7a
7b
7c
7d
7e
7f
7g
7h
AmpB
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
43.6 ± 3.86
92.58 ± 4.81
100
18.60 ± 8.59
67.84 ± 5.66
100
4.72 ± 1.39
57.14 ± 4.93
53.45 ± 4.42
67.59 ± 9.19
17.19 ± 0.91
9.34 ± 2.90
25.50 ± 5.35
22.76 ± 2.87
100
0
62.5
31.25
15.62
15.62
250
62.5
250
250
12.54 ± 0.36
48.41 ± 4.63
100
100
100
100
0
62.46 ± 0.30
41.46 ± 0.58
49.28 ± 3.20
90.90 ± 7.60
48.99 ± 3.00
100
38.79 ± 3.79
24.91 ± 0.15
50.44 ± 2.09
46.84 ±5.32
100
29.92 ± 6.13
19.01 ± 3.40
30.94 ± 5.61
35.32 ± 2.02
100
8.23 ± 3.18
100
0
0
94.15 ± 4.15
76.77 ± 1.91
100
2.52 ± 0.56
100
0.25
Figure 3. Dose–response curves of hybrids 7a–d (left) y 7e–h (right) against C. neoformans ATCC 32264.

Antibiotics 2019, 8, 239
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Table 2 shows that the hybrids derived from 2-alkoxy-quinolines (7a–d) were more potent than
those from 2-oxo-quinolines (7e–h), and this can clearly be seen in Figure 3.
The dotted lines show the percentages of inhibition of the hybrids at 31.25 µg/mL and is useful to
clearly observe the behavior of the hybrids at lower concentrations that are considered highly active. In
Figure 3, it can be corroborated that 7a–d possess higher anti-cryptococcal activity than 7e–h since the
inhibition percentages of 7a–d at concentrations ≤31.25 µg/mL are clearly higher than those of 7e–h.
From the above results, some preliminary structure–activity relationships can be drawn: (i) The
methoxy group in position C-6 (hybrid 7a), induced a relative lower activity than the methyl group
in the same position (hybrid 7b), as shown in Tables 1 and 2. (ii) The methyl group in position C-8
of the hybrid 7c seemed to confer a significant increase in antifungal potency compared with the
methyl group in position C-6 of hybrid 7b. (iii) Hybrids 7c and 7d showed the same MIC (15.62
values, although 7c seemed to be more potent than 7d, as evidenced by the much stronger inhibition
displayed by 7c at 3.9 g/mL, (see Table 2). This finding could suggest that either the chlorine atom at
µg/mL)
µ
position C-7 in 7d decreased the activity or the methyl group in position C-8 (hybrid 7c) increased the
antifungal effect, or that both played a significant role in the activity. (iv) The same detrimental trend
by the chlorine atom in position C-7 on the antifungal activity was also observed when comparing the
N-butylated hybrids 7f (6-Me) (MIC = 125 mg/L) with 7g (7-Cl) (MIC = 250 mg/L. (v) Nevertheless,
the methyl group in position C-6 (hybrid 7b) induced a lower activity even than the chlorine atom at
position C-7 in 7d, affording MIC values of 15.62 and 31.25, respectively, Table 2. (vi) Additionally,
comparison of the activity of the O-butylated hybrid 7b (MIC = 31.25) with the N-butylated hybrid
7f (MIC = 62.5), both possessing a 6-Me substituent, indicated that not only this substituent and its
position contributed to the activity of the synthesized hybrids 7.
2.3. Antibacterial Activity
The hybrids 7a–h were tested against two Gram-negative microorganisms; Escherichia coli and
Klebsiella pneumoniae, a Gram-positive bacteria, Staphylococcus aureus, and two acid-fast slow-growing
mycobacteria Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG. Results for each hybrid
were expressed as minimum inhibitory concentration values (MICs, µg/mL and µM), as shown in
Table 3. Chloramphenicol was used as a positive control for E. coli, K. pneumoniae, and S. aureus, and
isoniazid for mycobacteria. All the synthetic hybrids showed little activity against the Gram-negative
organisms with MIC values
the most potent, as evidenced by their moderate growth inhibition at high concentration (Figure 4).
