Synthesis, in vitro screening and docking studies of new thiosemicarbazide derivatives as antitubercular agents

Article abstract of DOI:10.3390/molecules24020251

A series of thiosemicarbazide derivatives was designed and synthesized by reaction of carboxylic acid hydrazide with isothiocyanates. The molecular structures of the investigated thiosemicarbazides were confirmed and characterized by spectroscopic analysis. The conformational preference of carbonylthiosemicarbazide chain and intra- and intermolecular interactions in the crystalline state were characterized using X-ray analysis. The antituberculosis activity of the target compounds were tested in vitro against four Mycobacterium strains: M. H37Ra, M. phlei, M. smegmatis, M. timereck. The most active compounds were those with 2-pyridine ring. They exhibited lower minimal inhibitory concentration (MIC) values in the range 7.81–31.25 μg/mL in comparison to the other isomers. Compound 5 had activity against M. smegmatis at a concentration of 7.81 μg/mL whereas compound 2 had activity against all tested strains at a concentration of 15.625 μg/mL. The molecular docking studies were performed for investigated compounds using the Mycobacterium tuberculosis glutamine synthetase MtGS as their molecular target.

Full text of DOI:10.3390/molecules24020251

molecules
Article
Synthesis, In Vitro Screening and Docking Studies of
New Thiosemicarbazide Derivatives as
Antitubercular Agents
Monika Pitucha 1,
* ,
, Zbigniew Karczmarzyk 2, Marta Swatko-Ossor 3, Waldemar Wysocki 2
Maciej Wos 1, Kamil Chudzik 1 , Grazyna Ginalska 3 and Andrzej Fruzinski 4
1
Independent Radiopharmacy Unit, Faculty of Pharmacy with Division of Medical Analytics, Medical
University of Lublin, 20-093 Lublin, Poland; wos.maciej@gmail.com (M.W.); kamilchudzik@umlub.pl (K.C.)
Faculty of Science, Siedlce University of Natural Sciences and Humanities, 08-110 Siedlce, Poland;
kar@uph.edu.pl (Z.K.); wwysocki@uph.edu.pl (W.W.)
Department of Biochemistry and Biotechnology, Faculty of Pharmacy with Division of Medical Analytics,
Medical University of Lublin, 20-093 Lublin, Poland; martaswatkoossor@umlub.pl (M.S.-O.);
grazyna.ginalska@umlub.pl (G.G.)
2
3
4
Institute of General and Ecological Chemistry, Technical University, 90-924 Łódz´, Poland;
andrzej.fruzinski@p.lodz.pl
*
Correspondence: monika.pitucha@umlub.pl; Tel.: +48-81-448-74-20
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 6 December 2018; Accepted: 8 January 2019; Published: 11 January 2019
Abstract: A series of thiosemicarbazide derivatives was designed and synthesized by reaction
of carboxylic acid hydrazide with isothiocyanates. The molecular structures of the investigated
thiosemicarbazides were confirmed and characterized by spectroscopic analysis. The conformational
preference of carbonylthiosemicarbazide chain and intra- and intermolecular interactions in the
crystalline state were characterized using X-ray analysis. The antituberculosis activity of the target
compounds were tested in vitro against four Mycobacterium strains: M. H37Ra, M. phlei, M. smegmatis,
M. timereck. The most active compounds were those with 2-pyridine ring. They exhibited lower
minimal inhibitory concentration (MIC) values in the range 7.81–31.25
to the other isomers. Compound had activity against M. smegmatis at a concentration of
7.81 g/mL whereas compound had activity against all tested strains at a concentration of
15.625 g/mL. The molecular docking studies were performed for investigated compounds using the
µg/mL in comparison
5
µ
2
µ
Mycobacterium tuberculosis glutamine synthetase MtGS as their molecular target.
Keywords: synthesis; thiosemicarbazide; X-ray analysis; tuberculosis; molecular docking
1. Introduction
Tuberculosis is the one of the most common infectious diseases worldwide [1]. Next to AIDS
(Acquired Immune Deficiency Syndrome), it is the leading cause of death in the world. Despite
the availability of tuberculosis treatment, about 9 million new cases of tuberculosis are reported
each year, and 1.5 million cases are fatal. The worst epidemiological situation is in Third World
countries, where the incidence rates range from 100–300 cases per 100,000 inhabitants [2]. More than
95% of cases fall to the developing countries of Asia, Africa, Brazil and Russia. The results of
treatment are still not satisfactory. The success rate for new cases and surgeries reported in 2013 was
76% [3]. Despite intensive efforts to prevent and treat tuberculosis, the problem is serious. MDR-TB
(multi-drug-resistant tuberculosis) is a disturbing phenomenon which embraces strains resistant to two
basic drugs: Isoniazid and Rifampicin. Moreover, mycobacterial resistance to Rifampicin, Isoniazid,
quinolones and aminoglycosides has increased—extensively drug-resistant tuberculosis (XD-TB) [4].
Molecules 2019, 24, 251; doi:10.3390/molecules24020251
www.mdpi.com/journal/molecules

