Synthesis and Antimicrobial Evaluation of Novel Derivatives of Semicarbazide and 1,2,4-triazole

Article abstract of DOI:10.1002/jhet.2893

Reaction of phenoxyacetic acid hydrazide with isocyanate was used to the synthesis of new semicarbazide derivatives. Cyclization of these compounds in a 2% aqueous solution of sodium hydroxide led to formation of 1,2,4-triazole-3-one. The chemical structure of synthesized compounds was confirmed by elemental analysis and spectroscopic methods (¹H and ¹³C NMR). On the basis of the NMR, spectra were found that cyclic compounds 1,2,4-triazole exist in the -one form. Moreover, all derivatives were examined for their in vitro activity against some species of bacteria. New compounds presented mild or moderate antimicrobial activity only against reference Gram-positive bacteria. Two derivatives (one semicarbazide and one triazole) showed bactericidal or bacteriostatic activity.

Full text of DOI:10.1002/jhet.2893

Month 2017
Synthesis and Antimicrobial Evaluation of Novel Derivatives of
Semicarbazide and 1,2,4-triazole
Anna Pachuta-Stec,a*
b
b
a
Anna Biernasiuk, Anna Malm, and Monika Pitucha
a
Department of Organic Chemistry, Faculty of Pharmacy, Medical University, 4a Chodźki Street, 20-093 Lublin, Poland
Department of Pharmaceutical Microbiology with Laboratory for Microbiological Diagnostics, Faculty of Pharmacy,
b
Medical University, 1 Chodźki Street, 20-093 Lublin, Poland
*
E-mail: anna.pachuta@umlub.pl
Additional Supporting Information may be found in the online version of this article.
Received December 5, 2016
DOI 10.1002/jhet.2893
Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com).
Reaction of phenoxyacetic acid hydrazide with isocyanate was used to the synthesis of new semicarbazide
derivatives. Cyclization of these compounds in a 2% aqueous solution of sodium hydroxide led to formation
of 1,2,4-triazole-3-one. The chemical structure of synthesized compounds was confirmed by elemental
1
13
analysis and spectroscopic methods ( H and C NMR). On the basis of the NMR, spectra were found that
cyclic compounds 1,2,4-triazole exist in the -one form. Moreover, all derivatives were examined for their
in vitro activity against some species of bacteria. New compounds presented mild or moderate antimicrobial
activity only against reference Gram-positive bacteria. Two derivatives (one semicarbazide and one triazole)
showed bactericidal or bacteriostatic activity.
J. Heterocyclic Chem., 00, 00 (2017).
INTRODUCTION
It is well known that there are drugs that contain 1,2,4-
triazole nucleus. Among them are nonsteroidal aromatase
inhibitors [21–23], antiviral [24], anxiolytic [25],
anticonvulsant [26], antidepressant [27], and antifungal
[28–31] drugs.
In the 1940s, the first antibiotics were discovered and
introduced to health care [1]. Then, they were very
effective against bacterial diseases and saved millions of
people’s lives. However, many antibiotics have lost their
effectiveness up till now, so bacterial infections are still
an alarming and challenging medicinal problem around
the world. This fact is connected especially with the
increasing number of drug resistant and multidrug
resistant microbial pathogens [1,2]. Bacterial diseases
caused by multidrug resistant strains are very dangerous
especially for children, the elderly, and people with a
weakened immune system. The problem is also the side
effects of drugs. These are very important reasons that
are still tend researchers to continually search and
synthesize new, less harmful, and more effective
biologically active compounds that could be used as
potential drug candidate.
Derivatives of semicarbazide and triazole are interesting
groups of compounds that meet these requirements.
It follows from the literature survey, derivatives of
semicarbazide and 1,2,4-triazole can be used as anti-
inflammatory [3,4], antioxidant [5,6], antiparasitic [7],
analgesic [8], anti-HIV [9], antiviral [10,11],
anticonvulsant [12], and antitumor [13,14] agents. In
addition, some of them also show antitubercular [4,15–17],
antifungal [18], and antibacterial properties [4,17,19,20].
