Synthesis and antimicrobial activity of some pyridinium salts

Article abstract of DOI:10.3390/molecules14125203

Some substituted benzylidenehydrazinylpyridinium derivatives bearing benzyl, ethylphenyl and propylphenyl groups on the pyridinium nitrogen were synthesized and screened for possible antibacterial and antifungal activities against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans using the microdilution method. Antimicrobial test results indicated that compounds containing a 3-phenylpropyl chain displayed the highest antimicrobial activity against Staphylococcus aureus and the compound 3d was the most active in the series against all tested bacteria and fungi strains.

Full text of DOI:10.3390/molecules14125203

Molecules 2009, 14, 5203-5215; doi:10.3390/molecules14125203
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Antimicrobial Activity of Some Pyridinium Salts
Vildan Alptüzün 1,*, Sülünay Parlar 1, Hüseyin Taşlı 2 and Ercin Erciyas 1
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ege University, 35100, Bornova,
İzmir, Turkey; E-Mails: sulunay.parlar@ege.edu.tr (S.P.); ercin.erciyas@ege.edu.tr (E.E.)
2
Department of Microbiology, Faculty of Pharmacy, Ege University, 35100 Bornova, İzmir, Turkey;
E-Mail: huseyin.tasli@ege.edu.tr (H.T.)
* Author to whom correspondence should be addressed; E-Mail: vildan.alptuzun@ege.edu.tr;
Tel.: +90 232 3399931; Fax: +90 232 3885258.
Received: 16 November 2009; in revised form: 4 December 2009 / Accepted: 10 December 2009 /
Published: 14 December 2009
Abstract: Some substituted benzylidenehydrazinylpyridinium derivatives bearing benzyl,
ethylphenyl and propylphenyl groups on the pyridinium nitrogen were synthesized and
screened for possible antibacterial and antifungal activities against Staphylococcus aureus,
Escherichia coli, Pseudomonas aeruginosa and Candida albicans using the microdilution
method. Antimicrobial test results indicated that compounds containing a 3-phenylpropyl
chain displayed the highest antimicrobial activity against Staphylococcus aureus and the
compound 3d was the most active in the series against all tested bacteria and fungi strains.
Keywords: hydrazones; pyridinium salts; synthesis; antimicrobial activity
Introduction
The shortage of new antibacterial drugs and increasing resistance of bacteria to antimicrobial agents
are important issues in drug development studies. Quaternized amine derivatives were previously
reported to exert antimicrobial properties [1–4], and several mono- or bis-quaternary ammonium
compounds, including various alkyl chain lengths or the same long alkyl chain with different
hydrophobic substituents were found to exhibit antimicrobial activity [5,6]. The activities of the
quaternary ammonium compounds were attributed to their effect on the cell wall resulting in a direct or
indirect lethal effect on the cell viability [7].

Molecules 2009, 14
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Pyridinium halides like quaternary nitrogen salts have antimicrobial properties and adsorption
properties on negatively charged solids. The polar heads are cationic pyridiniums, that were related to
their antimicrobial activities and characteristics [8–15]. The antimicrobial activity of 1-alkyl-
pyridinium salts depends on the adsorptive activities on the surface of bacterial cells as well as their
destruction [16] and the pKa values of the corresponding pyridines [17]. Factors controlling their
antimicrobial activity are molecular hydrophobicity [18,19], adsorbability [20], surface activity [19]
and electron density [21,22] of the ammonium nitrogen atom. These compounds possess one
hydrophobic alkyl chain and one hydrophilic quaternary nitrogen ion group in the same molecule,
which provides a greater surface activity and more profound antimicrobial potency, 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 compounds within the Schiff bases, are also
known as one of the most important classes of organic compounds, some of which show significant
biological activities such as antimicrobial [26–30] antitubercular [31–33], anticancer [34–36],
analgesic [37], anti-inflammatory [37], antiplatelet [38] and antiviral [39] effects.
Previously, we synthesized pyridinium oxime-ether derivatives and evaluated them for their
antimicrobial activities [40]. The results showed that the R groups attached to the oxime-ether
sturucture did not cause a significant change in the antimicrobial effect; however, the side chain
attached to the pyridinium nitrogen noticeably affected the antimicrobial activity. Among them,
compounds with 3-phenylpropyl chains displayed a remarkable activity and 1-(3-phenylpropyl)-3-
[([(naphthyl-1-il)methoxy]imino]methyl]pyridinium bromide (NF-MFE, Figure 1) having a naphthlene
ring and a 3-phenylpropyl chain had the lowest MIC values, in other words the highest antimicrobial
activity [40].
Figure 1. Structure of NF-MFE.
Based on these findings, ortho -methyl, -methoxyl, -hydroxyl benzylidenehydrazinylpyridinium
salts with benzyl, 2,6-dichlorobenzyl, 2-phenylethyl and 3-phenylpropyl groups on pyridinium
nitrogen were prepared to investigate the effects of such structural modifications of quaternary
pyridinium salts on the anticipated antimicrobial activity. It was also planned to replace the oxime-
ether function and hydrazone function bioisosterically. Our study also covered the relationship
between antimicrobial activity and length of side alkyl chains of the compounds.

