Photosynthesis-inhibiting activity of N-(Disubstituted-phenyl)-3-hydroxynaphthalene-2-carboxamides

Article abstract of DOI:10.3390/molecules26144336

A set of twenty-four 3-hydroxynaphthalene-2-carboxanilides, disubstituted on the anilide ring by combinations of methoxy/methyl/fluoro/chloro/bromo and ditrifluoromethyl groups at different positions, was prepared. The compounds were tested for their ability to inhibit photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. N-(3,5-Difluorophenyl)-, N-(3,5-dimethylphenyl)-, N-(2,5-difluorophenyl)-and N-(2,5-dimethylphenyl)-3-hydroxynaphthalene-2-carboxamides showed the highest PET-inhibiting activity (IC₅₀ ~ 10 μM) within the series. These compounds were able to inhibit PET in photosystem II. It has been found that PET-inhibiting activity strongly depends on the position of the individual substituents on the anilide ring and on the lipophilicity of the compounds. The electron-withdrawing properties of the substituents contribute towards the PET activity of these compounds.

Full text of DOI:10.3390/molecules26144336

molecules
Article
Photosynthesis-Inhibiting Activity of N-(Disubstituted-
phenyl)-3-hydroxynaphthalene-2-carboxamides †
Jiri Kos 1,
*
, Tomas Gonec 2 , Michal Oravec 3, Izabela Jendrzejewska 4 and Josef Jampilek 5
1
Department of Biochemistry, Faculty of Medicine, Masaryk University, Kamenice 5,
62500 Brno, Czech Republic
Department of Chemical Drugs, Faculty of Pharmacy, Masaryk University, Palackeho tr. 1946/1,
2
61200 Brno, Czech Republic; t.gonec@seznam.cz
Global Change Research Institute of the Czech Academy of Sciences, Belidla 986/4a,
3
60300 Brno, Czech Republic; oravec.m@czechglobe.cz
Institute of Chemistry, University of Silesia, Szkolna 9, 40007 Katowice, Poland;
4
izabela.jendrzejewska@us.edu.pl
Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University, Ilkovicova 6,
5
84215 Bratislava, Slovakia; josef.jampilek@gmail.com
*
Correspondence: jirikos85@gmail.com
†
Preliminary results presented at the 24th International Electronic Conference on Synthetic Organic Chemistry,
15 November–15 December 2020. Available online: https://ecsoc-24.sciforum.net/.
Abstract: A set of twenty-four 3-hydroxynaphthalene-2-carboxanilides, disubstituted on the anilide
ring by combinations of methoxy/methyl/fluoro/chloro/bromo and ditrifluoromethyl groups at dif-
ferent positions, was prepared. The compounds were tested for their ability to inhibit photosynthetic
electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. N-(3,5-Difluorophenyl)-, N-(3,5-
dimethylphenyl)-, N-(2,5-difluorophenyl)- and N-(2,5-dimethylphenyl)-3-hydroxynaphthalene-2-
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Kos, J.; Gonec, T.; Oravec,
M.; Jendrzejewska, I.; Jampilek, J.
Photosynthesis-Inhibiting Activity of
N-(Disubstituted-phenyl)-3-
carboxamides showed the highest PET-inhibiting activity (IC50 ~ 10 µM) within the series. These
compounds were able to inhibit PET in photosystem II. It has been found that PET-inhibiting activ-
ity strongly depends on the position of the individual substituents on the anilide ring and on the
lipophilicity of the compounds. The electron-withdrawing properties of the substituents contribute
towards the PET activity of these compounds.
hydroxynaphthalene-2-carboxamides.
Molecules 2021, 26, 4336. https://
doi.org/10.3390/molecules26144336
Keywords: hydroxynaphthalene-carboxamides; PET inhibition; spinach chloroplasts; structure-
activity relationships
Academic Editor: Julio A. A. Seijas
Vázquez
Received: 5 June 2021
Accepted: 14 July 2021
Published: 17 July 2021
1. Introduction
Due to population growth, there is a constant pressure on farmers to multiply yields
to ensure sufficient food. On the other hand, this challenge is difficult to meet due to deteri-
orating conditions for agriculture, such as the loss of quality agricultural land, desiccation
or, conversely, heavy rains and floods, climate change and the rise of many plant and crop
destroyers. One way to combat pathogens of plants is to use pesticides to help farmers
increase productivity per hectare by protecting plants from pests, diseases and weeds. For
example, food crops must compete with approximately 30,000 weed species. Herbicides
are still used widely around the world because manual weeding has never been an effective
method of weed control, especially when large-scale farming is used. Herbicides are often
used instead of tillage because the use of herbicides reduces erosion, fuel consumption,
greenhouse gas emissions and nutrient leakage, and saves water compared to plowing. Of
course, the question remains as to what extent the negative chemical effects of herbicides
harm non-target organisms and degrade soil and water resources [1–3].
