Investigation of Anti-Inflammatory Potential of N-Arylcinnamamide Derivatives

A series of sixteen ring-substituted N-arylcinnamanilides, previously described as highly antimicrobially effective against a wide spectrum of bacteria and fungi, together with two new derivatives from this group were prepared and characterized. Moreover, the molecular structure of (2E)-N-(2-bromo-5-fluorophenyl)-3-phenylprop-2-enamide as a model compound was determined using single-crystal X-ray analysis. All the compounds were tested for their anti-inflammatory potential, and most tested compounds significantly attenuated the lipopolysaccharide-induced NF-κB activation and were more potent than the parental cinnamic acid. (2E)-N-[2-Chloro-5-(trifluoromethyl)phenyl]-3-phenylprop-2-enamide, (2E)-N-(2,6-dibromophenyl)- 3-phenylprop-2-enamide, and (2E)-N-(2,5-dichlorophenyl)-3-phenylprop-2-enamide demonstrated the highest inhibition effect on transcription factor NF-κB at the concentration of 2 µM and showed a similar effectiveness as the reference drug prednisone. Several compounds also decreased the level of TNF-α. Nevertheless, subsequent tests showed that the investigated compounds affect neither IκBα level nor MAPKs activity, which suggests that the N-arylcinnamanilides may have a different mode of action to prednisone. The modification of the C(2,5)ʹ or C(2,6)ʹ positions of the anilide core by rather lipophilic and bulky moieties seems to be preferable for the anti-inflammatory potential of these compounds.


Introduction
Inflammation is physiological immune reaction against infectious agents or injury, and its main role is to eradicate the noxious agent and to restore tissue homeostasis [1]. Under normal conditions, inflammation is self-limiting, but when it is uncontrolled and continuous, chronic inflammatory phase can develop subsequently. Chronic inflammation is a hallmark of many diseases, including atherosclerosis [2], rheumatoid arthritis [3], psoriasis [4], cancer [5], chronic respiratory diseases [6,7], and type 2 diabetes mellitus [8].
To modulate and drive the inflammatory response and inflammation itself are big tasks of current medicine. One way of handling such conditions is the application of natural compounds and their derivatives. Natural material is a huge source of bioactive compounds [9,10], and many currently used

X-Ray Crystallography
Single crystal X-ray analysis revealed the molecular structure of (2E)-N-(2-bromo-5-fluorophenyl)-3phenylprop-2-enamide (15), which is depicted in Figure 1. The crystal data and structure refinement for 15 are given in Table 1. The crystal structure consists of individual molecules, which are connected through strong N-H···O and weak C-H···O hydrogen bonds and via Br···O non-covalent contacts into 1D polymeric chains, see Figure 2 and Table 2. Moreover, the crystal structure is further stabilized by weak C-H···C hydrogen bonds and other non-covalent interactions of C···Br and C···C types connecting individual molecules into a 3D supramolecular structure. Both benzene rings (ring 1 = C4, C5, C6, C7, C8, C9, and ring 2 = C10, C11, C12, C13, C14, C15) are nearly ideally planar with the maximal deviation of 0.010(4) Å for the C11 atom from the least-squares planes fitted through the mentioned carbon atoms. The rings are mutually oriented by the dihedral angle of 47.0(2) • , and this value is substantially lower as compared to the values of 73.89 (7) • and 79.46 (7) • found for N-(2-fluorophenyl)cinnamamide [28] (in two crystallographically independent molecules with the asymmetric unit) and the value of 24.6(1) • found for N-(3-chlorophenyl)cinnamamide [29]. Moreover, there are also next two known structures of halogenated N-phenylcinnamamides, i.e., N- (3,5-difluoro-4-chlorophenyl)cinnamide and N- (3,4-difluorophenyl)cinnamide [30], in which both aromatic rings are almost coplanar, with the dihedral angle of 0.54 • and 2.04 • , respectively. The selected bond lengths and angles of compound 15 are given in Table S1 in Supplementary Materials. Interestingly, the C1-O1 bond length is significantly longer (1.312(6) Å) than that of a typical double C=O bond (an average value of 1.235(7) Å was found by the analysis of 39 structures containing the N-phenylcinnamamide skeleton deposited in CSD, ver. 5.40, Aug 2019 update). This lengthening of this bond in compound 15 may be connected with the involvement of the O1 atom into non-covalent bonding (see Figure 2).
Molecules 2019, 24, x FOR PEER REVIEW 3 of 15 dihedral angle of 0.54° and 2.04°, respectively. The selected bond lengths and angles of compound 15 are given in Table S1 in Supplementary Materials. Interestingly, the C1-O1 bond length is significantly longer (1.312(6) Å) than that of a typical double C=O bond (an average value of 1.235 (7) Å was found by the analysis of 39 structures containing the N-phenylcinnamamide skeleton deposited in CSD, ver. 5.40, Aug 2019 update). This lengthening of this bond in compound 15 may be connected with the involvement of the O1 atom into non-covalent bonding (see Figure 2).