Molecules 2019, 24, 251
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The heterogeneous populations of mycobacteria (active and dormant) and ease of acquisition of
resistance requires a combination therapy with antibiotics of different molecular mechanisms of
action [
fails. Another problem is the interaction between antiviral drugs used in patients infected with the
HIV virus and anti-TB drugs [ ]. Therefore, interests and efforts of many research groups around
5]. This makes the treatment of multidrug-resistant tuberculosis long and costly, and it often
6
the world are focused on tuberculosis problems. Actions taken to reduce the effects of M. tuberculosis
resistance are multidirectional. Currently, an intensive search for new, effective anti-tuberculosis drugs
is under way. It is a long-lasting and costly process, so it is important to understand the mechanism of
action of proven and effective antituberculosis drugs. The development of counterparts with better
pharmacokinetic properties and less toxicity appears to be one of the methods of searching for new
potential drugs.
An excellent example of this type of solution seems to be the modification of Isoniazid,
a commonly used antituberculosis drug [7]. Isoniazid is a bactericidal germicide that is isonicotinic
acid hydrazide. It is used in the treatment and prevention of tuberculosis, in combination with other
antituberculosis agents.
In our work we propose the synthesis of Isoniazide analogs as compounds with potential
antitubercular activity. By using duality of biological action, we have designed compounds having a
pyridine ring and a thiosemicarbazide system as a well-known carrier antituberculosis agent [8,9].
2. Experimental
2.1. Synthesis
All chemicals used for the synthesis were purchased from Sigma-Aldrich, AlfaAesar and POCH
(Polish Chemical Reagents, Gliwice, Poland) companies and used without further purification. Melting
points were determined using Fisher–Johns block and presented without corrections. The 1H and l3
C
NMR spectra were recorded on a Bruker Avance 600 spectrometer (Bruker BioSpin GmbH, Rheinstetten,
Germany) in dimethyl sulfoxide (DMSO-d6). The chemical shifts are given in (ppm) scale using
δ
tetramethylsilane (TMS) as the standard reference. The attenuated total reflectance ATR-IR spectra were
−1
recorded over the range 4000–400 cm on the Thermo Scientific Nicolet 6700 FTIR spectrophotometer
equipped with ATR attachment with diamond crystal and Omnic software version 8.2. For accurate
mass measurements an Agilent Technologies liquid chromatograph 1290 coupled to an Agilent
Technologies 6550 iFunnel Q-TOF LC/MS equipped with Jet Stream Technology Ion Source was
employed. Flow injection analyses were performed using 0.1% formic acid in water:acetonitrile (50:50
v/v), mobile phase flow 0.5 mL min−1. Ions were acquired in positive polarity. Mass correction
was enabled, ions of m/z 121.0509 and 922.0098 were used as the reference ions. Instrument control,
data acquisition and analysis were performed using Agilent MassHunter Workstation B.07 Software.
Single-charged protonated ions for all examined compounds were detected.
The homogeneity of the newly obtained compounds was confirmed by TLC method on aluminum
oxide 60 F254 plates (Merck) in a CHCl3/C2H5OH (10:1 and 10:2) solvent system with UV visualization.
Compounds 1–4, 7, 8, 11, 13, 15, 16, 18, 20, 21 have been synthesized in our laboratory and reported in
literature [10–12]. Compounds 5, 6, 12, 14, and 19 have been published previously [13–17].
2.1.1. General Procedure for the Synthesis of 1-(pyridin-2-,3-,4-yl)carbonyl-4-substituted
Thiosemicarbazide (1–19)
The pyridine 2-, 3- or 4-pyridinecarboxylic acid hydrazide (0.01 mol) was dissolved in 15 mL
methanol and 0.01 mol of appropriate isothiocyanate was added. Mixtures were heated for 0.5–1 h
at reflux temperature. After this time mixture was cooled and appropriate thiosemicarbazide was
crystallized from methanol. (Supplementary Material includes experimental data of all compounds).

Molecules 2019, 24, 251
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2.1.2. General Procedure for the Synthesis of 1-(pyridin-4-ylacetyl)-4-substituted
Thiosemicarbazide (20–25)
A mixture of 4-pyridine acetic acid hydrazide (0.01 mol) and appropriate isothiocyanate (0.01 mol)
in anhydrous diethyl ether (10 mL) was kept at room temperature for 24 h. The product was filtered off
and crystallized from ethanol. (Supplementary Material includes experimental data of all compounds).
2.2. X-ray Structure Determination
X-ray data of
300K diffractometer; crystal sizes 0.45
0.28 0.14 mm for 11, 0.58 0.27
= 1.54178 Å) radiation, scans, T = 293 K, absorption correction: multi-scan [18], Tmin/Tmax
of 0.344/1.000 for , 0.419/1.000 for , 0.282/1.000 for 11, 0.349/1.000 for 13 and 0.759/1.000 for
4,
7
,
11
,
13 and 14 were collected on the Rigaku Oxford XtaLAB Synergy, Pilatus
0.20 0.13 mm for , 0.59 0.07 0.07 mm for , 0.59
0.06 mm for 13 and 0.50 0.16 0.03 mm for 14; CuK
×
×
4
×
×
7
×
×
×
×
×
×
α
(
λ
ω
4
7
14. The structures were solved by direct methods using SHELXS97 [19] and refined by full-matrix
least-squares with SHELXL97 [19]. The N-bound H atoms were located by difference Fourier synthesis
and refined freely. The remaining H atoms were positioned geometrically and treated as riding
on their parent C atoms with C–H distances of 0.93 Å (aromatic), 0.98 Å (CH), 0.97 Å (CH2) and
0.96 Å (CH3) and O–H distance of 0.82 Å. All H atoms were refined with isotropic displacement
parameters taken as 1.5 times those of the respective parent atoms. The compound
7 crystallizes
in non-centrosymmetric space group Cc. The absolute structure of crystal was confirmed by Flack
parameter of 0.00(2) with 1116 Friedel pairs [20]. The residual electron density map for 14 reveals
the molecule of dimethyl-diazene-N,N’-dioxide lying on 2-fold axis passing through the center of the
central N=N bond. In this molecule the N–C and N–O bond lengths were fixed to 1.507(9) and 1.430(9)
Å, respectively, due to the unstable geometry of this molecule in the refinement procedure. Moreover,
the final residual electron-density maps showed that the C14 and C15 atoms of the benzene ring are
disordered over two sites designed as A and B in the refinement procedure. The occupancy factors
(sof) for the split carbon atoms were refined and finally fixed at 0.5. Additionally, the geometry (bond
lengths and angles) of disordered fragments of benzene ring was corrected using appropriate DFIX
instructions of SHELXL97.
All calculations were performed using WINGX version 1.64.05 package [21]. CCDC-1864683 for
4
, 1,864,679 for 7, 1,864,681 for 11, 1,864,682 for 13 and 1,864,680 for 14 contain the supplementary
crystallographic data for this paper. These data can be obtained free of charge at www.ccdc.cam.ac.uk/
conts/retrieving.html [or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44(0) 1223 336 033; email: deposit@ccdc.cam.ac.uk]. (Supplementary
Material includes experimental data of compounds 4, 7, 11, 13 and 14 in CIF files).
Crystal data of
c = 14.3223(2) Å,
dcalc = 1.542 Mg m−3, F(000) = 348,
range 6.31–78.65◦ ), 3041 unique reflections, final R = 0.034, wR = 0.092, S = 1.068 for 2964 reflections
4
: C13H10N4OSCl2,M = 341.21, triclinic, space group P◦ 1, a = 7.4539(2), b = 7.8084(2),
= 104.442(2), = 92.188(2),
= 112.898(2) , V = 735.05(3) Å3, Z = 2,
(Cu K
) = 5.339 mm−1, T = 293 K, 14,710 measured reflections
α
β
γ
µ
α
(
θ
with I > 2σ(I).
Crystal data of
c = 7.9636(2) Å,
7
: C14H14N4OS2,M = 318.41, monoclinic, space group Cc, a = 7.9236(4), b = 25.2269(2),
= 113.018(2)o, V = 1465.09(9) Å3, Z = 4, dcalc = 1.444 Mg m−3, F(000) = 664,
range 6.32–78.94◦ ), 2622 unique
β
µ
(Cu K θ
α
) = 3.332 mm−1, T = 293K, 7385 measured reflections (
reflections, final R = 0.039, wR= 0.107, S = 1.076 for 2597 reflections with I > 2σ(I).
Crystal data of 11: C13H10N4OSCl2,M = 341.21, monoclinic, space group P21/c, a = 4.5176(1),
b = 27.8468(4), c = 11.8424(1) Å,
F(000) = 696, (Cu K
) = 5.328 mm−1, T = 293K, 15,369 measured reflections (
3028 unique reflections, final R = 0.040, wR = 0.111, S = 1.044 for 2761 reflections with I > 2σ(I).
β ,
= 98.573(1)◦ , V = 1473.14(4) Å3, Z = 4, dcalc = 1.538 Mg m−3
µ
α
θ
range 4.09–78.76◦ ),