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Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Herbicides can be classified according to the type/chemical structure of the active
ingredient, mechanism of action, method and time of application, mobility, type of formula-
tion or residual effect [
4–6
]. There are currently about 20 different mechanisms of action of
Molecules 2021, 26, 4336. https://doi.org/10.3390/molecules26144336
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 4336
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herbicides [
4–
11], but over 50% of commercially available herbicides act by reversibly bind-
ing to photosystem II (PS II), resulting in disruption of photosynthetic electron transport
(PET) [ ]. PS II uses light energy to oxidize water and reduce plastoquinone, which
4,6–8
consists of parts QA and QB. The plastoquinone QA acting as a single electron acceptor
is permanently bound to PS II; the plastoquinone QB acting as a two-electron acceptor
is loosely bound; after reduction, it separates from the reaction center and diffuses into
the hydrophobic membrane nucleus, the QB binding site being occupied by the oxidized
molecule plastoquinone [12]. Herbicides belonging to inhibitors of PS II inhibit photosyn-
thetic electron transfer (PET) by binding to the QB binding niche on the D1 protein of the
PS II complex in chloroplast thylakoid membranes, leading to inhibition of PET from QA
to QB, blocking CO2 fixation and inhibition of ATP production [6,9–11].
Studies of large libraries of structurally diverse PS II inhibitors have confirmed the
hydrophobic nature of the binding domain, with lipophilicity being the dominant deter-
minant of Hill inhibitory activity [12–18]. Significant amounts of herbicides acting as PET
inhibitors in PS II contain an amide (–CONH–) and/or carbamate (–HNCOO–) bond in
their structure that is capable of forming hydrogen bonds between the amide/carbamate
group and target proteins in the photosynthetic centers of thylakoid membranes, lead-
ing to conformational changes and PET inhibition [19–25]. Both the N- and O-terminal
ends of the CONH linker are substituted and the substituents further modify the bond
properties and strength of the basic scaffold [26]. Amides are thought to be inhibitors
of PS II, causing the displacement of QB from its binding pocket in the D1 protein [27],
and halogenated substituents have been found to contribute to increased PET inhibitory
activity [18,23,24,28–30].
Our team has long been investigating the effects of a wide range of variously substi-
tuted napthalenecarboxanilides [23,24,27,29–31] and quinolinecarboxanilides [32] on PS II.
A series of ring-monosubstituted anilides of 3-hydroxynaphthalene-2-carboxylic acid was
published by Kos et al. [29] and some interesting biological activity was found, including
herbicidal activity. Since monosubstituted derivatives of 3-hydroxy-N-arylnaphthalene-2-
carboxanilides showed PET inhibition in spinach chloroplasts (Spinacia oleracea L.), select
new, variously disubstituted, derivatives were evaluated for their PET-inhibiting activity.
2. Results and Discussion
2.1. Chemistry
All compounds were prepared by the reaction of 3-hydroxynaphthalene-2-carboxylic
acid with appropriate disubstituted anilines with the addition of phosphorus trichloride in
dry chlorobenzene under microwave conditions (Scheme 1) [29,31], which resulted in a
series of target N-(disubstituted-phenyl)-3-hydroxynaphthalene-2-carboxamides 1–24, see
Table 1.
Scheme 1. Synthesis of 3-hydroxy-N-arylnaphthalene-2-carboxanilides
tions: (a) PCl3, chlorobenzene, MW, 45 min [29,31].
1–24. Reagents and condi-

Molecules 2021, 26, 4336
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Table 1. Structure of ring-disubstituted 3-hydroxynaphthalene-2-carboxanilides
1–
24, calculated
values of Clog P for compounds, electronic parameters of anilide (Ar) and IC50 [µM] values related
σ
to photosynthetic electron transport (PET) inhibition in spinach chloroplasts of tested compounds in
comparison with the 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) standard.
PET Inhibition
3
Comp.
