Synthesis and Antimicrobial Evaluation of New Schiff Base Hydrazones Bearing 1,2,4-Triazole Moiety

Article abstract of DOI:10.1080/10426507.2013.789878

This study presents the synthesis and spectral analysis of new Schiff base hydrazone derivatives. New compounds were prepared by the reaction of [(4-phenyl-5-substituted-4H-1,2,4-triazol-3-yl)sulfanyl] acetohydrazide with various aldehydes. The structures

Full text of DOI:10.1080/10426507.2013.789878

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Phosphorus, Sulfur, and Silicon and the Related
Elements
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Synthesis and antimicrobial evaluation of new Schiff
base hydrazones bearing 1,2,4-triazole moiety
Łukasz Popiołek a , Urszula Kosikowska b , Monika Wujec a & Anna Malm b
a Department of Organic Chemistry, Faculty of Pharmacy , Medical University , 4A Chodzki
Str, 20–093 , Lublin , Poland
b Department of Pharmaceutical Microbiology, Faculty of Pharmacy , Medical University , 1
Chodzki Str, 20–093 , Lublin , Poland
Accepted author version posted online: 02 Sep 2013.
To cite this article: Phosphorus, Sulfur, and Silicon and the Related Elements (2013): Synthesis and antimicrobial evaluation of
new Schiff base hydrazones bearing 1,2,4-triazole moiety, Phosphorus, Sulfur, and Silicon and the Related Elements
To link to this article: http://dx.doi.org/10.1080/10426507.2013.789878
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Synthesis and antimicrobial evaluation
of new Schiff base hydrazones bearing 1,2,4-triazole moiety
Łukasz Popiołek1, Urszula Kosikowska2, Monika Wujec1 and Anna Malm2
1Department of Organic Chemistry, Faculty of Pharmacy, Medical University, 4A Chodzki Str.,
20-093 Lublin, Poland;
2Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University,
1 Chodzki Str, 20-093 Lublin, Poland;
Email: lukasz.popiolek@umlub.pl
Abstract
This study presents the synthesis and spectral analysis of new Schiff base hydrazone derivatives.
New compounds were prepared by the reaction of [(4-phenyl-5-substituted-4H-1,2,4-triazol-3-
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yl)sulfanyl]acetohydrazide with various aldehydes. The structures of the prepared compounds
1
was confirmed by means of H NMR, 13C NMR and elemental analyses. All synthesized
compounds were screened for their in vitro antimicrobial activity by using the agar dilution
technique against 4 reference strains of Gram-positive and Gram-negative bacteria and 12 yeasts
belonging to Candida spp.
Keywords: antimicrobial activity, Schiff base hydrazone derivatives, 1,2,4-triazole derivatives
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Introduction
The Schiff base hydrazone nucleus has attracted great and growing interest in chemistry and
biology for many years due to its facile synthesis and wide applications [1-4].
We report here the synthesis and spectral, as well as antimicrobial characterization, of new
Schiff base hydrazones obtained by the condensation reaction of substituted acetohydrazides
containing 1,2,4-triazole system with various aldehydes.
Schiff bases have gained importance because of their pharmacological properties such as
antibacterial [5-7], antifungal [8-10], anticancer [11, 12] and antiviral agents [13, 14]. Similarly,
the occurrence of 1,2,4-triazole system in numerous biologically active molecules has been
recognized to possess activities such as analgesic [15], antibacterial [16-18], antifungal [19, 20],
anti-inflammatory [21], antitumor [22, 23] and antitubercular [24].
Additionally, some of the present day drugs such as Ribavirin (antiviral agent) [25],
Rizatriptan (anitmigraine agent) [26], Alprazolam (anxiolytic agent) [27], Fluconazole and
Intraconazole (antifungal agents) [28] are the best examples of potent molecules possessing
triazole nucleus.
Since the 1,2,4-triazole moiety seems to be a possible pharmacophore in various
pharmacologically active agents, we decided to synthesize compounds with this function coupled
with Schiff bases. The choice of these ligands was motivated by two objectives. First, to provide
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new 1,2,4-triazole derivatives and to enrich the biological study of Schiff base hydrazones
containing 1,2,4-triazole moieties.
In the view of these facts, the aim of this study is to obtain new Schiff base hydrazone
derivatives incorporating the 1,2,4-triazole moiety as antimicrobial agents with a view to obtain
better therapeutic results.
Results and discussion
Chemistry
The synthetic pathway leading to the new Schiff base hydrazone derivatives 5-28 was carried
out according to the steps shown in Scheme 1. The substituents of compounds 5-28 are presented
in Table 1. In the initial step, ethyl [(4-phenyl-5-substituted-4H-1,2,4-triazol-3-
yl)sulfanyl]acetates were synthesized via reaction of 4-phenyl-5-substituted-4H-1,2,4-triazol-3-
thiones with ethyl bromoacetate in the presence of sodium ethanolate. The treatment of esters
with hydrazine hydrate afforded the corresponding acetohydrazides 1-4. The condensation
reaction of [(4-phenyl-5-substituted-4H-1,2,4-triazol-3-yl)sulfanyl] acetohydrazides with various
alkyl and aryl aldehydes, in the presence of glacial acetic acid in ethanol, gave good yield of new
products 5-28. Compounds 11, 12, 13, 16, 17, 18, 20, 22, 23, 24, 25, 27, 28 are registered in CAS
(Chemical Abstracts Service) database but without references and methods of synthesis.