Our team is also interested in search, synthesis, and
investigation of bioactive derivatives of thio/semicarbazide
and 1,2,4-triazole [14,19,20,32,33]. Among them, there
were derivatives of 1-phenoxyacetyl-4-substituted
thiosemicarbazide and 3-phenoxymethyl-4-substituted-
1,2,4-triazoline-5-thione, which were obtained by our team
[32]. One of these compounds displayed interesting
antinociceptive activity [32]. Some of the 3-
phenoxymethyl-1,2,4-triazoline-5-thione having phenyl, 4-
methylphenyl, and 4-methoxyphenyl group in position 4
were prepared earlier as bactericidal, fungicidal, and anti-
inflammatory agents [34–36]. From the literature, it can be
stated that 5-phenoxymethyl-1,2,4-triazoline-3-one also
displayed pharmacological properties like antidepressant
activity [37]. Based on all the aforementioned reports, and
in continuation of our early study, we also decided to
synthesize new derivatives of 5-phenoxymethyl-1,2,4-
triazoline-3-one. Such compounds were prepared
previously in several steps (etherification, chlorination,
thionation, and cyclocondensation) with 45% yield by
Bansal et al. [37] and also by cyclization reaction with
bases [38]. In our research, two-step synthesis was used to
prepare 1,2,4-triazole. First, new 1-phenoxyacetyl-4-
©
2017 Wiley Periodicals, Inc.

A. Pachuta-Stec, A. Biernasiuk, A. Malm, and M. Pitucha
Vol 000
substitutedsemicarbazidewasobtained,andnextcyclization
reaction of these compounds was used to prepare
corresponding 1,2,4-triazoline-3-one. Cyclization proceeded
with a yield of 75–89%. One of the cyclic derivatives,
Bacillus subtilis ATTC 6633, Bacillus cereus ATTC
10876, and Staphylococcus aureus ATTC 43300 with
MIC = 500 μg/mL and MBC ≥ 1000 μg/mL. The
remaining staphylococci were less susceptible to 6
(MIC = 1000 μg/mL and MBC ≥ 1000 μg/mL) (Table 1).
The substance 15 possessed also moderate activity
towards M. luteus ATTC 10240, B. subtilis ATTC 6633,
and B. cereus ATTC 10876 with MIC = 250–500 μg/mL
and MBC ≥ 1000 μg/mL and mild effect towards
staphylococci (except S. aureus ATTC 25923) with
MIC = 1000 μg/mL and MBC > 1000 μg/mL. The
compounds 6 and 15 showed bactericidal or bacteriostatic
activity (Table 1).
The other examined compounds, 11 and 16–18 indicated
mainly mild bioactivity (MIC = 1000 μg/mL and
MBC > 1000 μg/mL) or had no any effect towards these
bacteria (Table 1).
In turn, compounds 1–5, 7–9, 12–14, and 19–20 were
inactive against reference microorganisms. Unfortunately,
all synthesized derivatives were inactive against fungi
belonging to yeasts.
5
-phenoxymethyl-4-(4-brmophenyl)-1,2,4-triazole-3-one,
was obtained earlier [37]. Additionally, in vitro activity
against some species of bacteria were presented in this
paper.
RESULTS AND DISCUSSION
Chemistry. As the starting compound for the synthesis
of acyl derivatives of semicarbazide was used hydrazide of
phenoxyacetic acid, which was prepared by the reaction of
ethyl ester of phenoxyacetic acid with 100% hydrazine
hydrate [32]. New linear semicarbazide derivatives were
obtained by the reaction of phenoxyacetic acid hydrazide
with appropriate isocyanates in room temperature. Next,
the semicarbazide derivatives were cyclized by heating in a
2
% aqueous solution of sodium hydroxide, and the
corresponding 5-phenoxymethyl-4-substituted-1,2,4-triazole-
-one were obtained. The reaction conditions were
Structure–activity relationship.
From the in vitro
3
antimicrobial assay, it can be stated that only one
semicarbazide 6 with 2-chlorophenyl group attached to
semicarbazide at position 4 presented moderate effect
against all examined species of bacteria. In this case,
antimicrobial activity can be connected with the presence
of electron-withdrawing group (Cl) attached to phenyl
ring at position 2. Other semicarbazide derivatives (7–9)
possessing also electron-withdrawing substituent (2-Br, 4-
Br, and 3-Cl) were absolutely inactive. The rest of
established experimentally. The reactions were performed
according to Scheme 1.
Biological activity. According to our results, among of
examined compounds, 6, 11, and 15–18 indicated some
antibacterial activity only against reference Gram-positive
bacteria. The substance 6 had a spectrum of activity
towards all these bacteria. This compound showed
moderate effect against Micrococcus luteus ATTC 10240,
Scheme 1. Synthesis of semicarbazide (1–10) and 1,2,4-triazole derivatives (11–20).
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Synthesis and Antimicrobial Evaluation of Novel Derivatives of Semicarbazide
and 1,2,4--triazole
Table 1
The activity data expressed as MIC (MBC/MFC) [μg/mL] against the reference strains of microorganisms.
Compound number
Species
6
11
15
16
17
18
CIP
Staphylococcus aureus ATCC 25923
Staphylococcus aureus ATCC 6538
1000 (1000)
1000
—
1000
(>1000)
1000
(>1000)
1000
(>1000)
1000
(>1000)
1000
>1000)
1000
—
1000
(>1000)
1000
(>1000)
1000
(>1000)
250 (1000)
—
1000
(>1000)
1000
(>1000)
1000
(>1000)
1000
(>1000)
1000
(>1000)
1000
—
—
—
0.488
0.244
—
—
—
(>1000)
500
Staphylococcus aureus ATCC 43300
Staphylococcus epidermidis ATCC
—
—
0.244
0.122
0.976
0.030
0.061
(>1000)
1000
12228
(>1000)
500
Micrococcus luteus ATCC 10240
Bacillus subtilis ATCC 6633
Bacillus cereus ATCC 10876
1000
(>1000)
—
1000
(>1000)
—
(>1000)
500 (1000)
500
(>1000)
500 (1000)
(
500 (1000)
—
—
(>1000)
(>1000)
The standard chemotherapeutics agents: ciprofloxacin (CIP) used as positive control for bacteria. Em dash, “—,” indicates no activity.
