4-Substituted picolinohydrazonamides as a new class of potential antitubercular agents

Article abstract of DOI:10.1016/j.ejmech.2020.112106

The series of new 4-substituted picolinohydrazonamides were synthesized (6-25) and evaluated for tuberculostatic activity. Compounds having a hydrophilic cyclic amine such as morpholine and pyrrolidine at the end of the thiosemicarbazide chain, exhibited the highest antimycobacterial activity. The antimycobacterial activity of compounds 6, 11, and 15 (MIC 0.4–0.8 μg/mL) was higher than that of reference drugs. Moreover, derivative 15 exhibited lower activity against other tested microorganism such as bacteria gram-positive, gram-negative or fungi. Thus, this compound is characterized by the selectivity of antimicrobial activity. Antiproliferative study conducted against human dermal fibroblasts (HDF) and mouse melanoma cell line (B16-F10) revealed low cytotoxicity of compound 15. Conducted research allowed to identify compound 15 as leading for further research.

Full text of DOI:10.1016/j.ejmech.2020.112106

Journal Pre-proof
4-Substituted picolinohydrazonamides as a new class of potential antitubercular
agents
Malwina Krause, Henryk Foks, Dagmara Ziembicka, Ewa Augustynowicz-Kopeć,
Agnieszka Głogowska, Izabela Korona-Głowniak, Krzysztof Bojanowski, Danuta
Siluk, Katarzyna Gobis
PII:
S0223-5234(20)30073-8
DOI:
https://doi.org/10.1016/j.ejmech.2020.112106
EJMECH 112106
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 November 2019
Revised Date: 27 January 2020
Accepted Date: 27 January 2020
Please cite this article as: M. Krause, H. Foks, D. Ziembicka, E. Augustynowicz-Kopeć, A. Głogowska,
I. Korona-Głowniak, K. Bojanowski, D. Siluk, K. Gobis, 4-Substituted picolinohydrazonamides as a new
class of potential antitubercular agents, European Journal of Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.ejmech.2020.112106.
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4-Substituted picolinohydrazonamides as a new class of potential antitubercular agents
Malwina Krause1, Henryk Foks1, Dagmara Ziembicka1, Ewa Augustynowicz-Kopeć2,
Agnieszka Głogowska2, Izabela Korona-Głowniak3, Krzysztof Bojanowski4, Danuta
Siluk5, Katarzyna Gobis1
1Department of Organic Chemistry, Medical University of Gdańsk, Gdańsk, Poland;
2Department of Microbiology, Institute of Tuberculosis and Pulmonary Diseases, Warsaw,
Poland;
3Department of Pharmaceutical Microbiology, Medical University of Lublin, Lublin, Poland
4Sunny BioDiscovery Inc., Santa Paula, CA 93060, USA
5Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdańsk,
Poland
Abstract
The series of new 4-substituted picolinohydrazonamides were synthesized (6-25) and evaluated for
tuberculostatic activity. Compounds having a hydrophilic cyclic amine such as morpholine and
pyrrolidine at the end of the thiosemicarbazide chain, exhibited the highest antimycobacterial activity.
The antimycobacterial activity of compounds 6, 11, and 15 (MIC 0.4-0.8 µg/mL) was higher than that
of reference drugs. Moreover, derivative 15 exhibited lower activity against other tested
microorganism such as bacteria gram-positive, gram-negative or fungi. Thus, this compound is
characterized by the selectivity of antimicrobial activity. Antiproliferative study conducted against
human dermal fibroblasts (HDF) and mouse melanoma cell line (B16-F10) revealed low cytotoxicity
of compound 15. Conducted research allowed to identify compound 15 as leading for further research.
Keywords: synthesis; tuberculosis; thiosemicarbazide; cytotoxic activity; microscopic observation
1. Introduction
Tuberculosis (TB) is an infectious disease caused by the bacteria Mycobacterium tuberculosis
complex[1,2]. It seemed that the discovery of streptomycin, isoniazid, and rifampicin and the
development of combination therapy will eliminate TB[3–6]. However, the growing resistance of M.
tuberculosis and the emergence of strains resistant to first- and second-line drugs have increased the
incidence of TB, thereby increasing mortality caused by this disease[7–12]. TB is the ninth leading
cause of death in the world and leading cause from a single infectious agent[13]. The World Health
Organization estimates that 558 000 people developed drug-resistant TB in 2017. Moreover, 1.7
billion people have a latent TB infection and the risk of developing TB during their lifetime[14,15].
Although there is a dramatic increase in bacterial resistance to the antibiotics used, the number of
newly approved antibacterial drugs has declined[16–20]. Drug development for TB has stagnated for
decades, until 2012, when the FDA approved bedaquiline as a new antitubercular drug[21–23]. The
current situation could be called a crisis of antibiotic therapy. It is necessary to urgently obtain novel
drugs with a new mechanism of action[24–26].
Thiosemicarbazides, which due to the presence of sulfur and nitrogen atoms can easily form hydrogen
bonds with proteins are in the focus of scientists[27]. Compounds of this structure are known for their
wide biological activity: antibacterial, antiviral, cytotoxic, anticonvulsant, analgesic[28–30].
Thiosemicarbazides are also known as compounds active against tuberculosis. These include
vanadium and manganese complexes, conjugates with 4-nitropyrrole or hybrids with an imidazole and
piperazine moiety[31–33].

Figure 1. Project concept
Our team's long-term study of heterocyclic compounds exhibiting antitubercular activity has also
established thiosemicarbazides as promising tuberculostatic agents (Figure 1). First,
thiosemicarbazides containing a ring of pyrazine, which is also present in pyrazinamide first-line drug,
were obtained. The activity of these derivatives was varied with minimum inhibitory concentrations
(MICs) in the range 8.8–100 µg/mL. Substituents belonging to the group of secondary cyclic amines
(morpholine, hexamethyleneimine or phenylpiperazine) increased tuberculostatic activity while
substituents being primary alkylamines (aniline, 4-aminomorpholine or allylamine) caused a decrease
in activity. Subsequently, derivatives with the thioester moiety were obtained, but their activity was
negligible (MIC 50–100 µg/mL)[34,35]. Those results prompted us to convert pyrazine to pyridine
ring, which is found in isoniazid, another first-line chemotherapeutic. The obtained derivatives
inhibited the growth of mycobacteria over two times more strongly than their pyrazine analogs[36].
The substitution of the pyridine ring at the C4 position with a methyl or phenyl group resulted in an
increase in activity. Some of the obtained compounds were characterized by good antitubercular
activity with MICs in the range 0.75–3.1 µg/mL. It is noteworthy that the morpholine substituent is
essential for antitubercular activity because the derivatives possessing this moiety exhibited the
highest activity. Based on the obtained results, we synthesized cycloalkylaminothiosemicarbazides to
check the effect of other substituents on the activity of this group of compounds.
2. Results and discussion
2.1. Chemistry
Derivatives with methylenehydrazinecarbothioamide moiety at the C2 position and the nucleophilic
substituent at the C4 position were synthesized. The desired derivatives possessed cycloalkylamine
rings (morpholine, piperidine, pyrrolidine, and phenylpiperazine) in thiosemicarbazide moiety. The
synthetic route used in this study is outlined in Scheme 1. The starting material for the synthesis was
commercially available 4-chloropicolinonitrile. In the first step, the chlorine atom was substituted in a
nucleophilic reaction. The process was carried out in dioxane with the addition of DBU (1,8-
diazabicyclo[5.4.0]undec-7-ene) at reflux for 1 h. Phenol, thiophenol, morpholine, pyrrolidine, and
phenylpiperazine were used as nucleophilic agents. Subsequently, the nitrile group was converted into

