Synthesis, structural studies and biological evaluation of connections of thiosemicarbazide, 1,2,4-triazole and 1,3,4-thiadiazole with palmitic acid

Article abstract of DOI:10.3390/molecules23040822

Thirty new derivatives of palmitic acid were efficiently synthesized. All obtained compounds can be divided into three groups of derivatives: Thiosemicarbazides (compounds 1–10), 1,2,4-triazoles (compounds 1a–10a) and 1,3,4-thiadiazoles (compounds 1b–10b) moieties. ¹H-NMR, 13C-NMR and MS methods were used to confirm the structure of derivatives. All obtained compounds were tested in vitro against a number of microorganisms, including Gram-positive cocci, Gram-negative rods and Candida albicans. Compounds 4, 5, 6, 8 showed significant inhibition against C. albicans. The range of MIC values was 50–1.56 μg/mL. The halogen atom, especially at the 3rd position of the phenyl group was significantly important for antifungal activity. The biological activity against Candida albicans and selected molecular descriptors were used as a basis for QSAR models, that have been determined by means of multiple linear regression. The models have been validated by means of the Leave-One-Out Cross Validation. The obtained QSAR models were characterized by high determination coefficients and good prediction power.

Full text of DOI:10.3390/molecules23040822

molecules
Article
Synthesis, Structural Studies and Biological
Evaluation of Connections of Thiosemicarbazide,
1,2,4-Triazole and 1,3,4-Thiadiazole with Palmitic Acid
Michał Józ´wiak 1,2,3, Karolina Ste˛pien´ 3,4, Małgorzata Wrzosek 1,3, Wioletta Olejarz 1,3
,
1
5
Grazyna Kubiak-Tomaszewska , Anna Filipowska , Wojciech Filipowski 6 and
˙
Marta Struga 3,7,
*
1
Department of Biochemistry and Pharmacogenomics, Faculty of Pharmacy, Medical University of Warsaw,
02-097 Warsaw, Poland; michal.jozwiak@wum.edu.pl (M.J.); malgorzata.wrzosek@wum.edu.pl (M.W.);
wioletta.olejarz@wum.edu.pl (W.O.); grazyna.kubiak-tomaszewska@wum.edu.pl (G.K.-T.)
Department of Biochemistry, Second Faculty of Medicine, Medical University of Warsaw, 02-097 Warsaw,
Poland
Laboratory of Centre for Preclinical Research, Medical University of Warsaw, 02-097 Warsaw, Poland;
karolina.stepien@wum.edu.pl
Department of Pharmaceutical Microbiology, Medical University, 02-007 Warsaw, Poland
Faculty of Automatic Control, Electronics and Computer Science, Silesian University of Technology,
44-100 Gliwice, Poland; wojciech.filipowski@polsl.pl
2
3
4
5
6
7
Department of Biosensors and Processing of Biomedical Signals, Silesian University of Technology,
44-800 Zabrze, Poland; anna.filipowska@polsl.pl
Department of Biochemistry, First Faculty of Medicine, Medical University of Warsaw, 02-097 Warsaw,
Poland
*
Correspondence: marta.struga@wum.edu.pl
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 20 March 2018; Accepted: 29 March 2018; Published: 3 April 2018
Abstract: Thirty new derivatives of palmitic acid were efficiently synthesized. All obtained
compounds can be divided into three groups of derivatives: Thiosemicarbazides (compounds
1–10), 1,2,4-triazoles (compounds 1a–10a) and 1,3,4-thiadiazoles (compounds 1b–10b) moieties.
1H-NMR, 13C-NMR and MS methods were used to confirm the structure of derivatives. All obtained
compounds were tested in vitro against a number of microorganisms, including Gram-positive cocci,
Gram-negative rods and Candida albicans. Compounds
4, 5, 6, 8 showed significant inhibition against
C. albicans. The range of MIC values was 50–1.56 g/mL. The halogen atom, especially at the 3rd
µ
position of the phenyl group was significantly important for antifungal activity. The biological activity
against Candida albicans and selected molecular descriptors were used as a basis for QSAR models,
that have been determined by means of multiple linear regression. The models have been validated
by means of the Leave-One-Out Cross Validation. The obtained QSAR models were characterized by
high determination coefficients and good prediction power.
Keywords: palmitic acid derivatives; thiosemicarbazides; 1,2,4-triazoles; 1,3,4-thiadiazoles;
antimicrobial activity; quantitative structure-activity relationship
1. Introduction
In recent years bacterial and fungal infections have become a clinical challenge due to issues such
as transplantation, cancer therapy, HIV infections and the use of immunosuppressive agents [1,2]. In the
treatment of infections azoles are widely used as antifungal agents. A triazole derivative, Fluconazole,
was the first agent which had a high antifungal activity and showed low toxicity. Azoles inhibit the
activity of lanosterol 14 α-demethylase enzyme (CYP51), which prevents the ergosterol synthesis in
Molecules 2018, 23, 822; doi:10.3390/molecules23040822
www.mdpi.com/journal/molecules

Molecules 2018, 23, 822
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yeast and fungi. This depletion of ergosterol, and accumulation of lanosterol and other 14
α
-methyl
sterols causes the inhibition of growth of fungal cells [
of pathogenic fungi, Candida albicans, to fluconazole has been emerging rapidly [
serious clinical problem and to enhance the activity of compounds, different derivatives of heterocyclic
compounds are being synthesized containing not only 1,2,4-triazole arrangements, but also tetrazole [ ],
indole, benzimidazole, imidazole or 1,3,4-thiadiazole moieties [ ]. To increase the antifungal activity
of azoles, a piperazine ring can be introduced as side chains [9,10].
3,4
]. The resistance of the most common strains
5,6]. To overcome this
7
8
All mentioned heterocyclic moieties are common structures found in a large number of
compounds which display biological activity. Chemical molecules containing triazole and thiadiazoles
core play an important role in the medicinal chemistry and are known to be a very effective antibacterial
and antifungal factors used to treat diseases caused by different microorganisms [11]. Compounds
bearing heterocyclic moieties such as 1,2,4-triazole show, despite antifungal and antibacterial activity,
anti-inflammatory properties [12] or can act as antioxidants [13]. Those containing 1,2,4-triazole and
1,3,4-thiadiazole had been also reported to exhibit significant anticancer properties [14–16].
To consider these chemical substances to be active compounds against different types of cells,
the molecules must either interfere with plasma membranes or must enter the cells to affect metabolic
reaction within it. Due to high membrane selectivity, numerous potentially active compounds cannot
be transported across the membrane and therefore they do not display their properties.
Fatty acids and their derivatives have potential pharmacological activity [17–20]. The integrity
of a fungal cell is provided by its plasma membrane, therefore insertion of fatty acids into
bilayer can physically disrupt it and increase the fluidity [21]. Fatty acid derivatives containing
heterocyclic moieties also tend to be biologically beneficial in combating microorganisms [22,23].
It is important to evaluate the antibacterial and antifungal effect of these five-membered heterocyclic
compounds containing long alkyl chain as they show versatile pharmacological and biological activities.
These compounds are expected to have advantages over the parent heterocyclic molecules; they may
easily penetrate the cell membrane and they may disrupt the integrity of the cell [24].
Considering mentioned information on azoles and fatty acids properties, a series of palmitic acid
derivatives was synthesized bearing 1,2,4-triazole and 1,3,4-thiadiazole moieties. The antibacterial and
antifungal properties were evaluated and MIC values for all compounds were established.
2. Results and Discussion
2.1. Chemistry
In this study, three series of compounds, series (
1
–
10), which contain thiosemicarbazide, series (1a
–10a),
with 1,2,4-triazole moiety, and series (1b–10b), with 1,3,4-thiadiazole structure, were synthesized
(Scheme 1).
In a first step, the ester (ethyl palmitate) was transformed into the palmitohydrazide (
It was a starting material to obtain thiosemicarbazides. Derivatives of palmitic acid thiosemicarbazides
were prepared in reaction of hydrazide ( ) with an appropriate aryl isothiocyanate. In short,
palmitohydrazide ( ) and different isothiocyanates were allowed to react in boiling acetonitrile for 6 h.
Obtained N-(phenylsubstituted)-2-palmitoyl hydrazinecarbothioamides ( 10) were a starting material
to synthesize the 1,2,4-triazole (1a 10a) and 1,3,4-thiadiazole (1b 10b) palmitic acid derivatives.
1,2,4-Triazole (1a 10a) and 1,3,4-thiadiazole (1b 10b) were obtained using a method of cyclisation in
basic (2% NaOH) and acidic (conc. H2SO4) conditions, respectively [27,28].
A) [25,26].
A
A
1–
–
–
–
–

