Electrochemistry of ring-substituted 1-hydroxynaphthalene-2-carboxanilides: Relation to structure and biological activity

Article abstract of DOI:10.1016/j.electacta.2019.135485

Twenty-two novel antimycobacterial agents, 1-hydroxynaphthalene-2-carboxanilides, were studied by cyclic voltammetry on a glassy carbon electrode in a phosphate buffer pH 7.2 – dimethyl sulfoxide (DMSO) mixed medium (9:1; v/v). All compounds exhibited similar voltammetric behavior with one irreversible anodic signal in the range 100–300 mV corresponding to the oxidation of hydroxyl group on the naphthalene moiety. A shift of the oxidation potential was caused solely by electron donating or withdrawing effects of substituents and their position on the benzene moiety. Mechanism of oxidation in the studied medium was briefly outlined. Values of oxidation potentials exhibited very good linear correlation with calculated Hammett σ substituent constants. For all active compounds, a relationship between oxidation potentials and MIC or IC₅₀ values obtained from in vitro screening was investigated in detail. Primary in vitro screening of synthesized compounds was previously performed against three species of Mycobacterium pathogens. Additionally, their activity related to the inhibition of photosynthetic electron transport (PET) in spinach chloroplasts was tested in previous publications. In vitro screening against Mycobacterium tuberculosis was performed here for the first time with 1-hydroxy-N-(3-trifluoromethylphenyl)naphthalene-2-carboxamide being the most effective (MIC = 11.7 μmol L^?1). Furthermore, several other compounds showed higher antimycobacterial activity than the standard isoniazid. Relation of biological activities and oxidation potentials was successfully found in some cases; however, final correlations must also be considered with other physical and chemical factors contributing to the biological activity. Relation of structure, biological activity and electrochemical potential was also studied by cyclic voltammetry in cathodic area for three compounds containing reducible nitro moiety.

Full text of DOI:10.1016/j.electacta.2019.135485

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Electrochemistry of ring-substituted 1-hydroxynaphthalene-2-carboxanilides:
Relation to structure and biological activity
Július Gajdár, Konstantina Tsami, Hana Michnová, Tomáš Goněc, Marie
Brázdová, Zuzana Soldánová, Miroslav Fojta, Josef Jampílek, Jiří Barek, Jan Fischer
PII:
S0013-4686(19)32357-6
DOI:
https://doi.org/10.1016/j.electacta.2019.135485
EA 135485
Reference:
To appear in:
Electrochimica Acta
Received Date:
Accepted Date:
19 September 2019
09 December 2019
Please cite this article as: Július Gajdár, Konstantina Tsami, Hana Michnová, Tomáš Goněc, Marie
Brázdová, Zuzana Soldánová, Miroslav Fojta, Josef Jampílek, Jiří Barek, Jan Fischer,
Electrochemistry of ring-substituted 1-hydroxynaphthalene-2-carboxanilides: Relation to structure
and biological activity, Electrochimica Acta (2019), https://doi.org/10.1016/j.electacta.2019.135485
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Electrochemistry of ring-substituted 1-hydroxynaphthalene-2-carboxanilides: Relation
to structure and biological activity
1
2
3
4
5
Július Gajdár , Konstantina Tsami , Hana Michnová , Tomáš Goněc , Marie Brázdová ,
Zuzana Soldánová 5, Miroslav Fojta 5, Josef Jampílek 3,6, Jiří Barek 1, and Jan Fischer 1
1
Charles University, Faculty of Science, Department of Analytical Chemistry, UNESCO
Laboratory of Environmental Electrochemistry, Albertov 6, Prague 2, 12843, Czech Republic
2
National and Kapodistrian University of Athens, Department of Chemistry, Laboratory of
Analytical Chemistry, Athens, 15771, Greece
3
Palacký University, Faculty of Science, Regional Centre of Advanced Technologies and
Materials, Šlechtitelů 27, Olomouc, 78371, Czech Republic
4
University of Veterinary and Pharmaceutical Sciences Brno, Faculty of Pharmacy,
Department of Chemical Drugs, Palackého 1, Brno, 61242, Czech Republic
5 Institute of Biophysics of the Czech Academy of Sciences, Královopolská 135, Brno, 61265,
Czech Republic
6
Comenius University, Faculty of Natural Sciences, Department of Analytical Chemistry,
Ilkovičova 6, Bratislava, 84215, Slovakia
Abstract
Twenty-two novel antimycobacterial agents, 1-hydroxynaphthalene-2-carboxanilides, were
studied by cyclic voltammetry on a glassy carbon electrode in a phosphate buffer pH 7.2 –
dimethyl sulfoxide (DMSO) mixed medium (9:1; v/v). All compounds exhibited similar
voltammetric behavior with one irreversible anodic signal in the range 100-300 mV
corresponding to the oxidation of hydroxyl group on the naphthalene moiety. A shift of the
oxidation potential was caused solely by electron donating or withdrawing effects of
substituents and their position on the benzene moiety. Mechanism of oxidation in the studied
medium was briefly outlined. Values of oxidation potentials exhibited very good linear
correlation with calculated Hammett σ substituent constants. For all active compounds, a
relationship between oxidation potentials and MIC or IC50 values obtained from in vitro
screening was investigated in detail. Primary in vitro screening of synthesized compounds
was previously performed against three species of Mycobacterium pathogens. Additionally,
their activity related to the inhibition of photosynthetic electron transport (PET) in spinach
chloroplasts was tested in previous publications. In vitro screening against Mycobacterium
tuberculosis was performed here for the first time with 1-hydroxy-N-(3-
trifluoromethylphenyl)naphthalene-2-carboxamide being the most effective (MIC = 11.7
µmol L−1). Furthermore, several other compounds showed higher antimycobacterial activity
than the standard isoniazid. Relation of biological activities and oxidation potentials was
successfully found in some cases; however, final correlations must also be considered with
1

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other physical and chemical factors contributing to the biological activity. Relation of
structure, biological activity and electrochemical potential was also studied by cyclic
voltammetry in cathodic area for three compounds containing reducible nitro moiety.
