New 3-hydroxy-2-naphthoic hydrazide derivatives: thiosemicarbazides and 1,2,4-triazole-3-thiones, their synthesis and in vitro antimicrobial evaluation

Article abstract of DOI:10.1007/s13738-016-0911-1

Abstract: In this paper, we describe twelve 3-hydroxy-2-naphthoic hydrazide derivatives (six thiosemicarbazides and six 1,2,4-triazole-3-thiones) which we have synthesized. Thiosemicarbazide derivatives were prepared by condensation reaction of 3-hydroxy-

Full text of DOI:10.1007/s13738-016-0911-1

J IRAN CHEM SOC
DOI 10.1007/s13738-016-0911-1
ORIGINAL PAPER
New 3‑hydroxy‑2‑naphthoic hydrazide derivatives:
thiosemicarbazides and 1,2,4‑triazole‑3‑thiones, their synthesis
and in vitro antimicrobial evaluation
Łukasz Popiołek1 · Kinga Paruch1 · Paweł Patrejko1 · Anna Biernasiuk2 ·
Monika Wujec1
Received: 25 January 2016 / Accepted: 10 June 2016
© The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract In this paper, we describe twelve 3-hydroxy-
2-naphthoic hydrazide derivatives (six thiosemicarbazides
and six 1,2,4-triazole-3-thiones) which we have synthe-
sized. Thiosemicarbazide derivatives were prepared by
condensation reaction of 3-hydroxy-2-naphthoic hydrazide
with appropriate isothiocyanates. The 1,2,4-triazole-
3-thiones were synthesized by the cyclization reaction of
corresponding thiosemicarbazide derivatives in alkaline
media. All obtained compounds have been analyzed by
1H NMR and 13C NMR spectra and evaluated for in vitro
antimicrobial activity against a panel of reference strains of
microorganisms, including Gram-positive bacteria, Gram-
negative bacteria and fungi belonging to yeasts Candida
spp. Our antimicrobial screening results indicated that
some of the newly obtained compounds showed high anti-
bacterial activity, especially against Gram-positive bacteria.
Among them, the activity of compounds 1, 2 and 4 was
moderate to strong against staphylococci. In addition to
this, compounds 1 and 2 showed two times higher activity
than cefuroxime and four times higher activity than ampi-
cillin against Bacillus cereus ATCC 10876.
Graphical Abstract
H
N
S
S
N
O
NH
NH
N
NH
R
R
2% NaOH
OH
OH
= Alk, Ar
R
Keywords 3-Hydroxy-2-naphthoic hydrazide ·
1,2,4-Triazole derivatives · Thiosemicarbazide derivatives ·
Antimicrobial activity · MIC · MBC
Introduction
The development of drug resistance to clinically used anti-
infective agents reveals that there is an urgent need for the
discovery of new antimicrobial compounds to treat multi-
resistant infections [1]. Considering this, our attention
focused on thiosemicarbazide and 1,2,4-triazole deriva-
tives, which have been incorporated into a wide range of
therapeutically interesting molecules and possess many
medical applications [2].
Electronic supplementary material The online version of this
article (doi:10.1007/s13738-016-0911-1) contains supplementary
material, which is available to authorized users.
It is known that thiosemicarbazides act as antibacterial
[3–5], antifungal [6, 7], antitubercular [8], anticonvulsant
[9, 10] and antitumor agents [11, 12]. In turn, the 1,2,4-tri-
azoles display a broad range of biological activities, such
as antibacterial [13–19], antifungal [20–22], antitubercular
[23–26], antiviral [27] and antiproliferative properties [28–
30]. Several compounds possessing the triazole scaffold are
clinically useful medicines, such as ribavirin [31], tercona-
zole [32] or fluconazole [33].
* Łukasz Popiołek
lukasz.popiolek@umlub.pl
1
Department of Organic Chemistry, Faculty of Pharmacy,
Medical University of Lublin, 4A Chodz´ki Street,
20-093 Lublin, Poland
2
Department of Pharmaceutical Microbiology, Faculty
of Pharmacy, Medical University of Lublin, 1 Chodz´ki Street,
20-093 Lublin, Poland
1 3

J IRAN CHEM SOC
Looking for the interesting starting material for new
research project, our group found 3-hydroxy-2-naph-
thoic hydrazide, which caught our attention. The choice
of 3-hydroxy-2-naphthoic hydrazide as a starting mate-
rial for our synthesis was based on the literature findings
concerning this hydrazide. This hydrazide is very useful
of itself in the histochemical detection of peroxidation in
animal tissues. According to Pompella and Comporti [34],
3-hydroxy-2-naphthoic hydrazide/Fast Blue B reaction is
a sensitive and reliable tool for the revelation of lipid per-
oxidation-derived carbonyl compounds in tissue. Pompella
and Comporti [34] suggested that in the tissues of hetero-
geneous cell composition its use can allow the approach
of choice for the recognition of the cell types involved in
the lipid peroxidation process in vivo. Dogan et al. [35]
using this hydrazide, synthesized novel thiosemicarbazides,
1,2,4-triazoles and 1,3,4-thiadiazoles with significant anti-
bacterial activity against Staphylococcus aureus, Escheri‑
chia coli and Pseudomonas aeruginosa, as well as anti-
fungal activity against Candida albicans. Similarly, Khalil
et al. [36] obtained benzenobenzo[g]phthalazine-1,4-dione
derivatives from this hydrazide, which showed interesting
antibacterial activity against Bacillus thuringiensis and
E. coli, comparable to commonly used chemotherapeu-
tics like ampicillin and chloramphenicol. In another study,
1,2,4-triazoles derived from this hydrazide showed promis-
ing in vitro anticancer activity against 60 human tumor cell
lines [37].