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Scheme 1. Synthesis route for derivatives of palmitic acid thiosemicarbazides. Reagents and conditions:
) NH2NH2 80%, EtOH, rt. 2 h ( ) appropriate isothiocyanate, acetonitrile, reflux, 6 h ( ) 2% NaOH,
reflux (d) H2SO4(conc.)
(
a
b
c
.
Spectral data (NMR, HRMS) of all compounds were in full agreement with the proposed structures
(supplementary). The 1H-NMR spectrum for thiosemicarbazides (
10) exhibited three broad singlets
at 169.41–169.69 ppm for C=S and peaks at
10.61–8.55 ppm. 13C-NMR revealed peaks at
1–
δ
δ
δ
177.14–177.27 ppm for C=O.
1H-NMR spectra for triazole derivatives (1a
–10a) are characterized by peaks for NH groups in
the range
the range δ 168.34–168.42 ppm for groups C=S.
1,3,4-Thiadiazol-2-amine derivatives (1b
spectrum in the range δ 10.16–8.76 ppm.
δ
11.88–10.47 ppm, while 13C-NMR spectra for these derivatives show characteristic peaks in
–
10b) have a NH group that is present in the 1H-NMR
2.2. Biological Activity
All obtained compounds were tested in vitro against 12 strains of bacteria, including Gram-positive
cocci, Gram-negative rods and against two standard and two clinical isolates of Candida albicans using
the serial microdilution assay. Microorganism used in this study were chosen on the basis of their
common application in the antimicrobial tests for many substances, like antiseptic drugs, antibiotics or
in the search of new antimicrobial agents [29
all derivatives were obtained.
,30]. All compounds were screened and MIC values for

Molecules 2018, 23, 822
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2.2.1. Antimicrobial Study
Results show, that all 30 tested compounds were inactive as antibacterial factors (MIC values above
g/mL). Only two substances (5b and 8b) from 1,3,4-thiadiazole series show moderate antibacterial
activity against Gram-positive cocci. Derivative 5b and 8b MIC values were 50–12.5 g/mL and
100
µ
µ
50–25 µg/mL, respectively. None of the compounds were active against Gram-negative bacteria.
2.2.2. Antifungal Study
The antifungal activity of all obtained compounds was tested against standard strains and clinical
isolates of Candida albicans.
Among three groups of tested compounds (thiosemicarbazides, 1,2,4-triazoles, 1,3,4-thiadiazoles)
the thiosmicarbazide group (
were highly active against all tested Candida strains (compounds
found to be the most active with MIC value of 1.56 g/mL for all strains, while the three remaining
derivatives ( and ) showed weaker antifungal activity (MIC values of: 1.56 g/mL against ATCC
10231, 50–25 g/mL towards ATCC 30028, 12.5–3.125 g/mL for clinical isolate 26 and 6.25–3.125
against clinical isolate 18).
Candida strain ATCC 10231 was the most sensitive against thiosemicarbazide derivatives (
The MIC values were in the range of 12.5–1.56 g/mL. The second strain, in terms of sensitivity,
was the clinical isolate 18 (MIC range: 100–1.56 µg/mL).
All thiosemicarbazide derivatives contained a phenyl group in their structure. Compound
had unmodified phenyl ring, while compounds 10 were additionally substituted with at least one
selected from the group consisting of chlorine, bromine and methyl group in the 2, 3 or 4 position.
Each of the four active thiosemicarbazides ( ) had a halogen substituent in the 3-position
in the phenyl ring. Derivative containing bromine was the least active. Remaining three compounds
had chlorine in position 3. Derivative contained an additional chlorine substituent in position 4,
additionally had an electron donor substituent—methyl group in
1
–
10) showed promising antifungal activity, but only four compounds
4,
5,
6
and ). Compound was
8
5
µ
4,
6
8
µ
µ
µ
1–10).
µ
1
2
–
4, 5, 6, 8
4
8
while the most active compound
position 4.
5
Considering two other groups of compounds (1,2,4-triazoles, 1,3,4-thiadiazoles), only the
1,2,4-triazole derivative 5a, obtained by cyclization of the most active thiosemicarbazide
low level of antifungal activity (MIC 100–12.5 µg/mL).
5, showed
All discussed results are presented in Table 1.
Table 1. Antifungal activity against Candida albicans stains (MIC values in µg/mL).
MIC/µg/mL
Compound
Candida albicans
Candida albicans
Candida albicans
Candida albicans
ATCC 10231
ATCC 30028
Clinical Isolate 26
Clinical Isolate 18
1
2
3
4
5
6
7
8
9
12.5
1.56
1.56
1.56
1.56
1.56
1.56
1.56
1.56
12.5
100
100
100
50
1.56
25
>100
25
50
>100
>100
50
12.5
1.56
3.125
25
12.5
50
25
>100
25
12.5
6.25
1.56
3.125
25
6.25
50
12.5
10
25
1a
2a
3a
4a
5a
6a
7a
8a
9a
10a
50
>100
>100
>100
>100
25
50
>100
100
>100
>100
>100
>100
100
>100
>100
100
>100
>100
>100
>100
25
>100
>100
>100
>100
>100
>100
>100
>100
12.5
12.5
>100
100
100
50
>100
100
>100
100

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Table 1. Cont.
MIC/µg/mL
Compound
Candida albicans
Candida albicans
Candida albicans
Candida albicans
ATCC 10231
ATCC 30028
Clinical Isolate 26
Clinical Isolate 18
1b
2b
3b
4b
5b
6b
7b
8b
9b
10b
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Ref. *
0.25–0.125
0.25–0.125
0.25–0.125
0.25–0.125
* Ref.—Fluconazole.
2.2.3. Cytotoxic Studies
Cytotoxicity tests against three human cell lines were conducted. These were normal cells—human
immortal keratinocyte cell line from adult human skin (HaCaT) and two tumor cell lines—human
epithelial lung carcinoma cell line (A549) and human melanoma cell line (HTB-140). Two commonly
used anticancer drugs—cisplatin and doxorubicin were used in this study as reference compounds.
Assays were performed only for the three most active compounds (5, 6, 8) and for their corresponding
derivatives 5a, 6a, 8a and 5b, 6b, 8b from each group of 1,2,4-triazoles and 1,3,4-thiadiazoles.
The cytotoxic activities of tested derivatives were specified by determining the IC50 (inhibitory
concentrations), i.e., the concentrations of compounds, which inhibited cells’ viability and growth by
50% as compared to the control cells.
All tested derivatives were non-cytotoxic (IC50 > 100), which means that the antibacterial and
antifungal activity was not related to cytotoxicity.
2.3. Quantitative Structure-Activity Relationships
For all tested derivatives of thiosemicarbazides, 1,2,4-triazoles and 1,3,4-thiadiazoles 19 molecular
descriptors characterizing their physicochemical properties were determined.
Based on the calculated physicochemical parameters and microbiological assays, the similarity
of tested compounds was analyzed and quantitative structure-activity relationships (QSAR) models
describing the relationship between antifungal activity against Candida albicans ATCC 10231,
Candida albicans ATCC 30028 and Candida albicans clinical isolate 26 and clinical isolate 18 strains
were determined.
Quantitative structure-activity relationships (QSAR) modeling was performed using the multiple
linear regression (MLR) regression method. The minimal inhibitory concentrations (MIC) obtained
for tested fungal strains expressed as log (1/MIC (µM)) were dependent variables and molecular
descriptors were independent variables. Obtained QSAR models were internally validated by
Leave-One-Out Cross Validation (LOO) and Y-scrambling and externally validated by several metrics,
including variance explained in external prediction Q2(F1), Q2(F2), Q2(F3), and r2 metrics based on
m
the correlation of the observed and predicted response data with and without the intercept, and the
criteria recommended by Golbraikh and Tropsha. The results of QSAR models are presented in Table 2.