[Insert Scheme 1]
[Insert Table 1]
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All the newly obtained compounds are air stable solids and soluble in DMSO at ambient
temperature. The purity of the synthesized compounds was checked by elemental analyses and
thin layer chromatography. The structures of prepared derivatives were determined on the basis
1
of H NMR and 13C NMR spectra and all of the synthesized compounds have satisfactory
1
analyses for their proposed structures. H NMR spectral results for all compounds together with
hydrogen assignments and 13C NMR spectra results are presented in Experimental Section.
1
In the H NMR spectra of the compounds 5-28 the singlet peak due to the hydrazone proton
(NH) appeared in the region of δ 8.11-11.98 ppm, which confirmed the successful formation of
the desired products, whereas the singlet signal for -S-CH2- protons was observed in the region
of δ 3.43-4.55 ppm. All other aliphatic and aromatic protons were observed at the expected
regions.
13
In the C NMR spectra of 5-28 derivatives the carbon of -S-CH2- group had a typical signal
at about 30 ppm. Similarly, the presence of the =CH group was also confirmed by a signal at
about 150 ppm and for C=O group at 166 ppm. All other aliphatic and aromatic protons were
observed at expected regions.
Microbiology
According to our screening results obtained by agar dilution method with 1000 mg/L of the
newly synthesized compounds, no activity against the reference strains of Gram-positive or
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Gram-negative bacteria was found. Among the tested compounds, only compound 5 exhibited
moderate inhibitory effect against the reference strains of yeasts, as monitored by an appearance
of visible growth reduction. On the basis of the MIC values obtained by microdilution method it
was shown that compound 5 had some, incubation-time dependent activity against both reference
strains and clinical isolates of Candida albicans (Table 2). After 24 h incubation, MIC ranged
from 62.5 to 250 mg/L, while after 48 h incubation - from 250 to ≥1000 mg/L. C. parapsilosis
was included as a recommended reference strain for testing antifungals with activity against
Candida spp. MIC values in the case of this strain were 125 or 250 mg/L after 24 or 48 h
incubation, respectively.
The opportunistic yeasts belonging to Candida spp., mainly Candida albicans, are the source
of invasive or non-invasive yeast infections in patients, especially those with compromised
immune system function and those hospitalized with serious underlying diseases and their high
mortality [29-35]. In general, the use of the available antifungals is limited because of their
toxicity, side effects, drug interactions, limited routes, and the emergence of drug-resistant and
drug-low-susceptible strains.
MIC is mainly used to establish the activity of potentially antimicrobial agents against e.g. the
tested fungi and is important for clinical or epidemiological studies. According to CLSI and
EUCAST definitions and literature data [25-38], MIC obtained after only 24 h of incubation
should be used to determine the antifungal effect of widely used antifungal agents (e.g.
fluconazole, voriconazole). According to Pfaller et al. [36, 39, 40] and Ostrosky-Zeichner et al.
[41], the earlier reading of e.g. fluconazole predicted therapeutic outcomes and success as
accurately as the 48 h MICs. Based on the experimental data, the Subcommittees of CLSI and
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EUCAST have included the option to read MICs for Candida species after 24 h incubation.
According to Pfaller et al. [39], the 24h MIC test reliably identifies fluconazole resistance among
Candida spp.
Data presented in this paper indicate that compound 5 possessed some activity against the
tested C. albicans strains. However, after the prolonged to 48 h incubation, a significant decrease
of the antifungal activity of derivative 5 was found, as monitored by an increase of MIC values
from 4 to >16 times in the second reading, depending on the strain. The effect of the prolonged
incubation in the presence of antifungal agents increasing the MIC value has already been
described in the literature [42, 43]. As found by other authors [42, 43], the influence of the
incubation time as a test variable may depend on the nature of the preparation, e.g. the various
liposomal formulations of amphotericin B. The specific formulation of this antifungal may
influence the mechanism of action and/or the intracellular concentration at the site of antifungal
action. It is probable that our observation may be due to a decrease of the compound
concentration within yeast cells belonging to C. albicans, leading to an increase of MIC values.
This problem requires further studies, essential for searching new derivatives showing better and
stable antifungal activity.
Conclusions
1
In the present paper, we synthesized and characterized by H NMR, 13C NMR and elemental
analyses a new series of acetohydrazide derivatives. All the newly prepared compounds were
screened for their antimicrobial activity by agar dilution technique against 4 reference strains of
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Gram-positive and Gram-negative bacteria and 12 yeasts belonging to Candida spp.
Antimicrobial study revealed that compounds had no activity against the reference strains of
Gram-positive or Gram-negative bacteria, but derivative - 2-[(4-phenyl-4H-1,2,4-triazol-3-
yl)sulfanyl]-N-[(2-hydroxyphenyl)methylidene]acetohydrazide (5) exhibited moderate inhibitory
effect against the reference strains of Candida spp. yeasts.
Experimental
General
All reagents were purchased from Sigma-Aldrich (Munich, Germany) and Merck Co.
(Darmstadt, Germany) and used without further purification. Melting points were determined in
Fisher-Johns blocks (Fisher Scientific, Schwerte, Germany) and presented without any
corrections. The IR spectra (ν, cm-1) were recorded in KBr tablets using a Specord IR-75
spectrophotometer (Carl Zeiss, Jena, Germany). The 1H NMR spectra were recorded on a Bruker
Avance 300 apparatus (Bruker BioSpin GmbH, Rheinstetten/Karlsruhe, Germany) in DMSO-d6
13
with TMS as internal standard. The C NMR spectra were recorded on a Bruker Avance 300
apparatus. Chemical shifts are given in ppm (δ-scale). The purity of obtained compounds was
checked by TLC on aluminium oxide 60 F254 plates (Merck Co. Whitehouse Station, New
Jersey, USA), in a CHCl3/C2H5OH (10:1, v/v) solvent system. The spots were detected by
exposure to a UV lamp at 254 nm. Elemental analyses of the obtained compounds was
performed for C, H, N on AMZ 851 CHX analyser (PG, Gdańsk, Poland). The maximum
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percentage differences between calculated and found values for each element were within the
error and amounted to ± 0.4%.