Molecules 2019, 24, 251
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Crystal data of 13: 2(C13H11N4OSF)
·
C3H8O,M = 640.72, monoclinic, space group P21/n, a = 13.4658(2),
b = 9.8028(1), c = 23.9666(3) Å,
F(000) = 1336, (Cu K
) = 2.070 mm−1, T = 293 K, 32,030 measured reflections (
6328 unique reflections, final R = 0.057, wR = 0.166, S = 1.062 for 5725 reflections with I > 2σ(I).
β ,
= 104.402(1)◦ , V = 3064.23(7) Å3, Z = 4, dcalc = 1.389 Mg m−3
µ
α
θ
range 3.81–78.86◦ ),
Crystal data of 14: C13H11N4OSCl 0.5 C2H6N2O2,M = 351.81, monoclinic, space group C2/c,
a = 24.0692(5), b = 9.7448(2), c = 13.4379(3) Å,
= 103.383(2)◦ , V = 3066.26(12) Å3, Z = 8, dcalc = 1.524 Mg
−3, F(000) = 1456, ) = 3.640 mm−1, T = 293 K, 8718 measured reflections (
(Cu K
range 3.77–78.55◦ ),
·
·
β
m
µ
α
θ
3143 unique reflections, final R = 0.066, wR = 0.189, S = 1.125 for 2901 reflections with I > 2σ(I).
2.3. Antimycobacterial Assay
Four strains of mycobacteria, M. tuberculosis H37Ra, M. phlei, M. smegmatis and M. timereck,
were used for the study. The strains were donated by National Tuberculosis and Lung Diseases
Research Institute (Warsaw, Poland). The microorganisms were stored on Lewenstein–Jensen media
◦
at 4 C, then transferred to vials containing Middlebrook 7H9 broth supplemented with sodium
chl◦oride, bovine albumin, dextrose and catalase (ADC-BBL/Becton-Dickinson, USA) and grown at
37 C. The MIC values (minimal inhibitory concentration) for the tested compounds were determined
by log2 dilution method (Turnidge and Jorgensen, 1999).
The stock solutions of the tested compounds were freshly prepared in dimethyl sulfoxide (DMSO).
The concentrations of test substances were in the range of 500–1.95 µg/mL.
Inoculum for susceptibility testing was prepared by scraping microorganisms stored on
Lewnstein’s slants followed by their transfer to the vial containing Middlebrook 7H9 broth with
ADC enrichment. The bacterial suspension was then disrupted by vortexing with glass beads for
1 min. The supernatant was collected and equalized to 1.0 McFarland with fresh medium. Turbidity of
the suspensions was measured using nephelometer (Becton, Dickinson and Company, Franklin Lake,
MD, USA). Then, 12 µL of inoculum was added to each tube containing the tested compounds and
incubated at 37 ◦C. The observations were carried out for 21 days for M. 37Ra and 7 days for other
strains. Rifampicin was used as a control.
The growth of mycobacteria was assessed using a visual method. The MIC value was determined
as the lowest concentration of the drug at which no growth of microorganisms was observed
(no turbidity).
A sample containing the same volume of an inoculum (but cultured in the absence of drug) served
as a positive control, the medium alone (without the bacteria) was a negative control. Controls were
incubated in the same condition as a test tube. In addition, a control containing DMSO at the highest
concentration used in the experiment (1.25%) was prepared to exclude the bactericidal action of the
solvent itself.
The zones of growth inhibition were determined by a diffusion well test on Middlebrook 7H10
medium containing the enrichment of OADC (oleic acid, bovine albumin fraction, dextrose and
catalase (Becton, Dickinson and Company, Franklin Lake, MD, USA). Petri dishes (
ϕ 10 cm) were filled
with 25 mL of sterilized medium and allowed to solidify. Then, 200 microliters of bacterial suspension
with a density corresponding to 0.5 McFarland was spread over the surface and wells with a diameter
of 4 mm were cut out (3 per plate). Afterwards, 50 microliters of the tested drugs were introduced into
the wells. The tested compounds were dissolved in DMSO. The concentration of the stock solution
was 5 mg/mL. Rifampicin was used as◦a control.
The plates were incubated at 37 C for 21 days for M. H37Ra and 7 days for the remaining
strains. Zones of inhibition were evaluated using colony counter (SCAN® 500, Interscience, St Nom la
Breteche, France).