Inhibition of NF-κB Activity and Cell Signaling In Vitro
Cinnamic acid alone and several CA derivatives demonstrated the ability to influence the activity or expression of the pro-inflammatory transcription factor NF-κB in previous studies [15,33]. The effect of N-arylcinnamamide derivatives with the modified cinnamyl phenyl ring on NF-κB activity was studied by Chen et al. [23] as well as by Jan et al. [34], but the effect of derivatives with the unchanged cinnamyl moiety is described here for the first time. Apart from compounds 1 and 4, all anilides were able to significantly attenuate the lipopolysaccharide (LPS)-induced NF-κB activation by 10% to 27% in the non-toxic concentrations of 2 and 0.5 µM (used for compounds 10 (R = 3,4-Cl), 11 (R = 3,5-Cl), 13 (R = 3,5-CF3) with the lowest cell viability), see Figure 3. Moreover, most N-arylcinnamamide derivatives were more active than the parental CA. The highest inhibitory effect on NF-κB activity was observed for (2E)-N-[2-chloro-5-(trifluoromethyl)phenyl]-3-phenylprop-2-enamide (17), which was comparable with prednisone (PDS) applied at the same concentration level of 2 µM. The overall results indicate that monosubstituted N-arylcinnamanilides show lower inhibitory effect on NF-κB activity than the di-substituted ones. (6), with bulky CF3 substitution of the anilide ring, is the only exception.