Chemistry
Preparation of the compounds
Preparation of [(4-phenyl-5-substituted-4H-1,2,4-triazol-3-yl)sulfanyl]acetohydrazide (1-4)
The 100% hydrazine hydrate (10 mmol) was added to ethyl [(4-phenyl-5-substituted-4H-1,2,4-
triazol-3-yl)sulfanyl]acetate (10 mmol) in anhydrous ethanol (10 mL). The mixture was put to a
refrigerator for 24 h. After that, the precipitation of hydrazide (1-4) was filtered off, dried and
crystallized from ethanol [44-47].
[(4-Phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetohydrazide (1)
1
Yield: 78%; mp 132°C–134°C (dec.). H NMR (DMSO-d6): δ (ppm) = 3.89 (s, 2H, CH2); 4.29
(s, 2H, NH2); 7.51–7.61 (m, 5H, Ar-H); 8.86 (s, 1H, CH); 9.33 (s, 1H, NH). Anal. calcd. for
C10H11N5OS (249.29): C, 48.14; H, 4.44; N, 28.08; Found: C, 48.16; H; 4.41; N, 28.05%.
[(4-Phenyl-5-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetohydrazide (2)
Yield: 72.8%; mp. 86°C–88°C (dec.). IR (KBr), ν (cm−1): 3089 (CH aromatic), 2955, 1444 (CH
1
aliphatic), 1711 (C=O), 1601 (C=N), 1503 (C-N), 688 (C-S). H NMR (DMSO-d6) δ (ppm) =
2.19 (s, 3H, CH3), 3.79 (s, 2H, CH2), 4.21 (s, 2H, NH2), 7.35-7.64 (m, 5H, Ar-H), 9.30 (s, 1H,
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NH). Anal. calcd. for C11H13N5OS (263.32: C, 50.17; H, 4.98; N, 26.60; Found: C, 50.04; H,
4.96; N, 26.51%.
[(4,5-Diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetohydrazide (3)
Yield: 91.4%, mp. 196°C–198°C (dec.). IR (KBr), m (cm-1): 3105 (CH aromatic), 2980, 1423
(CH aliphatic), 1698 (C=O), 1611 (C=N), 1522 (C–N), 699 (C–S). 1H NMR (DMSO-d6) δ (ppm)
= 3.91 (s, 2H,CH2), 4.31 (s, 2H, NH2), 7.31-7.57 (m, 10H, Ar-H), 9.40 (brs, 1H, NH). Anal.
calcd. for C16H15N5OS (325.39); C, 59.06; H, 4.65; N, 21.52; found: C, 59.10; H, 4.63; N,
21.49%.
[(4-Phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]acetohydrazide (4)
Yield: 90.2%; mp. 202°C–206°C (dec.). IR (KBr), ν (cm−1): 3079 (CH aromtic), 2977, 1455 (CH
aliphatic), 1672 (C=O), 1608 (C=N), 1502 (C-N), 673 (C-S). 1H NMR (DMSO-d6): δ = 3.93 (s,
2H, CH2), 4.31 (s, 2H, NH2), 7.37–8.58 (m, 9H, Ar-H), 9.38 (s, 1H, NH). Anal. calcd. for
C15H14N6OS (326.4): C, 55.20; H, 4.32; N, 25.75; Found: C, 55.16; H, 4.35; N, 25.80%.
Preparation of acetohydrazide derivatives
To a suspension of hydrazide (1-4) (10 mmol) in ethanol (20 mL), an equimolar of various
aldehydes (10 mmol) was added. The suspension was heated until clear solution was obtained.
Then few drops of glacial acetic acid were added as a catalyst. The solution was refluxed for 4
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hrs. After the completion of the reaction, the solution was cooled to room temperature. The
obtained precipitate was filtered off and crystallized from ethanol.
2-[(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[(2-hydroxyphenyl)
methylidene]acetohydrazide (5)
1
Yield: 66%; mp. 124°C–126°C (dec.). H NMR (DMSO-d6): δ (ppm) = 4.15 (s, 2H, CH2); 4.54
(s, 1H, OH); 6.90-7.73 (m, 9H, Ar-H); 8.40 (s, 1H, CH); 8.46 (s, 1H, CH); 8.94 (s, 1H, NH). 13C
NMR: δ (ppm) = 30.1 (-S-CH2-), 117.4, 120.4, 121.2, 125.6, 128.5, 128.70, 129.2, 129.7, 137.5
(11Car), 147.5 (C-3 triazole), 150.2 (=CH), 154.5 (C-5 triazole), 158.3 (Car), 166.5 (C=O). Anal.
calcd. for C17H15N5O2S (353.4): C, 57.78; H, 4.28; N, 19.82; Found: C, 57.82; H, 4.22; N,
19.85%.