the methyl imidate group. The reaction was performed in the presence of methanol and a catalytic
amount of DBU. The reaction mixture was refluxed for 4 h and monitored by TLC (thin layer
chromatography). To obtain final products, the corresponding methyliminoesters were condensed with
various cycloalkylamino-1-carbothiohydrazides by heating for 15–60 minutes at boiling temperature.
Carbohydrazides were obtained in the reaction between cyclic amines and methyl
hydrazinecarbodithioate in alcohol or water. The synthesis method of cycloalkylamino-1-
carbothiohydrazides is described by Klayman et al[37]. From the synthesis process, 20 new final
derivatives were obtained. Syntheses of the desired cycloalkylaminethiosemicarbazide derivatives 6–
25 were achieved with 36–96% yields. All the obtained compounds were purified by crystallization.
1
13
All the newly synthesized compounds were characterized by IR, H NMR, and C NMR spectra and
elemental analysis. The results of the spectral analysis were in accordance with the assigned structures.
Scheme 1. Synthesis of cycloalkylaminethiosemicarbazide derivatives 6-25. a) dioxane, DBU, reflux, 1 h; b)
methanol, DBU, reflux, 4 h; c) cycloalkylamino-1-carbothiohydrazides (1 eq), reflux, 15–60 min
2.2. Biological activity
All the obtained compounds were evaluated for their in vitro tuberculostatic activity against M.
tuberculosis H37Rv strain and “wild-type” strain isolated from patients with TB (Spec. 210) resistant to
p-aminosalicylic acid (PAS), isonicotinic acid hydrazide (INH), ethambutol (ETB), and rifampicin
(RMP). MICs were determined as the minimum concentration inhibiting the growth of tested TB
strains in relation to the probe with no tested compound. INH and PZA were used as reference drugs.
Extensive microbiological tests were conducted for five compounds with the highest antitubercular
activity ( ,
6 7, 11, 15,
and ). This experiment aimed to verify the selectivity of tested compounds.
19
The study was performed on gram-positive bacteria Staphylococcus epidermis, Staphylococcus
aureus, Micrococcus luteus, Bacillus subtilis, Bacillus cereus, Streptococcus pyogenes, Streptococcus
pneumonia, and Streptococcus mutans; gram-negative bacteria Escherichia coli, Klebsiella
pneumonia, Pseudomonas aeruginosa, and Proteus mirabilis; and fungi Candida albicans and
Candida parapsilosis. Ciprofloxacin (CIP), vancomycin (VAN), and fluconazole (FCZ) were used as
reference drugs.
The most potent compounds were also tested for cytotoxic activity against human dermal fibroblasts
(ATCC PCS-201-010) and mouse melanoma cells (ATCC CRL-6375). The obtained results allowed

to determine whether the tested compounds in a concentration showing antimycobacterial activity also
show a toxic effect against the selected cell lines. MAP and bFGF were used as positive controls. All
values are means of at least two independent experiments, each performed in duplicate. Microscopic
images of cell lines B16-F10 and HDF showing changes caused by the addition of compounds 1 and
10 at a concentration of 5 µg/mL were also obtained. The bioactive data are summarized in Tables 1–4
and Figure 3.
2.2.1. Tuberculostatic activity
The obtained compounds exhibited very good tuberculostatic activity with MICs in the range of 0.4–
25 µg/mL (Table 1). All compounds exhibited the same activity against sensitive and resistant strains.
The presence of the highest hydrophilic cycloamines such as pyrrolidine and morpholine in the
thiosemicarbazide chain showed a positive effect on antitubercular activity. In particular, derivatives
having a morpholine ring (7, 11, 15, and 19) showed the highest tuberculostatic activity among the
tested compounds with MICs in the range of 0.4–3.1 µg/mL. Their antimycobacterial activity was
equal or higher than that of reference drugs. Excellent antitubercular potency (MIC 0.4 µg/mL) was
also observed for compound 6, which had pyrrolidine in the thiosemicarbazide chain and phenoxy
group at the C4 position of the pyridine ring. The presence of highly lipophilic phenylpiperazine
moiety (9, 13, 17, 21, and 25) significantly decreased the antitubercular potency (MIC 6.25–25
µg/mL). Similarly, the presence of phenylpiperazine at the pyridine ring resulted in a decrease in
antitubercular activity. The phenylpiperazine substituent negatively affected tuberculostatic activity
probably as a result of high lipophilicity and high molecular weight. The piperidine substituent in the
thiosemicarbazide chain did not have a significant effect on the antimycobacterial activity.
Presumably, the basicity of the substituent occurring at the C4 position of the pyridine ring influenced
the tuberculostatic activity of the tested compounds. Derivatives having a pyrrolidine moiety at the
pyridine ring (14–17), characterized by the highest basicity, exhibited high antitubercular potency
(MIC 0.4–6.25 µg/mL). In contrast, derivatives having a thiophenoxy group (10–13) of low basicity
showed significantly lower activity (MIC 0.8–25 µg/mL).
Table 1. In vitro tuberculostatic activity of compounds 6–25.
Minimal inhibitory concentration (µg/mL)
Compound
H37Rv
0.4
Spec. 210
0.4
6
7
3.1
3.1
8
12.5
25
12.5
25
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
6.25
0.8
6.25
0.8
12.5
25
12.5
25
6.25
0.4
6.25
0.4
6.25
6.25
6.25
3.1
6.25
6.25
6.25
3.1
6.25
6.25
12.5
6.25
6.25
6.25
12.5
6.25

24
25
25
25
3.1
25
25
25
INH
PZA
12.5
>400
2.2.2. Antimicrobial activity
Table 2. Antimicrobial activity against gram-positive bacteria
MIC (µg/mL)
Compound
6
7
11
15
19
CIP
VAN
Microorganism
Gram-positive
bacteria
S. aureus
ATCC 25923
0.49
0.98
0.24
15.6
7.8
0.98
3.9
15.6
7.8
15.6
15.6
15.6
0.49
0.24
0.24
0.98
0.49
0.49
S. aureus
ATCC 6538
S. aureus
ATCC 43300
125
15.6
31.3
S. epidermidis
ATCC 12228
0.12
3.9
0.49
1.95
15.6
0.49
0.98
M. luteus
ATCC 10240
1.95
0.49
0.98
1.95
0.49
7.8
0.24
1.95
7.8
0.49
0.98
1.95
7.8
7.8
31.3
31.3
1000
0.98
0.03
0.12
-
0.12
0.24
0.98
0.24
B. subtilis
ATCC 6633
B. cereus
ATCC 10876
31.3
7.8
S. pyogenes
ATCC 19615
3.9
S. pneumoniae
ATCC 49619
7.8
31.3
7.8
31.3
1.95
250
>1000
1000
-
-
0.24
0.98
S. mutans
ATCC 25175
0.98
1.95
Compound 6 showed a higher bacteriostatic activity against S. aureus ATCC 43300 (MIC 0.24
µg/mL) and S. epidermidis ATCC 12228 (MIC 0.12 µg/mL) than against the tested M. tuberculosis
strains (MIC 0.4 µg/mL) (Table 2). The potency of S. epidermidis ATCC 12228 growth inhibition was
higher than for reference drugs ciprofloxacin and vancomycin. The MIC of this compound for all
tested bacteria of the genus Bacillus and Staphylococcus was below 1 µg/mL. Moreover, this
compound was characterized by very good activity against S. mutans ATCC 25175 with MIC at the
same level as that of vancomycin (0.98 µg/mL). Bacteriostatic and bactericidal activity against gram-
positive bacteria of compound 7 was at a moderate level. Only for the M. luteus strain, MIC was lower
(0.49 µg/mL) than for the tested M. tuberculosis strains (3.1 µg/mL). Compound 11 showed higher
bacteriostatic activity than tuberculostatic activity against M. luteus ATCC 10240 and S. epidermidis
ATCC 12228 strains. The bacteriostatic activity of compounds 15 and 19 was not greater than their
tuberculostatic activity for all the tested gram-positive bacteria strains. Compound 15 exhibited good
antimicrobial activity against M. luteus ATCC 10240 and B. subtilis ATCC 6633 strains with MICs
below 1 µg/mL. All tested compounds had very weak or no bacteriostatic activity against gram-
negative bacteria (Table 3). The antifungal activity of the tested derivatives was much lower than the
reference drug fluconazole. The most sensitive strains on the test group of compounds were S.
epidermidis ATCC 12228 and M. luteus ATCC 10240.