Keywords:
Hydroxynaphthalene-2-carboxanilides,
Antimycobacterial
activity,
Photosynthetic electron transport inhibition, Hammett correlation, Structure-activity
relationship
1. Introduction
Tuberculosis (TB) is still one of the top ten causes of death worldwide and it is a leading
cause of death caused by a single infectious agent; a pathogen called Mycobacterium
tuberculosis. There were around 10 million new cases of TB in 2017 and approximately 0.6
million of them were caused by emerging multidrug-resistant strains of pathogens. Treatment
in those cases by the most common first-line drugs used for treatment of TB, isoniazid and
rifampicin, is ineffective. It complicates and prolongs the healing process and an individual
therapy lasting several months is necessary [1]. All of these facts are the reason for rapid and
intense research into novel molecular scaffolds [2]. Other nontuberculous Mycobacteria are
also responsible for a broad spectrum of diseases and are especially dangerous for
immunocompromised patients [3, 4].
Emergence of drug-resistant strains of Mycobacterium tuberculosis has led to the
development of novel antimycobacterial agents based on the structure of
hydroxynaphthalenecarboxanilide. These novel agents demonstrated spectrum of anti-
infectious [5-12] and/or antiproliferative properties [13, 14]. These structurally simple
compounds, regarded as the ring analogues of salicylanilides (e.g., niclosamide [7]) can be
considered as privileged structures. Their activity depends on the mutual position of
carboxamide and phenolic moieties [5-7, 9, 12], therefore analogues with various position of
phenolic moiety show different spectrum of biological activities as well as potency. A
carboxamide linker (CONH) mimicking a peptide bond is crucial for the activity, because by
the means of this moiety, a compound is bound to their supposed targets. Following the
polypharmacology idea, these compounds can be classified as multi-target compounds, since
they are able to affect various target structures, especially in microbial pathogens, similarly as
salicylanilides [15-18]. It is assumed that anilide part of the molecule is responsible for
influencing the physicochemical properties and binding strength of the test compounds to the
potential target. Moreover, variously substituted anilide rings cause an excess or lack of
electrons in the amide bond (in connection with the nature of the substituent and its electron
donating or withdrawing properties), which causes various acidity/oxidizability of a spatially
close phenolic moiety, resulting in the wide spectrum of activity and potency of the individual
compounds. These subtle changes in oxidizability can be presumably studied by
electrochemical methods in which case a change of measured oxidation potential would be an
indicator of different structure and, therefore, possibly also a different activity and potency.
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This study concerns only one subgroup of these compounds, 1-hydroxynaphthalene-2-
carboxanilides, that were prepared and tested by Gonec et al. [5]. It should be noted that this
type of compounds is able to inhibit photosynthetic electron transport (PET) in spinach
(Spinacia oleracea L.) chloroplasts. The formation of hydrogen bonds between the CONH
moiety and the target proteins in photosynthetic centers of thylakoid membranes changes
protein conformation resulting in PET inhibition. This amide moiety is present in many
herbicides acting as PET inhibitors in photosystem (PS) II, where they cause displacement of
plastoquinone (QB) from its binding pocket in one of the protein subunits of PS II, the D1
protein
[15,
19].
It
can
be
hypothesized
that
above-mentioned
hydroxynaphthalenecarboxanilides are able to inhibit cell growth by the inhibition of the
respiration by several possible targets/mechanisms of action, i.e. by the inhibition of ATP
synthase or cytochrome bc1, or by the inhibition of the electron transport [4, 7, 9, 20-22].
Thus, it was observed that antimycobacterial activity correlates with PET inhibition profile [5-
11, 15, 23-25].
A pilot electrochemical study of a model compound 1-hydroxy-N-(4-nitrophenyl)
naphthalene-2-carboxamide was carried out by our team in very small sample volumes. This
compound provided one anodic and one cathodic signal corresponding to the oxidation of
hydroxyl group and reduction of nitro group, respectively. Mechanisms of redox processes
were outlined and studied by cyclic voltammetry [26]. Findings included in that work were
used as a baseline for this current study. Phenols are electrochemically oxidized to radicals
which then undergo coupling reactions to create dimers or they can be further
electrochemically oxidized (at more positive potentials) to form quinone-like structures [27-
29]. Reducible nitro substituted aromatic compounds have been investigated extensively by
electrochemical methods [27, 30-32].
This study aims to investigate the relationship between structure and electrochemical potential
based on application of Hammett substituent constants that were at first collected by Zuman
[33]. Investigation of salicylaldehyde anils [34] and some boron heterocycles [35] even
reveals an effect of substituents that are connected to the oxidation center through more atoms
and delocalized systems. The aim of this work is also to investigate relationship between
electrochemistry and biological activity based on recent studies that have shown the
correlation between electrochemical behavior, structure and biological activity for some
groups of compounds [36-40]. Relations between electrochemical data, biological activity and
biochemical mechanism of action of antibacterial agents, derivatives of quinoxalines di-N-
oxides, was investigated [36, 41]. Another group of antibiotic compounds based on arylazoxy
derivatives was studied and a correlation between Hammett σ substituent constant and
electrochemical potential of reduction of studied compounds was proved [37]. However, it
must be noted that biological processes are complex and thus an absolute correlation with
electrochemistry cannot be expected and numbers of other parameters must be considered like
lipophilicity, diffusion, solubility, metabolism, membrane permeability, absorption, active site
binding, etc. [36, 38]. Alternatively, many biological processes are based on redox reactions
and studying electron transfer mechanism of those reactions by electrochemical methods can
be useful for observing and predicting biological phenomena [42, 43]. Correlation between
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electrochemistry and biological activity can also point to bio-reduction (bio-oxidation)
playing an important role in the mode of action [39, 40]. The prediction of substituent effect
can even possibly aid designing more powerful derivatives [44].