(doublet), dd (doublet of doublets), t (triplet), q (quartet),
m (multiplet) and b (broad). The progress of the reaction
and purity of obtained compounds were monitored by TLC,
using pre-coated aluminum sheets plates 60 F254 (Merck
Co. USA) in a CHCl3/C2H5OH (10:1, v/v) solvent sys-
tem. The spots were detected by exposure to a UV lamp at
254 nm. Elemental analyses of obtained compounds were
carried out with AMZ 851 CHX analyser (PG, Gdan´sk,
Poland). The results of elemental analyses (C, H, N) were
within 0.4 % of the calculated values.
Preparation of thiosemicarbazide derivatives (1–6)
3-Hydroxy-2-naphthoic hydrazide (2.02 g, 10 mmol) was
added to the flask with 15 ml of ethanol and heated under
reflux until hydrazide was dissolved. Then 11 mmol of
appropriate isothiocyanate was added, and the mixture was
heated under reflux for 3 h. Subsequently, the content of the
flask was left for 24 h. The obtained precipitate was filtered
off, dried and crystallized from ethanol.
N‑Ethyl‑2‑[(3‑hydroxynaphthalen‑2‑yl)carbonyl]hydrazi‑
necarbothioamide (1)
CAS Registry Number 219538-16-6. Yield: 95 %; M.p.:
196–198 °C. Analysis for C14H15N3O2S (289.35) Cal-
culated: C: 58.11 %, H: 5.23 %, N: 14.52 %; Found: C:
1
58.22 %, H: 5.26 %, N: 14.49 %. H NMR (DMSO-d6) δ
As a continuation of our research interest in the field of
synthesis and antimicrobial screening of heterocyclic com-
pounds, we have undertaken this research, in which we
have synthesized and test for in vitro antimicrobial activity
a series of thiosemicarbazides and 1,2,4-triazole-3-thiones
obtained form 3-hydroxy-2-naphthoic hydrazide. This
seemed reasonable in the view of the above mentioned facts.
(ppm) = 1.07–1.11 (t, 3H, CH3), 3.44–3.50 (q, 2H, CH2,
J = 6 Hz), 7.32–7.39 (m, 2H, ArH), 7.50–7.54 (m, 1H,
ArH), 7.75–7.78 (d, 2H, ArH, J = 9 Hz), 7.87–7.90 (d, 2H,
ArH, J = 9 Hz), 8.12–8.14 (m, 1H, ArH), 8.50 (s, 1H, OH),
9.54 (s, 1H, NH), 10.71 (s, 1H, NH), 11.51 (s, 1H, NH);
13C NMR (DMSO) δ (ppm) = 14.9 (CH3), 41.1 (CH2),
111.0, 119.1, 124.3, 126.3, 127.1, 128.9, 129.2, 130.9,
136.4 (10Car), 154.8 (C=O), 181.3 (C=S).
Experimental
Chemistry
2‑[(3‑Hydroxynaphthalen‑2‑yl)
carbonyl]‑N‑(prop‑2‑en‑1‑yl)hydrazinecarbothioamide (2)
CAS Registry Number 224456-14-8. Yield: 80 %; M.p.:
197–199 °C. Analysis for C15H15N3O2S (301.36) Cal-
culated: C: 59.78 %, H: 5.02 %, N: 13.94 %; Found: C:
All required chemicals and solvents were purchased from
Sigma-Aldrich (Munich, Germany) and Merck Co. (Darm-
stadt, Germany) and used without further purification.
Melting points were determined with Fisher–Johns blocks
1
59.85 %, H: 5.05 %, N: 13.90 %. H NMR (DMSO-d6) δ
(ppm) = 4.12–4.15 (m, 2H, CH2), 5.05–5.20 (m, 2H, CH2),
5.79–5.91 (m, 1H, CH), 7.32–7.39 (m, 2H, ArH), 7.49–7.55
(m, 1H, ArH), 7.75–7.78 (d, 1H, ArH, J = 9 Hz), 7.87–7.90
(d, 1H, ArH, J = 9 Hz), 8.31–8.34 (m, 1H, ArH), 8.51 (s,
1H, OH), 9.63 (s, 1H, NH), 10.77 (s, 1H, NH), 11.49 (s,
1H, NH); 13C NMR (DMSO) δ (ppm) = 31.2 (CH2), 46.4
(CH2), 111.0, 115.8, 119.0, 124.3126.3, 127.1, 128.9,
129.2, 130.9, 135.3, 136.5 (10Car), 132.6 (CH), 165.1
(C=O), 183.9 (C=S).
