​There is an increasing prevalence of multidrug-resistant parasites which contributes to a further rise in infections caused by opportunistic pathogens, particularly those of the genera Trypanosoma and Leishmania, which represent key etiological agents in zoonotic infections. Unfortunately, current therapeutic options for these diseases are very limited and non-specific. Our working group has previously reported numerous novel naphthalene and quinoline derivatives, some of them with anti-parasitic activity. In this study we report new structures with potent activity against both Leishmania mexicana and Trypanosoma brucei; some of them exhibited inhibitory effects comparable to or even greater than the reference compounds and do not show significant cytotoxic effects. The most active molecules in this series fulfill the criteria of Lipinski's Rule of Five and the Veber Rule, making these compounds as potential hit structures and certainly as interesting candidate compounds for starting structures for further developments. Regarding the possible mechanism of action of these compounds at the molecular level, we conducted a comprehensive molecular modeling study by using various combined techniques. This included docking studies, molecular dynamics simulations (analysis per residue), and Quantum Theory of Atoms and Molecules (QTAIM) calculations. Our results allow us to propose two possible molecular targets for Leishmania mexicana: i) Trypanothione reductase and ii) Arginase; whereas for Trypanosoma brucei, our simulations suggest that it is highly probable that these compounds act as inhibitors of its Trypanothione reductase enzyme.

As Schkuhria pinnata is a food supplement commonly available on the European market, the aqueous-methanol extract from the aerial parts of S. pinnata was subjected to chromatographic separation to isolate compounds with antiproliferative and pro-apoptotic properties. This procedure led to the isolation of two previously undescribed sesquiterpene lactones (1 and 2) bearing an unusually modified germacranolide skeleton, four known heliangolides (3-6), together with flavonoids hispidulin and pectolinarigenin. The structures of isolated compounds were elucidated by a comprehensive analysis of HRMS and 1D and 2D NMR data. Sesquiterpene lactones (1-6) were evaluated for their antiproliferative activities in the THP-1, DU-145, and MCF-7 cancer cell lines. Compounds 3-6 showed an antiproliferative effect with the highest intensity in the THP-1 leukemic cell line. The most effective compound 5, 2″-dehydroeucannabinolide semiacetal, induced apoptosis and loss of mitochondrial membrane potential, increased mitochondrial superoxide production, and suppressed mitochondrial ATP production in THP-1 cells.

Background and purpose: The increasing prevalence of multidrug-resistant parasites, particularly Trypanosoma and Leishmania, together with the limited efficacy of current therapies, highlights the need for new antiprotozoal agents. Current therapeutic options remain limited by toxicity and insufficient efficacy. Building on our previous work on naphthalene and quinoline derivatives with antiparasitic activity, we report new derivatives with potent antiprotozoal activity against both Leishmania mexicana and Trypanosoma brucei. Experimental approach: The synthesized compounds were evaluated for their antiprotozoal activity against Leishmania mexicana and Trypanosoma brucei, cytotoxicity, and metabolic stability in rat and human liver microsomes. To investigate their possible mechanism of action, a comprehensive molecular modelling study was performed, including molecular docking, molecular dynamics simulations (per-residue analysis), and quantum theory of atoms in molecules (QTAIM) calculations. Key results: Some synthesized compounds exhibited inhibitory effects comparable to or greater than those of the reference compounds and showed no significant cytotoxicity. The most active molecules demonstrated high metabolic stability in both rat and human liver microsomes. Molecular modelling provided mechanistic insight into the observed antiparasitic activity and identified trypanothione reductase and arginase as possible molecular targets in Leishmania mexicana. For Trypanosoma brucei, our simulations suggest that trypanothione reductase is the most probable molecular target and that these compounds inhibit this enzyme. Conclusion: The synthesized hydroxynaphthalene and quinoline derivatives represent promising antiprotozoal compounds with favourable metabolic stability and low cytotoxicity. Molecular modelling provided a possible explanation for their biological activity and suggested potential molecular targets that may support their further optimization.