2-[(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[(4-methylphenyl)
methylidene]acetohydrazide (6)
1
Yield: 63%; mp. 130°C–132°C (dec.). H NMR (DMSO-d6): δ (ppm) = 2.39 (s, 3H, CH3); 4.13
(s, 2H, CH2); 7.22-8.20 (m, 9H, Ar-H); 8.20 (s, 1H, CH); 8.93 (s, 1H, NH); 8.98 (s, 1H, CH). 13C
NMR: δ (ppm) = 21.2 (-CH3), 30.2 (-S-CH2), 125.6, 127.3, 127.8, 129.1, 131.9, 137.5, 138.5
(12Car), 147.5 (C-3 triazole), 148.6 (=CH), 154.4 (C-5 triazole), 166.0 (C=O). Anal. calcd. for
C18H17N5OS (351.42): C, 61,52; H, 4.88; N, 19.93; Found: C, 61.55; H, 4.89; N, 19.90%.
2-[(4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[(4-methoxyphenyl)
methylidene]acetohydrazide (7)
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1
Yield: 68%; mp. 166°C–168°C (dec.). H NMR (DMSO-d6): δ (ppm) = 3.88 (s, 3H, CH3); 4.13
(s, 2H, CH2); 6.98-8.07 (m, 9H, Ar-H); 8.18 (s, 1H, CH); 8.69 (s, 1H, NH); 8.94 (s, 1H, CH). 13C
NMR: δ (ppm) = 30.1 (-S-CH2-), 56.1 (-CH3), 114.6 (2Car), 125.6, 127.1, 128.7, 129.1, 129.2,
137.5 (9Car), 147.5 (C-3 triazole), 148.6 (=CH), 154.5 (C-5 triazole), 160.1 (Car), 166.1 (C=O).
Anal. calcd. For C18H17N5O2S (367.42): C, 58.84; H, 4.66; N, 19.06; Found: C, 58.87; H, 4.63;
N, 19.09%.
N-[(4-bromophenyl)methylidene]-2-[(4-phenyl-4H-1,2,4-triazol-3-
yl)sulfanyl]acetohydrazide (8)
Yield: 75%; mp. 140°C–142°C (dec.). 1H NMR (DMSO-d6): δ (ppm) = 4.07 (s, 2H, CH2); 7.50-
8.03 (m, 9H, Ar-H); 8.21 (s, 1H, CH); 8.85 (s, 1H, CH); 11.71 (s, 1H, NH). 13C NMR: δ (ppm) =
30.1 (-S-CH2-), 123.9, 125.6, 128.8, 129.1, 129.3, 132.4, 132.5, 137.5 (12Car), 147.5 (C-3
triazole), 148.7 (=CH), 154.6 (C-5 triazole), 166.1 (C=O). Anal. calcd. for C17H14BrN5OS
(416.29): C, 49.05; H, 3.39; N, 16.82; Found: C, 49.01; H, 3.40; N, 16.85%.
2-[(5-methyl-4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]-N-(propylidene)acetohydrazide
(9)
Yield: 25%; mp. 100°C–102°C (dec.). 1H NMR (DMSO-d6): δ (ppm) = 1.02 (t, J = 7.5 Hz, 3H,
CH3), 2.16 (s, 3H, CH3), 2.18-2.26 (m, 2H, CH2), 4.23 (s, 2H, CH2), 7.28-7.62 (m, 5H, Ar-H),
7.89 (t, J = 5.0 Hz, 1H, =CH), 11.20 (s, 1H, NH). 13C NMR: δ (ppm) = 9.5 (-CH3), 12.4 (-CH3),
12.3 (-CH2-), 30.2 (-S-CH2), 128.3, 129.9, 130.2, 136.2 (6Car), 145.6 (=CH), 156.0 (C-5
triazole); 158.4 (C-3 triazole), 166.1 (C=O). Anal. calcd. for C14H17N5OS (303.38): C, 55.42; H,
5.65; N, 23.08; Found: C, 55.38; H, 5.69; N, 23.15%.
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2-[(5-methyl-4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]-N-(2-methylpropylidene)
acetohydrazide (10)
Yield: 70%; mp. 132°C–134°C (dec.). 1H NMR (DMSO-d6): δ (ppm) = 1.06 (d, J = 2.5 Hz, 3H,
CH3), 1.10 (d, J = 2,5 Hz, 3H, CH3), 2.25 (s, 3H, CH3), 2.42-2.48 (m, 1H, CH), 4.29 (s, 2H,
13
CH2), 7.30-7.69 (m, 5H, Ar-H), 7.99 (d, J = 5.0 Hz, 1H, =CH), 11.21 (s, 1H, NH). C NMR: δ
(ppm) = 12.4 (-CH3), 18.2 (2xCH3), 29.7 (-CH), 30.1 (-S-CH2-), 128.4, 129.9, 130.3, 136.3
(6Car), 150.8 (=CH), 156.1 (C-5 triazole), 158.5 (C-3 triazole), 166.0 (C=O). Anal. calcd. for
C15H19N5OS (317.41): C, 56.76; H, 6.03; N, 22.06; Found: C, 56.71; H, 6.01; N, 22.09%.
2-[(5-methyl-4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[(2-hydroxyphenyl)methylidene]
acetohydrazide (11)
CAS registry number: 62372-96-7. Yield: 68%; mp. 200°C–202°C (dec.). 1H NMR (DMSO-d6):
δ (ppm) = 2.26 (s, 3H, CH3), 4.04 (s, 2H, CH2), 4.41 (s, 1H, OH), 6.89-7.76 (m, 9H, Ar-H), 8.52
(s, 1H, =CH), 9.65 (s, 1H, NH). 13C NMR: δ (ppm) = 12.5 (-CH3), 30.6 (-S-CH2-), 117.4, 120.4,
128.3, 128.5, 129.9, 130.2, 136.2 (11Car), 156.0 (C-5 triazole), 158.3 (Car), 158.4 (C-3 triazole),
166.1 (C=O). Anal. calcd. for C18H17N5O2S (367.42): C, 58.84; H, 4.66; N, 19.06; Found: C,
58.89; H, 4.70; N, 19.04%.