Table 3. Antimicrobial activity against gram-negative bacteria and yeasts
MIC (µg/mL)
Compound
6
7
11
15
19
CIP
FCZ
Microorganism
Gram-negative bacteria
E. coli
ATCC 25922
125
31.3
500
125
125
62.5
>1000
>1000
>1000
>1000
>1000
>1000
31.3
0.004
0.03
0.12
0.49
-
-
-
-
P. mirabilis
ATCC 12453
K. pneumoniae
ATCC 13883
>1000
250
500
>1000
>1000
P. aeruginosa
ATCC 9027
>1000
>1000
Yeasts
C. albicans ATCC 2091
C. albicans ATCC 102231
7.8
7.8
7.8
15.6
15.6
15.6
15.6
15.6
15.6
15.6
15.6
15.6
31.3
31.3
31.3
-
-
-
0.25
0.98
1.95
C. parapsilosis
ATCC 22019
2.2.3. Cytotoxic activity
The IC50 values for the examined derivatives were in the range of 2.56–38.83 µg/mL (Table 4). All
tested compounds showed weaker antiproliferative activity on HDF cells than on tumor cells B16-F10.
The determined IC50 values of obtained derivatives did not exceed the MIC against M. tuberculosis.
IC50 for compound 19 for human dermal fibroblasts could not be calculated due to the weak
antiproliferative activity of the compound across the tested concentrations. The selectivity index (IC50
HDF/IC50 B16-F10) indicated that compounds 6, 7, 11, and 15 had efficacy against tumor cells
greater than toxicity against normal cells. The calculated IC50-HDF/MIC-MT selectivity index for
compounds 6, 7, 11, and 15 revealed their lack of cytotoxicity. The MIC of M. tuberculosis growth for
compound 15 with the highest tuberculostatic activity was 90.45 times lower than the concentration
causing antiproliferative effect against noncancer cells.
Table 4. Cytotoxic activity of compounds 6, 7, 11, 15, and 19
SI
SI’
IC50 (µg/mL)
IC50-HDF/
IC50-B16-F10
10.39
IC50-HDF/
MIC-MT
67.27
Cell line B16-F10 HDF
6
2.56
9.38
26.91
38.83
32.65
36.18
NA
4.13
2.91
3.29
-
12.52
40.38
90.45
-
7
11
15
19
11.10
10.98
17.85
NA – not available in the range of concentrations used, IC50 could not be calculated due to the weak antiproliferative activity of compound
across the tested concentrations.
The selectivity index (SI) was calculated for each compound using the formula SI = IC50 for normal cell line (HDF)/IC50 for the respective
cancer cell line (B16-F10). A beneficial SI > 1.0 indicates a drug with efficacy against tumor cells greater than toxicity against normal cells.
SI was also calculated using the formula SI = IC50 for normal cell line (HDF)/MIC against M. tuberculosis. An SI value > 1.0 indicates that
the compound is nontoxic.
For two compounds with the highest tuberculostatic activity, cytotoxicity tests were extended by
microscopic examination (Figure 2). Observations allowed us to see morphological changes in the

presence of tested compounds at a concentration of 5 µg/mL. In the image showing control, three-
dimensional and compact growth of mouse melanoma cells was observed. After the addition of
compounds 6 and 15, drastic morphological changes were observed. Three-dimensional structures
disappeared, while only necrotic cells remained. Compound 6 also caused morphological changes in
human dermal fibroblasts. In the presence of derivative 15, such changes, however, did not occur.
Figure 2. Microscopic pictures of the cell lines HDF and B16-F10.
3. Conclusions
In conclusion, a series of novel cycloalkylaminothiosemicarbazide derivatives were successfully
synthesized. Their tuberculostatic activity in vitro was evaluated against M. tuberculosis H37Rv and
Spec. 210 strains using the twofold serial dilution MIC method. Compounds 7, 11, 15, and 19
possessing a morpholine ring at the end of the thiosemicarbazide chain exhibited the highest
antitubercular activity. The most potent compound 15 exhibited antimycobacterial activity with a MIC
0.4 µg/mL, which is higher than that for reference drugs. Activity at the same level was also observed
for compound 6. Despite the same antimycobacterial potency, compound 15 exhibited greater
selectivity than compound 6. In addition, both compounds did not show a cytotoxic effect on HDF
cells at concentrations exhibiting an antitubercular effect. However, extended microscopic studies
showed that compound 6 caused morphological changes in fibroblast cells, whereas in the presence of
compound 15, such changes were not observed. From the results obtained, we can conclude that the
synthesized derivatives have a promising anti-TB activity, and compound 15 may constitute a leading
structure for further research on antitubercular drugs.
4. Experimental
4.1. Chemistry

All materials and solvents were of analytical reagent grade. Thin-layer chromatography (TLC) was
performed on Merck silica gel 60F254 plates and visualized with UV light. The results of elemental
analyses (%C, H, N) for all the obtained compounds that were determined on Perkin-Elmer PE 2400
Series II CHNS analyzer (Perkin-Elmer, Shelton, CT) were in agreement with the calculated values
1
13
within ±0.4% range. H and C NMR spectra in CDCl3 or DMSO-d6 were recorded on Varian Unity
Plus (500 MHz) and Varian Gemini (200 MHz) instruments. IR spectra (KBr) were determined as KBr
pellets of the solids on a Satellite FT-IR spectrophotometer. Melting points were determined on a
Stuart SMP30 apparatus and retained without any corrections. Confirmation of compounds
identification was performed with the use of 6224 LC-TOF/MS system (Agilent Technologies,
Waldronn, Germany) equipped with electrospray ionisation source (ESI). 2 µl of sample was injected
directly to the Mass spectrometry detector. The mobile phase consisted of 0.1 % formic acid in
methanol (Sigma-Aldrich, USA) and 0.1 % formic acid in deionized water (70:30, v/v) from Milli-Q
water system (Millpore Inc., Bedford, MA, USA). The analysis was carried out under isocratic
condition with a flow rate at 0.3 ml/min at ambient temperature. The study was performed in positive
polarity in a scan mode in a 60-1200 m/z mass range. Ion source parameters were as follows:
temperature of drying gas (nitrogen), its flow rate and nebulizer pressure were set at 350 ºC, 11 L/min
and 45 psi, respectively. The capillary voltage was set at 3500 V. Besides, skimmer and octopole
voltages were 65 V and 750 V, respectively. Together with analysed sample reference masses were
analysed simultaneously (121.0509 m/z and 922.0098 m/z; Agilent Technologies, Waldbronn,
Germany). The reference masses were measured due to control accuracy of the measurement of
masses detected in the analysed sample. All compounds were analysed in 3 different fragmentor
voltages: 120, 150 and 200 V, in order to monitor compounds fragmentation pattern.
4.1.1. General procedure for the synthesis of nitriles 1–5
In a round-bottom flask, 40 mmol of 4-chloropicolinonitrile, 48 mmol of an appropriate nucleophilic
agent, 25 mL of dioxane, and 6 mL of DBU were added. The mixture was heated at reflux for 1 h.
Then, the solvent was evaporated, and ice was added. Precipitated products were filtered and
recrystallized from cyclohexane or methanol.
4-phenoxypicolinonitrile (1)
Starting from 4-chloropicolinonitrile (5.6 g) and phenol (4.51 g), the title compound 1 was obtained as
beige crystals (7.23 g, 88%): mp 90–92 °C (cyclohexane); IR (KBr): 3086, 3060 (ʋ CAr-H); 2234 (ʋ
CN); 1647 (ʋ C=N); 1580, 1489, 1413 (ʋ C=C); 1279, 1206 (δ C-H) 957, 849, 781, 697 (γ C-H) cm-1;
1H NMR (500 MHz, DMSO-d6): δ 7,15 (dd, 1H, Pyr, J1=3 Hz, J2=3 Hz); 7,23 (d, 2H, ArH, J=8 Hz);
7,34 (t, 1H, ArH, J=7 Hz); 7,51 (t, 2H, ArH, J=8 Hz); 7,67 (d, 1H, Pyr, J=8 Hz); 8,57 (d, 1H, Pyr, J=6
Hz) ppm; Anal. Calcd for C12H8N2O (196.06): C, 73.46; H, 4.11; N, 14.28; Found: C, 73.14; H, 4.07;
N, 14.16.
4-phenylthiopicolinonitrile (2)
Starting from 4-chloropicolinonitrile (5.6 g) and thiophenol (4.91 g), the title compound 2 was
obtained as light yellow crystals (8.67 g, 97.5%): mp 51–53 °C (cyclohexane); IR (KBr): 3047 (ʋ CAr-
1
H); 2234 (ʋ CN); 1647 (ʋ C=N); 1570, 1437 (ʋ C=C); 835, 759, 691 (ɣ C-H) cm-1; H NMR (500
MHz, DMSO-d6): δ 7,19 (dd, 1H, Pyr, J1=2 Hz, J2=3 Hz); 7,54-7,63 (m, 5H, ArH); 7,68 (d, 1H, Pyr,
J=2 Hz); 8,47 (d, 1H, Pyr, J=5 Hz) ppm; Anal. Calcd for C12H8N2S (212.04): C, 67.90; H, 3.80; N,
13.20; Found: C, 67.73; H, 3.79; N, 13.02.
4-pyrrolidinopicolinonitrile (3)

