Synthesis and Structural Study of Amidrazone Derived Pyrrole-2,5-Dione Derivatives: Potential Anti-Inflammatory Agents

1H-pyrrole-2,5-dione derivatives are known for their wide range of pharmacological properties, including anti-inflammatory and antimicrobial activities. This study aimed to synthesize new 3,4-dimethyl-1H-pyrrole-2,5-dione derivatives 2a–2f in the reaction of N3-substituted amidrazones with 2,3-dimethylmaleic anhydride and evaluate their structural and biological properties. Compounds 2a–2f were studied by the 1H-13C NMR two-dimensional techniques (HMQC, HMBC) and single-crystal X-ray diffraction (derivatives 2a and 2d). The anti-inflammatory activity of compounds 2a–2f was examined by both an anti-proliferative study and a production study on the inhibition of pro-inflammatory cytokines (IL-6 and TNF-α) in anti-CD3 antibody- or lipopolysaccharide-stimulated human peripheral blood mononuclear cell (PBMC) cultures. The antibacterial activity of compounds 2a–2f against Staphylococcus aureus, Enterococcus faecalis, Micrococcus luteus, Esherichia coli, Pseudomonas aeruginosa, Yersinia enterocolitica, Mycobacterium smegmatis and Nocardia corralina strains was determined using the broth microdilution method. Structural studies of 2a–2f revealed the presence of distinct Z and E stereoisomers in the solid state and the solution. All compounds significantly inhibited the proliferation of PBMCs in anti-CD3-stimulated cultures. The strongest effect was observed for derivatives 2a–2d. The strongest inhibition of pro-inflammatory cytokine production was observed for the most promising anti-inflammatory compound 2a.

The aim of this study was the synthesis of six new 3,4-dimethyl-1H-pyrrole-2,5-diones, N(1)-substituted by the imino moieties derived from N 3 -substituted amidrazones (1a-1f, Figure 1) [41] which have the general formula shown in Figure 2 (2a-2f). Thereafter, the main goal was the investigation of their structural and spectroscopic ( 1 H, 13 C NMR) properties in their solid state and solution, together with the evaluation of their biological activity. This was done to gain insight into the influence of the R 1 and R 2 substituents on the molecular conformation and intermolecular interactions and the anti-inflammatory and antibacterial properties of the compounds.

The Syntheses of Compounds 2a-2f
In this work, a series of N(1)-substituted derivatives of 3,4-dimethyl-1H-pyrrole-2,5dione 2a-2f were prepared from the respective N 3 -substituted amidrazones 1a-1f and 2,3-dimethylmaleic anhydride 3 (Scheme 1). The syntheses of 2a-2c and 2e-2f were carried out in toluene, chloroform or diethyl ether. The best yields (75-95%) were obtained at the boiling points of chloroform or toluene in a much shorter time than at room temperature. The exception was compound 2d, which was obtained only in diethyl ether at room temperature. Possibly the presence of two 2-pyridyl substituents hinders the formation of this product. The detailed dependencies of 2a-2f yields with the solvent, temperature and time are presented in Tables S2-S7 (Supplementary Data, part B).
The structures of 2a-2f were confirmed by elemental analyses, mass spectra and 1 H, 13 C NMR spectra with the application of two-dimensional HMQC and HMBC techniques (Supplementary Data, parts C-D, including Figures S25-S48). The 1 H-13 C NMR correlation spectroscopy allowed us to assign all 1 H and 13 C signals for each 2a-2f molecule exhibiting the presence of two isomeric forms (denoted generally as A and B; these symbols correspond to the species with the higher and the lower chemical shift of the most deshielded H(8) proton, i.e., NH) in DMSO-d 6 solutions. The assigned 1 H and 13 C NMR chemical shifts for A and B isomers of 2a-2f, compared to those for the parent amidra- A detailed description of our attempt to identify and attribute the A and B forms of 2a-2f, based on the comparative analysis of their 1 H and 13 C NMR spectra in solution and partly on the single-crystal X-ray data for 2a (R 1 = R 2 = phenyl) and 2d (R 1 = R 2 = 2-pyridyl), is presented, in the form of comments below Tables S8 and S9. The resulting general conclusion is that A and B are most likely geometric isomers differing in the position of R 1 and N(8)H-R 2 substituents at the C(7) carbon. The lack of rotation around the N(6)=C (7) double bond probably results in cis-/transisomerism: in one stereomer, R 1 is trans to N(1), and N(8)H-R 2 is cis to N(1), whereas in the other one R 1 is cis to N(1) and N(8)H-R 2 is trans to N(1). Taking into account the spatial orientation of the N(1) and N(8) atoms, these are Z and E stereomers ( Figure 3). The hypothesis of Z/E isomerism is supported by the fact that, in the solid phase of 2a or 2d, where only one stereomer is observed, the crystal structures correspond to such distinct isomeric species: 2a to Z and 2d to E. The hypothesis of Z/E isomerism is supported by the fact that, in the solid phase of 2a or 2d, where only one stereomer is observed, the crystal structures correspond to such distinct isomeric species: 2a to Z and 2d to E.

