SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF SOME ADAMANTYL-CONTAINING HYDRAZONES

Danish Scientific Journal No101, 2025 19 SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF SOME ADAMANTYL-CONTAINING HYDRAZONES Koshchii I., PhD in Chemistry, Associate Professor Department of Organic Chemistry and Technology of Organic Substances National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute” Vasilkevich O., PhD in Chemistry, Associate Professor Department of Organic Chemistry and Technology of Organic Substances National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute” Klimko Yu. PhD in Chemistry, Associate Professor Department of Organic Chemistry and Technology of Organic Substances National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute” https://doi.org/10.5281/zenodo.17493452 Abstract Several adamantyl-containing quaternary salts of benzylidenehydrazinylpyridine, based on benzyl, ethylphenyl, and propylphenyl groups on the pyridinium nitrogen, were synthesized and tested for possible antibacterial and antifungal activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans using a microdilution method. The results of the antimicrobial tests showed that compounds containing the 3-phenylpropyl chain exhibited the highest antimicrobial activity against Staphylococcus aureus, and compound 3d was the most active in the series against all tested bacterial and fungal strains. Keywords: hydrazones, pyridinium salts, adamantyl, antimicrobial activity. Introduction The shortage of new antibacterial drugs due to growing bacterial resistance to antimicrobial agents is a major challenge in the development of new drugs. Quaternary amine derivatives have previously been reported to exhibit antimicrobial properties [1–4]. Certain monoand bis-quaternary ammonium compounds, including various alkyl chain lengths with various hydrophobic substituents, have been found to exhibit antimicrobial activity [5–7]. Pyridinium halides, like quaternary nitrogen salts, exhibit antimicrobial properties and adsorption properties on negatively charged solids. The antimicrobial activity of 1-alkylpyridinium salts depends on their adsorption activity on the surface of bacterial cells [8–16] and the pKa values of the corresponding pyridines [17]. Factors controlling their antimicrobial activity include molecular hydrophobicity [18,19], adsorbability [20], surface activity [19], and the electron density [21,22] of the quaternary nitrogen atom. These compounds possess one hydrophobic alkyl chain and one hydrophilic quaternary nitrogen ion group per molecule, which provides greater surface activity and more pronounced antimicrobial activity compared to conventional antimicrobial agents [23]. On the other hand, Schiff bases are important in medicine, and many studies have reported their biological activity [24,25]. Hydrazones, a special group of Schiff bases, are also known as one of the most important classes of organic compounds, some of which exhibit significant biological activities such as antimicrobial [26–30], antituberculosis [31–33], anticancer [34–36], analgesic [37], anti-inflammatory [37], antiplatelet [38], and antiviral [39, 40] activities. The data reported in these studies showed that the side chain attached to the pyridinium