R
Clog P 2
σ(Ar)
IC50 [µM]
1 1
2 1
2,5-OCH3
3,5-OCH3
2,5-CH3
2,6-CH3
3,5-CH3
2,5-F
2,6-F
3,5-F
2,5-Cl
2,6-Cl
3.9563
4.5463
4.7942
4.1442
5.4442
4.4799
3.8799
5.0799
5.3699
4.5199
6.0999
6.2199
5.6399
5.6399
6.8207
4.2725
3.6725
4.8625
4.5825
5.0499
5.7999
6.0131
5.7331
5.8531
–
0.08
0.93
0.59
0.58
0.59
1.24
1.44
1.12
1.22
1.33
1.19
1.11
1.11
1.23
1.05
0.14
0.16
0.99
1.13
1.17
1.16
1.04
1.19
1.32
–
183
24.5
11.6
28.5
9.9
11.2
78.7
9.8
321
156
47.5
39.2
296
161
15.9
79.1
507
31.6
171
1405
527
31.0
13.2
621
2.1
3 1
4 1
5 1
6 1
7 1
8 1
9 1
10 1
11 1
12 1
13
3,4-Cl
3,5-Cl
2,4-Br
2,5-Br
14
15 1
16
3,5-CF3
2-OCH3-5-F
2-F-6-OCH3
3-F-5-OCH3
2-Cl-5-OCH3
2-F-4-Cl
3-F-4-Br
3-F-5-CF3
2-Cl-5-CF3
2-Br-4-CF3
–
17
18
19
20
21 1
22 1
23 1
24 1
DCMU
1
3
2
Compounds described in [31]; ChemBioDraw Ultra 13.0 (CambridgeSoft, PerkinElmer Inc., MA, USA);
calculated using ACD/Percepta ver. 2012 (Advanced Chemistry Development, Toronto, ON, Canada).
Lipophilicity is an extremely important parameter in the design of any biologically ac-
tive compound, as it primarily ensures sufficient penetration across biological membranes
to reach the target site of action [33]. In general, it can be stated that a higher value of
lipophilicity is required for agrochemicals acting in plant leaves due to the permeability
of a stronger and more lipophilic cuticle [34]. Lipophilicity, expressed as Clog P values
(predicted by ChemBioDraw Ultra 13.0), of the investigated compounds is listed in Table 1
.
Clog P values ranged from 3.6 (compound 17, R = 2-F-6-OCH3) to 6.8 (compound 15
R = 3,5-CF3). When comparing the general effect of substituents at the same positions
on lipophilicity, the order of the groups with respect to the increasing contribution of
lipophilicity is as follows: OCH3 < F < CH3 < Cl < Br < CF3; thus, in general, any substi-
tution by a fluorine or methoxy moiety significantly decreases lipophilicity. In addition
to the type of substituent, their mutual position on the aniline ring also has a significant
effect on the lipophilicity value. For example, in series with dichlorinated derivatives,
lipophilicity increases as follows: 2.6 < 2.5 < 3.4 < 3.5. In series with different moieties of
disubstituted compounds, the Clog P values are significantly decreased when a fluorine or
,
0
methoxy moiety is introduced, especially in positions C(2) and C(6)0, as mentioned above.
The predicted Clog P values are presented in the illustrated order in Figure 1, where they

Molecules 2021, 26, 4336
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are simultaneously divided into three groups according to the nature of the substitution.
The first group consists of methoxy-, methyl- and fluoro-disubstituted compounds
second group consists of dichloro, dibromo and 3,5-CF3 derivatives 15; and derivatives
16 24, disubstituted by two different substituents, are in the third group. This division
proves important for the description of PET inhibition, see below.
1–8; the
9–
–
Figure 1. Graphical comparison of lipophilicity of investigated compounds expressed as Clog P, predicted by ChemBioDraw
Ultra 13.0.
Electronic contributions of substituents are another important parameter, especially
for substituted aromatic rings (anilines, phenols). The electron
substituted anilide ring, predicted by the ADC/Percepta program, are listed in Table 1. As
with the lipophilicity values, the values are in a wide range. Based on the results of the
prediction program, the weakest electron-withdrawing properties have the substitution 2,5-
OCH3-Ph of compound = 0.08), while the strongest electron-withdrawing properties
have fluoro-substituted derivative (2,6-F-Ph, = 1.44). These values affect the electron
σ parameters of the whole
σ
1 (σ
7
σ
density at the amide linker and thus the overall binding to the putative site of action
of these compounds, which is on the acceptor side of PS II, at the section between P680
(primary donor of PS II) and QB [23–25,29].