Starting from 4-chloropicolinonitrile (5.6 g) and thiophenol (4 mL), the title compound 3 was obtained
as white crystals (6.55 g, 90%): mp 132–133 °C (methanol); IR (KBr): 2979-2856 (ʋ C-H); 2230 (ʋ
1
CN); 1602, 1530 (ʋ C=C); 1290, 1235, 1212, 1116 (δ C-H); 850, 829 (γ C-H) cm-1; H NMR (500
MHz, DMSO-d6): 1.95-1.97 (m, 4H, CH2); 3.28-3.30 (m, 4H, CH2); 6,65 (d, 1H, Pyr, J=4 Hz); 7.05 (s,
1H, Pyr); 8.15 (d, 1H, Pyr J=5 Hz) ppm; Calcd for C10H11N3 (173.10): C, 69.34; H, 6.40; N, 24.26;
Found: C, 68.98; H, 6.38; N, 23.99.
4-morpholinopicolinonitrile (4)
Starting from 4-chloropicolinonitrile (5.6 g) and morpholine (4.18 mL), the title compound 4 was
obtained as white crystals (7.92 g, 89%): mp 206-208 °C (methanol); IR (KBr): 3055 (ʋ CAr-H); 2975,
1
2855 (ʋ C-H); 2234 (ʋ CN); 1597 (ʋ C=C); 1258, 1116 (δ C-H); 990 (γ C-H) cm-1; H NMR (500
MHz, DMSO-d6): δ 3,36 (t, 4H, CH2, J=5 Hz); 3,68 (t, 4H, CH2, J=5 Hz); 7,05 (dd, 1H, Pyr, J1=3 Hz,
J2=3 Hz); 7,47 (d, 1H, Pyr, J=7 Hz); 8,24 (d, 1H, Pyr, J=7 Hz) ppm; Anal. Calcd for C10H11N3O
(189.09): C, 63.48; H, 5.86; N, 22.21; Found: C, 63.39; H, 5.80; N, 22.17.
4-(4-phenylpiperazin-1-yl)picolinonitrile (5)
Starting from 4-chloropicolinonitrile (5.6 g) and 4-phenylpiperazine (6.4 mL), the title compound 5
was obtained as beige crystals (9.80 g, 92%): mp 179-183 °C (methanol); IR (KBr): 3074-2847 (ʋ C-
1
H), 1647 (ʋ C=N), 1594, 1504, 1452 (ʋ C=C), 1390, 1271, 1236 (δ C-H), 837, 762 (ɣ C-H) cm-1; H
NMR (500 MHz, DMSO-d6): δ 3.22-3.24 (m, 4H, CH2); 3.56-3.58 (m, 4H, CH2); 6.79 (t, 1H, ArH,
J=7 Hz); 6.96 (d, 2H, ArH, J=8 Hz); 7.10 (d, 1H, Pyr, J=5 Hz); 7.22 (t, 2H, ArH, J=7 Hz); 7.53 (s, 1H,
Pyr); 8.24 (d, 1H, Pyr, J=6 Hz) ppm; Anal. Calcd for C16H16N4 (264.14): C, 72.70; H, 6.10; N, 21.20;
Found: C, 73.05; H, 5,93; N, 20,83.
4.1.2. General procedure for the synthesis of cycloalkylamine-1-thiosemicarbazides 6–25
Method A (6–13, 15, 18, 20, 21, 23)
To a solution of nitrile (2 mmol) in methanol (15 mL), DBU (2.7 mmol, 0.4 mL) was added, and the
mixture was refluxed for 4 h. After this time, 2 mmol of cycloalkylamino-1-carbothiohydrazides was
added and heated at reflux for 15–60 min. Then, the mixture was poured onto 40 g of ice and acidified
with acetic acid to give a precipitate. The resulting precipitate was filtered, dried, and recrystallized
from an appropriate solvent.
Method B (14, 16, 17, 19, 22, 24, 25)
To a solution of nitrile (2 mmol) in methanol (15 mL), DBU (2.7 mmol, 0.4 mL) was added, and the
mixture was refluxed for 4 h. After this time, 2 mmol of cycloalkylamino-1-carbothiohydrazides was
added and heated at reflux for 15–60 min. After cooling, the precipitate was filtered, dried, and
recrystallized from an appropriate solvent.
4-phenoxy-N'-(pyrrolidine-1-carbonothioyl)picolinohydrazonamide (6)
Starting from 4-phenoxypicolinonitrile (0.392 g) and pyrrolidine-1-carbothiohydrazide (0.290 g), the
title compound 6 was obtained as yellow crystals (0.370 g, 54%): mp 99–101 ˚C (ethanol); IR (KBr):
1
3420 (ʋ N-H); 2965, 2868 (ʋ C-H); 1581 (δ N-H); 1432 (δ C-H); 1232 (ʋ C-N) cm-1; H NMR (500
MHz, DMSO-d6): δ 1,72-1,81 (m, 4H, CH2); 3,53-3,54 (m, 4H, CH2); 6,75 (s, 1H, NH); 7,07 (d, 1H,
Pyr; J=3 Hz); 7,19-7,34 (m, 3H, ArH); 7,50 (t, 2H, ArH, J=8 Hz); 7,82 (s, 1H, Pyr); 8,44 (d, 1H, Pyr,