Molecules 2009, 14
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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
various substituted aromatic aldehydes in ethanol at room temperature to obtain the corresponding
hydrazone derivatives 2-4. In the last step, the final compounds 2a-4d were obtained by quaternation
of hydrazone derivatives 2-4 with the appropriate substituted alkyl halide in ethanol under reflux. All
title compounds are novel. With the exception of compound 4, the intermediate compounds 2-4 were
reported previously [42,43], but their spectral data have not been described in the literature.
Scheme 1. Synthesis of the title compounds.
The structures of the final compounds were determined by spectral analyses and the spectroscopic
properties were in accord with the proposed structures. The IR spectra of the final compounds showed
intense absorption bands within 3,315-3,446 cm-1 range and 1,436–1,644 cm-1 range that were
attributed to NH and C=N function vibrations, respectively. In the 1H NMR spectra, the proton signals

Molecules 2009, 14
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due to the NH group were recorded between 12.26–13.00 ppm. The proton signals belonging to the
N=CH group appeared as singlets at 8.52–8.65 ppm. This chemical shift suggested the E-isomer of the
compounds. Both IR and 1H-NMR data confirmed the condensation between the amino groups and the
carbonyl groups. As expected, while the pyridine hydrogens at the 2 and 6 positions of compounds 2–4
showed peaks between 8.19–8.21 ppm, the pyridinium hydrogen at these positions in the final
compounds 2a–4d showed peaks between 8.30–8.55 ppm [44]. The shifts of these peaks to a higher
frequency indicated that compounds 2–4 were quaternized with alkyl halide. All the 13C-NMR findings
confirmed the structures proposed as indicated in the Experimental section. The mass spectra of the
final compounds 2a–4d displayed the correct molecular ion peaks.
Antimicrobial Activity
The new series of benzylidenehydrazinylpyridinium salts with the hydroxyl, methyl, methoxyl
substituent in ortho position of benzene ring were evaluated for antimicrobial activity toward the Gram
positive and Gram negative bacteria and fungus. The bacterial strains represent important Gram
positive and Gram negative species, which are Staphylococcus aureus, Escherichia coli, and
Pseudomonas aeruginosa. Their antibacterial activities were assessed by measuring minimum
inhibitory concentration (MIC) with standard broth dilution assay (Table 1).
Table 1. Antimicrobial activity of compounds 2a–4d.
Minimum Inhibitory Concentration (MIC) (μg/mL)
Compound No
E. coli
P. aeruginosa
S. aureus
C. albicans
2a
64
1024
64
128
3a
32
512
16
64
4a
64
512
32
128
2b
>2,048
>2,048
>2,048
>2,048
512
32
>2,048
3b
>2,048
64
1,024
4b
>2,048
128
2,048
2c
64
32
64
3c
32
256
16
64
4c
2d
64
512
32
64
64
>2,048
256
8
32
3d
32
4
32
4d
128
1,024
1 (1–4)*
-
8
64
Ceftazidime
Fluconazole
<0.125 (0.06–0.5)*
-
4 (4–16)*
-
-
(0.25–1.0)*
* Acceptable quality control ranges of minimum inhibitory concentrations (MICs) (μg/mL) for reference
strains [45,46].
According to the antimicrobial activity results, all compounds were found to possess high
antimicrobial activity against Staphylococcus aureus and low antimicrobial activity against
Pseudomonas aeruginosa. Among the tested compounds, 2d, 3d, 4d were found to be the most active
derivatives against Staphylococcus aureus, being as effective against this organism as the standard