1
(Fisher Scientific, Germany) and are uncorrected. The H
NMR spectra were recorded on a Bruker Avance 300 appa-
ratus (Bruker BioSpin GmbH, Germany) in DMSO-d6
with TMS as internal standard. The 13C NMR spectra were
recorded on a Bruker Avance 300 apparatus in DMSO. The
chemical shifts are expressed on the δ (ppm) scale using
TMS as the standard reference. The coupling constants (J)
are given in Hertz. Spin multiples are given as s (singlet), d
1 3

J IRAN CHEM SOC
2‑[(3‑Hydroxynaphthalen‑2‑yl)carbonyl]‑N‑(2‑methoxy‑
phenyl)hydrazinecarbothioamide (3)
5.02 %, N: 11.02 %; Found: C: 62.91 %, H: 5.06 %, N:
11.05 %. H NMR (DMSO-d6) δ (ppm) = 1.28–1.32 (t,
1
3H, CH3, J = 6 Hz), 3.93–4.00 (q, 2H, CH2, J = 6 Hz),
6.83–6.86 (d, 2H, ArH, J = 9 Hz), 7.33–7.36 (d, 2H, ArH,
J = 9 Hz), 7.38–7.39 (m, 2H, ArH), 7.50–7.55 (m, 1H,
ArH), 7.76–7.79 (d, 1H, ArH, J = 9 Hz), 7.88–7.91 (d,
1H, ArH, J = 9 Hz), 8.39 (s, 1H, ArH), 8.52 (s, 1H, OH),
8.76 (s, 1H, NH), 10.55 (s, 1H, NH), 11.51 (s, 1H, NH);
13C NMR (DMSO) δ (ppm) = 15.1 (CH3), 63.7 (CH2),
109.6, 114.5, 117.0, 124.0, 126.3, 127.2, 127.3, 128.1,
128.6, 129.6, 132.7, 135.8, 150.3, 153.4 (16Car), 158.8
(C=O), 168.4 (C=S).
Yield: 94 %; M.p.: 196–197 °C. Analysis for C19H17N3O3S
(367.42) Calculated: C: 62.11 %, H: 4.66 %, N: 11.44 %;
Found: C: 62.15 %, H: 4.63 %, N: 11.47 %. H NMR
1
(DMSO-d6) δ (ppm) = 3.83 (s, 3H, CH3), 6.92–6.97 (m,
1H, ArH), 7.05–7.07 (m, 1H, ArH), 7.14–7.19 (m, 1H,
ArH), 7.32 (s, 1H, OH), 7.34–7.39 (m, 1H, ArH), 7.50–7.55
(m, 1H, ArH), 7.76–7.78 (d, 1H, ArH, J = 6 Hz), 7.93–7.94
(m, 1H, ArH), 8.08–8.09 (m, 1H, ArH), 8.47–8.48 (m, 1H,
ArH), 8.63–8.64 (m, 1H, ArH), 9.48 (s, 1H, NH), 11.20
(s, 1H, NH), 11.71 (s, 1H, NH); 13C NMR (DMSO) δ
(ppm) = 56.18 (CH3), 111.8, 113.3, 120.3, 121.6, 122.5,
124.3, 126.3, 128.8, 130.2, 132.5, 136.3, 150.1, 155.2
(16Car), 165.1 (C=O), 180.8 (C=S).
Preparation of 4,5‑disubstituted‑2,4‑di‑
hydro‑3H‑1,2,4‑triazole‑3‑thiones (7–12)
A mixture of appropriate thiosemicarbazide 1–6 (10 mmol)
and 20–40 ml of 2 % aqueous solution of sodium hydrox-
ide was refluxed for 2 h. Then, the solution was neutralized
with diluted hydrochloric acid, and the formed precipitate
was filtered off and crystallized from ethanol.
2‑[(3‑Hydroxynaphthalen‑2‑yl)carbonyl]‑N‑(3‑methoxy‑
phenyl)hydrazinecarbothioamide (4)
Yield: 93 %; M.p.: 198–200 °C. Analysis for C19H17N3O3S
(367.42) Calculated: C: 62.11 %, H: 4.66 %, N: 11.44 %;
1
Found: C: 62.18 %, H: 4.62 %, N: 11.41 %. H NMR
4‑Ethyl‑5‑(3‑hydroxynaphthalen‑2‑yl)‑2,4‑di‑
(DMSO-d6) δ (ppm) = 3.75 (s, 3H, CH3), 6.73–6.77 (m,
2H, ArH), 7.06–7.09 (d, 1H, ArH, J = 9 Hz), 7.23–7.28
(m, 2H, ArH), 7.33 (s, 1H, OH), 7.34–7.39 (m, 2H,
ArH), 7.49–7.55 (m, 1H, ArH), 7.76–7.79 (d, 1H, ArH,
J = 9 Hz), 7.90–7.93 (m, 1H, ArH), 8.53 (s, 1H, NH),
9.86 (s, 1H, NH), 11.56 (s, 1H, NH); 13C NMR (DMSO)
δ (ppm) = 55.56 (CH3), 110.9, 124.4, 121.6, 126.3, 128.9,
129.3, 130.4, 132.5, 136.4, 140.6, 155.2 (16Car), 165.1
(C=O), 179.4 (C=S).
hydro‑3H‑1,2,4‑triazole‑3‑thione (7)
CAS Registry Number 219538-20-2. Yield: 92 %; M.p.:
223–225 °C. Analysis for C14H13N3OS (271.34) Calcu-
lated: C: 61.97 %, H: 4.83 %, N: 15.49 %; Found: C:
1
61.92 %, H: 4.87 %, N: 15.53 %. H NMR (DMSO-d6) δ
(ppm) = 1.03–1.07 (t, 3H, CH3, J = 6 Hz), 3.86–3.94 (q,
2H, CH2, J = 9 Hz), 7.34–7.39 (m, 2H, ArH), 7.49–7.54
(m, 1H, ArH), 7.79–7.81 (d, 2H, ArH, J = 6 Hz), 7.88–
7.90 (d, 2H, ArH, J = 6 Hz), 8.03 (s, 1H, ArH), 10.65
(s, 1H, OH), 13.93 (s, 1H, NH); 13C NMR (DMSO) δ
(ppm) = 13.7 (CH3), 45.2 (CH2), 110.1, 116.7, 124.2,
126.5, 127.5, 128.3, 128.7, 132.6, 136.0, 150.0 (10Car),
166.7 (C=S).