J=5 Hz); 9,34 (s, 1H, NH); 12,57 (s, 1H, NH) ppm; Anal. Calcd for C17H19N5OS: C, 59.80; H, 5.61; N,
20.51; Found: C, 59.73; H, 5.41; N, 20.83; LC-MS m/z: 342.1385 [M+H]+.
N'-(morpholine-4-carbonothioyl)-4-phenoxypicolinohydrazonamide (7)
Starting from 4-phenoxypicolinonitrile (0.392 g) and morpholine-4-carbothiohydrazide (0.322 g), the
title compound 7 was obtained as yellow crystals (0.332 g, 62%): mp 159–160 ˚C (methanol-water
1:1); IR (KBr): 3419 (ʋ N-H), 2922, 2852 (ʋ C-H), 1671 (ʋ C=N), 1580 (δ N-H), 1463, 1417 (δ C-H),
1238 (ʋ C-N), 1112 (ʋ C-O-C) cm-1; 1H NMR (500 MHz, DMSO-d6): δ 3.56-3.58 (m, 4H, CH2); 3.81-
3.85 (m, 4H, CH2); 7.01 (s, 1H, NH); 7.23-7.25 (m, 2H, 1xArH+1xPyr); 7.33 (t, 1H, ArH, J=7 Hz);
7.51 (t, 3H, ArH, J=7Hz); 7.86 (s, 1H, Pyr); 8.60-8.61 (m, 2H, 1xPyr+1xNH); 12.60 (s, 1H, NH) ppm;
13C NMR (175 MHz, DMSO-d6): δ 47.17 (2C), 66.69 (2C), 110.96, 113.99, 121.11, 121.40, 126.39,
131.06 (2C), 144.76, 146.60, 152.18, 153.78, 166.02; 179.32 ppm; Anal. Calcd for C17H19N5O2S: C,
57.12; H, 5.36; N, 19.59; Found: C, 57.13; H, 5.02; N, 19.28; LC-MS m/z: 358.1334 [M+H]+.
4-phenoxy-N'-(piperidine-1-carbonothioyl)picolinohydrazonamide (8)
Starting from 4-phenoxypicolinonitrile (0.392 g) and piperidine-1-carbothiohydrazide (0.318 g), the
title compound 8 was obtained as yellow crystals (0.326 g, 46%): mp 74-75 ˚C (ethanol); IR (KBr):
3393, 3276, 3144 (ʋ N-H); 3050 (ʋ CAr-H); 2971, 2924 (ʋ C-H); 1671 (ʋ C=N); 1576 (δ N-H); 1471 (δ
1
C-H); 1233 (ʋ C-N) cm-1; H NMR (500 MHz, DMSO-d6): δ 1.21-1.57 (m, 6H, CH2); 3.40-3.83 (m,
4H, CH2); 6.81 (s, 1H, NH); 6.99 (d, 1H, Pyr, J=3 Hz); 7.13-7.53 (m, 5H, ArH); 7.84 (s, 1H, Pyr) 8.59
(d, 1H, Pyr, J=5 Hz); 9.21 (s, 1H, NH); 12.79 (s, 1H, NH) ppm; Anal. Calcd for C18H21N5OS: C,
60.82; H, 5.95; N, 19.70; Found: C, 60.76; H, 5.70; N, 19.32; LC-MS m/z: 356,1538 [M+H]+.
4-phenoxy-N'-(4-phenylpiperazine-1-carbonothioyl)picolinohydrazonamide (9)
Starting from 4-phenoxypicolinonitrile (0.392 g) and 4-phenylpiperazine-1-carbothiohydrazide
(0.472), the title compound 9 was obtained as yellow crystals (0.836 g, 96%): mp 147-150 °C
(ethanol-water 1:1); IR (KBr): 3410 (ʋ N-H); 3056 (ʋ CAr-H); 2926 (ʋ C-H); 1645, 1581 (δ N-H);
1
1448, 1462 (δ C-H); 1225 (ʋ C-N) cm-1; H NMR (500 MHz, DMSO-d6): δ 3.11-3.13 (m, 4H, CH2);
3.97-3.99 (m, 4H, CH2); 6.78-7.02 (m, 5H, 3xArH+1xNH2); 7.20-7.34 (m, 6H, 5xArH+1xPyr); 7.50-
7.51 (m, 2H, ArH); 7.87 (s,1H, Pyr); 8.60 (d, 1H, Pyr, J=4 Hz); 12.65 (s,1H, NH) ppm; 13C NMR (175
MHz, DMSO-d6): δ 46.30 (2C), 49.01 (2C), 110.95, 113.99, 116.22 (2C), 119.51, 121.11 (2C),
126.38, 129.41 (2C), 131.06 (2C), 144.64, 146.64, 151.68, 152.18, 153.79, 166.02, 179.09 ppm; Anal.
Calcd for C23H24N6OS: C, 63.87; H, 5.59; N, 19.43; Found: C, 63.66; H, 5.62; N, 19.44; LC-MS m/z:
433.1805 [M+H]+.
4-(phenylthio)-N'-(pyrrolidine-1-carbonothioyl)picolinohydrazonamide (10)
Starting from 4-phenylthiopicolinonitrile (0.424 g) and pyrrolidine-1-carbothiohydrazide (0.290 g), the
title compound 10 was obtained as yellow crystals (0.285 g, 40%): mp 160-162 °C (ethanol); IR
(KBr): 3417, 3228, 3130 (ʋ N-H); 3018(ʋ CAr-H); 2961, 2863 (ʋ C-H); 1665 (ʋ C=N); 1570 (δ N-H);
1
1422 (δ C-H); 1277 (ʋ C-N) cm-1; H NMR (500 MHz, DMSO-d6): δ 1.75-1.77 (m, 4H, CH2); 3.42-
3.54 (m, 4H, CH2); 6.73 (s, 2H, NH2); 7.42 (s, 1H, Pyr); 7.55-7.61 (m, 5H, ArH); 8.34 (d, 1H, Pyr,
J=5Hz); 9.24 (s, 1H, Pyr); 12.52 (s, 1H, NH) ppm; 13C NMR (175 MHz, DMSO-d6): δ 115.83, 121.49,
128.54, 130.64 (2C), 130.76 (3C), 130.89, 135.27, 135.76 (2C), 148.31 (2C), 150.61, 151.40, 177.81
ppm; Anal. Calcd for C17H19N5S2: C, 57.11; H, 5.36; N, 19.59; Found: C, 57.17; H, 5.12; N, 19.29;
LC-MS m/z: 358.1155 [M+H]+.

N'-(morpholine-4-carbonothioyl)-4-(phenylthio)picolinohydrazonamide (11)
Starting from 4-phenylthiopicolinonitrile (0.424 g) and morpholine-4-carbothiohydrazide (0.322 g),
the title compound 11 was obtained as yellow crystals (0.311 g, 41%): mp 74-76 °C (ethanol); IR
(KBr): 3422 (ʋ N-H); 3052 (ʋ CAr-H); 2923, 2852 (ʋ C-H); 1575 (δ N-H); 1439, 1416 (δ C-H); 1112 (ʋ
C-O-C) cm-1; 1H NMR (500 MHz, DMSO-d6): δ 3.55-3.80 (m, 8H, CH2); 6.95 (s, 1H, Pyr); 7.55-7.63
(m, 6H, 5xArH+1xNH); 8.04 (s, 1H, Pyr); 8.49 (d, 1H, Pyr, J=5Hz); 8.60 (s, 1H, NH); 12.56 (s, 1H,
NH) ppm; Anal. Calcd for C17H19N5OS2: C, 54.67; H, 5.13; N, 18.75; Found: C, 54.72; H, 5.03; N,
18.75; LC-MS m/z: 374.1104 [M+H]+.
4-(phenylthio)-N'-(piperidine-1-carbonothioyl)picolinohydrazonamide (12)
Starting from 4-phenylthiopicolinonitrile (0.424 g) and piperidine-1-carbothiohydrazide (0.318 g), the
title compound 12 was obtained as yellow crystals (0.500 g, 67%): mp 150-152 °C (ethanol); IR
(KBr): 3412, 3269, 3134 (ʋ N-H); 3031 (ʋ CAr-H); 2920, 2848 (ʋ C-H); 1668 (ʋ C=N); 1572 (δ N-H);
1
1473 (δ C-H); 1245 (ʋ C-N) cm-1; H NMR (500 MHz, DMSO-d6): δ 1.36-1.56 (m, 6H, CH2); 3.59-
3.82 (m, 4H, CH2); 6.77 (s, 1H, NH); 6.93 (d, 1H, Pyr, J=4 Hz); 7.51-7.62 (m, 5H, ArH); 8.02 (s, 1H,
Pyr); 8.48 (d, 1H, Pyr, J=5 Hz); 9.17 (s, 1H, NH); 12.67 (s, 1H, NH) ppm; 13C NMR (175 MHz,
DMSO-d6): δ 25.28, 26.15 (2C), 47.36 (2C), 118.67, 122.36, 128.41, 130.73, 130.89 (2C), 135.30,
135.59, 143.71, 144.74, 150.30, 152.31, 178.81 ppm; Anal. Calcd for C18H21N5S2: C, 58.19; H, 5.70;
N, 18.85; Found: C, 57.87; H, 5.58; N, 18.55; LC-MS m/z: 372.1311 [M+H]+.
N'-(4-phenylpiperazine-1-carbonothioyl)-4-(phenylthio)picolinohydrazonamide (13)
Starting from 4-phenylthiopicolinonitrile (0.424 g) and 4-phenylpiperazine-1-carbothiohydrazide
(0.472), the title compound 13 was obtained as yellow crystals (0.860 g, 95%): mp 164-166 °C
(methanol); IR (KBr): 3413, 3232 (ʋ N-H); 3034 (ʋ CAr-H); 2926 (ʋ C-H); 1598, 1572 (δ N-H); 1225
(ʋ C-N) cm-1; 1H NMR (500 MHz, DMSO-d6): δ 3.10 (bs, 4H, CH2); 3.98 (bs, 4H, CH2); 6.78-6.81 (m,
1H, ArH); 6.95-6.97 (m, 3H, 2xArH+1xPyr); 7.21 (t, 2H, ArH, J=8 Hz); 7.56-7.64 (m, 5H, ArH); 8.06
(s, 1H, Pyr); 8.38 (s, 1H, NH); 8.51 (d, 1H, Pyr, J=5 Hz); 9.50 (s, 1H, NH); 12.56 (s, 1H, NH) ppm;
Anal. Calcd for C23H24N6S2: C, 61.58; H, 5.39; N, 18.73; Found: C, 61.24; H, 5.33; N, 18.54; LC-MS
m/z: 449.1576 [M+H]+.
4-(pyrrolidin-1-yl)-N'-(pyrrolidine-1-carbonothioyl)picolinohydrazonamide (14)
Starting from 4-pyrrolidinopicolinonitrile (0.346 g) and pyrrolidine-1-carbothiohydrazide (0.290 g),
the title compound 14 was obtained as yellow crystals (0.230 g, 36%): mp 229-230 °C (ethanol); IR
(KBr): 3446, 3287, 3114 (ʋ N-H); 3021 (ʋ CAr-H); 2966, 2866 (ʋ C-H); 1669 (ʋ C=N); 1608 (δ N-H);
1
1427 (δ C-H); 1287 (ʋ C-N) cm-1; H NMR (500 MHz, DMSO-d6): δ 1.81-1.98 (m, 8H, CH2); 3.32-
3.54 (m, 8H, CH2); 6.55-6.57 (m, 1H, Pyr); 7.08 (s, 1H, NH); 7.23 (s, 1H, Pyr); 8.17 (d, 1H, Pyr, J=6
Hz); 8.40 (s, 1H, NH); 12.41 (s, 1H, NH) ppm; Anal. Calcd for C15H22N6S: C, 56.58; H, 6.96; N,
26.39; Found: C, 56.68; H, 6.73; N, 26.14; LC-MS m/z: 319.1703 [M+H]+.
N'-(morpholine-4-carbonothioyl)-4-(pyrrolidin-1-yl)picolinohydrazonamide (15)
Starting from 4-pyrrolidinopicolinonitrile (0.346 g) and morpholine-4-carbothiohydrazide (0.322 g),
the title compound 15 was obtained as yellow crystals (0.430 g, 64%): mp 208-210 °C (methanol); IR
(KBr): 3399, 3157 (ʋ N-H); 2923, 2852 (ʋ C-H); 1672 (ʋ C=N); 1599 (δ N-H); 1459, 1418 (δ C-H);
1
1256 (ʋ C-N); 1111 (ʋ C-O-C) cm-1; H NMR (500 MHz, DMSO-d6): δ 1.95 (t, 4H, CH2, J=6 Hz);
3.32-3.33 (m, 4H, CH2); 3.56 (t, 4H, CH2, J=6 Hz); 3.78 (t, 4H, CH2, J=5 Hz); 6.57-6.58 (m, 1H, Pyr);
7.25 (d, 1H, Pyr, J=2 Hz); 7.54 (s, 1H, NH); 8.18 (d, 1H, Pyr, J=6 Hz); 8.56 (s, 1H, NH); 12.44 (s, 1H,