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2.4. Computational Details
The crystal structure of Mycobacterium tuberculosis glutamine synthetase MtGS in complex with
magnesium, adenosine-5’-diphosphate (ADP) and L-methionine-s-sulfoximine phosphate MSO-P was
downloaded from Protein Data Bank (PDB ID: 2BVC [22]). Docking studies were performed using
the GOLD Suite v.5.5 software [23]. Preparation of enzyme (addition of hydrogens, removal of water
molecules, extraction of original ligands from the protein active site,) were done with GOLD as per
default settings. Binding site was determined using the previous knowledge of the original ligand’s
interaction site. In docking stimulations, each ligand was kept flexible but the amino acid residues of
the enzyme were held rigid. For the simulation runs default parameter values were taken. The selection
of atoms in the active site within 6 Å of original ligands was chosen as default. ChemPLP was selected
as the scoring function to rank the compounds to be investigated. Protein–ligand interactions were
analyzed using Hermes v1.8.2 [23]. The molecular structures of ligands were fully optimized without
any constraint at the DFT/B3LYP [24
The conformational preferences of
in GAUSSIAN 09.
,
25] level with 6–311++G(d,p.) basis set using GAUSSIAN 09 [26].
4 were calculated using semiempirical AM1 method implemented
3. Results and Discussion
3.1. Chemistry
The substrates for the planned synthesis were hydrazides of carboxylic acids: 2-pyridinecarboxylic
acid hydrazide ( ), 3-pyridinecarboxylic acid hydrazide ( ), 4-pyridinecarboxylic acid hydrazide (
and 4-pyridineacetic acid hydrazide ( ). Hydrazide C is available commercially. Other hydrazides
were prepared by the method described in the literature [10 12]. The title compounds were obtained by
A
B
C)
D
,
reaction of the corresponding hydrazide with commercially available isothiocyanates: 2-fluorophenyl,
2-chlorophenyl, 4-methyltiophenyl, 2,4-dichlorophenyl, phenyl, 3,4-dichlorophenyl, 4-nitrophenyl,
4-bromophenyl, morpholinoethyl, and methoxycarbonylomethyl isothiocyanate. The reaction was
carried out at the boiling point of methanol for 0.5–1 h (Scheme 1). Although the methods of synthesis
of thiosemicarbazide derivatives are known and described in the literature [27–29] they require
continuous modifications depending on the substrates used. Two methods for the preparation of
thiosemicarbazide derivatives are commonly used: in solution and at the melting point. The reaction
carried out at high temperature melts the substrates and yields the reaction product in good yield.
◦
The disadvantage of this method is high temperature (about 180 C), which can cause charring of
products or cyclization of intermediates of thiosemicarbazide. The process is carried out by heating
for several hours (about 20 h), prolonging the synthesis time. In addition, this method causes the
substance to be baked and the difficulty in its isolation, especially with poor solubility of the product.
In turn, the preparation of thiosemicarbazide derivatives in the solution requires the selection of a
suitable solvent. Using N,N-dimethylacetamide or acetonitrile the reaction can be carried out at room
temperature or by heating the starting materials. The desired product is usually isolated by distilling
off the solvent or adding water (precipitation). Unfortunately, the separation of the product is often
difficult, which limits the use of this method. Too high heating temperature can direct ring closure.
The cyclization problem can be solved by using diethyl ether (lower boiling solvent) as the solvent.
The disadvantage of this method is the poor solubility of substrates, which causes the reaction to occur
at low yield, and the product obtained requires multiple crystallization in order to get rid of unreacted
substrates (mainly hydrazide). In this research work it was decided to use methanol as solvate and
to heat substrates at boiling point. Proposed method of obtaining thiosemicarbazide derivatives is
a modification of the procedures described in the literature. Its advantage is the short heating time
(0.5–1 h) and the ease of isolation of the product from the mixture by filtration or partial evaporation
of the solvent (methanol). The solubility difference between hydrazide and thiosemicarbazide in
methanol is not insignificant. The applied method is characterized by satisfactory efficiency, and the
obtained product often does not require crystallization. In addition, the reaction environment excludes