2.2. Inhibition of Photosynthetic Electron Transport (PET) in Spinach Chloroplasts
The PET-inhibition of the studied compounds was expressed by the negative loga-
rithm of the IC50 value (concentration (in µM) of the compounds causing a 50% decrease
in the oxygen evolution rate relative to the untreated control). The evaluated disubsti-
tuted 3-hydroxynaphthalene-2-carboxanilides showed a wide range of PET inhibition
in spinach (Spinacia oleracea L.) chloroplasts with the IC50 values ranging from 9.8 to
1405
difluorophenyl)-(
demonstrated the highest PET-inhibiting activity (IC50 ~ 10
gated series. Acceptable activity was also found for N-(2-chloro-5-trifluoromethylphenyl)-
23) and N-(3,5-ditrifluoromethylphenyl)-3-hydroxynaphthalene-2-carboxamides (15) with
IC50 13.2 and 15.9 M, respectively. On the other hand, derivatives 21 (3-F-4-Br), 24 (2-
µ
M, see Table 1. N-(3,5-Difluorophenyl)-(
8
) and N-(3,5-dimethylphenyl)-(
) 3-hydroxynaphthalene-2-carboxamides
M) within the whole investi-
5), N-(2,5-
6) and N-(2,5-dimethylphenyl)-(
3
µ
(
µ
Br-4-CF3) and 20 (R = 2-F-4-Cl) were completely inactive (IC50 = 527, 621 and 1405 µM,
respectively).
The results of this screening indicate that the position of the substituents is crucial for
the activity, with the 3,5 positions being the most preferred (i.e., both meta positions are
substituted). However, 2,5-disubstituted derivatives also showed PET-inhibiting activity
when substituted with moieties with suitable properties, including electronic properties
and lipophilicity. As mentioned above, lipophilicity tends to affect biological activity. The
dependence of the PET-inhibiting activity, expressed as log(1/IC50 [M]), of the investigated

Molecules 2021, 26, 4336
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compounds in spinach chloroplasts on lipophilicity (Clog P) is shown in Figure 2A. It
can be stated that most of the evaluated compounds substituted by OCH3/CH3/F can
be traced to a quasi-parabolic dependence with the optimum Clog P ca. 5. The active
compounds have a range of lipophilicity values from 4.4 to 5.7. On the other hand, a
linear dependence can be observed for the dichloro-, dibromo- and bis(trifluoromethyl)-
substituted compounds, i.e., markedly lipophilic groups. The inhibition of PET increases
with increasing lipophilicity.
Figure 2B shows the dependence of the PET-inhibiting activity, expressed as log(1/IC50
[M]), on the electronic σ(Ar) properties of the whole anilide substituents. As can be seen,
electronic properties play a secondary role compared to lipophilicity and substituent
position; however, the quasi-parabolic (for OCH3/CH3/F substituted compounds) or
linear (for disubstituted compounds by Cl/Br/CF3 moieties) trend is evident. It can be
stated that electron-withdrawing properties in the range of σ(Ar) from approximately 0.6 to
1.2 are preferred.
A
B
5.2
5.2
3,5-F
3,5-CH3
2,5-CH3
3,5-CH3
3,5-F
2,5-CH3
2,5-F
3,5-CF3
3,5-CF3
3,5-OCH3
4.9
4.6
4.3
4.0
3.7
3.4
3.1
2.8
2.5
4.9
2-Cl-5-CF3
3-F-5-CF3
3,4-Cl
2,5-Br
2,5-F
2,6-CH3
2-Cl-5-CF3
3,5-OCH3
3-F-5-CF3
4.6
4.3
4.0
3.7
3.4
3.1
2.8
2.5
2,6-CH3
3,4-Cl
3-F-5-OCH3
3-F-5-OCH3
3,5-Cl
3,5-Cl
2,6-F
2,6-F
2-OCH3-5-F
2,6-Cl
2-Cl-5-OCH3
2-OCH3-5-F
2,5-Br
2-Cl-5-OCH3
2,4-Br
2,6-Cl
2,5-Cl
3-F-4-Br
2,5-OCH3
2,5-OCH3
2-F-6-OCH3
2,4-Br
3-F-4-Br
2-Br-4-CF3
2,5-Cl
2-F-6-OCH3
2-Br-4-CF3
2-F-4-Cl
2-F-4-Cl
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
0.0
0.2
0.4
0.6
0.8
σ(Ar)
1.0
1.2
1.4
1.6
Clog P
diOCH3/CH3/F diCl/Br/CF3 X-Y
diOCH3/CH3/F diCl/Br/CF3 X-Y
Figure 2. Dependence of PET-inhibiting activity log(1/IC50 [M]) of all discussed compounds
on lipophilicity expressed as Clog P ( ) and electronic parameters of whole N-aryl part of individual anilides (
squares are not involved in SAR discussion due to their inactivity.