NH) ppm; 13C NMR (175 MHz, DMSO-d6): δ 25.27 (2C), 47.18 (2C), 47.59 (2C), 66.72 (2C), 105.33,
108.84, 144.20, 146.27, 149.47, 152.63, 178,85 ppm; Anal. Calcd for C15H22N6OS: C, 53.87; H, 6.63;
N, 25.13; Found: C, 54.10; H, 6.56; N, 24.78; LC-MS m/z: 335.1650 [M+H]+.
N'-(piperidine-1-carbonothioyl)-4-(pyrrolidin-1-yl)picolinohydrazonamide (16)
Starting from 4-pyrrolidinopicolinonitrile (0.346 g) and piperidine-1-carbothiohydrazide (0.318 g), the
title compound 16 was obtained as yellow crystals (0.309 g, 47%): mp 198-200 °C (ethanol); IR
(KBr): 3377, 3259, 3135 (ʋ N-H); 3025 (ʋ CAr-H); 2926, 2850 (ʋ C-H); 1611, 1585 (δ N-H); 1421 (δ
1
C-H); 1244 (ʋ C-N) cm-1; H NMR (500 MHz, DMSO-d6): δ 1.44-1.57 (m, 6H, CH2); 1.91-1.98 (m,
4H, CH2); 3.29-3.32 (m, 4H, CH2); 3.80-3.88 (m, 4H, CH2); 6.54-6.56 (m, 1H, Pyr); 7.23 (s, 1H, Pyr);
7.38 (s, 1H, NH); 8.17 (d, 1H, Pyr, J=6 Hz); 8.35 (s, 1H, NH); 12.56 (s, 1H, NH) ppm; 13C NMR (175
MHz, DMSO-d6): δ 25.27 (4C), 25.33, 26.13 (2C), 47.33, 47.58, 105.17, 108.65, 144.42, 145.28,
149.46, 152.63, 178.36 ppm; Anal. Calcd for C16H24N6S: C, 57.80; H, 7.28; N, 25.28; Found: C,
57.45; H, 7.29; N, 24.96; LC-MS m/z: 333.1856 [M+H]+.
N'-(4-phenylpiperazine-1-carbonothioyl)-4-(pyrrolidin-1-yl)picolinohydrazonamide (17)
Starting from 4-pyrrolidinopicolinonitrile (0.346 g) and 4-phenylpiperazine-1-carbothiohydrazide
(0.472), the title compound 17 was obtained as yellow crystals (0.590 g, 72%): mp 200-202 °C
(ethanol); IR (KBr): 3419, 3296 (ʋ N-H); 3081 (ʋ C-H); 2960, 2854 (ʋ C-H); 1646 (ʋ C=N); 1602 (δ
1
N-H); 1420 (δ C-H); 1222 (ʋ C-N) cm-1; H NMR (500 MHz, DMSO-d6): δ 1.97-1.98 (m, 4H, CH2);
2.48-2.49 (m, 4H, CH2); 3.33-3.35 (m, 4H, CH2); 3.97-3.99 (m, 4H, CH2); 6.57 (d, 1H, Pyr, J=6 Hz);
6.78 (t, 1H, ArH, J=7 Hz); 6.96-6.97 (m, 2H, ArH); 7.21 (t, 2H, ArH, J=7 Hz); 7.27 (s, 1H, Pyr); 7.60
(s, 1H, NH); 8.19 (d, 1H, Pyr, J=6 Hz); 8.58 (s, 1H, NH); 12.47 (s, 1H, NH) ppm; 13C NMR (175
MHz, DMSO-d6): δ 46.10 (2C), 46.29 (2C), 49.01 (2C), 66.06 (2C), 106.06, 109.85, 116.22 (2C),
119.50, 129.41 (2C), 144.87, 145.80, 150.28, 151.70, 156.17, 178.73 ppm; Anal. Calcd for C21H27N7S:
C, 61.59; H, 6.64; N, 23.94; Found: C, 61.30; H, 6.70; N, 23.66; LC-MS m/z: 410.2120 [M+H]+.
4-morpholino-N'-(pyrrolidine-1-carbonothioyl)picolinohydrazonamide (18)
Starting from 4-morpholinopicolinonitrile (0.378 g) and pyrrolidine-1-carbothiohydrazide (0.290 g),
the title compound 18 was obtained as yellow crystals (0.382 g, 57%): mp 228-230 °C (methanol); IR
(KBr): 3390 (ʋ N-H); 3059 (ʋ CAr-H); 2964, 2858 (ʋ C-H); 1663 (ʋ C=N); 1602, 1579 (δ N-H); 1426
(δ C-H); 1234 (ʋ C-N); 1123 (ʋ C-O-C) cm-1; 1H NMR (500 MHz, DMSO-d6): δ 1.81 (t, 4H, CH2, J=6
Hz); 3.39-3.40 (m, 4H, CH2); 3.55-3.57 (m, 4H, CH2); 3.72 (t, 4H, CH2, J=5 Hz); 6.94 (t, 1H, Pyr, J=4
Hz); 7.20 (s, 1H, NH); 7.55 (s, 1H, Pyr); 8.27 (d, 1H, Pyr, J=5 Hz); 8.40 (s, 1H, NH); 12.45 (s, 1H,
NH) ppm; Anal. Calcd for C15H22N6SO: C, 53.87; H, 6.63; N, 25.13; Found: C, 54.02; H, 6.52; N,
24.99; LC-MS m/z: 335.1651 [M+H]+.
N'-(morpholine-4-carbonothioyl)-4-morpholinopicolinohydrazonamide (19)
Starting from 4-morpholinopicolinonitrile (0.378 g) and morpholine-4-carbothiohydrazide (0.322 g),
the title compound 19 was obtained as yellow crystals (0.365 g, 52%): mp 223-224 °C (methanol); IR
(KBr): 3397, 3234 (ʋ N-H); 3088 (ʋ CAr-H); 2960-2853 (ʋ C-H); 1662 (ʋ C=N); 1605 (δ N-H); 1412
(δ C-H); 1244 (ʋ C-N); 1124 (ʋ C-O-C) cm-1; 1H NMR (500 MHz, DMSO-d6): δ 3.40 (t, 4H, CH2, J=5
Hz); 3.56 (t, 4H, CH2, J=4Hz); 3.73 (t, 4H, CH2, J=4 Hz); 3.79 (t, 4H, CH2, J=4 Hz); 6.98 (s, 1H, Pyr);
7.58-7,60 (m, 2H, 1xPyr+1xNH); 8.29 (d, 1H, Pyr, J=6 Hz); 8.54 (s, 1H, NH); 12.48 (s, 1H, NH) ppm;
13C NMR (175 MHz, DMSO-d6): δ 46.09 (2C), 47.17 (2C), 66.05 (2C), 66.71 (2C), 106.06, 109.87,