Molecules 2019, 24, 251
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the possibility of cyclization which guarantees obtaining homogeneous compounds. As a result of the
syntheses carried out, 25 derivatives of thiosemicarbazide contained a pyridine ring were obtained.
The reaction and the purity of the compounds obtained were determined by thin layer chromatography
(TLC) using ready-made Merck chromatographic plates (aluminum oxide 60 F-254).As mobile phases
a mixture of chloroform and ethanol in a ratio of 10:1 or 8:2 was used. Chromatograms were visualized
in UV light with a wavelength of 254 nm. The structure of the obtained compounds was confirmed by
spectral analysis, mass spectrometry and crystallography analysis. Experimental data of all compounds
are described in Supplementary Material.
O
O
O
H
N
H
N
C H OH
O
NH2
2
5
N
R-NCS
N
H
NH2-NH2
N
H
R
+
N
S
N
A - D
1 - 25
No
1
R
No
14
R
Cl
F
pyridin-2-yl
pyridin-2-yl
pyridin-2-yl
pyridin-4-yl
pyridin-4-yl
pyridin-4-yl
Cl
NO2
2
3
15
16
SCH3
SCH3
Cl
N
O
4
5
pyridin-2-yl
pyridin-2-yl
17
18
pyridin-4-yl
pyridin-4-yl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
6
7
8
pyridin-3-yl
pyridin-3-yl
pyridin-3-yl
19
20
21
pyridin-4-yl
SCH3
pyridin-4-ylmethyl
pyridin-4-ylmethyl
F
NO2
Br
N
O
9
pyridin-3-yl
pyridin-3-yl
22
23
pyridin-4-ylmethyl
pyridin-4-ylmethyl
CH2COOCH
CH2COOCH
3
3
10
Cl
Cl
11
12
pyridin-3-yl
pyridin-3yl
24
pyridin-4-ylmethyl
Cl
Cl
Cl
Cl
Cl
Cl
25
pyridin-4-ylmethyl
F
13
pyridin-4-yl
Scheme 1. Synthesis of title compounds.

Molecules 2019, 24, 251
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3.2. X-Ray Analysis
The X-ray analyses for
4,
7
,
11
,
13 and 14 as the model compounds were performed in order to
confirm the synthesis pathway, assumed molecular structures, and identification of the tautomeric
form, and to find their conformational preferences observed in crystalline state.
The compound 13 crystallizes with one 2-propanol solvent molecule were attributable to two
molecules of the base. The structures and conformations of the molecules 4, 7, 11, 13 and 14
in the crystal are shown in Figure 1. In the crystalline state, all investigated molecules exist in
N1-amino/N3-amino/N4-amino/S2-thione/O5-keto tautomeric form, which is confirmed by the
C2–S2 and C5–O5 bond length of 1.6730(1) and 1.238(2) Å in 4, 1.695(4) and 1.226(4) Å in 7, 1.6733(17)
and 1.217(2) Å in 11, 1.695(2) and 1.226(3) Å in molecule A and 1.690(2) and 1.226(3) Å in molecule B of
13 and 1.728(4) and 1.220(3) Å in 14, respectively, typical for the carbonyl and thione group in amides
(1.234(12) Å) and thioureas (1.681(20) Å), respectively [30]. Moreover, the positions of the amino-H
atoms are localized at the difference electron-density maps in the vicinity of N1, N3 and N4 atoms for
all investigated crystals.
Figure 1. The molecular structures of compounds
4, 7, 11, 13 and 14 with atom labelling and
displacement ellipsoids drawing at the 30% probability level.

Molecules 2019, 24, 251
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The conformation of carbonylthiosemicarbazide spacer depends on the nature of substituents
at the ends of this chain. The torsion angles C11–N1–C2–N3, N1–C2–N3–N4, C2–N3–N4–C5 and
N3–N4–C5–C51 with values presented in Table 1 show that the carbonylthiosemicarbazide chain adopts
trans-cis-gauche-trans conformation in
conformation in with 2-pyridine group. This difference in conformation causes that the thione C2–S2
group has trans and cis conformation with respect to N3–N4 (torsion angle C11–N1–C2–S2) and N1–C11
(torsion angle N4–N3–C2–S2) bonds in 11 13 and 14, while the cis and cis conformation is observed in
(Table 1). Consequently, the torsion angles N52–C51–C5–O5 in and O5–C5–C51–C52(C56) in 13 and
7, 11, 13 and 14 with 3- and 4-pyridine substituent and all trans
4
7,
,
4
4
14 show that the carbonyl group is practically coplanar with the pyridine ring, while the torsion angles
O5–C5–C51–C52(C56) indicate that this group is slightly distorted from coplanarity. The analysis
of the differences in conformation of investigated molecules shows two possible conformations cis
and trans on C2–N3 bond and conformation trans and gauche on N3–N4 bond. The energy effect of
the free-rotation at C2–N3 and N3–N4 bonds for
The energies of conformations were minimized and all geometrical parameters optimized for each
rotation with a 10◦ increment from 180◦ to 180◦ of ϕ1 = N1–C2–N3–N4 and ϕ2 = C2–N3–N4–C5
4 was calculated using AM1 semiempirical method.
−
torsion angles as shown in Figure S1 and Figure S2, respectively (Supplementary Material). One can
see for ϕ1, that the minima of energy cis (ϕ1
barriers estimated about 6.9 kcal/mol, and within range of ϕ1 from
not exceed 2.8 kcal/mol. In the case of ϕ2, two minima of energy (gauche; ϕ2 = 100 and ϕ2
separated by the energy barriers not exceeding 9.5 kcal/mol. It should be noted that calculated energy
barriers do not prevent molecules against conformational changes in physiological conditions.
=
−
20◦) and trans (ϕ1 = 160◦) are separated by the energy
◦
−
20 to 180 the energy changes do
◦
◦
=
−
90 ) are
◦
Table 1. Selected torsion angles ( ) for 4, 7, 11, 13 and 14.
Torsion Angle
4
7
11
13A
13B
14
C11–N1–C2–N3
N1–C2–N3–N4
C2–N3–N4–C5
N3–N4–C5–C51
C11–N1–C2–S2
N4–N3–C2–S2
N52–C51–C5–O5
C52–C51–C5–O5
C56–C51–C5–O5
166.21(15)
162.99(14)
−154.75(15)
176.16(13)
−15.1(2)
−15.7(2)
−177.25(15)
-
179.4(3)
−11.0(5)
−69.1(4)
169.8(3)
1.0(5)
167.5(3)
-
35.0(5)
−148.9(4)
−
176.89(19)
3.5(2)
92.1(2)
176.47(15)
4.4(3)
177.69(12) 171.92(16)
172.9(2)
−10.7(3)
−87.1(3)
−171.1(2)
−178.8(3)
9.6(3)
5.1(4)
86.2(3)
179.79(19) 179.15(19)
94.4(3)
178.3(2)
3.1(4)
−
−
−
−9.9(3)
6.0(3)
−
174.28(17) −176.7(2)
-
-
-
-
150.14(17)
−29.1(2)
8.7(3)
−175.5(2)
−7.0(3)
177.2(3)
−7.3(4)
5.1(2)
177.9(2)
The tautomeric form and the conformations of the investigated molecules are stabilized by the
intramolecular hydrogen bonds: N1–H1 . . . N4 in 11 13 and 14, N1–H1 . . . Cl12 in and 11 and
N3–H3...O5, N4–H4...S2 and N4–H4...N52 in 4 (Figure 1, Table 2).
In the crystal structures of 11 13 and 14 the packing of the molecules is determined by a
7,
,
4
4
,
7,
,
net of intermolecular hydrogen bonds N–H . . . X (X = O, S, N), as a consequence of the tautomeric
form observed in crystals, where amino N–H groups are the proton donors and O-carbonyl, S-thione
and N-pyridine atoms interact as proton acceptors (Table 2). Moreover, the overlapping of parallel
benzene rings in
4
and pyridine rings in 13 and 14 is observed with the π . . . π distances characteristic
11 13 and 14 with the
for -stacking. A detailed description of the crystal structures of
π
4,
7,
,
structural motifs formed by molecules interacting via intermolecular hydrogen bonds is presented in
Supplementary Material. The analysis of the intermolecular interactions in the crystal may be helpful
in prediction of the possible interactions of the molecules of investigated compounds with the binding
site of potential molecular target related to biological activity.