X-ray Crystallography
The molecular plots of 2a and 2d with the atom labelling schemes (modification of the general numbering presented in Figure 2) are shown in Figure 4. The selected geometric parameters of 2a and 2d are listed in Table S11 (Supplementary Data, part F) together with those for the previously reported, closely related derivative of hexahydro-2H-isoindole-1,3dione (CSD refcode: LUZGUJ) [43,50], corresponding to the already mentioned analogue of 2a. The hypothesis of Z/E isomerism is supported by the fact that, in the solid phase of 2a or 2d, where only one stereomer is observed, the crystal structures correspond to such distinct isomeric species: 2a to Z and 2d to E.

X-ray Crystallography
The molecular plots of 2a and 2d with the atom labelling schemes (modification of the general numbering presented in Figure 2) are shown in Figure 4. The selected geometric parameters of 2a and 2d are listed in Table S11 (Suppl. Data, part F) together with those for the previously reported, closely related derivative of hexahydro-2H-isoindole-1,3-dione (CSD refcode: LUZGUJ) [43,50], corresponding to the already mentioned analogue of 2a.
2a 2d The yellow (2a) and orange (2d) prismatic crystals, suitable for diffraction studies, were grown by recrystallization of the originally synthesized compounds from pure ethanol (99.8%) using the standard solvent evaporation technique.
The single-crystal X-ray diffraction analysis revealed that both 2a and 2d crystallize in the same centrosymmetric space group P21/c with one molecule in the asymmetric part of the unit cell.
Both 2a and 2d have the same -N(6)=C(7)-N(8)H-bond system, as exhibited by the bond lengths proving the presence of the N(6)=C(7) double bond and the C(7)-N(8) single bond (Table S11, Suppl. Data, part F). This conclusion is consistent with the 1 H-13 C NMR correlation analysis results for all 2a-2f compounds in the DMSO-d6 solutions (paragraph 2.1). However, these two molecules adopt different configurations in the solid state: Z for 2a and E for 2d (Figure 3), as confirmed by the corresponding N(1)−N(6)=C(7)−N(8) torsion angles of −13.0(2) and 171.5(1). The latter value is very close to that found in LUZGUJ (173.1(3) o ), which adopted the E geometry in its solid state [43]. The yellow (2a) and orange (2d) prismatic crystals, suitable for diffraction studies, were grown by recrystallization of the originally synthesized compounds from pure ethanol (99.8%) using the standard solvent evaporation technique.
The single-crystal X-ray diffraction analysis revealed that both 2a and 2d crystallize in the same centrosymmetric space group P2 1 /c with one molecule in the asymmetric part of the unit cell.
Both 2a and 2d have the same -N(6)=C(7)-N(8)H-bond system, as exhibited by the bond lengths proving the presence of the N(6)=C(7) double bond and the C(7)-N(8) single bond (Table S11, Supplementary Data, part F). This conclusion is consistent with the 1 H-13 C NMR correlation analysis results for all 2a-2f compounds in the DMSO-d 6 solutions (paragraph 2.1). However, these two molecules adopt different configurations in the solid state: Z for 2a and E for 2d (Figure 3), as confirmed by the corresponding N(1)−N(6)=C(7)−N(8) torsion angles of −13.0(2) • and 171.5(1) • . The latter value is very close to that found in LUZGUJ (173.1(3) o ), which adopted the E geometry in its solid state [43].