144.82, 145.92, 150.28, 156.17, 178.97 ppm; Anal. Calcd for C15H22N6O2S: C, 51.41; H, 6.33; N,
23.98; Found: C, 51.46; H, 6.14; N, 23.90; LC-MS m/z: 351.1597 [M+H]+.
4-morpholino-N'-(piperidine-1-carbonothioyl)picolinohydrazonamide (20)
Starting from 4-morpholinopicolinonitrile (0.378 g) and piperidine-1-carbothiohydrazide (0.318 g), the
title compound 20 was obtained as yellow crystals (0.424 g, 61%): mp 208-210 °C (ethanol); IR
(KBr): 3399 (ʋ N-H); 3069 (ʋ CAr-H); 2922, 2852 (ʋ C-H); 1666 (ʋ C=N); 1605 (δ N-H); 1427 (δ C-
1
H); 1242 (ʋ C-N); 1111 (ʋ C-O-C) cm-1; H NMR (500 MHz, DMSO-d6): δ 1.44-1.57 (m, 6H, CH2);
3.31-3.40 (m, 4H, CH2); 3.73-3.82 (m, 8H, CH2); 6.95 (d, 1H, Pyr, J=4 Hz); 7.40 (s, 1H, NH); 7.56 (s,
1H, Pyr); 8.28 (d,1H, Pyr, J=6 Hz); 8.40 (s, 1H, NH); 12.60 (s, 1H, NH) ppm; Anal. Calcd for
C16H24N6OS: C, 55.15; H, 6.94; N, 24.12; Found: C, 55.34; H, 6.67; N, 23.78; LC-MS m/z: 349.1805
[M+H]+.
4-morpholino-N'-(4-phenylpiperazine-1-carbonothioyl)picolinohydrazonamide (21)
Starting from 4-morpholinopicolinonitrile (0.378 g) and 4-phenylpiperazine-1-carbothiohydrazide
(0.472), the title compound 21 was obtained as yellow crystals (0.600 g, 71%): mp 143-145 °C
(methanol); IR (KBr): 3424, 3299, 3161 (ʋ N-H); 3051 (ʋ CAr-H); 2957, 2846 (ʋ C-H); 1676 (ʋ C=N);
1595 (δ N-H); 1426 (δ C-H); 1225 (ʋ C-N); 1113 (ʋ C-O-C) cm-1; 1H NMR (500 MHz, DMSO-d6): δ
3.11 (t, 4H, CH2, J=4 Hz); 3.41 (t, 4H, CH2, J=4 Hz); 3.73 (t, 4H, CH2, J=4 Hz); 3.97 (t, 4H, CH2, J=4
Hz); 6.78 (t, 1H, ArH, J=7 Hz); 6.96-6.97 (m, 3H, 2xArH+1xPyr); 7.22 (t, 2H, ArH, J=7 Hz); 7.59-
7.61 (m, 2H, 1xPyr+1xNH); 8.30 (d, 1H, Pyr, J=6 Hz); 8.54 (s, 1H, NH); 12.51 (s, 1H, NH) ppm;
Anal. Calcd for C21H27N7OS: C, 59.27; H, 6.40; N, 23.04; Found: C, 59.18; H, 6.30; N, 23.06; LC-MS
m/z: 426.2068 [M+H]+.
4-(4-phenylpiperazin-1-yl)-N'-(pyrrolidine-1-carbonothioyl)picolinohydrazonamide (22)
Starting from 4-(4-phenylpiperazin-1-yl)picolinonitrile (0.528 g) and pyrrolidine-1-carbothiohydrazide
(0.290 g), the title compound 22 was obtained as yellow crystals (0.602 g, 74%): mp 208-212 °C
(ethanol); IR (KBr): 3437, 3310 (ʋ N-H), 2855 ( ʋ C-H), 1670 (ʋ C=N), 1646 (δ N-H), 1599 (ʋ C=C),
1
1580 (δ N-H), 1421, 1361 (ʋ C=C), 1231, 988 (δ C-H), 757, 692 (ɣ C-H) cm-1; H NMR (500 MHz,
DMSO-d6): δ 1.81-1.82 (m, 4H, CH2); 3.27 (t, 4H, CH2, J=4 Hz); 3.54-3.55 (m, 4H, CH2); 3.59-3.60
(m, 4H, CH2); 6.80 (t, 1H, ArH, J=7.3 Hz); 7.00 (d, 3H, 2ArH+Pyr, J=7 Hz); 7.18 (s, 1H, NH); 7.23 (t,
2H, ArH, J=7 Hz); 7.62 (s, 1H, Pyr); 8.28 (d, 1H, Pyr, J=5 Hz); 8.46 (s, 1H, NH); 12.47 (s, 1H, NH)
ppm; Anal. Calcd for C21H27N7S: C, 61.59; H, 6.64; N, 23.94; Found: C, 61.35; H, 6.68; N, 23.85; LC-
MS m/z: 410.2123 [M+H]+.
N'-(morpholine-4-carbonothioyl)-4-(4-phenylpiperazin-1-yl)picolinohydrazonamide (23)
Starting
from 4-(4-phenylpiperazin-1-yl)picolinonitrile
(0.528
g)
and
morpholine-4-
carbothiohydrazide (0.322 g), the title compound 23 was obtained as yellow crystals (0.380 g, 45%):
mp 150-152 °C (ethanol); IR (KBr): 3401 (ʋ N-H), 3088, 2922, 2850 (ʋ C-H), 1666 ʋ C=N), 1594 (ʋ
C=C), 1540 (δ N-H), 1414, 1347 (ʋ C=C), 1230, 1112, 988 (δ C-H), 890, 760 (ɣ C-H) cm-1; 1H NMR
(500 MHz, DMSO-d6): δ 3.28 (t, 4H, CH2, J=4 Hz); 3.56 (t, 4H, CH2, J=4 Hz); 3.60 (t, 4H, CH2, J=4
Hz); 3.79 (t, 4H, CH2, J=4 Hz); 6.81 (t, 1H, ArH, J=7 Hz); 7.00 (d, 3H, 2ArH+1xPyr, J=8 Hz); 7.24 (t,
2H, ArH, J=7 Hz); 7.60 (s, 1H, NH); 7.64 (s, 1H, Pyr); 8.30 (d, 1H, Pyr, J=6 Hz); 8.60 (s, 1H, NH);
12.50 (s, 1H, NH) ppm; Anal. Calcd for C21H27N7OS: C, 59.27; H, 6.40; N, 23.04; Found: C, 58.97; H,
6.40; N, 22.76; LC-MS m/z: 426.2070 [M+H]+.
4-(4-phenylpiperazin-1-yl)-N'-(piperidine-1-carbonothioyl)picolinohydrazonamide (24)