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Table 2. Formulas describing the relationship of activity against Candida albicans ATCC 10231, Candida
albicans clinical isolate 18 and molecular descriptors (training set = 80% of whole dataset, test set = 20%
of whole dataset).
C. albicans
Strain
Modeling
ntr = 24
Internal
Validation
External Validation
ntest = 6
QSAR Equation
Golbraikh&Tropsha
R = 0.889
Q2
Q2
= 0.890
= 0.890
(F1)
log(1/MICC. alb.ATCC10231) =
Q2
= 0.733
R2 = 0.790
R2adj.= 0.758
F = 25.1
k = 1.013
k’ = 0.964
R2 = 0.891
R2LOO = 0.146
Q2(F2) = 0.867
0.0001(±0.00005)EIA
0.19 (±0.05)Rf −
0.039(±0.006)HF −
11.84(±3.9)
+
Yscr
(F3)
Q2
=
−
0.097
Yscr
R’20 = 0.838
RMSEext = 0.321
cR2p = 0.713
p = 5.6·10-7
0
r2m = 0.717
Candida
albicans ATCC
10231
∆r2 = 0.111
RMSEtr = 0.419
m
R = 0.901
Q2
Q2
= 0.879
= 0.879
(F1)
Q2
= 0.750
R2 = 0.812
R2adj.= 0.784
F = 28.8
k = 1.005
k’ = 0.989
R2 = 0.845
R2LOO = 0.089
log(1/MICC. alb.ATCC10231) =
Q2(F2) = 0.904
−0.11(±0.01)logP2
+
Yscr
(F3)
Q2
=
−
0.167
0.00090(±0.0001)Rf2
+
Yscr
R’20 = 0.778
RMSEext = 0.208
cR2p = 0.766
0.030(±0.01)µ2 − 8.96(±1.8)
−7
0
r2m = 0.687
p = 1.9·10
∆r2 = 0.145
RMSEtr = 0.419
m
R = 0.918
Q2
Q2
= 0.879
= 0.879
(F1)
log(1/MICC. alb.clin.18) =
Q2
= 0.774
R2 = 0.843
R2adj.= 0.819
F = 35.7
k = 1.005
k’ = 0.989
R2 = 0.880
R2LOO = 0.122
−0.003(±0.0007)logP3
+
Q2(F2) = 0.904
Yscr
0.0003(±0.0001)Rf2
3.9·10−12(±1·10−12)ICC
7.89 (±1.1)
+
(F3)
Q2
=
−
0.115
Yscr
R’20 = 0.902
RMSEext = 0.208
2
−
cR2p = 0.814
−8
0
r2m = 0.806
p = 3.2·10
Candida
albicans clinical
isolate 18
∆r2 = 0.095
RMSEtr = 0.292
m
R = 0.831
Q2
Q2
= 0.640
= 0.641
(F1)
Q2
= 0.673
R2 = 0.691
R2adj.= 0.677
F = 49.2
k = 1.009
k’ = 0.967
R2 = 0.641
R2LOO = 0.057
Q2(F2) = 0.712
log(1/MICC. alb.clin.18) =
Yscr
2
4.9·10−12(±7·10-13)EE
6.94(±0.7)
−
(F3)
Q2
=
−
0.041
Yscr
R’20 = 0.605
RMSEext = 0.360
cR2p = 0.630
−7
0
r2m = 0.539
p = 4.87·10
∆r2 = 0.064
RMSEtr = 0.390
m
ntr—the number of samples in training set, R—multilinear correlation coefficient, R2—determination coefficient,
R2 —adjusted determination coefficient, RMSEtr—root mean square error in fitting, F—F-value; R2Yscr—average
adj
p
R2 of 25 random models, Q2Yscr—average R2 of 25 random models, cRp2
coefficient of LOO validation, ntest—the number of samples in test set, RMSEext—root mean square error in test set,
=
R2 − RYscr2, Q2LOO—determination
Q2
Q2
and Q2(F3)—variance explained in test set, concordance correlation coefficient; rm and
∆
r2m—average
2
(F1),
(F2)
and delta r2 values, respectively (criteria described by Roy), k and k0—slopes of the regression line over external
m
data, R2 and R’20—R2 values in Golbraikh and Tropsha criteria.
0
4 QSAR models were determined for all tested strains, where antifungal activity depends on
the following physicochemical parameters: logP—partition coefficient octanol/water, Rf—molar
refractivity, µ—dipole moment, EIA—isolated atomic energy, EE—electronic energy, ICC—core-core
interaction, HF—heat of formation.
For all the tested strains equations were obtained, where antimicrobial activity depends on molar
refractivity (Rf) and on one descriptor characterizing electron properties from the group of descriptors
described by the dependence ET = EB + EIA = ICC + EE. For each tested strain, simple linear equations
can be obtained for each of above mentioned electron descriptors at the R > 0.6 level. Molar refractivity
is, among others, considered as a measure of the mobility of electrons of a molecule or a measure
of steric interactions of compound [31] and it defines the volume occupied by an atom or group of
atoms. The presence of a strong correlation between Rf and biological activity of tested compounds
may indicate, that the factor affecting this activity is the strength of binding of the compound to the
polar surface [32,33].
Models, where one of the independent variables is logP, were obtained only for two of C. albicans
strains: ATCC 10231 and clinical isolate 18. This parameter describes the lipophilicity of compounds
and characterizes the ability of a compound to pass through biological membranes, i.e., the speed
of distribution and absorption processes [31]. According to the Lipinski’s rule, the logP should not
be greater than five (logP < 5) [34], however all compounds tested in this work show logP values
above seven.

Molecules 2018, 23, 822
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There is a positive linear correlation for the QSAR models obtained of such descriptors as Rf,
HF, ICC2, EE, EIA, µ2 and negative linear correlation of logP2 descriptors against biological activity
for selected fungal strains. All equations presented in Table 1 satisfy the acceptability and prediction
conditions of the R2 > 0.6, Q2
> 0.6, cR2p > 0.5, R2
−
Q2
< 0.3, Q2(F1), Q2
R’02)/R2 < 0.1, rm2 > 0.5 and ∆r2 < 0.2
and Q2
> 0.6,
LOO
LOO
(F2)
(F3)
0.85
models [35
≤
k
≤
1.15 or 0.85
≤
k
≤
1.15, (R2
−
R02)/R2 < 0.1 or (R2
−
m
–38]. The Figures 1 and 2 show correlation between predicted and observed values for C.
albicans strains characterized by the highest R2 determination coefficients.
Figure 1. Correlation of activity against Candida albicans clinical isolate 18 and logP3, Rf2, ICC2. Dashed
lines mark the area of predictability of the received models, which are described by the equations
ꢀ
featured above the graphs. —test set, ◦—training set.

Molecules 2018, 23, 822
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Figure 2. Correlation of activity against Candida albicans ATCC 10231 and logP2, RF2, 2. Dashed lines
µ
mark the area of predictability of the received models, which are described by the equations featured
ꢀ
above the graphs. —test set, ◦—training set.
Dendrogram in Figure 3 presents the division of the tested compounds based on their biological
activity for all 4 tested strains expressed as the minimum inhibitory concentration MIC (
Based on 33% Sneath criterion compounds were divided into four groups (A–D). Group A consisted
of compounds 10 15 showing strong activity for all tested strains (the average MIC
value for all tested strains was lower than 62 g/mL). In group B there were two compounds: and
µg/mL).
5,
,
8,
6,
4
,
, 9, 3
µ
7
2
characterized by high biological activity, however not for all tested fungal strains (mean MIC value
for all tested strains was in the range from 62.83 µg/mL to 81.64 µg/mL). Groups A and B contained
almost all of the thiosemicarbazide derivatives tested and one triazole derivative (5a). In group C,
there were compounds showing very low activity or having activity only against Candida albicans
ATCC 10231 strain (average MIC value for all tested strains was in the range from 112.5
128.1 g/mL). Group D contained all compounds that do not show biological activity for the fungal
strains tested.
µg/mL to
µ

Molecules 2018, 23, 822
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Figure 3. Dendrogram presenting the similarity of compounds in terms of their biological activity
against the tested fungal strains: Candida albicans ATCC 10231, Candida albicans ATCC 3002, Candida
albicans clinical isolate 26 and Candida albicans clinical isolate 18.
3. Materials and Methods
3.1. Chemistry
Ethyl palmitate was supplied from Sigma-Aldrich. Isothiocyanates were purchased from Alfa
Aesar or Sigma Aldrich. Acetonitrile, chloroform and methanol were supplied from POCh (Polskie
Odczynniki Chemiczne, Gliwice, Poland). All chemicals were of analytical grade and were used
without any further purification. Prior usage, dry acetonitrile was kept in crown cap bottles over
anhydrous phosphorus pentoxide (Carl Roth, Karlsruhe, Germany). The NMR spectra were recorded
on Varian VNMRS 300 Oxford NMR spectrometer (IET, Mundelein, IL, USA), operating at 300 MHz
(1H-NMR, relax. delay 1000 s, pulse 30.0 degrees) and 75.4 MHz (13C-NMR, relax. delay 3700 s,
pulse 45.0 degrees, proton-decoupled: Waltz-16 modulated). Chemical shifts (δ) were expressed in parts
per million relative to tetramethylsilane used as the internal reference. Mass spectral ESI measurements
were carried out on Waters ZQ Micro-mass instruments (Waters, Milford, MA, USA) with quadruple
mass analyzer. The spectra were performed in the negative ion mode at a declustering potential of
40–60 V. The sample was previously separated on a UPLC column (C18) using UPLC ACQUITYTM
system by Waters (Milford, MA, USA) connected with DPA detector. Flash chromatography was
performed on Merck silica gel 60 (200–400 mesh) (Merck, Darmstadt, Germany) using chloroform
eluent. Analytical TLC was carried out on silica gel F254 (Merck) plates (0.25 mm thickness).
3.1.1. Palmitohydrazide (A)
Compound was synthesized and described previously [39,40].
3.1.2. General Procedure for the Preparation of Palmitic Acid Thiosemicarbazide (1–10)
Palmitohydrazide (A 0.01 mol, 2.7 g) and 0.01 mol of appropriate isothiocyanate were refluxed
in acetonitrile for 6 h. The product was filtered and dried. The solid residue was purified by column
chromatography (chloroform:methanol 9.5:0.5 vol.).
◦
1
2-Palmitoyl-N-phenylhydrazinecarbothioamide (
(DMSO-d6) (ppm): 9.81 (s, 1H, NH), 9.21 (s, 1H, NH), 8.72 (s, 1H, NH), 7.44 (d, J = 7.8 Hz, 2H,
Carom.), 7.37 (t, J = 7.65 Hz, 2H, Carom.), 7.23 (t, J = 4.05 Hz, 1H, Carom.), 2.35 (t, J = 7.5 Hz, 2H, CH2),
1) Yield: 264 mg, 65%. Mp. 102–103 C. H-NMR
δ

