Synthesis and Antibacterial Evaluation of Mannich Bases Derived from 1,2,4-Triazole

Article abstract of DOI:10.1002/cbdv.201900377

The series of novel Mannich bases were synthesized and evaluated for their in vitro antibacterial activity against Gram-positive and Gram-negative bacterial strains. The results showed that all compounds were less active than the drugs used as reference, but some of them had moderate potency against Staphylococcus epidermidis ATCC 12228 and Bacillus subtilis ATCC 6633. The presence of a phenyl ring in the position 4 of piperazine seems to be necessary for antibacterial activity in this class of compounds.

Full text of DOI:10.1002/cbdv.201900377

Title: Synthesis and antibacterial evaluation of Mannich bases derived
from 1,2,4-triazole
Authors: Monika Wujec, Agata Paneth, Nazar Trotsko, Łukasz
Popiołek, Agnieszka Grzegorczyk, Tomasz Krzanowski, Sara
Janowska, and Anna Malm
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.201900377
Link to VoR: http://dx.doi.org/10.1002/cbdv.201900377

Chemistry & Biodiversity
10.1002/cbdv.201900377
Synthesis and antibacterial evaluation of Mannich bases derived from 1,2,4-triazole.
a
a
a
b
Agata Paneth , Nazar Trotsko , Łukasz Popiołek , Agnieszka Grzegorczyk , Tomasz
a
a
b
a*
Krzanowski , Sara Janowska , Anna Malm , Monika Wujec
aDepartment of Organic Chemistry, Faculty of Pharmacy, Medical University of Lublin, 4A
Chodzki Street, 20-093 Lublin, Poland, e-mail: monika.wujec@umlub.pl
bChair and Department of Pharmaceutical Microbiology with Laboratory for Microbiological
Diagnostics, Faculty of Pharmacy, Medical University of Lublin, 1 Chodzki Street, 20-093
Lublin, Poland
Abstract
The series of novel Mannich bases were synthesized and evaluated for their in vitro
antibacterial activity against Gram-positive and Gram-negative bacterial strains. The results
showed that all compounds were less active than the drugs used as reference, but some of
them had moderate potency against Staphylococcus epidermidis ATCC 12228 and Bacillus
subtilis ATCC 6633. The presence of a phenyl ring in the position 4 of piperazine seems to be
a necessary for antibacterial activity in this class of compounds.
Keywords: 1,2,4-triazole, Mannich base, antibacterial activity
Introduction
The problem of increasing microbial resistance to antibiotics has become a priority for public
health both in Poland and around the world. It is closely related to nosocomial infections and
closed care institutions (nursing homes, orphanages, etc.). According to estimations,
infectious diseases are still one of the leading causes of mortality. Development of civilization,
the progress of medical science has brought antibiotics as well as achievements in diagnostics.
However, immediately after the introduction of penicillin resistant strains appeared, and so
was the case after each new antibiotic resistance was also recorded immediately before the
registration.
Currently, microorganisms repel effectively the most massive attack that threatens their
existence. An extremely dynamic development of microbial resistance to anti-infective agents
has been observed. The situation is dramatic in hospitals. Every day, patients are identified to
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Chemistry & Biodiversity
10.1002/cbdv.201900377
be carriers or diagnosed with infections of resistant bacterial strains (strains which have
several resistance mechanisms).
Today, few companies express interest in researching new anti-infective drugs. This type of
research is expensive and less cost-effective than the drugs for the treatment of chronic
diseases.
In recent years, research into new antibacterial agents (which we really need) has been carried
out in many academic laboratories in the world.
Literature studies have shown that in various fields, 1,2,4-triazoles, have an important place
among nitrogen containing heterocyclic compounds due to their different pharmacological
activities such as anticancer,[1-3] antifungal,[4-7] antibacterial,[8-14] anti-inflammatory,[15]
anticonvulsant,[16-19] antiviral[20] and antioxidant.[21] In addition, studies have shown that the 2-
position of the triazole was the target of many reactions that lead to the formation of
biologically active compounds. One of them is Mannich's reaction. Many studies have shown
that Mannich bases possess potent biological activities: antibacterial, antifungal, anti-
inflammatory and many other effect.
In our previous study, we synthesized some novel Mannich bases bearing chlorophenyl
moiety.[22] The antibacterial activity of the synthesized compounds was similar or higher than
the activity of commonly used antibiotics such as ampicillin and cefuroxime.