nitrogen significantly influenced the antimicrobial activity. Among them, compounds with 3-phenylpropyl chains showed remarkable activity [40]. Based on these results, ortho-methyl, - methoxyl, -hydroxyl benzylidenehydrazinylpyridinium salts with benzyl, 2,6-dichlorobenzyl, 2-phenylethyl, and 3-phenylpropyl groups on the pyridine nitrogen were prepared to study the effect of such structural modifications of quaternary pyridinium salts on the expected antimicrobial activity. In addition to the above, the objective of this study was to introduce a bulky hydrocarbon radical, adamantane, into the Schiff base molecules. The latter is widely known to enhance the lipophilicity of the drug substance, which prolongs its therapeutic effect. Results and Discussion Chemistry Benzylidenehydrazinylpyridinium salts were prepared in three steps according to the reported procedure [41] as shown in Scheme 1. In the first step, 4-chloropyridine was refluxed with hydrazine hydrate in 1-propanol to give 4-hydrazinylpyridine (1). This compound was then condensed with 4-(1-adamantyl)benzaldehyde (2) in ethanol at room temperature to give hydrazone (3). In the last step, the final compounds (4a–d) were obtained by quaternization of hydrazone derivatives (3) with the appropriate substituted alkyl halides in ethanol under reflux. All the key compounds are new. Except for compounds (3) and (4), compounds (1) and (2) were previously described [42,43]. 20 Danish Scientific Journal No101, 2025 Scheme 1. Synthesis of the title compounds. The structures of the final compounds were determined by spectral methods. The IR spectra of the final compounds showed intense absorption bands in the range of 3315–3446 cm−1 and the range of 1436–1644 cm−1, which were assigned to vibrations of the NH and C=N functions, respectively. In the 1H NMR spectra, the signals of the NH group protons were recorded in the range of 12.26–13.00 ppm. The signals of the protons belonging to the N=CH group were observed as singlets in the range of 8.52–8.65 ppm. The IR and 1H NMR data confirmed the condensation between the amino and carbonyl groups. As expected, while the pyridine hydrogens at positions 2 and 6 of compound (3) showed peaks in the 8.19–8.21 ppm range, the signals of the hydrogens at these positions were in the final compounds (4a–d) in the 8.30–8.55 ppm range [44]. The shifts of these peaks to higher frequency indicated that compounds (4) were quaternized with an alkyl halide. All 13C NMR results supported the proposed structures as stated in the experimental section. The mass spectra of the final compounds (4a–d) showed molecular ion peaks that were consistent with the proposed structures. Antimicrobial Activity A series of benzylidenehydrazinylpyridinium salts with an adamantyl substituent at the para-position of the benzene ring were evaluated for antimicrobial activity against Gram-positive and Gram-negative bacteria and fungi. The bacterial strains represented important Gram-positive and Gram-negative species, including Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Their antibacterial activity was assessed by measuring the minimum inhibitory concentration (MIC) using a standard broth dilution assay (Table 1). Table 1. Antimicrobial activity of compounds 4а-d. Compound No Minimum