Molecules 2019, 24, 251
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◦
Table 2. Hydrogen-bond geometry (Å, ).
D–H . . . A
D–H
H . . . A
D . . . A
D–H . . . A
4
N1–H1 . . . Cl12
N3–H3 . . . O5
N4–H4 . . . S2
N4–H4 . . . N52
N1–H1 . . . O5 i
N3–H3 . . . O5 i
0.79(2)
0.85(2)
0.83(2)
0.83(2)
0.79(2)
0.85(2)
2.69(2)
2.34(2)
2.56(2)
2.31(2)
2.12(2)
2.02(2)
2.9681(16)
2.6787(19)
2.9497(16)
2.644(2)
2.849(2)
2.806(2)
103.2(18)
104.3(17)
109.9(17)
104.8(17)
155(2)
153.2(19)
i = −x, 2-y, −z
7
N1–H1 . . . N4
N1–H1 . . . O5 i
N3–H3 . . . N53 ii
N4–H4 . . . S2 i
0.86(2)
0.86(5)
0.80(6)
0.94(5)
2.27(5)
2.11(5)
2.07(5)
2.44(5)
2.658(4)
2.904(4)
2.842(5)
3.337(3)
107(4)
154(4)
162(5)
160(4)
1
i = x, −y, + z; ii = 1 + x, y, z
2
11
N1–H1 . . . N4
N1–H1 . . . Cl12
N1–H1 . . . O5 i
N3–H3 . . . N53 ii
N4–H4 . . . O5 i
0.82(2)
0.82(2)
0.82(2)
0.89(2)
0.85(3)
2.21(2)
2.49(2)
2.49(2)
1.98(2)
2.04(3)
2.637(2)
2.9154(17)
3.159(2)
2.860(2)
2.792(2)
113(2)
114(2)
140(2)
172(2)
147(2)
1
1
i = −1 + x, y, z; ii = 1 + x, − y, + z
2
2
13
N1A–H1A . . . N4A
N1B–H1B . . . N4B
N1A–H1A . . . N54B
N1B–H1B . . . N54A
O2–H1 . . . S2A i
0.89(4)
0.81(4)
0.89(4)
0.81(4)
0.82
0.84(3)
0.82(3)
0.78(3)
0.84(3)
2.31(3)
2.38(3)
2.05(4)
2.12(4)
2.55
2.48(3)
2.49(3)
2.17(3)
2.09(3)
2.697(3)
2.701(3)
2.883(3)
2.896(3)
3.364(2)
3.297(2)
3.291(2)
2.867(2)
2.849(3)
106(3)
105(3)
155(3)
160(3)
171
166(3)
166(3)
149(3)
151(3)
N3A–H3A . . . S2B ii
N3B–H3B . . . S2A iii
N4A–H4A . . . O5A i
N4B–H4B . . . O5B iv
1
1
1
1
i = 3/2−x, + −y, − z; ii = 1 + x, y, z; iii = −1 + x, y, z; iv = − x, −1/2 + y; − z
2
2
2
2
14
N1–H1 . . . N4
0.89(4)
0.89(4)
1.03(4)
0.86(5)
2.37(4)
2.07(4)
2.31(4)
2.06(5)
2.711(4)
2.930(3)
3.327(3)
2.851(4)
103(3)
163(4)
170(3)
151(4)
N1–H1 . . . N54 i
N3–H3 . . . S2 ii
N4–H4 . . . O5 iii
i = 12 -x, 3/2-y, 1-z; ii = 21 -x, 3/2-y, -z; ii = 12 -x, 21 +y, 12 -z
3.3. Antimycobacterial Activity
All obtained ( 25) compounds were tested in vitro against four Mycobacterium strains: M. H37Ra,
1–
M. phlei, M. smegmatis, M. timereck. The tests were performed by disc diffusion and broth microdilution.
The obtained results indicate the biological potential and varying activity depending on the type of
substituent in the position of the thiosemicarbazide and the pyridine ring substitution site. Of the
substances tested, the most promising seems to be compound
4 for which the growth inhibition zones
were respectively 29.6 mm (M. H37Ra), 30.1 mm (M. phlei), 31.0 mm (M. smegmatis), and 29.8 mm
(M. timereck). For the remaining compounds, the growth inhibition zone ranged from 0 to 30.5 mm
(M. H37Ra), 0 to 30.1 mm (M. phlei, M. smegmatis) and 0 to 29.8 mm (M. timereck). Analysis of the activity