1
–
24 in spinach chloroplasts
A
σ
B). Empty
Based on the structural similarity of the test compounds to previously performed
experiments with salicylanilides or hydroxynaphthanilides, the same mechanism of action
can be supposed, i.e., inhibition on the acceptor side of PS II, at the section between
P680 (primary donor of PS II) and plastoquinone QB [20
should be noted that plastoquinone QB on the acceptor side of PS II has been found
to be the site of inhibitory action of other amide-based derivatives [ 13 15 25 35], such
–24,27,29–31]. Furthermore, it
6, – , ,
as N-phenylpyrazine-2-carboxamides [19], N-substituted 2-aminobenzothiazoles [22] or
8-hydroxyquinoline-2-carboxanilides [32].
3. Materials and Methods
3.1. General Information
All reagents were purchased from Merck (Sigma-Aldrich, St. Louis, MO, USA) and
Alfa (Alfa-Aesar, Ward Hill, MA, USA). Reactions were performed using a CEM Discover
SP microwave reactor (CEM, Matthews, NC, USA). The melting points were determined
on a Kofler hot-plate apparatus HMK (Franz Kustner Nacht KG, Dresden, Germany) and
are uncorrected. Infrared (IR) spectra were recorded on a Smart MIRacle
™ ATR ZnSe for
Nicolet
™
Impact 410 Fourier-transform IR spectrometer (Thermo Scientific, West Pal−m1
Beach, FL, USA). The spectra were obtained by the accumulation of 256 scans with 2 cm
resolution in the region of 4000–650 cm−1. All 1H- and 13C-NMR spectra were recorded in
dimethyl sulfoxide-d6 (DMSO-d6) at 600 MHz for 1H and 150 MHz for 13C, on an Agilent
VNMRS 600 MHz system (Agilent Technologies, Santa Clara, CA, USA). The 1H and 13
C
chemical shifts (δ) are reported in ppm. High-resolution mass spectra were measured using

Molecules 2021, 26, 4336
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a high-performance liquid chromatograph Dionex UltiMate® 3000 (Thermo Scientific, West
Palm Beach, FL, USA) coupled with an LTQ Orbitrap XLTM Hybrid Ion Trap-Orbitrap
Fourier Transform Mass Spectrometer (Thermo Scientific) equipped with a HESI II (heated
electrospray ionization) source in positive and negative mode.
3.2. Synthesis
General Procedure for the Synthesis of N-(Disubstituted phenyl)-3-hydroxynaphthalene-
2-carboxamides 1–24
3-Hydroxynaphthalene-2-carboxylic acid (0.5 g, 2.65 mM) was suspended in dry
chlorobenzene (20 mL) at ambient temperature and phosphorus trichloride (0.12 mL,
1.35 mM), and the corresponding substituted aniline (2.65 mM) was added dropwise.
The reaction mixture was transferred to the microwave reactor, where the synthesis was
◦
◦
◦
performed (1st phase: 10 min, 100 C; 2nd phase: 15 min, 120 C; 3rd phase: 20 min, 130 C;
max 500 W). The mixture was then cooled to 60 ◦C, and the solvent was removed under
reduced pressure. The residue was washed sequentially with hydrochloric acid and water,
and the crude product was recrystallized from EtOH. All the compounds are presented in
Table 1.
The synthesis and analytical data for anilides 1–12, 15 and 21–24 were described
previously [31].
