Synthesis, Biological Activity and Molecular Docking Studies of Novel Nicotinic Acid Derivatives

In our research, we used nicotinic acid as a starting compound, which was subjected to a series of condensation reactions with appropriate aldehydes. As a result of these reactions, we were able to obtain a series of twelve acylhydrazones, two of which showed promising activity against Gram-positive bacteria (MIC = 1.95–15.62 µg/mL), especially against Staphylococcus epidermidis ATCC 12228 (MIC = 1.95 µg/mL). Moreover, the activity of compound 13 against the Staphylococcus aureus ATCC 43300 strain, i.e., the MRSA strain, was MIC = 7.81 µg/mL. Then, we subjected the entire series of acylhydrazones to a cyclization reaction in the acetic anhydride, thanks to which we were able to obtain twelve new 3-acetyl-2,5-disubstituted-1,3,4-oxadiazoline derivatives. Obtained 1,3,4-oxadiazolines were also tested for antimicrobial activity. The results showed high activity of compound 25 with a 5-nitrofuran substituent, which was active against all tested strains. The most promising activity of this compound was found against Gram-positive bacteria, in particular against Bacillus subtilis ATCC 6633 and Staphylococcus aureus ATCC 6538 (MIC = 7.81 µg/mL) and ATCC 43300 MRSA strains (MIC = 15.62 µg/mL). Importantly, the best performing compounds did not show cytotoxicity against normal cell lines. It seems practical to use some of these compounds or their derivatives in the future in the prevention and treatment of infections caused by some pathogenic or opportunistic microorganisms.