2-[(5-methyl-4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[(3-nitrophenyl)methylidene]
acetohydrazide (12)
1
CAS registry number: 62373-03-9.Yield: 93%; mp. 184°C–186°C (dec.). H NMR (DMSO-d6):
δ (ppm) = 2.24 (s, 3H, CH3), 4.47 (s, 2H, CH2), 7.51-8.58 (m, 9H, Ar-H), 8.96 (s, 1H, =CH),
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10.20 (s, 1H, NH). 13C NMR: δ (ppm) = 12.6 (-CH3), 30.1 (-S-CH2-), 122.3, 124.1, 128.3, 129.9,
130.2, 132.6, 136.2, 137.4, 147.4 (12Car), 147.9 (=CH), 156.2 (C-5 triazole), 158.4 (C-3
triazole), 166.2 (C=O). Anal. calcd. for C18H16N6O3S (396.42): C, 54.54; H, 4.07; N, 21.20;
Found: C, 54.69; H, 4.02; N, 21.27%.
2-[(5-methyl-4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[(4-methylphenyl)methylidene]
acetohydrazide (13)
CAS registry number: 62373-03-9. Yield: 73%; mp. 186°C–188°C (dec.). 1H NMR (DMSO-d6):
δ (ppm) = 2.24 (s, 3H, CH3); 2.38 (s, 3H, CH3); 4.04 (s, 2H, CH2); 7.21-8.19 (m, 9H, Ar-H); 8.71
13
(s, 1H, CH); 11.60 (s, 1H, NH). C NMR: δ (ppm) = 12.5 (-CH3), 21.1 (-CH3), 30.1 (-S-CH2-),
127.3, 128.4, 129.1, 129.9, 130.2, 131.9, 136.2, 138.5 (12Car), 148.6 (=CH), 156.1 (C-5 triazole),
158.4 (C-3 triazole), 166.1 (C=O). Anal. calcd. for C19H19N5OS (365,45): C, 62.44; H, 5.24; N,
19.16; Found: C, 62.49; H, 5.22; N, 19.11%.
2-[(5-methyl-4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[(4-methoxyphenyl)
methylidene]acetohydrazide (14)
1
Yield: 74%; mp. 118°C–120°C (dec.). H NMR (DMSO-d6): δ (ppm) = 2.24 (s, 3H, CH3); 3.84
(s, 3H, CH3); 4.04 (s, 2H, CH2); 6.96-7.92 (m, 9H, Ar-H); 8.17 (s, 1H, CH); 8.68 (s, 1H, NH).
13C NMR: δ (ppm) = 12.4 (-CH3), 30.1 (-S-CH2), 50.0 (-CH3), 114.3 (2Car), 127.1, 128.3, 129.1,
129.9, 130.2, 136.2 (9Car), 148.6 (=CH), 156.0 (C-5 triazole), 158.4 (C-3 triazole), 160.1 (Car),
166.0 (C=O). Anal. calcd. for C19H19N5O2S (381.45): C, 59.82; H, 5.02; N, 18.36; Found: C,
59.85; H, 5.05; N, 18.41%.
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N-[(4-bromophenyl)methylidene]-2-[(5-methyl-4-phenyl-4H-1,2,4-triazol-3-
yl)sulfanyl]acetohydrazide (15)
1
Yield: 65%; mp. 126°C–128°C (dec.). H NMR (DMSO-d6): δ (ppm) = 2.26 (s, 3H, CH3); 4.03
(s, 2H, CH2); 7.49-8.22 (m, 9H, Ar-H); 8.75 (s, 1H, CH); 10.04 (s, 1H, NH). 13C NMR: δ (ppm)
= 12.4 (-CH3), 30.0 (-S-CH2-), 123.9, 128.3, 129.3, 129.9, 130.2, 132.3, 132.5, 136.2 (12Car),
148.7 (=CH), 156.1 (C-5 triazole), 158,4 (C-3 triazole), 166.0 (C=O). Anal. calcd. for
C18H16BrN5OS (430.32): C, 50.24; H, 3.75; N, 16.27; Found: C, 50.29; H, 3.70; N, 16.31%.
2-[(5-methyl-4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[(pyridin-4-
yl)methylidene]acetohydrazide (16)
CAS registry number 351510-77-5. Yield: 42%; mp. 186°C–188°C (dec.). 1H NMR (DMSO-d6):
δ (ppm) = 2.29 (s, 3H, CH3); 4.46 (s, 2H, CH2); 7.46-8.95 (m, 9H, Ar-H); 8.56 (s, 1H, =CH);
10.15 (s, 1H, NH). 13C NMR: δ (ppm) = 12.5 (-CH3), 30.0 (-S-CH2-), 122.3, 128.3, 129.9, 130.2,
136.2, 140.7 (9Car), 148.6 (=CH), 150, 1 (2Car), 156.0 (C-5 triazole), 158.4 (C-3 triazole), 166.1
(C=O). Anal. calcd. for C17H16N6OS (352.41): C, 57.94; H, 4.58; N, 23.85; Found: C, 23.85; H,
4.61; N, 23.90%.