Molecules 2018, 23, 822
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1.73–1.6 (m, 2H, CH2), 1.3–1.23 (m, 24H, CH2), 0.89 (t, J = 6.6 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
(ppm): 14.1, 22.7, 25.3, 29.1, 29.2, 29.35, 29.40, 29.57, 29.61, 29.66 (2C), 29.68, 31.9 (2C), 34.3, 124.2
δ
(2C), 126.5, 129.3 (2C), 137.1, 169.7, 177.1. HRMS (ESI) calcd for C23H39N3OS [M
found 404.2769.
−
H]−: 404.2770;
◦
2
) Yield: 240 mg, 60%. Mp. 120–121 C. 1H-NMR
N-(2-Chlorophenyl)-2-palmitoylhydrazinecarbothioamide (
(DMSO-d6) (ppm): 10.61 (s, 1H, NH), 9.47 (s, 1H, NH), 8.86 (s, 1H, NH), 8.02 (d, J = 7.5 Hz, 1H,
δ
Carom.), 7.44 (d, J = 7.8 Hz, 1H, Carom.), 7.35–7.28 (m, 2H, Carom.), 7.19 (t, J = 7.5 Hz, 1H, Carom.), 2.37 (t,
J = 7.2 Hz, 2H, CH2), 1.71–1.61 (m, 2H, CH2), 1.29–1.23 (m, 24H, CH2), 0.89 (t, J = 6.3 Hz, 3H, CH3).
13C-NMR (DMSO-d6)
δ (ppm): 14.1, 22.7, 25.3, 29.1, 29.2, 29.35, 29.40, 29.57, 29.61, 29.66 (2C), 29.68,
31.9 (2C), 34.3, 124.2, 126.4, 129.4, 132.6 (2C) 137.1, 169.4, 177.2. HRMS (ESI) calcd for C23H38ClN3OS
[M − H]−: 438.2346; found 438.2361.
N-(3-Chloro-4-fluorophenyl)-2-palmitoylhydrazinecarbothioamide (
1H-NMR (DMSO-d6)
(ppm): 8.91 (s, 1H, NH), 8.77 (s, 1H, NH), 7.62 (t, J = 7.65 Hz, 1H, Carom.), 7.42 (d,
J = 8.1 Hz, 1H, Carom.), 7.11 (t, J = 8.1 Hz, 1H, Carom.), 5.26 (s, 1H, NH), 2.34 (t, J = 8.4 Hz, 2H, CH2),
1.76–1.60 (m, 2H, CH2), 1.32–1.23 (m, 24H, CH2), 0.87 (t, J = 6.45 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
(ppm): 14.1, 22.7, 25.8, 28.8, 28.9, 29.30, 29.31, 29.5, 29.58, 29.62 (2C), 29.64, 31.9 (2C), 34.3, 122.5, 124.7,
3
) Yield: 284 mg, 62%. Mp. 119–120 ◦C.
δ
δ
131.1, 134.5, 136.1, 148.4, 169.4, 177.2. HRMS (ESI) calcd for C23H37ClFN3OS [M
found 456.2297.
−
H]−: 456.2286;
◦
4
) Yield: 310 mg, 64%. Mp. 113–114 C. 1H-NMR
N-(3-Bromophenyl)-2-palmitoylhydrazinecarbothioamide (
(DMSO-d6) (ppm): 9.81 (s, 1H, NH), 9.05 (s, 1H, NH), 8.78 (s, 1H, NH), 7.68 (s,1H, Carom.), 7.46 (d,
δ
J = 8.1 Hz, 1H, Carom.), 7.34 (d, J = 8.1 Hz, 1H, Carom.), 7.22 (t, J = 7.5 Hz, 1H, Carom.), 2.34 (t, J = 7.65 Hz,
2H, CH2), 1.7–1.65 (m, 2H, CH2), 1.29–1.23 (m, 24H, CH2), 0.88 (t, J = 6.25 Hz, 3H, CH3). 13C-NMR
(DMSO-d6)
δ
(ppm): 14.1, 22.7, 25.3, 29.1, 29.2, 29.35, 29.40, 29.57, 29.61, 29.66 (2C), 29.68, 31.9 (2C),
−
34.3, 123.4, 125.8 (2C), 127.6, 129.3, 137.1, 169.5, 177.2. HRMS (ESI) calcd for C23H38BrN3OS [M
482.1875; found 482.1867.
−
H] :
N-(3-Chloro-4-methylphenyl)-2-palmitoylhydrazinecarbothioamide (
1H-NMR (DMSO-d6)
(ppm): 9.90 (s, 1H, NH), 9.18 (s, 1H, NH), 8.77 (s, 1H, NH), 7.48 (s, 1H, Carom.),
5
) Yield: 304 mg, 67%. Mp. 139–140 ◦C.
δ
7.29 (d, J = 8.1 Hz, 1H, Carom.), 7.19 (d, J = 8.1 Hz, 1H, Carom.), 2.35 (t, J = 8.4 Hz, 2H, CH2), 2.34 (s,
3H, CH3), 1.76–1.60 (m, 2H, CH2), 1.32–1.23 (m, 24H, CH2), 0.87 (t, J = 6.45 Hz, 3H, CH3). 13C-NMR
(DMSO-d6)
δ (ppm): 14.1, 20.0, 22.7, 25.8, 28.8, 28.9, 29.29, 29.31, 29.5, 29.58, 29.62 (2C), 29.64, 31.9
(2C), 34.3, 122.5, 123.8, 124.7, 131.1, 134.5, 136.1, 169.4, 177.2. HRMS (ESI) calcd for C24H40ClN3OS
[M − H]−: 452.2237; found 452.2528.
N-(3-Chlorophenyl)-2-palmitoylhydrazinecarbothioamide (
(DMSO-d6) (ppm): 10.19 (s, 1H, NH), 9.28 (s, 1H, NH), 9.04 (s, 1H, NH), 7.56 (s, 1H, Carom.), 7.41 (d,
6
) Yield: 246 mg, 56%. Mp. 98–99 ◦C. 1H-NMR
δ
J = 7.8 Hz, 1H, Carom.), 7.31 (d, J = 6.6 Hz, 1H, Carom.), 7.18 (t, J = 7.8 Hz, 1H, Carom.), 2.36 (t, J = 7.5 Hz,
2H, CH2), 1.7–1.55 (m, 2H, CH2), 1.29–1.22 (m, 24H, CH2), 0.87 (t, J = 6.15 Hz, 3H, CH3). 13C-NMR
(DMSO-d6) δ (ppm): 14.1, 22.7, 25.3, 29.1, 29.2, 29.35, 29.40, 29.57, 29.61, 29.66 (2C), 29.68, 31.9 (2C), 34.3,
124.6, 126.5, 128.3, 129.3, 136.1, 137.1, 169.5, 177.3. HRMS (ESI) calcd for C23H38ClN3OS [M − H]−
:
438.2346; found 438.2361.
◦
7
) Yield: 334 mg, 69%. Mp. 105–106 C. 1H-NMR
N-(2-Bromophenyl)-2-palmitoylhydrazinecarbothioamide (
(DMSO-d6) (ppm): 10.0 (s, 1H, NH), 9.15 (s, 1H, NH), 8.58 (s, 1H, NH), 7.92 (d, J = 7.5 Hz, 1H, Carom.),
δ
7.6 (d, J = 8.1 Hz, 1H, Carom.), 7.36 (t, J = 7.8 Hz, 1H, Carom.), 7.12 (t, J = 7.8 Hz, 1H, Carom.), 2.35 (t,
J = 7.35 Hz, 2H, CH2), 1.7–1.6 (m, 2H, CH2), 1.3–1.21 (m, 24H, CH2), 0.88 (t, J = 6.6 Hz, 3H, CH3).
13C-NMR (DMSO-d6)
δ (ppm): 14.1, 22.7, 25.2, 29.1, 29.2, 29.35, 29.40, 29.57, 29.61, 29.66 (2C), 29.68,
31.9 (2C), 34.3, 122.4, 128.1, 130.5, 132.2, 132.9, 137.1, 169.7, 177.1. HRMS (ESI) calcd for C23H38BrN3OS
[M − H]−: 482.1875; found 482.1867.