Inhibition of NF-κB Activity and Cell Signaling In Vitro
Cinnamic acid alone and several CA derivatives demonstrated the ability to influence the activity or expression of the pro-inflammatory transcription factor NF-κB in previous studies [15,33]. The effect of N-arylcinnamamide derivatives with the modified cinnamyl phenyl ring on NF-κB activity was studied by Chen et al. [23] as well as by Jan et al. [34], but the effect of derivatives with the unchanged cinnamyl moiety is described here for the first time. Apart from compounds 1 and 4, all anilides were able to significantly attenuate the lipopolysaccharide (LPS)-induced NF-κB activation by 10% to 27% in the non-toxic concentrations of 2 and 0.5 µM (used for compounds 10 (R = 3,4-Cl), 11 (R = 3,5-Cl), 13 (R = 3,5-CF 3 ) with the lowest cell viability), see Figure 3. Moreover, most N-arylcinnamamide derivatives were more active than the parental CA. The highest inhibitory effect on NF-κB activity was (17), which was comparable with prednisone (PDS) applied at the same concentration level of 2 µM. The overall results indicate that monosubstituted N-arylcinnamanilides show lower inhibitory effect on NF-κB activity than the di-substituted ones. (2E)-3-Phenyl-N-[3-(trifluoromethyl)phenyl]prop-2-enamide (6), with bulky CF 3 substitution of the anilide ring, is the only exception.
The electron σ parameters of individual substituents (listed in Pospisilova et al. [25]) play only a secondary role in this matter; however, it can be stated that the electron-withdrawing properties of the anilide substituent seem to be more advantageous. On the other hand, the position of the substituents on the anilide ring, lipophilicity, and bulkiness influence the activity of the compounds significantly. C (2,5) or C (2,6) di-substitutions by bulky substituents are preferred. The dependences of the inhibition of NF-κB on the lipophilicity expressed by distributive π parameters of the whole anilide ring and on  Figure 4, where the discussed compounds are shown, with the exception of toxic compounds 10, 11, 13, and compound 18 (R = 2-OCH 3 -5-NO 2 ), which is excluded due to the completely different nature of the substituents compared to the rest (that are methyls or halogens). Figure 4A,B show the trends of the increasing ability to inhibit NF-κB with increasing distributive π parameters (correlation coefficient r = 0.7892, n = 14) as well as anilide ring bulkiness (r = 0.8118, n = 14). The electron σ parameters of individual substituents (listed in Pospisilova et al. [25]) play only a secondary role in this matter; however, it can be stated that the electron-withdrawing properties of the anilide substituent seem to be more advantageous. On the other hand, the position of the substituents on the anilide ring, lipophilicity, and bulkiness influence the activity of the compounds significantly. C(2,5)ʹ or C(2,6)ʹ di-substitutions by bulky substituents are preferred. The dependences of the inhibition of NF-κB on the lipophilicity expressed by distributive π parameters of the whole anilide ring and on the molar volume of the whole anilide ring are illustrated in Figure 4, where the discussed compounds are shown, with the exception of toxic compounds 10, 11, 13, and compound 18 (R = 2-OCH3-5-NO2), which is excluded due to the completely different nature of the substituents compared to the rest (that are methyls or halogens). Figures 4A and 4B show the trends of the increasing ability to inhibit NF-κB with increasing distributive π parameters (correlation coefficient r = 0.7892, n = 14) as well as anilide ring bulkiness (r = 0.8118, n = 14).  The electron σ parameters of individual substituents (listed in Pospisilova et al. [25]) play only a secondary role in this matter; however, it can be stated that the electron-withdrawing properties of the anilide substituent seem to be more advantageous. On the other hand, the position of the substituents on the anilide ring, lipophilicity, and bulkiness influence the activity of the compounds significantly. C(2,5)ʹ or C(2,6)ʹ di-substitutions by bulky substituents are preferred. The dependences of the inhibition of NF-κB on the lipophilicity expressed by distributive π parameters of the whole anilide ring and on the molar volume of the whole anilide ring are illustrated in Figure 4, where the discussed compounds are shown, with the exception of toxic compounds 10, 11, 13, and compound 18 (R = 2-OCH3-5-NO2), which is excluded due to the completely different nature of the substituents compared to the rest (that are methyls or halogens). Figures 4A and 4B show the trends of the increasing ability to inhibit NF-κB with increasing distributive π parameters (correlation coefficient r = 0.7892, n = 14) as well as anilide ring bulkiness (r = 0.8118, n = 14). To confirm the effect of the most potent compound 17 on NF-κB, the analysis of its nuclear translocation was performed (see Figure 5). There is visible nuclear translocation of NF-κB after LPS stimulation. This movement was slightly affected by 17. It is in agreement with observed inhibition of NF-κB activity and it can delineate the possible mechanisms of action. between the NF-κB inhibition and unobserved alteration of the above-mentioned signaling pathways could be also explained by a different mode of action as compared to known anti-inflammatory drugs, like PDS. It can be hypothesized that the tested N-arylcinnamanilides might act either via the inhibition of the nuclear translocation of NF-κB (as was described above), or influence its binding to DNA [41,42], or act by epigenetic regulation [43]. A combination of more than one mechanism of action is not excluded as well.  Having discovered the high ability of compounds 17 (R = 2-Cl-5-CF 3 ) and 8 (R = 2,5-Cl) to moderate the activity of NF-κB, we decided to elucidate further their mechanism of action and compare it with CA used as the reference compound. The activity of NF-κB is driven by the level of its inhibitor IκB and by the activity of several mitogen-activated protein kinases (MAPKs), especially p38 and c-Jun N-terminal kinase (JNK) [35,36], as well as ERK [37]. The influence of CA on NF-κB and MAPKs expression and activity was described previously [38,39], the same as for some cinnamides [23]. Our results showed no effect of compounds 17, 8, and CA on the IκBα levels and MAPKs activity, see Figure 6. Similar effects were also referred for a synthetic derivative of CA, WK2-16 ((E)-N-hydroxy-4-methoxy-2-(biphenyl-4-yl)cinnamide) [34,40]. It was proposed that WK2-16 could influence transcription factors YY-1 and STAT-1/-3. However, the discrepancy between the NF-κB inhibition and unobserved alteration of the above-mentioned signaling pathways could be also explained by a different mode of action as compared to known anti-inflammatory drugs, like PDS. It can be hypothesized that the tested N-arylcinnamanilides might act either via the inhibition of the nuclear translocation of NF-κB (as was described above), or influence its binding to DNA [41,42], or act by epigenetic regulation [43]. A combination of more than one mechanism of action is not excluded as well.