2. Experimental
2.1. Chemicals
Compounds 1-8c were synthetized according to methods described in detail in [5] and
schematically depicted in Scheme 1.
Scheme 1: Synthesis of ring-substituted 1-hydroxynaphthalene-2-carboxanilides 1-8c: R = H
(1), OCH3 (2a-c), CH3 (3a-c), F (4a-c), Cl (5a-c), Br (6a-c), CF3 (7a-c), NO2 (8a-c); (a) PCl3,
chlorobenzene, microwave (adapted from [5]).
2.2 Electrochemical procedures
Appropriate amount of solid compound 1-8c was weighted and dissolved in 1 mL of dimethyl
sulfoxide (DMSO, p.a., Penta, Czech Republic) to the concentration 1 mmol L−1. 0.1 mol L−1
phosphate buffer was used as a supporting electrolyte and it was prepared by mixing disodium
hydrogenphosphate and sodium dihydrogen phosphate adjusted to pH 7.2 by concentrated
1 mol L−1 sodium hydroxide solution (all p.a., Lach-ner, Czech Republic). Prepared solutions
were stored in a fridge in the dark.
All voltammetric measurements were carried out on Eco-Tribo Polarograph controlled by
Polar Pro 5.1 software (Polaro-Sensors, Czech Republic). A three-electrode system was used
with a working glassy carbon disc electrode (GCE, 2 mm diameter, Metrohm, Switzerland),
an Ag|AgCl|3 mol L−1 KCl reference electrode (Elektrochemicke detektory, Turnov, Czech
Republic), and an auxiliary platinum wire electrode (0.5 mm diameter). pH was measured
with pH-meter Jenway 3510 (Bibby Scientific Ltd, United Kingdom) with Jenway combined
glass electrode (924 005) calibrated using standard aqueous buffers. The working GCE had to
be pre-treated before every measurement by polishing to mirror-like appearance with an
aqueous slurry of alumina powder (1.1 µm) on a polishing pad and then it was sonicated for
30s in methanol and for 30s in deionized water.
Cyclic voltammetry (CV) was usually carried out at a scan rate 50 mV s−1. Daily variations of
the reference electrode potential were resolved by evaluating the peak of 50 µmol L−1
K4[FeII(CN)6] in the separate solution containing the same supporting electrolyte that was
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measured in regular intervals. Measured potentials are usually reported after the subtraction of
[FeII(CN)6]4− oxidation potential. Solutions for measurements were prepared as follows: 20
(or 10) µL of 1 mmol L−1 stock solution of studied compound and 80 (or 90) µL of DMSO
were pipetted into a vial and filled with phosphate buffer pH 7.2 to the final volume 1.0 mL.
Solution was then transferred to a voltammetric cell. In the case of reducible substances 8a-c
oxygen was removed by 5min purging with nitrogen. CV scans were repeated at least 3 times
for every single compound with the pre-treatment of GCE in-between scans. Potentials were
evaluated at the point of maximum peak height for every single peak in CV. Parameters of
fitted curves were evaluated by OriginPro 2016 software and they are reported at 0.95
confidence level.
2.3. In vitro antimycobacterial evaluation
The methodology of in vitro antimycobacterial activity screening of the investigated
compounds 1-8c against Mycobacterium marinum CAMP 5644, M. kansasii DSM 44162 and
M. smegmatis ATCC 700084 was described in [5] and minimum inhibitory concentration
(MIC) values are shown in Table 1.
Mycobacterium tuberculosis ATCC 25177/H37Ra was grown in Middlebrook broth (MB),
supplemented with Oleic-Albumin-Dextrose-Catalase (OADC) supplement (Difco, Lawrence,
KS, USA). At log phase growth, a culture sample (10 mL) was centrifuged at 15,000 rpm/20
min using a bench top centrifuge (MPW-65R, MPW Med Instruments, Warszawa, Poland).
Following the removal of the supernatant, the pellet was washed in fresh Middlebrook
7H9GC broth and resuspended in fresh OADC-supplemented MB (10 mL). The turbidity was
adjusted with MB to match McFarland standard No. 1 (3×108 CFU). A further 1:10 dilution
of the culture was then performed in MB. The antimicrobial susceptibility of M. tuberculosis
was investigated in a 96-well plate format. In these experiments, sterile deionized water (300
µL) was added to all outer-perimeter wells of the plates to minimize evaporation of the
medium in the test wells during incubation. Each evaluated compound (100 µL) was
incubated with M. tuberculosis (100 µL). Dilutions of each compound were prepared in
duplicate. For all synthesized compounds, final concentrations ranged from 256 to 2 μg mL−1.
All compounds were dissolved in DMSO, and subsequent dilutions were made in
supplemented MB. The plates were sealed with Parafilm and incubated at 37 °C for 14 days.
Following the incubation, a 10% addition of alamarBlue (Difco) was mixed into each well,
and readings at 570 nm and 600 nm were taken, initially for background subtraction and
subsequently after 24 h reincubation. The background subtraction is necessary for strongly
colored compounds, where the color may interfere with the interpretation of any color change.
For noninterfering compounds, a blue color in the well was interpreted as the absence of
growth, and a pink color was scored as growth. Isoniazid (Sigma) was used as the positive
control, as it is a clinically used antitubercular drug. The results are shown in Table 1.
2.4. Study of inhibition of photosynthetic electron transport (PET) in spinach chloroplasts
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The methodology of the evaluation of ring-substituted 1-hydroxynaphthalene-2-carboxanilides
1-8c related to the inhibition of photosynthetic electron transport (PET) in spinach (Spinacia
oleracea L.) chloroplasts was described in [5]. Inhibitory efficiency was expressed as IC50
values, see Table 1.