The bond lengths in 2a and 2d are comparable, being in good agreement with those in LUZGUJ (Table S11, Supplementary Data, part F). This similarity is mainly observed within the N(1)-N(6)=C(7)-N(8) chain, as exemplified by the clear distinction between the N(1)-N(6) and C(7)-N(8) single bonds versus the N(6)=C(7) double bond. However, the C(7)−C(11) single bond is shorter in 2a and LUZGUJ than in 2d, whereas the N(8)-C(21)bond is longer in 2a than in 2d and LUZGUJ (Table S11, Supplementary Data, part F). On the other hand, in 2a and 2d, one can observe the elongation of the N(1)−N(6) and N(6)=C(7) bonds and the shortening of the C(7)−N(8) bond in comparison to those in the eight previously reported N 1 -acylamidrazones derived from 1d (PAZDIF [48] and RIBVEG, RICGUI, RICHAP, RICHET, RICHIX, RICHOD, and RICHUJ [49] (Table S12, Supplementary Data, part G). These phenomena are well-exemplified when compared to 2a vs. N 1 -acylamidrazones (as all have the same Z geometry, see Table S12) or 2d vs.  [48,49], reflecting the above relationships as predominant. Moreover, in both 2a and 2d, the N(6)=C (7) bonds are longer, and the C(7)−N(8) bonds are shorter than the respective standard Nsp 2 =Csp 2 (1.28 Å) and Csp 2 −NH(−C ar ) (1.38 Å) bonds [51]. This suggests an extended π-electron delocalization in 2a and 2d molecules and can explain the propensity of all 2a-2f compounds to exist in the solutions as various geometric (Z/E) isomers. The bond angles within the N(1)-N(6)=C(7)-N(8) chain in 2a, 2d and LUZGUJ are largely variable (Table S11, Supplementary Data, part F). From these data, it can be seen that there is a greater similarity between 2d and LUZGUJ (having the same E geometry) than between 2a and LUZGUJ (having the same R 1 = R 2 = phenyl substituents). Thus, the spatial arrangement of substituents seems to depend mainly on the molecule configuration. On the other hand, an important role is also played by the type of a substituent at N(1), as the differences between the N(1)-N(6)-C(7) and N(6)-C(7)-N(8) bond angles in 2a or 2d and the corresponding ones in already mentioned N 1 -acylamidrazones derived from 1d (Table S12, [15,24,35,37,39,40] (Table S13 Supplementary Data, part F).
The formal sp 2 hybridization of N(1) in 2a results in near co-planarity of the N(6) atom with the 3,4-dimethyl-1H-pyrrole-2,5-dione ring, as revealed by only a slight N(6) displacement from the N(1)>>C(5) best plane, being 0.059 Å. In contrast, the same parameter in 2d is much greater, being as much as 0.380 Å due to the partial sp 3 N(1) hybridization. This difference between N(1) atoms in both compounds is also reflected by the sum of bond angles around this atom, which in 2a is 359. Similarly, in 2a, the R 1 and R 2 substituents are noticeably twisted with respect to the N(6)−C(7)−N(8) moiety, as shown by the dihedral angles between the C(11) >> C(16) best plane or the C(21) >> C(26) best plane and the N(6)−C(7)−N(8) plane, being 29.5 • and 61.4 • , respectively. In 2d, the R 1 substituent is even more twisted, but the R 2 one is much less twisted, as revealed by the relevant dihedral angles of 74.  Table S14, Supplementary Data, part F), resulting in the S(6) ring motif [52].