Inhibitory Concentration (MIC) (μg/mL) E. coli P. aeruginosa S. aureus C. albicans 4a 4b 4c 4d Ceftazidime Fluconazole 35 >2048 66 131 <0.125 (0.06-0.5)* 512 >2048 512 1030 1 (1–4)*_ 20 67 32 4 4 (4–16)*_ 66 1030 66 66 (0.25–1.0)* * Acceptable quality control ranges of minimum inhibitory concentrations (MICs) (μg/mL) for strains [45,46]. Danish Scientific Journal No101, 2025 21 Based on the antimicrobial activity results, all compounds exhibited high antimicrobial activity against Staphylococcus aureus and low antimicrobial activity against Pseudomonas aeruginosa. Among the tested compounds, 4d was the most active derivative against Staphylococcus aureus, being as effective against this microorganism as the standard compound ceftazidime. The results showed that the longer the side chain of a compound, the greater its antimicrobial activity. In the series of adamantyl-substituted phenylpropyl derivatives, the antimicrobial activity of 4d was followed by 4c, 4a, and 4b, respectively. No significant difference in the antibacterial activity of compounds 4c and 4a was observed. Both compounds had identical MIC values against the test microorganisms, with the exception of Pseudomonas aeruginosa. According to the MIC values of the compounds, 4d had the lowest MIC values (4 μg/mL) compared to other pyridinium salts. This bioisosteric substitution resulted in a slight increase in antimicrobial activity. On the other hand, compounds with a 2,6-dichlorobenzyl side chain on the pyridinium nitrogen showed no activity against Gramnegative bacteria. Our study showed that all compounds exhibited stronger antibacterial activity against Gram-positive bacteria than Gram-negative bacteria. These data suggest that pyridinium salts act on cell membranes, and the surface activity of these compounds may be primarily responsible for their antibacterial properties. However, all tested compounds exhibited low antifungal activity against Candida albicans. The reason for the weaker antifungal activity compared to the antibacterial effect can be considered the difference in the mechanism of action of the compounds on the inhibition of the respiratory system of fungal cells, and not the destruction of the cell wall. Materials and methods. The melting points (mp) of the synthesized compounds were recorded on a Boetius bench. 1H and 13C NMR spectra were recorded on a Bruker AM300 spectrometer (300 and 75 MHz, respectively) in DMSO-d6 and CDCl3 at 25 °C. Chemical shifts were measured in DMSO-d6 with TMS as an internal standard. IR spectra were recorded using the thin film method and in KBr tablets on a Bruker Alpha spectrometer. Electrospray ionization (ESI) mass spectra were measured on an Agilent 1100 LC/MSD instrument. Reagents and solvents used for the synthesis were purchased from Aldrich. 4-(1Adamantyl)benzaldehyde was provided by AKos GmbH. Thin-layer chromatography was performed on silica gel-coated 60 F254 plates (Merck). The spots were developed using UV light or iodine. 4-Hydrazinopyridine Hydrochloride (1). Compound (1) was obtained according to the method described in [47]. A solution of 4-chloropyridine (0.01 mol) and hydrazine monohydrate (0.15 mol) in 1-propanol (30 ml) was refluxed for 18 h. The solution was cooled to 0 °C, the precipitate was filtered off, washed with cold 1-propanol and crystallized from ethanol. M.p. 242–243 °C (ref. [47,48] 242–243 °C). 