Introduction
Antibiotic resistance describes the ability of bacteria to survive when exposed to antibiotics [1,2]. Once bacteria are exposed to antibiotics, there are three possibilities-the bacteria will die, stagnate (will not multiply) or will multiply. The third possibility is called antibiotic resistance, which poses a serious danger to public health worldwide. It leads to higher health care costs, longer hospitalization, patient failures and even deaths [3]. It turned out that it was necessary to introduce information and education activities in relation to medical professionals and to the entire society, as well as rigorous compliance with the rules of infection control and prevention of infections in all health care facilities (hospitals, outpatient facilities and nursing homes) [4]. Unfortunately, these actions are not sufficient and scientists are still constantly searching for new chemotherapeutic agents to help the immune system, while bacteria are constantly developing mechanisms that allow them to survive [5]. Bacterial strains resistant to all available antibiotics are already bloodstream infections, urinary tract infections, surgical site infections, meningitis, catheter-related infections [17,22] Streptococcus spp. (e.g., S. pneumoniae) penicillin (PRP-penicillin-resistant S. pneumoniae) bloodstream infections, surgical site infections, pneumonia, upper respiratory tract infections, meningitis, ear and sinus infections [17,22] Enterobacterales (e.g., E. coli, K. pneumoniae, K. oxytoca, Enterobacter spp.) beta-lactams (ESBL-extended spectrum beta-lactamase producing Enterobacterales), carbapenems (CRE-carbapenem resistant Enterobacterales) intra-abdominal infections and diseases of abdomen, bloodstream infections, urinary tract infections, surgical site infections, pneumonia, upper respiratory tract infections, meningeal, eye, bone infections, skin and soft-tissue infections, febrile neutropenia, surgical wound infections [17][18][19]22,27,28] Non-fermenting Gram-negative rods (e.g., Pseudomonas aeruginosa, Acinetobacter spp.) carbapenems (multi-drug resistant P. aeruginosa, carbapenem resistant Acinetobacter) pneumonia, bloodstream infections, skin and soft-tissue infection (burns), complicated urinary tract infections and abdominal infections, heart, brain, catheter-related, and at surgical sites [17,19,22,28] Candida spp. (e.g., C. auris, C. glabrata) azoles bloodstream infections, urinary tract infections, pneumonia, superficial and mucosal infections, life-threatening disseminated candidiasis [17,19,22,29,30] Candida albicans is one of the species of microorganisms that belong to the natural physiological flora of the human body. This fungus is isolated from the mucous membranes of the digestive tract, respiratory tract, mouth and skin. Unfortunately, these species can also cause opportunistic infections. First of all, these are non-dangerous, but troublesome to heal, surface yeasts [31]. Systemic candidiasis appears more and more often and constitutes a bigger problem. They are observed in patients with severe immunodeficiency, caused by long-term therapy or diseases which cause a decrease of the immune response, such as cancer or AIDS [32]. Unfortunately, in recent years, an increase in the number of isolated strains resistant to the antifungal agents used so far has been additionally observed. Moreover, some of them show resistance to many pharmaceutics at once, this is called multidrug resistance (MDR). This makes the candidiasis treatments ineffective [33].
Invasive Candida infections remain an important cause of morbidity and mortality, especially in hospitalized and immunocompromised or critically ill patients. Currently, there are only three major classes of medicines approved for the treatment of serious candidiasis. Moreover, the efficacy of these antimycotics is also compromised by the development of drug resistance in this pathogen population [29,30].
That is why it is so important to search for new molecules that will be able to fight even with the most resistant strains of bacteria or fungi and at the same time will be safe for the human body.
Having in mind these facts, in this research we decided to design, synthetize, evaluate for antimicrobial activity and cytotoxicity and perform molecular docking studies of acylhydrazones and 1,3,4-oxadiazole derivatives obtained from nicotinic acid hydrazide.
The structure of all obtained compounds was confirmed by spectroscopic methods: 1 H NMR, 13 C NMR and FT-IR spectra as well as elemental analysis.
The first group of compounds, i.e., acylhydrazones (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13), showed the following characteristic signals in the 1 H NMR spectra: singlet for the NH group in the range of 11.73-12.91 ppm and the signal for the =CH group at δ 8.23-9.11 ppm. In the case of the 13 C NMR spectra, the signals for the carbon atom of =CH and C=O groups appeared in the range of δ 137.11-138.61 ppm and 161.18-185.41 ppm, respectively. Additionally, we observed characteristic signals in expected regions in the FT-IR spectra. The remaining fragments of the examined compounds gave characteristic signals in the expected ranges of the chemical shift. the carbon atom of 1,3,4-oxadiazoline ring in the 13 C NMR spectra were found at δ 88.41-125.08 ppm and δ 150.83-155.08 ppm, respectively. Similarly, the signal for the carbon atom of the methyl group of the acetyl substituent appeared around 20 ppm. Additionally, we observed characteristic signals for C=O, C=N and C-OC bonds at the expected values in FT-IR spectra. Scheme 1. Reaction leading to novel 3-acetyl-2,5-disubstituted-1,3,4-oxadiazoline derivatives.

Microbiology
All synthesized compounds  were examined for their antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria and fungi belonging to yeasts Candida spp. A panel of reference strains of microorganisms also included some resistant staphylococci, that is the methicillin-resistant Staphylococcus aureus MRSA ATCC 43300. On the basis of MIC and MBC values, we discovered that new compounds showed interesting antibacterial activity. Table 2 presents the compounds which displayed antimicrobial activity.

Microbiology
All synthesized compounds  were examined for their antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria and fungi belonging to yeasts Candida spp. A panel of reference strains of microorganisms also included some resistant staphylococci, that is the methicillin-resistant Staphylococcus aureus MRSA ATCC 43300. On the basis of MIC and MBC values, we discovered that new compounds showed interesting antibacterial activity. Table 2 presents the compounds which displayed antimicrobial activity.
The obtained results showed a very high antibacterial effect of compounds 5 and 13 against Gram-positive bacteria ( Table 2). Their activity was strong or very strong with minimal inhibitory concentrations (MIC) ranging from 7.81 µg/mL to 15.62 µg/mL and from 1.95 µg/mL to 15.62 µg/mL, respectively. In turn, ranges of minimal bactericidal concentrations (MBC) values were 7.81-31.25 µg/mL for 5 and 3.91-31.25 µg/mL for compound 13. Moreover, these substances showed a bactericidal effect (MBC/MIC = 1-4).