semicarbazide derivatives with aliphatic substituents and
electron donating (ethoxy) group at phenyl moiety had no
any activity, too.
assay was performed. Our data showed that some of the
newly synthesized compounds indicated mild or moderate
antimicrobial activity only against reference Gram-
positive bacteria. The most active was one semicarbazie 6
with 2-chlorophenyl group attached to semicarbazide at
position 4. Among triazole, the best activity presented
derivative possessing cyclohexyl substituent at position 4
of triazole ring. These compounds 6 and 15 showed
bactericidal or bacteriostatic activity.
In the case of the derivatives of 1,2,4-triazole, five
compounds (11 and 15–18) displayed moderate-to-mild
bioactivity. The best activity against M. luteus showed
derivative 15 heaving aliphatic substituent (cyclohexyl) at
position 4 of triazole moiety, whereas the presence of this
substituent in semicarbazide 5 can be connected with the
lack of activity. Second, compound 11 with aliphatic
substituent (allil) indicated only mild activity. Triazole
derivatives heaving aromatic substituents with electron-
withdrawing group at positions 2 and 3 of phenyl ring (2-
Cl, 2-Br, and 3-Cl) attached to triazole moiety also
presented mild bioactivity.
In conclusion, the introduction of aromatic (2-
chlorophenyl) substituent to semicarbazie 6 was the most
favorable in terms of antimicrobial activity, in contrast to
triazole where derivative heaving aliphatic group 15 was
the most active. In the case of both semicarbazide and
triazole, substituent 2-chlorophenyl was favorable for
antibacterial activity.
EXPERIMENTAL
Chemistry.
Melting points were determined in a
Fisher-Johns block and presented without any
corrections. Elemental analysis was made on a Perkin-
Elmer 2400 CHN Analyser. All results were in good
agreement with calculated values. The error range of
1
13
±
0.4% for each element analyzed. The H and C NMR
spectra were recorded on a Bruker Avance 300-MHz
spectrometer in DMSO-d and with TMS as an internal
6
standard. Chemicals were purchased from Sigma-Aldrich
or Lancaster Synthesis and used without further
purification.
CONCLUSION
Preparation
of
1-phenoxyacetyl-4-substituted
semicarbazide 1–10.
A total of 1.7-g (10 mmol)
We designed and synthesized new derivatives of
semicarbazide and 1,2,4-triazole with potential biological
activity in this paper. For all compounds, detailed
analysis of the H and C NMR spectra was performed.
Taking into account the possibility of existing of keto-
enole tautomeric forms in the cyclic products, we
determined on the basis of the NMR spectra that
compounds 11–20 exist in the -one form. The synthesized
products was isolated with medium and good yields.
Additionally, for all compounds, in vitro antimicrobial
phenoxyacetic acid hydrazide, 10 mmol of appropriate
isocyanate, and 10-mL diethyl ether were mixed carefully
and left in room temperature for 24 h. Then, the product
was washed with diethyl ether, dried, and crystallized
from ethanol.
1
13
1-Phenoxyacetyl-4-allilsemicarbazide (1). Yield 95%, mp
1
174–176°C. H NMR (DMSO-d ) δ: 9.84, 7.89 (2s, 2H,
6
2NH), 7.27–6.87 (m, 5H, arom.), 6.61 (t, J = 6.0 Hz, 1H,
NH), 5.81–5.67 (m, 1H, CH CH═CH ), 5.23–4.95 (m,
2
2
2H, CH CH═CH ), 4.56 (s, 2H, OCH ), 3.74–3.58 (m,
2
2
2
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Pachuta-Stec, A. Biernasiuk, A. Malm, and M. Pitucha
Vol 000
1
3
1
2
1
(
H, CH CH═CH ). C NMR (DMSO-d ) δ: 168.14,
H NMR (DMSO-d ) δ: 10.43, 9.89, 9.63 (3s, 3H, 3NH),
2
2
6
6
58.29
(C═O),
158.22
(i-PhC-OCH2),
136.78
7.49–7.24 (m, 5H, arom.), 7.18–6.93 (m, 4H, arom.),
4.70 (s, 2H, OCH2). C NMR (DMSO-d ) δ: 182.33,
1
3
CH CH═CH ), 129.92 (m-Ph2C), 121.58 (p-PhC),
2
2
6
1
17.54 (CH CH═CH ), 115.10 (o-Ph2C), 66.42 (OCH ),
168.07 (C═O), 158.22 (i-PhC-OCH ), 137.12 (C-1),
2
2
2
2
4
1.91 (CH CH═CH ). Anal. Calcd for C H N O
132.01 (C-3), 131.58 (C-5), 129.93 (m-Ph2C), 128.64
(C-2), 127.61 (C-4, C-6), 121.61 (p-PhC), 114.98
(o-Ph2C), 66.38 (OCH ). Anal. Calcd for C H ClN O
3 3
2
2
12 15 3 3
(249.26): C, 57.82; H, 6.06; N, 16.86; found: C, 57.86;
H, 6.09; N, 16.81%.