A new group of potent histone deacetylase inhibitors (HDACis) capable of inhibiting cell growth and affecting cell-cycle progression in Tohoku Hospital Pediatrics-1 (THP-1) monocytic leukaemia cells was synthesized. The inhibitors belong to a series of hydroxamic acid derivatives. We designed and synthesized a series of 22 N-hydroxycinnamamide derivatives, out of which 20 are new compounds. These compounds contain various substituted anilides as the surface recognition moiety (SRM), a p-hydroxycinnamate linker, and hydroxamic acids as the zinc-binding group (ZBG). The whole series of synthesized hydroxamic acids inhibited THP-1 cell proliferation. Compounds 7d and 7p, which belong to the category of derivatives with the most potent antiproliferative properties, exert a similar effect on cell-cycle progression as vorinostat and induce apoptosis in THP-1 cells. Furthermore, compounds 7d and 7p were demonstrated to inhibit HDAC class I and II in THP-1 cells with comparable potency to vorinostat and increase acetylation of histones H2a, H2b, H3, and H4. Molecular modelling was used to predict the probable binding mode of the studied HDACis in class I and II histone deacetylases in terms of Zn2+ ion chelation by the hydroxamate group.

Background and purpose: New compounds and innovative therapeutic approaches are trying to prevent antimicrobial resistance, which has become a global health challenge. Experimental approach: This study includes a series of twelve mono-, di- and trichlorinated 1-hydroxynaphthalene-2-carboxanilides designed as multitarget agents. All compounds were evaluated for their antistaphylococcal activity. Furthermore, MTT assay and chemoproteomic analysis of selected compounds were performed. Cytotoxicity in human cells was also tested. Key results: N-(3,5-Dichlorophenyl)-1-hydroxynaphthalene-2-carboxamide (10) demon-strated activity comparable to or higher than clinically used drugs, with minimum inhibitory concentrations (MICs) of 0.37 μM. The compound was equally effective against clinical isolates of methicillin-resistant S. aureus. On the other hand, compound 10 showed 96 % inhibition of S. aureus respiration only at a concentration of 16× MIC. Chemoproteomic analysis revealed that the effect of agent 10 on staphylococci resulted in the downregulation of four proteins. This compound expressed no in vitro cytotoxicity up to a concentration of 30 μM. Conclusion: From the set of tested mono-, di- and trisubstituted derivatives, it is evident that the position of chlorine atoms is decisive for significant antistaphylococcal activity. Inhibition of energy metabolism does not appear to be one of the main mechanisms of action of compound 10; on the contrary, the antibacterial effect may likely be contributed by downregulation of proteins (especially ATP-dependent protease ATPase subunit HslU) involved in processes essential for bacterial survival and growth, such as protein, nucleotide/nucleic acid synthesis and efficient protein repair/degradation.

Background and purpose: Many new compounds are being prepared to overcome the problem of increasing microbial resistance and the increasing number of infections. Experimental approach: This study includes a series of twenty-seven mono-, di- and trisubstituted 2-hydroxynaphthalene-1-carboxanilides designed as multitarget agents. The compounds are substituted with methoxy, methyl, and nitro groups, as well as additionally with chlorine, bromine, and trifluoromethyl at various positions. All the compounds were evaluated for antibacterial activities against Gram-positive and Gram-negative bacteria and mycobacteria. Cytotoxicity on human cells was also tested. Key results: Three compounds showed activity comparable to clinically used drugs. N-(3,5-Dimethylphenyl)-2-hydroxynaphthalene-1-carboxamide (13) showed only anti¬sta¬phylococcal activity (minimum inhibitory concentration (MIC) = 54.9 µM); 2-hydroxy-N-[2-methyl-5-(tri¬fluoro¬methyl)phenyl]naphthalene-1-carboxamide (22) and 2-hydroxy-N-[4-nitro-3-(trifluoromethyl)phe¬nyl]na¬phtha¬lene-1-carboxamide (27) were active across the entire spectrum of tested bacteria/mycobacteria, both against the sensitive set and against resistant isolates (MICs range 0.3 to 92.6 µM). Compound 22 was even active against E. coli (MIC = 23.2 µM). The active agents showed no in vitro cytotoxicity up to a concentration of 30 μM. Conclusion: Compounds with trifluoromethyl in the meta-anilide position, experimental lipophilicity expressed as log k (logarithm of the capacity factor) in the range of 0.31 to 0.34 and calculated electron σ parameter for the anilide substituent higher than 0.59 were effective. The investigated compounds meet the definition of Michael acceptors. Based on ADME screening, the investigated compounds 13, 22 and 27 should have suitable physico¬chemical parameters for good bioavailability in the organism. Therefore, these are promising agents for further study.