Starting from 4-(4-phenylpiperazin-1-yl)picolinonitrile (0.528 g) and piperidine-1-carbothiohydrazide
(0.318 g), the title compound 24 was obtained as yellow crystals (0.370 g, 44%): mp 168-170 °C
(ethanol); IR (KBr): 3402, 3241 (ʋ N-H), 3058, 2928, 2848 (ʋ C-H), 1665 (ʋ C=N), 1958, 1541 (δ N-
H), 1479, 1417 (ʋ C=C), 1339, 1231, 987 (δ C-H), 755 (ɣ C-H) cm-1; 1H NMR (500 MHz, DMSO-d6):
δ 1.43-1.44 (m, 4H, CH2); 1.56-1.57 (m, 2H, CH2); 3.26-3.27 (m, 4H, CH2); 3.60-3.61 (m, 4H, CH2);
3.82-3.83 (m, 4H, CH2); 6.81 (t, 1H, ArH, J=7.3); 7.00 (d, 3H, 2ArH+Pyr, J=8 Hz); 7.23 (t, 2H, ArH,
J=8 Hz); 7.42 (s, 1H, NH); 7.63 (s, 1H, Pyr); 8.29 (d, 1H, Pyr, J=5 Hz); 8.47 (s, 1H, NH); 12.61 (s,
1H, NH) ppm; Anal. Calcd for C22H29N7S: C, 62.38; H, 6.90; N, 23.15; Found: C, 62.43; H, 6.73; N,
23.13; LC-MS m/z: 424.2277 [M+H]+.
4-(4-phenylpiperazin-1-yl)-N'-(4-phenylpiperazine-1-carbonothioyl)picolino hydrazonamide (25)
Starting from 4-(4-phenylpiperazin-1-yl)picolinonitrile (0.528 g) and 4-phenylpiperazine-1-
carbothiohydrazide (0.472), the title compound 25 was obtained as yellow crystals (0.370 g, 44%): mp
177-181 °C ethanol); IR (KBr): 3429, 3228 (ʋ N-H), 3139-2829 (ʋ C-H), 1674 (ʋ C=N), 1599, 1558 (δ
1
N-H), 1497, 1425, 1350 (ʋ C=C), 1230, 988 (δ C-H), 753, 693 (ɣ C-H) cm-1; H NMR (500 MHz,
DMSO-d6): δ 3.11 (t, 4H, CH2, J=4 Hz); 3.27 (t, 4H, CH2, J=4 Hz); 3.61 (t, 4H, CH2, J=4 Hz); 3.99 (t,
4H, CH2, J=4 Hz); 6.77-6.83 (m, 2H, ArH); 6.96-7.03 (m, 5H, 4xArH+1xPyr); 7.20-7.26 (m, 4H,
ArH); 7.42 (s, 1H, NH); 7.66 (s, 1H, Pyr); 8.31 (d, 1H, Pyr, J=6 Hz); 8.61 (s, 1H, NH); 12.53 (s, 1H,
NH) ppm; Anal. Calcd for C27H32N8S: C, 64.77; H, 6.44; N, 22.38; Found: C, 64.47; H, 6.46; N,
22.02; LC-MS m/z: 501.2545 [M+H]+.
4.2. Biological assays
4.2.1. Antimycobacterial activity assay
The synthesized compounds were examined in vitro for their tuberculostatic activity against the M.
tuberculosis H37Rv strain and “wild-type” strain isolated from patients with TB: Spec. 210 resistant to
p-aminosalicylic acid (PAS), isonicotinic acid hydrazide (INH), ethambutol (ETB), and rifampicin
(RMP). Investigations were performed by a classical test tube method of successive dilution in
Youmans’ modification of the Proskauer and Beck liquid medium containing 10% of bovine
serum[38,39]. Bacterial suspensions were prepared from 14-day-old cultures of slowly growing strains
and from 48-h-old cultures of saprophytic strains[40,41]. Solutions of compounds in ethylene glycol
were tested. Stock solutions contained 10 mg of compounds in 1 mL. Dilutions (in geometric
progression) were prepared in Youmans’ medium. The medium containing no investigated substances
and containing isoniazid (INH), pyrazinamide (PZA), or rifampicin (RMP) as reference drugs was
used for comparison. Incubation was performed at a temperature of 37 °C. MICs were determined as
the minimum concentration that inhibits the growth of tested TB strains in relation to the probe with
no tested compound. The influence of the compound on the growth of bacteria at concentrations 0.75,
1.5, 3.1, 6.2, 12.5, 25, 50, and 100 µg/mL was evaluated.
4.2.2. Antimicrobial assay
The compounds were screened for antibacterial and antifungal activities by the microdilution broth
method using Mueller-Hinton broth and Mueller-Hinton broth with 5% lysed sheep blood for the
growth of nonfastidious and fastidious bacteria, respectively, or Mueller-Hinton broth with 2%
glucose for the growth of fungi. MICs of the tested derivatives were evaluated for the panel of
reference microorganisms from American Type Culture Collection (ATCC), including gram-negative
bacteria (E. coli ATCC 25922, P. mirabilis ATCC 12453, K. pneumoniae ATCC 13883, P.
aeruginosa ATCC 9027), gram-positive bacteria (S. aureus ATCC 25923, S. aureus ATCC 43300, S.

aureus ATCC 6538, S. epidermidis ATCC 12228, M. luteus ATCC 10240, B. subtilis ATCC 6633, B.
cereus ATCC 10876, S. pyogenes ATCC 19615, S. pneumoniae ATCC 49619, S. mutans ATCC
25175), and fungi (C. albicans ATCC 10231, C. albicans ATCC 2091, C. parapsilosis ATCC 22019).
The compounds dissolved in dimethyl sulfoxide (DMSO) were first diluted to a concentration of 1000
µg/mL in an appropriate broth medium recommended for bacteria or yeasts. Then, using the same
media, serial twofold dilutions were made to obtain final concentrations of the tested derivatives that
ranged from 0.98 to 1000 µg/mL. Sterile 96-well polystyrene microtitrate plates (Nunc, Denmark)
were prepared by dispensing 200 µL of appropriate dilution of the tested derivatives in broth medium
per well. The inocula were prepared with fresh microbial cultures in sterile 0.85% NaCl to match the
turbidity of 0.5 McFarland standard, and 2 µL was added to wells to obtain a final density of 1.5 × 106
colony forming units (CFU)/mL for bacteria and 5 × 104 CFU/mL for yeasts. After incubation
(bacterial strains at 35oC for 24 h and yeast strains at 30oC for 48 h), the MICs were visually assessed
as the lowest concentration showing complete growth inhibition of the reference microbial strains.
Appropriate DMSO control (at a final concentration of 10%), a positive control (containing inoculum
without the tested derivatives), and a negative control (containing the tested derivatives without
inoculum) were included on each microplate. Ciprofloxacin, vancomycin, and fluconazole were used
as the standard drugs. Each experiment was performed in triplicate.
4.2.3. Cytotoxicity assay
Compounds were dissolved in DMSO at 20 mg/mL, and further dilutions were made in deionized
double-distilled water. Samples were tested at 1, 5, 20, and 100 µg/mL. Magnesium ascorbyl
phosphate (MAP, 100 µg/mL) and basic fibroblast growth factor (bFGF, 15 ng/mL) were used as
positive controls. Human dermal fibroblasts (ATCC PCS-201-010) and B16-F10 mouse melanoma
cells (ATCC CRL-6475, Manassas, VA) were plated at 6000 cells/well in a 96-well plate with phenol-
free DMEM supplemented with 10% fetal bovine serum (FBS) and CnT-57 medium (Zen-Bio), and
the samples were added immediately thereafter in triplicates. The culture was incubated for 72 h.
Deionized double-distilled water was the negative control. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, Sigma, St. Louis, MO) was added to cell cultures at the end of the 72-h
incubation period, and incubation was pursued for additional 2.5 h. The culture media were then
discarded, and the intracellular MTT reduction product formazan was solubilized in isopropanol.
Funding: This work was supported by the National Science Centre (Cracow, Poland) on the basis of
decision number DEC-2017/25/B/NZ7/00124.
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Highlights
•
•
•
A series of 4-substituted picolinohydrazonamide derivatives were synthesized.
Compounds were tested for their tuberculostatic and antimicrobial activity.
Cytotoxic activity against human dermal fibroblasts and mouse melanoma cells was
determined.
•
Compound 15 is selected as good leading structure for further studies.