Cytotoxicity Studies
The analysis of cells' sensitivity to the tested compounds was performed with the use of the MTT method-analysis of the metabolic activity of cells and NR uptake assay analysing the stability of cell membranes and thus direct cytotoxicity.
The MTT method showed a higher sensitivity of colon epithelial tumour cells to the analysed compounds than normal cells of the colonic epithelium. Compound 21 had the weakest effect on tumour cells, reducing their metabolic activity to 80% at the compound concentration of 200 µg/mL. The strongest activity was shown by compound 17, which reduced the metabolic activity of cells to 41% at the concentration of 75 µg/mL in comparison to the control (100% activity), not treated with the tested substances. The IC 50 value, in this case, was 61.18 µg/mL. In the case of normal cells, the highest decrease in cell metabolic activity was also demonstrated after compound 17 application. However, at the concentration of 200 µg/mL, cell metabolism decreased only to 71.6% as compared with the untreated control ( Figure 1).
Carrying out the analysis by the neutral red uptake (NR), there were slight differences in the sensitivity of the cells to the tested compounds in comparison to the results obtained with the MTT method. In the case of tumour cells, all tested compounds showed similar cytotoxic activity. At the highest concentration of the compounds (200 µg/mL), HT29 cell viability oscillated between 70% and 78%. In the case of normal colonic epithelial cells, the compound numbered 20 maintained cell viability over the control value at the entire range of tested concentrations. Compounds 21 and 22 at the concentration of 200 µg/mL decreased cell viability to 85 and 80%, respectively. Compound 17 showed the strongest cytotoxic activity against these cells, reducing their viability to 46% at the highest applied concentration (200 µg/mL). This allowed the calculation of an IC 50 value for this compound against normal cells at 179.81 µg/mL ( Figure 2). weakest effect on tumour cells, reducing their metabolic activity to 80% at the compound concentration of 200 µg/mL. The strongest activity was shown by compound 17, which reduced the metabolic activity of cells to 41% at the concentration of 75 µg/mL in comparison to the control (100% activity), not treated with the tested substances. The IC50 value, in this case, was 61.18 µg/mL. In the case of normal cells, the highest decrease in cell metabolic activity was also demonstrated after compound 17 application. However, at the concentration of 200 µg/mL, cell metabolism decreased only to 71.6% as compared with the untreated control ( Figure 1).    The reduction abilities of the tested compounds (17,20,21,22) were assessed by DPPH and FRAP methods. The results of the obtained studies were compared to known compounds with reductive activity, water-soluble synthetic vitamin E (Trolox) and ascorbic acid, respectively. In general, the tested compounds (17, 20, 21, 22) showed weak but detectable reductive activity. Compound 17 was the most effective, reducing the DPPH free radical at a concentration of 150 µg/mL which is equivalent to that of synthetic vitamin E at a concentration of 3.558 ± 0.717 µg/mL. The weakest reducing activity was observed for compound 20. On the other hand, compound 20 showed the strongest ferric-reducing power (FRAP), equal to 3.51 ± 1.35 µg/mL of ascorbic acid activity. The The reduction abilities of the tested compounds (17,20,21,22) were assessed by DPPH and FRAP methods. The results of the obtained studies were compared to known compounds with reductive activity, water-soluble synthetic vitamin E (Trolox) and ascorbic acid, respectively. In general, the tested compounds (17, 20, 21, 22) showed weak but detectable reductive activity. Compound 17 was the most effective, reducing the DPPH free radical at a concentration of 150 µg/mL which is equivalent to that of synthetic vitamin E at a concentration of 3.558 ± 0.717 µg/mL. The weakest reducing activity was observed for compound 20. On the other hand, compound 20 showed the strongest ferric-reducing power (FRAP), equal to 3.51 ± 1.35 µg/mL of ascorbic acid activity. The weaker reducing activity of the Fe 3+ ion was found for compound 22 (Tables 3 and 4).  May-Grünwald-Giemsa (MGG) cell staining was designed to evaluate cell morphology after incubation for 24 h with compounds 17, 20, 21 and 22. The performed observations are in line with the quantitative results obtained with the NR method. The activity of compounds determined to be cytotoxic in NR uptake assay, in MGG staining in both normal and neoplastic cells was observed as a decrease in the number of stained cells due to the detachment of dying cells from the carrier surface. In addition, you can also observe the detachment of cells from each other, their shrinkage as well as loss of intercellular interaction ( Figure S9; Supplementary Materials).