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compound ceftazidime. The results indicated that the longer the side chain of a compound, the more
antimicrobial activity it possesses. Among the benzene ring ortho-substitution series, methyl
derivatives are more active than the hydroxyl and methoxyl substituted derivatives. In the methyl
substituted series, the antimicrobial activity of the phenylpropyl derivative 3d was followed by the
phenylethyl derivative 3c, benzyl derivative 3a and dichlorobenzyl derivative 3b, respectively. No
meaningful difference in antibacterial activities were observed for compounds 3c and 3a. Both of them
had the same MIC values against test microorganisms, except against Pseudomonas aeruginosa.
According to the MIC values of the compounds, 3d had lowest MIC values (4 μg/mL) compared to the
other pyridinium salts. Moreover, the compounds 2d, 3d, 4d having the phenylpropyl side chain
showed higher activity than oxime-ether derivatives with the same side chain against Staphylococcus
aureus. This bioisosteric replacement resulted in a slightly increased antimicrobial activity. On the
other hand, compounds with a 2,6-dichlorobenzyl side chain on the pyridinium nitrogen displayed no
activity against the Gram negative bacteria. Our study revealed that all the compounds had stronger
antibacterial activity against Gram positive bacteria when compared to Gram negative bacteria. The
findings suggest that the pyridinium salts act on the cell membranes and surface activity of these
compounds may be chiefly responsible for the antibacterial properties of the compounds. However, all
the tested compounds exhibited low antifungal activity against Candida albicans. The reason for the
weaker antifungal activity according to antibacterial effect might be postulated as different action in
the mechanism of the compounds such as inhibition effect on respiratory systems of fungus cells,
rather than cell wall destruction.
Experimental
General
Melting points were determined with an Electrothermal IA9100 melting point apparatus and are not
1
corrected. H- and 13C-NMR spectra were recorded on a Varian AS 400 Mercury Plus NMR
instrument. Chemical shifts were measured in DMSO-d6 with TMS as internal reference.
Abbreviations for data quoted are: s, singlet; d, doublet; t, triplet; quin, quintet; dd, doublet of
doublets; m, multiplet. IR spectra of compounds were recorded as potassium bromide pellets on a
Jasco FT/IR-400 spectrometer. The electrospray ionization (ESI) mass spectra were measured on an
Agilent 1100 LC/MSD Trap. The conditions of the spray chamber were as follows: ion polarity,
positive; drying gas temperature, 300 °C; nebulizer pressure, 10 psi; drying gas flow, 5.00 L min−1.
Reagents and solvents used for synthesis were purchased from Aldrich, Fluka, and Merck companies.
Thin-layer chromatographies were carried out on pre-coated silica gel 60 F254 plates (Merck). The
spots were visualized with UV light or iodine.
Synthesis of 4-Hydrazinopyridine Hydrochloride (1)
Compound 1 was prepared according to the method reported by Mann et al. [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 and the precipitate was filtered, washed with cold 1-propanol