2‑[(3‑Hydroxynaphthalen‑2‑yl)carbonyl]‑N‑(4‑methoxy‑
phenyl)hydrazinecarbothioamide (5)
CAS Registry Number 224456-22-8. Yield: 86 %; M.p.:
194–196 °C. Analysis for C19H17N3O3S (367.42) Cal-
culated: C: 62.11 %, H: 4.66 %, N: 11.44 %; Found:
5‑(3‑Hydroxynaphthalen‑2‑yl)‑4‑(prop‑2‑en‑1‑yl)‑2,4‑dihy‑
dro‑3H‑1,2,4‑triazole‑3‑thione (8)
1
C: 62.15 %, H: 4.63 %, N: 11.48 %. H NMR (DMSO-
d6) δ (ppm) = 3.75 (s, 3H, CH3), 6.90–6.93 (d, 2H, ArH,
J = 9 Hz), 7.27–7.36 (m, 3H, ArH), 7.37 (s, 1H, OH),
7.47–7.54 (m, 1H, ArH), 7.75–7.78 (d, 2H, ArH, J = 9 Hz),
7.81–7.92 (m, 2H, ArH), 8.53 (s, 1H, NH), 9.76 (s, 1H,
NH), 11.54 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 55.7
(CH3), 110.9, 113.8, 119.3, 124.3, 126.3, 127.2, 128.9,
129.3, 132.3, 136.4 (16Car), 157.2 (C=O), 179.4 (C=S).
CAS Registry Number 29213-07-8 Yield: 92 %; M.p.:
199–200 °C. Analysis for C15H13N3OS (283.30) Calcu-
lated: C: 63.58 %, H: 4.62 %, N: 14.83 %; Found: C:
1
63.52 %, H: 4.65 %, N: 14.79 %. H NMR (DMSO-d6) δ
(ppm) = 4.61–4.62 (d, 2H, CH2, J = 3 Hz), 4.69–4.95 (dd,
2H, CH2, J = 9 Hz), 5.59–5.71 (m, 1H, CH), 7.32–7.37
(m, 2H, ArH), 7.47–7.53 (m, 1H, ArH), 7.77–7.80 (d, 1H,
ArH, J = 9 Hz), 7.86–7.88 (d, 2H, ArH, J = 6 Hz), 7.98 (s,
1H, ArH), 10.68 (s, 1H, OH), 14.01 (s, 1H, NH); 13C NMR
(DMSO) δ (ppm) = 46.18 (CH2), 110.1, 116.6, 124.2,
126.5, 127.5, 128.3, 128.7, 131.9, 136.0, 150.3 (10Car)
117.6 (= CH2), 132.6 (CH), 167.4 (C=S).
N‑(4‑Ethoxyphenyl)‑2‑[(3‑hydroxynaphthalen‑2‑yl)car‑
bonyl]hydrazinecarbothioamide (6)
Yield: 94 %; M.p.: 195–197 °C. Analysis for
C20H19N3O3S (381.45) Calculated: C: 62.97 %, H:
1 3

J IRAN CHEM SOC
5‑(3‑Hydroxynaphthalen‑2‑yl)‑4‑(2‑methoxyphenyl)‑2,4‑di‑
hydro‑3H‑1,2,4‑triazole‑3‑thione (9)
(DMSO-d6) δ (ppm) = 1.23–1.29 (t, 3H, CH3, J = 9 Hz),
3.90–3.97 (q, 2H, CH2, J = 6 Hz, J = 9 Hz), 6.83–6.86 (d,
2H, ArH, J = 9 Hz), 7.07 (s, 1H, ArH), 7.17–7.20 (d, 2H,
ArH, J = 9 Hz), 7.28–7.33 (m, 1H, ArH), 7.41–7.47 (m,
1H, ArH), 7.65–7.68 (d, 1H, ArH, J = 9 Hz), 7.79–7.81 (d,
1H, ArH, J = 6 Hz), 8.02 (s, 1H, ArH), 14.08 (s, 1H, NH);
13C NMR (DMSO) δ (ppm) = 15.0 (CH3), 63.7 (CH2),
109.6, 114.5, 117.0, 124.0, 126.3, 127.2, 127.3, 128.1,
128.6, 129.6, 132.7, 135.8, 150.3, 153.3, 153.4, 158.8
(16Car), 168.4 (C=S).
Yield: 93 %; M.p.: 220–222 °C. Analysis for C19H15N3O2S
(349.41) Calculated: C: 65.31 %, H: 4.33 %, N: 12.03 %;
1
Found: C: 65.38 %, H: 4.31 %, N: 12.06 %. H NMR
(DMSO-d6) δ (ppm) = 3.54 (s, 3H, CH3), 6.92–6.99 (m,
2H, ArH), 7.05 (s, 1H, ArH), 7.25–7.35 (m, 3H, ArH),
7.39–7.45 (m, 1H, ArH), 7.62–7.65 (d, 2H, ArH, J = 9 Hz),
7.79–7.81 (d, 2H, ArH, J = 6 Hz), 7.87 (s, 1H, ArH),
10.19 (s, 1H, OH), 14.00 (s, 1H, NH); 13C NMR (DMSO)
δ (ppm) = 55.9 (CH3), 109.4, 112.8, 116.8, 120.5, 123.0,
123.9, 126.2, 127.0, 128.0, 128.5, 130.8, 131.3, 132.3,
135.7, 150.8, 153.5 (16Car), 168.7 (C=S).