Inhibition of TNF-α Secretion
To verify the effect of tested compounds on NF-κB activity, the secretion of TNF-α, which is under its transcription control [44], was measured. CA is known for its ability to attenuate the production of this pro-inflammatory cytokine in vitro and in vivo [45,46]. In our experiment, pure CA did not affect the TNF-α production, see Figure 7. This could be caused by the usage of a very low, but pharmacologically relevant concentration of 2 µM. On the other hand, compounds 12 (R = 2,6-Br), 6 (R = 3-CF3), and 17 (R = 2-Cl-5-CF3) that showed the highest inhibitory effect on NF-κB were able to significantly decrease the level of TNF-α by 10%, 10.4%, and 12.4%, respectively. This correlates with the observed attenuation of NF-κB, when compound 17 reached the greatest effect. However, the effect of PDS was notably higher-the reduction of TNF-α by 70.3%. In case of NF-κB inhibition, the tested compounds are comparable with PDS, but PDS is more effective in TNF-α decreasing. This indicates that the modes of action of PDS and the discussed N-arylcinnamanilides are different. Figure 7. Effect of N-arylcinnamamide derivatives on the secretion of TNF-α. THP1-Blue™ NF-κB cells were differentiated to macrophages by PMA. Such cells were pretreated by compounds or PDS (2 µM) for 1 h, then LPS (1 µg/mL) was added to trigger the inflammatory signalization. Cultivation medium was collected 24 h after LPS stimulation, and the amount of TNF-α was measured by the ELISA technique. Graph represents mean ± SEM. * indicates statistical significance (p < 0.05) to DMSO group; ** indicates statistical significance (p < 0.01) to DMSO group **** indicates statistical significance (p < 0.0001) to DMSO group.

Inhibition of TNF-α Secretion
To verify the effect of tested compounds on NF-κB activity, the secretion of TNF-α, which is under its transcription control [44], was measured. CA is known for its ability to attenuate the production of this pro-inflammatory cytokine in vitro and in vivo [45,46]. In our experiment, pure CA did not affect the TNF-α production, see Figure 7. This could be caused by the usage of a very low, but pharmacologically relevant concentration of 2 µM. On the other hand, compounds 12 (R = 2,6-Br), 6 (R = 3-CF 3 ), and 17 (R = 2-Cl-5-CF 3 ) that showed the highest inhibitory effect on NF-κB were able to significantly decrease the level of TNF-α by 10%, 10.4%, and 12.4%, respectively. This correlates with the observed attenuation of NF-κB, when compound 17 reached the greatest effect. However, the effect of PDS was notably higher-the reduction of TNF-α by 70.3%. In case of NF-κB inhibition, the tested compounds are comparable with PDS, but PDS is more effective in TNF-α decreasing. This indicates that the modes of action of PDS and the discussed N-arylcinnamanilides are different.

