Synthesis and spectrum of biological activities of novel N-arylcinnamamides

Article abstract of DOI:10.3390/ijms19082318

A series of sixteen ring-substituted N-arylcinnamamides was prepared and characterized. Primary in vitro screening of all the synthesized compounds was performed against Staphylococcus aureus, three methicillin-resistant S. aureus strains, Mycobacterium tuberculosis H37Ra, Fusarium avenaceum, and Bipolaris sorokiniana. Several of the tested compounds showed antistaphylococcal, antitubercular, and antifungal activities comparable with or higher than those of ampicillin, isoniazid, and benomyl. (2E)-N-[3,5-bis(trifluoromethyl)phenyl]-3-phenylprop-2-enamide and (2E)-3-phenyl-N-[3-(trifluoromethyl)phenyl]prop-2-enamide showed the highest activities (MICs = 22.27 and 27.47 μM, respectively) against all four staphylococcal strains and against M. tuberculosis. These compounds showed an activity against biofilm formation of S. aureus ATCC 29213 in concentrations close to MICs and an ability to increase the activity of clinically used antibiotics with different mechanisms of action (vancomycin, ciprofloxacin, and tetracycline). In time-kill studies, a decrease of CFU/mL of >99% after 8 h from the beginning of incubation was observed. (2E)-N-(3,5-Dichlorophenyl)-and (2E)-N-(3,4-dichlorophenyl)-3-phenylprop-2-enamide had a MIC = 27.38 μM against M. tuberculosis, while a significant decrease (22.65%) of mycobacterial cell metabolism determined by the MTT assay was observed for the 3,5-dichlorophenyl derivative. (2E)-N-(3-Fluorophenyl)-and (2E)-N-(3-methylphenyl)-3-phenylprop-2-enamide exhibited MICs = 16.58 and 33.71 μM, respectively, against B. sorokiniana. The screening of the cytotoxicity of the most effective antimicrobial compounds was performed using THP-1 cells, and these chosen compounds did not shown any significant lethal effect. The compounds were also evaluated for their activity related to the inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. (2E)-N-(3,5-dichlorophenyl)-3-phenylprop-2-enamide (IC₅₀ = 5.1 μM) was the most active PET inhibitor. Compounds with fungicide potency did not show any in vivo toxicity against Nicotiana tabacum var. Samsun. The structure–activity relationships are discussed.

Full text of DOI:10.3390/ijms19082318

International Journal of
Molecular Sciences
Article
Synthesis and Spectrum of Biological Activities of
Novel N-arylcinnamamides
Sarka Pospisilova 1,2, Jiri Kos 1,*, Hana Michnova 1,2, Iva Kapustikova 1, Tomas Strharsky 1,
Michal Oravec 3, Agnes M. Moricz 4, Jozsef Bakonyi 4, Tereza Kauerova 5, Peter Kollar 5,
Alois Cizek 2 and Josef Jampilek 1
ID
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Comenius University, Odbojarov 10,
83232 Bratislava, Slovakia; sharka.pospisilova@gmail.com (S.P.); michnova.hana@gmail.com (H.M.);
kapustikova@fpharm.uniba.sk (I.K.); strharsky2@uniba.sk (T.S.); josef.jampilek@gmail.com (J.J.)
Department of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine,
2
University of Veterinary and Pharmaceutical Sciences, Palackeho 1, 61242 Brno, Czech Republic;
cizeka@vfu.cz
Global Change Research Institute CAS, Belidla 986/4a, 60300 Brno, Czech Republic; oravec.m@czechglobe.cz
3
4
Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences,
Herman Otto Str. 15, 1022 Budapest, Hungary; moricz.agnes@agrar.mta.hu (A.M.M.);
bakonyi.jozsef@agrar.mta.hu (J.B.)
Department of Human Pharmacology and Toxicology, Faculty of Pharmacy,
5
University of Veterinary and Pharmaceutical Sciences, Palackeho 1, 61242 Brno, Czech Republic;
tereza.kauerova@gmail.com (T.K.); kollarp@vfu.cz (P.K.)
*
Correspondence: jirikos85@gmail.com; Tel.: +421-2-5011-7224
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 9 July 2018; Accepted: 3 August 2018; Published: 7 August 2018
Abstract: A series of sixteen ring-substituted N-arylcinnamamides was prepared and
characterized. Primary in vitro screening of all the synthesized compounds was performed
against Staphylococcus aureus, three methicillin-resistant S. aureus strains, Mycobacterium tuberculosis
H37Ra, Fusarium avenaceum, and Bipolaris sorokiniana. Several of the tested compounds showed
antistaphylococcal, antitubercular, and antifungal activities comparable with or higher than those of
ampicillin, isoniazid, and benomyl. (2E)-N-[3,5-bis(trifluoromethyl)phenyl]-3-phenylprop-2-enamide
and (2E)-3-phenyl-N-[3-(trifluoromethyl)phenyl]prop-2-enamide showed the highest activities
(
MICs = 22.27 and 27.47 µM, respectively) against all four staphylococcal strains and against
M. tuberculosis. These compounds showed an activity against biofilm formation of S. aureus
ATCC 29213 in concentrations close to MICs and an ability to increase the activity of clinically
used antibiotics with different mechanisms of action (vancomycin, ciprofloxacin, and tetracycline).
In time-kill studies, a decrease of CFU/mL of >99% after 8 h from the beginning of incubation was
observed. (2E)-N-(3,5-Dichlorophenyl)- and (2E)-N-(3,4-dichlorophenyl)-3-phenylprop-2-enamide
had a MIC = 27.38 µM against M. tuberculosis, while a significant decrease (22.65%) of mycobacterial
cell metabolism determined by the MTT assay was observed for the 3,5-dichlorophenyl
derivative.
exhibited MICs = 16.58 and 33.71
(2E)-N-(3-Fluorophenyl)- and (2E)-N-(3-methylphenyl)-3-phenylprop-2-enamide
M, respectively, against B. sorokiniana. The screening of the
µ
cytotoxicity of the most effective antimicrobial compounds was performed using THP-1 cells, and
these chosen compounds did not shown any significant lethal effect. The compounds were also
evaluated for their activity related to the inhibition of photosynthetic electron transport (PET) in
spinach (Spinacia oleracea L.) chloroplasts. (2E)-N-(3,5-dichlorophenyl)-3-phenylprop-2-enamide
(
IC50 = 5.1 µM) was the most active PET inhibitor. Compounds with fungicide potency did not show
any in vivo toxicity against Nicotiana tabacum var. Samsun. The structure–activity relationships
are discussed.
Int. J. Mol. Sci. 2018, 19, 2318; doi:10.3390/ijms19082318
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2018, 19, 2318
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Keywords: cinnamamides; antistaphylococcal activity; time-kill assay; biofilm; antitubercular activity;
MTT assay; antifungal activity; PET inhibition; toxicity; structure–activity relationship
1. Introduction
Cinnamic acids and other hydroxy- or phenyl-substituted derivatives of cinnamic acids have been
widely investigated by scientists due to their significant and varied biological effects. Cinnamic
acids occur naturally in all plants [
phenyl-propanoids, coumarins, lignans, isoflavonoids, flavonoids, stilbenes, aurones, anthocyanins,
spermidines, and tannins [ ]. The spectrum of their biological activities include anti-inflammatory,
antioxidant, hepatoprotective, antidiabetic, antidepressant/anxiolytic, antifungal, antibacterial,
1]. They are formed in the biochemical pathway providing
2
antiviral, and anticancer effects [
as well [17].
3–16]. Derivatives of cinnamic acids are used as agriculture fungicides
The adaptation of microorganisms to external influences and, thus, the development of their
resistance against antimicrobial agents is not a surprise; unfortunately, this process is faster and faster.
Thus, the emerging resistance of microbial pathogens to clinically used drugs, including second-
and third-choice drugs, and the development of cross-resistant or multidrug-resistant strains are
alarming. Microbial pathogens have developed a number of mechanisms to adapt to the effects of
the environment. In addition, the increase in the number of infections and the occurrence of new,
especially opportunistic species are also caused by the general immunosuppression of patients, and
this fact makes these diseases extremely serious. Since the 1990s, only an inconsiderable number
of really new drugs for systemic administration have been marketed for the treatment of infections,
although the discovery of new molecules has been a priority [18].
Thus, in the light of the above mentioned facts, new simple anilides of cinnamic acid were
designed as antimicrobial multitarget agents, synthesized using a modern microwave-assisted
method and screened against a battery of bacterial/mycobacterial and fungal pathogens. These
compounds were designed based on the experience with naphthalenecarboxamides—simple
molecules with a number of biological activities, and in fact, the new ring-substituted
(2E)-N-phenyl-3-phenylprop-2-enamides can be considered as open analogues of recently described
naphthalene-2-carboxanilides [19,20].
As an amide moiety is able to affect photosystem II (PS II) by reversible binding [21], resulting
in the interruption of the photosynthetic electron transport (PET) [22–24], and can be found in many
herbicides acting as photosynthesis inhibitors [25 30], these N-arylcinnamamides were additionally
–
tested on the inhibition of PET in spinach (Spinacia oleracea L.) chloroplasts using the Hill reaction.
The idea of this screening is based on the fact that both drugs and pesticides are designed to target
particular biological functions. These functions/effects may overlap at the molecular level, which
causes a considerable structural similarity between drugs and pesticides. Since different classes of
herbicides are able to bind to different mammalian cellular receptors, the majority of pharmaceutical
companies have pesticide divisions, and developed biologically active agents are investigated as
both pesticides and drugs. Previously, several successful pesticides became pharmaceuticals and vice
versa [31–35].
2. Results and Discussion
2.1. Chemistry and Physicochemical Properties
All the studied compounds 1–16 were prepared according to Scheme 1. The carboxyl group of
starting cinnamic acid was activated with phosphorus trichloride. In the reaction with an appropriate
ring-substituted aniline, the generated acyl chloride subsequently gave the final amide in dry
chlorobenzene via microwave-assisted synthesis. All the compounds were recrystallized from ethanol.