2
15 14
1
-Phenoxyacetyl-4-ethylsemicarbazide (2).
Yield 90%,
(319.74): C, 56.34; H, 4.41; N, 13.14; found: C, 56.29;
H, 4.37; N, 13.07%.
1
mp 184–186°C. H NMR (DMSO-d ) δ: 9.85, 7.85 (2s,
6
2
H, 2NH), 7.49–6.88 (m, 5H, arom.), 6.45 (t,
1-Phenoxyacetyl-4-(3-chlorophenyl)semicarbazide (7). CAS
J = 6.0 Hz, 1H, NH), 4.61 (s, 2H, OCH ), 3.20 (q,
Registry Number 544424-61-5. Yield 91%, mp 206–208°C.
2
1
J = 6.0, 9.0 Hz, 2H, CH ), 1.05 (t, J = 9.0 Hz, 3H,
H NMR (DMSO-d ) δ: 10.10, 9.05, 8.39 (3s, 3H, 3NH),
2
6
13
CH3). C NMR (DMSO-d ) δ: 168.07, 158.30 (C═O),
7.85–7.24 (m, 5H, arom.), 7.21–6.90 (m, 4H, arom.),
6
13
1
58.21 (i-PhC-OCH2), 129.92 (m-Ph2C), 121.59
4.68 (s, 2H, OCH2). C NMR (DMSO-d ) δ: 168.32,
6
(
p-PhC), 115.10 (o-Ph2C), 66.41 (OCH ), 34.47 (CH ),
158,18 (C═O), 155.61 (i-PhC-OCH ), 141.67 (C-1),
2
2
2
1
5
1
6.01 (CH ). Anal. Calcd for C H N O (237.25): C,
5.68; H, 6.37; N, 17.71; found: C, 55.71; H, 6.39; N,
7.68%.
133.47 (C-3), 130.74 (C-5), 129.95 (m-Ph2C), 122.03
(C-4), 121.67 (C-2), 118.21 (C-6), 117.46 (p-PhC), 115.13
(o-Ph2C), 66,48 (OCH ). Anal. Calcd for C H ClN O
3
11 15 3 3
2
15 14
3 3
1
-Phenoxyacetyl-4-butylsemicarbazide (3). CAS Registry
(319.74): C, 56.34; H, 4.41; N, 13.14; found: C, 56.39;
H, 4.39; N, 13.17%.
1
Number 896536-67-7. Yield 92%, mp 166–168°C.
H
NMR (DMSO-d ) δ: 9.79, 7.77 (2s, 2H, 2NH), 7.41–6.84
1-Phenoxyacetyl-4-(2-bromophenyl)semicarbazide (8). Yield
6
1
(m, 5H, arom.), 6.37 (t, J = 6.0 Hz, 1H, NH), 4.52 (s, 2H,
88%, mp 200–202°C. H NMR (DMSO-d ) δ: 10.23,
6
OCH ), 3.01 (q, J = 9.0, 15.0 Hz, 2H, CH CH CH CH ),
8.99, 8.17 (3s, 3H, 3NH), 8.12–7.23 (m, 5H, arom.),
2
2
2
2
3
13
1
3
.50–1.16 (m, 4H, CH CH CH CH ), 0.87 (t, J = 9.0 Hz,
7.22–6.85 (m, 4H, arom.), 4.78 (s, 2H, OCH
NMR (DMSO-d ) δ: 158.16 (2C═O), 155.20 (i-PhC-
OCH ), 137.32 (C-1, C-2), 132.96 (C-3), 129.96 (m-
Ph2C), 128.54 (C-4, C-5), 124.75 (C-6), 121.92 (p-
PhC), 115.17 (o-Ph2C), 66.44 (OCH ). Anal. Calcd for
C H14BrN O (364.19): C, 49.47; H, 3.87; N, 11.54;
15 3 3
);
2
C
2
2
2
3
13
H, CH3). C NMR (DMSO-d ) δ: 168.05, 158.39
6
6
(
(
(
(
C═O), 158.21 (i-PhC-OCH ), 129.91 (m-Ph2C), 121.58
p-PhC), 115.10 (o-Ph2C), 66.40 (OCH2), 39.29
2
2
CH CH CH CH ), 32.45 (CH CH CH CH ), 19.89
2
2
2
2
3
2
2
2
3
CH CH CH CH ), 14.21 (CH3). Anal. Calcd for
2
2
2
3
C H N O (265.31): C, 58.85; H, 7.22; N, 15.84;
found: C, 49.39; H, 3.83; N, 11.47%.
1
3 19 3 3
found: C, 58.79; H, 7.19; N, 15.89%.