Finally, in both 2a and 2d, the phenyl or 2-pyridyl substituents are almost perpendicular to each other, as shown by the dihedral angle between the C(11) << C(16) and the C(21) << C(26) best planes, which are 88.9 • and 81.3 • , respectively.
The studied molecules are proton-deficient, as each possesses one HB donor (N(8)−H(8)) and three or five potential HB acceptors (O(1), O(2) and N(6), as well as N (12) and  N(22), optionally). The presence of numerous acceptor atoms, aromatic rings and 'active' methyl groups stimulates the formation of weak hydrogen bonds. Among the intermolecular interactions involved in the stabilization of 2a and 2d crystals, a number of weak C−H···O/N/π hydrogen bonds (their full list, including geometric parameters and the symmetry codes, together with the selected C−H···C short contacts, is presented in Table S14 (Supplementary Data, part F)) and dipolar C=O···C contacts play an important role.
Generally, it must be noted that some substantial differences in molecular packing occur between 2a and 2d (Figures 5 and 6).
Finally, in both 2a and 2d, the phenyl or 2-pyridyl substituents are almost perpendicular to each other, as shown by the dihedral angle between the C(11) << C(16) and the C(21) << C(26) best planes, which are 88.9° and 81.3, respectively.
The studied molecules are proton-deficient, as each possesses one HB donor (N(8)−H(8)) and three or five potential HB acceptors (O(1), O(2) and N(6), as well as N (12) and N(22), optionally). The presence of numerous acceptor atoms, aromatic rings and 'active' methyl groups stimulates the formation of weak hydrogen bonds. Among the intermolecular interactions involved in the stabilization of 2a and 2d crystals, a number of weak C−HO/N/π hydrogen bonds (their full list, including geometric parameters and the symmetry codes, together with the selected C−HC short contacts, is presented in Table S14 (Suppl. Data, part F)) and dipolar C=OC contacts play an important role.
Generally, it must be noted that some substantial differences in molecular packing occur between 2a and 2d (Figures 5 and 6).  Finally, in both 2a and 2d, the phenyl or 2-pyridyl substituents are almost perpendicular to each other, as shown by the dihedral angle between the C(11) << C(16) and the C(21) << C(26) best planes, which are 88.9° and 81.3°, respectively.
The studied molecules are proton-deficient, as each possesses one HB donor (N(8)−H(8)) and three or five potential HB acceptors (O(1), O(2) and N(6), as well as N (12) and N (22), optionally). The presence of numerous acceptor atoms, aromatic rings and 'active' methyl groups stimulates the formation of weak hydrogen bonds. Among the intermolecular interactions involved in the stabilization of 2a and 2d crystals, a number of weak C−H⋅⋅⋅O/N/π hydrogen bonds (their full list, including geometric parameters and the symmetry codes, together with the selected C−H⋅⋅⋅C short contacts, is presented in Table S14 (Suppl. Data, part F)) and dipolar C=O⋅⋅⋅C contacts play an important role.
The main forces promoting the self-assembly of molecules in the crystal lattice of 2d seem to result from hydrogen bonding involving amine and pyridine functions (  (Figure 6b), π-stacking contacts between the overlapping C(21) << C(26) pyridyl rings, and electrostatic C=O···π interactions involving the 1H-pyrrole-2,5-dione system.