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◦
N-(3,4-Dichlorophenyl)-2-palmitoylhydrazinecarbothioamide (
8
) Yield: 342 mg, 72%. Mp. 103–104 C.
1H-NMR (DMSO-d6)
δ
(ppm): 9.31 (s, 1H, NH), 9.09 (s, 1H, NH), 8.94 (s, 1H, NH), 7.71 (d, J = 2.4 Hz,
1H, Carom.), 7.43 (d, J = 2.1 Hz, 1H, Carom.), 7.38 (s, 1H, Carom.), 2.35 (t, J = 9 Hz, 2H, CH2), 1.73–1.62 (m,
2H, CH2), 1.32–1.22 (m, 24H, CH2), 0.88 (t, J = 6.75 Hz, 3H, CH3).13C-NMR (DMSO-d6)
(ppm): 14.1,
δ
22.7, 25.3, 29.1, 29.2, 29.35, 29.40, 29.57, 29.61, 29.66 (2C), 29.68, 31.9 (2C), 34.3, 121.1, 129.3, 129.4, 131.3,
135.4, 137.2, 169.6, 177.2. HRMS (ESI) calcd for C23H37Cl2N3OS [M − H]−: 472.1991; found 472.1985.
◦
9
) Yield: 339 mg, 70%. Mp. 120–121 C. 1H-NMR
N-(4-Bromophenyl)-2-palmitoylhydrazinecarbothioamide (
(DMSO-d6) (ppm): 9.8 (s, 1H, NH), 9.09 (s, 1H, NH), 8.82 (s, 1H, NH), 7.47 (d, J = 6.9 Hz, 2H, Carom.),
7.38 (d, J = 6.9 Hz, 2H, Carom.), 2.34 (t, J = 7.65 Hz, 2H, CH2), 1.7–1.6 (m, 2H, CH2), 1.32–1.21 (m, 24H,
CH2), 0.88 (t, J = 6.75 Hz, 3H, CH3).13C-NMR (DMSO-d6)
(ppm): 14.1, 22.7, 25.3, 29.1, 29.2, 29.35,
δ
δ
29.40, 29.57, 29.61, 29.66 (2C), 29.68, 31.9 (2C), 34.3, 127.7, 131.7 (2C), 132.1 (2C), 137.4, 169.5, 177.1.
HRMS (ESI) calcd for C23H38BrN3OS [M − H]−: 482.1875; found 482.1867.
◦
N-(4-Chlorophenyl)-2-palmitoylhydrazinecarbothioamide (10) Yield: 268 mg, 61%. Mp. 115–116 C.
1H-NMR (DMSO-d6)
δ
(ppm): 10.05 (s, 1H, NH), 9.15 (s, 1H, NH), 8.55 (s, 1H, NH), 7.43 (d, J = 7.1 Hz
2H, Carom.), 7.32 (d, J = 7.1 Hz, 2H, Carom.), 2.34 (t, J = 7.5 Hz, 2H, CH2), 1.68–1.58 (m, 2H, CH2),
1.32–1.19 (m, 24H, CH2), 0.88 (t, J = 6.75 Hz, 3H, CH3).13C-NMR (DMSO-d6)
(ppm): 14.1, 22.7, 25.2,
,
δ
29.1, 29.2, 29.35, 29.41, 29.57, 29.61, 29.66 (2C), 29.68, 31.9 (2C), 34.3, 129.1 (2C), 131.1 (2C), 133.7, 137.1,
169.6, 177.2. HRMS (ESI) calcd for C23H38ClN3OS [M − H]−: 438.2346; found 438.2361.
3.1.3. General Procedure for Derivatives of 3-Pentadecyl-1H-1,2,4-Triazole-5(4H)-Thione (1a–10a)
N-(Substituted-phenyl)-2-palmitoyl-hydrazinecarbothioamide (0.01 mol) was refluxed with 2%
NaOH solution (40–50 mL) for 4 h. After cooling the solution was neutralized with dilute hydrochloric
acid. The precipitated compound was filtered and then crystalized from ethanol.
3-Pentadecyl-4-phenyl-1H-1,2,4-triazole-5(4H)-thione (1a) Yield: 279 mg, 72%. Mp. 89–90 ◦C. 1H-NMR
(DMSO-d6)
J = 1.5 Hz, 1H, Carom.), 2.46 (t, J = 7.65 Hz, 2H, CH2), 1.59–1.5 (m, 2H, CH2), 1.25–1.19 (m, 24H, CH2),
0.87 (t, J = 6.6 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
(ppm): 14.1, 22.7, 25.8, 26.0, 28.8, 29.0, 29.3, 29.3,
δ (ppm): 11.79 (s, 1H, NH), 7.59–7.53 (m, 3H, Carom.), 7.34 (d, J = 2.1 Hz, 1H, Carom.), 7.31 (d,
δ
29.5, 29.6 (2C), 29.6 (2C), 31.9 (2C), 127.9 (2C), 129.8 (2C), 130.0, 133.5, 153.0, 168.3. HRMS (ESI) calcd
for C23H37N3S [M − H]−: 386.2630; found 386.2629.
4-(2-Chlorophenyl)-3-pentadecyl-1H-1,2,4-triazole-5(4H)-thione (2a) Yield: 338 mg, 80%. Mp. 139–140 ◦C.
1H-NMR (DMSO-d6)
δ
(ppm): 10.47 (s, 1H, NH), 7.64 (d, J = 7.8 Hz, 1H, Carom.), 7.56–7.46 (m, 2H,
Carom.), 7.39 (d, J = 7.2 Hz, 1H, Carom.), 2.48–2.31 (m, 2H, CH2), 1.68–1.53 (m, 2H, CH2), 1.26–1.21 (m,
24H, CH2), 0.87 (t, J = 6.75 Hz, 3H, CH3).13C-NMR (DMSO-d6)
(ppm): 14.0, 22.7, 25.8, 26.0, 28.8,
δ
29.0, 29.27, 29.32, 29.5, 29.61 (2C), 29.63 (2C), 31.9 (2C), 124.3, 126.4, 129.4, 132,6 (2C) 137.1, 153.1, 168.4.
HRMS (ESI) calcd for C23H36ClN3S [M − H]−: 420.2240; found 420.2223.
4-(3-Chloro-4-fluorophenyl)-3-pentadecyl-1H-1,2,4-triazole-5(4H)-thione (3a) Yield: 321 mg, 73%. Mp. 90–91 ◦C.
1H-NMR (DMSO-d6)
J = 7.65 Hz, 1H, Carom.), 7.27
CH2), 1.26–1.20 (m, 24H, CH2), 0.87 (t, J = 6.75 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
δ
(ppm): 11.88 (s, 1H, NH), 7.43 (dd, J = 2.2 Hz, J = 2.2 Hz, 1H, Carom.), 7.34 (t,
7.22 (m, 1H, Carom.), 2.46 (t, J = 7.65 Hz, 2H, CH2), 1.63–1.53 (m, 2H,
(ppm): 14.1, 22.7,
−
δ
25.8, 25.9, 28.8, 29.0, 29.29, 29.32, 29.5, 29.58 (2C), 29.62 (2C), 31.9 (2C), 126.2, 128.4, 131.9, 135.6, 138.6,
148.8, 152.9, 168.4. HRMS (ESI) calcd for C23H35ClFN3S [M − H]−: 438.2146; found 438.2157.
4-(3-Bromophenyl)-3-pentadecyl-1H-1,2,4-triazole-5(4H)-thione (4a) Yield: 317 mg, 68%. Mp. 101–102 ◦C.
1H-NMR (DMSO-d6)
δ (ppm): 11.80 (s, 1H, NH), 7.43 (dd, J = 1.5 Hz, J = 1.5 Hz, 1H, Carom.), 7.50 (t,
J = 1.8 Hz, 1H, Carom.), 7.45 (t, J = 8.1 Hz, 1H, Carom.), 7.30 (dd, J = 1.5 Hz, J = 1.5 Hz, 1H, Carom.), 2.46
(t, J = 7.65 Hz, 2H, CH2), 1.63–1.53 (m, 2H, CH2), 1.26–1.20 (m, 24H, CH2), 0.87 (t, J = 6.75 Hz, 3H,
CH3). 13C-NMR (DMSO-d6)
δ (ppm): 14.0, 22.7, 25.8, 26.0, 28.8, 29.0, 29.27, 29.32, 29.5, 29.61 (2C), 29.63