The hybrid 7b demonstrated a potent anti-staphylococcal activity with a MIC value of 2 g/mL (5 M).
The hybrid 7h also demonstrated a significant inhibition against S. aureus with a MIC value of 20 g/mL
(47 M) and should also be considered as a hit molecule for exploring related chemical space. Hybrids
7a and 7b showed significant inhibition against M. tuberculosis H37Rv with MIC values of 20 and
10 g/mL (46 and 24 M), respectively. The remaining hybrids 7d 7e, and 7h displayed moderate
growth inhibition against the virulent H37Rv strain with MIC values of 50 g/mL (–115 M). A similar
trend of activity was found against M. bovis BCG, confirming their antimycobacterial effect.
≥
50 µg/mL. Against K. pneumoniae, hybrids 7a and 7b showed to be
µ
µ
µ
µ
µ
µ
,
µ
µ

Antibiotics 2019, 8, 239
6 of 11
Table 3. Minimum inhibitory concentration values against E. coli, K. pneumoniae, S. aureus, M. tuberculosis
H37Rv and M. bovis BCG for the synthetic hybrids 7a–h.
MICs in µg/mL (µM)
Hybrid
E. coli
K. pneumoniae
S. aureus
M. bovis BCG
M. tuberculosis H37Rv
7a
7b
7c
7d
7e
7f
7g
7h
100 (231)
200 (480)
200 (480)
100 (229)
200 (462)
200 (478)
200 (228)
100 (228)
20 (62)
100 (231)
50 (120)
200 (480)
200 (457)
100 (231)
200 (478)
100 (114)
100 (228)
20 (62)
100 (231)
2 (5)
50 (115)
50 (120)
200 (480)
50 (114)
50 (115)
200 (478)
100 (228)
50 (114)
nd
20 (46)
10 (24)
100 (240)
100 (229)
100 (231)
200 (478)
100 (114)
20 (47)
200 (480)
50 (114)
50 (115)
100 (239)
100 (228)
50 (114)
nd
chloramphenicol
isoniazid
20 (62)
nd
nd
nd
0.05 (0.36)
0.05 (0.36)
nd = not determined.
Figure 4. Antibacterial activity of hybrids 7a–h. Comparative dose–response curves of hybrids 7a–h
against ( ) E. coli, ( ) K. pneumoniae, and ( ) S. aureus. ( ) Comparison of the concentrations required for
a
b
c
d
anti-mycobacterial and cytotoxic effects against M. tuberculosis H37Rv, M. bovis BCG, and (Verda Reno)
VERO cell line.
The anti-mycobacterial response of the hybrids towards the non-virulent M. bovis BCG and
the virulent M. tuberculosis H37Rv was compared (Table 3). A correlation in MIC between the two
organisms was found to be 0.8 for this dataset. Substitution at C-6 as in 7a and 7b seemed to be
essential for increasing the anti-mycobacterial effect, whereas benzyl substitution on the nitrogen
atom, as in 7h, seemed to promote anti-staphylococcal activity. A number of antimicrobial quinolone
hybrids have been published in recent literature [24,25], and although some of these hybrids were

Antibiotics 2019, 8, 239
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active against Gram-negative bacteria, only the hybrids with chloroquine and triclosan [26], showed
inhibition of S. aureus at concentrations comparable (MIC–2 mg/L) to our best results. Against
Mycobacterium tuberculosis, the hybrids of quinolone and pyrazole displayed inhibitory values (MIC
12.5–50 mg/L) similar to our results [27]. However, evidently other quinoline derivatives are more
potent antimycobacterial agents [28] and were developed, for instance, as the currently approved
drug bedaquiline.