Molecular Docking
In order to predict the target enzymes for most active compounds (5, 13, 17, 25) and to evaluate the possible interactions, a molecular docking study was carried out with the use of the Autodock Vina 4.2 [52]. Structures of compounds 13 and 25 are chemically related to widespread chemotherapeutic agents, which contain 5-nitrofuran moieties, such as Furazolidone, Nitrofurazone, and Nifuroxazide [53]. That is why it is logical to suppose that synthesized compounds may have the same mode of antibacterial action. This purpose is also confirmed by the fact that the structurally related compounds with 5-nitrophenyl moiety 10 and 22 do not possess any antimicrobial activity. The 5-nitrofuran derivatives have to be activated before mediating its cytotoxic effects. Reactions of activations are catalyzed by nitroreductase (NTR) enzymes. As a result, the production of free radicals is observed. They can readily react with cellular macromolecules, and they are directly responsible for antibacterial action. As a result, lipids oxidation, cell membrane damages, enzyme inactivation, and finally fragmentation of the DNA sequence is observed [54]. That is why we evaluated the binding energy of compounds 5 and 17 for Escherichia coli Nitroreductase (PDB code: 1YKI) and compare them with such energy for native ligand Nitrofurazone. Additionally, we made a cross-docking simulation with the nitrofurantoin, which was used as the reference compound in the microbiology assays. The dihydrofolate reductase (S. aureus, PDB code: 5ISP) and tyrosyl-tRNA synthetase (S. aureus, PDB code: 1JIJ) were also selected for in silico docking simulations of antibacterial activity of the most active compounds (5, 13, 17, 25).
Docking demonstrated that compounds 13 and 25 have a better affinity to Nitroreductase compared to Nitrofurazone (Table 5). As shown in Figure 4, compound 13 linked tightly with amino acid residues of Escherichia coli Nitroreductase by hydrogen bonds with LYS74 (2.54Å), LYS14 (2.42 Å), GLU165 (2.11Å), THR41 (2.15Å), ARG121 (2.57Å) and ASN117 (2.30Å). Binding energy also increases owing to additional stabilized forces, such as van der Waals, attractive charge and amide-Pi stacked interactions between molecule and ASN42, GLU165 and THR41, respectively.  As shown in Figure 4, compound 13 linked tightly with amino acid residues of Escherichia coli Nitroreductase by hydrogen bonds with LYS74 (2.54Å), LYS14 (2.42 Å), GLU165 (2.11Å), THR41 (2.15Å), ARG121 (2.57Å) and ASN117 (2.30Å). Binding energy also increases owing to additional stabilized forces, such as van der Waals, attractive charge and amide-Pi stacked interactions between molecule and ASN42, GLU165 and THR41, respectively. It is quite similar to the interaction between Nitrofurazone and the Nitroreductase active site, where planar medicine molecule also makes several hydrogen bonds with the protein [55]. Docking results allowed us to suppose that compounds 13 and 25 would be more attractive substrates for Nitroreductase of S. aureus and interesting scaffold for new 5-nitrofuran derivatives with a wide spectrum of antimicrobial activities ( Figure 5). It is quite similar to the interaction between Nitrofurazone and the Nitroreductase active site, where planar medicine molecule also makes several hydrogen bonds with the protein [55]. Docking results allowed us to suppose that compounds 13 and 25 would be more attractive substrates for Nitroreductase of S. aureus and interesting scaffold for new 5-nitrofuran derivatives with a wide spectrum of antimicrobial activities ( Figure 5).