Int. J. Mol. Sci. 2018, 19, 2318
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Many different molecular parameters/descriptors are used to determine structure-activity
relationships (SAR). Lipophilicity and electronic properties are among the most frequent ones.
Hammett’s
σ parameters were used for the description of electronic properties. They were calculated
for the whole substituted anilide ring using ACD/Percepta ver. 2012 (Advanced Chemistry
Development Inc., Toronto, ON, Canada, 2012), see Table 1. The lipophilicity of the studied compounds
was predicted as log P using ACD/Percepta software and Clog P using ChemBioDraw Ultra 13.0
(CambridgeSoft, PerkinElmer Inc., Cambridge, MA, USA). Log P is the logarithm of the partition
coefficient for n-octanol/water. Clog P is the logarithm of n-octanol/water partition coefficient based
on the established chemical interactions. In addition, the lipophilicity of studied compounds 1–16
was investigated by means of reversed-phase high-performance liquid chromatography (RP-HPLC)
determination of capacity factors k with the subsequent calculation of log k [36]. The analysis was
made under isocratic conditions with methanol as an organic modifier in the mobile phase using an
end-capped nonpolar C18 stationary RP column. The results are shown in Table 1.
Scheme 1. Synthesis of (2E)-N-aryl-3-phenylprop-2-enamides 1–16. Reagents and conditions: (a) PCl3,
chlorobenzene, and MW.
Table 1. Structure of ring-substituted (2E)-N-aryl-3-phenylprop-2-enamides 1–16, experimentally
determined values of lipophilicity log k, calculated values of log P/Clog P, and electronic Hammett’s
σ parameters.
Clog P a
b
log P b
σAr
Comp.
R
log k
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
H
0.1146
0.2729
0.2640
0.1330
0.2327
0.4859
0.2691
0.5799
0.0632
0.6821
0.8155
0.0992
0.9814
0.4875
0.4588
3.6640
4.1630
4.1630
3.4646
4.0646
4.9978
4.0120
4.5878
3.7378
5.3178
5.4378
3.9778
6.0386
4.4178
4.1378
4.9509
3.18
3.40
3.40
3.17
3.32
4.26
3.57
4.65
4.56
4.70
4.79
4.80
5.68
4.07
4.12
4.88
0.60
0.48
0.46
1.02
0.82
0.89
0.59
1.22
1.33
1.19
1.11
1.33
1.05
1.28
1.19
1.19
3-CH3
4-CH3
2-F
3-F
3-CF3
2,5-CH3
2,5-Cl
2,6-Cl
3,4-Cl
3,5-Cl
2,6-Br
3,5-CF3
2-F-5-Br
2-Br-5-F
2-Cl-5-CF3 0.6178
a
calculated using ChemBioDraw Ultra 13.0; b calculated using ACD/Percepta ver. 2012.
The results obtained with the discussed compounds show that the experimentally-determined
lipophilicities (log k) are in accordance with the calculated Clog P values as illustrated in Figure 1A;
correlation coefficient r = 0.9513, n = 16. On the other hand, log P values calculated by ACD/Percepta
show differences for compounds
9 (2,6-Cl) and 12 (2,6-Br), see Figure 1B. When these two compounds

Int. J. Mol. Sci. 2018, 19, 2318
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are excluded, r = 0.9774 (n = 14) is observed. This poor match for 2,6-disubstituted anilides
9 and
12 may be caused by intramolecular interactions that are probably caused by the steric effect of
spatially-close moieties, which was not included in prediction by ACD/Percepta. The proximity
of the di-ortho-substituents to the carboxamide group on the aniline ring leads to the twist of
the aniline ring plane towards the carboxamide group, i.e., to the plane of the benzene ring of
cinnamic acid. The described process resulted in the planarity violation of the molecule. Otherwise,
(2E)-N-[3,5-bis(trifluoromethyl)phenyl]-3-phenylprop-2-enamide (13) is the most lipophilic, while
compounds
9, 12 and N-phenylcinnamamide (1) are characterized by the lowest lipophilicity. It can be
stated that log k values specify lipophilicity within the series of the studied compounds.
B
A
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
r = 0.9774
r = 0.9513
2,6-Br
2,6-Cl
2,6-Br
2,6-Cl
3.0
3.5
4.0
4.5
Clog P
5.0
5.5
6.0
6.5
3.0
3.5
4.0
4.5
log P
5.0
5.5
6.0
Figure 1. Comparison of experimentally found log k values of ring-substituted N-arylcinnamamides
16 with Clog P calculated using ChemBioDraw Ultra ( ) and log P calculated using
ACD/Percepta (B).
1–
A
2.2. In Vitro Antibacterial Susceptibility Testing
All the cinnamanilides were tested on their antistaphylococcal activity against three clinical
isolates of methicillin-resistant Staphylococcus aureus (MRSA) [37 38] and S. aureus ATCC 29213
as the reference and quality control strain. Although various derivatives of cinnamic acid were
described as promising antibacterial agents [ 14 15], the compounds showed only limited activity
(MICs > 256 g/mL), except for (2E)-3-phenyl-N-[3-(trifluoromethyl)phenyl]prop-2-enamide ( ) and
,
4–
6,
8,
9,
,
µ
6
3,5-bis(trifluoromethyl)phenyl derivative 13, see Table 2. As minimum inhibitory concentrations
(MICs) of these compounds are the same against the reference and the MRSA strains (27.47 and
22.27
These compounds were also tested against Enterococcus faecalis ATCC 29212 as the reference strain
and three isolates from American crows of vanA-carrying vancomycin-resistant E. faecalis (VRE) [39
but without any effect in the tested concentrations, which may indicate a specific mechanism of
action [37 40]. From Table 2 it is obvious that compounds and 13 exhibited activities comparable with
µM, respectively), it can be speculated about the specific effectivity against Staphylococcus sp.
]
,
6
those of the standards. Due to the small number of active compounds, no SAR could be established.
2.2.1. Synergy Effect with Clinically Used Drugs against MRSA
The most effective compounds
6 and 13 were tested for their ability of synergic activity with
clinically used antibacterial drugs tetracycline, ciprofloxacin, and vancomycin. These antibiotics
have different mechanisms of actions and different mechanisms of resistance to them, thus the
prospective synergism could give an idea of the mechanism of action of the cinnamic derivatives.
The investigation of synergistic activity was performed according to the methodology [41]. The method
of fractional inhibitory concentration (FIC) was used [42]. For all the wells of the microtitration plates
that corresponded to a MIC value, the sum of the FICs (
equation FIC = FICA + FICB = (CA/MICA) + (CB/MICB), where MICA and MICB are the MICs of drugs
A and B alone, respectively, and CA and CB are the concentrations of the drugs in the combination,
ΣFIC) was calculated for each well, using the
Σ

Int. J. Mol. Sci. 2018, 19, 2318
5 of 25
respectively [42]. Synergy was defined as a
Σ
FIC
≤
0.5; additivity was defined as 0.5 < ΣFIC < 1;
indifference was defined as 1 ≤ ΣFIC < 4; and antagonism was defined as ΣFIC ≥ 4 [41]. As the FIC
index was evaluated for every single well corresponding to the MIC value, the results are presented as
a range. The test was made with all 3 methicillin-resistant isolates, MRSA 63718, SA 3202, and SA 630.
The isolates were also resistant to used antibiotics. Note that isolate MRSA SA 630 is susceptible to
tetracycline. The results are mentioned in Table 3.
Table 2.
M) values related to PET inhibition in spinach chloroplasts in comparison with
3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) standard, in vitro anti-Staphylococcus activities
MIC ( M) in comparison with standard ampicillin (AMP), in vitro antitubercular activity MIC (
g/mL)) in comparison with standard isoniazid (INH), in vitro antifungal activity MIC ( M ( g/mL))
of compounds 16 compared to standard benomyl (BNM), and in vitro antiproliferative (Tox) assay
Structure of ring-substituted (2E)-N-aryl-3-phenylprop-2-enamides
1–
16,
IC50
(
µ
µ
µM
(µ
µ
µ
1
–
(IC50 (µM)) of chosen compounds compared to standard camptothecin (CMP).
MIC (µM (µg/mL))
PET
IC50
(µM)
Tox IC50
(µM)
Comp.
R
MRSA MRSA MRSA SA
SA
Mtb
FA
BS
63718
SA 630
3202
>1146
(>256)
>1078
(>256)
>1078
(>256)
>1061
(>256)
>1061
(>256)
27.47
(8)
>1018
(>256)
>876
(>256)
>876
(>256)
438
(128)
438
(128)
>671
(256)
22.27
(8)
>799
(>256)
>799
(>256)
>785
(>256)
5.72
>1146
(>256)
>1078
(>256)
>1078
(>256)
>1061
(>256)
>1061
(>256)
27.47
(8)
>1018
(>256)
>876
(>256)
>876
(>256)
876
(256)
876
(256)
>671
(256)
22.27
(8)
>1146
(>256)
>1078
(>256)
>1078
(>256)
>1061
(>256)
>1061
(>256)
27.47
(8)
>1018
(>256)
>876
(>256)
>876
(>256)
438
>1146
(>256)
>1078
(>256)
>1078
(>256)
>1061
(>256)
>1061
(>256)
27.47
(8)
>1018
(>256)
>876
(>256)
>876
(>256)
876
(256)
438
(128)
>671
(256)
22.27
(8)
286
(64)
67.43
(16)
134
(32)
265
(64)
66.31
(16)
27.47
(8)
254
(64)
876
(256)
876
(256)
27.38
(8)
27.38
(8)
167
(64)
22.27
(8))
199
(64)
199
(64)
785
(256)
1146
(256)
270
143
(32)
33.71
(8)
1
2
H
3-CH3
4-CH3
2-F
–
250
343
320
223
165
189
338
67.1
1380
54.9
5.1
>30
(64)
1078
(256)
1061
(256)
531
(128)
54.93
(16)
1019
(256)
876
(256)
876
(256)
219
(64)
219
(64)
671
(256)
713
(256)
799
(256)
799
539
3
–
(128)
66.32
(16)
16.58
(4)
54.93
(16)
1019
(256)
876
(256)
876
(256)
110
(32)
110
(32)
671
(256)
356
(128)
49.98
(16)
799
4
–
5
3-F
>30
6
3-CF3
2,5-CH3
2,5-Cl
2,6-Cl
3,4-Cl
3,5-Cl
2,6-Br
3,5-CF3
2-F-5-Br
2-Br-5-F
2-Cl-5-CF3
–
22.72 ± 1.73
7
–
8
–
9
–
10
11
12
13
14
15
16
AMP
INH
BNM
29.81 ± 0.31
(128)
109
(64)
29.44 ± 1.73
>671
(256)
22.27
(8)
>799
(>256)
>799
(>256)
>785
(>256)
45.81
(16)
–
732
111
188
205
63.2
–
22.59 ± 1.88
>799
(>256)
>799
(>256)
>785
(>256)
45.81
(16)
>799
(>256)
>799
(>256)
>785
(>256)
45.81
(16)
–
–
–
–
–
–
(256)
785
(256)
(256)
785
(256)
–
–
–
(2)
36.55
(5)
–
–
–
–
–
–
–
–
1.94
(0.5)
–
17.22
(5)
–
–
–
–
–
–
–
–
CMP
DCMU
–
–
–
–
–
–
–
–
–
–
–
–
0.16 ± 0.07
–
2.1
–
–
–
SA = Staphylococcus aureus ATCC 29213; MRSA = clinical isolates of methicillin-resistant S. aureus 63718, SA 630, and
SA 3202 (National Institute of Public Health, Prague, Czech Republic); Mtb = Mycobacterium tuberculosis H37Ra;
FA = Fusarium avenaceum (Fr.) Sacc. IMI 319947; BS = Bipolaris sorokiniana (Sacc.) Shoemaker H-299 (NCBI GenBank
accession No. MH697869).