Microbiology
In vitro antimicrobial assay
5‑(3‑Hydroxynaphthalen‑2‑yl)‑4‑(3‑methoxyphenyl)‑2,4‑di‑
hydro‑3H‑1,2,4‑triazole‑3‑thione (10)
The examined compounds 1–12 were screened in vitro for
antibacterial and antifungal activity. Procedure for antimi-
crobial assay is presented in Supplemental Material.
Yield: 94 %; M.p.: 271–273 °C. Analysis for C19H15N3O2S
(349.41) Calculated: C: 65.31 %, H: 4.33 %, N: 12.03 %;
1
Found: C: 65.37 %, H: 4.37 %, N: 12.00 %. H NMR
Results and discussion
Chemistry
(DMSO-d6) δ (ppm) = 3.56 (s, 3H, CH3), 6.82–6.95 (m,
3H, ArH), 7.09 (s, 1H, ArH), 7.20–7.33 (m, 2H, ArH),
7.42–7.47 (m, 1H, ArH), 7.66–7.68 (d, 1H, ArH, J = 6 Hz),
7.78–7.81 (d, 1H, ArH, J = 9 Hz), 8.03 (s, 1H, ArH), 10.30
(s, 1H, OH), 14.15 (s, 1H, NH); 13C NMR (DMSO) δ
(ppm) = 55.65 (CH3), 109.6, 114.5, 116.9, 120.4, 124.0,
126.4, 127.2, 128.1, 128.6, 129.8, 132.7, 135.7, 135.9,
150.0, 153.5, 159.3 (16Car), 169.1 (C=S).
The main purpose of this work was the synthesis, spectral
identification and in vitro antimicrobial screening of thio-
semicarbazide (1–6) and 1,2,4-triazole-3-thione derivatives
(7–12). The compounds 1–12 were synthesized according
to the steps presented in Scheme 1. Compounds 3, 4, 6, 9,
10 and 12 are unknown in scientific literature.
5‑(3‑Hydroxynaphthalen‑2‑yl)‑4‑(4‑methoxyphenyl)‑2,4‑di‑
hydro‑3H‑1,2,4‑triazole‑3‑thione (11)
1,2,4-Triazole derivatives (7–12) were prepared in two-
step synthesis. In the first step of synthesis, 3-hydroxy-
2-naphthoic hydrazide was subjected to condensation
reaction with appropriate isothiocyanates to afford 1,4-dis-
ubstituted thiosemicarbazide derivatives (1–6). Then new
1,2,4-triazole-3-thiones (7–12) were synthesized by the
cyclization reaction of corresponding thiosemicarbazide
(1–6) derivatives in alkaline media.
CAS Registry Number 910875-05-7. Yield: 95 %; M.p.:
272–274 °C. Analysis for C19H15N3O2S (349.41) Cal-
culated: C: 65.31 %, H: 4.33 %, N: 12.03 %; Found: C:
1
65.37 %, H: 4.31 %, N: 12.07 %. H NMR (DMSO-d6)
δ (ppm) = 3.68 (s, 3H, CH3), 6.85–6.88 (d, 2H, ArH,
J = 9 Hz), 7.08 (s, 1H, ArH), 7.19–7.22 (d, 2H, ArH,
J = 9 Hz), 7.28–7.33 (m, 1H, ArH), 7.41–7.46 (m, 1H,
ArH), 7.65–7.68 (d, 1H, ArH, J = 9 Hz), 7.79–7.81 (d,
1H, ArH, J = 6 Hz), 8.02 (s, 1H, ArH), 10.27 (s, 1H, OH),
14.09 (s, 1H, NH); 13C NMR (DMSO) δ (ppm) = 55.7
(CH3), 109.6, 114.2, 116.9, 124.0, 126.3, 127.2, 127.4,
128.1, 128.6, 129.6, 132.7, 135.8, 150.3, 153.4, 159.5
(16Car), 168.4 (C=S).
All synthesized compounds are stable solids and dis-
solve in DMSO in room temperature. The spectral data
(1H NMR, 13C NMR) confirm the successful synthesis of
desired compounds (7–12).
1
In the H NMR spectra of 1,4-disubstituted thiosemi-
carbazides (1–6), three typical proton singlet signals for
the three NH groups were observed in the range of δ 8.53–
11.71 ppm. Whereas in the 13C NMR two signals for C=O
group and C=S group were observed at δ 154.8–165.1 ppm
and δ 168.4–183.0 ppm, respectively. The 1,2,4-triazole-
4‑(4‑Ethoxyphenyl)‑5‑(3‑hydroxynaphthalen‑2‑yl)‑2,4‑di‑
hydro‑3H‑1,2,4‑triazole‑3‑thione (12)
1
3-thiones (7–12) in H NMR spectra showed characteristic
singlet signal for NH group at δ 13.93–14.15 ppm. Beside
this, in 13C NMR spectra for these compounds (7–12) a sig-
nal for C=S was observed at δ 166.7–169.1 ppm. All other
aliphatic and aromatic signals were found at usual regions.