Molecules 2018, 23, 822
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(2C), 31.9 (2C), 123.4, 125.8 (2C), 127.6, 129.3, 137.1, 153.2, 168.4. HRMS (ESI) calcd for C23H36BrN3S
[M − H]−: 464.1735; found 464.1727.
4-(3-Chloro-4-methylphenyl)-3-pentadecyl-1H-1,2,4-triazole-5(4H)-thione (5a) Yield: 270 mg, 62%. Mp.
94–95 ◦C. 1H-NMR (DMSO-d6)
δ (ppm): 11.85 (s, 1H, NH), 7.43 (dd, J = 0.3 Hz, J = 0.3 Hz, 1H, Carom.),
7.33 (d, J = 2.1 Hz, 1H, Carom.), 7.14 (dd, J = 2.4 Hz, J = 2.4 Hz,1H, Carom.), 2.46 (s, 3H, CH3), 2.46 (t,
J = 7.65 Hz, 2H, CH2), 1.62–1.52 (m, 2H, CH2), 1.26–1.20 (m, 24H, CH2), 0.87 (t, J = 6.75 Hz, 3H, CH3).
13C-NMR (DMSO-d6)
δ (ppm): 14.1, 20.0, 22.7, 25.8, 25.9, 28.8, 29.0, 29.29, 29.32, 29.5, 29.58 (2C), 29.62
(2C), 31.9 (2C), 126.2, 128.4, 131.9, 135.6 (2C), 138.6, 152.9, 168.4. HRMS (ESI) calcd for C24H38ClN3S
[M − H]−: 434.2397; found 434.2388.
4-(3-Chlorophenyl)-3-pentadecyl-1H-1,2,4-triazole-5(4H)-thione (6a) Yield: 312 mg, 74%. Mp. 97–98 ◦C.
1H-NMR (DMSO-d6)
δ
(ppm): 11.79 (s, 1H, NH), 7.53–7.51 (m, 1H, Carom.), 7.35 (s, 1H, Carom.), 7.28–7.24
(m, 2H, Carom.), 2.46 (t, J = 7.65 Hz, 2H, CH2), 1.62–1.52 (m, 2H, CH2), 1.24–1.21 (m, 24H, CH2), 0.87 (t,
J = 6.6 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
(ppm): 14.1, 22.7, 25.8, 25.9, 28.8, 29.0, 29.29, 29.32, 29.5,
δ
29.58 (2C), 29.62 (2C), 31.9 (2C), 124.6, 126.5, 128.4, 129.4, 136.2, 137.2, 153.0, 168.4. HRMS (ESI) calcd
for C23H36ClN3S [M − H]−: 420.2240; found 420.2223.
◦
4-(2-Bromophenyl)-3-pentadecyl-1H-1,2,4-triazole-5(4H)-thione (7a) Yield: 327 mg, 70%. Mp. 128–129 C.
1H-NMR (DMSO-d6)
δ
(ppm): 11.63 (s, 1H, NH), 7.8 (d, J = 7.8 Hz, 1H, Carom.), 7.56–7.36 (m, 3H,
Carom.), 2.51–2.27 (m, 2H, CH2), 1.64–1.55 (m, 2H, CH2), 1.23–1.21 (m, 24H, CH2), 0.87 (t, J = 6.6 Hz, 3H,
CH3). 13C-NMR (DMSO-d6)
(ppm): 14.1, 22.7, 25.8, 25.9, 28.8, 29.0, 29.29, 29.32, 29.5, 29.58 (2C), 29.62
δ
(2C), 31.9 (2C), 122.5, 128.2, 130.6, 132.2, 133.0, 137.1, 153.1, 168.4. HRMS (ESI) calcd for C23H36BrN3S
[M − H]−: 464.1735; found 464.1727.
4-(3,4-Dichlorophenyl)-3-pentadecyl-1H-1,2,4-triazole-5(4H)-thione (8a) Yield: 338 mg, 74%. Mp. 84–85
◦C. 1H-NMR (DMSO-d6)
δ (ppm): 11.7 (s, 1H, NH), 7.66 (d, J = 8.4 Hz, 2H, Carom.), 7.46 (s, 1H, Carom.),
7.23 (d, J = 8.4 Hz, 1H, Carom.), 2.46 (t, J = 7.5 Hz, 2H, CH2), 1.6–1.53 (m, 2H, CH2), 1.23–1.21 (m, 24H,
CH2), 0.87 (t, J = 6 Hz, 3H, CH3). 13C-NMR (DMSO-d6) δ (ppm): 14.1, 22.7, 25.8, 25.9, 28.8, 29.0, 29.29,
29.32, 29.5, 29.58 (2C), 29.62 (2C), 31.9 (2C), 122.5, 128.2, 130.6, 132.2, 13.0, 137.1, 153.1, 168.4. HRMS
(ESI) calcd for C23H35Cl2N3S [M − H]−: 454.1851; found 454.1845.
4-(4-Bromophenyl)-3-pentadecyl-1H-1,2,4-triazole-5(4H)-thione (9a) Yield: 294 mg, 63%. Mp. 79–80 ◦C.
1H-NMR (DMSO-d6)
δ
(ppm): 11.68 (s, 1H, NH), 7.7 (d, J = 8.4 Hz, 2H, Carom.), 7.21 (d, J = 8.4 Hz,
2H, Carom.), 2.46 (t, J = 7.65 Hz, 2H, CH2), 1.68–1.51 (m, 2H, CH2), 1.23–1.21 (m, 24H, CH2), 0.87 (t,
J = 6.6 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
(ppm): 14.1, 22.7, 25.8, 25.9, 28.8, 29.0, 29.29, 29.32, 29.5,
δ
29.58 (2C), 29.62 (2C), 31.9 (2C), 127.7, 131.8 (2C), 132.2 (2C), 137.5, 153.0, 168.4. HRMS (ESI) calcd for
C23H36BrN3S [M − H]−: 464.1735; found 464.1727.
4-(4-Chlorophenyl)-3-pentadecyl-1H-1,2,4-triazole-5(4H)-thione (10a) Yield: 291 mg, 69%. Mp. 66–67 ◦C.
1H-NMR (DMSO-d6)
δ
(ppm): 11.62 (s, 1H, NH), 7.24 (d, J = 8.4 Hz, 2H, Carom.), 6.57 (d, J = 8.4 Hz,
2H, Carom.), 2.46 (t, J = 7.65 Hz, 2H, CH2), 1.68–1.51 (m, 2H, CH2), 1.23–1.21 (m, 24H, CH2), 0.88 (t,
J = 6.6 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
(ppm): 14.1, 22.7, 25.8, 25.9, 28.8, 29.0, 29.2, 29.3, 29.5,
δ
29.58 (2C), 29.62 (2C), 31.9 (2C), 129.2 (2C), 131.2 (2C), 133.8, 137.1, 153.0, 168.4. HRMS (ESI) calcd for
C23H36ClN3S [M − H]−: 420.2240; found 420.2223.
3.1.4. General Procedure for Derivatives of 5-Pentadecyl-N-(Substituted
Phenyl)-1,3,4-Thiadiazol-2-Amine (1b–10b)
N-(Substituted-phenyl)-2-palmitoyl-hydrazinecarbothioamide (0.01 mol) was mixed for 4 h with
concentrated sulfuric acid (0.5 mL). Then crushed ice was added to the solution. After cooling the
solution neutralized with dilute NaOH. The precipitated compound was filtered and dried. The solid
residue was purified by column chromatography (chloroform:methanol 9.5:0.5 vol.).