Int. J. Mol. Sci. 2018, 19, 2318
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Table 3. Combined effect of most potent N-arylcinnamamides and tetracycline (TET), ciprofloxacin
(CPX), and vancomycin (VAN).
Concentration
(µg/mL) Causing
Synergistic Effect
Concentration
(µg/mL) Causing
Additive Effect
Combination
of Compds.
Separate MIC
Isolate
FIC Index
(µg/mL)
6/TET
6/CPX
6/VAN
8/128
16/16
32/2
1.004–2.250
0.75–1.125
1.000–1.250
–
–
–
2/64; 8/32
8/4; 4/8
–
MRSA
63718
6/TET
6/CPX
6/VAN
13/TET
13/CPX
13/VAN
16/64
8/8
8/1
32/64
32/8
32/1
1.002–1.25
1.000–1.250
0.750–1.256
0.500–1.125
0.375–1.250
0.750–1.25
–
–
–
–
–
4/0.25
16/16; 4/32; 2/64
2/4
MRSA
SA 3202
8/16
8/1
–
16/0.25
6/CPX
6/VAN
13/CPX
13/VAN
8/256
8/1
8/256
4/1
0.625–1.125
0.750–1.250
0.375–1.004
0.562–1.250
–
–
4/64; 1/128
2/0.5
4/8
0.25/0.5
MRSA
SA 630
2/32; 1/64
–
Although the activity of cinnamic acid derivatives is known for a long time, the exact mechanism
of action is still unknown. The most reported mechanism of action is interaction with plasmatic
membrane. The compounds can cause disruption of the membrane, damage the membrane proteins,
etc. [43–46]. There are also specific targets for cinnamic acid derivatives [46]. Nevertheless, it is possible
that the wide spectrum of effects to cells is caused by the primary activity of the compounds, which is
membrane destabilization [46].
Both compounds 6 and 13 tested for synergy showed additivity with vancomycin against MRSA
SA 630 and SA 3202. A similar effect was reported by Hemaiswarya et al. [47]; compound 13 had
synergistic effect with ciprofloxacin against both tested strains. The effect of derivative 13 was
also synergistic with tetracycline against MRSA SA 3202. The rest combinations with compound
13 had additive effect. Whereas compound 13 had a potential to increase the activity of all tested
antibiotics, which have different mechanisms of actions and to which bacteria develop different
resistance mechanisms, it can be expected that compound 13 acts by its own mechanism of action or
increases the availability of the antibiotics by interaction with the membrane.
2.2.2. Dynamics of Antibacterial Activity
Within the pre-test subcultivation aliquots on agar, antistaphylococcal-effective compounds
and 13 showed bactericidal activity, i.e., minimal bactericidal concentrations were MIC. These
facts were verified using the time-kill curve assay for testing the bactericidal effect. The dynamics
of antibacterial activity was tested against S. aureus ATCC 29213 for the most active compounds
(Figure 2A) and 13 (Figure 2B). Both compounds showed concentration-dependent activity that was
bactericidal in concentration 4 MIC in the case of compound 13 or very close to the bactericidal level
for compound after 8 h from the beginning of incubation. The increase of bacterial growth at 24 h
could be caused by the selection of resistant mutants, as observed previously [37].
6
≤4×
6
×
6

Int. J. Mol. Sci. 2018, 19, 2318
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B
A
10
9
10
9
8
7
6
8
7
6
5
5
mr
4
4
3
2
3
2
0
5
10
15
20
25
0
5
10
15
20
25
Time [h]
log 1× MIC
Time [h]
Growth control
log 4× MIC
log 2× MIC
Growth control
log 2× MIC
log 1× MIC
Bactericidal effect
Bactericidal effect
Figure 2. Time-kill curve of compound 6 (A) and compound 13 (B) against S. aureus ATCC 29213.
2.2.3. Inhibition of Bofilm Formation
There are many evidences in literature that cinnamic acid derivatives are inhibitors of biofilm
formation [47 51]. The most studied derivate is cinnamaldehyde that interacts with quorum sensing
system in bacterial biofilms [46 52 53]. Thus, selected compounds were also tested for their ability to
inhibit biofilm formation. Compounds and 13 were tested as inhibitors of biofilm formation against
S. aureus ATCC 29213. MRSA strains were not producers of biofilm.
The activity of compound does not depend on concentration in concentrations above 8 µg/mL;
–
,
,
6
6
only the highest concentration showed lower inhibition effect. This could be caused the higher
lipophilicity of the compound and potential formation of precipitates, which could decrease the
antibacterial activity of the compound. The lowest concentration of the compound, which inhibited
≥
80% of biofilm formation, was 8 µg/mL, then the activity sharply decreased. Interestingly, on the
other hand, concentrations of compound 13 close to MIC against planktonic cells had the lowest
inhibition activities against biofilm forming, and the activity increased for sub-MIC values. These
conditions could be potentially toxic for planktonic cells, but they can induce biofilm formation [54].
In general, the inhibition activity against biofilm formation was comparable with the activity against
planktonic cells. Despite many studies reported a higher resistance of biofilm, there were also studies
that proved a similar or only little lower antibiofilm activity of tested compounds compared to
planktonic cells. [13,55]. Budzynska et al. [55] described high antibiofilm activity of plant essential oils
compared to MICs. De Vita et al. [13] studied the activity of cinnamic acid derivatives against candida
biofilm. These compounds had good effect against biofilm formation, and the effective concentrations
were lower than 10
structure, based on cinnamic acid.
Ampicillin (16–0.125 g/mL), vancomycin (32–0.25
were used as positive controls. Ciprofloxacin and vancomycin caused the induction of biofilm
×
MIC. Thus, the high activity of our compounds can be explained due to their
µ
µg/mL), and ciprofloxacin (8–0.063 µg/mL)
formation in sub-MIC concentrations, which is in line with already published results [56
,57]. All the
results are shown in Figure 3.
2.3. In Vitro Antitubercular Activity
The evaluation of the in vitro antitubercular activity of the compounds was performed against
Mycobacterium tuberculosis ATCC 25177/H37Ra, see Table 2. In order to reduce risks, a replacement
of model pathogens is commonly used in basic laboratory screening. For M. tuberculosis, avirulent
strain H37Ra is used that has a similar pathology as M. tuberculosis strains infecting humans and, thus,
represents a good model for testing antitubercular agents [58]. The potency of the compounds was
expressed as the MIC that is defined for mycobacteria as 90% or greater (IC90) reduction of growth in
comparison with the control.

Int. J. Mol. Sci. 2018, 19, 2318
8 of 25
115
95
75
55
35
15
-5
256
128
64
32
16
8
4
2
1
0.5
0.25 0.125 0.063
-25
-45
-65
Concentration [µg/mL]
comp. 13 CPX VAN
comp. 6
AMP
Figure 3. Inhibition of bacterial film formation. (CPX = ciprofloxacin, AMP = ampicillin,
VAN = vancomycin).
In comparison with antibacterial activities, the investigated compounds exhibited much higher
effect against M. tuberculosis. Compounds 13 (R = 3,5-CF3), 10 (R = 3,4-Cl), 11 (R = 3,5-Cl), and
6 (R = 3-CF3) were the most effective; their activity ranged from 22.27 to 27.47 µM. The dependences of
the antitubercular activity of the compounds against M. tuberculosis expressed as log (1/MIC (M)) on
lipophilicity expressed as log k are illustrated in Figure 4A. When inactive compounds 8 (R = 2,5-Cl),
9
(R = 2,6-Cl), and 16 (R = 2-Cl-5-CF3) are eliminated from the SAR study (illustrated by empty
symbols), two different dependences in relation to the position and the type of substituents can
be observed. Based on Figure 4A, it can be stated that compounds substituted in positions C(3)’,
C(3,4)’, C(3,5)’, or C(2,6)’ showed an increasing trend of activity with the lipophilicity increase up to
compound 6 (R = 3-CF3), at which the activity achieved plateau and from approximately log k
had an insignificant increase. The second, in fact, a linear, insignificantly increasing dependence
can be found for the compounds substituted in positions C(2)’ and C(2,5)’. It is important to note
≈
0.5
that the antitubercular activity of the discussed cinnamanilides is also dependent on electronic
σ
parameters, see Figure 4B. As mentioned above, the linear insignificantly increasing dependence can
be found for the derivatives substituted on the anilide in positions C(2)’ and C(2,5)’, while a bilinear
dependence of activity on
σ (for derivatives substituted in positions C(3)’, C(3,4)’, C(3,5)’, and C(2,6)’) can
be observed. The activity increases with the increasing electron-withdrawing effect with r = 0.8803
(n = 5) to optimum σAr ca. 1 (compound 13, R = 3,5-CF3) and then decreases (r = 0.9162, n = 4) with
increasing values of the electron-withdrawing parameter.
Additionally, a standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay was performed on selected compounds that were the most effective against M. tuberculosis
H37Ra and the MICs of which were previously determined, see Table 2. The MTT test can be used to
assess cell growth by measuring respiration. The MTT measured viability of M. tuberculosis H37Ra less
than 70% after exposure to the MIC values for each test agent is considered as a positive result of this
assay. This low level of cell viability indicates inhibition of cell growth by inhibition of respiration [59].
All the selected compounds, i.e.,
and 13 (R = 3,5-CF3, 66.09%) showed less than 70% viability of M. tuberculosis H37Ra at the tested
concentration equal to MICs (i.e., 8 g/mL or 22 and 27 M). At MIC = 16 g/mL (33 M) compound
6 (R = 3-CF3, 40.99%), 10 (R = 3,4-Cl, 59.65%), 11 (R = 3,5-Cl, 22.65%),
µ
µ
µ
µ