3. Results and discussion
3.1 Voltammetry in anodic area: Mechanism of oxidation
The phosphate buffer pH 7.2 – DMSO (9:1, v/v) medium was mostly used in this
voltammetric study. It was the optimal medium for the voltammetric determination of the
studied compound 8c from our pilot study [26]. These conditions also provide similar pH that
was used for in vitro screening of studied compounds, and it is also very close to
physiological conditions. DMSO had to be used because of a limited solubility of compounds
in aqueous media.
All the compounds gave one anodic oxidation response (Ia) ranging from ca. 200 to 400 mV
vs. Ag/AgCl. Mechanism of the oxidation was closely studied with an unsubstituted
compound 1 and conclusions from our previous study of the compound 8c were also utilized
[26]. The cyclic voltammogram in anodic area of the compound 1 is shown in Fig. 1. The
compound 1 gave the peak Ia at 417 mV in the forward scan. In the reverse scan, products of
the oxidation were reduced at the potential 181 mV at the peak Ic. In the second sweep a new
peak IIa appeared at 208 mV and the height of the peak Ia was significantly decreased.
Heights of all peaks were slowly decreasing during multiple consecutive scans probably due
to the passivation of the electrode surface. A study by cyclic voltammetry with scan rates 10 –
1000 mV s−1 suggested that process Ia was diffusion controlled (until scan rate 200 mV s−1)
and the process IIa was controlled by adsorption. The current of the peak Ic did not correlate
well with either scan rate or its square root, therefore mixed adsorption/diffusion control is
presumed. The potential of the peak Ia was shifted to more positive potentials with the
increasing scan rate as expected from an irreversible process. However, potentials of peaks Ic
and IIa remained the same. The current ratio of i(Ia):i(Ic) was around 2:1 and it was
independent on the scan rate.
All the compounds should exhibit the same pH dependency of the voltammetric response
similar to the compound 8c from our previous study [26]. The potential of the peak Ia
depended on pH in neutral and slightly alkaline media with a slope close to −59 mV pH−1
(1e−/1H+ oxidation) suggesting a deprotonation step in the oxidation mechanism [26]. The
oxidation potential was independent on pH in alkaline media, meaning that the compound
(hydroxyl group) was already in the deprotonated form (given by pKa of the compound).
Cyclic voltammetry of the compound 1 carried out in an alkaline buffer pH 11 – DMSO (9:1,
v/v) medium gave only two peaks during multiple scans. There was the peak Ia in the forward
scan and the peak Ic in the reverse scan. However, peak IIa for adsorbed species in the second
sweep was not observed.
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From the literature concerning the oxidation of phenolic compounds [27-29] we can suggest,
that the process Ia should be an irreversible process corresponding to the first step of the
oxidation to a radical according to Scheme 2. Generated radicals in following reactions can
either react with components of the solution or create polymeric films that contribute to the
passivation of the electrode surface which is a common phenomenon observed during
electrooxidation of compounds containing phenolic moiety [45, 46]. Peaks Ic, and IIa
therefore correspond to adsorption processes involving adsorbed molecules created from
previous reactions.
I, A
0.6
Ia
0.3
IIa
0.0
Ic
-0.3
0
200
400
600
E vs Ag/AgCl, mV
Fig. 1: Cyclic voltammogram (scan rate 50 mV s−1) of compound 1 (c= 20 µmol L−1)
obtained on GCE in phosphate buffer pH 7.2 and DMSO (9:1, v/v) medium. The first cycle
(red line) and forward scan of the second cycle (blue) are shown (reverse scan of the second
cycle is the same as the first one). Forward scan of the supporting electrolyte is illustrated as a
black dotted line. The arrow denotes the start and the direction of a scan from 0 V.
Scheme 2: Proposed first step of mechanism of the oxidation of compounds 1-8c on GCE in
the phosphate buffer pH 7.2 and DMSO (9:1, v/v) medium.
3.2 Voltammetry in anodic area: Relation of oxidation potential and Hammett σ values
As mentioned before, compounds 1-8c were studied by the method of CV in the phosphate
buffer pH 7.2 – DMSO medium (9:1, v/v). Experimentally obtained values of peaks Ia and Ic
are shown in Table 1. Scan rate 50 mV s−1 was used because at that scan rate process Ia is still
diffusion controlled which is a requirement for application of Hammett correlations.
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All the studied compounds gave peaks corresponding to the signals obtained for the
compound 1 described in the previous chapter, but respective potentials of observed peaks
were different and characteristic for every substituent and its position. The proposed
mechanism of oxidation was the same for every compound and no irregularities were
observed, concluding that substituents do not participate in the redox reaction (apart from
compounds 8a-c that contain a reducible nitro group at far more negative potentials; see
chapter 3.4). The only difference in oxidation potentials of the hydroxyl moiety on the
naphthalene ring was therefore caused by electron withdrawing or electron donating effects of
various substituents on the benzene ring (or sum of inductive and mesomeric effects).