Toxic Activity of 2a-2f
The effect of different concentrations of 2a-2f or ibuprofen (as a reference drug) on the viability of PBMCs in 24 h cell culture was studied. Compounds 2a-2f and ibuprofen induced no apoptosis or necrosis of the analyzed cells at low (10 µg/mL) or medium (50 µg/mL) concentrations (data not shown). However, in the highest dose (100 µg/mL), 2a and 2f appeared to be slightly toxic (79% and 64% of viable cells, respectively), as shown in Figure S49 (Supplementary Data, part G).

Anti-Inflammatory Activity of 2a-2f 2.4.1. Antiproliferative Activity of 2a-2f
The effect of different concentrations of 2a-2f or ibuprofen on soluble anti-CD3 antibody-induced PBMC proliferation in 72 h cell culture is shown in Figure 7. Generally, all compounds 2a-2f inhibited this process (except for 2c in the lowest 10 µg/mL dose). Derivative 2d significantly suppressed PBMC proliferation in each dose (39-77% of inhibition compared to 18-39% for ibuprofen). Significant differences were obtained for compounds 2a-2c in the selected concentrations, while derivatives 2e and 2f inhibited PBMC proliferation only in the medium dose. The strongest inhibitory effect was observed for 2c in the highest 100 µg/mL concentration (85% inhibition).

The Effects of Compounds 2a-2f on Pro-Inflammatory and Anti-Inflammatory Cy tokine Production
The effect of different concentrations of 2a-2f or ibuprofen (as a reference compoun on the LPS-induced production of pro-inflammatory (IL-6 and TNF-α) and anti-inflam matory (IL-10) cytokines in 24 h PBMC culture is presented in Figures 8-10. LPS is a endotoxin of Gram-negative bacteria, used extensively for inducing an immune respon in vitro. It promotes cytokine production in PBMC cultures, including pro-inflammato TNF-α and IL-6 and anti-inflammatory IL-10 [33]. TNF-α is the early pro-inflammato cytokine produced by monocytes, macrophages and lymphocytes in response to inflam matory stimuli, which, together with IL-6, has a broad spectrum of action. Production TNF-α and IL-6 induces basic symptoms of inflammation such as heat, swelling, redne and pain. In contrast, IL-10, also produced by monocytes, macrophages and lymphocyt (especially type 2 T helper cells, regulatory T and B cells), has anti-inflammatory prope ties, and LPS could also mediate its production.
The strongest inhibition of pro-inflammatory IL-6 production in LPS-stimulate PBMC culture (Figure 8) was also observed for 2a in the highest 100 µ g/mL dose (64% inhibition compared to 11% for ibuprofen). At this concentration, 2b and 2c exhibited tendency to inhibit IL-6 production (by 28% and 18%, respectively).