In summary, the hybrid 7b represents a promising molecule showing significant antibacterial
effect against S. aureus (5 µM) and M. tuberculosis H37R (24 µM), for which the study of its mechanism
of action should be undertaken to assess its potential development as an anti-infective agent. In this
direction, some previous reports indicated that quinoline hybrids, along with other nitrogen-containing
heterocycles have shown to bind to heme and hemozoin unities, but also DNA, acetylcholinesterase,
or to affect prostaglandin production [29–33]. The hits discovered in this study open the door for
the exploration of a focused library of compounds and to deepen the study of their antimicrobial
mechanism of action.
2.4. Cytotoxicity and Selective Index
The cytotoxic activity of the hybrids 7a–h was evaluated by an MTT (3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide) assay on VERO Cells (ATCC® CCL-81
™) with peroxide as a
positive control and dimethyl sulfoxide (DMSO) as a negative control. VERO cell line growth was
expressed as half-lethal concentration (LC50) values and are summarized in Table 4.
Table 4. Cytotoxic activity (LC50) in VERO cells and selectivity index (SI) of the active synthetic hybrids
7a–h, for E. coli, K. pneumoniae, S. aureus, M. tuberculosis H37Rv and M. bovis BCG.
Selectivity Index (SI) (SI = LC50/MIC)
Hybrid
Cytotoxicity
LC50-Vero cells
M. bovis
BCG
M. tuberculosis
E. coli
K. pneumoniae
S. aureus
H37Rv
7a
7b
7c
7d
7e
7f
277 ± 14.6
234 ± 4
2.77
1.17
1.34
1.89
0.62
0.75
1.44
2.35
2.77
4.68
1.34
0.94
1.25
0.75
1.44
2.35
2.77
117
5.54
4.68
1.34
3.78
2.5
0.75
1.44
4.70
13.9
23.4
1.34
3.78
2.50
1.50
1.44
4.70
268 ± 5.69
189 ± 14.5
125 ± 22
2.68
1.89
1.25
0.75
1.44
11.8
150 ± 12.2
144 ± 8.9
235 ± 6.5
7g
7h
Examination of cytotoxic activity of the hybrids 7a–h on the green monkey VERO cell line showed,
in general, a low effect with LC50 values higher than 125
showed to be slightly less cytotoxic (LC50 ~ 189–277
125–235
MIC against bacteria. As the cytotoxic concentrations were relatively similar, the hybrids with lower
MIC values such as 7a 7b, and 7h, showed higher selectivity. Specifically, the hybrid 7b showed
µ
M (Table 4). The 2-alkoxy-quinolines (7a–d
)
µM) than the 2-oxo-quinolines (7e–h) (LC50
~
µM). The selectivity index (SI) was calculated as the ratio between LC50 on VERO cells and
,
high SI values of 117 and 23.4 for S. aureus and M. tuberculosis H37Rv. The SI is an indirect measure
of the therapeutic window, and it can serve as a predictor of safety during in vivo trials for a given
bacterial infection.
3. Materials and Methods
3.1. Antifungal Activity
3.1.1. Microorganisms and Media
For the antifungal evaluation, standardized strains from the American type culture collection
(ATCC, Manassas, VA, USA), and CEREMIC (CCC, Centro de Referencia en Micología, Facultad

Antibiotics 2019, 8, 239
8 of 11
de Ciencias Bioqu
í
micas y Farmacéuticas, Rosario, Argentina) were used: C. albicans ATCC 10231,
C. neoformans ATCC 32264, A. flavus ATCC 9170, A. fumigatus ATTC 26934, A. niger ATCC 9029. Strains
◦
were grown on Sabouraud-chloramphenicol agar slants for 48h at 30 C, maintained on slopes of
Sabouraud-dextrose agar (SDA, Oxoid, Cambridge, UK) and sub-cultured every 15 days to prevent
pleomorphic transformations. Inocula were obtained according to reported procedures [22,23] and
adjusted to 1–5 × 103 cells with colony-forming units (CFU)/mL.