Int. J. Mol. Sci. 2018, 19, 2318
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2
(R = 3-CH3) showed inhibition of viability 13.23%, and compound
5
(R = 3-F) showed inhibition of
viability 11.04% at MIC = 32 µg/mL (33 µM).
A
B
4.9
4.6
4.3
4.0
3.7
3.4
3.1
2.8
2.5
4.9
3,5-CF3
3,5-Cl
3-CF3
3,4-Cl 3,5-Cl
3,5-CF3
4.6
4.3
4.0
3.7
3.4
3.1
2.8
2.5
3,4-Cl
3-CF3
3-F
3-CH3
3-F
3-CH3
4-CH3
2,6-Br
H
2-F-5-Br
2,6-Br
2-Br-5-F 2-F-5-Br
2,5-CH3
H
4-CH3
2-Br-5-F
2-F
2,5-CH3
2-F
2-Cl-5-CF3
2-Cl-5-CF3
2,6-Cl
2,5-Cl
2,6-Cl
2,5-Cl
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0.3
0.5
0.7
0.9
σAr
1.1
1.3
1.5
log k
Figure 4. Relationships between in vitro antitubercular activity against M. tuberculosis log (1/MIC (M))
and lipophilicity expressed as log k ( ) and electronic Hammett’s parameters of ring-substituted anilide
A
σ
ring (B) of studied compounds. (Derivatives excluded from SAR are illustrated by empty symbols.).
Similar effects were observed previously, for example, with ring-substituted 6-hydroxynaphthalene-
2-carboxanilides, where 3-Cl, 4-Cl, 3-Br, and 3-CF3 substituted derivatives decreased the
viability of M. tuberculosis H37Ra in the range from 41.2% to 46.5% at the lowest tested
concentration (MICs = 8
amide, where the decrease of the viability of M. tuberculosis H37Ra was to 18.8% at MIC = 8
and with N-alkoxyphenylhydroxynaphthalenecarboxanilides substituted in C(3)’ position of the anilide
core by a longer alkoxy tail [61 62]. Since the MTT assay was positive, it can be stated that the tested
µg/mL) [20], with 8-hydroxy-N-(3-trifluoromethylphenyl)quinoline-2-carbox-
µg/mL [60],
,
compounds caused a decrease of mycobacterial cell metabolism. Thus, based on the structure analogy
of (2E)-N-aryl-3-phenylprop-2-enamides with naphthalene-2-carboxanilides, it may be hypothesized
that the mechanism of action of these ring-substituted anilides of cinnamic acid could be connected
with the affection of mycobacterial energy metabolism [59,63–66]; nevertheless, another possible site
of action of the studied compounds in the mycobacteria cannot be excluded [67–70].
2.4. In Vitro Activity against Plant Pathogenic Fungi
Fungal infections are not only a problem in human and veterinary medicine, but also an important
problem in agriculture. Plants diseases in general are a major factor limiting the crop quality. Fungal
pathogens cause production losses and also can product mycotoxins, which are dangerous for
consumers. The widespread use of fungicides increases food availability and safety, but it can leads
to the selection of resistant pathogens and an increase of the production of mycotoxins [71]. As
cinnamic acid and its derivatives do not have only antibacterial and antimycobacterial activity, but
also activity against plant pathogens [7,72], all the prepared compounds were tested for their potency
against Fusarium avenaceum (Fr.) Sacc. IMI 319947 and Bipolaris sorokiniana (Sacc.) Shoemaker H-299.
B. sorokiniana is a wide-spread wheat and barley pathogen. It causes many diseases, such as head
blight, seedling blight, common root rot, spot blotch, etc. [73]. The last one is a big problem, especially
in Southern Asia, where 20% of crop yield is lost because of leaf blight disease [74]. F. avenaceum is one
of the most common Fusarium species causing head blight disease of cereals. It can be isolated from
cereal seeds and feed products [75]. Fusarium spp. produces a wide spectrum of mycotoxins. The most
important are the trichothecenes, zearalenone, moniifromin, and the fumonisins. These compounds
have toxic effect on humans and animals [76].
Only compound
F. avenaceum within the series of compounds, see Table 2. On the other hand, the investigated
compounds demonstrated higher effect against B. sorokiniana. (2E)-N-(3-Fluorophenyl)- ( ) and
(2E)-N-(3-methylphenyl)-3-phenylprop-2-enamide ( ) had MICs = 16.58 and 33.71 M, respectively,
6 (R = 3-CF3) showed moderate activity (MIC = 54.93 µM) against
5
2
µ

Int. J. Mol. Sci. 2018, 19, 2318
10 of 25
which is comparable with the benomyl standard. Also compounds
6
,
4
(R = 2-F), and 14 (2-F-5-Br)
demonstrated moderate activity (MIC range 49.98–66.32 µM) against B. sorokiniana. Surprising was the
inactivity of compound 13 (R = 3,5-CF3) against both fungal pathogens.
The dependences of the antifungal activity of the compounds against B. sorokiniana expressed as
log (1/MIC (M)) on lipophilicity expressed as log k are illustrated in Figure 5. In general, effective
compounds are preferentially substituted in positions C(3)’, C(3,5)’, or C(3,4)’. When inactive compounds
(illustrated by empty symbols) substituted in C(4)’, C(2,6)’, or C(2,5)’ are eliminated from the SAR
study, a bilinear dependence can be found. The activity increases with increasing lipophilicity from
unsubstituted derivative
1 to compound 5 (R = 3-F) with the supposed lipophilicity optimum log
k = 0.23 and then decreases to derivative 13 (R = 3,5-CF3); r = 0.9678, n = 7. It can be stated that it is an
opposite trend in comparison with antitubercular findings, which can be caused by differences in the
composition and structure of mycobacterial and fungal cell walls [77]. A similar trend can be found for
electronic properties of substituents in individual derivatives. The activity increases with an increase
of electron-withdrawing effect from compound
(compound , R = 3-F) and then decreases with increasing electron-withdrawing effect as follows:
σAr = 0.89 (compound
2 (R = 3-CH3, σAr = 0.48) to an optimum σAr = 0.82
5
6, R = 3-CF3), 1.02 (compound
4, R = 2-F), 1.11 (compound 11, R = 3,5-Cl), and
1.19 (compound 10, R = 3,4-Cl).
4.9
4.6
4.3
4.0
3.7
3.4
3-F
2-F-5-Br
3-CH3
2-F
H
3-CF3
3,5-Cl
3,4-Cl
3,5-CF3
2,6-Br
2,6-Cl
2-Br-5-F
4-CH3
0.2
2-Cl-5-CF3
0.8
3.1
2.8
2,5-Cl
2,5-CH3
0.4
0.0
0.6
1.0
1.2
log k
Figure 5. Relationships between in vitro antifungal activity against B. sorokiniana log (1/MIC (M)) and
lipophilicity expressed as log k of studied compounds. (Derivatives excluded from SAR are illustrated
by empty symbols.).
Inhibition of B. sorokiniana Germination
All the compounds were additionally evaluated for the inhibition of B. sorokiniana conidium
germination at two concentrations (128 and 256
that at both concentrations, the compounds showed the inhibition of germination. The effect
was concentration-dependent for compounds 10 15, and 16, and the rest of compounds had
concentration-independent effect on the germination. Interestingly, compound that was one of the
µg/mL), see results in Table 4. It can be stated
7,
,
6
most active against all bacterial cells including M. tuberculosis and displayed strong inhibition on the
mycelial growth of both investigated fungi (F. avenaceum and B. sorokiniana) (Table 2), showed the
weakest effect in the germination test. Apart from compound
highest B. sorokiniana mycelial growth inhibitory effect and had characteristic anti-germination activity.
Moreover, compound was the most effective in both assays (IC50 = 16.58 M in mycelial growth test
6, compounds 2, 4, 5, and 14 exerted the
5
µ
and 95.4% germination inhibition at 128 µg/mL).
2.5. In Vitro Antiproliferative Assay
The preliminary in vitro screening of the antiproliferative activity of the most effective
antimicrobial compounds was performed using a Water Soluble Tetrazolium salts-1 (WST-1) assay
kit [78] and the human monocytic leukemia THP-1 cell line by means of the method described

Int. J. Mol. Sci. 2018, 19, 2318
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recently [20,79]. The principle of the WST-1 assay kit is that antiproliferative compounds inhibit
mitochondrial dehydrogenases. The activity of this enzyme directly correlates with the number
of metabolically active cells in the culture. Antiproliferative effect was evaluated as IC50 value
(concentration of compound causing 50% inhibition of cell proliferation). It can be stated that a
compound is considered cytotoxic if it shows a toxic effect on cells up to 10
compound concentration used for the toxicity test was 3-fold higher than this.
µM [80]. The highest
IC50 values of the most effective compounds
10 (R = 3,4-Cl), 11 (R = 3,5-Cl), and 13 (R = 3,5-CF3) ranged from ca. 22 to >30
For comparison, the IC50 of camptothecin was 0.16 0.07 M. Both compounds
2
(R = 3-CH3),
5
(R = 3-F),
6
(R = 3-CF3),
M, see Table 2.
and effective
µ
±
µ
5
2
against B. sorokiniana as well as compounds 10 and 11 potent against M. tuberculosis showed IC50
approximately 30 M and higher, and compounds and 13 showed IC50 = 22 M, i.e., the treatment
µ
6
µ
with these concentrations did not lead to significant antiproliferative effect on THP-1 cells, and these
compounds inhibited selectively vital processes in B. sorokiniana, M. tuberculosis, or Staphylococcus
strains. Based on these observations, it can be concluded that all the tested compounds can be
considered as nontoxic agents for subsequent design of novel therapeutic agents.
Table 4. Inhibition (%) of Bipolaris sorokiniana conidium germination by compounds 1–16 in comparison
to negative control. Benomyl (BNM) was used as positive control.
Inhibition (%)
Compared to
Negative Control
Inhibition (%)
Compared to
Negative Control
Concentration
(µg/mL)
Concentration
Comp.
Comp.
(µg/mL)
256
128
59.6
74.2
256
128
82.5
76.3
1
9
10
256
128
64.7
59.1
256
128
84.3
51.3
2
256
128
88.7
89.1
256
128
84.8
82.6
3
11
256
128
60.8
58.7
256
128
91.3
82.1
4
12
256
128
93.6
95.4
256
128
76.3
75.5
5
13
256
128
18.4
26.7
256
128
81.8
77.6
6
7
14
256
128
73.5
35.1
256
128
100
60.9
15
256
128
92.7
88.7
256
128
86.2
61.2
8
16
10
5
100
100
10
5
100
100
BNM
BNM
2.6. Inhibition of Photosynthetic Electron Transport (PET) in Spinach Chloroplasts
The activity of the evaluated cinnamamides related to the inhibition of photosynthetic electron
transport (PET) in spinach (Spinacia oleracea L.) chloroplasts was moderate or low relative to the
standard, see Table 2, except for compound 11 (R = 3,5-Cl) that expressed the highest PET-inhibiting
activity comparable with the Diuron® standard (IC50 = 5.1
µM). With respect to these low activities, no
detailed SAR study can be proposed; nevertheless, from the results listed in Table 2, bilinear trends
for both PET inhibition vs. lipophilicity and PET inhibition vs. electronic properties of the anilide
core can be suggested. Thus, PET inhibition activity increases with the lipophilicity (log k) increase as
follows: 0.063 (
<<< 0.815 (11, R = 3,5-Cl) and then decreases to 0.981 (13, R = 3,5-CF3). Also PET inhibition activity
increases with the increase of electron-withdrawing (σAr) properties as follows: 0.46 ( , R = 4-CH3) <
1.05 (13, 3,5-CF3) <<< 1.11 (11, R = 3,5-Cl) and decreases as follows: >>> 1.19 (10, R = 3,4-Cl) > 1.28 (14
9, R = 2,6-Cl) < 0.264 (3, R = 4-CH3) < 0.487 (14, R = 2-F-5-Br) < 0.682 (10, R = 3,4-Cl)
3
,