Organofluorine compounds are virtually unheard of in natural products, but 20-25% of drugs
contain a fluorine atom. The introduction of a fluorine atom into the molecule greatly changes
both the physical and chemical properties of the organic compounds. The widely used
antibacterial agents are fluoroquinolones, which were obtained by introducing fluoride into
the quinolone skeleton, most commonly in position 6. An equally important chemotherapeutic
agent, having a fluorine atom in its structure is linezolid, used mainly in infections caused by
multi-drug resistant strains of bacteria.
The above premises prompted us to synthesize novel Mannich bases bering fluorophenyl
moiety.
Results and Discussion
Chemistry
4
-Fluorobenzoic acid hydrazide 1 was added to 3-chlorophenylisothiocyanate 2 and 1-(4-
fluorobenzoyl)-4-(3-chlorophenyl)thiosemicarbazide 3 was obtained with a good yield (90%).
Alkaline cyclization of compound 3 using 2% solution of sodium hydroxide afforded 4-(3-
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Chemistry & Biodiversity
10.1002/cbdv.201900377
chlorophenyl)-5-(4-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 4. Finally, Mannich
reaction of s-triazoles, suitable amines and 37% aqueous solution of formaldehyde was
performed in 99,8% ethanol for 2h in room temperature to give compounds 5a-5j. The
compound 5k was formed after heating the solution under reflux for 30 min. The precipitates
were filtered off, dried and crystallized from 96% ethanol.
The desired 2-aminosubstituted 4-(3-chlorofenylo)-5-(4-fluorofenylo)-2,4-dihydro-3H-1,2,4-
triazole-3-thione 5a-5k were prepared according synthetic route depicted in Scheme 1. The
substituent R derived from amines RH are presented in the Table 1.
O
NCS
O
EtOH, reflux 30 min
1. 2% NaOH, reflux 2h
. 3N HCl
NH
HN
NH
+
2
S
NH2
F
Cl
F
NH
2
1
3
Cl
R
H
N
N
N
N
S
37% HCHO, Amine (RH), EtOH, rt
S
N
N
F
F
Cl
Cl
5a
5k
4
Scheme 1. Synthetic route for the new compounds 5a-5k.
Table 1. The substituent R derived from amines RH
Compound
R
Compound
R
Cl
5
a
5g
Cl
N
N
N
N
5
b
5h
N
N
N
N
N
F
N
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Chemistry & Biodiversity
10.1002/cbdv.201900377
5
5
5
c
d
e
5i
N
O
N
N
O
N
N
N
CH3
Cl
F
5j
5k
CF3
NH
Cl
O
N
N
N
OH
5
f
N
N
Antibacterial Activity
The in vitro antimicrobial activity of the compounds 5a-5k was evaluated using the broth
microdilution method against Gram-positive and Gram-negative reference bacteria (Table 2).
The MIC values indicate that the most of the compounds exhibited antibacterial activity
towards Gram-positive bacterial strains in the range 60-250 µg/mL. Among all tested
compounds the best activity with MIC value 30 µg/mL against M. luteus and 60 µg/mL
against B. subtilis, S. aureus and S. epidermidis was observed for derivatives of 4-
chlorophenylpiperazin 5e. Introduction of a second chlorine atom into the phenyl ring 5g
resulted in a decrease in activity against Gram-positive strains but in the case of Gram-
negative bacteria the 2-fold increases. The second active compound was 5k with morpholin
moiety. The MIC values for M. luteus, B. subtilis and S. epidermidis were 60 µg/mL. The
most sensitive strain of Gram-positive bacteria was S. epidermidis ATCC 12228. Six
compounds inhibited the growth of this strain with MIC value 60 µg/mL. The application of
furanopiperazine to Mannich reaction causes a significant decrease in activity compared to
phenylpiperazine derivatives.
From these results it is clear that i) new compounds were more active against Gram-positive
than against Gram-negative bacteria; ii) the compound with monochlorosubstituted phenyl
ring was the most active; iii) the compound with two chlorine atoms was the most active
against Gram-negative bacteria; iv) piperazine derivatives were more active than the aniline
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Chemistry & Biodiversity
10.1002/cbdv.201900377
derivative; v) the presence of a phenyl ring in the position 4 of piperazine seems to be a
necessary condition for antibacterial activity; vi) MBC/MIC ratio indicates that all
compounds showed bacteriostatic activity against Gram-positive bacteria but few of them
showed bactericidal activity towards Gram-negative bacteria strains as E. coli and P.
aeruginosa. This fact suggests that these compounds have more than one mechanism of
antimicrobial action.