Inhibition of TNF-α Secretion
To verify the effect of tested compounds on NF-κB activity, the secretion of TNF-α, which is under its transcription control [44], was measured. CA is known for its ability to attenuate the production of this pro-inflammatory cytokine in vitro and in vivo [45,46]. In our experiment, pure CA did not affect the TNF-α production, see Figure 7. This could be caused by the usage of a very low, but pharmacologically relevant concentration of 2 µM. On the other hand, compounds 12 (R = 2,6-Br), 6 (R = 3-CF3), and 17 (R = 2-Cl-5-CF3) that showed the highest inhibitory effect on NF-κB were able to significantly decrease the level of TNF-α by 10%, 10.4%, and 12.4%, respectively. This correlates with the observed attenuation of NF-κB, when compound 17 reached the greatest effect. However, the effect of PDS was notably higher-the reduction of TNF-α by 70.3%. In case of NF-κB inhibition, the tested compounds are comparable with PDS, but PDS is more effective in TNF-α decreasing. This indicates that the modes of action of PDS and the discussed N-arylcinnamanilides are different. Figure 7. Effect of N-arylcinnamamide derivatives on the secretion of TNF-α. THP1-Blue™ NF-κB cells were differentiated to macrophages by PMA. Such cells were pretreated by compounds or PDS (2 µM) for 1 h, then LPS (1 µg/mL) was added to trigger the inflammatory signalization. Cultivation medium was collected 24 h after LPS stimulation, and the amount of TNF-α was measured by the ELISA technique. Graph represents mean ± SEM. * indicates statistical significance (p < 0.05) to DMSO group; ** indicates statistical significance (p < 0.01) to DMSO group **** indicates statistical significance (p < 0.0001) to DMSO group. Figure 7. Effect of N-arylcinnamamide derivatives on the secretion of TNF-α. THP1-Blue™ NF-κB cells were differentiated to macrophages by PMA. Such cells were pretreated by compounds or PDS (2 µM) for 1 h, then LPS (1 µg/mL) was added to trigger the inflammatory signalization. Cultivation medium was collected 24 h after LPS stimulation, and the amount of TNF-α was measured by the ELISA technique. Graph represents mean ± SEM. * indicates statistical significance (p < 0.05) to DMSO group; ** indicates statistical significance (p < 0.01) to DMSO group **** indicates statistical significance (p < 0.0001) to DMSO group.

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 of 16 and 18 were determined on an apparatus STA 449 F1 Jupiter (NETZSCH, Selb, Germany). Infrared (IR) spectra were recorded on an UATR Zn/Se for a Spectrum Two™ Fourier-transform IR spectrometer (PerkinElmer, Waltham, MA, USA). The spectra were obtained by the accumulation of 32 scans with 4 cm −1 resolution in the region of 4000-400 cm −1 . All 1 H-and 13 C-NMR spectra were recorded on a JEOL JNM-ECA 600II NMR spectrometer (600 MHz for 1 H and 150 MHz for 13 C, Jeol, Tokyo, Japan) in dimethyl sulfoxide-d 6 (DMSO-d 6 ). 1 H and 13 C 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 XL TM 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 derivative (3.37 mM) were added dropwise. Following this step, a magnetic stirrer was added to the tube 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, the solvent was evaporated in vacuum. A solid residue was washed with 2 M HCl, and a crude product was recrystallized, using 96% ethanol first, and then using 50% ethanol.

X-Ray Crystallography
Crystals of (2E)-N-(2-bromo-5-fluorophenyl)-3-phenylprop-2-enamide (15) suitable for a single X-ray analysis were prepared by the slow evaporation of ethanol from the solution of compound 15 at 4 • C. The diffraction data were collected on a D8 QUEST diffractometer (Bruker) equipped with a PHOTON 100 CMOS detector, using Mo-Kα radiation (λ = 0.71073 Å). The data collection and reduction were performed using the APEX3 software [47]. The structure was solved by a direct method (SHELXS) and refined using the Bruker SHELXTL software package [48]. The benzene ring, involving C4, C5, C6, C7, C8, and C9 atoms, was refined with a riding group fitting (AFIX 66) with the fixed C-C bonds (1.39 Å). All H-atoms were found from difference Fourier maps and refined using a riding model, with C-H = 0.95 Å for (CH) and 0.88 Å for (NH) and with U iso (H) = 1.2 U eq (CH, NH). The graphics were drawn, and additional structural calculations were performed by the DIAMOND [49] software.
Crystallographic data has been deposited with the Cambridge Crystallographic Data Centre under CCDC deposition number 1957819. Copies of this information may be obtained free of charge from the Director, CCDC, 12 Union Road, Cambridge CB2 1EY, UK (fax: +44-1223-336033; e-mail: deposit@ccdc.cam.ac.uk or www:http://www.ccdc.cam.ac.uk).