Monosubstituted derivatives are going to be discussed in three groups based on the position of
the substituent (ortho, meta, para) for the purpose of finding a relation between the structure
and the electrochemical potential. Hammett σ substituent values were adapted from [5] and
they were correlated with experimentally obtained values of potentials for the peak Ia and the
peak Ic from the reverse scan. As can be seen from plots for ortho- (Fig. 2A), meta- (Fig. 2B),
and para- (Fig. 2C) substituted compounds, it is possible to establish a linear correlation
between σ values and the potential of oxidation. The potential of the peak Ia of ortho- and
para- derivatives gave linear dependency with similar slopes: −104±24 mV (r = −0.8866);
−99±24 mV (r = −0.8789), respectively. However, unsubstituted compound 1 had to be
excluded from both of correlations as an outlier. Meta- derivatives gave linear dependency
with a higher slope value −163±35 mV (r = −0.8855), but with the unsubstituted compound 1
included in the correlation. All calculated slopes were significantly different from 0 (at 0.95
confidence level); therefore, there is a very good correlation in measured data between
Hammett σ substituent constants and oxidation potential. Electron donating substituents -CH3
and -OCH3 caused a peak shift to more positive potentials, while on the other side electron
withdrawing substituents -NO2 and -CF3 caused a peak shift towards 0 V, facilitating the
oxidation. Halogen derivatives having -F, -Cl, and -Br as a weak electron withdrawing
substituent had the oxidation potential in-between. Unsubstituted compound 1 gave the most
positive potential. The value of a slope indicates how strongly a substituent affects the
oxidation of a compound [33]. These findings suggest that electron withdrawing and donating
effects of substituents on the benzene ring are significantly influencing electron density at the
hydroxyl group on the naphthalene ring connected to the benzene ring with the –CONH–
bridge. Additionally, a significant difference between slopes of ortho/para and meta
derivatives was caused by well-known difference of inductive effect causing a change in
electron density which is similar for ortho/para position contrary to meta position.
Similarly, dependencies of more positive cathodic peak Ic were evaluated (Fig. 2) but they
gave lower slope values; in the case of meta-derivatives, a slope that was not even
significantly different from 0. Therefore, for further discussion only potentials of the peak Ia
are investigated.
Slight deviations from linear trend might be caused by varying adsorption or sterical effects of
the respective compounds. In an ideal case, it should only be applied to diffusion processes
[33]. In this case, adsorption processes also have to be considered based on a significant
hydrophobicity of compounds. However, very good correlations were observed (r ~ 0.88),
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even though the studied substituents were on the secondary aromatic structure connected by –
CONH– bridge.
300
-H
A
-OCH3
-Br
-Cl
-F
200
100
0
-CH3
-NO2
-CF3
-CF3
peak Ia
-H
-Br
-Cl
-NO2
peak Ic
-CH3
-OCH3
-F
-0.4
0.0
0.4
0.8

300
200
100
0
-H
B
-OCH3
-CH3
-F
-CF3
-Cl
-NO2
-Br
peak Ia
peak Ic
-F -Cl
-H
-CH3
-NO2
-CF3
-OCH3
-Br
0.0
0.4
0.8

300
200
100
0
-H
C
-CH3
-F
-F
-Br
-OCH3
-CF3
peak Ia
-Cl
-NO2
-NO2
-CF3
peak Ic
-Cl
-Br
-H
-OCH3
-CH3
-0.4
0.0
0.4
0.8

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Fig. 2: Plot of potentials of anodic peak Ia (red) and cathodic reverse peak Ic (blue) vs.
Hammett σ substituent constants of ortho- (A); meta- (B); and para- (C) substituted
compounds 1-8c obtained from Table 1. Linear straight lines correspond to linear fits; shaded
areas illustrate confidence intervals at 0.95 level. Crossed-out unsubstituted compound 1 in A
and C was not included in a linear fit. Linear fit parameters are summarized in Table 2.
Table 1: Measured anodic potentials of signals Ia and Ic from CVs of ring-substituted 1-
hydroxynaphthalene-2-carboxanilides 1-8c and their calculated Hammett σ substituent
constants, in vitro antimycobacterial activities (MICs) of compounds adapted from [5], new
MICs against M. tuberculosis and IC50 values related to PET inhibition in spinach
chloroplasts.
Eb, mV
Ia
MIC, µmol L−1
MKd MSe
15.2
>873
>873
>873
115
PET IC50,
µmol L−1
31.3
199.6
23.5
79.5
62.8
20.0
28.7
57.0
15.6
20.3
29.4
7.9
Compd
R-
σa
Ic
43
MMc
60.7
>873
54.5
>873
28.8
28.8
115.0
56.8
MTf
60.8
>873
54.5
>873
231
28.8
462
56.9
28.4
114
430
26.9
430
>748
93.6
>748
23.4
11.7
23.4
415
>830
>830
1
H
0
279
224
240
199
207
241
241
197
196
182
186
180
143
192
147
185
104
195
145
132
147
117
243
>873
>873
>873
231
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
8a
8b
8c
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
2-CF3
3-CF3
4-CF3
2-NO2
3-NO2
4-NO2
−0.28
0.12
−0.27
−0.17
−0.07
−0.17
0.06
0.34
0.06
0.22
0.37
0.23
0.22
0.39
0.23
0.51
0.43
0.51
0.77
0.71
0.78
−11
28
40
−7
28
27
−6
44
43
−7
44
47
6
28.8
57.7
>910
57.7
923
228
28.4
28.4
14.2
860
910
>910
860
114.0
107.0
107.0
215.0
>748
>748
46.7
96.6
46.7
187.0
>830
>830
104.0
26.8
26.8
>860
215
10.8
61.9
8.2
9.6
126.1
5.3
36.5
NDg
19.9
24.0
>748
>748
23.3
>748
>748
29
30
26
39
55
33
44
57
46.7
>773
>748
23.3
>830
>830
>773
>748
>748
>830
>830
>830
51.9
a- calculated using ACD/Percepta ver. 2012 (Advanced Chemistry Development, Inc.,
Toronto, ON, Canada, 2012); adapted from [5]
b- Potentials reported vs [FeII(CN)6]4−
c- MM = M. marinum CAMP 5644; adapted from [5]
d- MK = M. kansasii DSM 44162; adapted from [5]
e- MS = M. smegmatis ATCC 700084; adapted from [5]
f- MT = M. tuberculosis ATCC 25177/H37Ra
g- ND = not determined, see chapter 3.3
3.3 Relation between oxidation potential and biological activity
The evaluation of the in vitro antimycobacterial activity of compounds 1-8c was performed
against Mycobacterium marinum CAMP 5644, M. kansasii DSM 44162 and M. smegmatis
10

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ATCC 700084, see Table 1. The MIC values (minimum inhibitory concentration; lowest
concentration at which no visible bacterial growth is observed) were taken from Gonec et al.