N-(2,4-Dibromophenyl)-3-hydroxynaphthalene-2-carboxamide (13). Yield 56%; mp 241–243 ◦C;
IR (cm−1): 3221; 1641; 1625; 1603; 1575; 1524; 1462; 1448; 1398; 1363; 1345; 1321; 1290;
1240; 1206; 1175; 1146; 1081; 1035; 951; 913; 878; 867; 846; 825; 791; 767; 737; 688; 1H NMR
(DMSO-d6), δ: 11.97 (s, 1H), 11.07 (s, 1H), 8.70 (s, 1H), 8.42 (d, J = 8.8 Hz, 1H), 7.99 (d,
J = 8.2 Hz, 1H), 7.97 (d, J = 2.2 Hz, 1H), 7.78 (d, J = 8.3 Hz, 1H), 7.66 (dd, J = 2.2 Hz,
J = 8.8 Hz, 1H), 7.53 (ddd, J = 1.2 Hz, J = 6.8 Hz, J = 8.3 Hz, 1H), 7.38 (s, 1H), 7.37 (ddd,
J = 1.1 Hz, J = 6.8 Hz, J = 8.2 Hz, 1H); 13C NMR (DMSO-d6),
δ: 163.6, 152.6, 136.2, 136.1,
134.4, 132.8, 131.3, 129.1, 128.6, 127.2, 125.7, 124.4, 124.0, 120.4, 116.3, 114.9, 110.8; HR-MS
C17H12O2NBr2 [M + H]+ calculated 419.9229 m/z, found 419.9237 m/z.
N-(2,5-Dibromophenyl)-3-hydroxynaphthalene-2-carboxamide (14). Yield 49%; mp 233–235 ◦C;
IR (cm−1): 3190; 1636; 1622; 1597; 1568; 1506; 1447; 1393; 1360; 1344; 1250; 1192; 1174; 1147;
1080; 1069; 1029; 962; 915; 902; 868; 848; 796; 770; 750; 736; 1H NMR (DMSO-d6),
δ: 12.02
(s, 1H), 11.14 (s, 1H), 8.74 (d, J = 2.3 Hz, 1H), 8.71 (s, 1H), 8.00 (d, J = 8.2 Hz, 1H), 7.79 (d,
J = 8.3 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.54 (ddd, J = 1.2 Hz, J = 6.8 Hz, J = 8.3 Hz, 1H),
7.39 (s, 1H), 7.38 (ddd, J = 1.1 Hz, J = 6.8 Hz, J = 8.2 Hz, 1H), 7.32 (dd, J = 2.4 Hz, J = 8.5 Hz,
1H); 13C NMR (DMSO-d6),
δ: 163.6, 152.5, 138.1, 136.2, 134.2, 133.0, 129.1, 128.7, 128.1,
127.2, 125.7, 125.0, 124.0, 120.9, 120.3, 112.7, 110.8; MS C17H12O2NBr2 [M + H]+ calculated
419.9229 m/z, found 419.9239 m/z.
N-(5-Fluoro-2-methoxyphenyl)-3-hydroxynaphthalene-2-carboxamide (16). Yield 80%; mp 198–
203 ◦C; IR (cm−1): 3194, 1640, 1625, 1615, 1601, 1538, 1488, 1432, 1393, 1356, 1346, 1249,
1214, 1176, 1148, 1065, 1038, 975, 866, 838, 786, 731, 711; 1H-NMR (DMSO-d6),
δ: 11.86 (s,
1H), 11.25 (s, 1H), 8.70 (s, 1H), 8.40 (dd, J = 11.0 Hz, J=3.3 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H),
7.78 (d, J = 8.4 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 7.37 (s, 1H), 7.36 (t, J = 7.5 Hz, 1H), 7.12
(dd, J = 9.2 Hz, J = 5.1 Hz, 1H), 6.92 (td, J = 8.6 Hz, J = 3.3 Hz, 1H), 3.92 (s, 3H); 13C-NMR
(DMSO-d6),
δ: 163.0, 156.0 (d, J = 232.2 Hz), 152.4, 144.8 (d, J = 1.8 Hz), 136.0, 132.9, 129.1,
129.0 (d, J = 12.9 Hz), 128.5, 127.2, 125.7, 123.9, 121.0, 111.7 (d, J = 9.1 Hz), 110.8, 109.0
(d, J = 22.8 Hz), 106.9 (d, J = 29.6 Hz), 57.0; HR-MS: C18H13FNO3 [M
310.0885 m/z, found 310.0881 m/z.