Salicylanilides are pharmacologically active compounds with a wide spectrum of biological effects. Halogenated salicylanilides, which have been used for decades in human and veterinary medicine as anthelmintics, have recently emerged as candidates for drug repurposing in oncology. The most prominent example of salicylanilide anthelmintic, that is intensively studied for its potential anticancer properties, is niclosamide. Nevertheless, recent studies have discovered extensive anticancer potential in a number of other salicylanilides. This potential of their anticancer action is mediated most likely by diverse mechanisms of action such as uncoupling of oxidative phosphorylation, inhibition of protein tyrosine kinase epidermal growth factor receptor, modulation of different signaling pathways as Wnt/β-catenin, mTORC1, STAT3, NF-κB and Notch signaling pathways or induction of B-Raf V600E inhibition. Here we provide a comprehensive overview of the current knowledge about the proposed mechanisms of action of anticancer activity of salicylanilides based on preclinical in vitro and in vivo studies, or structural requirements for such an activity.

Pattern 1-hydroxy-N-(2,4,5-trichlorophenyl)-2-naphthamide and the thirteen original carbamates derived from it were prepared and characterized. All the compounds were tested against Staphylococcus aureus ATCC 29213 as a reference and quality control strain and in addition against three clinical isolates of methicillin-resistant S. aureus (MRSA). Moreover, the compounds were evaluated against Enterococcus faecalis ATCC 29212, and preliminary in vitro cytotoxicity of the compounds was assessed using the human monocytic leukemia cell line (THP-1). The lipophilicity of the prepared compounds was experimentally determined and correlated with biological activity. While pattern anilide had no antibacterial activity, the prepared carbamates demonstrated high antistaphylococcal activity comparable to the used standards (ampicillin and ciprofloxacin), which unfortunately were ineffective against E. feacalis. 2-[(2,4,5-Trichlorophenyl)carba- moyl]naphthalen-1-yl ethylcarbamate (2) and 2-[(2,4,5-trichlorophenyl)carbamoyl]naphthalen-1-yl butylcarbamate (4) expressed the nanomolar minimum inhibitory concentrations (MICs 0.018–0.064 μM) against S. aureus and at least two other MRSA isolates. Microbicidal effects based on the minimum bactericidal concentrations (MBCs) against all the tested staphylococci were found for nine carbamates, while 2-[(2,4,5-trichlorophenyl)carbamoyl]naphthalen-1-yl heptylcarbamate (7) and 2-[(2,4,5-trichlorophenyl)carbamoyl]naphthalen-1-yl (4-phenylbutyl)carbamate (14) demonstrated MBCs in the range of 0.124–0.461 μM. The selectivity index (SI) for most investigated carbamates was >20 and for some derivatives even >100. The performed tests did not show an effect on the damage to the bacterial membrane, while the compounds were able to inhibit the respiratory chain of S. aureus.

Aminopeptidase N (APN), also known as CD13 antigen or membrane alanyl aminopeptidase, belongs to the M1 family of the MA clan of zinc metallopeptidases. In cancer cells, the inhibition of aminopeptidases including APN causes the phenomenon termed the amino acid deprivation response (AADR), a stress response characterized by the upregulation of amino acid transporters and synthetic enzymes and activation of stress-related pathways such as nuclear factor kB (NFkB) and other pro-apoptotic regulators, which leads to cancer cell death by apoptosis. Recently, APN inhibition has been shown to augment DR4-induced tumor cell death and thus overcome resistance to cancer treatment with DR4-ligand TRAIL, which is available as a recombinant soluble form dulanermin. This implies that APN inhibitors could serve as potential weapons for overcoming cancer treatment resistance. In this study, a series of basically substituted acetamidophenones and the semicarbazones and thiosemicarbazones derived from them were prepared, for which APN inhibitory activity was determined. In addition, a selective anti-proliferative activity against cancer cells expressing APN was demonstrated. Our semicarbazones and thiosemicarbazones are the first compounds of these structural types of Schiff bases that were reported to inhibit not only a zinc-dependent aminopeptidase of the M1 family but also a metalloenzyme.