Table 2. Values MIC [μg/mL] and MBC [μg/mL] of compounds 5a-5k against Gram-positive
(1-5) and Gram-negative (6-8) references microorganisms
Microorganisms
Compounds
1
2
3
4
5
6
7
8
MIC;
MBC
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
250;
250;
4000
125;
4000
125;
4000
250;
2000
30;
2000
125;
2000
60;
4000
250;
4000
250;
4000
250;
4000
60;
250;
4000
60;
4000
60;
2000
500;
2000
60;
4000
60;
4000
250;
4000
60;
4000
1000;
4000
250;
4000
60;
500;
4000
500;
4000
125;
4000
500;
2000
60;
4000
125;
2000
250;
4000
125;
4000
1000;
4000
100;
4000
250;
4000
20
250;
2000
500;
4000
250;
4000
500;
2000
125;
4000
250;
4000
250;
4000
500;
4000
1000;
4000
250;
4000
250;
4000
4.9
500;
4000
500;
4000
500;
4000
500;
2000
250;
2000
500;
2000
125;
4000
500;
2000
1000;
4000
500;
2000
250;
4000
nd
500;
4000
500;
2000
500;
2000
500;
1000
250;
2000
500;
500
125;
2000
500;
500
1000;
2000
500;
1000
500;
2000
nd
500;
2000
500;
2000
500;
2000
500;
1000
250;
2000
500;
1000
125;
2000
500;
2000
1000;
2000
500;
1000
500;
2000
nd
2
000
200;
000
60;
1
2
000
250;
1
000
60;
2
000
60;
1
000
g
h
i
125;
2
000
60;
00
1000;
000
250;
000
60;
5
2
j
1
k
2
000
2.4
nd
4000
1.2
nd
4000
0.98
nd
VAN
CIP
nd
nd
0.6
0.04
0.48
Legend: 1-Bacillus subtilis ATCC 6633, 2-Micrococcus luteus ATCC 10240, 3-
Staphylococcus epidermidis ATCC 12228, 4-Staphylococcus aureus ATCC 29213, 5-
Enterococccus faecalis ATCC 29212, 6-Salmonella typimurium ATCC 14028, 7-Escherichia
coli ATCC 25922, 8-Pseudomonas aeruginosa ATCC 27853, nd-not determined
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Chemistry & Biodiversity
10.1002/cbdv.201900377
Conclusion
In summary, a series of novel Mannich bases were synthesized and evaluated for their in vitro
antimicrobial activity against Gram-positive and Gram-negative bacteria. The results showed
that all compounds were less active than the drugs used as reference. The best bioactivity was
noted
for
4-(3-chlorophenyl)-5-(4-fluorophenyl)-1-[(4-(4-chlorophenyl)piperazin-1-
yl)methyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione.
Experimental Section
Substances used in this research were purchased from Sigma-Aldrich (Munich, Germany) and
Merck Co.(Darmstadt, Germany) and were used without further purification. Melting points
were determined on Fisher-Johns blocks melting point apparatus (Fisher Scientific, Germany)
1
13
and left uncorrected. The H and C NMR spectra were recorded on the Bruker Avance 300
or 600 apparatus (Bruker BioSpin GmbH, Germany) in (D )DMSO with TMS as the internal
6
standard. Chemical shifts were given in ppm (δ). TMS was used as the standard reference.
The IR spectra were recorded in KBr discs using Nicolet 6700 FTIR spectrometer (Thermo
Scientific). The progress of the reaction and the purity of obtained compounds were
monitored by TLC using aluminum sheet 60 F254 plates (Merck Co., USA), in a
CHCl /EtOH (10:1, v/v) solvent system. The spots were detected by exposure to the UV lamp
3
at 254 nm. The elemental analysis of obtained compounds was carried out with the AMZ 851
CHX analyser (PG, Gdansk, Poland). The results of elemental analysis (C, H, N) were within
0
.4% of the calculated values.