Yield: 94 %; M.p.: 274–276 °C. Analysis for C20H17N3O2S
(363.43) Calculated: C: 66.10 %, H: 4.71 %, N: 11.56 %;
Found: C: 66.15 %, H: 4.70 %, N: 11.59 %. H NMR
1
1 3

J IRAN CHEM SOC
H
N
S
N
S
SH
N
N
O
NH
NH
O
NH2
NH
N
N
NH
R
R
R
2% NaOH
+
R-NCS
OH
OH
OH
OH
EtOH
( 7 - 12 )
( 1 - 6 )
Compound No
R
1, 7
2, 8
C2H5
CH2=CHCH2
2-CH3OC6H4
3-CH3OC6H4
4-CH3OC6H4
4-CH3CH2OC6H4
3, 9
4, 10
5, 11
6, 12
Scheme 1 Synthesis scheme of 1,2,4-triazole derivatives (7–12)
Table 1 Activity data expressed as MIC [µg/ml] against the reference strains of bacteria and fungi
Species/compound
MIC (MBC/MFC) [µg/ml] of the tested compounds
CPX CFX APC FLU
1
2
3
4
5
7
8
9
11
Gram-positive bacteria
Staphylococcus aureus ATCC 25923
Staphylococcus aureus ATCC 6538
Staphylococcus aureus ATCC 43300
15.62 15.62
31.25 31.25
15.62 7.81
–
–
15.62
15.62
–
–
–
–
62.5 62.5 125
250 0.488 0.49
250 0.244 0.98
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
125 125 62.5
250 7.81
62.5 125 31.25 250 0.244 nd
62.5 125 62.5 250 0.122 0.24
Staphylococcus epidermidis ATCC 12228 7.81
3.91
250
–
–
–
–
15.62
Micrococcus luteus ATCC 10240
Bacillus subtilis ATCC 6633
Bacillus cereus ATCC 10876
Gram-negative bacteria
62.5
31.25 1000 125 125 31.25 125 0.976 0.98
31.25 15.62
15.62 15.62
31.25
–
–
125 125 62.5
125 125 62.5
250 0.031 15.62 nd
1000
250 0.061 31.25 62.5 nd
Bordetella bronchiseptica ATCC 4617
Fungi
–
–
–
250
–
500 500
–
–
0.976 nd
nd
nd
Candida albicans ATCC 2091
Candida albicans ATCC 10231
Candida parapsilosis ATCC 22019
–
–
–
–
–
–
–
1000
1000
–
–
–
–
500 500 1000
500 500
500 500 1000
–
–
–
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.244
0.976
1.953
1000
1000
–
The standard compounds used as positive controls: ciprofloxacin (CPX), cefuroxime (CFX), ampicillin (APC) for bacteria and fluconazole
(FLU*) for fungi
Compounds with bactericidal effect (MBC/MIC ≤ 4) are marked with bold
“–” no activity, nd not determined, MIC minimal inhibitory concentration, MBC minimal bactericidal concentration
The above mentioned spectral data successfully confirmed
the formation of expected products.
The synthesized compounds (1–12) were screened
in vitro for antibacterial and antifungal activity.
Proteus mirabilis ATCC 12453, Salmonella typhimurium
ATCC 14028 and P. aeruginosa ATCC 9027. Moreover,
compounds 6, 10 and 12 had no activity against the other
reference strains of bacteria (Table 1).
On the basis of minimal inhibitory concentration (MIC)
values presented in Table 1, obtained by the broth microdi-
lution method, it was shown that some of the tested com-
pounds showed high influence on the growth of Gram-pos-
itive bacteria.
Among them, compounds 1, 2 and 4 possessed the highest
activity against staphylococci with MIC = 3.91–31.25 µg/
Microbiology
The results of our study indicated that examined com-
pounds 1–3, 5, 6, 9–12 had no inhibitory effect on the
growth of reference strains of Gram-negative bacteria, i.e.,
E. coli ATCC 25922, Klebsiella pneumoniae ATCC 13883,
1 3

J IRAN CHEM SOC
ml. S. epidermidis ATCC 12228 and S. aureus ATCC 43300
were especially sensitive to them.
(MIC = 500 µg/ml) with fungicidal effect. The other sub-
stances had mild or no activity toward yeasts (Table 1).
Compound 1 showed highest activity toward S. epider‑
midis ATCC 12228 (MIC = 7.81 µg/ml) and S. aureus ATCC
43300 (MIC = 15.62 µg/ml) with bactericidal effect. The
activity against other Staphylococcus species was strong
(MIC = 15.62–31.25 µg/ml). Compound 2 showed the high-
est activity of all synthesized compounds against S. epider‑
midis ATCC 12228 (MIC = 3.91 µg/ml) but with bacte-
riostatic effect. The activity against S. aureus ATCC 43300
was also very strong (MIC = 7.81 µg/ml) or strong against
S. aureus ATCC 25923 and S. aureus ATCC 6538 with
MIC = 15.62 µg/ml and 31.25 µg/ml, respectively. Com-
pound 4 showed the strongest activity with bactericidal effect
against S. aureus ATCC 43300. Its activity against other
staphylococci was also very good (MIC = 15.62 µg/ml).