The Effects of Compounds 2a-2f on Pro-Inflammatory and Anti-Inflammatory Cytokine Production
The effect of different concentrations of 2a-2f or ibuprofen (as a reference compound) on the LPS-induced production of pro-inflammatory (IL-6 and TNF-α) and antiinflammatory (IL-10) cytokines in 24 h PBMC culture is presented in Figures 8-10. LPS is an endotoxin of Gram-negative bacteria, used extensively for inducing an immune response in vitro. It promotes cytokine production in PBMC cultures, including pro-inflammatory TNF-α and IL-6 and anti-inflammatory IL-10 [33]. TNF-α is the early pro-inflammatory cytokine produced by monocytes, macrophages and lymphocytes in response to inflammatory stimuli, which, together with IL-6, has a broad spectrum of action. Production of TNF-α and IL-6 induces basic symptoms of inflammation such as heat, swelling, redness and pain. In contrast, IL-10, also produced by monocytes, macrophages and lymphocytes (especially type 2 T helper cells, regulatory T and B cells), has anti-inflammatory properties, and LPS could also mediate its production.
The strongest inhibition of pro-inflammatory IL-6 production in LPS-stimulated PBMC culture (Figure 8) was also observed for 2a in the highest 100 µg/mL dose (64% of inhibition compared to 11% for ibuprofen). At this concentration, 2b and 2c exhibited a tendency to inhibit IL-6 production (by 28% and 18%, respectively).
In regards to pro-inflammatory TNF-α production (Figure 9), a strong inhibitory effect in LPS-stimulated PBMC culture was observed for 2a, only in the highest 100 µg/mL dose (65% inhibition, in comparison to 6% for ibuprofen). In contrast, 2c produced a 19% inhibition of TNF-α, while 2b and 2d-f revealed only small or even negligible impacts in all doses compared to LPS alone or ibuprofen.
Finally, we observed a significant inhibition of anti-inflammatory IL-10 production ( Figure 10) for derivatives 2a-2c, 2e and 2f in medium (50 µg/mL) or their highest (100 µg/mL) doses (76-92% and 71-95% inhibition, in comparison to 57% and 77% for ibuprofen). However, compound 2d showed a similar inhibitory profile to ibuprofen (42 and 75% inhibition, respectively). All tested derivatives and ibuprofen elevated IL-10 production in the lowest concentration. In regards to pro-inflammatory TNF-α production ( Figure 9), a strong inhibitory effect in LPS-stimulated PBMC culture was observed for 2a, only in the highest 100 µ g/mL dose (65% inhibition, in comparison to 6% for ibuprofen). In contrast, 2c produced a 19% inhibition of TNF-α, while 2b and 2d-f revealed only small or even negligible impacts in all doses compared to LPS alone or ibuprofen. Finally, we observed a significant inhibition of anti-inflammatory IL-10 production ( Figure 10) for derivatives 2a-2c, 2e and 2f in medium (50 µ g/mL) or their highest (100 µ g/mL) doses (76-92% and 71-95% inhibition, in comparison to 57% and 77% for ibuprofen). However, compound 2d showed a similar inhibitory profile to ibuprofen (42 and 75% inhibition, respectively). All tested derivatives and ibuprofen elevated IL-10 production in the lowest concentration.  In regards to pro-inflammatory TNF-α production (Figure 9), a strong inhibitory effect in LPS-stimulated PBMC culture was observed for 2a, only in the highest 100 µ g/mL dose (65% inhibition, in comparison to 6% for ibuprofen). In contrast, 2c produced a 19% inhibition of TNF-α, while 2b and 2d-f revealed only small or even negligible impacts in all doses compared to LPS alone or ibuprofen. Finally, we observed a significant inhibition of anti-inflammatory IL-10 production ( Figure 10) for derivatives 2a-2c, 2e and 2f in medium (50 µ g/mL) or their highest (100 µ g/mL) doses (76-92% and 71-95% inhibition, in comparison to 57% and 77% for ibuprofen). However, compound 2d showed a similar inhibitory profile to ibuprofen (42 and 75% inhibition, respectively). All tested derivatives and ibuprofen elevated IL-10 production in the lowest concentration.

Antibacterial Activity of 2a-2f
The results of MIC determination, presented in Table S15 (Suppl. Data, part H), exhibited the best antibacterial activity for 2a and 2c against Staphylococcus aureus, as well as for 2d against Yersinia enterocolitica (all MICs = 128 µ g/mL). Moreover, 2b inhibited the growth of S. aureus, 2c inhibited Y. enterocolitica and M. smegmatis, and 2d inhibited Esch-

Peripheral Blood Mononuclear Cell Preparation
After informed consent, fresh blood (18 mL) was obtained from five healthy donors at the Occupational Medicine Clinic located in Dr. Antoni Jurasz University Hospital in Bydgoszcz, Poland.