The procedure for the synthesis of 4-(3-chlorophenyl)-5-(4-fluorophenyl)-2,4-dihydro-3H-
,2,4-triazole-3-thione 4
1
A solution of 0.01 mol of 4-fluorobenzoic acid hydrazide 1 and equimolar amount of 3-
chlorophenyl isothiocyanate 2 in 25 mL of anhydrous ethanol was heated under reflux for 15
min. Next, the solution was cooled and the solid formed was filtered off, washed with diethyl
ether, dried, and crystallized from ethanol. The obtained thiosemicarbazide 3 (0.01 mol)
dissolved in 30 mL of 2% aqueous NaOH was refluxed for 2 h. After cooling, the solution
was neutralized with dilute hydrochloric acid. The precipitate was filtered off and crystallized
from ethanol.
General procedures for the synthesis of compounds 5a-5k
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Chemistry & Biodiversity
10.1002/cbdv.201900377
1
mmol of the triazole (4) was dissolved in 10 mL anhydrous ethanol and then appropriate
amine (1 mmol) and formaldehyde solution (aq. 37%, 1 mmol) were added. The mixture was
stirred at room temperature for 2h. The precipitate was filtered off, dried and crystallized from
9
3
6% ethanol. If morpholine was used, it was necessary to heat the substrates under reflux for
0 minutes. Next steps were the same as for other amines.
4
3
-(3-Chlorophenyl)-5-(4-fluorophenyl)-1-[(4-fenylopiperazin-1-yl)methyl]-2,4-dihydro-
1
H-1,2,4-triazole-3-thione (5a). Yield: 68%. M.p.: 156-158˚C. H NMR (300 MHz, (D
6
)
DMSO, ppm): 2.98 (s, 2CH
2
piperazine, 4 H), 3.16 (s, 2CH
2
piperazine, 4 H), 5.25 (s, N-CH
) DMSO, ppm) δ= 50.3, 50.8, 69.5,
16.4, 116.5, 119.8, 122.0, 125.3, 128.9, 129.6, 130.3, 131.4, 131.6, 131.7, 133.8, 136.7,
2
,
13
2
1
1
H), 6.75-7.69 (m, ArH, 13 H); C NMR (600 MHz, (D
6
-1
40.4, 140.5, 148.8, 162.1, 169.9. IR (KBr, cm ): 3065 (CHarom.), 2885, 2815 (CHaliph.), 1323
23ClFN S (480.00): C 62.56, H 4.83, N 14.59; found: C 62.24, H
(C=S). Anal. calc. for C25
H
5
4
.71, N 14.42.
4
-(3-Chlorophenyl)-5-(4-fluorophenyl)-1-[(4-(2-fluorophenyl)piperazin-1-yl)methyl]-2,4-
1
dihydro-3H-1,2,4-triazole-3-thione (5b). Yield: 67%. M.p.: 176-178˚C. H NMR (300 MHz,
(
D
6
) DMSO, ppm): 3.02; 3.03 (d, J = 2.7, 4CH
.71 (m, ArH, 12 H); 13C NMR (600 MHz, (D
16.5, 119.8, 122.3, 125.3, 128.3, 129.6, 130.2, 131.4, 131.6, 131.7, 133.8, 136.7, 140.3,
2
piperazine , 8 H), 5.25 (s, N-CH
2
, 2 H), 7.08-
7
1
1
6
) DMSO, ppm) δ= 50.3, 50.7, 69.5, 116.2,
-1
40.4, 148.8, 162.0, 169.9. IR (KBr, cm ): 3068 (CHarom.), 28840, 2820 (CHaliph.), 1324
S (497.99): C 60.30, H 4.45, N 14.06; found: C 60.34, H
(C=S). Anal. calc. for C25
H22ClF
2
N
5
4
.62, N 14.10.
4
2
-(3-Chlorophenyl)-5-(4-fluorophenyl)-2-{[4-(3-metoksyphenyl)piperazin-1-yl]methyl}-
1
,4-dihydro-3H-1,2,4-triazole-3-thione (5c). Yield: 92%. M.p.: 145-147˚C. H NMR (300
MHz, (D
s, O-CH
NMR (600 MHz, (D
6
) DMSO, ppm): 2.97 (s, 2CH
, 3 H), 5.25 (s, N-CH , 2 H), 6.34-6.54 (m, ArH, 3 H), 7.09-7.69 (m, ArH, 9 H); C
) DMSO, ppm) δ= 48.8, 50.3, 50.8, 69.3, 102.2, 104.7, 108.7, 116.2,
16.5, 122.3, 128.2, 131.6, 131.7, 133.8, 136.6, 140.3, 140.5, 148.7, 152.9, 160.6, 161.9,
2
piperazine, 4 H), 3.15 (s, 2CH
2
piperazine, 4 H), 3.70
1
3
(
3
2
6
1
1
-1
69.9. IR (KBr, cm ): 2950 (CHarom.), 2832 (CHaliph.), 1320 (C=S). Anal. calc. for
C
26
H
25ClFN
5
OS (510.03): C 61.23, H 4.94, N 13.73; found: C 61.15, H 4.78, N 13.64.