Cell Cultivation
THP1-Blue™ NF-κB cell line was purchased from Invivogen (San Diego, CA, USA). Cells were cultured in RPMI 1640 medium (Merck, Darmstadt, Germany) containing stabilized 2 mM l-glutamine (Merck) supplemented with antibiotics [100 U/mL penicillin and 100 mg/mL streptomycin (Merck)] and 10% FBS (Merck). The cultures were passaged twice a week. Cells were kept in an incubator at 37 • C in a water-saturated atmosphere of air containing 5% CO 2 .
The viability of the used cell lines (5th-18th passage) was over 95% for each experiment. The cell number and viability were determined by staining with Trypan Blue solution. Cells were counted manually using a haemocytometer and an optical microscope. Cells that remained unstained were considered viable, while light red cells were considered non-viable. The viability percentage was calculated as the ratio of the number of all viable cells to the number of all cells.

Cell Viability Determination
The cell viability was determined as was described previously [50]. Briefly, THP1-Blue™ NF-κB cells (500,000 cells/mL) were washed by PBS, resuspended in a serum-free RPMI 1640 medium and seeded into 96-well plates (100 µL/well, i.e., 50,000 cells per each well). After 2 h, tested compounds (30-0.47 µM) dissolved in DMSO were added to the cells. The final concentration of DMSO was 0.1% (v/v) in each well. Measurements were taken after 24 h incubation with the tested substances. Viability was measured by the Cell Proliferation Reagent kit WST-1 (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's manual. The amount of formazan formed, which corresponded to the number of metabolically active cells in the culture, was calculated as a percentage of the control cells, which were treated only with serum-free RPMI 1640 medium and were assigned as 100%. The IC 50 values were calculated by four-parameter logistic (4PL) analysis from obtained viability curves.

Determination of NF-κB Activity
The activity of NF-κB transcription factors was evaluated on THP1-Blue™ NF-κB cells as described previously [50,51]. Briefly, cells were transferred into serum-free RPMI 1640 medium as was delineated above in Section 3.4. All tested compounds were added 2 h later in non-toxic concentration 2 µM but compounds 10, 11, and 13 in concentration 0.5 µM. After 1 h of incubation of the treated cells with the samples, cells were stimulated by lipopolysaccharide (LPS) from E. coli 0111:B4 (Merck) dissolved in serum-free RPMI 1640 medium (1 µg/mL). After 24 h of incubation, 20 µL of cultivation medium was mixed with 200 µL of Quanti-Blue™ medium (Invivogen) and incubated according to the manufacturer's instructions at 37 • C for 30-40 min. The activity of NF-κB was determined as secretion of embryonic alkaline phosphatase spectrophotometrically in a Tecan Infinite M200 (Tecan, Männedorf Switzerland) at 650 nm and compared with an untreated control (100%).

Immunocytochemical Analysis of NF-κB Nuclear Migration
To differentiate THP1-Blue™ NF-κB monocytes into macrophages, the cells were stimulated with phorbol myristate acetate (PMA) at the final concentration of 50 ng/mL, as was described previously [51,52]. Cells were seeded into 8-well cell culture glass slide (SPL, Korea) at the concentration of 500,000 cells/mL, 150,000 cells per well. The compound 17 was added at the final concentration of 2 µM, and 1 h later, the cells were activated by LPS (1 µg/mL). After 1h, the culture slide was washed with PBS and fixed with 4% formaldehyde. The cells were permeabilized by 0.1% Triton X-100 (v/v; Merck) and blocked with 3% bovine serum albumin (BSA, Merck) dissolved in PBS. The primary antibody NF-κB p65 (Abcam, Cambridge, UK; product No. ab16502) at concentration 2.5 µg/mL was used for overnight incubation at 4 • C. After that, secondary anti-rabbit antibody labeled with Alexa Fluor ® 488 (Cell Signaling; product No. 4412; dilution 1:1000) was added for 1 h in dark, and the nuclei were stained using 0,5 µg/mL DAPI (Merck) for 5 min. The wells were washed three-times between the mentioned steps with PBS. The coverslip was mounted with VectaShield mounting (Vector laboratories, Burlingame, CA, USA) medium and sealed with the nail polish. The nail polish was allowed to dry at 4 • C and the translocation of NF-κB was observed by fluorescence microscopy.