4-(2-(4-(1-Adamantyl)benzylidene)hydrazinyl)pyridine(3). 4-Hydrazinylpyridine (0.01 mol) and benzaldehyde derivative (2) (0.01 mol) were stirred in ethanol (30 ml) at room temperature for 4–9 h. The precipitate was filtered off, washed with cold ethanol, and crystallized from ethanol. Yield 73%. M.p. 232 °C. IR (ν, cm-1): 1427, 1452, 1486, 1527, 1538 (Ar-C=C and N=C), 2886, 2948 (aliphatic C-H), 3014 (Ar-C-H), 3210 (N-H); 1H-NMR (δ, mmp): 1.76 (6CH2, Ad), 1.87 (3CH, Ad), 6.97 (2H, d, J = 6.2 Hz, Py-H), 7.23 (2H, d, J = 8.2 Hz, Ar-H), 7.83 (2H, d, J = 8.2 Hz, Ar-H), 8.19 (1H, s, N=CH), 8.21 (2H, d, J = 6.2 Hz, Py-H), 10.80 (1H, s, NH). General Synthesis Method for Compounds (4a-d). Hydrazone (3) (0.01 mol) and the corresponding alkyl halide (0.02 mol) were refluxed in ethanol (30 mL) for 10-40 h. The mixture was cooled to 0°C, and the resulting precipitate was filtered off and washed with cold ethanol. The crude products were crystallized from ethanol, yielding compounds (4a-d). 1-Benzyl-4-(2-(4-(1-adamantyl)benzylidene)hydrazinyl)pyridine chloride (4a). Yield 68%. M.p.= 267 °C. IR (ν, cm-1): 1455, 1482, 1517, 1544, 1600, (ArC=C and N=C), 1644 (N+=C), 2838, 2911, 2979 (aliphatic C-H), 3037 (ArC-H), 3397 (N-H). 1H-NMR (δ, mmp.): 1.76 (6CH2, Ad), 1.87 (3CH, Ad), 5.48 (2H, s, N+-CH2-Ph), 7.16 (1H, dd, J = 7.0, 2.3 Hz, Py-H), 7.23 (2H, d, J = 8.2 Hz, Ar-H), 7.83 (2H, d, J = 8.2 Hz, ArH), 7.36-7.42 (5H, m, Ar-H), 7.56 (1H, d, J = 7.4 Hz, Py-H), 8.47 (1H, d, J = 7.4 Hz, Py-H), 8.55 (1H, d, J = 7.4 Hz, Py-H), 8.62 (1H, s, N=CH), 13.00 (1H, s, NH). 13C-NMR (δ, mmp.): 60.75 107.80, 109.64, 126.96, 127.47, 128.78, 129.46, 129.78, 130.9, 131.7, 132.18, 136.20, 137.92, 143.72, 144.89, 147.99, 154.38. MS (m/z): 458 (M•+), 91.135, 77. 1-(2,6-Dichlorobenzyl)-4-(4-(1-adamantyl)benzylidene)hydrazinyl)pyridinium chloride (4b). Yield 72%. M.p. 298 °C; IR (ν, cm-1): 1436, 1513, 1581 (ArC=C and N=C), 1644 (N+=C), 2694, 2886 (aliphatic CH), 3052 (Ar-C-H), 3442 (N-H). 1H-NMR (δ, mmp): 1.76 (6CH2, Ad), 1.87 (3CH, Ad), 5.70 (2H, s, N+- CH2-Ph), 7.14 (1H, dd, J = 7.2, 2.8 Hz, Py-H), 7.267.30 (2H, m, Ar-H), 7.34 (1H, td, J = 7.4, 1.2 Hz, ArH), 7.52-7.56 (2H, m, Py -H), 7.23 (2H, d, J = 8.2 Hz, Ar-H), 7.83 (2H, d, J = 8.2 Hz, Ar-H), 8.22 (1H, dd, J = 7.0, 1.6 Hz, Py-H), 8.34 (1H, dd, J = 7.2, 1.6 Hz, PyH), 8.60 (1H, s, N=CH), 13.00 (1H, s, NH). 13C-NMR (δ, mmp): 56.44, 108.29, 110.28, 125.29, 128.30, 129.22, 129.72, 130.00, 131.74, 133.16, 134.16, 137.00, 143.50, 144.66, 144.74, 148.69, 154.80. MS (m/z): 527 (M•+), 135, 159, 145. 4-(4-(1-Adamantyl)benzylidene)hydrazinyl)-1phenethylpyridinium bromide (4c). Yield 87%. M.p.190 °C. IR (ν, cm-1): 1455, 1513, 1546, 1577 (ArC=C and N=C), 1644 (N+=C), 2836, 2908 (aliphatic CH), 3054 (Ar-C-H), 3415 (N-H); 1H NMR (δ, mmp): 1.76 (6СН2, Ad), 1.87 (3CH, Ad), 3.13 (2H, t, J = 7.2 Hz, N+-CH2-CH2-Ph), 4.50 (2H, t, J = 7.2 Hz, N+-CH2CH2-Ph), 7.03 (1H, dd, J = 7.0, 2.3 Hz, Py-H), 7.197.36 (5H, m, Ar-H), 7.23 (2H, d, J = 8.2 Hz, Ar-H), 7.83 (2H, d, J = 8.2 Hz, Ar-H), 7.50 (1H, dd, J = 7.0, 2.3 Hz, Py-H), 8.30 (1H, d, J = 7.4 Hz, Py-H), 8.35 (1H, d, J = 7.0 Hz, Py-H), 8.53 (1H, s, N=CH), 12.38 (1H, s, NH). 