-Phenoxyacetyl-4-benzylsemicarbazide (4). Yield 89%,
1-Phenoxyacetyl-4-(4-bromophenyl)semicarbazide (9). CAS
Registry Number 905242-61-7. Yield 93%, mp 230–232°C.
1
1
1
mp 196–198°C. H NMR (DMSO-d ) δ: 9.88, 7.97 (2s,
H NMR (DMSO-d ) δ: 10.02, 8.91, 8.25 (3s, 3H,
6
6
2
H, 2NH), 7.33–6.94 (m, 11H, arom. + NH), 4.57 (s, 2H,
3NH), 7.45 (s, 4H, arom.), 7.32–6.98 (m, 5H, arom.),
1
3
13
OCH ), 4,27 (d, J = 6.0 Hz, 2H, CH ). C NMR
4.61 (s, 2H, OCH ). C NMR (DMSO-d ) δ: 168.30,
2 6
2
2
(
DMSO-d ) δ: 168.21, 158.57 (C═O), 158.22 (i-PhC-
158.18 (C═O), 155.64 (i-PhC-OCH ), 139.52 (C-1),
2
6
OCH ), 140.99 (C-1), 129.92 (m-Ph2C), 128.58 (C-3, C-
131.84 (C-3, C-5), 129.96 (m-Ph2C), 121.67 (p-PhC),
121.04 (C-2, C-6), 115.38 (o-Ph2C), 113.88 (C-4) 66.47
2
5
), 127.36 (C-2, C-6), 126.99 (C-4), 121.59 (p-PhC),
15.10 (o-Ph2C), 66.45 (OCH2), 43.04 (CH2).
Anal. Calcd for C H N O (299.32): C, 64.20; H, 5.72;
1
(OCH ). Anal. Calcd for C15H14BrN O (364.19): C,
2 3 3
49.47; H, 3.87; N, 11.54; found: C, 49.51; H, 3.92; N,
11.49%.
1
6 17 3 3
N, 14.04; found: C, 63.99; H, 5.67; N, 14.15%.
1
-Phenoxyacetyl-4-cyclohexylsemicarbazide (5).
CAS
1-Phenoxyacetyl-4-(4-ethoxyphenyl)semicarbazide (10). CAS
Registry Number 560106-25-4. Yield 90%, mp 192–194°C.
Registry Number 902632-86-4. Yield 93%, mp 214–216°C.
1
1
H NMR (DMSO-d ) δ: 9.80, 7.72 (2s, 2H, 2NH),
H NMR (DMSO-d ) δ: 9.99, 8.56, 8.08 (3s, 3H, 3NH),
6
6
7
.45–6.82 (m, 5H, arom.), 6.17 (d, J = 6.0 Hz, 1H,
7.37–7.31 (m, 4H, arom.), 7.04–6.97 (m, 5H, arom.), 4.63
(s, 2H, OCH ), 3.96 (q, J = 7.0, 14.0 Hz, 2H,
OCH CH ), 1.32 (t, J = 7.0 Hz, CH
(DMSO-d ) δ: 168.29, 158.24 (C═O), 156.00 (C-1),
154.29 (i-PhC-OCH ), 132.95 (C-4), 129.99 (m-Ph2C),
121.67 (p-PhC), 121.23, 120.97 (C-2, C-6), 115.16
(o-Ph2C), 114.85 (C-3, C-5), 66.50 (OCH ), 63.54
(OCH CH ), 15.22 (CH ). Anal. Calcd for C17
NH), 4.52 (s, 2H, OCH ), 1.88–1.00 (m, 11H, C H ).
2
2
6 11
13
13
C NMR (DMSO-d ) δ: 167.93, 158.20 (C═O), 157.54
2
3
). C NMR
3
6
(
(
(
(
i-PhC-OCH ), 129.92 (m-Ph2C), 121.59 (p-PhC), 115.09
o-Ph2C), 66.37 (OCH ), 48.54 (CH), 33.47 (2CH ), 25.71
CH ), 25.09 (2CH ). Anal. Calcd for C H N O
291.34): C, 61.84; H, 7.26; N, 14.42; found: C, 61.69; H,
.27; N, 14.35%.
-Phenoxyacetyl-4-(2-chlorophenyl)semicarbazide (6). CAS
Registry Number 894315-61-8. Yield 86%, mp 136–138°C.
6
2
2
2
2
2
2
15 21 3 3
2
7
2
3
3
H N O
19 3 4
1
(329.35): C, 61.99; H, 5.81; N, 12.76; found: C, 61.90;
H, 5.74; N, 12.71%.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Synthesis and Antimicrobial Evaluation of Novel Derivatives of Semicarbazide
and 1,2,4--triazole
13
Preparation
of
5-phenoxymethyl-4-substituted-1,2,4-
1.73–1.08 (m, 11H, C H ). C NMR (DMSO-d ) δ:
6 11 6
triazole-3-one 11–20. Semicarbazide 1–10 (10 mol) was
dissolved in 2% aqueous NaOH (40–50 mL) and refluxed
for 2 h. After cooling, the solution was filtered and
treated with diluted hydrochloric acid (3 M) until acidic
reaction was obtained. The isolated product was collected
by filtration, dried, and crystallized from ethanol.