The growth of Micrococcus luteus ATCC 10240 was
inhibited strongly by compounds 4 and 9 (MIC = 31.25 µg/
ml) with bacteriostatic effect. The MIC values of other
compounds were much higher for this microorganism
(MIC = 62.5–125 µg/ml), with bacteriostatic or bacteri-
cidal effect.
Considering Bacillus subtilis ATCC 6633, the highest
antibacterial activity against this microorganism was shown
by compound 2 (MIC = 15.62 µg/ml) with bacteriostatic
effect. The activity of this compound was equal to the
activity of cefuroxime (MIC = 15.62 µg/ml). Compounds
1 and 4 showed also high activity against this bacterium
(MIC = 31.25 µg/ml) with bacteriostatic and bactericidal
effect, respectively.
It is worth to underline that compounds 1 and 2
showed two times higher activity than cefuroxime
(MIC = 31.25 µg/ml) against Bacillus cereus ATCC
10876. The activity of these compounds (MIC = 15.62 µg/
ml) was also 4 times higher than the activity of ampicillin
(MIC = 62.5 µg/ml) against this pathogen. The sensitive-
ness of B. cereus to synthesized compounds is very impor-
tant due to the fact that this pathogen is a common source
of severe infections [38].
Structure–activity relationship
On the basis of our antimicrobial assay results, we can con-
clude that the introduction of 3-methoxyphenyl substituent
in 1,2,4-triazole derivative (10) can be connected with the
disappearance of the activity of this compound, whereas the
same substituent in thiosemicarbazide (4) did not revealed
such attribute and was active against many bacterial strains.
The thiosemicarbazide derivatives 1 and 2 substituted
with aliphatic substituents: ethyl (1) and allyl (2) as well as
compound 4 substituted with meta-methoxyphenyl showed
the strongest activity among all of synthesized compounds.
Ortho-methoxyphenyl and para-methoxyphenyl thiosemi-
carbazide derivatives (3, 5) showed the weakest activity
among all thiosemicarbazide derivatives. In the case of
thiosemicarbazide (6) and 1,2,4-triazole derivative (12), the
presence of 4-ethoxyphenyl substituent can be connected
with the lack of antimicrobial activity. The 1,2,4-triazole
derivative 9, which is substituted with ortho-methoxyphe-
nyl substituent in comparison to the thiosemicarbazide with
the same substituent (3), showed moderate to good activity
against bacterial strains.
In general, the introduction of alkyl (1, 2) and 3-meth-
oxyphenyl substituents (4) was the most beneficial in terms
of bioactivity in the case of thiosemicarbazides, whereas in
1,2,4-triazoles the compounds with alkyl (7, 8), 2-methoxy-
phenyl (9), 4-methoxyphenyl (11) substituents displayed
the most significant activity.
Conclusions
In this work, we synthesized and analyzed 6 thiosemicar-
bazide derivatives (1–6) and 6 1,2,4-triazole derivatives
(7–12) and tested them against a panel of bacterial and fun-
gal strains. Our antimicrobial screening results indicate that
some of the newly obtained compounds showed significant
antibacterial activity, especially against Gram-positive bac-
teria. Among them, the activities of compounds 1, 2 and 4
were moderate to strong against staphylococci. In addition
to these compounds 1 and 2 showed two times higher activ-
ity than cefuroxime and 4 times higher activity than ampi-
cillin against B. cereus ATCC 10876.
The activity of compounds 7–9 against Gram-positive
bacteria was good with MIC = 62.5–125 µg/ml, and had
bactericidal or bacteriostatic effect (MBC/MIC = 1–32).
In turn, the minimum concentrations of 11, which inhib-
ited the growth of these microorganisms were lower
(MIC = 125–250 µg/ml).
Besides, compounds 4, 7 and 8 were found to exhibit
some activity against B. bronchiseptica ATCC 4617,
belonging to Gram-negative bacteria (MIC = 250–500 µg/
ml). Compounds 7 and 8 had bactericidal effect against this
microorganism.
Acknowledgments Dr Łukasz Popiołek is awarded with the Fellow-
ship for Young Scientists with Outstanding Scientific Achievements
from the Medical University of Lublin (Poland).
The obtained compounds indicated also some activ-
ity against Candida spp. ATCC, belonging to yeasts.
The substances 7 and 8 exhibited moderate activity
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
1 3

J IRAN CHEM SOC
Open Access This article is distributed under the terms of the Crea-
tive Commons Attribution 4.0 International License (http://crea-
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
18. R. Singh, G.V. Pujar, M.N. Purohit, V.M. Chandrashekar, Med.
Chem. Res. 22, 2163–2173 (2013)
19. H. Bendaha, L. Yu, R. Touzani, R. Souane, G. Giaever, C.
Nislow, C.H. Boone, S.E. Kadiri, G.W. Brown, M. Bellaoui, Eur.