The superimposition of the X-ray complexes of cyclohexanediones (i.e., TUB015), described by our research group, and nocodazole, within the colchicine binding site of tubulin provided an almost perfect overlap of both ligands. This structural information led us to propose hybrids of TUB015 and nocodazole using a salicylanilide core structure. Interestingly, salicylanilides, such as niclosamide, are well-established signal transducers and activators of transcription (STAT3) inhibitors with anticancer properties. Thus, different compounds with this new scaffold have been synthesized with the aim to identify compounds inhibiting tubulin polymerization and/or STAT3 signaling. As a result, we have identified new salicylanilides (6 and 16) that showed significant antiproliferative activity against a panel of cancer cells. Both compounds were able to reduce the levels of p-STAT3Tyr705 without affecting the total expression of STAT3. While compound 6 inhibited tubulin polymerization and arrested the cell cycle of DU145 cells at G2/M, similar to TUB015, compound 16 showed a more potent effect on inhibiting STAT3 phosphorylation and arrested the cell cycle at G1/G0, similar to niclosamide. In both cases, no toxicity towards PBMC cells was detected. Thus, the salicylanilides described here represent a new class of antiproliferative agents affecting tubulin polymerization and/or STAT3 phosphorylation.

Sulfate-reducing bacteria (SRB) are a heterogeneous group of anaerobic microorganisms that play an important role in producing hydrogen sulfide not only in the natural environment, but also in the gastrointestinal tract and oral cavity of animals and humans. The present review was written with the inclusion of 110 references including the time period from 1951 to 2021. The following databases were evaluated: Web of Science, Scopus and Google Scholar. The articles chosen to be included in the review were written mainly in the English and Czech languages. The molecular mechanisms of microbial cryoprotection differ depending on the environment where microorganisms were initially isolated. It was observed that the viability of microorganisms after cryopreservation is dependent on a number of factors, primarily colony age, amount of inoculum, cell size or rate of cooling, and their molecular inventory. Therefore, this paper is devoted to assessing the performance and suitability of various cryopreservation methods of intestinal bacteria, including molecular mechanisms of their protection. In order to successfully complete the cryopreservation process, selecting the correct laboratory equipment and cryopreservation methodology is important. Our analysis revealed that SRB should be stored in glass vials to help mitigate the corrosive nature of hydrogen sulfide, which can affect their physiology on a molecular level. Furthermore, it is recommended that their storage be performed in distilled water or in a suspension with a low salt concentration. From a molecular biological and bioengineering perspective, this contribution emphasizes the need to consider the potential impact associated with SRB in the medical, construction, and environmental sectors.

Prenylated or geranylated flavonoids have been studied for their promising antiproliferative and cytotoxic activities. Twelve natural geranylated flavonoids (1-12) were isolated from the fruit of Paulownia tomentosa Steud. Their structures were elucidated using UV and IR spectroscopy, mass spectrometry, and 1D and 2D NMR spectroscopy. The absolute configurations were determined using NMR and circular dichroism. Seven of the compounds were characterized as new geranylated derivatives isolated from a natural source for the first time, namely 3 '-O-methyl-5 '-hydroxyisodiplacone (3), paulodiplacone A (5), tomentone II (6), tomentone B (7), tomentodiplacone P (8), paulodiplacone B (9), and tomentoflavone A (12). After 24 h of incubation at concentrations in the range 1-30 mu M, the isolated compounds were tested for their antiproliferative and cytotoxic potentials against the human monocytic leukaemia cell line THP-1, using WST-1 and LDH assays, respectively. Almost all of the test compounds induced a concentration-dependent reduction in the metabolic activity of THP-1 cells and a concentration-dependent reduction in the cell viability. Diplacone (1) was the most potent antiproliferative and cytotoxic agent (IC50 9.31 +/- 0.72 mu M, LC50 18.01 +/- 1.19 mu M). 3 '-O-Methyl-5 '-hydroxydiplacone (2) showed relatively strong antiproliferative effect (IC50 12.61 +/- 0.90 mu M) and weaker cytotoxic activity (LC50 > 30 mu M), indicating that it may serve as a potential lead compound for further testing. The structure-activity relationship for the 12 isolated compounds is discussed.