It is quite similar to the interaction between Nitrofurazone and the Nitroreductase active site, where planar medicine molecule also makes several hydrogen bonds with the protein [55]. Docking results allowed us to suppose that compounds 13 and 25 would be more attractive substrates for Nitroreductase of S. aureus and interesting scaffold for new 5-nitrofuran derivatives with a wide spectrum of antimicrobial activities ( Figure 5). Compounds 5 and 17 have quite a good affinity to target enzymes, nevertheless, those energies are smaller when compared to binding energies of initial ligands. Dock- Compounds 5 and 17 have quite a good affinity to target enzymes, nevertheless, those energies are smaller when compared to binding energies of initial ligands. Docking results allow suggesting that antimicrobial activity of substances 5 and 17 is connected mainly with inhibition of tyrosyl-tRNA synthetase. The most active compounds (13 and 25) showed good affinity to tyrosyl-tRNA synthetase and it is correlated with their wide activity against either Gram-positive or Gram-negative bacteria strains. The binding energies are shown in Table 6. The planar molecule of compound 5 occupies hydrophobic pockets, made by LEU8, VAL31 and LEU54. Additionally, three amino acids have alkyl interactions with iodine substituent of the phenyl ring (TYR98, ILE14 and VAL6) ( Figure 6). ing results allow suggesting that antimicrobial activity of substances 5 and 17 is connected mainly with inhibition of tyrosyl-tRNA synthetase. The most active compounds (13 and 25) showed good affinity to tyrosyl-tRNA synthetase and it is correlated with their wide activity against either Gram-positive or Gram-negative bacteria strains. The binding energies are shown in Table 6. The planar molecule of compound 5 occupies hydrophobic pockets, made by LEU8, VAL31 and LEU54. Additionally, three amino acids have alkyl interactions with iodine substituent of the phenyl ring (TYR98, ILE14 and VAL6) ( Figure 6). Compound 17 makes the several alkyl interactions with aliphatic aminoacids LEU20, ALA7, VAL20 and VAL31, also by the iodine atoms, but the lack of hydrogen bond interaction decreases the affinity of compounds 5 and 17 with tyrosyl-tRNA synthetase, compared to the reference ligand SB-239629 (Figure 7) [56]. Compound 17 makes the several alkyl interactions with aliphatic aminoacids LEU20, ALA7, VAL20 and VAL31, also by the iodine atoms, but the lack of hydrogen bond interaction decreases the affinity of compounds 5 and 17 with tyrosyl-tRNA synthetase, compared to the reference ligand SB-239629 (Figure 7) [56]. Compound 17 makes the several alkyl interactions with aliphatic aminoacids LEU20, ALA7, VAL20 and VAL31, also by the iodine atoms, but the lack of hydrogen bond interaction decreases the affinity of compounds 5 and 17 with tyrosyl-tRNA synthetase, compared to the reference ligand SB-239629 (Figure 7) [56]. An appropriate ADMET profile for new compounds is often crucial for further development as new potential antimicrobial agents. Modern in silico techniques allow simplifying the search for new chemical entities with suitable physicochemical properties for efficient and safe oral administration. The Lipinski rule of five become the nonofficial standard in medicinal chemistry. Therefore, ADMET profile predictions were made for 5, 13, 17 and 25 using freely accessible portals SwissADME and ProTOX. Addi- An appropriate ADMET profile for new compounds is often crucial for further development as new potential antimicrobial agents. Modern in silico techniques allow simplifying the search for new chemical entities with suitable physicochemical properties for efficient and safe oral administration. The Lipinski rule of five become the non-official standard in medicinal chemistry. Therefore, ADMET profile predictions were made for 5, 13, 17 and 25 using freely accessible portals SwissADME and ProTOX. Additionally, ciprofloxacin and nitrofurantoin were chosen as the reference compounds for comparison. Results are highlighted in Table 7. According to obtained data, compounds 5, 13, 17 and 25 possess suitable ADMET profiles, which allows us to suggest them as the perspective antimicrobial agents.