J. Med. Chem. 46, 4117–4124 (2011)
20. P.Z. Zhang, S.F. Zhou, T.R. Li, J. Jiang, Chin. Chem. Lett. 23,
1381–1384 (2012)
21. Y. Zou, Q. Zhao, J. Liao, H. Hu, S. Yu, X. Chai, M. Xu, Q. Wu,
Bioorg. Med. Chem. Lett. 22, 2959–2962 (2012)
22. M. Shiradkar, G.V.S. Kumar, V. Dasari, S. Tatikonda, K.C.H.
Akula, R. Shah, Eur. J. Med. Chem. 42, 807–816 (2007)
23. N.B. Patel, I.H. Khan, S.D. Rajani, Eur. J. Med. Chem. 45,
4293–4299 (2010)
24. C.H. Menendez, A. Chollet, F. Rodriguez, C. Inard, M.R. Pasca,
C.H. Lherbet, M. Baltas, Eur. J. Med. Chem. 52, 275–283 (2012)
25. K.M. Krishna, B. Inturi, G.V. Pujar, M.N. Purohit, G.S. Vijayku-
mar, Eur. J. Med. Chem. 84, 516–529 (2014)
References
1. R.C. Moellering Jr., Int. J. Antimicrob. Agents 37(1), 2–9 (2011)
2. S. Singhal, S. Arora, S. Agarwal, R. Sharma, N. Singhal, J.
Pharm. Pharm. Sci. 2(6), 4661–4681 (2013)
3. M. Sheikhy, A.R. Jalilian, A. Novinrooz, F. Motamedi-Sedeh, J.
Biomed. Sci. Eng. 5, 39–42 (2012)
26. Y.A. Al-Soud, M.N. Al-Dweri, N.A. Al-Masoudi, Farmaco 59,
775–783 (2004)
27. Y.A. Al-Soud, N.A. Al-Masoudi, A.E.-R.S. Ferwanah, Bioorg.
Med. Chem. 11, 1701–1708 (2003)
28. P.P. Kattimani, R.R. Kamble, M.Y. Kariduraganavar, A. Dor-
ababu, R.K. Hunnur, Eur. J. Med. Chem. 62, 232–240 (2013)
29. G.E.D.A.A. Abuo-Rahma, M. Abdel-Aziz, E.A.M. Beshr, T.F.S.
Ali, Eur. J. Med. Chem. 71, 185–198 (2014)
30. A. Krajczyk, K. Kulinska, T. Kulinski, B.L. Hurst, C.W. Day,
D.F. Smee, T. Ostrowski, P. Januszczyk, J. Zeidler, Antivir.
Chem. Chemother. 23(4), 161–171 (2014)
4. P. Umadevi, K. Deepti, I. Srinath, G. Vijayalakshmi, M. Tarakar-
amji, Int. J. Pharm. Pharm. Sci. 4(3), 379–383 (2012)
5. M.A.M.S.H. El-Sharief, S.Y. Abbas, K.A.M. El-Bayouki, E.W.
El-Gammal, Eur. J. Med. Chem. 67, 263–268 (2013)
6. P.C. Jain, N.P.S. Prakash, M.M.S. Kinthada, M.C. Jain, Int. J.
PharmTech. 2(3), 726–734 (2010)
7. Z.C. Li, L.H. Chen, X.J. Yu, Y.H. Hu, K.K. Song, X.W. Zhou
et al., J. Agric. Food Chem. 58, 12537–12540 (2010)
8. V. Alagarsamy, P. Parthiban, Rasǎyan J. Chem. 4(4), 547–553
(2011)
9. S. Rastogi, H. Rastogi, Ind. J. Chem. 49B, 547–553 (2010)
10. R.J. Nevagi, A.S. Dhake, H.I. Narkhede, R. Kaur, Bioorg. Chem.
54, 68–72 (2014)
31. J. Heeres, R. Hendrickx, J.J. Van Cutsem, Med. Chem. 26(4),
611–613 (1983)
32. H. Washton, Diagn. Microbiol. Infect. Dis. 12(4 Suppl), 229–233
(1989)
11. H.J. Zhang, Y. Qian, D.D. Zhu, X.G. Yang, H.L. Zhu, Eur. J.
Med. Chem. 46, 4702–4708 (2011)
33. A. Kotiranta, K. Lounatmaa, M. Haapasalo, Microbes Infect. 2,
189–198 (2000)
12. S. Padhye, Z. Afrasiabi, E. Sinn, J. Fok, K. Mehta, N. Rath,
Inorg. Chem. 44, 1154–1156 (2005)
34. A. Pompella, M. Comporti, Histochemistry 95, 255–262 (1991)
35. H.N. Dogan, S. Rollas, H. Erdeniz, Farmaco 53, 462–467 (1998)
36. A.El-G.M. Khalil, A.M. Berghot, M.A. Gouda, J. Saudi Chem.
Soc. 20, 165–172 (2016)
13. B.S. Patil, G. Krishnamurthy, H.S.B. Naik, P.R. Latthe, M.
Ghate, Eur. J. Med. Chem. 45, 3329–3334 (2010)
14. G. Turan-Zitouni, Z.A. Kaplancikli, M.T. Yildiz, P. Chevallet, D.
Kaya, Eur. J. Med. Chem. 40, 607–613 (2005)
37. H.N. Dogan, A. Duran, S. Rollas, Ind. J. Chem. 44B, 2301–2307
(2005)
15. D. Ji, J. Lu, B. Lu, C.H. Xin, J. Mu, J. Li, C.H. Peng, X. Bao,
Bioorg. Med. Chem. Lett. 23, 1997–2000 (2013)
38. E.J. Bottone, Clin. Microbiol. Rev. 23, 382–398 (2010)
16. M. Koparir, C. Orek, A.E. Parlak, A. Soylemez, P. Koparir, M.
Karatepe, S.D. Dastan, Eur. J. Med. Chem. 63, 340–346 (2013)
17. Y.J. Mange, A.M. Isloor, S. Malladi, S. Isloor, H.-K. Fun, Arab.
J. Chem. 26, 177–181 (2013)
1 3