[5]. In addition, all the compounds were evaluated against M. tuberculosis ATCC
25177/H37Ra. Avirulent strain M. tuberculosis ATCC 25177/H37Ra has a similar pathology
as M. tuberculosis strains infecting humans [47], and simultaneously with M. marinum, both
strains are commonly used model pathogens in basic laboratory screening for reduction of
risks of laboratory workers [5]. M. kansasii DSM 44162 was used as a representative of non-
tuberculous mycobacteria, causing the most frequent non-tuberculous mycobacterial lung
infections [48]. All these strains belong to slow-growing species contrary to M. smegmatis,
which is an ideal representative of a fast-growing non-pathogenic microorganism particularly
useful in studying basic cellular processes of special relevance to pathogenic mycobacteria [5,
48].
Antimycobacterial activities of the compounds against individual tested strains were
expressed as log(1/MIC) and were plotted against anodic oxidation potentials expressed as
E(Ia), illustrated in Figs. 3 and 4 for the purpose of finding a relationship between the
antimycobacterial activity and the potential. Note that only compounds with exact MIC values
are illustrated in graphs. All the compounds were completely inactive against fast-growing M.
smegmatis with the exception of compounds 6c (R = 4-Br) and 3b (R = 3-CH3), that showed
approx. 2.5-fold higher potency than isoniazid (commonly used drug for TB treatment); their
E(Ia) values are 185 and 241 mV, respectively. Due to a small number of effective
compounds, any dependence of the activity on the anodic potential was difficult to predict.
After elimination of all inactive compounds (MIC > 100 µmol L−1) from following
correlations (activities against M. marinum, M. kansasii and M. tuberculosis), only around 10
active compounds were left to investigate their relation to oxidation potentials. This amount
proved to be too small to provide any definitive conclusions; anyway, it was still possible to
outline some trends that could be useful in establishing a relation between activity and
potential. This was illustrated on dependencies of activity against M. marinum and M.
kansasii (after elimination of inactive compounds and unsubstituted 1). Dependence of
potential on activity against M. marinum (Fig. 3A) gave a quasi-parabolic trend (r = 0.7530, n
= 9). This trend had a maximum around potentials 195-206 mV (compounds 3a and 4b).
Dependency of the potential and the activity against M. kansasii (Fig. 3B) was also fitted with
a quasi-parabolic trend (r = 0.7716, n = 9) with a maximum around potential 182 mV
(compound 4c).
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A
4.6
4.4
4.2
4.0
3.8
4b
3a
3b
7b
4a
6c
2b
1
ortho
meta
para
H
7a
100
150
200
250
300
E vs [Fe(CN)6]4-, mV
5.0
4.8
4.6
4.4
4.2
4.0
B
4c
1
7c
6c
5b
5c
4b
3b
3c
8c
meta
para
H
100
150
200
E vs [Fe(CN)6]4-, mV
250
Fig. 3. Plot of log(1/MIC [mol L−1]) of in vitro antimycobacterial activity against M. marinum
(A) and M. kansasii (B) vs. potential of anodic peak E(Ia) of selected compounds 1-8c; values
obtained from Table 1. Dashed line represents quasi-parabolic trend. Polynomial fit
parameters are given in Table 2.
1-Hydroxy-N-(3-trifluoromethylphenyl)naphthalene-2-carboxamide (7b) demonstrated the
highest effectivity (MIC = 11.7 µmol L−1) against M. tuberculosis. Additionally, other
compounds, such as 7a (R = 2-CF3), 7c (R = 4-CF3), 5b (R = 3-Cl), 3b (R = 3-CH3) and 4b
(R = 3-F) also showed high potency in the range from 23.4 to 28.8 µmol L−1. Since MIC of
isoniazid against M. tuberculosis is 36.6 µmol L−1, it can be stated that the above-mentioned
compounds expressed higher potency than the standard. Nevertheless, it is evident that the
activity against M. tuberculosis is, in general, connected with CF3 substitution of the anilide
ring. On the other hand, meta position of substitution was the most advantageous similar to
the case of M. marinum, but contrary to M. kansasii, where para position of substitution on
the anilide ring is preferable [5]. A quasi-parabolic trend with a good correlation (r = 0.8620,
12

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n = 6) was found (Fig. 4) only for a subset of meta derivatives with a maximum at the
potential 195 mV (compound 7b). The only active ortho and para compounds in the order 7a,
7c, 4a, 1 had a decreasing activity with higher oxidation potential; however, no reliable
conclusions can be drawn from this trend.
5.2
ortho
meta
para
H
7b
4.8
7a
7c
5b
4b
4a
3b
2b
4.4
4.0
1
6b
100
150
200
250
300
E vs [Fe(CN)6]4-, mV
Fig. 4. Plot of log(1/MIC [mol L−1]) of in vitro antimycobacterial activity against M.
tuberculosis vs. potential of anodic peak E(Ia) of selected compounds 1-8c; values obtained
from Table 1. Dashed line represents quasi-parabolic trend only for meta subset. Polynomial
fit parameters are given in Table 2.
As mentioned above, these compounds were additionally tested for their ability to inhibit
photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. PET
inhibiting activity expressed as IC50 value (compound concentration causing 50% inhibition
of PET) was taken from Gonec et al. [5] (Table 1). Compound 7b was the most effective
(IC50 = 5.3 µmol L−1), slightly worse than the selective herbicide 3-(3,4-dichlorophenyl)-1,1-
dimethylurea, DCMU (Diurone®) with IC50 = 1.9 µmol L−1. It was not possible to determine
IC50 value for the compound 8a (R = 2-NO2) due to its interactions during the measurement.