2-[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[(2-hydroxyphenyl)
methylidene]acetohydrazide (17)
1
CAS Registry Number: 62372-96-7. Yield: 85%; mp. 212°C–214°C (dec.). H NMR (DMSO-
d6): δ (ppm) = 4.15 (s, 2H, CH2); 4.54 (s, 1H, OH); 6.90-7.74 (m, 14H, Ar-H); 8.37 (s, 1H, CH);
8.48 (s, 1H, NH). 13C NMR: δ (ppm) = 30.2 (-S-CH2), 117.6, 120.7, 121.3, 125.8, 128.5, 128.6,
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128.8, 130.1, 137.4 (17Car), 150.7 (=CH), 158.5 (Car) 159.5 (C-3 triazole), 166.5 (C=O), 167.4
(C-5 triazole). Anal. calcd. for C23H19N5O2S (429.49): C, 64.32; H, 4.46; N, 16.31; Found: C,
64.35; H, 4.48; N, 16.35%.
2-[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[(4-methylphenyl)
methylidene]acetohydrazide (18)
1
CAS Registry Number: 93300-42-6. Yield: 84%; mp. 214°C–216°C (dec.). H NMR (DMSO-
d6): δ (ppm) = 2.39 (s, 3H, CH3); 4.14 (s, 2H, CH2); 7.30-7.67 (m, 14H, Ar-H); 8.04 (s, 1H, CH);
13
8.22 (s, 1H, NH). C NMR: δ (ppm) = 21.1 (-CH3), 30.0 (-S-CH2), 125.6, 127.3, 128.6, 128.8,
129.1, 129.5, 130.1, 130.6, 131.9, 137.4, 138.5 (18Car), 148.5 (=CH), 159.5 (C-3 triazole), 166.1
(C=O), 167.4 (C-5 triazole). Anal. calcd. for C24H21N5OS (427.52): C, 67.43; H, 4.95; N, 16.38;
Found: C, 67.48; H, 4.98; N, 16.42%.
2-[(4,5-diphenyl-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[(4-methoxyphenyl)
methylidene]acetohydrazide (19)
1
Yield: 93%; mp. 210°C–212°C (dec.). H NMR (DMSO-d6): δ (ppm) = 3.76 (s, 3H, CH3); 4.04
(s, 2H, CH2); 6.96-7.60 (m, 14H, Ar-H); 7.93 (s, 1H, CH); 8.11 (s, 1H, NH). 13C NMR: δ (ppm)
= 30.2 (-S-CH2-), 56.8 (-CH3), 114.8 (2Car), 125.6, 128.4, 128.6, 128.8, 129.1, 129.5, 130.1,
137.4 (15Car), 148.2 (=CH), 159.5 (C-3 triazole), 160.3 (Car), 166.1 (C=O), 167.4 (C-5 triazole).
Anal. calcd. for C24H21N5O2S (443.52): C, 64.99; H, 4.77; N, 15.79; Found: C, 64.96; H, 4.72; N,
15.75%.
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N-[(4-bromophenyl)methylidene]-2-[(4,5-diphenyl-4H-1,2,4-triazol-3-
yl)sulfanyl]acetohydrazide (20)
1
CAS Registry Number: 306756-39-8. Yield: 87%; mp. 202°C–204°C (dec.). H NMR (DMSO-
d6): δ (ppm) = 4.08 (s, 2H, CH2); 7.31-7.97 (m, 14H, Ar-H); 8.16 (s, 1H, CH); 9.00 (s, 1H, NH).
13C NMR: δ (ppm) = 30.0 (-S-CH2-), 124.0, 125.6, 128.4, 128.6, 128.8, 129.3, 129.5, 130.1,
130.6, 132.4, 132.5, 137.4 (18Car), 148.7 (=CH), 159.5 (C-3 triazole), 166.1 (C=O), 167.5 (C-5
triazole). Anal. calcd. for C23H18BrN5OS (492.39): C, 56.10; H, 3.68; N, 14.22; Found: C, 56.13;
H, 3.72; N, 14.26%.
2-{[4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl]sulfanyl}-N-(propylidene)
acetohydrazide (21)
1
Yield: 44%; mp. 134°C–136°C (dec.). H NMR (DMSO-d6): δ (ppm) = 1,01 (t, J = 5,0 Hz, 3H,
CH3), 2,18-2,23 (m, 2H, CH2), 4,37 (s, 2H, CH2), 7,30-8,57 (m, 9H, Ar-H), 8,08 (t, J = 5,0 Hz,
13
1H, =CH), 11,24 (s, 1H, NH). C NMR: δ (ppm) = 9.5 (-CH3), 23.3 (-CH2-), 30.1 (-S-CH2-),
126.1, 128.4, 128.8, 130.1, 132.1, 134.6, 137.4 (9Car), 145.7 (=CH), 146.4, 146.9 (2Car), 159.5
(C-3 triazole), 162.4 (C-5 triazole), 166.2 (C=O). Anal. calcd. for C18H18N6OS (366,44): C,
59,00; H, 4,95; N, 22,93; Found: C, 59.03; H, 4,93; N, 22,98%.
2-{[4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl]sulfanyl}-N-(2-methylpropylidene)
acetohydrazide (22)
CAS registry number 901628-30-6. Yield: 60%; mp. 200°C–202°C (dec.). 1H NMR (DMSO-d6):
δ (ppm) = 0.95 (d, J = 2,5 Hz, 3H, CH3), 1.01 (d, J = 2,5 Hz, 3H, CH3), 2.46-2.52 (m, 1H, CH),
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4,33 (s, 2H, CH2), 7.24-8.55 (m, 9H, Ar-H), 8.43 (d, J = 5,0 Hz, 1H, =CH), 11.17 (s, 1H, NH).