and crystallized from ethanol. Mp: 242–243 °C (lit. [47,48] 242–243 °C).

Molecules 2009, 14
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General Procedure for Synthesis of Benzylidenehydrazinylpyridine Derivatives 2–4
4-Hydrazinylpyridine (0.01 mol) and the appropriate benzaldehyde derivative (0.01 mol) were
stirred in ethanol (30 mL) at room temperature for 5–10 h. The precipitate was filtered and washed
with cool ethanol and crystallized from ethanol.
4-(2-(2-Hydroxybenzylidene)hydrazinyl)pyridine (2): Yield 58 %; mp: 194 °C; IR (ν, cm-1): 1,425,
1
1,500, 1,542, 1,598 (aromatic C=C and N=C), 3,004 (aromatic C-H), 3,525 (N-H, O-H); H-NMR δ:
6.84-6.89 (4H, m, Ar-H, pyridine-H), 7.19 (1H, td, J = 7.8, 1.6 Hz, Ar-H), 7.64 (1H, dd, J = 7.8, 1.6
Hz, Ar-H), 8.20 (2H, d, J = 5.9 Hz, pyridine-H), 8.24 (1H, s, N=CH), 10.20 (1H, s, OH), 10.79
(1H, s, NH).
4-(2-(2-Methylbenzylidene)hydrazinyl)pyridine (3): Yield 65 %; mp: 202 °C IR (cm-1): 1,427, 1,452,
1,486, 1,527, 1,538 (aromatic C=C and N=C), 2,886, 2,948 (aliphatic C-H), 3,014 (aromatic C-H),
3,210 (N-H); 1H-NMR δ: 6.97 (2H, d, J = 6.2 Hz, pyridine-H), 7.19-7.23 (3H, m, Ar-H), 7.83 (1H, d,
J = 8.2 Hz, Ar-H), 8.19 (1H, s, N=CH), 8.21 (2H, d, J = 6.2 Hz, pyridine-H) 10.80 (1H, s, NH).
4-(2-(2-Methoxybenzylidene)hydrazinyl)pyridine (4): Yield 69 %; mp: 205 °C; IR (ν, cm-1): 1,465,
1,492, 1,533, 1,592, (aromatic C=C and N=C), 2,834, 2,886, 2,940, 2,992 (aliphatic C-H), 3,083
1
(aromatic C-H), 3,212 (N-H); H-NMR δ: 3.83 (3H, s, -OCH3), 6.92 (2H, d, J = 6.4 Hz, pyridine-H),
6.98 (1H, t, J = 7.2 Hz, Ar-H), 7.05 (1H, d, J = 8.0 Hz, Ar-H), 7.32 (1H, td, J = 7.8, 1.6 Hz, Ar-H),
7.86 (1H, dd, J = 8.0, 1.6 Hz, Ar-H), 8.19 (2H, d, J = 6.0 Hz, pyridine-H), 8.29 (1H, s, N=CH), 10.78
(1H, s, NH).
General Procedure for Synthesis of the Final Compounds 2a-4d
A mixture of 2-4 (0.01 mol) and the corresponding alkyl halide (0.02 mol) were refluxed in ethanol
(30 mL) for 6-50 h. The mixture was cooled to room temperature or 0 °C and the obtained precipitate
was filtered and washed with cool ethanol. The crude products were crystallized from ethanol to give
compounds 2a–4d.
1-Benzyl-4-(2-(2-hydroxybenzylidene)hydazinyl)pyridinium chloride (2a): Yield 73%; mp: 293 °C; IR
(ν, cm-1): 1,454, 1,517, 1,552, 1,604 (aromatic C=C and N=C), 1,644 (N+=C), 2,844, 2,915 (aliphatic
1
C-H), 3,075 (aromatic C-H), 3,401 (N-H, O-H); H-NMR δ: 5.46 (2H, s, N+-CH2-Ph), 6.86 (1H, t,
J = 7.6 Hz, Ar-H), 6.96 (1H, d, J = 8.2 Hz, Ar-H), 7.14 (1H, dd, J = 7.0, 2.0 Hz, Ar-H), 7.27 (1H, td,
J = 7.8, 1.6 Hz, Ar-H), 7.36-7.41 (5H, m, Ar-H, pyridinium-H), 7.55 (1H, dd, J = 7.0, 2.3 Hz,
pyridinium-H), 7.81 (1H, d, J = 7.8 Hz, Ar-H), 8.43 (1H, d, J = 7.0 Hz, pyridinium-H), 8.52 (1H, d,
13
J = 7.0 Hz, pyridinium-H), 8.64 (1H, s, N=CH), 10.30 (1H, s, OH), 12.93 (1H, s, NH); C-NMR δ:
60.79 (CH2), 107.74 (CH), 109.64 (CH), 117.05 (CH), 120.26 (CH), 120.36 (qC), 126.73 (CH), 129.03
(CH), 129.31 (2 CH), 129.50 (2 CH), 132.77 (CH), 136.20 (qC), 143.62 (CH), 144.84 (CH), 145.82
(CH), 154.17 (qC), 157.57 (qC); ESI-MS m/z: 304.35 (M+).