13C NMR (δ, mmp): 36.92, 59.03, 107.37, 108.99, 126.94, 127.36, 127.55, 129.23, 129.62, 130.96, 22 Danish Scientific Journal No101, 2025 131.73, 132.12, 137.33, 137.89, 143.71, 144.84, 147.53, 154.10. MS (m/z): 513(M•+), 77, 91, 135. 4-(4-(1-Adavantyl)benzylidene)hydrazinyl)-1-(3phenylpropyl)pyridiniumbromide (4d).Yield 49%; M.p.160 °C; IR (ν, cm-1): 1454, 1488, 1511, 1552, 1585, (Ar-C=C and N=C), 1644 (N+=C), 2827, 2915 (aliphatic C-H), 3058 (Ar-C-H), 3424 (N-H); 1H-NMR (δ, mmp): 1.76 (6CH2, Ad), 1.87 (3CH, Ad), 2.12 (2H, quin, J = 7.6 Hz, N+-CH2-CH2-CH2-Ph), 2.60 (2H, t, J = 7.8 Hz, N+-CH2-CH2-CH2-Ph), 4.28 (2H, t, J = 7.2 Hz, N+-CH2-CH2-CH2-Ph), 7.06 (1H, dd, J = 7.2, 2.4 Hz, Py-H), 7.16-7.30 (5H, m, Ar-H), 77.56 (1H, dd, J = 6.8, 2.4 Hz, Py-H), 7.23 (2H, d, J = 8.2 Hz, Ar-H), 7.83 (2H, d, J = 8.2 Hz, Ar-H) 8.39 (1H, d, J = 6.8 Hz, PyH), 8.46 (1H, d, J = 7.2 Hz, Py-H), 8.52 (1H, s, N=CH), 12.34 (1H, s, NH); 13C-NMR (δ, mmp): 32.28, 52.50, 58.08, 107.50, 109.15, 126.76, 126.98, 127.50, 128.89, 129.12, 131.00, 131.80, 132.14, 137.33, 141.15, 143.79, 144.80, 147.64, 154.29. MS(m/z): 527 (M•+), 77, 91, 135. Antimicrobial activity The minimum inhibitory concentration (MIC) was determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) [45,46]. The in vitro antimicrobial activity of the final compounds (4a–d) was assessed against the standard strains Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Candida albicans ATCC 90028. Antibacterial and antifungal assays were performed in Mueller–Hinton broth and Sabouraud dextrose broth, respectively. All synthesized compounds were weighed (10 mg), dissolved in DMSO (250 μL), and diluted with water (750 μL) to prepare stock solutions of 10 mg/mL. Serial dilutions from 2048 to 1 μg/mL were performed in a 96-well plate. Fifty μL of bacterial suspension obtained from a 24-hour culture (~106 CFU/mL) was added to each well with a final DMSO concentration of 1:16. The plates were incubated at 35°C for 24 hours. We tested Ceftazidime as an antimicrobial agent for quality control of the method. Each experiment was repeated twice. Conclusions We described the synthesis of a series of adamantyl-containing benzylidenehydrazinylpyridinium salts (4a-d) with antimicrobial activity. The final synthesized compounds were characterized by spectral data (IR, 1H-NMR, 13C-NMR, MS). Both the synthesis and antimicrobial activity of the final compounds (4a-d) were reported for the first time. Compounds (4a-d) were found to possess moderate activity against S. aureus. Notable activity was detected for compounds bearing a 3-phenylpropyl side chain on the nitrogen. The most active compound was 4-(4-(1-adamantylbenzylidene)hydrazinyl)-1-(3-phenylpropyl)pyridinium bromide (4d), with an MIC value of 4 μg/mL against S. aureus. The results showed that a longer side chain on the pyridinium nitrogen resulted in enhanced activity. References: 1. Thorsteinsson, T.; Masson, M.; Kristinsson, K.G.; Hjalmarsdottir, M.A.; Hilmarsson, H.; Loftsson, T. Soft antimicrobial agents: Synthesis and activity of labile environmentally.friendly long chain quaternary ammonium compounds. J. Med. Chem. 2003, 46, 4173–4181. 2. Vnutskikh, Z.A.; Shklyaev, Y.V.; Odegova, T.F.; Chekryshkin, Y.S.; Tolstikov, A.G.;El’chishcheva, N.V.; Syropyatov, B.Y. Synthesis and antimicrobial activity of mono-and biquaternized derivates of dipyridylethanes and dipyridylethylenes. Khim. Farm. Zh. 2006 40,19–22. 