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Chemistry & Biodiversity
10.1002/cbdv.201900377
4
-(3-Chlorophenyl)-5-(4-fluorophenyl)-1-[(4-(3-trifluoromethylphenyl)piperazin-1-
1
yl)methyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione (5d). Yield: 50%. M.p.: 155-158˚C. H
NMR (300 MHz, (D
H), 5.26 (s, N-CH
ppm) δ= 49.6, 50.3, 69.5, 116.2, 116.5, 119.8, 122.4, 125.3, 128.3, 129.6, 130.2, 131.4, 131.6,
6
) DMSO, ppm): 2.99 (s, 2CH
2
piperazine, 4 H), 3.27 (s, 2CH
2
piperazine,
1
3
4
2
, 2 H), 7.05-7.69 (m, ArH, 12 H); C NMR (600 MHz, (D
6
) DMSO,
-1
1
31.7, 133.8, 136.7, 140.4, 140.5, 148.8, 162.1, 169.9 IR (KBr, cm ): 2938 (CHarom.), 2828
(
CHaliph.), 1319 (C=S). Anal. calc. for C26 S (547.99): C 56.99, H 4.05, N 12.78;
H
22ClF
4
N
5
found: C 56.89, H 4.01, N 12.76.
4
-(3-Chlorophenyl)-5-(4-fluorophenyl)-1-[(4-(4-chlorophenyl)piperazin-1-yl)methyl]-2,4-
1
dihydro-3H-1,2,4-triazole-3-thione (5e). Yield: 65%. M.p.: 185-188˚C. H NMR (300 MHz,
) DMSO, ppm): 2.97 (s, 2CH piperazine, 4 H), 3.16 (s, 2CH piperazine, 4 H), 5.25 (s, N-
CH , 2 H), 6.93;6.96 (d, J = 9.0, 2ArH, 2 H), 7,21; 7.24 (d, J = 9.0, 2ArH, 2 H), 7.27-7.69 (m,
ArH, 8 H); C NMR (600 MHz, (D
(D
6
2
2
2
1
3
6
) DMSO, ppm) δ= 48.6, 50.2, 69.3, 116.3, 116.4, 117.5,
1
1
22.3, 128.2, 129.1, 129.6, 130.2, 131.4, 131.6, 131.7, 133.8, 136.6, 140.5, 140.6, 148.8,
-1
62.8, 169.9. IR (KBr, cm ): 2936 (CHarom.), 2837 (CHaliph.), 1325 (C=S). Anal. calc. for
FN S (514.44): C 58.37, H 4.31, N 13.61; found: C 58.42, H 4.31, N 13.42.
C
25
H22Cl
2
5
4
2
-(3-Chlorophenyl)-5-(4-fluorophenyl)-2-{4-[(4-hydroksyphenyl)piperazin-1-yl]methyl]-
1
,4-dihydro-3H-1,2,4-triazole-3-thione (5f). Yield: 80%. M.p.: 165-167˚C. H NMR (300
MHz, (D
6
) DMSO, ppm): 2.97 (s, 4CH
2
piperazine, 8 H), 5.235.25 (s, N-CH , 2 H), 6.62, 6.65
2
(d, J= 9.0, ArH, 2 H), 6.76, 6.79 (d, J= 9.0, ArH, 2 H), 7.24-7.70 (m, ArH, 8 H), 8.84 (s, OH,
1
3
1
1
1
H); C NMR (600 MHz, (D
6
) DMSO, ppm) δ= 48.2, 50.3, 69.3, 115.9, 116.3, 116.4, 119.8,
22.3, 128.3, 129.6, 130.3, 131.4, 131.7, 131.8, 133.8, 136.7, 140.3, 140.4, 148.8, 162.0,
-1
69.9. IR (KBr, cm ): 3030 (CHarom.), 2880, 2815 (CHaliph.), 1320 (C=S). Anal. calc. for
23ClFN OS (495.99): C 60.54, H 4.67, N 14.12; found: C 60.44, H 4.58, N 14.22.