1
1
57.79 (C═O), 155.26 (Ctriazole), 146.66 (i-PhC-OCH2),
29.92 (m-Ph2C), 121.59 (p-PhC), 115.09 (o-Ph2C),
66.35 (OCH ), 48.43 (CH), 33.43 (2CH ), 29.27 (CH ),
2
6
2
2
2
5.65 (2CH ). Anal. Calcd for C H N O (273.33): C,
2 15 19 3 2
5.61; H, 7.01; N, 15.37; found: C, 65.69; H, 7.07; N,
15.29%.
5
-Phenoxymethyl-4-allil-1,2,4-triazole-3-one (11).
Yield
5-Phenoxymethyl-4-(2-chlorophenyl)-1,2,4-triazole-3-one
1
1
(
16). Yield 84%, mp 164–166°C. H NMR (DMSO-d ) δ:
8
1
4%, mp 124–126°C. H NMR (DMSO-d ) δ: 11.96 (s,
H, NH), 7.49–6.94 (m, 5H, arom.), 6.03–5.81 (m, 1H,
6
6
1
5
1
1
1
4.15 (s, 1H, NH), 7.71–7.49 (m, 4H, arom.), 7.23–6.79 (m,
13
H, arom.), 4.97 (s, 2H, OCH2). C NMR (DMSO-d ) δ:
CH CH═CH ), 5.23–5.10 (m, 2H, CH CH═CH ), 5.04
6
2
2
2
2
13
(
s, 2H, OCH ), 4.43–4.23 (m, 2H, CH CH═CH ).
NMR (DMSO-d ) δ: 157.83 (C═O), 155.14 (Ctriazole),
43.78 (i-PhC-OCH ), 133.06 (CH CH═CH ), 130.04
m-Ph2C), 121.97 (p-PhC), 117.54 (CH CH═CH ),
15.27 (o-Ph2C), 61.35 (OCH ), 43.09 (CH CH═CH ).
Anal. Calcd for C H N O (231.25): C, 62.32; H, 5.67;
N, 18.17; found: C, 62.26; H, 5.59; N, 17.99%.
5
7%, mp 158–160°C. H NMR (DMSO-d ) δ: 11.81 (s,
H, NH), 7.43–6.93 (m, 5H, arom.), 5.04 (s, 2H, OCH2),
.65 (q, J = 9.0, 18.0 Hz, 2H, CH ), 1.19 (t, J = 9.0 Hz,
H, CH3). C NMR (DMSO-d ) δ: 157.86 (C═O),
55,36 (Ctriazole), 143.70 (i-PhC-OCH ), 130.09 (m-
Ph2C), 121,97 (p-PhC), 115.26 (o-Ph2C), 61.32 (OCH2),
6.21 (CH ), 14.72 (CH ). Anal. Calcd for C H N O
C
69.29 (C═O), 157.59 (Ctriazole), 148.34 (i-PhC-OCH2),
2
2
2
32.63 (C-1), 132.22 (C-3), 131.44 (C-2), 131.31 (C-5),
30.64 (C-4), 129.91 (m-Ph2C), 128.75 (C-6), 122.09 (p-
6
1
(
1
2
2
2
PhC), 115.11 (o-Ph2C), 60.43 (OCH ). Anal. Calcd for
2
2
2
C H ClN O (301.73): C, 59.71; H, 4.01; N, 13.93; found:
15 12
3 2
2
2
2
C, 59.59; H, 4.07; N, 13.87%.
1
2 13 3 2
5
-Phenoxymethyl-4-(3-chlorophenyl)-1,2,4-triazole-3-one
1
(
17). Yield 81%, mp 168–170°C. H NMR (DMSO-d ) δ:
6
-Phenoxymethyl-4-ethyl-1,2,4-triazole-3-one (12).
Yield
1
12.19 (s, 1H, NH), 7.61–7.46 (m, 4H, arom.), 7.28–6.84 (m,
8
1
3
3
1
6
13
5
1
H, arom.), 5.10 (s, 2H, OCH2). C NMR (DMSO-d ) δ:
6
57.64 (C═O), 154.39 (Ctriazole), 143.15 (i-PhC-OCH2),
2
13
134.58 (C-1), 133.78 (C-3), 131.29 (C-5), 129.96 (m-Ph2C),
29.05 (C-4), 127.36 (C-2), 126.14 (C-6), 122.07 (p-PhC),
15.36 (o-Ph2C), 61.57 (OCH2). Anal. Calcd for
C H ClN O (301.73): C, 59.71; H, 4.01; N, 13.93; found:
6
1
1
2
15
12
3 2
3
(
2
3
11 13 3 2
C, 59.79; H, 3.97; N, 13.97%.
219.24): C, 60.26; H, 5.98; N, 19.17; found: C, 60.17;
H, 6.05; N, 19.24%.