Discussion
The condensation reaction is a simple method by which acylhydrazones (2-13) can be obtained quickly. Then, the cyclization reaction in acetic anhydride of obtained compounds enabled the synthesis of 3-acetyl-2,5-disubstituted-1,3,4-oxadiazoline derivatives. The ease of this synthesis, good yield results and high biological potential of the obtained derivatives encourage the search for compounds of antimicrobial nature among them. Among the compounds from the group of acylhydrazones, a substance with the 5-nitrofuran substituent showed the highest activity. It showed high activity especially against Gram-positive bacterial strains, among them towards Staphylococcus epidermidis (MIC = 1.95 µg/mL), Staphylococcus aureus ATCC 6538 (MIC = 3.91 µg/mL) and also against the MRSA strain (MIC = 7.81 µg/mL). The 1,3,4-oxadiazoline derivative, i.e., the compound numbered as 25, showed half of the activity (MIC = 15.62 µg/mL) against the same strains. Similarly, compound 5 with the 2-hydroxy-3,5-diiodophenyl substituent after conversion to 1,3,4oxadiazoline derivative showed half of the activity against the same bacterial strains. On the basis of obtained results, it can be concluded that the nicotinic acid-derived acylhydrazones are more active than the corresponding 1,3,4-oxadiazoline derivatives in terms of antibacterial activity against Gram-positive bacteria. On the other hand, the activity against fungal strains was different. In this case, the 3-acetyl-1,3,4-oxadiazolines were more active towards yeasts than acylhydrazones. This fact can be observed in the example of the compounds with the 5-nitrofuran substituent, i.e., 13 (acylhydrazone) and 25 (1,3,4oxadiazoline derivative). The substance 25 showed good activity against Candida albicans ATCC 10231 (MIC = 15.62 µg/mL), while compound 13 showed no activity.
Analysis of the results of the conducted research in terms of the structure-activity relationship (SAR), it can be stated that acylhydrazones showed greater biological activity in relation to N-acetyl-1,3,4-oxadiazolines. The most active of the two groups were those derivatives that had the 5-nitrofuran substituent. We saw a similar situation in our previous articles [57-61, where, the cyclization process also resulted in a decrease in the activity of the tested compounds. Thus, it can be concluded that the introduction of a 1,3,4-oxadiazole ring into the molecule deteriorates the antimicrobial activity, and the 5-nitro-furoyl moiety significantly improves the effectiveness against bacteria and fungi.
Moreover, the tested acylhydrazones were significantly more active against Grampositive bacteria than against Gram-negative bacteria. This difference can be seen perfectly during the analysis of the activity of compound 5 with a 2-hydroxy-3,5-diiodophenyl substituent, where the activity against Gram-positive bacteria was within the limits of MIC = 7.81-15.62 µg/mL and the activity against Gram-negative was observed only for one strain-Bordetella bronchiseptica ATCC 4617 (MIC= 62.5 µg/mL), and the others substances were not active. A similar difference in activity was observed for the compound numbered 13 with a 5-nitrofuran substituent. It was the most active against Gram-positive bacteria Staphylococcus epidermidis ATCC 12228 (MIC = 1.95 µg/mL), and against the remaining Gram-positive, it also showed high activity (MIC =1.95-15.62 µg/mL). On the other hand, against Gram-negative strains, activity was either good or moderate. The activity of Nacetyl-1,3,4-oxadiazolines was similar to acylhydrazones. They also showed greater activity against Gram-positive than towards Gram-negative bacteria. The most active of this group was compound numbered as 25 against Gram-positive strains at (MIC = 7.81-62.5 µg/mL), while for Gram-negative bacteria the range of activity was MIC = 62.5-250 µg/mL. Additionally, for compound 17, the measured activity against Gram-positive bacteria was MIC = 7.81-62.5 µg/mL, and no activity was found towards Gram-negative strains.
In conclusion, the results showed slightly different activity of the newly synthesized compounds against Gram-positive and Gram-negative bacteria. Among them, compounds 5, 13, 17 and 25 had a satisfactory and beneficial activity with strong or good effect towards all reference Gram-positive bacteria. In turn, microorganisms belong to bacteria from Enterobacterales and non-fermenting rods were less sensitive to these substances. Only substances 13 and 25 showed some activity with moderate or good effects depending on the microorganism. Due to the distinctive structure of the cell wall, Gram-negative bacteria are usually more resistant to antibiotics or other antibacterial interventions than Gram-positive. Their cell membrane is thin but difficult to penetrate. Therefore, these bacteria are harder to kill [57]. Our results also confirm this relationship.