−
H]− calculated
N-(2-Fluoro-6-methoxyphenyl)-3-hydroxynaphthalene-2-carboxamide (17). Yield 66%; mp 138–
144 ◦C; IR (cm−1): 3259, 2836, 1651, 1622, 1596, 1532, 1515, 1506, 1466, 1438, 1279, 1249,
1216, 1167, 1146, 1087, 900, 873, 834, 789, 767, 747, 728; 1H-NMR (DMSO-d6),
δ: 11.76 (s, 1H),
10.22 (s, 1H), 8.69 (s, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.54 (t, J = 7.3 Hz,
1H), 7.30–7.40 (m, 3H), 6.99 (d, J = 8.4 Hz, 1H), 6.94 (t, J = 9.0 Hz, 1H), 3.84 (s, 3H); 13C-NMR
(DMSO-d6), δ: 166.3, 158.1 (d, J = 246.4 Hz), 155.8 (d, J = 5.3 Hz), 154.6, 136.2, 131.0, 128.9,

Molecules 2021, 26, 4336
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128.5, 128.2 (d, J = 10.7 Hz), 126.8, 125.8, 123.9, 118.7, 113.7 (d, J = 15.3 Hz), 110.9, 107.9 (d,
J = 26.4 Hz), 107.7, 56.3; HR-MS: C18H13FNO3 [M
310.0880 m/z.
−
H]− calculated 310.0885 m/z, found
N-(3-Fluoro-5-methoxyphenyl)-3-hydroxynaphthalene-2-carboxamide (18). Yield 59%; mp 227–
230 ◦C; IR (cm−1): 3147, 1644, 1622, 1595, 1557, 1520, 1456, 1448, 1359, 1261, 1224, 1212,
1191, 1141, 1129, 1063, 999, 987, 872, 858, 816, 767, 745, 690; 1H-NMR (DMSO-d6),
δ: 11.12
(s, 1H), 10.64 (s, 1H), 8.41 (s, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.76 (d, J = 8.1 Hz, 1H), 7.51
(t, J = 7.0 Hz, 1H), 7.32-7.40 (m, 3H), 7.21 (s, 1H), 6.62 (d, J = 11.0 Hz, 1H), 3.78 (s, 3H);
13C-NMR (DMSO-d6),
δ: 165.7, 162.9 (d, J = 238.5 Hz), 160.7 (d, J = 12.9 Hz), 153.3, 140.6 (d,
J = 13.7 Hz), 135.7, 130.5, 128.7, 128.1, 126.9, 125.8, 123.8, 122.5, 110.5, 102.0 (d, J = 2.0 Hz),
99.4 (d, J = 27.3 Hz), 97.0 (d, J = 25.0 Hz), 55.6; HR-MS: C18H13FNO3 [M
310.0885 m/z, found 310.0881 m/z.
−
H]− calculated
N-(2-Chloro-5-methoxyphenyl)-3-hydroxynaphthalene-2-carboxamide (19). Yield 58%; mp 187–
188 ◦C; IR (cm−1): 3177, 2954, 2834, 1638, 1624, 1598, 1539, 1462, 1447, 1427, 1358, 1305,
1274, 1262, 1220, 1167, 1147, 1135, 1063, 1028, 960, 916, 866, 845, 787, 771, 745, 719; 1H-NMR
(DMSO-d6), δ: 11.97 (s, 1H), 11.17 (s, 1H), 8.73 (s, 1H), 8.25 (d, J = 2.9 Hz, 1H), 7.99 (d,
J = 8.2 Hz, 1H), 7.78 (d, J = 8.3 Hz, 1H), 7.53 (ddd, J = 8.3 Hz, J = 6.8 Hz, J = 1.2 Hz, 1H), 7.46
(d, J = 8.8 Hz, 1H), 7.38 (ddd, J = 8.2 Hz, J = 6.8 Hz, J = 1.2 Hz, 1H), 7.38 (s, 1H), 6.78 (dd,
J = 8.8 Hz, J = 3.0 Hz, 1H), 3.80 (s, 3H); 13C-NMR (DMSO-d6),
δ: 163.4, 158.5, 152.5, 136.1,
132.9, 129.6, 129.1, 128.6, 127.2, 125.7, 124.0, 120.6, 114.2, 110.8, 110.4, 108.0, 55.5; HR-MS:
C18H15ClNO3 [M + H]+ calculated 328.0735 m/z, found 328.0737 m/z.