1-[2-[({[2-/3-(Alkoxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]pyrrolidinium/azepan- ium oxalates or dichlorides (alkoxy = butoxy to heptyloxy) were recently described as very promising antimycobacterial agents. These compounds were tested in vitro against Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 (reference and control strains), three methicillin-resistant isolates of S. aureus, and three isolates of vancomycin-resistant E. faecalis. 1-[3-(Dipropylammonio)-2-({[3-(pentyloxy-/hexyloxy-/heptyloxy)phenyl]carbamoyl}oxy)propyl]pyrrolidinium dichlorides showed high activity against staphylococci and enterococci comparable with or higher than that of used controls (clinically used antibiotics and antiseptics). The screening of the cytotoxicity of the compounds as well as the used controls was performed using human monocytic leukemia cells. IC50 values of the most effective compounds ranged from ca. 3.5 to 6.3 µM, thus, it can be stated that the antimicrobial effect is closely connected with their cytotoxicity. The antibacterial activity is based on the surface activity of the compounds that are influenced by the length of their alkoxy side chain, the size of the azacyclic system, and hydro-lipophilic properties, as proven by in vitro experiments and chemometric principal component analyses. Synergistic studies showed the increased activity of oxacillin, gentamicin, and vancomycin, which could be explained by the direct activity of the compounds against the bacterial cell wall. All these compounds demonstrate excellent antibiofilm activity, when they inhibit and disrupt the biofilm of S. aureus in concentrations close to minimum inhibitory concentrations against planktonic cells. Expected interactions of the compounds with the cytoplasmic membrane are proven by in vitro crystal violet uptake assays.

Sulfate is present in foods, beverages, and drinking water. Its reduction and concentration in the gut depend on the intestinal microbiome activity, especially sulfate-reducing bacteria (SRB), which can be involved in inflammatory bowel disease (IBD). Assimilatory sulfate reduction (ASR) is present in all living organisms. In this process, sulfate is reduced to hydrogen sulfide and then included in cysteine and methionine biosynthesis. In contrast to assimilatory sulfate reduction, the dissimilatory process is typical for SRB. A terminal product of this metabolism pathway is hydrogen sulfide, which can be involved in gut inflammation and also causes problems in industries (due to corrosion effects). The aim of the review was to compare assimilatory and dissimilatory sulfate reduction (DSR). These processes occur in some species of intestinal bacteria (e.g., Escherichia and Desulfovibrio genera). The main attention was focused on the description of genes and their location in selected strains. Their coding expression of the enzymes is associated with anabolic processes in various intestinal bacteria. These analyzed recent advances can be important factors for proposing possibilities of metabolic pathway extension from hydrogen sulfide to cysteine in intestinal SRB. The switch from the DSR metabolic pathway to the ASR metabolic pathway is important since toxic sulfide is not produced as a final product.

Ring-substituted 1-hydroxynaphthalene-2-carboxanilides were previously investigated for their antimycobacterial properties. In our study, we have shown their antiproliferative and cell death-inducing effects in cancer cell lines. Cell proliferation and viability were assessed by WST-1 assay and a dye exclusion test, respectively. Cell cycle distribution, phosphatidylserine externalization, levels of reactive oxygen or nitrogen species (RONS), mitochondrial membrane depolarization, and release of cytochrome c were estimated by flow cytometry. Levels of regulatory proteins were determined by Western blotting. Our data suggest that the ability to inhibit the proliferation of THP-1 or MCF-7 cells might be referred to meta- or para-substituted derivatives with electron-withdrawing groups -F, -Br, or -CF3 at anilide moiety. This effect was accompanied by accumulation of cells in G1 phase. Compound 10 also induced apoptosis in THP-1 cells in association with a loss of mitochondrial membrane potential and production of mitochondrial superoxide. Our study provides a new insight into the action of salicylanilide derivatives, hydroxynaphthalene carboxamides, in cancer cells. Thus, their structure merits further investigation as a model moiety of new small-molecule compounds with potential anticancer properties.