Molecules 2019, 24, 251
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within the strains showed that compound
3
effectively inhibited the growth of M. H37Ra (30.5 mm),
compound
4 inhibited the growth of M. phlei and M. smegmatis (30.0 mm) and M. timereck (29.8 mm).
Detailed data are given in Table 3.
Table 3. Inhibition zone for tested compounds (250 µg of compound was used per well).
Inhibition Zone (mm) 250 µg of Compound Per Well
No
M. H37Ra
M. phlei
M. smegmatis
M. timereck
1
2
3
4
5
6
7
8
28.5
26.4
30.5
29.6
8.3
20.7
20.0
0.0
28.1
23.6
22.3
30.1
14.8
10.6
21.0
0.0
28.3
24.1
21.4
31.0
10.7
17.4
10.5
0.0
29.2
25.4
27.6
29.8
16.5
18.8
20.0
0.0
9
10
11
0.0
26.7
0.0
0.0
20.7
0.0
0.0
21.3
0.0
0.0
19.6
0.0
12
13
25.9
0.0
23.4
0.0
22.4
0.0
18.4
0.0
14
15
16
20.5
25.5
0.0
18.4
21.5
0.0
19.6
18.4
0.0
21.0
8.3
0.0
17
18
19
20
9.5
9.0
9.8
18.6
22.2
16.3
0.0
24.0
13.5
0.0
21.2
23.8
8.3
22.3
18.5
0.0
21
22
23
24
23.3
20.1
0.0
0.0
11.1
9.9
0.0
0.0
21.0
10.2
0.0
22.9
23.3
0.0
0.0
0.0
25
8.9
35.4
10.8
36.1
8.8
33.2
0.0
28.0
Rifampicin
The determined minimal inhibitory concentration with 2-pyridine ring exhibited lower MIC
values (7.81–31.25 g/mL) in comparison to the other isomers (Figure 2). The exception is compound
, which caused the inhibition of growth of M. phlei at a concentration of 125 g/mL. The lowest MIC
value (7.81 g/mL) was obtained for compound against M. smegmatis. For the remaining strains, these
values ranged within 15.625 and 31.25 g/mL. Compound 2 (having a 2-chlorophenyl substituent) had
activity against all tested strains at a concentration of 15.625 g/mL. Replacing chlorine with fluorine
) results in an increase in the minimum inhibitory concentration twice against M. pheli. In turn,
the additional chlorine atom ( ) causes an increase in the inhibitory concentration against M. phlei,
M. smegmatis, M. timereck. Less promising were the compounds associated with the pyridine ring in the
para position, with MIC values above 250 g/mL. Only compound 19 showed inhibition of growth of
microorganisms at concentrations of 31.25–62.5 g/mL (Figure 3). No antituberculosis activity was
µ
3
µ
µ
5
µ
µ
(1
4
µ
µ
observed for compounds linked to the pyridine ring with a methylene linker (20–25). MIC values
above 250 µg/mL were observed for these derivatives. Detailed data are shown in Table 4.

Molecules 2019, 24, 251
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Figure 2. Antimycobacterial activity of active compounds.
Figure 3. Antimycobacterial activity of the most active compounds.
Table 4. Minimal inhibitory concentration for tested compounds.
M. H37Ra
M. phlei
M. smegmatis
M. timereck
No
MIC
MIC
MIC
MIC
(µg/mL)
(µM/L)
(µg/mL)
(µM/L)
(µg/mL)
(µM/L)
(µg/mL)
(µM/L)
1
2
3
4
5
6
7
10
12
19
15.625
15.625
15.625
15.625
15.625
62.5
15.625
125
62.5
53.82
50.93
49.07
45.79
45.79
203.73
49.07
465.91
183.17
91.58
1.18
31.25
15.625
125
31.25
31.25
500
15.625
250
31.25
62.5
107.64
50.93
392.56
91.58
91.58
1629.88
49.07
931.82
91.58
183.17
18.99
15.625
15.625
31.25
31.25
7.81
500
125
62.5
62.5
53.82
50.93
98.14
91.58
22.89
1629.88
392.56
232.95
183.17
183.17
18.99
15.625
15.625
31.25
31.25
15.625
250
15.625
125
62.5
53.82
50.93
98.14
91.58
45.78
814.94
49.07
465.91
183.17
183.17
4.74
31.25
62.5
15.625
62.5
3.9
Rifampicin 0.976
15.625
* Compounds 8, 9, 13–18 and 20–25 MIC > 250 µg/mL.