N-(4-Chloro-2-fluorophenyl)-3-hydroxynaphthalene-2-carboxamide (20). Yield 75%; mp 267–
◦
269 C; IR (cm−1): 3194, 1647, 1627, 1601, 1552, 1489, 1449, 1414, 1393, 1357, 1338, 1259, 1207,
1174, 1147, 1118, 1064, 951, 918, 897, 870, 841, 820, 767, 740, 722, 667; 1H NMR (DMSO-d6)
δ:
11.85 (s, 1H), 10.97 (s, 1H), 8.66 (s, 1H), 8.37 (t, J = 8.7 Hz, 1H), 7.97 (d, J = 8.2 Hz, 1H), 7.77
(d, J = 8.3 Hz, 1H), 7.56 (dd, J = 2.4 Hz, J = 10.8 Hz, 1H), 7.52 (ddd, J = 1.2 Hz, J = 6.8 Hz,
J = 8.3 Hz, 1H), 7.37 (ddd, J = 1.1 Hz, J = 6.8 Hz, J = 8.2 Hz, 1H), 7.36 (s, 1H), 7.34 (ddd,
J = 1.2 Hz, J = 2.4 Hz, J = 8.8 Hz, 1H). 13C NMR (DMSO-d6),
δ: 163.8, 152.8 (d, J = 247.5 Hz),
152.8, 136.1, 132.4, 129.0, 128.5, 127.9 (d, J = 10.0 Hz), 127.1, 125.7, 125.7 (d, J = 10.7 Hz),
124.9 (d, J = 3.4 Hz), 124.0, 123.6, 120.4, 115.9 (d, J = 23.1 Hz), 110.9; HR-MS: C17H12ClFNO2
[M + H]+ calculated 316.0535 m/z, found 316.0535 m/z.
3.3. Study of Inhibition of Photosynthetic Electron Transport (PET) in Spinach Chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.) according to Kralova
et al. [36]. Screening was performed as described previously [e.g., 19–25,31]. A selective
herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea, DCMU (Diuron®, Merck, Darmstadt,
Germany) was used as a standard. The results are summarized in Table 1.
4. Conclusions
A series of 3-hydroxynaphthalene-2-carboxanilides substituted with two similar or
different atoms or groups on the anilide ring was prepared under microwave-assisted
conditions and tested for their ability to inhibit photosynthetic electron transport (PET) in
spinach (Spinacia oleracea L.) chloroplasts. N-(3,5-Difluorophenyl)-3-hydroxynaphthalene-2-
carboxamide (
difluorophenyl)-3-hydroxynaphthalene-2-carboxamide (
3-hydroxynaphthalene-2-carboxamide ( ) exhibited the highest PET-inhibiting activity
with their IC50 values ranging from 9.8 to 11.6
8
), N-(3,5-dimethylphenyl)-3-hydroxynaphthalene-2-carboxamide (
5), N-(2,5-
6) and N-(2,5-dimethylphenyl)-
3
0 0
M. The C(3,5) and C(2,5) disubstituted
µ
isomers were found to be the most active among the test compounds. Furthermore,
for diOCH3/diCH3/diF substituted derivatives, a Clog P value of approximately 5 is
important, while for diCl/diBr/diCF3 substituted derivatives, PET inhibition increases
with increasing lipophilicity to a Clog P value of 6.8 of N-(3,5-ditrifluoromethylphenyl)-3-
hydroxynaphthalene-2-carboxamides (15) with IC50 = 15.9
µM. The electronic properties
of the substituents play a complementary role and the electron-withdrawing properties

Molecules 2021, 26, 4336
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(σ(Ar) ca. 0.6 to 1.2) for PET activity seem to be more advantageous. Based on the structural
similarity of the investigated compounds with previously published isomers, it can be
concluded that these hydroxynaphthanilides inhibit PET in photosystem II.
Author Contributions: J.K., T.G. and I.J. synthesized and characterized the compounds. M.O.
performed analytical measurement. J.K. performed biological screening. J.J. designed the compounds.
J.K., T.G. and J.J. wrote the paper. All authors have read and agreed to the published version of
the manuscript.
Funding: This study was supported by the Slovak Research and Development Agency (projects
APVV-17-0373 and APVV-17-0318). This work is based on use of Large Research Infrastructure
CzeCOS supported by the Ministry of Education, Youth and Sports of the Czech Republic within the
CzeCOS program, grant number LM2018123; M.O. was supported by SustES—Adaptation strategies
for sustainable ecosystem services and food security under adverse environmental conditions, project
no. CZ.02.1.01/0.0/0.0/16_019/0000797.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Conflicts of Interest: The authors declare no conflict of interest.
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