3.Ovchinnikova,I.G.Fedorova,O.V.;Rusinov,G.L.;Zueva,M.N.;Mordovskoi,G.G.S ynthesis and antimicrobial activity of N-alkylpyridinium podands. Khim. Farm. Zh.2003, 37, 17–19. 4. Burke, J.R. and Frey, P.A. Correlation of electronic effects in N-alkylnicotinamides with NMR chemical shifts and hydride transfer reactivity. J. Org. Chem. 1996, 61, 530–533. 5. Clarissa, K.L.N.; Singhal, V.; Widmer, F.; Wright, L.C.; Sorrell, T.C.; Jolliffe, K.A. Synthesis, antifungal and haemolytic activity of a series of bis(pyridinium)alkanes. Bioorg. Med. Chem.2007, 15, 3422– 3429. 6. Zhao, T.; Sun, G. Hydrophobicity and antimicrobial activities of quaternary pyridinium salts. J.Appl. Microbiol. 2008, 104, 824–830. 7. Kourai, H.; Yabuhara, T.; Shirai, A., Maeda, T.; Nagamune, H. Synthesis and antimicrobial activities of a series of new bis-quaternary ammonium compounds. Eur. J. Med. Chem. 2006, 41,437–444. 8. Okazaki, K.; Maeda, T.; Nagamune, H.; Manabe, Y.; Kourai, H. Synthesis and antimicrobial characteristics of 4,4'(α,ωpolymethylenedithio)bis(1alkylpyridinium iodide)s. Chem. Pharm. Bull. 1997, 45, 1970–1974. 9. Maeda, T.; Okazaki, K.; Nagamune, H.; Manabe, Y.; Kourai, H. Bactericidal action of 4,4'- (α,ω-polymethylenedithio)bis-(1-alkylpyridinium iodide)s. Biol. Pharm. Bull. 1998, 21, 1057–1061. 10. Maeda, T.; Manabe, Y.; Yamamoto, M.; Yoshida, M.; Okazaki, K.; Nagamune, H.; Hiroki, K. Synthesis and antimicrobial characteristics of novel biocides. 4,4'-(1,6-Hexamethylene dioxydicarbonyl)bis(1alkylpyridinium iodide)s. Chem. Pharm. Bull. 1999, 47, 1020–1023. 11. Yoshida, M.; Maeda, T.; Okazaki, K.; Nagamune, H.; Kunikata, K.; Tsuchiya, H.; Tetsuto, N.; Hiroki, K. Synthesis and antimicrobial characteristics of N,N'-Hexamethylenebis(4-carbamoyl-1decylpyridinium bromide). Biocontrol Sci. 2000, 5, 65–71. 12. Yoshida, M.; Maeda, T.; Okazaki, K.; Nagamune, H.; Kunikata, K.; Tsuchiya, H.; Namba, T.;Kourai, H. Synthesis of 4,4'-(tetramethylenedicarbonyldiamino)-bis(1-decylpyridinium bromide) and its antimicrobial and deodorant characteristics. Biocontrol Sci. 2001, 6, 75–80. 13. Shirai, A.; Maeda, T.; Hara, I.; Yoshinari, A.; Nagamune, H.; Kourai, H. Antimicrobial characteris- Danish Scientific Journal No101, 2025 23 tics of bis-quaternary ammonium compounds possessing a p-phenylene group in their spacer chains. Biocontrol Sci. 2003, 8, 151–157. 14. Pernak, J.; Rogoz, J.; Mirska, I. Synthesis and antimicrobial activities of new pyridinium and benzimidazolium chlorides. Eur. J. Med. Chem. 2001, 36, 313320. 15. Viscardi, G.; Quagliotto, P.; Barolo, C.; Savarino, P.; Barni, E.; Fisicaro, E. Synthesis and surface and antimicrobial properties of novel cationic surfactants. J. Org. Chem. 2000, 65, 8197–8203. 16. Kourai, H.; Takechi, H.; Horie, T.; Uchiwa, N.; Takeichi, K.; Shibasaki, I. The antimicrobial characteristics of quaternary ammonium salts. Part X. Antimicrobial characteristics and a mode of action of N-alkylpyridinium iodides against Escherichia coli. J. Antibact. Antifung. Agents 1985, 13, 3–10. 17. Kourai, H.; Takechi, H.; Horie, T.; Takeichi, K.; Shibasaki, I. The antimicrobialcharacteristics of quaternary ammonium salts. Part XI. Quantitative structure-activity relationship of antimicrobial N-laurylpyridinium iodides. J. Antibact., Antifung. Agents 1985, 13, 245–253. 