3.1.2. Antifungal Susceptibility Testing
Minimum inhibitory concentration (MIC) of each hybrid was determined by using broth
microdilution techniques according to the guidelines of the Clinical and Laboratory Standards
Institute for yeasts (M27-A3) [22] and for filamentous fungi (M38 A2) [23]. MIC values were
determined in RPMI-1640 (Sigma-Aldrich, St Louis, MO, USA) buffered to pH 7.0 with MOPS
◦
(3-(N-morpholino)propanesulfonic acid). Microtiter trays were incubated at 35 C for yeasts and
Aspergillus spp. MICs were visually recorded at 48h for yeasts, and at a time according to the control
fungus growth, for Aspergillus spp. For the assay, stock solutions of the hybrids were 2-fold diluted
with RPMI-1640 from 250 to 3.90
µg/mL (final volume = 100 µL) and a final DMSO concentration
≤
1%. A volume of 100 L of inoculum suspension was added to each well, with the exception of the
µ
sterility control where sterile water was added to the well instead. Amphotericin B (Sigma-Aldrich,
St Louis, MO, USA) was used as positive control. Endpoints were defined as the lowest concentration
of drug resulting in total inhibition (MIC) of visual growth compared to the growth in the control wells
containing no antifungal drug.
3.1.3. Fungal Growth Inhibition Percentage Determination
This determination was performed with the yeast C. neoformans ATCC 32264. For the assay,
compound test wells (CTWs) were prepared with stock solutions of each hybrid in DMSO (maximum
concentration
suspension (100
≤
1%), diluted with RPMI-1640, to final concentrations of 250–3.90
µ
g/mL. An inoculum
µL) was added to each well (final volume in the well = 200
µL). A growth control
well (GCW) (containing medium, inoculum, and the same amount of DMSO used in a CTW,
but compound-free) and a sterility control well (SCW) (sample, medium, and sterile water instead
of inoculum) were included for each fungus tested. Microtiter trays were incubated in a moist, dark
chamber at 30 ◦C for 48h for both yeasts. Microplates were read in a Versa Max microplate reader
(Molecular Devices, Sunnyvale, CA, USA). Amphotericin B was used as positive control. Tests were
performed in triplicate. Reduction of growth for each compound concentration was calculated as
follows: % of inhibition = 100−(OD 405 CTW−OD405 SCW)/(OD405 GCW−OD405 SCW).
3.2. Antituberculosis and Antibacterial Activity
The agar dilution spot culture growth inhibition (SPOTi) assay [34,35] was performed to evaluate
the minimum inhibitory concentration (MIC) values of the synthetic hybrids against the laboratory
strain M. tuberculosis H37Rv in a biosafety level 3 laboratory of the National Health Institute in Bogota.
A stock solution of the hybrids was prepared in DMSO at a concentration of 200 mg/mL. Dilutions of
the compounds were prepared in DMSO in 24 well plates, 2 mL of each dilution was dispensed in
each well at 200, 100, 50, 20, and 10 mg/mL. The final volume was 2 mL of molten Middlebrook 7H10
medium (HiMedia, Mumbai, India) supplemented with 0.5% glycerol and 10% oleic acid, albumin,
dextrose, and catalase (OADC, BD, USA) were added to the wells. An inoculum having a cell density
of 106 CFU/mL was prepared from a 2-week culture of M. tuberculosis H37Rv (ATCC 27294) grown in
Löwenstein–Jensen medium slants at 37 ◦C. Two microliters of the diluted inoculum were dispensed in
the middle of the agar from each well, and the plates were incubated for 2–3 weeks at 37 ◦C. Isoniazid
was included as a positive control at 10, 1, 0.1, 0.05, and 0.01 µg/mL concentrations. After the incubation
period, the plates were observed, and the MIC was determined as the minimum concentration on
which growth was not observed. The experiment was repeated on a different day observing exactly

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the same results. The SPOTi agar dilution method was also employed for MIC determination against
M. bovis BCG. The cells were passaged first in Middlebrook 7H9 and then spotted into Middlebrook
7H10 medium with supplement albumin, 0.5% glycerol, dextrose, and sodium chloride (NaCl). The
plates were incubated for 1 week at 37 ◦C. The concentrations tested were 200, 100, 50, 20 and 10
and isoniazid was used as a positive control.