Int. J. Mol. Sci. 2018, 19, 2318
12 of 25
R = 2-F-5-Br) >> 1.33 (
9
, R = 2,6-Cl). It can be concluded, as mentioned above, that the substitution of
the anilide core in C(3,5)’ or C(3,4)’ positions is preferable for high PET-inhibiting activity.
The inhibition of electron transport in PS II at the QB site plastoquinone, i.e., at the acceptor side of PS II
was observed for ring-substituted salicylanilides and carbamoylphenylcarbamates [26,28], ring-substituted
hydroxynaphthalene-2-carboxanilides [27 30], N-alkoxyphenylhydroxynaphthalene-carboxamides [29],
,
8-hydroxyquinoline-2-carboxamides [81], and N-substituted 2-aminobenzothiazoles [82]. Based on the
structural analogy and the presence of the amide bond, it can be hypothesized that the mechanism of action
of the investigated compounds is not different from the mechanism of action of the above compounds.
Moreover, as mentioned previously [26
–
29
,83
,
87], a good correlation between antimycobacterial
activity and herbicidal effect was found.
2.7. In Vivo Toxicity against Plant Cells
The most potent antifungal compounds
2,
5
,
6, and 14 were tested for in vivo toxicity against
plant cells. Nicotiana tabacum var. Samsun was used for this test [88]. The results of treatment of plant
leaves with injected water solutions of each compound as well as dimethyl sulfoxide (DMSO) are
illustrated in Figure 6, where photographs of leaves are shown. The negative DMSO control did not
have any toxic effect to the plant cells as well. On the other hand, the 5% aqueous solution of DMSO
showed a significant toxic effect on the leaves demonstrated as a loss of chlorophyll in the injected area
(Figure 6A). Based on Figure 6B,C, it can be concluded that the most effective antifungal compounds
had no visible influence on the plant tissue.
These results correspond to above-mentioned PET-inhibiting activity, when all the tested
compounds showed no PET inhibition, and thus, they will not be toxic for plants at their prospective
application as a plant fungicide.
Figure 6. Toxic effect of 5% DMSO (
compounds (blue ring), (red ring), and
ring) and 14 (red ring) ( ). The black spots in the injected area of compound
of the leaf tissue caused by a needle.
A
) to leaf of Nicotiana tabacum compared to nontoxic effect of
(black ring) ( ) and nontoxic effect of compounds (black
in Figure 6B are damages
2
5
6
B
6
C
5

Int. J. Mol. Sci. 2018, 19, 2318
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3. Materials and Methods
3.1. Chemistry—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). Melting points were determined on an apparatus Stuart SMP10
(Stone, UK) and are uncorrected. Infrared (IR) spectra were recorded on an UATR Zn/Se for a
Spectrum Two
™
Fourier-transform IR spectrometer (Per−k1inElmer, Waltham, MA, USA). The spectra
−1
were obtained by the accumulation of 32 scans with 4 cm resolution in the region of 4000–400 cm
.
All 1H- and 13C-NMR spectra were recorded on a JEOL ECZR 400 MHz NMR spectrometer (400 MHz
1
for H and 100 MHz for 13C, Jeol, Tokyo, Japan) in dimethyl sulfoxide-d6 (DMSO-d6). 1H and
13C chemical shifts (
δ) are reported in ppm. High-resolution mass spectra were measured using
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 the positive mode.
Synthesis
Cinnamic acid (3.37 mM) was suspended at room temperature in dry chlorobenzene (20 mL)
inside a microwave tube, where phosphorus trichloride (1.7 mM) and the corresponding aniline
(3.37 mM) were added dropwise. Then a ◦magnetic stirrer was used, and the reaction mixture was
transferred to the microwave reactor at 120 C for 20 min, where the synthesis at elevated pressure was
performed. After the mixture was cooled to 60 ◦C, and solvent was evaporated in vacuum. A solid
was washed with 2 M HCl, and a crude product was recrystallized, first using 96% ethanol and then
using 50% ethanol.
(2E)-N-Phenyl-3-phenylprop-2-enamide (
1626, 1594, 1578, 1543, 1493, 1442, 1347, 1288, 1248, 1188, 976, 904, 864, 759, 737, 704, 690, 676, 620, 592,
553, 512, 484, 454; 1H-NMR (DMSO-d6),
: 10.22 (s, 1H), 7.72 (d, J = 7.3 Hz, 2H), 7.64–7.59 (m, 3H),
7.47–7.36 (m, 3H), 7.34 (t, J = 8 Hz, 2H), 7.09–7.05 (m, 1H), 6.85 (d, J = 16 Hz, 1H); 13C-NMR (DMSO-d6),
: 163.53, 140.16, 139.28, 134.73, 129.77, 129.03, 128.81, 127.72, 123.36, 122.29, 119.23; HR-MS: for
C15H14NO [M + H]+ calculated 224.1070 m/z, found 224.1065 m/z.
1
) [89]. Yield 91%; Mp 116–118 ◦C; IR (cm−1): 3062, 3028, 1661,
δ
δ
(2E)-N-(3-Methylphenyl)-3-phenylprop-2-enamide (
) [89]. Yield 80%; Mp 113–115 ◦C; IR (cm−1): 3259,
2
3136, 3082, 3064, 3028, 2918, 1658, 1620, 1541, 1488, 1447, 1408, 1342, 1292, 1201, 987, 978, 864,
1
775, 763, 729, 714, 685, 676, 556, 487; H-NMR (DMSO-d6),
δ: 10.14 (s, 1H), 7.64–7.60 (m, 2H), 7.58
(
d, J = 15.6 Hz, 1H), 7.53 (m, 1H), 7.5 (d, J = 8.2 Hz, 1H), 7.47–7.40 (m, 3H), 7.21 (t, J = 7.8 Hz, 1H), 6.89
(d, J=7.8 Hz, 1H), 6.84 (d, J = 15.6 Hz, 1H), 2.3 (s, 3H); 13C-NMR (DMSO-d6),
δ: 163.48, 140.06, 139.23,
137.99, 134.75, 129.78, 129.04, 128.67, 127.73, 124.11, 122.38, 119.73, 116.45, 21.25; HR-MS: for C16H16NO
[M + H]+ calculated 238.1226 m/z, found 238.1222 m/z.
(2E)-N-(4-Methylphenyl)-3-phenylprop-2-enamide (3
) [89]. Yield 82%; Mp 166–168 ◦C; IR (cm−1): 3240,
3187, 3128, 3085, 3028, 2954, 1660, 1622, 1597, 1538, 1493, 1448, 1405, 1342, 1252, 1187, 984, 973, 813, 781,
762, 720, 674, 533, 510, 485; 1H-NMR (DMSO-d6) δ: 10.14 (s, 1H), 7.63–7.55 (m, 5H), 7.47–7.40 (m, 3H),
7.15–7.13 (m, 2H), 6.83 (d, J = 16 Hz, 1H), 2.26 (s, 3H); 13C-NMR (DMSO-d6)
δ: 163.33, 139.90, 136.81,
134.79, 132.32, 129.73, 129.22, 129.03, 127.70, 122.40, 119.22, 20.51; HR-MS: for C16H16NO [M + H]+
calculated 238.1226 m/z, found 238.1222 m/z.
(2E)-N-(2-Fluorophenyl)-3-phenylprop-2-enamide (
3182, 3126, 3085, 3059, 3020, 1661, 1627, 1539, 1491, 1448, 1342, 1257, 1188, 1105, 973, 760, 731, 719,
701, 687, 659, 557, 535, 495, 479; 1H-NMR (DMSO-d6)
: 9.69 (s, 1H), 8.11 (td, J = 7.9 Hz, 2.1 Hz, 1H),
7.64–7.57 (m, 3H), 7.48–7.40 (m, 4H), 7.31–7.26 (m, 1H), 7.20–7.16 (m, 1H), 7.09 (d, J=15.6 Hz, 1H);
4
) [89]. Yield 85%; Mp 114–116 ◦C; IR (cm−1): 3238,
δ