C
25
H
5
4
2
-(3-Chlorophenyl)-5-(4-fluorophenyl)-1-[(4-(3,4-dichlorophenyl)piperazin-1-yl)methyl]-
1
,4-dihydro-3H-1,2,4-triazole-3-thione (5g). Yield: 52%. M.p.: 181-183˚C. H NMR (300
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Chemistry & Biodiversity
10.1002/cbdv.201900377
MHz, (D
6
) DMSO, ppm): 2.95 (s, 2CH
, 2 H), 6.92-7.68 (m, ArH, 11 H); C NMR (600 MHz, (D
0.0, 69.3, 115.9, 116.4, 116.8, 122.3, 128.6, 129.1, 130.2, 131.0, 131.6, 131.7, 135.9, 136.6,
2
piperazine, 4 H), 3.22 (s, 2CH
2
piperazine, 4 H), 5.25
1
3
(s, N-CH
2
6
) DMSO, ppm) δ= 48.1,
5
1
-1
48.7, 151.2, 162.8, 164.3, 169.9, IR (KBr, cm ): 3050 (CHarom.), 2822 (CHaliph.), 1325 (C=S).
FN S (548.89): C 54.70, H 3.86, N 12.76; found: C 54.60, H 3.76, N
Anal. calc. for C25
H21Cl
3
5
1
2.68.
4
-(3-Chlorophenyl)-5-(4-fluorophenyl)-2-[4-(pyrimidin-2-yl)piperazin-1-yl)methyl]-2,4-
1
dihydro-3H-1,2,4-triazole-3-thione (5h). Yield: 70%. M.p.: 151-153˚C. H NMR (300 MHz,
(D
6
) DMSO, ppm): 2.89 (s, 2CH
2
piperazine, 4 H), 3.78 (s, 2CH piperazine, 4 H), 5.25 (s, N-
2
CH , 2 H), 6.58, 6.60, 6.61 (t, J = 4.8, ArH pyrimidine, 1 H), 7.22-7.68 (m, ArH, 11 H), 8.33,
2
1
3
8
1
1
.35 (d, J = 4.5, CH, 2 H); C NMR (600 MHz, (D
6
) DMSO, ppm) δ= 43.6, 50.2, 69.6, 110.5,
16.2, 116.4, 119.6, 122.3, 128.2, 129.2, 130.2, 131.4, 131.6, 131.7, 133.7, 136.6, 148.7,
-1
58.3, 161.5, 161.6, 169.9. IR (KBr, cm ): 3064 (CHarom.), 2925, 2825 (CHaliph.), 1327 (C=S).
21ClFN S (481.97): C 57.32, H 4.39, N 20.34; found: C 57.30, H 4.36, N
Anal. calc. for C23
H
7
2
0.41.
4
-(3-Chlorophenyl)-5-(4-fluorophenyl)-2-{4-[(furan-2-yl)piperazin-1-yl]metyl}-2,4-
1
dihydro-3H-1,2,4-triazole-3-thione (5i). Yield: 53%. M.p.: 145-147˚C. H NMR (300 MHz,
(
D
6
) DMSO, ppm): 2.89 (s, 2CH
CH , 2 H), 6.60-6.62 (m, ArH, 1 H), 6.97-6.99 (m, ArH, 1 H), 7.22-7.83 (m, ArH, 9 H); C
NMR (600 MHz, (D ) DMSO, ppm) δ= 43.5, 50.3, 69.6, 113.5, 116.3, 116.4, 120.1, 122.4,
2
piperazine, 4 H), 3.71 (s, 2CH
2
piperazine, 4 H), 5.24 (s, N-
1
3
2
6
-1
1
3
28.2, 130.2, 131.4, 131.6, 131.7, 136.6, 148.6, 158.3, 161.4, 161.6, 169.9. IR (KBr, cm ):
060 (CHarom.), 2880, 2825 (CHaliph.), 1320 (C=S). Anal. calc. for C23 OS (469.96):
H21ClFN
5
C 58.78, H 4.50, N 14.90; found: C 58.76, H 4.60, N 14.86.