Molecules 2009, 14
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1-(2,6-Dichlorobenzyl)-4-(2-(2-hydroxybenzylidene)hydrazinyl)pyridinium chloride (2b): Yield 68%;
mp: 288 °C; (ν, cm-1): 1,440, 1,477, 1,513, 1,548 (aromatic C=C and N=C), 1,643 (N+=C), 2,825,
2,892 (aliphatic C-H), 3,062 (aromatic C-H), 3,446 (N-H, O-H); 1H-NMR δ: 5.70 (2H, s, N+-CH2-Ph),
6.87 (1H, t, J = 7.8 Hz, Ar-H), 6.94 (1H, d, J = 8.4 Hz, Ar-H), 7.06 (1H, dd, J = 7.2, 2.4 Hz,
pyridinium-H), 7.28 (1H, td, J = 7.6, 1.6 Hz, Ar-H), 7.52-7.56 (2H, m, Ar-H, pyridinium-H), 7.64 (2H,
d, J = 8.0 Hz, Ar-H), 7.80 (1H, dd, J = 7.6, 1.6 Hz, Ar-H), 8.17 (1H, d, J = 7.6 Hz, pyridinium-H),
8.34 (1H, d, J = 7.6 Hz, pyridinium-H), 8.59 (1H, s, N=CH), 10.25 (1H, s, OH), 12.70 (1H, s, NH);
13C NMR δ: 56.16 (CH2), 107.52 (CH), 109.61 (CH), 117.10 (CH), 120.13 (CH), 120.33 (qC), 126.92
(CH), 130.12 (CH), 130.15 (2 CH), 132.79 (CH), 133.23 (qC), 136.92 (qC), 143.08 (CH), 144.62
(CH), 146.20 (CH), 154.19 (qC), 157.66 (qC); ESI-MS m/z: 372.08 (M+), 374.07 (M+2).
4-(2-(2-Hydroxybenzylidene)hydrazinyl)-1-phenethylpyridinium bromide (2c): Yield 83%; mp:
228 °C; IR (ν, cm-1): 1,455, 1,513, 1,550 (aromatic C=C and N=C), 1,644 (N+=C), 2,931, 2,971
(aliphatic C-H), 3,070 (aromatic C-H), 3,264 (O-H), 3,415 (N-H); 1H-NMR δ: 3.12 (2H, t, J = 7.0 Hz,
N+-CH2-CH2-Ph), 4.48 (2H, t, J = 7.0 Hz, N+-CH2-CH2-Ph), 6.85-6.97 (3H, m, Ar-H, pyridinium-H),
7.19-7.31 (6H, m, Ar-H), 7.49 (1H, dd, J = 7.0, 2.3 Hz, pyridinium-H), 7.83 (1H, dd, J = 7.8, 1.2 Hz,
Ar-H), 8.25 (1H, d, J = 7.0 Hz, pyridinium-H), 8.30 (1H, d, J = 7.0 Hz, pyridinium-H), 8.56 (1H, s,
13
N=CH), 10.16 (1H, s, OH), 12.27 (1H, s, NH); C-NMR δ: 36.92 (CH2), 59.05 (CH2), 107.33 (CH),
109.07 (CH), 117.00 (CH), 120.18 (CH), 120.34 (qC), 126.98 (CH), 127.59 (CH), 129.27 (2 CH),
129.59 (2 CH), 132.79 (CH), 137.34 (qC), 143.58 (CH), 144.28 (CH), 145.53 (CH), 153.96 (qC),
157.48 (qC). ESI-MS m/z: 318.42 (M+).
4-(2-(2-Hydroxybenzylidene)hydrazinyl)-1-(3-phenylpropyl)pyridinium bromide (2d): Yield 41%; mp:
197 °C; IR (ν, cm-1): 1,459, 1,492, 1,513, 1,515, 1,552 (aromatic C=C and N=C), 1,644 (N+=C), 2,832,
1
2,904 (aliphatic C-H), 3,031 (aromatic C-H), 3,419 (N-H, O-H); H-NMR δ: 2.12 (2H, quin,
J = 7.6 Hz, N+-CH2-CH2-CH2-Ph), 2.59 (2H, t, J = 8.0 Hz, N+-CH2-CH2-CH2-Ph), 4.26 (2H, t,
J = 7.4 Hz, N+-CH2-CH2-CH2-Ph), 6.88 (1H, t, J = 7.6 Hz, Ar-H), 6.93 (1H, d, J = 8.0 Hz, Ar-H), 7.03
(1H, d, J = 6.8 Hz, pyridinium-H), 7.15-7.30 (6H, m, Ar-H), 7.54 (1H, dd, J = 7.6, 2.4 Hz,
pyridinium-H), 7.84 (1H, dd, J = 7.6, 1.6 Hz, Ar-H), 8.37 (1H, d, J = 7.2 Hz, pyridinium-H), 8.44 (1H,
d, J = 7.6 Hz, pyridinium-H), 8.58 (1H, s, N=CH), 10.17 (1H, s, OH), 12.32 (1H, s, NH);
13C-NMR δ:32.24 (CH2), 32.50 (CH2), 58.01 (CH2), 107.50 (CH), 109.22 (CH), 117.00 (CH), 120.26
(CH), 120.36(qC), 126.73 (CH), 127.04 (CH), 128.89 (2 CH), 129.09 (2 CH), 132.73 (CH), 141.15
(qC), 143.50 (CH), 144.69 (CH), 145.41 (CH), 153.98 (qC), 157.42 (qC); ESI-MS m/z: 332.18 (M+).
1-Benzyl-4-(2-(2-methylbenzylidene)hydazinyl)pyridinium chloride (3a): Yield 71%; mp: 257 °C; IR
(ν, cm-1): 1,455, 1,482, 1,517, 1,544, 1,600, (aromatic C=C and N=C), 1,644 (N+=C), 2,838, 2,911,
2,979 (aliphatic C-H), 3,037 (aromatic C-H), 3,397 (N-H); 1H-NMR δ: 2.45 (3H, s, CH3), 5.48 (2H, s,
N+-CH2-Ph), 7.16 (1H, dd, J = 7.0, 2.3 Hz, pyridinium-H), 7.28 (1H, t, J = 6.6 Hz, Ar-H), 7.33 (1H, d,
J = 7.8 Hz, Ar-H), 7.36-7.42 (6H, m, Ar-H), 7.56 (1H, d, J = 7.4 Hz, pyridinium-H), 7.86 (1H, d,
J = 7.0 Hz, Ar-H), 8.47 (1H, d, J = 7.4 Hz, pyridinium-H), 8.55 (1H, d, J = 7.4 Hz, pyridinium-H),
13
8.62 (1H, s, N=CH), 13.00 (1H, s, NH); C-NMR δ: 20.13 (CH3), 60.75 (CH2), 107.80 (CH), 109.64
(CH), 126.96 (CH), 127.47 (CH), 128.78 (2 CH), 129.46 (CH), 129.78 (2 CH), 130.97 (CH), 131.78