Compound 2a (R = 2-OCH3) expressed the lowest PET-inhibiting activity
(IC50 = 199.6 µmol L−1), although, in general, it can be stated that ortho-substituted subset
expressed the lowest PET inhibition in the comparison with meta- and para-substituted
subsets. Within these two subsets, compounds 2c (R = 4-OCH3) and 7c (R = 4-CF3) showed
the lowest potency (IC50 = 79.5 and 36.5 µmol L−1, respectively), therefore, similarly as in
Gonec et al. [5], these compounds were excluded from the search of relationship between
PET-inhibiting activity and the oxidation potential. The dependences of PET inhibition
expressed as log(1/IC50) on the anodic potential E(Ia) of the compounds are shown in Fig. 5.
Even though ortho subset exhibits lowest activity, it gives very clear steep linear dependency
on the potential (r = 0.9373, n = 5). In addition, both meta and para substituted compounds
gave quasi-parabolic trend (r = 0.6925, n = 12) with maximum activity between potentials 180
to 195 mV. Furthermore, an above-mentioned presumption about a correlation between PET
13

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inhibition and the antimycobacterial activity [5-11, 15, 23-25] can be outlined based on these
results. This can be seen for effective compounds 7b (R = 3-CF3), 5b (R = 3-Cl), 4b (R = 3-F)
and 3b (R = 3-CH3); while, more importantly, compounds 3b and 4b were active in all
performed tests.
5.5
7b
5b
6c
6b
8b
5c
5.0
4.5
4.0
3.5
4b
4c
5a
3b
2b
8c
1
3c
7c
4a
6a
3a
2c
ortho
meta
para
H
7a
2a
100
150
200
250
300
E vs [Fe(CN)6]4-, mV
Fig. 5. Plot of log(1/IC50 [mol L−1]) of PET inhibition in spinach chloroplasts vs. potential of
anodic peak E(Ia) of selected compounds 1-8c; values obtained from Table 1. Eliminated
compounds are marked by an empty symbol. Dashed lines represent quasi-parabolic trend
only for meta and para subset (black) and linear trend for ortho subset only (red). Fit
parameters are given in Table 2.
Table 2: Parameters of calculated linear dependencies of potentials E(Ia) and E(Ic) on
Hammett σ substituent constants and polynomial dependencies of antimycobacterial activity
MIC values for M. marinum (M.m), M. kansasii (M.k.) and M. tuberculosis (M.t.) and PET
IC50 values on the potential E(Ia).
Hammett correlations: x = a × σ + b
From Fig.
2A
x, mV
a, mV
−104 (±24)
43.7 (±8.3)
−163 (±35)
14 (±11)
b, mV
r
E(Ia, ortho)
E(Ic, ortho)
E(Ia, meta)
E(Ic, meta)
E(Ia, para)
E(Ic, para)
197 (±10)
−3.4 (±3.2)
250 (±13)
33.1 (±4.0)
192.3 (±9.4)
38.6 (±3.2)
−0.8866
0.9204
−0.8855
0.4606
−0.8789
0.7260
2A
2B
2B
2C
−99 (±24)
22.5 (±8.7)
2C
Activity-structure correlations: x = a ×10−5 × E2(Ia) + b ×10−2 × E(Ia) + c
From Fig.
x
a, mV−2
−3.6 (±1.5)
−8.2 (±2.8)
−23 (±8.0)
−9.5 (±3.6)
-
b, mV−1
1.52 (±0.58)
3.0 (±1.0)
9.2 (±3.2)
3.4 (±1.3)
c
r
3A
3B
4
5
5
log(1/MIC(M.m.))
log(1/MIC(M.k.))
log(1/MIC(M.t.), meta)
log(1/IC50, meta+para)
log(1/IC50, ortho)
2.81 (±0.55)
1.97 (±0.91)
−4.6 (±3.1)
2.0 (±1.2)
8.03 (±0.83)
0.7530
0.7716
0.8621
0.6925
−0.9373
−1.92 (±0.41)
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As previously stated, these compounds can be considered as multi-target agents. One of the
supposed mechanisms of actions seems to be the inhibition of bacterial respiration by the
inhibition of ATP synthase (F0F1 complex) or cytochrome bc1 [4-10, 21]. Since enzymatic
systems and principles of these redox processes consisting in electron transfer across the
membrane are similar to each other, these compounds were also evaluated for their ability to
inhibit photosynthetic electron transport [49] (observed bond and inhibition of PSII target site
[5]) in chloroplasts. The most active substances have anodic potentials ranged 180 – 200 mV.
In relation to this fact, it can only be hypothesized that these compounds can possess a
specific (higher) ability to be subject to redox reaction, thus having a higher physical affinity
for these enzyme systems than other natural substrates. Binding to the enzyme system results
in its inhibition, and thus the disruption of the energy state of the cell, which is manifested by
an antiproliferative effect on mycobacteria and the inhibition of photosynthesis. Of course, in
addition to this physical affinity (characterized by an anodic potential ranged 180–200 mV),
the primary requirement for successful compound binding to enzyme systems is the steric
arrangement of molecules that unequivocally favor derivatives substituted at the meta position
of the anilide ring [5-10, 50-52]. It should be noted that compounds substituted by halogen
moieties with anodic potential ranging from 117 to 147 mV showed significant toxicity
against the normal human fibroblast cell line (NHDF) [14] and no antimycobacterial or PET-
inhibiting activity [5].
3.4 Voltammetry in cathodic area: Relation of potential on structure and biological activity of
compounds 8a-c
Only 3 of studied compounds 8a-c contain a nitro moiety (reducible at GCE), therefore they
will be discussed only briefly. Cyclic voltammogram of the compound 8b is shown in Fig. 6.
Compounds 8a-c gave one cathodic response (IIIc) corresponding to the well-known 4-
electron reduction to the hydroxylamine that is reversibly oxidized to the nitroso compound in
a reverse scan (pair of peaks IVa/IVc) [27, 31]. This mechanism of reduction was investigated
in detail in our previous publication with the compound 8c [26].