13C NMR: δ (ppm) = 18.2 (2xCH3), 29.7 (-CH), 30.0 (-S-CH2-), 126.2, 128.4, 128.8, 130.1,
132.1, 134.6, 137.4, 146.4, 146.7 (11Car), 150.8 (=CH), 159.6 (C-3 triazole), 162.5 (C-5
triazole), 166.0 (C=O). Anal. calcd. for C19H20N6OS (380.47): C, 59.98; H, 5.30; N, 22.09;
Found: C, 59.91; H, 5.34; N, 22.13%.
2-{[4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl]sulfanyl}-N-[(2-
hydroxyphenyl)methylidene]acetohydrazide (23)
CAS registry number 903116-56-3. Yield: 90%; mp. 210°C–212°C (dec.). 1H NMR (DMSO-d6):
δ (ppm) = 3.43 (s, 2H, CH2), 4.11 (s, 1H, OH), 6.82-8.56 (m, 13H, Ar-H), 8.41 (s, 1H, =CH),
10.23 (s, 1H, NH). 13C NMR: δ (ppm) = 30.1 (-S-CH2), 117.3, 120.4, 121.2, 126.3, 128.5, 128.6,
128.8, 129.8, 130.2, 132.3, 134.7, 137.9, 146.4, 146.9 (16Car), 150.9 (=CH), 158.4 (Car), 159.5
(C-3 triazole), 162.4 (C-5 triazole), 166.2 (C=O). Anal. calcd. for C22H18N6O2S (430.48): C,
61.38; H, 4.21; N, 19.52; Found: C, 61.43; H, 4.28; N, 19.58%.
2-{[4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl]sulfanyl}-N-[(3-
nitrophenyl)methylidene]acetohydrazide (24)
CAS registry number 903107-17-5. Yield: 98%; mp. 202°C–204°C (dec.). 1H NMR (DMSO-d6):
δ (ppm) = 4.55 (s, 2H, CH2), 7.38-8.71 (m, 13H, Ar-H), 8.91 (s, 1H, =CH), 11.98 (s, 1H, NH).
13C NMR: δ (ppm) = 30.1 (-S-CH2-), 122.3, 124.1, 128.4, 128.7, 129.9, 130.0, 132.6, 134.6,
137.4, 137.8, 146.3, 146.9, 147.4 (17Car), 147.9 (=CH), 159.4 (C-3 triazole), 162.4 (C-5
triazole), 166.1 (C=O). Anal. calcd. for C22H17N7O3S (459.48): C, 57.51; H, 3.73; N, 21.34;
Found: C, 57.54; H, 3.76; N, 21.36%.
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2-[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[(4-methylphenyl)
methylidene]acetohydrazide (25)
1
CAS Registry Number: 903408-50-4. Yield: 65%; mp. 184°C–186°C (dec.). H NMR (DMSO-
d6): δ (ppm) = 2.31 (s, 3H, CH3); 4.10 (s, 2H, CH2); 7.22-8.57 (m, 13H, Ar-H); 8.15 (s, 1H, CH);
9.93 (s, 1H, NH). 13C NMR: δ (ppm) = 21.0 (-CH3), 30.1 (-S-CH2-), 126.1, 127.4, 128.4, 128.7,
129.2, 130.1, 131.9, 132.2, 134.6, 137.4, 138.5, 146.3, 146.9 (17Car), 148.8 (=CH), 159.6 (C-3
triazole), 162.3 (C-5 triazole), 166.0 (C=O). Anal. calcd. for C23H20N6OS (428.50): C, 64.47; H,
4.70; N, 19.61; Found: C, 64.50; H, 4.72; N, 19.63%.
2-[(4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)sulfanyl]-N-[(4-methoxyphenyl)
methylidene]acetohydrazide (26)
1
Yield: 44%; mp. 193°C–195°C (dec.). H NMR (DMSO-d6): δ (ppm) = 3.76 (s, 3H, CH3); 4.07
(s, 2H, CH2); 6.88-8.55 (m, 13H, Ar-H); 8.00 (s, 1H, CH); 8.12 (s, 1H, NH). 13C NMR, δ (ppm):
30.1 (-S-CH2), 56.2 (-CH3), 114.4 (2Car), 126.1, 127.1, 128.4, 128.8, 129.1, 130.1, 132.1, 134.6,
137.4, 146.4, 146.9 (14Car), 148.6 (=CH), 159.5 (C-3 triazole), 160.1 (Car), 162.4 (C-5 triazole),
166.7 (C=O). Anal. calcd. for C23H20N6O2S (444.5089): C, 62.15; H, 4.54; N, 18.91; Found: C,
62.18; H, 4.57; N, 18.79%.
N-[(4-bromophenyl)methylidene]-2-{[4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-
yl]sulfanyl}acetohydrazide (27)
CAS registry number 903407-49-8. Yield: 76%; mp. 228°C–230°C (dec.). 1H NMR (DMSO-d6):
δ (ppm) = 4.10 (s, 2H, CH2), 7.36-8.57 (m, 13H, Ar-H), 7.98 (s, 1H, =CH), 10.16 (s, 1H, NH).
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13C NMR: δ (ppm) = 30.2 (-S-CH2-), 124.0, 126.2, 128.3, 128.7, 129.3, 130.0, 132.1, 132.5,
134.6, 137.4, 146.5, 146.8 (17Car), 148.6 (=CH), 159.4 (C-3 triazole), 162.3 (C-5 triazole), 166.1
(C=O). Anal. calcd. for C22H17BrN6OS (493.38): C, 53.56; H, 3.47; N, 17.03; Found: C, 53.62;
H, 3.41; N, 17.09%.