Differentiation Monocytes into Macrophages and Evaluation of TNF-α Secretion
Differentiated THP1-Blue™ NF-κB macrophages (see the chapter 3.6) were pre-treated for 1 h with 2 µM solutions of the tested compounds 8, 12, 6, 17, cinnamic acid, and prednisone dissolved in DMSO, or only the vehicle [0.1% (v/v) DMF solution]. The inflammatory-like response was triggered by adding 1 µg/mL of LPS from E. coli 0111:B4 (Sigma-Aldrich) to the pre-treated macrophages; the control cells were left without LPS treatment. After 24 h, the supernatants were collected and the concentration of TNF-α was measured using a Human TNF-α ELISA Kit (Diaclone, Besançon, France), according to the manufacturer's manual.

Statistical Analysis
Statistical analysis was carried out in GraphPad Prism 8.0.1 (San Diego, CA, USA). Outliers were identified by the ROUT statistical method (Q = 5%) and excluded for further analysis. Data were expressed as mean ± SEM. Groups were compared with the help of the one-way ANOVA test followed by Fisher's LSD multiple comparison test. The value p < 0.05 was assigned as statistically significant.

Conclusions
A series of eighteen ring-substituted N-arylcinnamanilides was designed. The molecular structure of (2E)-N-(2-bromo-5-fluorophenyl)-3-phenylprop-2-enamide (15) as a model compound was determined by single-crystal X-ray diffraction. All the compounds were screened for their anti-inflammatory potential at the pharmacologically relevant, non-toxic concentration of 2 µM, only least viable compounds 10 (R = 3,4-Cl), 11 (R = 3,5-Cl), and 13 (3,5-CF 3 ), which IC 50 ranged from 2.17 to 6.28 µM on THP1-Blue™ NF-κB cell line, were tested at the concentration level of 0.5 µM. It seems that cell viability is mainly affected by di-substitution on the C (3,5) or C (3,4) positions by Cl and/or CF 3 , i.e., groups with highly lipophilic and strong electron-withdrawing properties. Thus, although it would be advantageous to obtain compounds with both antimicrobial and anti-inflammatory activity, compounds 10, 11, and 13 were excluded from further study because of this undesirable cytotoxic effect. Most tested compounds significantly attenuated the LPS-induced NF-κB activation. They were more potent than the parental cinnamic acid. (2E)-N-[2-Chloro-5-(trifluoromethyl)phenyl]-3-phenylprop-2-enamide (17), (2E)-N-(2,6-dibromophenyl)-3-phenylprop-2-enamide (12), and (2E)-N-(2,5-dichlorophenyl)-3-phenyl-prop-2-enamide (8) demonstrated the highest activity within the series that was comparable with prednisone. Compounds 12 and 17 also decreased the level of TNF-α, which correlates with the observed attenuation of NF-κB. On the other hand, subsequent tests showed that the investigated compounds did not affect IκBα level and MAPKs activity as expected. Based on above-mentioned fact, it can be hypothesized that the investigated N-arylcinnamanilides may have a different mode of action, which may consist of the nuclear translocation of NF-κB inhibition, or affect its binding to DNA, or act by epigenetic regulation, while the combinations of these effects are not excluded. In conclusion, it can be stated that anti-inflammatory activity (while minimizing antiproliferative activity) is positively influenced by di-substitution on the C (2,5) or C (2,6) positions by rather lipophilic and preferably bulky moieties that causes the anilide ring to rotate, resulting in a non-planar configuration of the entire system, as confirmed by the crystal structure.