Int. J. Mol. Sci. 2018, 19, 2318
14 of 25
13C-NMR (DMSO-d6)
δ: 163.96, 153.34 (d, J = 244.7 Hz), 140.72, 134.74, 129.90, 129.05, 127.82, 126.41
(
d, J = 10.6 Hz), 125.09 (d, J = 7.7 Hz), 124.44 (d, J = 2.9 Hz), 121.88, 119.23, 115.47 (d, J = 19.3 Hz);
HR-MS: for C15H13FNO [M + H]+ calculated 242.0976 m/z, found 242.0971 m/z.
(2E)-N-(3-Fluorophenyl)-3-phenylprop-2-enamide (
). Yield 84%; Mp 112–114 ◦C; IR (cm−1): 3298, 3061,
3031, 2979, 1625, 1596, 1529, 1489, 1420, 1334, 1186, 974, 943, 858, 777, 768, 756, 727, 714, 688, 679, 658,
556, 492; 1H-NMR (DMSO-d6)
: 10.44 (s, 1H), 7.76–7.72 (m, 1H), 7.65–7.61 (m, 3H), 7.48–7.34 (m, 5H),
6.92–6.88 (m, 1H), 6.82 (d, J = 15.6 Hz, 1H); 13C-NMR (DMSO-d6)
: 163.87, 162.23 (d, J = 241.8 Hz),
5
δ
δ
141.00 (d, J = 10.6 Hz), 140.80, 134.61, 130.44 (d, J = 9.6 Hz), 129.95, 129.06, 127.85, 121.87, 115.02 (d,
J=2.9 Hz), 109.83 (d, J = 21.2 Hz), 106.07 (d, J = 27.3 Hz); HR-MS: for C15H13FNO [M + H]+ calculated
242.0976 m/z, found 242.0972 m/z.
◦
6
) [90]. Yield 75%; Mp 109–111 C; IR (cm−1):
(2E)-3-Phenyl-N-[3-(trifluoromethyl)phenyl]prop-2-enamide (
3400, 2905, 2360, 1678, 1630, 1526, 1491, 1331, 1154, 1112, 1100, 1072, 982, 886, 808; 1H-NMR (DMSO-d6)
: 10.57 (s, 1H), 8.22 (s, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.66–7.62 (m, 3H), 7,58 (t, J = 7.8 Hz, 1H), 7.48–7.40
(m, 4H), 6.82 (d, J = 15.6 Hz, 1H); 13C-NMR (DMSO-d6)
: 164.05, 140.98, 140.07, 134.54, 130.04, 129.99,
δ
δ
129.58 (q, J = 30.8 Hz), 129.05, 127.88, 124.16 (q, J = 272.6 Hz), 122.76, 121.72, 119.65 (q, J = 3.9 Hz),
115.29 (q, J = 3.9 Hz); HR-MS: for C16H13F3NO [M + H]+ calculated 292.0944 m/z, found 292.0938 m/z.
(2E)-N-(2,5-Dimethylphenyl)-3-phenylprop-2-enamide (7
). Yield 91%; Mp 174–176 ◦C; IR (cm−1): 3242,
3056, 3028, 2977, 2918, 1657, 1621, 1579, 1544, 1494, 1449, 1417, 1343, 1286, 1266, 1199, 1159, 990,
1
978, 878, 780, 764, 746, 734, 724, 707, 681, 624, 560, 492; H-NMR (DMSO-d6)
δ
: 9.42 (s, 1H), 7.63
(
d, J = 6.9 Hz, 2H), 7.58 (d, J = 16 Hz, 1H), 7.47–7.39 (m, 4H), 7.10 (d, J = 7.8 Hz, 1H), 6.99 (d, J = 16 Hz,
1H), 6.90 (d, J = 7.3 Hz, 1H), 2.26 (s, 3H), 2.20 (s, 3H); 13C-NMR (DMSO-d6)
δ: 163.58, 139.88, 136.17,
134.97, 134.85, 130.15, 129.67, 128.99, 127.94, 127.68, 125.70, 124.97, 122.36, 20.67, 17.54; HR-MS: for
C17H18NO [M + H]+ calculated 252.1383 m/z, found 252.1378 m/z.
◦
8
). Yield 83%; Mp 173–175 C; IR (cm−1): 3234,
(2E)-N-(2,5-Dichlorophenyl)-3-phenylprop-2-enamide (
3108, 3062, 3026, 1662, 1622, 1582, 1528, 1463, 1449, 1406, 1341, 1261, 1182, 1093, 1055, 992, 979, 917,
872, 856, 806, 761, 718, 693, 675, 580, 559, 488; 1H-NMR (DMSO-d6)
: 9.77 (s, 1H), 8.15 (d, J = 2.3 Hz,
1H), 7.66–7.62 (m, 3H), 7.54 (d, J = 8.7 Hz, 1H), 7.45–7.41 (m, 3H), 7.24 (dd, J = 8.7 Hz, 2.7 Hz, 1H),
δ
7.18 (d, J = 15.6 Hz, 1H); 13C-NMR (DMSO-d6)
δ: 164.20, 141.47, 136.28, 134.60, 131.61, 130.83, 130.05,
129.03, 127.94, 125.40, 124.11, 123.61, 121.51; HR-MS: for C18H12Cl2NO [M + H]+ calculated 292.0290
m/z, found 292.0289 m/z.
◦
(2E)-N-(2,6-Dichlorophenyl)-3-phenylprop-2-enamide (
1661, 1630, 1569, 1522, 1449, 1428, 1338, 1183, 971, 782, 758, 712, 697, 508; 1H-NMR (DMSO-d6)
(s, 1H), 7.67–7.62 (m, 3H), 7.58–7.56 (m, 2H), 7.48–7.41 (m, 3H), 7.39–7.35 (m, 1H), 6.89 (d, J = 16 Hz,
9
). Yield 85%; Mp 212–214 C; IR (cm−1): 3256,
δ: 10.09
1H); 13C-NMR (DMSO-d6)
δ: 163.67, 140.98, 134.51, 133.69, 133.03, 129.97, 129.19, 129.05, 128.56, 127.85,
120.72; HR-MS: for C15H12Cl2NO [M + H]+ calculated 292.0290 m/z, found 292.0288 m/z.
(2E)-N-(3,4-Dichlorophenyl)-3-phenylprop-2-enamide (10). Yield 78%; Mp 173–175 ◦C; IR (cm−1): 3269,
3095, 3025, 1663, 1627, 1585, 1526, 1474, 1378, 1339, 1289, 1229, 1182, 1126, 1025, 974, 863, 810, 761, 699,
677, 579, 564, 515, 484; 1H-NMR (DMSO-d6)
δ: 10.52 (s, 1H), 8.12–8.11 (m, 1H), 7.64–7.61 (m, 3H), 7.58
(s, 2H), 7.46–7.40 (m, 3H), 6.79 (d, J = 16 Hz, 1H); 13C-NMR (DMSO-d6)
δ: 163.63, 141.08, 139.38, 134.49,
131.09, 130.72, 130.03, 129.05, 127.88, 124.78, 121.55, 120.39, 119.25; HR-MS: for C15H12Cl2NO [M + H]+
calculated 292.0290 m/z, found 292.0288 m/z.
◦
(2E)-N-(3,5-Dichlorophenyl)-3-phenylprop-2-enamide (11) [89]. Yield 79%; Mp 139–141 C; IR (cm−1): 3246,
3177, 3085, 2967, 1663, 1622, 1584, 1532, 1442, 1408, 1338, 1180, 1109, 969, 937, 844, 802, 760,717, 668, 556,
484; 1H-NMR (DMSO-d6)
δ: 10.56 (s, 1H), 7.76 (d, J = 1.8 Hz, 2H), 7.65–7.61 (m, 3H), 7.48–7.40 (m, 3H),
7.29 (t, J = 2,1 Hz, 1H), 6.76 (d, J = 15.6 Hz, 1H); 13C-NMR (DMSO-d6)
δ: 164.06, 141.60, 141.35, 134.41,
134.12, 130.06, 129.02, 127.90, 122.48, 121.37, 117.30; HR-MS: for C15H12Cl2NO [M + H]+ calculated
292.0290 m/z, found 292.0288 m/z.

Int. J. Mol. Sci. 2018, 19, 2318
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(2E)-N-(2,6-Dibromophenyl)-3-phenylprop-2-enamide (12). Yield 81%; Mp 240–242 ◦C; IR (cm−1): 3251,
3180, 3028, 2902, 1660, 1627, 1558, 1516, 1441, 1422, 1337, 1263, 1180, 970, 781, 766, 759, 721, 710, 694,
1
686, 626, 561, 501; H-NMR (DMSO-d6)
δ
: 10.12 (s, 1H), 7.75 (d, J = 7.8 Hz, 2H), 7.67–7.64 (m, 2H),
7.60 (d, J = 16 Hz, 1H), 7.48–7.40 (m, 3H), 7.21 (t, J = 8 Hz, 1H), 6.87 (d, J = 15.6 Hz, 1H); 13C-NMR
(DMSO-d6) : 163.50, 140.85, 135.77, 134.50, 132.24, 130.19, 129.94, 129.05, 127.80, 124.33, 120.86; HR-MS:
for C15H12Br2NO [M + H]+ calculated 379.9280 m/z, found 379.9288 m/z.
δ
◦
(2E)-N-[3,5-bis(Trifluoromethyl)phenyl]-3-phenylprop-2-enamide (13). Yield 75%; Mp 143–145 C; IR (cm−1):
3272, 3085, 2967, 2938, 2879, 1663, 1622, 1575, 1472, 1440, 1377, 1276, 1168, 1129, 1110, 1096, 974, 937, 886,
859, 841, 728, 680, 627, 557, 486; 1H-NMR (DMSO-d6)
δ
: 10.89 (s, 1H), 8.36 (s, 2H), 7.77 (s, 1H), 7.70–7.65
(m, 3H), 7.48–7.40 (m, 3H), 6.78 (d, J = 16 Hz, 1H); 13C-NMR (DMSO-d6)
δ: 164.42, 141.78, 141.13,
134.28, 130.80 (q, J = 32.8 Hz), 130.22, 129.08, 127.98, 123.23 (q, J = 272.6 Hz), 121.07, 118.93–118.78 (m),
116.12–115.98 (m); HR-MS: for C17H12F6NO [M + H]+ calculated 360.0818 m/z, found 360.0811 m/z.
◦
(2E)-N-(2-Fluoro-5-bromophenyl)-3-phenylprop-2-enamide (14). Yield 83%; Mp 161–164 C; IR (cm−1): 3280,
2967, 2936, 2879, 1660, 1613, 1529, 1474, 1448, 1411, 1345, 1254, 1174, 985, 870, 800, 762, 722, 676, 617,
600, 564, 484; 1H-NMR (DMSO-d6)
δ
: 10.13 (s, 1H), 8.44 (dd, J = 7.1 Hz, 2.5 Hz, 1H), 7.65–7.60 (m, 3H),
7.48–7.40 (m, 3H), 7.35–7.27 (m, 2H), 7.11 (d, J = 15.6 Hz, 1H); 13C-NMR (DMSO-d6)
δ: 164.22, 152.03 (d,
J = 245.6 Hz), 141.30, 134.60, 130.02, 129.04, 128.25 (d, J = 12.5 Hz), 127.88, 127.10 (d, J = 7.7 Hz), 125.10,
121.50, 117.41 (d, J = 21.2 Hz), 115.91 (d, J = 2.9 Hz); HR-MS: for C15H12FBrNO [M + H]+ calculated
320.0081 m/z, found 320.0077 m/z.
◦
(2E)-N-(2-Bromo-5-fluorophenyl)-3-phenylprop-2-enamide (15). Yield 81%; Mp 164–166 C; IR (cm−1): 3230,
3072, 3045, 2967, 2937, 2880, 1661, 1623, 1591, 1538, 1420, 1342, 1198, 1155, 991, 977, 869, 854, 804, 761,
715, 668, 598, 589, 559, 482; 1H-NMR (DMSO-d6)
δ
: 9.64 (s, 1H), 7.83 (dd, J = 11 Hz, 3.2 Hz, 1H), 7.72
(dd, J = 8.7 Hz, 5.9 Hz, 1H), 7.67–7.62 (m, 3H), 7.48–7.42 (m, 3H), 7.15 (d, J = 15.6 Hz, 1H), 7.04 (ddd,
J = 8.9 Hz, 8 Hz, 3.2 Hz, 1H); 13C-NMR (DMSO-d6)
: 164.12, 161.05 (d, J = 243.7 Hz), 141.44, 137.74 (d,
δ
J = 11.6 Hz), 134.61, 133.85 (d, J = 8.7 Hz), 130.05, 129.04, 127.95, 121.55, 113.41 (d, J = 23.1 Hz), 112.44
(d, J = 27 Hz), 110.6 (d, J = 2.9 Hz); HR-MS: for C15H12FBrNO [M + H]+ calculated 320.0081 m/z, found
320.0077 m/z.
(2E)-N-[2-Chloro-5-(trifluoromethyl)phenyl]-3-phenylprop-2-enamide (16). Yield 77%; Mp 144–146 ◦C; IR
(cm−1): 3280, 2967, 2936, 2879, 1528, 1329, 1262, 1165, 1116, 1080, 964, 892, 814, 758, 707, 686, 668,
1
644, 605, 560, 534, 490, 452; H-NMR (DMSO-d6)
δ
: 9,22 (s, 1H), 8.40 (d, J = 1.8 Hz, 1H), 7.79–7.77
(m, 1H), 7.68–7.64 (m, 3H), 7.55–7.53 (m, 1H), 7.49–7.40 (m, 3H), 7.19 (d, J = 15.6 Hz, 1H); 13C-NMR
(DMSO-d6) : 164.42, 141.66, 135.99, 134.60, 130.70, 130.06, 129.02, 128.98, 128.09 (q, J = 38.2 Hz), 127.96,
δ
123.69 (q, J = 272.6 Hz), 121.98 (q, J = 3.9 Hz), 121.46, 120.95 (q, J = 3.9 Hz); HR-MS: for C16H12ClF3NO
[M + H]+ calculated 326.0554 m/z, found 326.0547 m/z.
3.2. Lipophilicity Determination by HPLC (Capacity Factor k/Calculated log k)
A HPLC separation module Waters® e2695 equipped with a Waters 2996 PDA Detector
(Waters Corp., Milford, MA, USA) were used. A chromatographic column Symmetry® C18
5 µm,
4.6 × 250 mm, Part No. W21751W016 (Waters Corp.) was used. The HPLC separation process was
monitored by the Empower™ 3 Chromatography Data Software (Waters Corp.). Isocratic elution
by a mixture of MeOH p.a. (72%) and H2O-HPLC Mili-Q grade (28%) as a mobile phase was used.
The total flow of the column was 1.0 mL/min, injection 5
µ
L, column temperature 40 ◦C, and sample
◦
temperature 10 C. The detection wavelength 214 nm was chosen. The KI methanolic solution was used
for the determination of dead time (tD). Retention times (tR) were measured in minutes. The capacity
factors k were calculated using the Empower™ 3 Chromatography Data Software according to the
formula k = (tR − tD)/tD, where tR is the retention time of the solute, while tD is the dead time obtained
using an unretained analyte. Each experiment was repeated three times. Log k, calculated from the