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3.4. Molecular Docking
The thiosemicarbazide derivatives containing pyridine ring
1
–25 were tested in virtual screening
to the Mycobacterium tuberculosis glutamine synthetase MtGS. Glutamine synthetase as an enzyme is a
central component of bacterial nitrogen metabolism [25]. It catalyzes the ligation of glutamate and
ammonia forming L-glutamine, ADP, phosphate and a proton with the hydrolysis of ATP being a
potential drug target [25,31–33]. An enzyme MtGS is of particular interest as a new molecular target
in a novel antibiotic strategy, due to its relationship to pathogenicity of mycobacteria [33]. Therefore,
we attempted to check in silico the affinity of the investigated thiosemicarbazide derivatives for the
enzyme MtGS as its potential inhibitors. In crystalline state, MtGS occurs in a complex with magnesium,
adenosine-5’-diphosphate ADP and L-methionine-s-sulfoximine phosphate MSO-P consisting of six
subunits that constitute a hexameric ring. Overall MtGS structure with location of active sites is
presented in Figure 4 [25].
Figure 4. Overall MtGS structure. The active sites represented by a ball-and-stick model of MSO-P and
ADP are located at subunit-subunit interfaces.
The molecular docking study performed for 1–25 showed that all investigated ligands bind to the
active site of the MtGS receptor with the scoring function ChemPLP presented in Table 5. The values
of scoring ChemPLP function vary in the narrow range from 56.13 for 11 to 70.97 for 25, which shows
that all ligands have similar affinity to MtGS. Moreover, the way of connection with the active site
of MtGS are similar for all ligands. Most of the investigated compounds, with the exceptions of
7, 8, 11 and 23, show interaction with enzyme via intermolecular hydrogen bonds with the amine
group NH of the thiosemicarbazide part of the molecule as a donor of proton and carbonyl group of
Glu214 as an acceptor of proton. Other active amino acids in the binding domain in MtGS interacting
with analyzed ligands are: Arg364 via N–H . . . O5 for
Lys215 via N–H . . . O5 for 10 and 23; and Ser280 via O–H . . . N(pyridine) for
O–H . . . O (nitro group) for 15. The most active against Mycobacterium tuberculosis compounds
interacts with the binding domain of MtGS by Glu214 with N4 . . . O(Glu214) distance of 2.722 Å for
2.948 Å for , 2.604 Å for and 2.628 Å for and N3 . . . O(Glu214) distance of 3.058 Å for (Figure 5).
It should be noted that the molecules of compounds 11 13 and 14 are linked with the active site of
MtGs in conformations differing from those observed in the crystalline state (Figure S3; Supplementary
1,
20 and 21; Arg352 via N–H . . . O5 for
8
;
7,
11 17 23 and
,
,
2–5
2,
3
4
5
3
4,
7
,
,

Molecules 2019, 24, 251
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Materials), which confirms high flexibility of the carbonylthiosemicarbazide spacer. The docking of the
native ligands MSO-P and ADP of MtGS show that they much better fit to the active site of this enzyme
by interacting with more amino acid residues in comparison to investigated thiosemicarbazide, as can
be seen in Table 5.
Table 5. The results of the molecular docking of ligands 1–25 to the MtGS.
Compound
ChemPLP Value
Amino Acid Involved in Hydrogen Bonds
1
2
3
4
5
6
7
8
9
57.60
56.89
59.84
63.25
64.43
58.49
58.71
57.73
64.38
66.12
56.13
67.24
56.94
56.74
64.50
63.33
65.92
64.68
65.80
62.09
67.00
65.40
67.89
65.72
70.97
127.47
137.10
Glu214, Arg364
Glu214
Glu214*2
Glu214
Glu214
Glu214
Ser280
Glu133, Arg352
Glu214
Glu214, Lys215
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
MSO-P
ADP
Ser280
Glu214
Glu214
Glu214
Glu214, Ser280
Glu214
Glu214, Ser280
Glu214
Glu214
Glu214, Arg364
Glu214, Arg364
Glu214*2
Lys215*2, Ser280
Glu214
Glu214*2
Lys215, Glu219*2, Glu227*2, Glu133*2, Arg352*2
Lys215, Glu133*2, Arg364, Arg347*2, Glu219.Glu227*2
Figure 5. A view of the interaction between (a) 2, (b) 3, (c) 4 and (d) 5 and the binding site of MtGS e.

Molecules 2019, 24, 251
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4. Conclusions
In conclusion, we synthesized several new thiosemicarbazides bearing pyridine ring by the
reaction of carboxylic acid hydrazide with appropriate isothiocyanates. The structures of obtained
compounds were confirmed by spectroscopic methods and X-ray investigations. The X-ray
analyses performed for 4, 7, 11, 13 and 14 show that all investigated compounds exist in
N1-amino/N3-amino/N4-amino/S2-thione/O5-keto tautomeric form. The conformations of the
molecules, stabilized by N–H...X (X = N, Cl, O, S) intramolecular hydrogen bonds, are connected
with 2-, 3- and 4-pyridine substituents of carbonylthiosemicarbazide chain. The consequence of
the tautomeric form observed in the crystals of 4, 7, 11, 13 and 14 is the net of the intermolecular
hydrogen bonds N–H . . . X (X = O, S, N), which play a key role in crystal packing. All newly
obtained thiosemicarbazides were tested in vitro against four Mycobacterium strains: M. H37Ra,
M. phlei, M. smegmatis, M. timereck. The antibacterial activity is strongly connected with the position
of the pyridine substituent in relation to the thiosemicarbazide skeleton. The most active against
Mycobacterium tuberculosis are compounds 2–5 with 2-pyridine ring. The molecular docking studies
investigating thiosemicarbazides using the enzyme MtGS as their potential biological target indicated
that the amino and carbonyl groups of carbonylthiosemicarbazide spacer play an important role in
interactions with the active site of MtGS, with Glu214, Glu133, Arg364, Arg352, Lys215 and Ser280 as
the most active amino acid residues.
Supplementary Materials: The Supplementary Materials are available online.
Author Contributions: M.P. created the concept and designed the study. Z.K. carried out quantum chemical
calculations and docking study. M.S.-O. and G.G. coordinated and conducted the tuberculostatic assay. A.F. and
W.W. performed x-ray analysis. M.W. performed the synthetic experiments. K.C. editing the manuscript and
language correction. All the authors read and approved the final manuscript.
Funding: The theoretical calculations were supported in part by PL-Grid Infrastructure. The results of the research
carried out in part under the research theme No. 9/S/91 were financed from the science grant granted by the
Ministry of Science and Higher Education.
Acknowledgments: The authors thank Emilia Fornal (Department of Pathophysiology, Medical University of
Lublin, Poland) for LC/QTOF data acquisition and analysis.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–25 are available from the correspondence author.
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