Molecules 2009, 14
5210
(CH), 132.18 (qC), 136.20 (qC), 137.92 (qC), 143.72 (CH), 144.89 (CH), 147.99 (CH), 154.38 (qC);
ESI-MS m/z: 302.38 (M+).
1-(2,6-Dichlorobenzyl)-4-(2-(2-methylbenzylidene)hydrazinyl)pyridinium chloride (3b): Yield 72%;
mp: 290 °C; IR (ν, cm-1): 1,436, 1,513, 1,581 (aromatic C=C and N=C), 1644 (N+=C), 2,694, 2,886
1
(aliphatic C-H), 3,052 (aromatic C-H), 3,442 (N-H); H-NMR δ: 2.46 (3H, s, CH3), 5.70 (2H, s, N+-
CH2-Ph), 7.14 (1H, dd, J = 7.2, 2.8 Hz, pyridinium-H), 7.26-7.30 (2H, m, Ar-H), 7.34 (1H, td, J = 7.4,
1.2 Hz, Ar-H), 7.52-7.56 (2H, m, Ar-H, pyridinium-H), 7.64 (2H, d, J = 8.0 Hz, Ar-H), 7.84 (1H, dd,
J = 8.4, 1.6 Hz, Ar-H), 8.22 (1H, dd, J = 7.0, 1.6 Hz, pyridinium-H), 8.34 (1H, dd, J = 7.2, 1.6 Hz,
pyridinium-H), 8.60 (1H, s, N=CH), 13.00 (1H, s, NH); 13C-NMR δ: 20.12 (CH3) 56.44 (CH2), 108.29
(CH), 110.28 (CH), 125.29 (CH), 128.30 (CH), 129.22 (CH), 129.72 (CH), 130.00 (2 CH), 131.74
(CH), 133.16 (qC), 134.16 (qC), 137.00 (qC), 143.50 (CH), 144.66 (CH), 144.74 (CH), 148.69 (qC),
154.80 (qC); ESI-MS m/z: 370.10 (M+), 372.09 (M+2).
4-(2-(2-Methylbenzylidene)hydrazinyl)-1-phenethylpyridinium bromide (3c): Yield 80%; mp: 190 °C;
IR (ν, cm-1): 1,455, 1,513, 1,546, 1,577 (aromatic C=C and N=C), 1,644 (N+=C), 2,836, 2,908
1
(aliphatic C-H), 3,054 (aromatic C-H), 3,415 (N-H); H NMR δ: 2.45 (3H, s, CH3), 3.13 (2H, t,
J = 7.2 Hz, N+-CH2-CH2-Ph), 4.50 (2H, t, J = 7.2 Hz, N+-CH2-CH2-Ph), 7.03 (1H, dd, J = 7.0, 2.3 Hz,
pyridinium-H), 7.19-7.36 (8H, m, Ar-H), 7.50 (1H, dd, J = 7.0, 2.3 Hz, pyridinium-H), 7.88 (1H, dd,
J = 8.2, 1.6 Hz, Ar-H), 8.30 (1H, d, J = 7.4 Hz, pyridinium-H), 8.35 (1H, d, J = 7.0 Hz, pyridinium-
H), 8.53 (1H, s, N=CH), 12.38 (1H, s, NH); 13C NMR δ: 20.06 (CH3), 36.92 (CH2), 59.03 (CH2),
107.37 (CH), 108.99 (CH), 126.94 (CH), 127.36 (CH), 127.55 (CH), 129.23 (2 CH), 129.62 (2 CH),
130.96 (CH), 131.73 (CH), 132.12 (qC), 137.33 (qC), 137.89 (qC), 143.71 (CH), 144.84 (CH), 147.53
(CH), 154.10 (qC). ESI-MS m/z: 316.46 (M+).
4-(2-(2-Methylbenzylidene)hydrazinyl)-1-(3-phenylpropyl)pyridinium bromide (3d): Yield 45%; mp:
160 °C; IR (ν, cm-1): 1,454, 1,488, 1,511, 1,552, 1,585, (aromatic C=C and N=C), 1,644 (N+=C),
1
2,827, 2,915 (aliphatic C-H), 3,058 (aromatic C-H), 3,424 (N-H); H-NMR δ: 2.12 (2H, quin,
J = 7.6 Hz, N+-CH2-CH2-CH2-Ph), 2.48 (3H, s, CH3), 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, pyridinium-H), 7.16-7.30
(7H, m, Ar-H), 7.35 (1H, td, J = 7.2, 1.6 Hz, Ar-H), 7.56 (1H, dd, J = 6.8, 2.4 Hz, pyridinium-H), 7.89
(1H, dd, J = 7.6, 2.0 Hz, Ar-H), 8.39 (1H, d, J = 6.8 Hz, pyridinium-H), 8.46 (1H, d, J = 7.2 Hz,
pyridinium-H), 8.52 (1H, s, N=CH), 12.34 (1H, s, NH); 13C-NMR δ: 20.13 (CH3), 32.28 (CH2), 52.50
(CH2), 58.08 (CH2), 107.50 (CH), 109.15 (CH), 126.76 (CH), 126.98 (CH), 127.50 (CH), 128.89 (2
CH), 129.12 (2 CH), 131.00 (CH), 131.80 (CH), 132.14 (qC), 137.33 (qC), 141.15 (qC), 143.79 (CH),
144.80 (CH), 147.64 (CH), 154.29 (qC); ESI-MS m/z: 330.21 (M+).
1-Benzyl-4-(2-(2-methoxybenzylidene)hydazinyl)pyridinium chloride (4a): Yield 73%; mp: 244 °C; IR
(ν, cm-1): 1,479, 1,513, 1,546, 1,602 (aromatic C=C and N=C), 1,643 (N+=C), 2,748, 2,908, 2,971
(aliphatic C-H), 3,037 (aromatic C-H), 3,428 (N-H); 1H-NMR δ: 3.85 (3H, s, -OCH3), 5.47 (2H, s, N+-
CH2-Ph), 7.02 (1H, t, J = 7.4 Hz, Ar-H), 7.11 (1H, d, J = 8.2 Hz, Ar-H), 7.16 (1H, dd, J = 7.4, 2.7 Hz,
pyridinium-H), 7.37-7.46 (6H, m, Ar-H), 7.60 (1H, dd, J = 7.2, 2.7 Hz, pyridinium-H), 7.94 (1H, dd,