2-{[4-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl]sulfanyl}-N-[(pyridin-4-
yl)methylidene]acetohydrazide (28)
CAS registry number 901624-83-7. Yield: 92%; mp. 198°C–200°C (dec.). 1H NMR (DMSO-d6):
δ (ppm) = 4.54 (s, 2H, CH2), 7.35-8.64 (m, 13H, Ar-H), 7.99 (s, 1H, =CH), 11.96 (s, 1H, NH).
13C NMR: δ (ppm) = 30.1. (-S-CH2-), 122.3, 126.1, 128.4, 130.1, 132.2, 134.6, 137.4, 140.7,
146.4, 146.9 (14Car), 148.7 (=CH), 150.1 (2Car), 159.5 (C-3 triazole), 162.5 (C-5 triazole), 166.2
(C=O). Anal. calcd. for: C21H17N7OS (415.47): C, 60.71; H, 4.12; N, 23.60; Found: C, 60.78; H,
4.07; N, 23.67%.
Microbiology
Materials and methods
The antimicrobial activity of the tested compounds was screened on the panel of American
Type Culture Collection (ATCC) reference strains of Gram-positive bacteria (Staphylococcus
aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Bacillus subtilis ATCC 6633,
Micrococcus luteus ATCC 10240), and of the Gram-negative bacteria (Escherichia coli ATCC
25922, Klebsiella pneumoniae ATCC 13883, Proteus mirabilis ATCC 12453, Pseudomonas
aeruginosa ATCC 9027). Moreover, we used the yeasts belonging to Candida spp. - two
reference strains (C. albicans ATCC 2091 and C. parapsilosis ATCC 22019) and ten clinical
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isolates of C. albicans from the Department of Pharmaceutical Microbiology of Medical
University of Lublin collection.
Microbial suspensions were prepared in sterile saline (0.85% NaCl) with an optical density of
0.5 McFarland standard - 150 x 106 CFU/mL (CFU - colony forming units). All stock solutions
of the tested compounds were prepared in dimethyl sulfoxide (DMSO). Mueller-Hinton agar (for
bacteria) or Mueller-Hinton agar supplemented with 2% glucose and buffered at pH 5.6 (for
fungi) were used for examination of antibacterial or antifungal activity. The Mueller-Hinton
medium plus 2% glucose was used instead of RPMI 1640. It should be noted that Mueller-
Hinton medium is readily available, possess as good batch-to-batch reproducibility and supports
the growth of most pathogenic yeasts [48].
In the first step the in vitro antimicrobial potency of all tested compounds was screened using
the agar dilution method on the basis of the microbial growth inhibition on the agar medium to
which the tested compounds at concentrations 1000 μg/mL were added, as described elsewhere
[49]. The medium with DMSO at the final concentration and without the tested compounds
served as a negative control; no microbial growth inhibition was observed. The plates were
poured on the day of testing. 10 μL of each microbial suspension was put onto the prepared solid
media. The plates were pre-incubated at room temperature for 1-1.5 h and then were incubated at
35oC for 18 h for bacteria and for 24-48 h for yeasts.
Subsequently, for compound 5 showing inhibitory effect on the growth of tested yeasts, MIC
was estimated by broth microdilution technique, according to the Clinical and Laboratory
Standards Institute (CLSI) standards M27-A3 for yeasts [36] and European Committee on
Antimicrobial Susceptibility Testing (EUCAST), Subcommittee of Antifungal Susceptibility
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Testing [37], with some modification. Microdilution technique was developed using sterile 96-
well microplates, which were inoculated with a yeast suspension of optical density of 0.5
McFarland standard. A 2 µL of the suspension was put into 198 µL of Mueller Hinton broth
buffered at pH 5.6 supplemented with 2% glucose containing two-fold dilution of tested
compound at the final concentration from 7.82 to 1000 mg/L. The medium without the tested
compound was used as negative control. After incubation (at 35oC for 24 or 48 h), the optical
density (OD600) measurements were determined spectrophotometrically for microbial culture in
broth medium. The MIC (minimal inhibitory concentration) values, defined as the lowest
concentration of a compound that prevents visible growth of the tested microorganisms, were
determined after 24 and 48 h by comparison with the fungal growth in control (compound free)
medium. The blank control wells with two-fold dilution of each of the tested compounds added
to broth medium (total volume - 200 µL) without the yeast suspension were incubated under the
same conditions. MIC is usually defined as the lowest concentration of compound that inhibit of
the growth of tested microorganisms [37].
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Table 1. Substituents of compounds 1 - 28.
Compounds
R1
R2
H
CH3
C6H5
3-C5H4N
H
1
2
-
-
-
3
-
4
2-OHC6H4
4-CH3C6H4
4-CH3OC6H4
4-BrC6H4
C3H7
5
H
6
H
7
H
8
CH3
CH3
9
(CH3)2CHCH2
10
28
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CH3
2-OHC6H4
3-NO2C6H4
4-CH3C6H4
4-CH3OC6H4
4-BrC6H4
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
CH3
CH3
CH3
CH3
CH3
4-C5H4N
C6H5
2-OHC6H4
4-CH3C6H4
4-CH3OC6H4
4-BrC6H4
C6H5
C6H5
C6H5
3-C5H4N
3-C5H4N
3-C5H4N
3-C5H4N
3-C5H4N
3-C5H4N
3-C5H4N
3-C5H4N
C3H7
(CH3)2CHCH2
2-OHC6H4
3-NO2C6H4
4-CH3C6H4
4-CH3OC6H4
4-BrC6H4
4-C5H4N
29
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