-Phenoxymethyl-4-butyl-1,2,4-triazole-3-one (13). Yield
5
-Phenoxymethyl-4-(2-bromophenyl)-1,2,4-triazole-3-one
1
(
18). Yield 87%, mp 126–128°C. H NMR (DMSO-d )
6
5
δ: 12.12 (s, 1H, NH), 7.83–7.41 (m, 4H, arom.), 7.24–
1
8
1
1%, mp 147–149°C. H NMR (DMSO-d ) δ: 11.87 (s,
H, NH), 7.52–6.61 (m, 5H, arom.), 5.17 (s, 2H,
OCH ), 3.81 (t, J = 9.0 Hz, 2H, CH CH CH CH ),
.78–1.52 (m, 2H, CH CH CH CH ), 1.31–1.07 (m, 2H,
13
6
6
(
(
5
1
(
5
1
.77 (m, 5H, arom.), 4.85 (s, 2H, OCH2). C NMR
DMSO-d ) δ: 157.75 (C═O), 154,13 (Ctriazole), 143.26
6
2
2
2
2
3
i-PhC-OCH ), 133.61 (C-3), 132.27 (C-1), 131.94 (C-
2
1
2 2 2 3
), 131.46 (C-4), 129.88 (m-Ph2C), 129.25 (C-6),
23.41 (C-2), 121.92 (p-PhC), 115.11 (o-Ph2C), 61.45
13
CH CH CH CH ), 0.71 (t, J = 9.0 Hz, 3H, CH ).
C
2
2
2
3
3
NMR (DMSO-d ) δ: 157.84 (C═O), 155.46 (Ctriazole),
43.82 (i-PhC-OCH ), 130.09 (m-Ph2C), 121.97 (p-
PhC), 115.20 (o-Ph2C), 61.33 (OCH2), 40.84
CH CH CH CH ), 31.04 (CH CH CH CH ), 19.77
6
OCH ). Anal. Calcd for C H BrN O (346.18): C,
2 15 12 3 2
1
2
2.04; H, 3.49; N, 12.14; found: C, 51.98; H, 3.43; N,
2.07%.
(
(
2 2 2 3 2 2 2 3
5
-Phenoxymethyl-4-(4-bromophenyl)-1,2,4-triazole-3-one
CH CH CH CH ), 13.97 (CH3). Anal. Calcd for
1
2
2
2
3
(19). Yield 89%, mp 176–178°C. H NMR (DMSO-d ) δ:
6
C H N O (247.29): C, 63.14; H, 6.93; N, 16.99;
13
17
3
2
12.14 (s, 1H, NH), 7.70–7.42 (m, 4H, arom.), 7.28–6.84 (m,
found: C, 63.21; H, 6.99; N, 16.89%.
13
5
1
H, arom.), 4.93 (s, 2H, OCH2). C NMR (DMSO-d ) δ:
6
5-Phenoxymethyl-4-benzyl-1,2,4-triazole-3-one (14).
Yield
57.66 (C═O), 154.41 (Ctriazole), 143.19 (i-PhC-OCH2),
1
8
1
4
1
1
6%, mp 148–150°C. H NMR (DMSO-d ) δ: 11.97 (s,
H, NH), 7.34–6.85 (m, 10H, arom.), 4.95 (s, 2H, OCH ),
.88 (s, 2H, CH2). C NMR (DMSO-d ) δ: 157.70 (C═O),
55.61 (Ctriazole), 143.86 (i-PhC-OCH ), 137.06 (C-1),
29.97 (m-Ph2C), 128.97 (C-3, C-5), 128.00 (C-4), 127.66
C-2, C-6), 121.94 (p-PhC), 115.18 (o-Ph2C), 61.29
OCH ), 44.30 (CH ). Anal. Calcd for C H N O
6
132.70 (C-3, C-5), 132.57 (C-1, C-4), 129.96 (C-2, C-6),
129.49 (m-Ph2C), 122.06 (p-PhC), 115.38 (o-Ph2C), 61.50
(OCH ). Anal. Calcd for C H BrN O (346.18): C,
2
13
6
2
15 12
3 2
2
52.04; H, 3.49; N, 12.14; found: C, 52.09; H, 3.53; N,
2.20%.
1
(
(
5-Phenoxymethyl-4-(4-ethoxyphenyl)-1,2,4-triazole-3-one
1
(20). Yield 80%, mp 112–114°C. H NMR (DMSO-d ) δ:
2
2
16 15
3
2
6
(281.31): C, 68.31; H, 5.37; N, 14.94; found: C, 68.23; H,
11.78 (s, 1H, NH), 7.37–7.12 (m, 4H, arom.), 7.04–6.98 (m,
5
.30; N, 14.88%.