Molecules 2009, 14
5211
J = 7.8, 1.6 Hz, Ar-H), 8.45 (1H, d, J = 7.0 Hz, pyridinium-H), 8.54 (1H, d, J = 7.0 Hz, pyridinium-
13
H), 8.65 (1H, s, N=CH), 13.00 (1H, s, NH); C-NMR δ: 56.55 (OCH3), 60.82 (CH2), 107.96 (CH),
109.76 (CH), 112.84 (CH), 121.52 (CH), 122.04 (qC), 126.49 (CH), 128.72 (2 CH), 129.48 (CH),
129.80 (2 CH), 133.05 (CH), 136.27 (qC), 143.74 (CH), 144.43 (CH), 144.86 (CH), 154.61 (qC),
158.65 (qC); ESI-MS m/z: 318.41 (M+).
1-(2,6-Dichlorobenzyl)-4-(2-(2-methoxybenzylidene)hydrazinyl)pyridinium chloride (4b): Yield 79%;
mp: 274 °C; IR (ν, cm-1): 1,434, 1,459, 1,479, 1,513, 1,542, 1,602, (aromatic C=C and N=C), 1,641
1
(N+=C), 2,724, 2,904 (aliphatic C-H), 3,064 (aromatic C-H), 3,315 (N-H); H-NMR δ: 3.86 (3H, s,
-OCH3), 5.71 (2H, s, N+-CH2-Ph), 7.01-7.04 (2H, m, Ar-H, pyridinium-H), 7.12 (1H, d, J = 8.0 Hz,
Ar-H), 7.45 (1H, td, J = 7.8, 1.6 Hz, Ar-H), 7.54 (1H, dd, J = 8.8, 7.2 Hz, Ar-H), 7.58 (1H, dd,
J = 7.6, 2.8 Hz, pyridinium-H), 7.64 (2H, d, J = 8.0 Hz, Ar-H), 7.91 (1H, dd, J = 7.6, 1.6 Hz, Ar-H),
8.17 (1H, d, J = 7.2 Hz, pyridinium-H), 8.34 (1H, dd, J = 7.2, 1.6 Hz, pyridinium-H), 8.59 (1H, s,
N=CH), 12.62 (1H, s, NH); 13C-NMR δ: 56.23 (CH2), 56.54 (OCH3), 107.77 (CH), 109.70 (CH),
112.76 (CH), 121.52 (CH), 122.11 (qC), 130.10 (CH), 130.16 (2 CH), 132.99 (CH), 133.09 (CH),
133.25 (qC), 136.92 (qC), 143.20 (CH), 144.68 (CH), 144.75 (CH), 154.33 (qC), 158.67 (qC). ESI-MS
m/z: 386.10 (M+), 388.09 (M+2).
4-(2-(2-Methoxybenzylidene)hydrazinyl)-1-phenethylpyridinium
bromide
(4c):
Yield
95%;
mp: 201 °C; IR (ν, cm-1): 1,477, 1,513, 1,548, 1,600, (aromatic C=C and N=C), 1,644 (N+=C), 2,836,
1
2,904 (aliphatic C-H), 3,064 (aromatic C-H), 3,421 (N-H); H-NMR δ: 3.13 (2H, t, J = 7.2 Hz, N+-
CH2-CH2-Ph), 3.86 (3H, s, -OCH3), 4.49 (2H, t, J = 7.2 Hz, N+-CH2-CH2-Ph), 6.94 (1H, dd, J = 7.2,
2.3 Hz, pyridinium-H), 7.02 (1H, t, J = 7.4 Hz, Ar-H), 7.12 (1H, d, J = 8.6 Hz, Ar-H), 7.18-7.31 (5H,
m, Ar-H), 7.45 (1H, td, J = 7.8, 1.2 Hz, Ar-H), 7.54 (1H, dd, J = 7.2, 2.3 Hz, pyridinium-H), 7.95 (1H,
dd, J = 7.6, 1.6 Hz, Ar-H), 8.25 (1H, d, J = 7.0 Hz, pyridinium-H), 8.31 (1H, d, J = 7.0 Hz,
pyridinium-H), 8.57 (1H, s, N=CH), 12.26 (1H, s, NH); 13C-NMR δ: 36.92 (CH2), 56.57 (OCH3),
59.06 (CH2), 107.45 (CH), 109.10 (CH), 112.73 (CH), 121.52 (CH), 122.10 (qC), 126.52 (CH), 127.59
(CH), 129.36 (2 CH), 129.61 (2 CH), 132.99 (CH), 137.34 (qC), 143.68 (CH), 144.13 (CH), 144.76
(CH), 154.09 (qC), 158.62 (qC); ESI-MS m/z: 332.46 (M+).
4-(2-(2-Methoxybenzylidene)hydrazinyl)-1-(3-phenylpropyl)pyridinium bromide (4d): Yield 59%;
°
mp: 165 C; IR (ν, cm-1): 1,457, 1,511, 1,546, 1,571, 1,598, (aromatic C=C and N=C), 1644 (N+=C),
1
2,832, 2,908, 2,962 (aliphatic C-H), 3,066 (aromatic C-H), 3,421 (N-H); H-NMR δ: 2.12 (2H, quin,
J = 7.6 Hz, N+-CH2-CH2-CH2-Ph), 2.59 (2H, t, J = 7.8 Hz, N+-CH2-CH2-CH2-Ph), 3.86 (3H, s,
-OCH3), 4.26 (2H, t, J = 7.4 Hz, N+-CH2-CH2-CH2-Ph), 6.98-7.05 (2H, m, Ar-H, pyridinium-H), 7.12
(1H, d, J = 8.0 Hz, Ar-H), 7.16-7.30 (5H, m, Ar-H), 7.45 (1H, td, J = 7.6, 1.6 Hz, Ar-H), 7.59 (1H, dd,
J = 7.0, 2.4 Hz, pyridinium-H), 7.96 (1H, dd, J = 7.4, 1.6 Hz, Ar-H), 8.36 (1H, d, J = 7.2 Hz,
13
pyridinium-H), 8.44 (1H, d, J = 7.6 Hz, pyridinium-H), 8.58 (1H, s, N=CH), 12.30 (1H, s, NH); C-
NMR δ: 32.25 (CH2), 32.55 (CH2), 56.53 (OCH3), 58.08 (CH2), 107.64 (CH), 109.25 (CH), 112.65
(CH), 121.51 (CH), 122.11 (qC), 126.46 (CH), 126.72 (CH), 128.89 (2 CH), 129.08 (2 CH), 132.94
(CH), 141.18 (qC), 143.59 (CH), 143.87 (CH), 144.72 (CH), 154.13 (qC), 158.55 (qC); ESI-MS m/z:
346.21 (M+).