4
-(3-Chlorophenyl)-2-[N-(4-chloro-3-fluorophenyl)aminomethyl]-5-(3-fluorophenyl)-2,4-
1
dihydro-3H-1,2,4-triazole-3-thione (5j). Yield: 85%. M.p.: 131-133˚C. H NMR (300 MHz,
(
D
6
) DMSO, ppm): 5.59, 5.61 (d, J=7.2, N-CH
2
, 2 H), 6.94-6.99 (m, NH, 1 H), 7.22-7.65 (m,
) DMSO, ppm) δ= 43.6, 50.3, 69.8, 116.4, 116.5, 120.1,
22.3, 128.4, 129.2, 130.2, 131.5, 131.7, 131.8, 136.6, 148.4, 159.6, 161.5, 161.6, 169.9. IR
1
3
ArH, 11 H); C NMR (600 MHz, (D
6
1
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Chemistry & Biodiversity
10.1002/cbdv.201900377
-1
(
KBr, cm ): 2950 (CHarom.), 2832 (CHaliph.), 1329 (C=S). Anal. calc. for C21
463.33): C 54.44, H 3.05, N 12.09; found: C 54.29, H 3.11, N 12.07.
H14Cl
2
F
2
4
N S
(
4
1
-(3-Chlorophenyl)-5-(4-fluorophenyl)-1-[(morpholin-4-yl)methyl)]-2,4-dihydro-3H-
1
,2,4-triazole-3-thione (5k). Yield: 41%. M.p.: 197-201˚C. H NMR (300 MHz, (D
morpholine, 4 H), 3.60 (s, 2CH morpholine, 4 H), 5.16 (s, N-CH
) DMSO, ppm) δ= 46.5, 50.3, 52.1, 62.2,
6
) DMSO,
ppm): 2.81 (s, 2CH
2
2
2
, 2 H),
1
3
7
6
2
5
.25-7.69 (m, ArH, 8 H); C NMR (600 MHz, (D
9.6, 116.3, 116.5, 122.4, 131.4, 131.6, 131.7, 158.3, 161.4, 161.6, 169.9. IR (KBr, cm ):
6
-1
968 (CHarom.), 2859 (CHaliph.), 1359 (C=S). Anal. calc. for C19
6.36, H 4.48, N 13.84; found: C 56.34, H 4.41, N 13.72.
H
18ClFN OS (404.88): C
4
Antibacterial Assay
The antimicrobial activity of the compounds was tested against microorganisms belonging to
the American Type Culture Collection (ATCC): Gram-positive bacteria (Bacillus subtilis
ATCC 6633, Micrococcus luteus ATCC 10240, Staphylococcus epidermidis ATCC 12228,
Staphylococcus aureus ATCC 29213, Enterococccus faecalis ATCC 29212) and Gram-
negative bacteria (Salmonella typhimurium ATCC 14028, Escherichia coli ATCC 25922,
Pseudomonas aeruginosa ATCC 27853). Bacterial strains from the collection of the Chair
and Department of Pharmaceutical Microbiology with Laboratory for Microbiological
Diagnostics of Medical University in Lublin were used in our experiments. Microbial
suspensions were prepared in sterile saline (0.85% NaCl) with an optical density of 0.5
8
McFarland standard – 1.5x10 CFU/ml (CFU – colony forming units). All the stock solutions
of the tested compounds were dissolved in DMSO. Mueller-Hinton medium was used with a
series of 2-fold dilutions of the tested substances in the range of final concentrations from
3
.91 to 1000 mg/mL. Two antibiotics Ciprofloxacin and Vancomycin were used as control
antimicrobial agents. The in vitro antibacterial activity of all tested compounds were screened
on the basis of minimal inhibitory concentration (MIC). It was determined by a broth
microdilution method, based on EUCAST (European Committee on Antimicrobial
Susceptibility Testing) guidelines.[23] MBC (minimal bactericidal concentration) was
estimated by a broth microdilution technique by plating out the contents of wells, that showed
no visible growth of bacteria, onto Mueller-Hinton Agar and incubating at 35ºC for 18 h. Both
MIC and MBC values were given in µg/mL, according to EUCAST references.[23]
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Chemistry & Biodiversity
10.1002/cbdv.201900377
Acknowledgments
We acknowledge two students: Krystian Pęzioł nad Maciej Bielecki for help in carrying out
this work.
Author Contribution Statement
M. Wujec designed the study, analyzed the data and drafted the manuscript.
A. Grzegorczyk, T. Krzanowski, Ł. Popiołek, S. Janowska conducted experiments and
collected the data.
1
3
N. Trotsko analyzed the IR and C NMR spectra
A. Paneth and A. Malm revised the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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