Intestinal sulfate-reducing bacteria are often isolated from patients with inflammatory bowel disease, including ulcerative colitis, and can be involved in the development of gut inflammation. A comparison of the metabolism of intestinal sulfate-reducing bacteria isolated from individuals with colitis and healthy controls using statistical analysis has never been studied and described before. The aim of our research was to evaluate the parameters of dissimilatory sulfate reduction in Desulfovibrio species that were isolated from the feces of healthy objects and individuals with colitis. Principal component analysis indicates that the strains that were isolated from individuals with colitis grouped in the same cluster by biomass accumulation and sulfide production, same as the strains isolated from healthy individuals. Sulfate and lactate consumption measured over time showed negative correlation (Pearson correlations, p<0.01), healthy: -0.760; colitis: -0.770; healthy: -0.828; colitis: -0.847, respectively. The calculated linear regression (R-2) was lower in biomass accumulation and hydrogen sulfide production, 0.531; 0.625 respectively. Thus, biomass accumulation and sulfide production, together with measured kinetic parameters play an important factor in bowel inflammation, including ulcerative colitis. Additionally, acetate production can also synergize with H2S, while sulfate consumption and lactate oxidation likely represent minor factors in bowel disease.

A series of twenty-six methoxylated and methylated N-aryl-1-hydroxynaphthalene- 2-carboxanilides was prepared and characterized as potential anti-invasive agents. The molecular structure of N-(2,5-dimethylphenyl)-1-hydroxynaphthalene-2-carboxamide as a model compound was determined by single-crystal X-ray diffraction. All the analysed compounds were tested against the reference strain Staphylococcus aureus and three clinical isolates of methicillin-resistant S. aureus as well as against Mycobacterium tuberculosis and M. kansasii. In addition, the inhibitory profile of photosynthetic electron transport in spinach (Spinacia oleracea L.) chloroplasts was specified. In vitro cytotoxicity of the most effective compounds was tested on the human monocytic leukaemia THP-1 cell line. The activities of N-(3,5-dimethylphenyl)-, N-(3-fluoro-5-methoxy-phenyl)- and N-(3,5-dimethoxyphenyl)-1-hydroxynaphthalene-2-carbox- amide were comparable with or even better than the commonly used standards ampicillin and isoniazid. All promising compounds did not show any cytotoxic effect at the concentration >30 µM. Moreover, an in silico evaluation of clogP features was performed for the entire set of the carboxamides using a range of software lipophilicity predictors, and cross-comparison with the experimentally determined lipophilicity (log k), in consensus lipophilicity estimation, was conducted as well. Principal component analysis was employed to illustrate noticeable variations with respect to the molecular lipophilicity (theoretical/experimental) and rule-of-five violations. Additionally, ligand-oriented studies for the assessment of the three-dimensional quantitative structure–activity relationship profile were carried out with the comparative molecular surface analysis to determine electron and/or steric factors that potentially contribute to the biological activities of the investigated compounds.

The small-large intestine axis in hydrogen sulfide accumulation and testing of sulfate and lactate in the gut-gut axis of the intestinal environment has not been well described. Sulfate reducing bacteria (SRB) of the Desulfovibrio genus reduce sulfate to hydrogen sulfide and can be involved in ulcerative colitis development. The background of the research was to find correlations between hydrogen sulfide production under the effect of an electron acceptor (sulfate) and donor (lactate) at different concentrations and Desulfovibrio piger Vib-7 growth, as well as their dissimilatory sulfate reduction in the intestinal small-large intestinal environment. Methods: Microbiological, biochemical, and biophysical methods, and statistical processing of the results (principal component and cross-correlation analyses) were used. Results: D. piger Vib-7 showed increased intensity of bacterial growth and hydrogen sulfide production under the following concentrations of sulfate and lactate: 17.4 mM and 35.6 mM, respectively. The study showed in what kind of intestinal environment D. piger Vib-7 grows at the highest level and produces the highest amount of hydrogen sulfide. Conclusions: The optimum intestinal environment of D. piger Vib-7 can serve as a good indicator of the occurrence of inflammatory bowel diseases; meaning that these findings can be broadly used in medicine practice dealing with the monitoring and diagnosis of intestinal ailments.

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.