Int. J. Mol. Sci. 2018, 19, 2318
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capacity factor k, is used as the lipophilicity index converted to log P scale [36]. The log k values of
individual compounds are shown in Table 1.
3.3. Biological Testing
3.3.1. In Vitro Antibacterial Evaluation
The synthesized compounds were evaluated for in vitro antibacterial activity against
representatives of multidrug-resistant bacteria and clinical isolates of methicillin-resistant
Staphylococcus aureus (MRSA) 63718, SA 630, and SA 3202 [37,38] that were obtained from the National
Institute of Public Health (Prague, Czech Republic). S. aureus ATCC 29213 was used as a reference
and quality control strain. In addition, all the compounds were tested for their activity against
vancomycin-susceptible Enterococcus faecalis ATCC 29212 as a reference strain and three isolates from
American crows of vanA-carrying vancomycin-resistant E. faecalis (VRE) 342B, 368, and 725B [39].
Ampicillin (Sigma) was used as the standard. Prior to testing, each strain was passaged onto nutrient
agar (Oxoid, Basingstoke, UK) with 5% of bovine blood, and bacterial inocula were prepared by
suspending a small portion of bacterial colony in sterile phosphate buffered saline (pH 7.2–7.3). The cell
density was adjusted to 0.5 McFarland units using a densitometer (Densi-La-Meter, LIAP, Riga, Latvia).
This inoculum was diluted to reach the final concentration of bacterial cells 5 × 105 CFU/mL in the
wells. The compounds were dissolved in DMSO (Sigma), and the final concentration of DMSO in the
Cation Adjusted Mueller-Hinton (CaMH) broth (Oxoid) or Brain-Heart Infusion for enterococci did not
exceed 2.5% of the total solution composition. The final concentrations of the evaluated compounds
ranged from 256 to 0.008 µg/mL. The broth dilution micro-method, modified according to the NCCLS
(National Committee for Clinical Laboratory Standards) guidelines [91] in Mueller–Hinton (MH) broth,
was used to determine the minimum inhibitory concentration (MIC). Drug-free controls, sterility
controls, and controls consisting of MH broth and DMSO alone were included. The determination of
results was performed visually after 24 h of static incubation in the darkness at 37 ◦C in an aerobic
atmosphere. The results are shown in Table 2.
3.3.2. Synergy Effect with Clinically Used Drugs
For synergy effect study, a method of fractional inhibitory concentration was used. The tested
compounds (A) and conventional used antibiotic (B) (tetracycline, ciprofloxacin, and vancomycin
(purchased from Sigma)) were diluted in the microtitration plate in CaMH broth (Oxoid) to get an
original combination of concentration in every well. The raw H was used for evaluation of MIC(A)
column 12 was used for evaluation of MIC(B). The plate was inoculated by the bacterial suspension to
reach final concentration 5
105 CFU/mL in the wells. The fractional inhibitory concentration (FIC)
index was calculated using the concentrations in the first nonturbid (clear) well found in each row and
column along the turbidity/nonturbidity interface [42]. A FIC 0.5 means synergy; 0.5 < FIC < 1
FIC 4 is antagonism [41]. The tests were made in
;
×
Σ
≤
Σ
is additivity; 1 ≤ ΣFIC < 4 is indifference; and
Σ
≥
duplicate, and the results were averaged. The results are summarized in Table 3.
3.3.3. Dynamics of Antibacterial Effect
The most active antistaphylococcal compounds were studied for their dynamics of antibacterial
effect. The method of time-kill curves were used [37
reach the final concentration equal to 1 MIC, 2 MIC, and 4
concentrations 1 MIC and 2 MIC were used, due to the higher MIC and dissolution problems in the
concentration equal to 4 MIC. The tubes were inoculated with the culture of S. aureus ATTC 29213
,40]. Compound
6 was diluted in CaMH to
×
×
×
MIC. For the compound 13, only
×
×
×
diluted to 1 McFarland in the exponential phase of growth. The final concentration of bacteria was
7.5 × 106 CFU/mL. Tubes were stored in an incubator at 37 ◦C without shaking. Immediately after 4,
6, 8, and 24 h of inoculation, 100
(PBS). From each dilution 2
µ
L of sample was serially diluted 1:10 in phosphate buffered sali◦ne
×
20 µL were put onto MH agar plates. The plates were incubated at 37 C

Int. J. Mol. Sci. 2018, 19, 2318
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for 24 h, and the colonies were counted. Bactericidal effect is defined as a
compared to the growth control in time 0. The test was made in duplicate on 2 separate occasions, and
the results were averaged. The results are illustrated in Figure 2.
−
3log decrease of CFU/mL
3.3.4. Biofilm Inhibition Assay
The most active antistaphylococcal compounds were studied for their ability to inhibit biofilm
formation. The compounds were diluted in a 96-well plate in tryptic soy broth (TSB) containing
2% of glucose to reach concentrations 256–2 µg/mL. S. aureus ATCC 29213 cultivated overnight on
blood agar was used for preparing the inoculum. A few colonies were put in a tube with 5 mL
of TSB + 2% glucose and cultivated to reach the exponential phase of growth. The inoculum was
diluted to 1 McFarland and then 1:1000 in TSB + 2% glucose. The final concentration of bacteria in
each well was 1 × 105. As positive controls, ampicillin, vancomycin, ciprofloxacin, and tetracycline
(Sigma) were used. As the compounds were dissolved in DMSO (up to 5%), the growth control
included 5% of DMSO for verification that the applied DMSO concentration did not possess bacterial
◦
growth-inhibiting activity. The plate was cultivated for 48 h at 37 C without shaking. After incubation,
the content of the wells was removed and the wells were washed 3-fold by PBS. After drying, 125 µL
of 0.1% crystal violet was put to every well. The plane was stained for 20 min at room temperature,
the content was removed, and the plate was again washed by PBS 3-fold. The coloured biofilm was
taken off from the wells by 33% acetic acid, and absorbance in 595 nm was measured. As a blank,
non-inoculated plate treated in the same way was used. The test was made in triplicates in 3 separated
occasions. The ability to inhibit biofilm formation was evaluated as a percentage inhibition of growth
compared to the growth control. The results are illustrated in Figure 3.
3.3.5. In Vitro Antimycobacterial Evaluation
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,
ODAC-supplemented MB (10 mL). The turbidity was adjusted to match McFarland standard No. 1
(3 × 108 CFU) with MB broth. A further 1:10 dilution of the culture was then performed in MB broth.
The antimicrobial susceptibility of M. tuberculosis was investigated in a 96-well plate format. In these
experiments, sterile deionised water (300
minimize evaporation of the medium in the test wells during incubation. Each evaluated compound
(100 L) was incubated with M. tuberculosis (100 L). Dilutions of each compound were prepared
in duplicate. For all synthesized compounds, final concentrations ranged from 128 to 4 g/mL.
µL) was added to all outer-perimeter wells of the plates to
µ
µ
µ
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 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 coloured compounds, where the colour may interfere with the
interpretation of any colour change. For noninterfering compounds, a blue colour in the well was
interpreted as the absence of growth, and a pink colour 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 2.
3.3.6. MTT Assay
Compounds were prepared as previously stated and diluted in Middlebrook media to achieve
the desired final concentration 128–1
µg/mL. Mycobacterium tuberculosis ATCC 25177/H37Ra was
suspended in ODAC supplemented Middlebrook broth at a MacFarland standard of 1.0 and