Molecules 2009, 14
5212
Antimicrobial Activity
The minimal inhibitory concentration (MIC) was determined by the broth microdilution method
according to the Clinical and Laboratory Standards Institute (CLSI ) [45,46]. In vitro antimicrobial
activity of the final compounds 2a–4d was evaluated against standard strains; Staphylococcus aureus
ATCC 29213, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Candida
albicans ATCC 90028. The antibacterial and antifungal assay were performed in Mueller–Hinton
broth and Sabouraud dextrose broth, respectively. All the synthesized compounds were weighed
(10 mg), dissolved in DMSO (250 μL) and diluted with water (750 μL) to prepare the stock solutions
of 10 mg/mL. The serial dilution from 2048 to 1 μg/mL was made in a 96-well plate. Fifty μL of a
bacterial suspension, obtained from a 24 h 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 h. We tested ceftazidime as
antimicrobial agents for quality control of the method. Each experiment was carried out in duplicate.
Conclusions
In summary, we have described the synthesis of a number of benzylidenehydrazinylpyridinium salts
2a–4d with antimicrobial activity. The final synthesized compounds were characterized by spectral
1
data (IR, H-NMR, 13C-NMR, ESI-MS). Both the synthesis and antimicrobial activity of the final
compounds 2a–4d are for the first time. The compounds 2a–4d were found to have reasonable activity
against S. aureus. A remarkable activity were found in compounds carrying a 3-phenylpropyl side
chain on the nitrogen. The most active compound was 4-(2-(2-methylbenzylidene)hydrazinyl)-1-(3-
phenylpropyl)pyridinium bromide (3d), having MIC value of 4 μg/mL against S. aureus. The results
indicated that the longer side chain on pyridinium nitrogen caused increased activity. Further
investigations of this effect are in progress.
Acknowledgements
This study was supported by Ege University Scientific Research Project Council (Project number:
07/ECZ/007).
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. Synthesis
and antimicrobial activity of N-alkylpyridinium podands. Khim. Farm. Zh. 2003, 37, 17–19.

Molecules 2009, 14
5213
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,ω-polymethylenedithio)bis-(1-alkylpyridinium 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(1-alkylpyridinium 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-1-
decylpyridinium 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
characteristics 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, 313–320.
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 antimicrobial characteristics 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.

Molecules 2009, 14
5214
19. Kourai, H.; Manabe, Y.; Matsutani, E.; Hasegawa, Y.; Nakagawa, K. Antimicrobial activities of
alkylallyldimethylammonium iodides and alkylallyldiethylammonium 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 of
antibacterial 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 activity of Schiff
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 1,2-benzisothiazole 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)-1-phenylalkylidene]-(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 4-quinolylhydrazone 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.

Molecules 2009, 14
5215
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-triazole-5(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 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 indolo-
and quinolino-derivatives. Part XIII. The cyclisation of certain 4-pyridyl- and 4-quinolyl-
hydraxones. 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.
Sample Availability: Samples of compounds 2a-2d, 3a-3d, 4a-4d are available from the corresponding
author.
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).