18. Kourai, H.; Machikawa, F.; Horie, T.; Takeichi, K.; Shibasaki, I. The antimicrobial characteristics of quaternary ammonium salts. Part IX. Quantitative structure-activity correlation on antimicrobial activity and hydrophobicity of N-alkylypyridinium iodide derivatives. J. Antibact.Antifung. Agents 1983, 11, 553–562. 19. Kourai, H.; Manabe, Y.; Matsutani, E.; Hasegawa, Y.; Nakagawa, K. Antimicrobial activities of alkylallyldimethylammonium iodides andalkylallyldiethylammonium iodides. J. Antibact. Antifung. Agents. 1995, 23, 271–280. 20. Kourai, H.; Machikawa, F.; Horie, T.; Takeichi, K.; Shibasaki, I. The antimicrobial characteristics of quaternary ammonium salts. Part VII. Quantitative relation between antimicrobial activity and adsorbability of N-octylquinolinium iodide on Escherichia Coli K12. J. Antibact. Antifung. Agents 1983, 11, 51–54. 21. Maeda, T.; Goto, S.; Manabe, Y.; Okazaki, K.; Nagamune, H.; Kourai, H. Bactericidal action of N-alkylcyanopyridinium bromides against Escherichia Coli K12 W3110. Biocontrol Sci. 1996, 1, 41–49. 22. Okazaki, K.; Maeda, T.; Nagamune, H.; Kourai, H. Quantitative structure-activity relationship ofantibacterial dodecylpyridinium iodide derivatives. Biocontrol Sci.1996, 1, 51–59. 23. Shirai, A.; Maeda, T.; Nagamune, H.; Matsuki, H.; Kaneshina, S.; Kourai, H. Biological and physicochemical properties of gemini quaternary ammonium compounds in which the positions of a cross-linking sulfur in the spacer differ. Eur. J. Med. Chem. 2005, 40, 113–123. 24. Sridhara, S.K.; Pandeyab, S.N.; Stablesc, J.P.; Ramesha, A. Anticonvulsant activity of hydrazones, Schiff and Mannich bases of isatin derivatives. Eur. J. Pharm. Sci. 2002, 16,129–132. 25. Pandeya, S.N.; Sriram, D.; Nath, G.; De Clercq, E. Synthesis and antimicrobial and Mannich bases of isatin and its derivatives with pyrimidine. Farmaco 1999, 54, 624–628. 26. Vicini, P.; Zani, F.; Cozzini, P.; Doytchinova, I. Hydrazones of 1,2benzisothiazole hydrazides: synthesis, antimicrobial activity and QSAR investigations. Eur. J. Med. Chem. 2002, 37,553–564. 27. Gössnitzera, E.; Feierlb, G.; Wagnerb, U. Synthesis, structure investigations, and antimicrobial activity of selected s-trans-6-aryl-4-isopropyl-2-h2-[(E)-1phenylalkylidene]-(E)-hydrazino 1,4-dihydropyrimidine hydrochlorides. Eur. J. Pharm. Sci. 2002, 15, 49– 61. 28. Küçükgüzel, Ş.G.; Mazi, A.; Şahin, F.; Öztürk, S.; Stables, J.P. Synthesis and biological activities of diflunisal hydrazide-hydrazones. Eur. J. Med. Chem. 2003, 38, 1005–1009. 29. Loncle, C.; Brunel, J.; Vidal, N.; Dherbomez, M.; Letourneux, Y. Synthesis and antifungal activity of cholesterol-hydrazone derivatives. Eur. J. Med. Chem. 2004, 39, 1067–1071. 30. Masunari, A.; Tavares, L.C. A new class of nifuroxazide analogues: Synthesis of 5-nitrophene derivatives with antimicrobial activity against multidrug-resistant Staphylococcus aureus. Bioorg.Med. Chem. 2007, 15, 4229–4236. 31. Savini, L.; Chiasserini, L.; Gaeta, A.; Pellerano, C. Synthesis and anti-tubercular evaluation of quinolylhydrazones. Bioorg. Med. Chem. 2002, 10, 2193–2198. 32. Kaymakçıoğlu, B.K.; Rollas, S. Synthesis, characterization and evaluation of antituberculosis activity of some hydrazones. Il Farmaco. 