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◦
1
5-Pentadecyl-N-phenyl-1,3,4-thiadiazol-2-amine (1b) Yield: 186 mg, 48%. Mp. 97–98 C. H-NMR
(DMSO-d6) (ppm): 9.52 (s, 1H, NH), 7.59–7.53 (m, 3H, Carom.), 7.40 (d, J = 2.1 Hz, 1H, Carom.),
7.38 (d, J = 1.5 Hz, 1H, Carom.), 2.46 (t, J = 7.65 Hz, 2H, CH2), 1.68–1.63 (m, 2H, CH2), 1.25–1.19 (m, 24H,
CH2), 0.87 (t, J = 6.6 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
(ppm): 14.1, 22.7, 25.7, 26.0, 28.5, 28.8, 29.1,
δ
δ
29.3, 29.5, 29.61 (2C), 29.63 (2C), 31.9 (2C), 127.4, 130.2 (2C), 135.4 (2C), 140.8, 158.1, 173.0. HRMS (ESI)
calcd for C23H37N3S [M − H]−: 386.2630; found 386.2629.
◦
N-(2-Chlorophenyl)-5-pentadecyl-1,3,4-thiadiazol-2-amine (2b) Yield: 224 mg, 53%. Mp. 66–67 C. 1H-NMR
(DMSO-d6)
Carom.), 7.10 (t, J = 8.7 Hz, 1H, Carom.), 2.89 (t, J = 7.65 Hz, 2H, CH2), 1.71–1.61 (m, 2H, CH2), 1.25–1.19
(m, 24H, CH2), 0.87 (t, J = 6.6 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
(ppm): 14.1, 22.7, 25.8, 26.0, 28.8,
δ (ppm): 9.59 (s, 1H, NH), 7.63 (dd, J = 2.7 Hz, J = 2.7 Hz, 1H, Carom.), 7.30–7.25 (m, 1H,
δ
29.0, 29.27, 29.32, 29.5, 29.61 (2C), 29.63 (2C), 31.9 (2C), 124.3, 126.5, 129.9, 133.0 (2C) 140.8, 158.1, 173.1.
HRMS (ESI) calcd for C23H36ClN3S [M − H]−: 420.2240; found 420.2223.
N-(3-Chloro-4-fluorophenyl)-5-pentadecyl-1,3,4-thiadiazol-2-amine (3b) Yield: 246 mg, 56%. Mp. 84–85 ◦C.
1H-NMR (DMSO-d6)
δ
(ppm): 8.75 (s, 1H, NH), 7.50 (dd, J = 1.8 Hz, J = 1.8 Hz, 1H, Carom.), 7.39–7.28
(m, 2H, Carom.), 7.38 (d, J = 1.5 Hz, 1H, Carom.), 2.87 (t, J = 7.65 Hz, 2H, CH2), 1.76–1.66 (m, 2H, CH2),
1.25–1.19 (m, 24H, CH2), 0.87 (t, J = 6.6 Hz, 3H, CH3).13C-NMR (DMSO-d6)
(ppm): 14.1, 22.7, 25.8,
δ
25.9, 28.5, 28.8, 29.1, 29.3, 29.57, 29.62 (2C), 29.7 (2C), 30.4, 31.9, 128.0, 132.3, 137.7, 138. 9, 139.1, 148.8,
158.5, 173.2. HRMS (ESI) calcd for C23H35ClFN3S [M − H]−: 438.2146; found 438.2157.
◦
N-(3-Bromophenyl)-5-pentadecyl-1,3,4-thiadiazol-2-amine (4b) Yield: 229 mg, 49%. Mp. 82–83 C. 1H-NMR
(DMSO-d6)
J = 2.4 Hz, 1H, Carom.), 2.85 (t, J = 7.65 Hz, 2H, CH2), 1.76–1.66 (m, 2H, CH2), 1.25–1.19 (m, 24H, CH2),
0.87 (t, J = 6.6 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
(ppm): 14.1, 22.6, 25.8, 26.0, 28.8, 29.0, 29.2, 29.3,
δ (ppm): 8.75 (s, 1H, NH), 7.87 (d, J = 2.2 Hz1H, Carom.), 7.67–7.55 (m, 2H, Carom.), 7.38 (d,
δ
29.5, 29.62 (2C), 29.63 (2C), 31.9 (2C), 123.4, 125.8 (2C), 127.7, 129.4, 140.8, 158.5, 173.2. HRMS (ESI)
calcd for C23H36BrN3S [M − H]−: 464.1735; found 464.1727.
N-(3-Chloro-4-methylphenyl)-5-pentadecyl-1,3,4-thiadiazol-2-amine (5b) Yield: 262 mg, 60%. Mp. 116–117
◦
C. 1H-NMR (DMSO-d6)
δ (ppm): 8.76 (s, 1H, NH), 7.43 (dd, J = 0.3 Hz, J = 0.3 Hz, 1H, Carom.), 7.38 (d,
J = 2.1 Hz, 1H, Carom.), 7.14 (dd, J = 2.4 Hz, J = 2.4 Hz,1H, Carom.), 2.79 (t, J = 7.65 Hz, 2H, CH2), 2.45 (s,
3H, CH3), 1.62–1.52 (m, 2H, CH2), 1.26–1.20 (m, 24H, CH2), 0.87 (t, J = 6.75 Hz, 3H, CH3). 13C-NMR
(DMSO-d6)
δ
(ppm): 14.1, 19.9, 22.7, 25.8, 25.9, 28.5, 28.8, 29.1, 29.3, 29.57, 29.62 (2C), 29.7 (2C), 30.−4,
H] :
31.9, 128.0, 132.3, 133.6, 137.8, 138. 9, 139.1, 158.5, 173.2. HRMS (ESI) calcd for C24H38ClN3S [M
434.2397; found 434.2388.
−
◦
N-(3-Chlorophenyl)-5-pentadecyl-1,3,4-thiadiazol-2-amine (6b) Yield: 241 mg, 57%. Mp. 94–95 C. 1H-NMR
(DMSO-d6) (ppm): 10.16 (s, 1H, NH), 7.45–7.44 (m, 1H, Carom.), 7.29–7.27 (m, 1H, Carom.), 7.05 – 7.01
(m, 1H, Carom.), 2.99 (t, J = 7.65 Hz, 2H, CH2), 1.83–1.73 (m, 2H, CH2), 1.29–1.21 (m, 24H, CH2), 0.87 (t,
J = 6.6 Hz, 3H, CH3).13C-NMR (DMSO-d6)
(ppm): 14.1, 22.7, 25.8, 26.0, 28.8, 29.0, 29.30, 29.34, 29.5,
δ
δ
29.59 (2C), 29.63 (2C), 31.9 (2C), 125.1, 127.5, 129.4, 130.4, 137.2, 139.2, 158.8, 173.2. HRMS (ESI) calcd
for C23H36ClN3S [M − H]−: 420.2240; found 420.2223.
◦
N-(2-Bromophenyl)-5-pentadecyl-1,3,4-thiadiazol-2-amine (7b) Yield: 238 mg, 51%. Mp. 66–67 C. 1H-NMR
(DMSO-d6)
(t, J = 7.8 Hz, 1H, Carom.), 7.00 (t, J = 7.65 Hz, 1H, Carom.), 2.95 (t, J = 7.65 Hz, 2H, CH2), 1.76–1.69 (m,
2H, CH2), 1.29–1.21 (m, 24H, CH2), 0.87 (t, J = 6.6 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
(ppm): 14.1,
δ (ppm): 8.76 (s, 1H, NH), 7.9 (d, J = 8.1 Hz, 1H, Carom.), 7.6 (d, J = 7.8 Hz, 1H, Carom.) 7.34
δ
22.7, 25.8, 26.0, 28.8, 29.0, 29.31, 29.34, 29.5, 29.62 (2C), 29.65 (2C), 31.9 (2C), 123.5, 129.1, 131.6, 133.2,
135.0, 140.1, 158.5, 173.2. HRMS (ESI) calcd for C23H36BrN3S [M − H]−: 464.1735; found 464.1727.
◦
N-(3,4-Dichlorophenyl)-5-pentadecyl-1,3,4-thiadiazol-2-amine (8b) Yield: 242 mg, 53%. Mp. 64–65 C.
1H-NMR (DMSO-d6)
δ
(ppm): 9.08 (s, 1H, NH), 7.60 (d, J = 8.4 Hz, 2H, Carom.), 7.48 (s, 1H, Carom.),
7.33 (d, J = 6.9 Hz, 1H, Carom.), 2.92 (t, J = 7.5 Hz, 2H, CH2), 1.69–1.67 (m, 2H, CH2), 1.23–1.21 (m, 24H,
CH2), 0.87 (t, J = 6 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
(ppm): 14.1, 22.7, 25.8, 26.0, 28.8, 29.0, 29.3,
δ

Molecules 2018, 23, 822
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29.4, 29.5, 29.59 (2C), 29.64 (2C), 31.7 (2C), 123.5, 129.2, 131.5, 133.2, 134.0, 139.1, 158.6, 173.2. HRMS
(ESI) calcd for C23H35Cl2N3S [M − H]−: 454.1851; found 454.1845.
◦
N-(4-Bromophenyl)-5-pentadecyl-1,3,4-thiadiazol-2-amine (9b) Yield: 229 mg, 49%. Mp. 67–68 C. 1H-NMR
(DMSO-d6)
2.87 (t, J = 7.65 Hz, 2H, CH2), 1.67–1.51 (m, 2H, CH2), 1.25–1.21 (m, 24H, CH2), 0.87 (t, J = 6.6 Hz, 3H,
CH3). 13C-NMR (DMSO-d6)
(ppm): 14.1, 22.7, 25.8, 26.0, 28.8, 29.0, 29.30, 29.32, 29.5, 29.61 (2C), 29.63
δ (ppm): 8.76 (s, 1H, NH), 7.68 (d, J = 8.4 Hz, 2H, Carom.), 6.79 (d, J = 8.4 Hz, 2H, Carom.),
δ
(2C), 31.9 (2C), 128.7, 132.8 (2C), 133.2 (2C), 140.1, 158.0, 173.1. HRMS (ESI) calcd for C23H36BrN3S
[M − H]−: 464.1735; found 464.1727.
◦
N-(4-Chlorophenyl)-5-pentadecyl-1,3,4-thiadiazol-2-amine (10b) Yield: 249 mg, 59%. Mp. 66–67 C.
1H-NMR (DMSO-d6)
δ
(ppm): 8.86 (s, 1H, NH), 7.34 (d, J = 8.4 Hz, 2H, Carom.), 6.67 (d, J = 8.4
Hz, 2H, Carom.), 2.86 (t, J = 7.65 Hz, 2H, CH2), 1.69–1.51 (m, 2H, CH2), 1.25–1.21 (m, 24H, CH2), 0.87 (t,
J = 6.6 Hz, 3H, CH3). 13C-NMR (DMSO-d6)
(ppm): 14.1, 22.7, 25.8, 26.0, 28.8, 29.0, 29.31, 29.34, 29.5,
δ
29.59 (2C), 29.64 (2C), 31.9 (2C), 123.2 (2C), 132.2 (2C), 134.8, 140.1, 158.46, 173.0. HRMS (ESI) calcd for
C23H36ClN3S [M − H]−: 420.2240; found 420.2223.
3.2. Biological Assay
3.2.1. In Vitro Evaluation of Antimicrobial Activity
Microorganisms used in this study were as follows: Gram-positive bacteria: Staphylococcus aureus
NCTC 4163, S. aureus ATCC 25923, S. aureus ATCC 6538, S. aureus ATCC 29213, S. epidermidis ATCC
12228, S. epidermidis ATCC 35984, Bacillus subtilis ATCC 6633, Enterococcus hirae ATCC 9341, Enterococcus
faecalis ATCC 29212; Gram-negative rods: E. coli ATCC 8196, Pseudomonas aeruginosa ATCC 15442,
Bordetella bronchiseptica ATCC 4617 and yeasts: Candida albicans ATCC 10231, C. albicans ATCC 30028,
C. albicans clinical isolate 26, C. albicans clinical isolate 18.
All microorganisms used were obtained from the collection of the Department of Pharmaceutical
Microbiology, Medical University of Warsaw, Poland.
3.2.2. Media, Growth Conditions and Antimicrobial Assay
Minimal Inhibitory Concentration (MIC) was tested by the twofold serial microdilution method
(in 96-well microtiter plates) using Mueller-Hinton Broth medium (Beckton Dickinson, Franklin Lakes,
NJ, USA) for bacteria or RPMI-1640 medium for Candida species according to CLSI guidelines [41,42]
The stock solution of tested compounds was prepared in DMSO and diluted in sterile water.
Concentrations of tested agents ranged from 1.56 to 200 g/mL for bacteria strains and from 1.56 to
100
g/mL for fungi. The final inoculum of all studied bacteria was 105 CFU/mL (colony forming
units per mL) and 0.5–2.5
105 CFU/mL for fungi. Minimal inhibitory concentrations (the lowest
.
µ
µ
×
concentration of the tested agent that prevents visible growth of a microorganism) were read after 18 h
(bacteria) or 24 h (yeasts) of incubation at 35 ◦C.
3.2.3. Cytotoxicity Studies
Human immortal keratinocyte cell line from adult human skin (HaCaT). Human epithelial lung
carcinoma cell line (A549) and Human melanoma cell line (HTB-140) were bought from American Type
Culture Collection (Rockville, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)
supplemented with 1% antibiotics (penicillin and streptomycin), and 1◦0% heat-inactivated FBS-fetal
bovine serum (Gibco Life Technologies, Waltham, MA, USA), at 37 C and 5% CO2 atmosphere.
Cells were passaged using trypsin-EDTA (Gibco Life Technologies, Waltham, MA, USA) and cultured
in 96-well plates (1 × 104 cells per well). Experiments were conducted in DMEM with 2% FBS.