Chemistry
All reagents used for the experiments were purchased from Merck Co. (Darmstadt, Germany) and used without further purification. They had a class of purity declared by the manufacturer. The melting points of the obtained compounds were determined with a Fisher-Johns apparatus (Fisher Scientific, Schwerte, Germany), and presented without any correction. The purity of the obtained compounds was assessed through thin layer chromatography (TLC) on plates covered with silica gel (aluminium oxide 60 F-254) by Merck. Chloroform-ethanol mixtures in the ratio 10:1 (v/v) were used as the mobile phase. Spots were developed by irradiation with UV light with a wavelength λ = 254 nm. The FT-IR spectra were recorded on a Nicolet 6700 spectrometer (Thermo Scientific, Madison, Wisconsin, USA) in cm −1 . The 1 H and 13 C NMR spectra were recorded on the Bruker Avance 300 and 600 apparatus (Bruker BioSpin GmbH, Ettlingen, Germany). The compounds were dissolved in dimethyl sulfoxide (DMSO-d6 ) before analysis. Tetramethylsilane (TMS) was used as an internal standard. Chemical shift values are given in ppm. Elemental analysis was determined by a Perkin Elmer 2400 Series II CHNS/O analyzer (Waltham, MA, USA), and the results were within ±0.4% of the theoretical values.