µg/mL
The evaluation of the hybrids against a panel of two Gram-negative microorganisms E. coli (see
Table S1 in Supplementary Materials) and K. pneumoniae (see Table S2 in Supplementary Materials),
and the Gram-positive bacteria S. aureus (see Table S3 in Supplementary Materials), with a varied
pattern of drug-susceptibility was performed on LB broth by two-fold serial dilution with a maximum
concentration of the synthetic hybrids at 200, 100, 50, 20, and 10 µg/mL. Growth was determined by
optical density measurements at 640 nm every 120 min for 36 h. Chloramphenicol was used as a
positive control, and the experiments were performed in duplicate.
3.3. Cytotoxicity on VERO Cell Line
The VERO cell line (ATCC® CCL-81
™) was cultured in Dulbecco Modified Eagle Medium (DMEM)
supplemented with 10% bovine fetal serum (FBS) and 1% streptomycin-penicillin and passaged twice
before the assay in 21 cm2 cell culture Petri dishes at 37 ◦C in 5% CO2 incubator and 100% humidity.
The cells were then cultured in 96 well plates for 24h before the assay to a cell density of 90.000 cells
per well. A 10 mg/mL hybrid stock solution was prepared in DMSO, following 2-fold serial dilution
until 0.01 mg/mL. Finally, 5
for 48h. Hydrogen peroxide was used as a positive control and DMSO as a negative control under the
same dilution conditions. After 24h of incubation, culture media was changed for 100 L of DMEM
media without FBS freshly prepared with MTT solution at 5 mg/mL, and the plates were further
incubated for 2h. The media was then removed, and 100 L of DMSO was added to each well. After
µL were transferred to each plate containing the VERO cells and incubated
µ
µ
30 min incubation, the absorbance was read at 540 nm on a microplate reader (FLUOstar Omega) [36].
The experiment was performed in duplicate on different days with different cell cultures and different
stock of the hybrids. The IC50 values were determined by interpolation from the mean absorbance
data of 100% viability (negative control) and 0% viability (positive control).
4. Conclusions
In summary, we report the antimicrobial activity of eight quinoline-based hydroxyimidazolium
hybrids 7a–h against panels of fungal and bacterial pathogens, including mycobacteria. Hybrid
compounds 7c–d showed the highest antifungal activity against C. with MIC values of 15.6
µg/mL
each one. Furthermore, all hybrids showed promising antibacterial activity, although, compound 7b
presented the strongest activity against S. aureus and M. tuberculosis H37Rv with MIC values of 2
(5 µM) and 10 µg/mL (24 µM), respectively.
µg/mL
Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6382/8/4/239/s1,
Table S1. Percentages of inhibition of Escherichia coli by hybrids 7a–h, Table S2. Percentages of inhibition of
Klebsiella pneumoniae by hybrids 7a–h, Table S3. Percentages of inhibition of Staphylococcus aureus by hybrids 7a–h
.
Figure S1. Copies of 1H NMR spectra for compounds 7a–h.
Author Contributions: D.I., B.I., J.Q., and R.A. designed the target compounds and performed the synthetic
experiments; S.Z. and L.S. planned and performed the antifungal assays and drew the graphics; D.I., O.V., A.B.,
E.M., J.G., and G.P. planned and performed the antibacterial assays and drew the graphics; all authors contributed
in writing the paper.
Funding: This research received no external funding.
Acknowledgments: The authors gratefully acknowledge financial support from COLCIENCIAS, Universidad del
Valle, Universidad del Norte. L.S. and S.Z. acknowledge Agencia Nacional de Promoción Científica y Tecnológica
(ANPCyT), PICT2016-1833, and the National University of Rosario, Argentina (UNR) for funds. L.S. is a member
of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) researcher career.
Conflicts of Interest: The authors declare no conflict of interest.

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