5H, arom.), 4.86 (s, 2H, OCH ), 4.03 (q, J = 7.0, 14.0 Hz,
2
13
5-Phenoxymethyl-4-cyclohexyl-1,2,4-triazole-3-one (15). Yield
2H, CH ), 1.33 (t, J = 7.0 Hz, CH ). C NMR (DMSO-d6)
2 3
1
7
5%, mp 180–182°C. H NMR (DMSO-d ) δ: 11.68 (s,
δ: 158.94 (C═O), 157,88 (Ctriazole), 155.12 (C-1), 143.69 (i-
6
1
H, NH), 7.36–7.20 (m, 5H, arom.), 4.55 (s, 2H, OCH2),
PhC-OCH ), 129.99 (m-Ph2C), 128.83 (C-2, C-6), 125.55
2
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Pachuta-Stec, A. Biernasiuk, A. Malm, and M. Pitucha
Vol 000
(
6
C-4), 121.99 (p-PhC), 119.26 (C-3, C-5), 115.40 (o-Ph2C),
Appropriate DMSO, growth, and sterile controls were
carried out. The medium with no tested substances was
used as control.
3.86 (OCH CH ), 61.45 (OCH ), 15.05 (CH ). Anal. Calcd
2
3
2
3
for C H N O (311.33): C, 65.58; H, 5.50; N, 13.50;
17 17 3 3
found: C, 65.63; H, 5.44; N, 13.57%.
The minimal bactericidal concentration (MBC) or
minimal fungicidal concentration (MFC) are defined as
the lowest concentration of the compounds that is
required to kill a particular bacterial or fungal species.
MBC or MFC was determined by removing the culture
using for MIC determinations from each well and
spotting onto appropriate agar medium. The plates were
incubated. The lowest compounds concentrations with no
visible growth observed was assessed as a bactericidal or
fungicidal concentration. All the experiments were
repeated three times, and representative data are
presented [41].
Microbiology. The examined compounds 1–20 were
screened in vitro for antibacterial and antifungal activities
using the broth microdilution method according to
European Committee on Antimicrobial Susceptibility
Testing (EUCAST) [39] and Clinical and Laboratory
Standards Institute guidelines [40] against a panel of
reference strains of microorganisms, including Gram-
positive bacteria (S. aureus ATCC 25923, S. aureus
ATCC 6538, Staphylococcus epidermidis ATCC 12228,
B. subtilis ATCC 6633, B. cereus ATCC 10876, and
M. luteus ATCC 10240), Gram-negative bacteria
(
Bordetella bronchiseptica ATCC 4617, Escherichia coli
In this study, no bioactivity was defined as an
MIC > 1000 μg/mL, mild bioactivity as an MIC in
the range 501–1000 μg/mL, moderate bioactivity with
MIC from 126 to 500 μg/mL, good bioactivity as an
MIC in the range 26–125 μg/mL, strong bioactivity
with MIC between 10 and 25 μg/mL, and very strong
bioactivity as a MIC < 10 μg/mL. The MBC/MIC
or MFC/MIC ratios were calculated in order to
determine bactericidal/fungicidal (MBC/MIC ≤ 4 and
MFC/MIC ≤ 4) or bacteriostatic/fungistatic (MBC/
MIC > 4 and MFC/MIC > 4) effect of the tested
compounds [42].
ATCC 25922, Klebsiella pneumoniae ATCC 13883,
Proteus mirabilis ATCC 12453, Salmonella typhimurium
ATCC 14028, and Pseudomonas aeruginosa ATCC
9
027), and fungi belonging to yeasts (Candida albicans
ATCC 10231 and Candida parapsilosis ATCC 22019).
The microorganisms belonging to ATCC came from
American Type Culture Collection, routinely used for the
evaluation of antimicrobials. All the used microbial
cultures were first subcultured on nutrient agar or
Sabouraud agar for bacteria and fungi, respectively.
The surface of Mueller-Hinton agar (for bacteria) and
RPMI 1640 with MOPS (for fungi) were inoculated with
the suspensions of bacterial or fungal species. Microbial
suspensions were prepared in sterile saline (0.85% NaCl)
with an optical density of McFarland standard scale 0.5.
Samples containing examined compounds were dissolved
in 1-mL DMSO. Furthermore, bacterial and fungal
suspensions were put onto Petri dishes with solid media
containing 2000 μg/mL of the tested compounds
followed incubation. The inhibition of microbial growth
was judged by comparison with a control culture
prepared without any sample tested. Ciprofloxacin or
fluconazole (Sigma) were used as a reference antibacterial
or antifungal compounds, respectively.
Subsequently, minimal inhibitory concentration (MIC)
of the compounds was examined by the microdilution
broth method, using their twofold dilutions in Mueller-
Hinton broth (for bacteria) and RPMI 1640 broth with
MOPS (for fungi) prepared in 96-well polystyrene plates.
Final concentrations of the compounds ranged from 1000
to 0.488 μg/mL. Microbial suspensions were prepared in
sterile saline (0.85% NaCl) with an optical density of 0.5
McFarland standard. Next, bacterial or fungal suspension
was added per each well containing 200-μL broth and
various concentrations of the examined compounds. After
incubation, the MIC was assessed spectrophotometric as
the lowest concentration of the samples showing
complete bacterial or fungal growth inhibition.
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