In Vitro Toxic Effects on PBMCs by APC Annexin V and Propidium Iodide Staining Assay and Flow Cytometry
The effects of compounds 2a-2f on cell viability were studied in PBMCs culture by flow cytometry. The cells (1 × 10 6 cells/mL) were seeded in 24-well polypropylene, nonadherent plates (Cytogen, Zgierz, Poland). After that, increasing amounts of 2a-2f in DMSO were added to the cells and incubated for 24 h at 37 • C at 5% CO 2 conditions. The final concentrations of 2a-2f were 10, 50 and 100 µg/mL. Control samples contained DMSO or ibuprofen. After stimulation, the tubes were centrifuged at 400 g at 4 • C for 5 min and washed once with PBS. Then, the cells were stained with allophycocyanin-conjugated Annexin V (APC Annexin V) and propidium iodide (PI) (both from BD Pharmingen, San Diego, CA, USA) in accordance with the manufacturer's manual. A total of 10,000 cells were acquired on an FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed with FlowJo software (v 7.6.1, Tree Star, Ashland, OR, USA).
3.6. Anti-Inflammatory Activity 3.6.1. In Vitro Antiproliferative Effects by VPD-450 Staining Assay and Flow Cytometry Antiproliferative effects were examined by flow cytometry in BD Horizon Violet Proliferation Dye 450 (VPD450, BD Pharmingen)-labeled PBMCs. Flow cytometry assay was employed to find the cytotoxic potential of compounds 2a-2f on the proliferation of soluble anti-human CD3 monoclonal antibody (mouse IgG2a, clone OKT3, Sigma-Aldrich)induced PBMCs. Briefly, freshly isolated PBMCs at a concentration of 10-20 × 10 6 cells/mL in PBS were labeled for 11 min with VPD450 (1uM) at 37 • C. The VPD450 labeling reaction was terminated with complete media containing 10% fetal bovine serum (FBS) and then resuspended at a 1 × 106 cells/mL concentration in 5% FBS/RPMI1640. VPD450-stained cells were cultured in conical polypropylene tubes (BD Bioscience) for 72 h in 37 • C at 5% CO 2 atmosphere with anti-CD3 (1 µg/mL, positive control) and/or increasing concentration of 2a-2f in DMSO (10, 50 and 100 µg/mL). Control samples contained DMSO or ibuprofen. The culture tubes were centrifuged at 400× g at RT for 5 min, washed once in PBS, and 10,000 cells from every sample were acquired on a FACSCanto II flow cytometer (Becton Dickinson) and analyzed with FlowJo software (v 7.6.1, Tree Star, Ashland, OR, USA).

Antibacterial Activity
The broth microdilution method determined the minimum inhibitory concentration (MIC), defined as the lowest concentration of the compounds 2a-2f that inhibited bacterial growth. The strains used in the study: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 came from the American Type Culture Collection (Manassas, VA, USA) and are the recommended reference strains for antibiotic susceptibility testing. Other strains, including Micrococcus luteus, Yersinia enterocolitica O3, Mycobacterium smegmatis and Nocardia corallina (currently Rhodococcus sp.), came from environmental sources, are deposited in the Department of Genetics and Microbiology collection, and have been used by us in previously published experiments [45,46].
Compounds 2a-2f were dissolved in DMSO, diluted tenfold in Mueller-Hinton broth (MHB) to the concentration of 1.024 mg/mL, and then serially diluted in MHB to concentrations ranging from 512 µg/mL to 0.25 µg/mL.
The wells were inoculated with bacterial cultures to the final concentration of 10 4 colony-forming units (CFU) per mL. Bacterial growth was assayed by measuring optical density at OD 550 nm after 18 h incubation at 37 • C. The wells containing only MHB and 2.5% dimethyl sulfoxide were applied as a negative control. All MIC determinations were carried out in triplicates.

Data Analysis
Data were analyzed in Statistica 13.3 software (StatSoft, Cracow, Poland) and graphed in Excel 2016 (Microsoft, Redmond, WA, USA). All p-values represent the nonparametric Mann-Whitney U test.
The comparative analysis of the 1 H-13 C NMR spectra of 2a-2f to those for the parent amidrazones 1a-1f demonstrated that they appeared in DMSO-d 6 as a mixture of distinct A and B forms, being most likely geometric Z and E isomers, respectively. This is consistent with the results of single-crystal X-ray diffraction studies of 2a and 2d, which revealed the respective Z and E isomers in their solid phase.
All studied compounds possess anti-inflammatory properties by inhibiting PBMC proliferation (especially 2c and 2d) as well as TNF-α and IL-6 production (only 2a). Additionally, 2a and 2c exhibit antibacterial activity, particularly against S. aureus.