Int. J. Mol. Sci. 2018, 19, 2318
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then diluted 1:10, using Middlebrook broth as a diluent. The diluted mycobacteria (50
µL) were
added to each well containing the compound to be tested. Diluted mycobacteria in broth free
from inhibiting compounds were used as a growth control. As positive controls, ciprofloxacin
and rifampicin were used. All compounds and controls were prepared in duplicate. Plates
were incubated at 37 ◦C for 7 days. After the incubation period, 10% well volume of MTT
(3-(4,5-dimethylthiazol-2-y◦l)-2,5-diphenyl-tetrazolium bromide) reagent (Sigma) was mixed into each
well and incubated at 37 C for 4 h in dark. Then 100 µL of 17% Sodium dodecyl sulfate in 40%
dimethylformamide was added to each well. The plates were read at 570 nm. The absorbance readings
from the cells grown in the presence of the tested compounds were compared with uninhibited cell
growth to determine the relative percent viability. The percent viability was determined through the
MTT assay. The percent viability is calculated through comparison of a measured value against that of
the uninhibited control: % viability = OD570E/OD570P
compound-exposed cells, while OD570P is the reading from the uninhibited cells (positive control).
×
100, where OD570E is the reading from the
Cytotoxic potential is determined by a percent viability of <70% [59,92]. Rifampicin a ciprofloxacin
(Sigma) were used as positive controls.
3.3.7. In Vitro Antifungal Activity
96-Well microplates were used for testing the inhibitory effect of compounds against mycelial
growth of Fusarium avenaceum (Fr.) Sacc. IMI 319947 and Bipolaris sorokiniana (Sacc.) Shoemaker
H-299 (NCBI GenBank accession No. MH697869). The compounds were diluted in DMSO to reach
the concentration of 10 mg/mL and then diluted in the microtiter plates in supplemented lysogeny
broth (LB, 10 g/L tryptone (Microtrade, Budapest, Hungary), 5 g/L yeast extract (Scharlau, Barcelona,
Spain), and 10 g/L NaCl (Reanal, Budapest, Hungary)) to get the final concentration of 256–2 µg/mL.
Positive (benomyl, Chinoin Fundazol 50WP®, Chinoin, Budapest, Hungary) and negative (solvents of
the compounds) controls were used. 50 mL LB medium was inoculated with the fungal culture grown
in an agar plate and shaken at 100 rpm at 22 ◦C for 3 days in dark; then mycelium was cut with a
sterile blender to small parts. The mycelium suspension was diluted to set OD600 = 0.2. This inoculum
was diluted 2-fold with the content of the wells. The absorbance at 600 nm was measured by a
spectrophotometer (Labsystems Multiscan MS 4.0, Thermo Scientific) immediately, and the plates
were incubated at 22 ◦C. The absorbance was measured again after 24, 48, and 72 h. The MIC was
counted in the time, when absorbance in the negative control tripled. The experiment was repeated on
3 separated occasions, and the results were averaged. The results are summarized in Table 2.
3.3.8. Inhibition of Bipolaris sorokiniana Germination
The inhibitory effect on the conidium germination was tested according to De Lucca et al. [93
with some modifications. The tested compounds were diluted in a 96-well microplate in 45 L sterile
distilled water to reach the final concentrations of 256 and 128 g/mL. As a positive control, benomyl
(10 and 5 g/mL) was used. As a negative control, nontreated wells with 5% DMSO were used.
Conidium suspension was prepared from sporulating culture grown on an agar plate. 5 L of the
]
µ
µ
µ
µ
aliquot was added to each well. The final concentration of conidia in each well was 3000 conidia in
1 mL. The plate was incubated at 22 ◦C for 48 h in dark. After the incubation, the aliquot from each
well was observed microscopically (400 ) for germination. A total of 50 conidia from each well were
×
observed. Germination was defined as the development of germ tube(s) of any size from a conidium.
The inhibition effect was counted as a ratio of nongerminated conidia compared to the negative control.
The experiment was performed in duplicate. The results are summarized in Table 4.
3.3.9. In Vitro Antiproliferative Assay
Human monocytic leukemia THP-1 cells were used for in vitro antiproliferative assay. Cells
were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK) and
routinely cultured in RPMI (Roswell Park Memorial Institute) 1640 medium supplemented

Int. J. Mol. Sci. 2018, 19, 2318
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with 10% fetal bovine serum, 2% L-glutamine, 1% penicillin, and streptomycin at 37 ◦C with
5% CO2. Cells were passaged at approximately one-week intervals. The antiproliferative
activity of the compounds was determined using a Water Soluble Tetrazolium Salts-1 (WST-1,
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) assay kit (Roche Diagnostics,
Mannheim, Germany) according to the manufacturer’s instructions. The tested compounds were
dissolved in DMSO and added in five increasing concentrations (0.37, 1.1, 3.3, 10, and 30 µM) to the
cell suspension in the culture RPMI 1640 medium. The maximum concentration of DMSO in the assays
◦
never exceeded 0.1%. Subsequently, the cells were incubated at 37 C with 5% CO2 for 24 h. For WST-1
assays, cells were seeded into 96-well plates (5
×
104 cells/well in 100
µL culture medium) in triplicate
in serum-free RPMI 1640 medium, and measurements were taken 24 h after the treatment with the
compounds. The median inhibition concentration values, IC50, were deduced through the production
of a dose-response curve. All data were evaluated using GraphPad Prism 5.00 software (GraphPad
Software, San Diego, CA, USA). The results are shown in Table 2.
3.3.10. Study of Inhibition of Photosynthetic Electron Transport (PET) in Spinach Chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.) according to Masarovicova and
Kralova [94]. The inhibition of photosynthetic electron transport (PET) in spinach chloroplasts was
determined spectrophotometrically (Genesys 6, Thermo Scientific), using an artificial electron acceptor
2,6-dichlorophenol-indophenol (DCIPP) according to Kralova et al. [95], and the rate of photosynthetic
electron transport was monitored as a photoreduction of DCPIP. The measurements were carried out
in phosphate buffer (0.02 mol/L, pH 7.2) containing sucrose (0.4 mol/L), MgCl2 (0.005 mol/L), and
NaCl (0.015 mol/L). The chlorophyll content was 30 mg/L in these experiments, and the samples
were irradiated (~100 W/m2 with 10 cm distance) with a halogen lamp (250 W) using a 4 cm water
filter to prevent warming of the samples (suspension temperature 22 ◦C). The studied compounds
were dissolved in DMSO due to their limited water solubility. The applied DMSO concentration (up to
4%) did not affect the photochemical activity in spinach chloroplasts. The inhibitory efficiency of the
studied compounds was expressed by IC50 values, i.e., by molar concentration of the compounds
causing a 50% decrease in the oxygen evolution rate relative to the untreated control. The comparable
IC50 value for the selective herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea, DCMU (Diuron®) was
about 2.1 µmol/L. The results are shown in Table 2.
3.3.11. In Vivo Toxicity against Plant Cells
Nicotiana tabacum var. Samsun was used for this test. The compounds were diluted in water to
reach concentrations equal to 1
same concentration were used. The positive control was 5% DMSO, which should be toxic to the plant
×
MIC and 4
×
MIC. As the negative control, solutions of DMSO in the
3
cells. The aqueous solutions were injected into the plant leaves by a syringe (needle 27G × ⁄
4”) to fill
area approx. 1 cm2 [88]. The test was made in triplicates in three different plants. The plants were kept
in a greenhouse on direct sunlight. The effect of the compounds was visually checked every day for
5 weeks. In the presence of tested compounds, no visible changes in the plant tissues were observed.
The results are illustrated in Figure 6.
4. Conclusions
A series of sixteen ring-substituted N-arylcinnamamides was prepared, characterized,
and evaluated against Staphylococcus aureus, three methicillin-resistant S. aureus strains,
Mycobacterium tuberculosis H37Ra, Fusarium avenaceum, and Bipolaris sorokiniana. Additionally,
the compounds were tested for their activity related to the inhibition of photosynthetic electron
transport in spinach chloroplasts. (2E)-3-Phenyl-N-[3-(trifluoromethyl)phenyl]prop-2-enamide
(6) and (2E)-N-[3,5-bis(tri-fluoromethyl)phenyl]-3-phenylprop-2-enamide (13) showed the highest
activity (MIC = 27.47 and 22.27
µM, respectively) against all four staphylococcal strains as
well as against M. tuberculosis.
These compounds also showed activity against bacterial

Int. J. Mol. Sci. 2018, 19, 2318
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biofilm forming, the ability to increase the effect of clinically used antibiotics such as
tetracycline, vancomycin, and ciprofloxacin and concentration-dependent antibacterial effect,
which led to killing >99% of bacteria. On the other hand, (2E)-N-(3-fluorophenyl)- (
(2E)-N-(3-methylphenyl)-3-phenylprop-2-enamide ( ) had MICs = 16.58 and 33.71 µM, respectively,
against B. sorokiniana, while both compounds did not show any in vivo toxicity against Nicotiana
tabacum var. Samsun. (2E)-N-(3,5-dichlorophenyl)-3-phenylprop-2-enamide (11, IC50 = 5.1 M) was the
5) and
2
µ
most active PET inhibitor. These compounds showed activities comparable with or higher than those of
standards. A significant decrease of mycobacterial cell metabolism (viability of M. tuberculosis H37Ra)
was observed using the MTT assay. The screening of the cytotoxicity of the selected compounds
was performed using THP-1 cells, and no significant lethal effect was observed for the most potent
compounds. The position of substituents on the anilide ring seems to be crucial for both antitubercular
and antifungal activity; positions C(3)’, C(3,4)’, and C(3,5)’ are preferable. Lipophilicity is another
important factor; antitubercular activity increases with increasing lipophilicity, while antifungal activity,
to the contrary, decreases from the most effective compound
5 (R = 3-F) with lipophilicity log k = 0.23
with increasing lipophilicity. The activity is also dependent on the electronic parameters of substituents
on the anilide core. Bilinear trends for effective compounds can be observed for both antitubercular and
antifungal dependences; nevertheless, for antitubercular effectivity, rather more electron-withdrawing
properties are preferred (σAr
≈
1, compound 13, R = 3,5-CF3), while for antifungal activity against
B. sorokiniana, less electron-withdrawing properties are preferred (σAr = 0.82, compound 5, R = 3-F).
Also the dependences of PET-inhibiting activity on lipophilicity and electronic properties showed
bilinear trends, as mentioned above, where C(3,5)’ or C(3,4)’ substitution of the anilide core is preferable.
Author Contributions: J.K. and T.S.—synthesis and characterization of the compounds and study of PET
inhibition. I.K. and M.O.—analysis of the compounds. S.P., H.M., A.C., A.M.M., and J.B.—antimicrobial and
pesticide evaluation and in vivo toxicity test. T.K. and P.K.—in vitro cytotoxicity assay. J.J.—conceived and
designed the experiments, SAR, and the writing of the paper.
Acknowledgments: This contribution was supported by grant of the Comenius University in Bratislava
No. UK/229/2018, grants of the Faculty of Pharmacy of Comenius University in Bratislava FaF UK/9/2018
and FaF UK/37/2018, VEGA project No.1/0040/17 and partially by SANOFI-AVENTIS Pharma Slovakia, s.r.o.
and the National Research, Development, and Innovation Office (NKFIH) of Hungary (grant
no. K119276). The HPLC/HRMS system forms a part of the National Infrastructure CzeCOS ProCES
CZ.02.1.01/0.0/0.0/16_013/0001609; Michal Oravec was supported by the National Sustainability Program
(NPU I; Grant No. LO1415).
Conflicts of Interest: The authors declare no conflicts of interest.
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