MTT Assay
After 24 h incubation of cells with the tested compounds in 100 µL of culture medium, MTT solution (5 mg/mL, 25 µL/well) was added and incubated for an additional 3 h. The purple crystals of formazan which formed in the medium were solubilized overnight in 10% sodium dodecyl sulphate (SDS) in a 0.01 M HCl mixture. The product was quantified spectrophotometrically by measuring its absorbance at 570 nm with the use of an Emax Microplate Reader (Molecular Devices Corporation, Menlo Park, CA).

Neutral Red (NR) Uptake Assay
Cells were grown for 24 h in a 96-well multiplate in 100 µL of culture medium (RPMI 1640) supplemented with 5% fetal bovine serum (FBS) and tested compounds. Subsequently, the medium was discarded and a 0.4% neutral red solution in 2% FBS medium was added to each well. The plate was incubated for 3 h at 37 • C in a humidified 5% CO 2 /95% air incubator. After incubation, the dye-containing medium was removed, the cells were fixed with 1% CaCl 2 in 4% paraformaldehyde, and the incorporated dye was solubilized using 1% acetic acetate in a 50% ethanol solution (100 µL). The plates were gently shaken for 20 min at room temperature and the absorbance of the extracted dye was measured spectrophotometrically at 540 nm.

Nitric Oxide (NO) Measurement
Nitrate, i.e., a stable end product of NO, was determined in culture supernatants with a spectrophotometric method based on the Griess reaction. Culture supernatants were collected from cell cultures treated with specific concentrations of the tested compounds. Briefly, 100 µL of the culture supernatant was placed in 96-well flat-bottomed plates in triplicate and incubated with 100 µL of Griess reagent (1% sulfanilamide/0.1% N-(1naphthyl)ethylenediamine dihydrochloride) in 3% H 3 PO 4 at room temperature for 10 min. The optical density was measured at 550 nm using a microplate reader. A standard curve was prepared with the use of 0.5-25 µM sodium nitrite (NaNO 2 ) for calibration.

DPPH Free Radical Scavenging Test
Free radical scavenging activity of terpene was measured by the DPPH assay. Briefly, 100 µL of DPPH solution (0.2 mg/mL in ethanol) was added to 100 µL of the tested compound concentrations (0-200 µg/mL). Trolox in increasing concentrations (1-50 µg/mL) was used as a standard. After 20 min of incubation at room temperature, the absorbances of the solutions were measured at 515 nm. The lower the absorbance, the higher the free radical scavenging activity of the compounds. The activity of each compound was determined by comparing its absorbance with that of the standard.
The ability of the compounds to scavenge the DPPH radical was calculated by the following formula: Xcontrol is the absorbance of the control and Xcompound is the absorbance in the presence of synthesized compounds.

Ferric-Reducing Antioxidant Power Assay
Each compound concentration was dissolved in Milli-Q water and mixed with an equal volume of 0.2 M sodium phosphate buffer (pH 6.6) and 1% potassium ferricyanide. The mixture was incubated for 30 min at 37 • C. Thereafter, 10% trichloroacetic acid (w/v) was added and the mixture was centrifuged at 1000× g for 5 min. One millilitre of the upper layer was mixed with an equal volume of Milli-Q water and 0.1% ferric chloride. The absorbance was read at 700 nm with the use of an EL800 Universal Microplate Reader (BioTek Instruments, Winooski, VT, USA). Ascorbic acid (0-150 µg/mL) was used as a positive control.

May-Grünwald-Giemsa (MGG) Staining
The cells were incubated in 24-well plates in 1 mL of culture medium supplemented with tested compounds. After 24 h of incubation (37 • C in a humidified 5% CO 2 /95% air), the medium was discarded and the cell cultures were rinsed with RPMI 1640 medium and stained with May-Grünwald (MG) stain for 5 min followed by staining for another 5 min in MG diluted in an equal quantity of water. The MG was removed and Giemsa reagent (diluted 1:20 in water) was added to the cells, which were next incubated at room temperature for 15 min. After that, the cells were rinsed three times with water, dried and subjected to microscopic observations (Olympus, BX51; Olympus).

Statistical Analysis
Results are presented as mean ± SD from three experiments. Data were analysed with the use of one-way ANOVA with Dunnett's post hoc test. Differences of p ≤ 0.05 were considered as significant.

Molecular Docking
A molecular docking study was carried out using the Autodock Vina 4.2. The X-ray crystal structures of target proteins were downloaded from the Protein Data Bank. The AutoDock tools were used to remove water molecules, add polar hydrogen atoms, merge nonpolar hydrogen atoms, define rotatable bonds, and add Kollman charges. The validations of selected docking parameters were performed by redocking of the initial ligands from the used enzyme structures.
The structure of nitrofurantoin was downloaded from the PubChem portal. The 3D structures of the synthesized compounds were prepared with the use of HyperChem 7.5 software. At first, the molecules were optimized by the method of molecular mechanics MM + with the achievement of an RMS gradient of less than 0.1 kcal/(mol Å). The final minimization of the energies of the investigated intermediates was carried out by the semi-empirical quantum chemical method PM3 until the RMS gradient was less than 0.01 kcal/(mol Å). The visualization and interpretation of the obtained data were performed with the use of Discovery Studio Visualizer.