Molecules 2018, 23, 822
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3.2.4. MTT Cytotoxicity Assay
The cell viability was assessed by determination of MTT salt 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, converted by mitochondrial dehydrogenase. The cells were incubated
for 72 h in 96-well plates with given concentration of tested compound, and subsequently for
another 4 h with 0.5 mg/mL of MTT solution, which in live cells—under the effect of mitochondrial
dehydrogenase—converts to insoluble formazan. The converted dye was then solubilized with the
use of 0.04 M HCl in absolute isopropanol. The absorbance of solubilized formazan was measured
spectrophotometrically at 570 nm (using Epoch microplate reader, BioTek Inc., Winooski, VT, USA),
equipped with Gen5 software (BioTech Instruments, Inc., Biokom, Winooski, VT, USA). Cell viability
was presented as a percent of MTT reduction in the treated cells versus the control (cells incubated
in serum-free DMEM without studied compounds). The relative MTT level (%) was calculated as
(A)/(B)
×
100, where (A) is the absorbance of the tested sample and (B) means the absorbance of the
control sample containing untreated cells. Decreasing of the relative MTT level indicates decreased
cell viability.
3.3. QSAR Modeling
3.3.1. Physicochemical Parameters
Conformational search and physicochemical parameters were calculated using HyperChem ver.
7.5 [43]. Optimized Potentials for Liquid Simulations (OPLS) force field were used for extensive
conformational search performed at molecular mechanics level. The most stable structures obtained
were subsequently optimized to the closest local minimum at the semiempirical level using PM3
parametrizations. Convergence criteria were set to 0.1 and 0.01 kcal
·
mol−1
·
Å−1 for OPLS and PM3
calculations, respectively [44,45]. The selected descriptors were then used to develop a QSAR models.
3.3.2. QSAR Models
QSAR models have been determined by means of multiple linear regression (MLR) [46].
Comparison of multiple linear regression equations (Y = B0 + B1 X1 + B2 X2 .... + BN XN) [47] have been
·
·
·
performed with the Statistica 12.0 software [48]. Equation coefficients were calculated by means of the
classic least squares method [49], in which the sum of squares of differences between the theoretical
value and deviations of empirical value for variable Y and is the lowest. The statistical calculations
were conducted at confidence level of 95% (p < 0.05). The analyses were performed by means of
multiple backward regression which entailed successively rejecting the least statistically significant
(having the highest p value) structural parameters (molecular descriptors). Statistical verification of
the model was performed by means of T-test and Fisher–Snedecor F-test. T-test is used to verify
significance of each parameter of a model (independent, explanatory variables), and F-test is used
for checking statistical significance of the entire model [50]. The following multiple linear regression
assumptions were verified for statistically significant equations:
•
•
•
•
Model linearity,
coincidence condition,
uniform dispersion (homoscedasticity) condition,
normal distribution of model residue.
The coincidence condition entails checking if the sign of the coefficient of correlation between the
explanatory variable Xj and the response variable Y is the same as the sign of the Bj coefficient in the
multiple linear regression model (with multiple explanatory variables) of the Xj variable.
The homoscedasticity condition has been analyzed based on diagrams of residual dispersion in
relation to observed variables and based on the Lagrange Multiplier Test.

Molecules 2018, 23, 822
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Normal distribution of the dependent variable Y and model residuals were verified by means of
the Shapiro–Wilk test [51]. Every independent variable used in a QSAR model is related to at least five
compounds [47].
3.3.3. Model Validation
The model has been internally validated by means of the Leave-One-Out Cross Validation (LOO
CV). Successively one compound, utilized to validate the resulting model, was eliminated from the
data set used as a basis for model (n
formula [52,53]:
−
1). The Q2LOO validation coefficient was calculated from the
ꢀ
ꢁ
2
n
yexp,i − ypred,i
yexp,i − yave,i
∑
i=1
Q2 = 1 −
(1)
ꢂ
ꢃ
2
n
∑
i=1
where: yexp,i—experimental output value for the i-th compound, ypred,i—predicted output value for the
i-th compound, yave,i—average value for the output variable without the i-th compound.
For all models, validation in the form of a Y-randomization test (Scrambling model) was also
performed to check, whether the selection of descriptors used to create QSAR models was not
random. The test is based on the multiple replacements of the biological activity values used in
MLR with random values. The deviation in the values of the squared mean correlation coefficient of
the randomized model (Rr2) from the squared correlation coefficient of the nonrandom model (R2) is
reflected in the value of cRp2 parameter computed from the following equation [54]:
q
cR2p = R R2 − R2r
(2)
2
The threshold value of cRp is 0.5. For a QSAR model having the corresponding value above the
stated limit, it might be considered that the model is not obtained by chance [37,38].
External validation of the models was also conducted. The parameters of predictive models such
as Q2(F1) [38], Q2(F2) and Q2(F3) [55] were calculated.
The parameters k, k’, R2 , R’2 for determining the external predictability of QSAR models were
0
0
calculated using methods described in Golbraikh and Tropsha [56].
2
2
Internally validation of the prediction model was also carried out by calculating the rm
, ∆rm
parameters described by Roy and et al. [57]. The obtained QSAR models should meet the assumptions
r2m > 0.5 and ∆rm2 < 0.2 [57].
3.3.4. Cluster Analysis
Cluster analysis is used to identify a relatively similar, homogeneous group of objects
(compounds) in the measured features space. In this paper, the agglomerative hierarchical cluster
analysis complete linkage method (squared Euclidean distance) was conducted to find a group
of compounds characterized by similar biological activity or similar physicochemical parameters.
Data standardization was the first stage of cluster analysis. Its purpose was to increase the influence
of data with minor deviation and decrease the influence of data with major deviation. Furthermore,
the standardization procedure eliminates the influence of different units of measurement and renders
the data dimensionless [58].
4. Conclusions
We successfully synthesized three groups of compounds: Thiosemicarbazides (compounds
1–10),
1,2,4-triazoles (compounds 1a 10a) and 1,3,4-thiadiazoles (compounds 1b 10b) to determine their
–
–
microbiological activity and cytotoxicity. Obtained compounds did not show significant activity
against Gram-positive cocci and Gram-negative bacteria.
Thiosemicarbazide derivatives exhibited potent to moderate activity against Candida albicans.
Compound
5 presented the strongest potency against all tested strains of C. albicans, with MIC values

Molecules 2018, 23, 822
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1.56
µg/mL. Derivatives
4,
6
and
8
showed diverse activity for different strains of fungi. The MIC
value for C. albicans ATCC 10231 was 1.56 µg/mL, for C. albicans 30028 was in the range 50–25 µg/mL,
for C. albicans isolate 26 it was 2.5–3.125 µg/mL and for C. albicans isolate 18—6.25–3.125 µg/mL.
Biological activity was closely related to the presence of a halogen substituent at the 3-position of
the aromatic ring; substituents in other positions did not affect the antimicrobial activity. Chloride
atoms turn out to have the strongest influence.
The statistical analysis allowed to determine 4 QSAR models, where the antifungal activity
depends on the following physicochemical parameters: EIA, EE, ICC, µ, logP, Rf, HF. The obtained
models can be used to predict the biological activity against the tested fungal strains for the new
thiosemicarbazide, triazole and thiadiazole derivatives, as the effect of EIA, EE, ICC descriptors on the
antifungal activity against all tested strains was indicated.
1
Supplementary Materials: The following are available online, Figures S1–S30: H-NMR spectra of the products
1–10, 1a–10a, 1b–10b. Figures S31–S36: 13C-NMR spectra of the selected products 1–10, 1a–10a, 1b–10b.
Acknowledgments: This work was supported by the Medical University of Warsaw and carried out with the use
of CePT infrastructure financed by the European Union—the European Regional Development Fund within the
Operational Programme Innovative Economy for 2007–2013.
Author Contributions: Michał Józ´wiak performed the synthesis of thiosmicarbazides, triazoles and thiadiazoles
and performed part of antimicrobial activity assays, wrote part of the paper. Karolina Ste˛pien´ performed part of
˙
antimicrobial activity assays. Małgorzata Wrzosek and Grazyna Kubiak-Tomaszewska collected part of biological
data and wrote part of the paper. Wioletta Olejarz performed part of cytotoxicity assay, Anna Filipowska
determined molecular descriptors, analyzed molecular orbitals, performed a statistical analysis, conducted cluster
analysis, determined QSAR models, validated part of models and wrote part of the paper. Wojciech Filipowski
performed conformational search, conducted geometry optimization, determined part of molecular descriptors,
analyzed part of data, validated part of models and wrote part of the paper. Marta Struga did spectral data
analysis and was the principal investigator of the project and provided the research funding. All authors approved
the final version of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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