2002, 57, 595– 599 33. Gemma, S.; Savini, L.; Altarelli, M.; Tripaldi, P.; Chiasserini, L.; Coccone, S.S.; Kumar, V.; Camodeca, C.; Campiani, G.; Novellino, E.; Clarizio, S.; Delogu, G.; Butini, S. Development of antitubercular compounds based on a 4quinolylhydrazone scaffold. Further structure–activity relationship studies. Bioorg. Med. Chem. 2009, 17, 6063–6072. 34. Pandey, J.; Pal, R.; Dwivedi, A.; Hajela, K. Synthesis of some new diaryl and triaryl hydrazone derivatives as possible estrogen receptor modulators. Arzneimittelforschung 2002, 52, 39–44. 35. Savini, L.; Chiasserini, L.; Travagli, V.; Pellerano, C.; Novellino, E.; Cosentino, S.; Pisano, M.B. New α-heterocyclichydrazones: evaluation of anticancer, anti-HIV and antimicrobial activity. Eur. J. Med. Chem. 2004, 39, 113–122. 36. Cocco, M.T.; Congiu, C.; Lilliu, V.; Onnis, V. Synthesis and in vitro antitumoral activity of new hydrazinopyrimidine-5-carbonitrile derivatives. Bioorg. Med. Chem. 2005, 14, 366–372. 37. Salgın-Gökşen, U.; Gökhan-Kelekçi, N.; Göktaş, Ö.; Köysal, Y.; Kılıç, E.; Işık, Ş.; Aktay, G.;Özalp, M. 1-Acylthiosemicarbazides, 1,2,4-triazole5(4H)-thiones, 1,3,4-thiadiazoles and hydrazones containing 5-methyl-2-benzoxazolinones: Synthesis, analgesic-anti-inflammatory and antimicrobial activities. Bioorg. Med. Chem. 2007, 15, 5738–5751. 38. Silva, G.A.; Costa, L.M.M.; Brito, F.C.F.; Miranda, A.L.P.; Barreiro, E.J.; Fraga, C.A.M. New class 24 Danish Scientific Journal No101, 2025 of potent antinociceptive and antiplatelet 10H-phenothiazine-1-acylhydrazone derivatives. Bioorg. Med. Chem. 2004, 12, 3149–3158. 39. Vicini, P.; Incerti, M.; La Colla, P.; Loddo, R. Anti-HIV evaluation of benzo[d]isothiazole hydrazones. Eur. J. Med. Chem. 2009, 44, 1801–1807. 40. Alptüzün, V.; Taşlı, H.; Erciyas, E. Synthesis and antimicrobial activities of some pyridinium salts. J. Fac. Pharm, Ankara. 2006, 35, 177–188. 41. Douglas, A.W.; Fisher, M.H.; Fishinger, J.J.; Gund, P.; Harris, E.E.; Olson, G.; Patchett, A.A.; Ruyle, W.V. Anticoccidial 1-Substituted 4(1H)-Pyridinone Hydrazones. J. Med. Chem. 1977, 20, 939–943. 42. Hegarty, A.F.; Moroney, P.J.; Scott, F.L. A change from rate-determining bromination to geometric isomerisation of pyridylhydrazones. J. Chem. Soc. Perkin Trans. 2 1973, 1466–1471. 43. Vereshchagina, N.N.; Postovskii, I. Trudy Ural’skogo Politekhnicheskogo Instituta im. S. M. Kirova. 1960, 94, 24–33. 44. Hesse, M.; Meier, H.; Zeeh, B. Spectroscopic Methods in Organic Chemistry; Enders, D., Noyori, R., Trost, B.M., Eds.; Georg Thieme Verlag Stuttgart: New York, NY, USA, 1997; p. 212. 45. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; eighteen informational supplement M100– S18. CLSI, Wayne, PA, USA, 2008. 46. The National Commmittee for Clinical Laboratory Standards (NCCLS). Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard-second edition, M27-A2. NCCLS, 2002, Wayne, PA, USA. 47. Mann, F.G.; Prior, A.F.; Willcox, T.J. The structure and properties of certain polycyclic indoloand quinolino-derivatives. Part XIII. The cyclisation of certain 4-pyridyland 4-quinolylhydraxones. J. Chem. Soc. 1959, 3830–3834. 48. Comrie, A.M.; Stenlake, J.B. Antituberculosis agens. Part III. Pyridine-4-sulphonamides and – sulphonhydrazides. J. Chem. Soc. 1958, 3514–3518.