Regrettably, nitro derivatives 8a-c did not show any antimicrobial activity (Table 3), despite
many different compounds with a nitro moiety exhibiting high antimycobacterial and/or
antibacterial effects such as 5-nitroimidazoles (e.g., metronidazole, ornidazol, tinidazol) and
5-nitrofurans (e.g., nitrofurantoin, nifurantel, nifuroxazid), where a nitro moiety is a crucial
structural factor for the high activity. Effectivity of a new class of antimycobacterial drugs
derived from nitroimidazole, such as delamanid and pretomanid, is also based on the presence
of a nitro moiety [4]. From this point of view, the biological activity of these derivatives was
disappointing. Therefore, the only valid conclusion into a relationship between the structure
and the reduction potential IIIc of compounds 8a-c is as follows. The reduction is facilitated
in a series para→meta→ortho, with ortho-substituted compound having the potential closest
to 0 V, as summarized in Table 3.
It should be noted that in the previous paper, where antiproliferative activity of these
compounds was investigated [14], the potential of reversible peaks IVa/IVc correlates with
15

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the cytotoxicity against normal human fibroblast cell line (NHDF), where the compound 8a
(R = 2-NO2) showed IC50 = 22.71±2.71 µmol L–1, the compound 8b (R = 3-NO2) IC50 > 25
µmol L–1 and the compound 8c (R = 4-NO2) IC50 = 12.30±1.49 µmol L–1 [14]. It can be
summarized that cytotoxic potential against human cells within these nitro derivatives
increases with decreasing potential E(IVa/IVc) as follows: −242 mV (R = 3-NO2, 8b) < −268
mV (R = 2-NO2, 8a) < −292 mV (R = 4-NO2, 8c).
IVa
0.5
I, µA
0.0
IVc
-0.5
IIIc
-1.0
-900
-600
-300
0
E vs Ag/AgCl, mV
Fig. 6: Cyclic voltammogram (scan rate 50 mV s−1) of compound 8b (c = 10 µmol L−1)
obtained on GCE in phosphate buffer pH 7.2 and DMSO (9:1, v/v) medium. The first cycle
(red line) and forward scan of the second cycle (blue) are shown (reverse scan of the second
cycle is the same as the first one). The arrow denotes the start and the direction of a scan from
0 V.
Table 3: Measured cathodic potentials of signals IIIc and reversible pair IVa/IVc from CV of
NO2-substituted 1-hydroxynaphthalene-2-carboxanilides 8a-c and their IC50 values related to
PET inhibition in spinach chloroplasts; in vitro antimycobacterial activity (MIC) of
compounds adapted from [5].
Ea, mV
MIC, µmol L−1
PET IC50,
µmol L−1
NDf
Compd
R-
IIIc
IVa/IVc
−268
MMb
>830
>830
104
MKc
MSd
MTe
415
>830
>830
8a
8b
8c
2-NO2
3-NO2
4-NO2
−690
−721
−755
>830
>830
>830
>830
>830
−242
19.9
24.0
−292
51.9
a- Potentials reported vs [FeII(CN)6]4−
b- MM = M. marinum CAMP 5644; adapted from [5]
c- MK = M. kansasii DSM 44162; adapted from [5]
d- MS = M. smegmatis ATCC 700084; adapted from [5]
e- MT = M. tuberculosis ATCC 25177/H37Ra
f- ND = not determined, see chapter 3.3
16

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4. Conclusion
Twenty-two novel antimycobacterial agents were investigated by cyclic voltammetry. The
hydroxyl group on the naphthalene moiety gave one irreversible oxidation signal and its
potential ranged from 104 mV (7a, R = 2-CF3) to 279 mV (1, R = H) (vs. [FeII(CN)6]4−).
Relationships between the structure (Hammett σ substituent constant) and the oxidation
potential were established with satisfactory correlation coefficients (r ~ 0.88), that was
associated with electron donating or withdrawing capabilities of given substituents and their
position. In general, electron donating substituents caused a shift to more positive potentials
and vice versa. Correlations between the oxidation potential and in vitro antimycobacterial
activity of selected mycobacterial pathogens mostly gave quasi-parabolic dependencies with
good correlation coefficients (approx. r ~ 0.7-0.8) and with maxima usually in the range 180-
195 mV. However, two most active compounds 3b (R = 3-CH3) and 4b (R = 3-F) gave
potential 196 mV and 241 mV, respectively, so in general, biological activity should be
positively affected by higher oxidation potential.
Based on these results we can prove that the ease of oxidation of phenolic group can be used
as one of many parameters that can help design of novel antibiotic agents. In the end, it is
essential to remind that also a lot of other factors and physicochemical parameters, i.e.
lipophilicity (as discussed in Gonec et al. [5]) have an undeniable effect on the drug
effectivity.
Acknowledgments
J.G., K.T., J.B., and J.F. acknowledge the support of the Czech Science Foundation (17-
03868S). J.G. thanks for the support of Specific University Research (SVV 260440). H.M.,
T.G., and J.J. acknowledge the support of the Ministry of Education of the Czech Republic
(LO1305) and the Slovak Research and Development Agency (APVV-17-0373 and APVV-
17-0318). M.B., Z.S. and M.F. acknowledge institutional support from the Czech Academy of
Sciences (No. 68081707).
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CRediT author statement
Július Gajdár: Investigation; Writing - Original Draft, Formal analysis, Project administration
Konstantina Tsami: Investigation
Hana Michnová: Investigation
Tomáš Goněc: Investigation
Marie Brázdová: Supervision
Zuzana Soldánová: Investigation
Miroslav Fojta: Writing - Review & Editing
Josef Jampílek: Conceptualization, Writing - Original Draft, Formal analysis, Project
administration
Jiří Barek: Conceptualization, Writing - Review & Editing, Supervision
Jan Fischer: Conceptualization, Writing - Review & Editing, Supervision

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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: