Synthesis, antiproliferative and antimicrobial activity of new Mannich bases bearing 1,2,4-triazole moiety

Article abstract of DOI:10.3109/14756366.2013.855926

This study presents the synthesis, antiproliferative and antimicrobial evaluation of a new series of Mannich base derivatives containing 1,2,4-triazole system. New compounds were prepared by the reaction of 4,5-disubstituted 1,2,4-triazole-3-thiones with

Full text of DOI:10.3109/14756366.2013.855926

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–10
2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.855926
!
RESEARCH ARTICLE
Synthesis, antiproliferative and antimicrobial activity of new Mannich
bases bearing 1,2,4-triazole moiety
1
2
3
4
Łukasz Popiołek , Jolanta Rzymowska , Urszula Kosikowska , Anna Hordyjewska , Monika Wujec , and Anna Malm
1
3
1
2
Department of Organic Chemistry, Faculty of Pharmacy, Department of Biology and Genetics, Faculty of Pharmacy, Department of
3
4
Pharmaceutical Microbiology, Faculty of Pharmacy, and Department of Medical Chemistry, Faculty of Medicine, Medical University of Lublin,
Lublin, Poland
Abstract
Keywords
,2,4-Triazole derivatives, antimicrobial
activity, antiproliferative activity, Mannich
base derivatives
This study presents the synthesis, antiproliferative and antimicrobial evaluation of a new series
of Mannich base derivatives containing 1,2,4-triazole system. New compounds were prepared
by the reaction of 4,5-disubstituted 1,2,4-triazole-3-thiones with formaldehyde and various
1
1
13
C
amines. The structures of the prepared compounds were confirmed by means of H NMR,
NMR and elemental analyses. Twelve compounds were evaluated for their in vitro
antiproliferative activities against six chosen cancer cell lines. All synthesized compounds
were screened for their in vitro antimicrobial activity by using the agar dilution technique. For
History
Received 11 July 2013
Revised 15 September 2013
Accepted 30 September 2013
Published online 5 February 2014
17 potentially active compounds, their antibacterial activity was confirmed on the basis of MIC
minimal inhibitory concentration) by broth microdilution method using the reference Gram-
(
positive and Gram-negative bacterial strains.
Introduction
Literature survey also reveals that piperazine or morpholine
2
2,23
ring is significant for antimicrobial activity
1
antimicrobial agents . Some in vitro cytotoxic activitiy of
. Mannich bases of
,2,4-triazole containing N-methylpiperazine moiety are known as
The search for novel antibacterial and antitumor agents devoid of
side effects continues to be an active area of research in medicinal
chemistry. Among all heterocyclic systems the therapeutic
importance of 1,2,4-triazoles and their derivatives is well
documented and they have been reported to possess various
24
Mannich bases on Maurine P388 lyphocytic leukemia have also
25,26
been reported
.
1
2,3
Prompted by these observations and in an attempt to
continue our studies on the synthesis of biologically active
nitrogen and sulfur containing heterocycles, we herein report
the synthesis, spectral data and evaluation of biological
activities of new Mannich bases containing 1,2,4-triazole
moiety.
biological activities such as analgesic , antifungal , antibacter-
7,8
4
,5
6
9
ial , antiviral , antiphlogistic , and antitubercular . Therefore,
,5-substituted 1,2,4-triazoles seems to be suitable candidates for
4
further chemical modifications and might be of interest as
pharmacologically active compounds.
Our interest was directed to Mannich bases which in recent
years have gained importance due to their application in
pharmaceutical chemistry and comprehensive bioactivities like
Experimental
Chemistry
General
1
0–13
14,15
16
17
antibacterial
, antifungal
anticonvulsant properties. They are also used in polymer
, anticancer , analgesic and
1
8
1
industry as paints and surface active reagents .
9
Mannich reaction is a three-component condensation reaction
involving an active hydrogen containing compound, formaldehyde
and a secondary amine. The scope of Mannich reaction has been
expanded in recent years to include a wide variety of amines,
All reagents were purchased from Sigma-Aldrich (Munich,
Germany) and Merck Co. (Darmstadt, Germany) and used
without further purification. Melting points were determined in
Fisher-Johns blocks (Fisher Scientific, Schwerte, Germany) and
presented without any corrections. The H NMR spectra were
recorded on a Bruker Avance 300 apparatus (Bruker BioSpin
2
0
ammonia equivalents, imines and acetylated imines . The
aminomethylation of aromatic substrates by Mannich reaction is
of considerable importance for the synthesis and modification of
1
2
1
GmbH, Rheinstetten/Karlsruhe, Germany) in DMSO-d with
6
biological active compounds .
1
3
TMS as internal standard. The C NMR spectra were recorded
on a Bruker Avance 300 apparatus. Chemical shifts are given in
ppm (ꢀ-scale). Multiplicities of NMR signals are represented as
singlet (s), doublet (d), doublet of doublet (dd), triplet (t), quartet
Address for correspondence: Łukasz Popiołek, Department of Organic
Chemistry, Faculty of Pharmacy, Medical University of Lublin, 4A
Chodzki Str., 20-093 Lublin, Poland. E-mail: lukasz.popiolek@umlub.pl (q) and multiplet (m). The purity of obtained compounds was

2
Ł. Popiołek et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
checked by TLC on aluminium oxide 60 F254 plates (Merck Co. (272.32); Calculated: C, 57.34; H, 4.44; N, 20.57; S, 11.77;
Whitehouse Station, NJ), in a CHCl /C H OH (10:1, v/v) solvent Found: C, 57.41; H, 4.40; N, 20.61; S, 11.81%.
3
2 5
system. The spots were detected by exposure to a UV lamp at
54 nm. Elemental analyses of the obtained compounds were Preparation of 1,2,4-triazole derivatives
2
performed for C, H, N, S on AMZ 851 CHX analyser (PG,
Gda n´ sk, Poland). The maximum percentage differences between
calculated and found values for each element were within the
error and amounted to ꢀ0.4%.
A mixture of appropriate thiosemicarbazide 1a–1d (10 mmol) and
0–40 ml of 2% aqueous solution of sodium hydroxide was
refluxed for 2 h. Then, the solution was neutralized with diluted
hydrochloric acid and the formed precipitate was filtered off and
crystallized from ethanol.
2
Preparation of thiosemicarbazide derivatives
4-(4-Chlorophenyl)-5-cyclopropyl-2,4-dihydro-3H-1,2,4-tri-
azole-3-thione (2a)
A mixture of appropriate hydrazide: cyclopropopanecarbohydra-
zide, cyclopentanecarbohydrazide, cyclohexanecarbohydrazide or
nicotinic hydrazide (10 mmol) and 10 mmol appropriate isothio-
CAS Registry Number: 1038358-95-0. Yield: 37%; m. p.
1
ꢁ
ꢁ
1
(
72 C–174 C (dec.). H NMR (DMSO-d ): ꢀ (ppm) ¼ 0.78–0.92
ꢁ
6
cyanate was heated in an oil bath at 50–90 C for 8–12 h. The
m, 4H, CH -cyclopropyl), 1.46–1.55 (m, 1H, CH-cyclopropyl),
2
product was washed with diethyl ether to remove unreacted
isothiocyanate. Then it was filtered off, dried and crystallized
from ethanol.
7
.52–7.54 (dd, 2H, Ar-H, J ¼ 6 Hz), 7.65–7.67 (dd, 2H, Ar-H,
1
3
J ¼ 6 Hz), 13.65 (s, 1H, NH). C NMR: ꢀ (ppm) ¼ 6.67 (CH-
cyclopropyl), 7.31 (2 ꢂ CH -cyclopropyl), 129.93, 130.84,
2
1
Analysis for C H ClN S (251.73); Calculated: C, 52.48; H,
33.20, 134.52 (6C ), 154.04 (C-5 triazole), 168.01 (C¼S).
11 10 3
N-(4-chlorophenyl)-2-(cyclopropylcarbonyl)hydrazinecar-
bothioamide (1a)
CAS Registry Number: 685113-56-8. Yield: 80%; m. p.
ar
4.00; N, 16.69; S, 12.74; Found: C, 52.47; H, 4.05; N, 16.57; S,
12.72%.
ꢁ
ꢁ
ꢁ
1
1
70 C–172 C (dec.). Temperature of reaction: 70 C for 12 h. H
NMR (DMSO-d ): ꢀ (ppm) ¼ 0.63–0.66 (m, 2H, CH -cyclopro-
6
2
5-Cyclopentyl-4-(2,4-dichlorophenyl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (2b)
pyl), 0.75–0.81 (m, 2H, CH -cyclopropyl), 1.42–1.50 (m, 1H,
2
CH-cyclopropyl), 7.32–7.58 (m, 4H, Ar-H), 9.17 (s, 1H, NH),
1
ꢁ
ꢁ
1
Yield: 89%; m. p. 203 C–205 C (dec.). H NMR (DMSO-d ):
3
6
9
(
1
(
4
1
.77 (s, 1H, NH), 10.11 (s, 1H, NH). C NMR: ꢀ (ppm) ¼ 10.76
ꢀ
1
1
(ppm) ¼ 1.44–1.78 (m, 8H, CH -cyclopentyl), 2.63–2.73 (m,
2
2 ꢂ 2 ꢂ CH -cyclopropyl), 16.93 (CH-cyclopropyl), 121.95,
2
H, CH-cyclopentyl), 7.67–7.73 (m, 2H, Ar-H), 7.96–7.99 (m,
28.20, 129.37 (5C ), 139.93 (C ), 176.35 (C¼O), 179.38
13
ar
ar
H, Ar-H), 13.83 (s, 1H, NH). C NMR: ꢀ (ppm) ¼ 26.12, 34.79
C¼S). Analysis for C H ClN OS (269.75); Calculated: C,
1
1
12
3
(
1
1
4 ꢂ CH -cyclopentyl), 38.72 (CH-cyclopentyl), 129.46, 130.82,
2
9.98; H, 4.48; N, 15.58; S, 11.89; Found: C, 49.96; H, 4.45; N,
5.62; S, 11.86%.
31.69, 135.53, 136.44, 137.07 (6C ), 156.90 (C-5 triazole),
ar
67.17 (C¼S). Analysis for C H Cl N S (314.23); Calculated:
1
3
13
2 3
C, 49.69; H, 4.17; N, 13.37; S, 10.20; Found: C, 49.72; H, 4.21;
N, 13.42; S, 10.08%.
2-(Cyclopentylcarbonyl)-N-(2,4-dichlorophenyl)hydrazinecar-
bothioamide (1b)
CAS Registry Number: 891381-19-4. Yield: 91%; m. p.
4-(4-Chlorophenyl)-5-cyclohexyl-2,4-dihydro-3H-1,2,4-tri-
azole-3-thione (2c)
ꢁ
ꢁ
ꢁ
1
62 C–164 C (dec.). Temperature of reaction: 90 C for 12 h. H
1
ꢁ
ꢁ
1
Yield: 93%; m. p. 212 C–214 C (dec.). H NMR (DMSO-d ):
NMR (DMSO-d ): ꢀ (ppm) ¼ 1.50–1.83 (m, 8H, CH -cyclopen-
6
2
6
tyl), 2.63–2.73 (m, 1H, CH-cyclopentyl), 7.39–7.72 (m, 3H, Ar-
H), 9.37 (s, 1H, NH), 9.79 (s, 1H, NH), 9.94 (s, 1H, NH).
ꢀ
7
(ppm) ¼ 1.04–1.80 (m, 10H, cykloheksyl), 2.84 (m, 1H, CH),
.52–7.55 (dd, 2H, Ar-H, J ¼ 9 Hz), 7.69–7.72 (dd, 2H, Ar-H,
1
3
C
1
3
NMR: ꢀ (ppm) ¼ 26.12, 32.26 (4 ꢂ CH -cyclopentyl), 46.12 (CH-
2
J ¼ 9 Hz), 11.04 (s, 1H, NH). C NMR: ꢀ (ppm) ¼ 25.15, 29.92,
cyclopentyl), 125.30, 128.73, 129.00, 129.96, 130.95, 134.63
2
130.25, 133.45, 135.65 (6C ), 157.97 (C-5 triazole), 167.24
9.47 (5 ꢂ CH -cyclohexyl), 34.20 (CH-cyclohexyl), 129.74,
2
(
6C ), 176.59 (C¼O), 180.86 (C¼S). Analysis for
ar
ar
C H Cl N OS (332.25); Calculated: C, 46.99; H, 4.55; N,
13 15 2 3
(C¼S). Analysis for C H ClN S (293.81); Calculated: C, 57.23;
1
4
16
3
1
2.65; S, 9.65; Found: C, 46.92; H, 4.57; N, 12.61; S, 9.62%.
H, 5.49; N, 14.30; S, 10.91; Found: C, 57.29; H, 5.53; N, 14.27; S,
0.87%.
1
N-(4-chlorophenyl)-2-(cyclohexylcarbonyl)hydrazinecar-
bothioamide (1c)
CAS Registry Number: 891077-72-8. Yield: 90%; m. p.
4
-Phenyl-5-(pyridin-3-yl)-2,4-dihydro-3H-1,2,4-triazole-3-
thione (2d)
CAS Registry Number: 57600-03-0. Yield: 89%; m. p.
ꢁ
ꢁ
ꢁ
1
1
72 C–174 C (dec.). Temperature of reaction: 90 C for 12 h. H
ꢁ
1
NMR (DMSO-d ): ꢀ (ppm) ¼ 1.04–2.28 (m, 10H, cyklohexyl),
6
222–223 C (dec.). H NMR (DMSO-d ): ꢀ (ppm) ¼ 7.16–7.18
6
2
3
.63 (m, 1H, CH), 7.35–7.57 (m, 4H, Ar-H), 9.07, 9.63, 9.84 (3s,
(
m, 1H, Ar-H), 7.34–7.38 (m, 2H, Ar-H), 7.46–7.50 (m, 2H, Ar-
H), 7.57–7.59 (m, 1H, Ar-H), 8.28 (m, 1H, Ar-H), 8.69–8.71
m, 1H, Ar-H), 8.72–8.74 (m, 1H, Ar-H), 10.98 (s, 1H, NH).
1
3
H, 3NH). C NMR: ꢀ (ppm) ¼ 25.15, 25.92, 27.93 (5 ꢂ CH -
2
cyclohexyl), 42.43 (CH-cyclohexyl), 121.95, 128.20, 129.37,
39.93 (6C ), 176.11 (C¼O), 179.38 (C¼S). Analysis for
(
1
3
1
C H ClN OS (311.83); Calculated: C, 53.92; H, 5.82; N,
ar
C NMR: ꢀ (ppm) ¼ 126.04, 128.71, 129.36, 131.48, 134.49,
1
4
18
3
136.86, 146.07, 146.57 (11C ), 157.02 (C-5 triazole), 168.22
ar
1
3.48; S, 10.28; Found: C, 53.89; H, 5.88; N, 13.45; S, 10.25%.
(
C¼S). Analysis for C H N S (254.31); Calculated: C, 61.40;
1
3 10 4
H, 3.96; N, 22.03; S, 12.61; Found: C, 61.51; H, 3.98; N, 21.98; S,
2.67%.
N-phenyl-2-(pyridin-3-ylcarbonyl)hydrazinecarbothioamide
(1d)
1
ꢁ
CAS Registry Number: 54584-49-5. Yield: 92%; m. p. 120 C–
ꢁ
ꢁ
1
22 C (dec.). Temperature of reaction: 50 C for 8 h. H NMR
Preparation of Mannich base derivatives
1
(
DMSO-d ): ꢀ (ppm) ¼ 7.13–7.15 (m, 1H, Ar-H), 7.28–7.32 (m, To a solution of corresponding compounds 2a–2d (10 mmol) in
6
2
8
1
H, Ar-H), 7.34–7.38 (m, 2H, Ar-H), 7.57–7.59 (m, 1H, Ar-H); 10 ml of 96% ethanol, appropriate amine (10 mmol) and formal-
.21–8.23 (m, 1H, Ar-H), 8.69–8.71 (m, 1H, Ar-H), 8.90–8.92 (m, dehyde (37%, 0.2 ml) were added. The mixture was stirred under
H, Ar-H), 9.34, 9.71, 10.09 (3s, 3H, 3NH). C NMR: ꢀ reflux at room temperature for 1h. Next, distilled water was added
1
3
(
ppm) ¼ 121.54, 122.68, 124.47, 132.64, 137.58, 149.45, 150.91 and the precipitate formed was filtered off and crystallized from
(11C ), 168.05 (C¼O), 179.39 (C¼S). Analysis for C H N OS ethanol.
ar
13 12 4

DOI: 10.3109/14756366.2013.855926
Mannich bases bearing 1,2,4-triazole moiety
3
1
3
4
2
-(4-Chlorophenyl)-5-cyclopropyl-2-(pyrrolidin-1-ylmethyl)-
,4-dihydro-3H-1,2,4-triazole-3-thione (3a)
2H, Ar-H, J ¼ 9 Hz), 7.67–7.70 (dd, 2H, Ar-H, J ¼ 9 Hz).
C
NMR: ꢀ (ppm) ¼ 10.50 (CH-cyclopropyl), 10.71 (2 ꢂ CH -
2 2
2
ꢁ
ꢁ
1
Yield: 65%; m. p. 108 C–110 C (dec.). H NMR (DMSO-d ): cyclopropyl), 50.36, 51.43 (4 ꢂ CH -piperazine), 63.51 (–CH –),
6
ꢀ
(
(ppm) ¼ 0.88–0.95 (m, 4H, 2 ꢂ CH -cyclopropyl), 1.53–1.62 116.77, 120.31 (2C ), 129.35, 129.72, 130.29, 133.71, 135.24
2 ar ar
2
ar
m, 1H, CH-cyclopropyl), 1.66–1.70 (m, 4H, 2 ꢂ CH -pyrroli- (8C ), 150.96 (C ), 154.66 (C-5 triazole), 170.69 (C¼S).
2 2 22 24 5
dine), 2.77–2.81 (m, 4H, 2 ꢂ CH -pyrrolidine), 5.10 (s, 2H, CH ), Analysis for C H ClN S (425.98); Calculated: C, 62.03; H,
7
.55–7.57 (dd, 2H, Ar-H, J ¼ 6 Hz), 7.68–7.70 (dd, 2H, Ar-H, 5.68; N, 16.44; S, 7.53; Found: C, 62.08; H, 5.61; N, 16.41; S,
1
3
J ¼ 6 Hz). C NMR: ꢀ (ppm) ¼ 6.50 (CH-cyclopropyl), 7.53 7.56%.
(
(
2 ꢂ CH -cyclopropyl), 29.31, 50.19 (4 ꢂ CH -pyrrolidine), 65.21
2 ar
2
2
5-Cyclopentyl-4-(2,4-dichlorophenyl)-2-(pyrrolidin-1-
ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (4a)
–CH –), 129.37, 130.87, 133.64, 134.64 (6C ), 152.70 (C-5
triazole), 168.84 (C¼S). Analysis for C H ClN S (334.87);
1
6
19
4
ꢁ
CAS Registry Number: 929980-59-6. Yield: 68%; m. p. 72 C–
1
Calculated: C, 64.18; H, 6.40; N, 18.71; S, 10.71; Found: C,
4.15; H, 6.45; N, 18.67; S, 10.65%.
ꢁ
7
4 C (dec.). H NMR (DMSO-d ): ꢀ (ppm) ¼ 1.49–1.59 (m, 2H,
6
6
CH -cyclopentyl), 1.69–1.71 (m, 4H, 2 ꢂ CH -pyrrolidine), 1.72–
2
2
4
2
-(4-Chlorophenyl)-5-cyclopropyl-2-(piperidin-1-ylmethyl)-
,4-dihydro-3H-1,2,4-triazole-3-thione (3b)
1.79 (m, 6H, 3 ꢂ CH -cyclopentyl), 2.68–2.75 (m, 1H, CH-
2
cyclopentyl), 2.76–2.81 (m, 4H, 2 ꢂ CH -pyrrolidine), 5.14 (s,
2
ꢁ
ꢁ
1
Yield: 93%; m. p. 132 C–134 C (dec.). H NMR (DMSO-d ): 2H, CH ), 7.40–7.76 (m, 2H, Ar-H), 8.00–8.02 (m, 1H, Ar-H).
6
2
1
3
ꢀ
(
(ppm) ¼ 0.85–0.95 (m, 4H, 2 ꢂ CH -cyclopropyl), 1.35–1.38
C NMR: ꢀ (ppm) ¼ 24.01, 25.31, 25.38, 30.47 (4 ꢂ CH -
2
2
m, 2H, CH -piperidine), 1.49–1.54 (m, 4H, 2 ꢂ CH -piperidine), cyclopentyl), 30.94 (CH -pyrrolidine), 35.70 (CH-cyclopentyl),
2
2
2
1
piperidine), 4.98 (s, 2H, CH ), 7.56–7.58 (dd, 2H, Ar-H, 65.18 (–CH –), 129.41, 130.60, 131.43, 133.19, 134.02, 136.27
.55–1.62 (m, 1H, CH-cyclopropyl), 2.66–2.70 (m, 4H, 2 ꢂ CH - 39.99, 40.27 (2 ꢂ CH -pyrrolidine), 50.09 (CH -pyrrolidine),
2
2
2
2
2
1
3
J ¼ 6 Hz), 7.67–7.69 (dd, 2H, Ar-H, J ¼ 6 Hz). C NMR: ꢀ (6C ), 154.40 (C-5 triazole), 167.12 (C¼S). Analysis for
ar
(
ppm) ¼ 10.50 (CH-cyclopropyl), 10.71 (2 ꢂ CH -cyclopropyl), C H Cl N S (397.36); Calculated: C, 54.41; H, 5.58; N,
2
18 22
2 4
2
1
3.42, 24.57, 53.90 (5 ꢂ CH -piperidine), 63.51 (–CH –), 129.72, 14.10; S, 8.07; Found: C, 54.45; H, 5.59; N, 14.13; S, 8.04%.
2
2
30.29, 133.71, 135.24 (6C ), 154.66 (C-5 triazole), 170.69
ar
5
-Cyclopentyl-4-(2,4-dichlorophenyl)-2-(piperidin-1-
ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (4b)
(
C¼S). Analysis for C H ClN S (348.89); Calculated: C, 58.52;
1
7
21
4
H, 6.07; N, 16.06; S, 9.19; Found: C, 58.53; H, 6.09; N, 16.11; S,
.22%.
ꢁ
ꢁ
1
Yield: 92%; m. p. 88 C–90 C (dec.). H NMR (DMSO-d ): ꢀ
6
9
(
ppm) ¼ 1.32–1.38 (m, 2H, CH -cyclopentyl), 1.47–1.54 (m, 6H,
2
4
2
-(4-Chlorophenyl)-5-cyclopropyl-2-(piperazin-1-ylmethyl)-
,4-dihydro-3H-1,2,4-triazole-3-thione (3c)
3 ꢂ CH -piperidine), 1.63–1.79 (m, 6H, 3 ꢂ CH -cyclopentyl),
2
2
2
2.63–2.70 (m, 4H, 2 ꢂ CH -piperidine), 2.72–2.79 (m, 1H, CH-
ꢁ
ꢁ
1
Yield: 15%; m. p. 222 C–224 C (dec.). H NMR (DMSO-d ): cyclopentyl), 5.05 (s, 2H, CH ), 7.67–7.76 (m, 2H, Ar-H), 7.99–
6
2
1
3
ꢀ
(ppm) ¼ 0.83–0.95 (m, 2H, CH -cyclopropyl), 1.03–1.13 (m, 8.01 (m, 1H, Ar-H). C NMR: ꢀ (ppm) ¼ 24.00, 25.29, 25.36,
2
2
2
2
H, CH -cyclopropyl), 1.51–1.60 (m, 1H, CH-cyclopropyl), 2.68– 25.99 (4 ꢂ CH -cyclopentyl), 30.94 (CH -piperidine), 35.68 (CH-
2
2
2
.82 (m, 8H, CH -piperazine), 5.12 (s, 2H, CH ), 7.54–7.58 (dd, cyclopentyl), 40.25, 51.61 (4 ꢂ CH -piperidine), 69.99 (–CH –),
2
2
2
2
H, Ar-H, J ¼ 12 Hz), 7.64–7.68 (dd, 2H, Ar-H, J ¼ 12 Hz), 8.78 129.40, 130.60, 131.43, 133.19, 134.04, 136.25 (6C ), 154.21 (C-
ar
1
3
(
(
(
s, 1H, NH). C NMR: ꢀ (ppm) ¼ 10.50 (CH-cyclopropyl), 10.71 5 triazole), 168.95 (C¼S). Analysis for C H Cl N S (411.39);
2 2
1
9
24
2 4
2 ꢂ CH -cyclopropyl), 45.59, 53.21 (4 ꢂ CH -piperazine), 63.51 Calculated: C, 55.47; H, 5.88; N, 13.62; S, 7.79; Found: C, 55.49;
–CH –), 129.72, 130.29, 133.71, 135.24 (6C ), 154.66 (C-5 H, 5.91; N, 13.60; S, 7.75%.
ar
2
triazole), 170.69 (C¼S). Analysis for C H ClN S (349.88);
1
6
20
5
5
-Cyclopentyl-4-(2,4-dichlorophenyl)-2-(piperazin-1-
ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (4c)
Calculated: C, 54.92; H, 5.76; N, 20.02; S, 9.16; Found: C, 54.95;
H, 5.79; N, 20.05; S, 9.14%.
ꢁ
ꢁ
1
Yield: 82%; m. p. 244 C–246 C (dec.). H NMR (DMSO-d ):
6
4-(4-Chlorophenyl)-5-cyclopropyl-2-{[4-(2-hydroxyethyl)
piperazin-1-yl]methyl}-2,4-dihydro-3H-1,2,4-triazole-3-thione
ꢀ (ppm) ¼ 1.23–1.29 (m, 2H, CH -cyclopentyl), 1.48–1.56 (m,
2
6H, 3 ꢂ CH -cyclopentyl), 2.05–2.13 (m, 1H, CH-cyclopentyl),
2
(3d)
2.70–2.78 (m, 8H, 4 ꢂ CH -piperazine), 5.09 (s, 2H, CH ), 7.66–
2
2
ꢁ
ꢁ
1
Yield: 73%; m. p. 154 C–156 C (dec.). H NMR (DMSO- 7.75 (m, 2H, Ar-H), 7.78–8.06 (m, 1H, Ar-H), 9.17 (s, 1H, NH).
1
3
d ): ꢀ (ppm) ¼ 0.74–0.97 (m, 4H, 2 ꢂ CH -cyclopropyl), 1.54–
C NMR: ꢀ (ppm) ¼ 26.12, 34.79 (4 ꢂ CH -cyclopentyl), 39.66
6
2
2
1
.63 (m, 1H, CH-cyclopropyl), 2.32–2.40 (m, 4H, 2 ꢂ CH - (CH-cyclopentyl), 45.59, 53.21 (4 ꢂ CH -piperazine), 63.51
2 2 ar
2
2
piperazine), 2.70–2.76 (m, 4H, 2 ꢂ CH -piperazine), 2.89–2.91 (–CH –), 129.56, 131.01, 131.62, 135.43, 135.98, 136.22 (6C ),
(
7
t, 2H, CH ), 3.46–3.48 (t, 2H, CH ), 4.99 (s, 2H, CH ), 7.34– 154.02 (C-5 triazole), 169.21 (C¼S). Analysis for C H Cl N S
2
2
2
18 23
2 5
1
3
.71 (m, 4H, Ar-H), 8.89 (s, 1H, OH).
C NMR: ꢀ (412.38); Calculated: C, 52.43; H, 5.62; N, 16.98; S, 7.78; Found:
(
5
(
ppm) ¼ 6.48 (CH-cyclopropyl), 7.50 (2 ꢂ CH -cyclopropyl), C, 52.46; H, 5.63; N, 16.96; S, 7.74%.
2
2.43, 53.58 (4 ꢂ CH -piperazine), 50.21 (–CH –), 59.25
2 2
2
2
5
piperazin-1-yl]methyl}-2,4-dihydro-3H-1,2,4-triazole-3-thione
-Cyclopentyl-4-(2,4-dichlorophenyl)-2-{[4-(2-hydroxyethyl)-
–CH –), 63.51 (–CH –), 120.32, 125.98, 129.13, 129.94,
1
Analysis for C H ClN OS (393.93); Calculated: C, 54.88; H,
6
8
30.85, 139.06 (6C ), 154.66 (C-5 triazole), 170.69 (C¼S).
18 24 5
ar
(4d)
ꢁ
ꢁ
1
Yield: 61%; m. p. 92 C–94 C (dec.). H NMR (DMSO-d ): ꢀ
6
.14; N, 17.78; S, 8.14; Found: C, 54.92; H, 6.17; N, 17.80; S,
.10%.
(
3
ppm) ¼ 1.47–1.57 (m, 2H, CH -cyclopentyl), 1.60–1.80 (m, 6H,
2
ꢂ CH -cyclopentyl), 2.33–2.37 (t, 2H, CH ), 2.69–2.74 (m, 4H,
2
2
4-(4-Chlorophenyl)-5-cyclopropyl-2-[(4-phenylpiperazin-1-
yl)methyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione (3e)
2 ꢂ CH -piperazine), 2.77–2.81 (m, 1H, CH-cyclopentyl), 3.44–
2
3.50 (m, 4H, 2 ꢂ CH -piperazine), 3.97 (s, 1H, OH), 4.38–4.42 (t,
2
CAS Registry Number: 1286639-67-5. Yield: 65%; m. p. 2H, CH ), 5.06 (s, 2H, CH ), 7.40–7.77 (m, 2H, Ar-H), 8.00–8.01
2
2
ꢁ
ꢁ
1
13
1
08 C–110 C (dec.). H NMR (DMSO-d ): ꢀ (ppm) ¼ 0.85–0.98 (m, 1H, Ar-H). C NMR: ꢀ (ppm) ¼ 26.12, 34.79 (4 ꢂ CH -
2 2
6
2
(
m, 4H, 2 ꢂ CH -cyclopropyl), 1.54–1.63 (m, 1H, CH-cyclopro- cyclopentyl), 39.66 (CH-cyclopentyl), 52.43, 53.58 (4 ꢂ CH -
2 2 2 2
pyl), 2.86–2.89 (t, 4H, 2 ꢂ CH -piperazine), 3.12–3.15 (t, 4H, piperazine), 58.02 (–CH –), 59.25 (–CH –), 63.51 (–CH –),
2
6
ꢂ CH -piperazine), 5.09 (s, 2H, CH ), 6.76–6.81 (m, 1H, Ar-H), 129.56, 131.01, 131.62, 135.43, 135.98, 136.22 (6C ), 154.02
2
2
ar
.93–6.95 (m 2H, Ar-H), 7.19–7.24 (m 2H, Ar-H), 7.58–7.61 (dd, (C5-triazole), 169.21 (C¼S). Analysis for C H Cl N OS
20 27 2 5

4
Ł. Popiołek et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
(
456.43); Calculated: C, 52.63; H, 5.96; N, 15.34; S, 7.03; Found: (m, 4H, 2 ꢂ CH -piperazine), 2.67–2.82 (m, 4H, 2 ꢂ CH -pipera-
2
2
C, 52.65; H, 5.98; N, 15.37; S, 7.01%.
zine), 2.84–2.89 (m, 1H, CH-cyclohexyl), 2.95 (t, 2H, CH ), 3.54
2
(
4
t, 2H, CH ), 3.77 (s, 1H, OH), 4.78 (s, 2H, CH ), 7.24–7.48 (m,
2
2
5-Cyclopentyl-4-(2,4-dichlorophenyl)-2-[(4-phenylpiperazin-1-
yl)methyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione (4e)
13
H, Ar-H).
C
NMR:
ꢀ
(ppm) ¼ 25.15, 25.92, 29.47
(5 ꢂ CH -cyclohexyl), 34.92 (CH-cyclohexyl), 52.43, 53.58
(
(
ꢁ
ꢁ
1
Yield: 87%; m. p. 67 C–69 C (dec.). H NMR (DMSO-d ): ꢀ
2
6
4 ꢂ CH -piperazine), 58.02 (–CH –), 59.25 (–CH –), 63.51
2
2
2
(
3
2
ppm) ¼ 1.49–1.56 (m, 2H, CH -cyclopentyl), 1.63–.179 (m, 6H,
2
–CH –), 129.72; 130.29, 133.71, 135.24 (6C ), 157.28 (C-5
ar
2
ꢂ CH -cyclopentyl), 2.59–2.62 (t, 4H, 2 ꢂ CH -piperazine),
2
2
2
triazole), 170.69 (C¼S). Analysis for C H ClN OS (436.01);
2
1
30
5
.84–2.94 (m, 1H, CH-cyclopentyl), 4.13–4.16 (t, 4H, 2 ꢂ CH -
Calculated: C, 57.85; H, 6.94; N, 16.06; S, 7.35; Found: C, 57.87;
H, 6.97; N, 16.11; S, 7.32%.
piperazine), 5.16 (s, 2H, CH ), 6.78–6.81 (m, 1H, Ar-H), 6.94–
2
6
.96 (m, 2H, Ar-H), 7.18–7.25 (m, 2H, Ar-H), 7.66–7.78 (m, 2H,
3
1
Ar-H), 8.00–8.02 (m, 1H, Ar-H). C NMR: ꢀ (ppm) ¼ 26.12,
4-(4-Chlorophenyl)-5-cyclohexyl-2-[(4-phenylpiperazin-1-
yl)methyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione (5e)
3
5
4.79 (4 ꢂ CH -cyclopentyl), 39.66 (CH-cyclopentyl), 50.36,
2
1
Yield: 79%; m. p. 68 C–70 C (dec.). H NMR (DMSO-d ):
1.43 (4 ꢂ CH -piperazine), 63.51 (–CH –), 116.77, 120.31
ꢁ
ꢁ
2
2
6
(
(
3C ), 129.35, 129.56, 131.01, 131.62, 135.43, 135.98, 136.22
ar
8C ), 150.96 (C ), 154.02 (C-5 triazole), 169.21 (C¼S).
ꢀ (ppm) ¼ 1.13–1.76 (m, 10H, 5 ꢂ CH -cyclohexyl), 2.39–
2
ar
ar
2.51 (m, 1H, CH-cyclohexyl), 2.68–2.72 (m, 4H,
2 ꢂ CH -cyclohexyl), 3.13–3.17 (m, 4H, 2 ꢂ CH -cyclohexyl),
Analysis for C H Cl N S (488.47); Calculated: C, 59.01; H,
2
4
27
2
5
2
2
5
6
.57; N, 14.34; S, 6.56; Found: C, 59.06; H, 5.61; N, 14.37; S,
.61%.
5
Ar-H), 7.19–7.25 (m, 3H, Ar-H), 7.53–7.56 (m, 1H, Ar-H),
.14 (s, 2H, CH ), 6.76–6.81 (m, 2H, Ar-H), 6.93–6.96 (m, 2H,
2
1
.66–7.70 (m, 1H, Ar-H), 8.72 (s, 1H, CH). C NMR: ꢀ
3
7
(
4
2
-(4-Chlorophenyl)-5-cyclohexyl-2-(pyrrolidin-1-ylmethyl)-
,4-dihydro-3H-1,2,4-triazole-3-thione (5a)
ppm) ¼ 25.15, 25.92, 29.47 (5 ꢂ CH -cyclohexyl), 34.92 (CH-
2
ꢁ
ꢁ
1
Yield: 73%; m. p. 91 C–93 C (dec.). H NMR (DMSO-d ): ꢀ
cyclohexyl), 50.36, 51.43 (4 ꢂ CH -piperazine), 63.51 (–CH –),
2
2
6
1
16, 120.31 (3C ), 129.35, 129.72, 130.29, 130.29, 133.71,
ar
(
ppm) ¼ 1.16–1.62 (m, 10H, 5 ꢂ CH -cyclohexyl), 1.70–1.92 (m,
2
1
Analysis for C H ClN S (468.06); Calculated: C, 64.15; H,
6.46; N, 14.96; S, 6.85; Found: C, 64.19; H, 6.48; N, 14.99; S,
6.88%.
35.24 (8C ), 150.96 (C ), 157.28 (C-5 triazole), 170.69 (C¼S).
ar
ar
4
2
7
H, 2 ꢂ CH -pyrrolidine), 2.38–2.46 (m, 1H, CH-cyclohexyl),
2
2
5
30
5
.69–2.78 (m, 4H, 2 ꢂ CH -pyrrolidine), 5.16 (s, 2H, CH ), 7.48–
2
2
.50 (dd, 2H, Ar-H, J ¼ 6 Hz), 7.66–7.68 (dd, 2H, Ar-H,
1
3
J ¼ 6 Hz). C NMR: ꢀ (ppm) ¼ 24.92 (2 ꢂ CH -pyrrolidine),
2
2
5.15, 25.92, 29.47 (5 ꢂ CH -cyclohexyl), 34.92 (CH-cyclo-
2 2
2
4-Phenyl-2-(pyrrolidin-1-ylmethyl)-2,4-dihydro-3H-1,2,4-tri-
azole-3-thione (6a)
CAS Registry Number: 142529-43-9. Yield: 23%; m. p.
hexyl), 53.65 (2 ꢂ CH -pyrrolidine), 62.10 (–CH –), 129.72,
1
30.29, 133.71, 135.24 (6C ), 157.28 (C-5 triazole), 170.69
ar
(
C¼S). Analysis for C H ClN S (376.94); Calculated: C, 60.54;
ꢁ
ꢁ
1
2
0
27
4
134 C–136 C (dec.). H NMR (DMSO-d ): ꢀ (ppm) ¼ 1.67–1.76
6
H, 6.68; N, 14.86; S, 8.51; Found: C, 60.58; H, 6.71; N, 14.82; S,
8
(
m, 4H, 2 ꢂ CH -pyrrolidine), 2.88–2.92 (t, 4H, 2 ꢂ CH -
2 2
2
2
.55%.
pyrrolidine), 5.29 (s, 2H, CH ), 5.29 (s, 2H, CH ), 7.44–7.54
(m, 6H, Ar-H), 7.69–7.73 (m, 1H, Ar-H), 8.52–8.63 (m, 2H, Ar-
4-(4-Chlorophenyl)-5-cyclohexyl-2-(piperidin-1-ylmethyl)-2,4-
dihydro-3H-1,2,4-triazole-3-thione (5b)
1
3
H). C NMR: ꢀ (ppm) ¼ 23.96, 50.26 (4 ꢂ CH -pyrrolidine),
2
ꢁ
ꢁ
1
Yield: 78%; m. p. 87 C–89 C (dec.). H NMR (DMSO-d ): ꢀ
65.79 (–CH
1
–), 122.45, 124.00, 129.28, 129.95, 130.21, 135.15,
2
6
36.51, 147.56, 149.24 (9C ), 150.28, 151.66 (2C ), 154.80 (C-5
ar
ar
(
4
2
ppm) ¼ 1.11–1.56 (m, 10H, 5 ꢂ CH -cyclohexyl), 1.59–1.64 (m,
2
triazole), 170.04 (C¼S). Analysis for C H N S (337.44);
1
8 19 5
H, 2 ꢂ CH -piperidine), 2.06–2.18 (m 4H, 2 ꢂ CH -piperidine),
2
2
2
Calculated: C, 64.07; H, 5.68; N, 20.75; S, 9.50; Found: C,
6
.39–2.44 (m, 1H, CH-cyclohexyl), 2.56–2.62 (m, 2H, CH -
4.11; H, 5.64; N, 20.81; S, 9.45%.
piperidine), 5.35 (s, 2H, CH ), 7.37–7.40 (dd, 2H, Ar-H,
2
1
3
J ¼ 9 Hz), 7.44–7.47 (dd, 2H, Ar-H, J ¼ 9 Hz). C NMR: ꢀ
4-phenyl-2-(piperidin-1-ylmethyl)-5-(pyridin-3-yl)-2,4-dihy-
dro-3H-1,2,4-triazole-3-thione (6b)
(
(
ppm) ¼ 24.57, 25.10 (4 ꢂ CH -piperidine), 25.15, 26.92, 29,47
2
5 ꢂ CH -cycohexyl), 34.92 (CH-cyclohexyl), 53.90 (CH -piperi-
2
2
CAS Registry Number: 142529-46-2. Yield: 89%; m. p.
1
dine), 63.51 (–CH –), 129.72, 130,29, 133.71, 135.24 (6C ),
2
ꢁ
ꢁ
ar
1
1
50 C–152 C (dec.). H NMR (DMSO-d ): ꢀ (ppm) ¼ 1.30–
6
1
57.28 (C-5 triazole), 170.69 (C¼S). Analysis for C H ClN S
2
0
27
4
.54 (m, 6H, 3 ꢂ CH -piperidine), 2.68–2.79 (m, 4H, 2 ꢂ CH -
2
2
(390.97); Calculated: C, 61.44; H, 6.96; N, 14.33; S, 8.20; Found:
C, 61.38; H, 6.99; N, 14.30; S, 8.17%.
13
C
piperidine), 5.12 (s, 2H, CH ), 7.30–8.63 (m, 9H, Ar-H).
2
NMR: ꢀ (ppm) ¼ 23.42, 24.57, 53.90 (5 ꢂ CH -piperidine), 63.51
2
(
(
–CH –), 126.15, 128.65, 128.83, 129.36, 132.06, 134.57, 136.59
2
4-(4-Chlorophenyl)-5-cyclohexyl-2-(piperazin-1-ylmethyl)-2,4-
dihydro-3H-1,2,4-triazole-3-thione (5c)
9C ), 146.42, 149.92 (2C ), 154.70 (C-5 triazole), 168.26
ar
ar
ꢁ
ꢁ
1
Yield: 92%; m. p. 182 C–184 C (dec.). H NMR (DMSO-d ):
(C¼S). Analysis for C H N S (351.47); Calculated: C, 64.93;
19 21 5
6
H, 6.02; N, 19.93; S, 9.12; Found: C, 64.98; H, 6.07; N, 19.88; S,
9
ꢀ
(ppm) ¼ 1.05–1.75 (m, 10H, 5 ꢂ CH -cyclohexyl), 2.38–2.45
2
.08%.
(m, 4H, 2 ꢂ CH -piperazine), 2.68–2.74 (m, 4H, 2 ꢂ CH -pipera-
2
2
zine), 2.77–2.83 (m, 1H, CH-cyclohexyl), 5.03 (s, 2H, CH ),
2
4-Phenyl-2-(piperazin-1-ylmethyl)-5-(pyridin-3-yl)-2,4-dihy-
dro-3H-1,2,4-triazole-3-thione (6c)
7
.52–7.54 (dd, 2H, Ar-H, J ¼ 6 Hz), 7.67–7.69 (dd, 2H, Ar-H,
1
3
J ¼ 6 Hz), 8.71 (s, 1H, NH). C NMR: ꢀ (ppm) ¼ 25.45, 25.62,
ꢁ
ꢁ
1
Yield: 78%; m. p. 242 C–244 C (dec.). H NMR (DMSO-d ):
6
3
0.41 (5 ꢂ CH -cyclohexyl), 34.52 (CH-cyclohexyl), 40.26; 40.54
2 2
2
ꢀ
(
5
13
(ppm) ¼ 1.04–1.14 (m, 2H, CH -piperazine), 1.25–1.31
2
(4 ꢂ CH -piperazine), 50.32 (–CH –), 129.72, 130.15, 133.02,
m, 2H, CH -piperazine), 3.20–3.30 (m, 4H, 2 ꢂ CH -piperazine),
2
2
1
C H ClN S (391.96); Calculated: C, 58.22; H, 6.69; N, 17.87;
35.24 (6C ), 157.28 (C-5 triazole), 170.69 (C¼S). Analysis for
19 26 5
ar
.24 (s, 2H, CH ), 7.46–8.56 (m, 9H, Ar-H), 9.23 (s, 1H, NH).
2
C
NMR:
ꢀ
3.51 (–CH –), 126.15, 128.65, 128.83, 129.36, 132.06, 136.59
(ppm) ¼ 45.59, 53.21 (4 ꢂ CH -piperazine),
2
S, 8.18; Found: C, 58.29; H, 6.72; N, 17.85; S, 8.15%.
6
2
4-(4-Chlorophenyl)-5-cyclohexyl-2-{[4-(2-hydroxyethyl)piper-
azin-1-yl]methyl}-2,4-dihydro-3H-1,2,4-triazole-3-thione (5d)
(9C ), 146.42, 146.92 (2C ), 154.70 (C-5 triazole), 168.26
ar ar
(C¼S). Analysis for C H N S (352.46); Calculated: C, 61.34;
1
8 20 6
ꢁ
ꢁ
1
Yield: 82%; m.p. 153 C–155 C (dec.). H NMR (DMSO-d ): H, 5.72; N, 23.84; S, 9.10; Found: C, 61.27; H, 5.78; N, 23.80; S,
6
ꢀ
(ppm) ¼ 1.22–1.48 (m, 10H, 5 ꢂ CH -cyclohexyl), 2.48–2.58 9.06%.
2

DOI: 10.3109/14756366.2013.855926
Mannich bases bearing 1,2,4-triazole moiety
5
2
-{[4-(2-Hydroxyethyl)piperazin-1-yl]methyl}-4-phenyl-5-
Microbiology
Materials and methods
(ppm) ¼ 2.89–2.95 (m, 4H, 2 ꢂ CH -piperazine), 3.21 (t, 2H, All the derivatives were screened for their in vitro antibacterial
(pyridin-3-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (6d)
ꢁ
ꢁ
1
Yield: 70%; m. p. 168 C–170 C (dec.). H NMR (DMSO-d ):
6
ꢀ
2
CH ), 3.36–3.43 (m, 4H, 2 ꢂ CH -piperazine), 3.55 (t, 2H, CH ), activity using agar plate method (with 1000 mg/ml concentration
2
2
2
4
.46 (s, 2H, CH ), 7.17–7.60 (m, 5H, Ar-H), 8.27–8.77 (m, 4H, of compounds) and next the compounds with potential antibac-
2
1
3
Ar-H), 8.87 (s, 1H, OH). C NMR: ꢀ (ppm) ¼ 52.43, 53.58 terial activity were tested using microdilution technique to
(
(
4 ꢂ CH -piperazine), 58.02 (–CH –), 59.25 (–CH –), 63.51 estimate minimal inhibitory concentration (MIC), as was
2
2
2
2
2
7
–CH –), 126.15, 128.65, 128.83, 129.36, 132.06, 134.57, described earlier . Eleven reference strains of aerobic bacteria
1
1
6
2
36.59 (9C ), 146.42, 146.92 (2C ), 154.70 (C-5 triazole), from American Type Culture Collection were used. There were
ar ar
68.26 (C¼S). Analysis for C H N OS (396.50); Calculated: C, representing Gram-positive bacteria Staphylococcus aureus
2
0 24 6
0.58; H, 6.10; N, 21.20; S, 8.09; Found: C, 60.62; H, 6.05; N, ATCC 6538, S. aureus ATCC 25923, S. aureus ATCC 43300,
1.24; S, 8.01%.
S. epidermidis ATCC 12228, Bacillus subtilis ATCC 6633,
Bacillus cereus ATCC 10876, Micrococcus luteus ATCC 10240
and representing Gram-negative bacteria Escherichia coli ATCC
25922, Klebsiella pneumoniae ATCC 13883, Proteus mirabilis
4
2
-Phenyl-5-(pyridin-3-yl)-2-[(4-phenylpiperazin-1-yl)methyl]-
,4-dihydro-3H-1,2,4-triazole-3-thione (6e)
CAS Registry Number: 915144-29-5. Yield: 95%; m. p. ATCC 12453 and Pseudomonas aeruginosa ATCC 27853. All the
ꢁ
ꢁ
1
1
2
3
54 C–156 C (dec.). H NMR (DMSO-d ): ꢀ (ppm) ¼ 2.62– bacterial cultures were adjusted to 0.5 McFarland standards
6
6
.65 (t, 2H, CH -piperazine), 3.00–3.04 (t, 2H, CH -piperazine), 150 ꢂ 10 CFU/ml (CFUs – colony forming units) in sterile 0.85%
2
2
.15–3.19 (t, 2H, CH -piperazine), 3.46–3.48 (t, 2H, CH - NaCl. All stock solutions of the tested compounds were prepared
2
2
piperazine), 5.32 (s, 2H, CH ), 6.79–6.84 (m, 2H, Ar-H), 6.96– in DMSO (dimethyl sulfoxide). The medium with DMSO at the
2
7
Ar-H), 7.73–7.77 (m, 1H, Ar-H), 8.57–8.65 (m, 1H, Ar-H).
.07 (m, 4H, Ar-H), 7.22–7.27 (m, 4H, Ar-H), 7.42–7.63 (m, 2H, final concentration and without the tested compounds served as
1
3
C
negative control – no microbial growth inhibition was observed.
NMR: ꢀ (ppm) ¼ 50.36, 51.43 (4 ꢂ CH -piperazine), 63.51 Cefuroxime (second generation of cephalosporins) was used as a
2
(
–CH –), 116.77 (2C ), 120.31 (C ), 126.15, 128.65, 128.83, positive control.
2 ar ar
1
29.35, 129.36, 132.06, 134.57, 136.59 (11Car), 146.42,
Using Mueller–Hinton agar plate method with 1000 mg/ml
1
46.92 (2C ), 150.96 (C ), 154.70 (C-5 triazole), 168.26 concentration of each derivative, 10 ml of each bacterial suspen-
ar
ar
(
C¼S). Analysis for C H N S (428.55); Calculated: C, 67.26; sion was put onto the prepared solid media and antibacterial
2
4 24 6
ꢁ
H, 5.64; N, 19.61; S, 7.48; Found: C, 67.34; H, 5.61; N, 19.64; S, activity was detected after incubation (37 C for 18 h) on the basis
7
.44%.
of the microbial growth inhibition. MIC of compounds, which
inhibited the growth of bacteria on agar medium, was tested
through the Mueller–Hinton broth microdilution method as
recommended by Clinical Laboratory Standards Institute . MIC
is usually defined as the lowest concentration of the compound at
which there was no visible growth of microorganisms. The 96-
2
8
Antiproliferative activity
Materials and methods
The experiment was conducted on reference cancer cell lines: well microplates and Mueller–Hinton broth with a series of two-
A549 (Adenocarcinomic human alveolar basal epithelial cells), fold dilution of the tested compound in the range of final
HeLa (Human cervical cancer cells), TOV-112D (Human concentrations from 7.82 to 1000 mg/ml were used. After incu-
ꢁ
ovarian cancer cell line), T47D (Human ductal breast bation (at 35 C for 18 h), spectrophotometric measurements of
epithelial tumor cell line) and L929 (Murine aneuploid optical density (OD600) of the bacterial cultures with the tested
fibrosarcoma cell line) and GMK (Green monkey kidney cell compounds were performed in order to determine MIC. OD600 of
line) as a normal cell lines. The control group was the chosen bacterial cultures in the medium without the tested compounds
cell line untreated with compounds. Cell lines were obtained and the blank wells with two-fold dilution of each of the tested
from European Collection of Cell Cultures (ECAACC). Cell compounds added to broth without bacterial suspension were used
ꢁ
cultures were grown at 37 C in a humidified atmosphere as controls. In our study, the bioactivity of tested compounds
consisting 5% CO in air. Cultures were maintained at density against bacteria was defined as mild (MIC4500 mg/ml), moderate
2
6
of 2–5 ꢂ 10 cell/ml in exponential growth serum free condi- (MIC in the range 4125–500 mg/ml), good (MIC in the range
2
9
tions containing RPMI-1640 medium supplemented with 10% ꢃ31.25–125 mg/ml), according to O’Donnell et al. .
fetal bovine serum (FBS, Sigma), 100 U/ml of penicillin
(Sigma), 100 mg/ml streptomycin (Sigma), and routinely Results and discussion
passaged twice a week. Cell viability was assessed by the
ability to exclude trypan blue dye (0.5% v/v, Sigma). Next
Chemistry
cultures were incubated in the presence of tested compounds The hydrazides of cyclopropionic acid, cyclopentanoic acid,
3a, 3b, 3c, 3e, 4a, 4b, 4d, 5c, 5e, 6a, 6b, 6e) at the cyclohexanoic acid and nicotinic acid were initial substrates for
(
concentrations of 50, 100 and 150 mM during 24, 48 and 72 h. the synthesis of new Mannich base derivatives 3a–3e, 4a–4e, 5a–
The investigated 12 compounds were dissolved in dimethyl- 5e, 6a–6e. In the first step, thiosemicarbazide derivatives 1a–1d
sulfoxide (DMSO, Sigma) and then diluted in cell culture were obtain by the reaction of corresponding carboxylic acid
media. The antiproliferative activity of novel compounds was hydrazide with approptiate isothiocyanate. The substracts were
0
assessed by 5-bromo-2 -deoxyuridine test (BrdU, Sigma) on heated in an oil bath, temperatures were selected experimentally
0
ꢁ
El 808 ELISA reader. In these method, 5-bromo-2 -deoxyur- (t ¼ 50–90 C). Subsequently, the reaction of thiosemicarbazide
x
iu
idine, a thymidine analog, replaces [3H] thymidine. BrdU is derivatives 1a–1d with 2% aqueous solution of sodium hydroxide
incorporated into newly synthesized DNA strands of actively lead to the formation of 4,5-disubstituted 1,2,4-triazole-3-thione
proliferating cells. Following partial denaturation of double derivatives 2a–2d. The treatment of 1,2,4-triazole derivatives
stranded DNA, BrdU is detected immunochemically allowing with formaldehyde and corresponding secondary amine in ethanol
the assessment of the population of cells, which are afforded new Mannich base derivatives 3a–3e, 4a–4e, 5a–5e, 6a–
synthesizing DNA. All results were done in triplicates.
6e in moderate to good yields.

6
Ł. Popiołek et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Compounds 1a, 1b, 1c, 2a, 3e, 4a, 6a, 6b are registered in CAS The synthetic pathway leading to the new Mannich base
Chemical Abstracts Service) database but without references and derivatives 3a–e, 4a–e, 5a–e, 6a–e was carried out according to
(
methods of synthesis. Compounds 1d , 2d , 6e
synthesized earlier.
3
0
31
32
were the steps shown in Scheme 1. The substituents of all obtained
compounds 1–6 are presented in Table 1.
All the newly obtained compounds are air stable solids and
soluble in DMSO at ambient temperature. The purity of the
synthesized compounds was checked by elemental analyses and
thin layer chromatography. The structures of prepared derivatives
Table 1. Substituents of compounds 1–6.
Compound
R
1
R
2
R
3
1
13
were determined on the basis of H NMR and C NMR spectra
and all of the synthesized compounds have satisfactory analyses
1
a
–
–
1
for their proposed structures. H NMR spectral results for all
1
3
compounds together with hydrogen assignments and C NMR
spectra results are presented in Experimental Section.
1b
–
–
1
H NMR spectra of the thiosemicarbazide derivatives 1a–d
show three proton signals typical for the NH group in the ꢀ 9.07–
1
0.11 ppm range, whereas in the H NMR spectra of the 1,2,4-
1
triazole compounds 2a–d the singlet peak due to the proton of NH
group appeared in the region of ꢀ 11.04–13.65 ppm, which
confirmed the successful formation of the desired products. All
the H NMR spectra of Mannich base derivatives derivatives 3a–
e, 4a–e, 5a–e, 6a–e confirmed adequate N-substitution of 1,2,4-
1
c
–
–
–
–
1
triazole-3-thiones. The singlet signal for CH group was observed
2
in the ꢀ 4.46-5.35 ppm range (compounds 3a–e, 4a–e, 5a–e, 6a– 1d
e), for NH group (compounds 3c, 4c, 5c, 6c) ꢀ 8.71–9.23 ppm, and
for OH group in the region of 3.77–3.97 and 8.87–8.89 ppm
(compounds 3d, 4d, 5d, 6d). All other aliphatic and aromatic
protons for obtained compounds were observed at expected
regions.
N
2
a
4-Cl
–
–
1
3
In the C NMR spectra of thiosemicarbazide derivatives 1a–d
the carbon of C¼S group had a typical signal at about 176 ppm
and for C¼O group at about 168–180 ppm. Similarly, in the 1,2,4-
triazole derivatives 2a–d, the presence of the C¼S group was also
confirmed by a signal at about 167–168 ppm and for the C-5 in
2
b
2,4-diCl
1,2,3-triazole system in the range of 154–157 ppm. The 2a–d
derivatives may occur in thione or thiol form. However, in our
case the signal of C¼S group in compounds 2a–d proved that they
were obtained in thione form. The thione-thiol tautomerism was 2c
not observed. It is consistent with the studies recently conducted
in our department which were focused on the cyclization
4-Cl
–
3
3,34
mechanism of thiosemicarbazide derivatives
.
In the
Mannich base derivatives 3a–e, 4a–e, 5a–e, 6a–e the CH2
group was observed at about 50–69 ppm. All other aliphatic and
aromatic signals were observed at expected regions.
(continued )
Scheme 1. Synthetic route for new Mannich base derivatives 3a–3e, 4a–4e, 5a–5e, 6a–6e.

DOI: 10.3109/14756366.2013.855926
Mannich bases bearing 1,2,4-triazole moiety
7
Compound
R
1
R
2
R
3
Compound
R
1
R
2
R
3
2
d
H
–
4d
2,4-diCl
N
OH
N
N
3
a
4-Cl
4-Cl
4
e
2,4-diCl
N
3
3
b
c
N
N
N
5
5
a
4-Cl
4-Cl
4-Cl
H
N
N
b
N
3
3
d
4-Cl
N
N
OH
5
c
4-Cl
H
N
N
e
4-Cl
N
5
5
d
4-Cl
N
N
OH
N
N
4
4
a
2,4-diCl
2,4-diCl
e
4-Cl
N
N
b
N
N
6
6
a
H
H
4
c
2,4-diCl
H
N
N
N
b
N
N
N
(
continued )
(continued )

8
Ł. Popiołek et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Table 1. Continued
Antiproliferative activity
The purpose of this study was to evaluate in vitro antiproliferative
activities against chosen cell lines of the 12 compound based on
of Mannich bases containing 1,2,4-triazole system. Evaluation of
cell cycle progression is essential for investigations in many
scientific fields. Measurement of [3H] thymidine incorporation as
cells enter S phase has been a traditional method for detection of
cell proliferation. Precentage of cells viability, before the
foundation of the culture, was about 87–96%. The amount of
BrdU was determined according to OD. The higher the OD, the
higher the BrdU concentration in the sample. Results were
presented as the percentage of cell viability in comparison to
control. The influence of various concentrations of new
synthesized compounds on the proliferative activity of chosen
cell cultures is summarized in Table 2.
Compound
R
1
R
2
R
3
6
c
H
H
N
N
N
6
6
d
H
N
OH
N
N
N
The investigated compounds proved to be low toxic for
checked cell lines, since applied in concentrations of 50, 100 and
150 mM caused only slight viability decreases in examined cell
cultures. Only 1 of the 12 investigated compounds was found to be
e
H
little biologically active in vitro. We have noticed, that compound
6
b evoked slight dose-dependent viability decreases in T47D,
N
HeLa and A549 cell lines (15–20% growth inhibition was
observed comparison to control). In case of HeLa cell line,
except compound 6b, it was noticed that compound 6a, in the
N
Table 2. Effects of tested compounds (3a, 3b, 3c, 3e, 4a, 4b, 4d, 5c, 5e, 6a, 6b, 6e) on the viability of chosen cell lines (%).
L929
GMK
A549
HeLa
TOV-112D
T47D
Number of derivative
and dose
24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h
Control
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0C
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0C
0
0
0C
0
0
0
0
0
0
0
0
0C
0
0
0
0
0
0
0
0
0
0
5
5
0
0
0
0
0
0
0
5
10
0
5
5
5
5
10
0
0
10
0
5
5
2
2
5
0
5
5
0
5
20
0
5
5
0
0
5
5
0
5
5
0
0
0
5
5
10
5
10
10
0
10
15
5
10
15
5
10
10
5
10
15
2
10
10
5
10
25
5
5
10
0
0
0
0
0
5
10
0
0
0
0
0
0
0
0
0
5
0
0
5
0
5
5
0
2
5
0
0
10
0
5
10
0
10
15
0
5
10
5
10
20
5
15
20
0
0
0
0
0
0
0
5
0
0
5
0
5
5
0
5
10
0
0
10
0
5
10
0
10
15
0
5
10
5
10
20
5
15
20
0
0
0
0
0
0
0
5
0
0
5
0
0
5
0
5
10
0
0
15
0
5
10
0
10
15
0
5
10
5
10
20
5
15
20
0
0
0
0
0
0
0
5
0
0
0
0
0
5
0
5
5
0
5
10
0
0
0
0
0
5
0
5
5
5
10
15
0
5
5
0
0
0
0
0
5
10
0
0
0
0
5
5
5
10
10
0
10
10
0
5
10
0
5
15
5
10
15
5
10
20
0
5
0
5
5
10
0
0
0
10
5
10
10
0
0
0
0
0
0
5
5
0
5
0
5
5
5
0
5
10
0
5
5
0
5
5
5
0
5
10
0
5
5
a–50 mM
3
3
a–100 mM
a–150 mM
3
b–50 mM
3
3
b–100 mM
b–150 mM
3
c–50 mM
3
3
c–100 mM
c–150 mM
0
0
5
10
5
10
15
5
10
15
5
15
20
10
15
15
5
10
20
15
20
20
5
10
25
5
15
20
10
15
20
3
e–50 mM
5
5
5
5
3
3
e–100 mM
e–150 mM
10
10
5
10
10
5
10
5
5
10
15
5
10
15
5
15
20
5
10
20
5
10
15
5
10
15
5
15
20
10
15
15
10
10
20
15
20
20
5
10
25
5
10
20
10
15
20
10
15
5
10
15
5
15
20
5
10
15
10
10
20
15
20
20
5
10
20
5
10
20
10
15
20
10
10
15
15
2
10
20
5
10
15
5
15
15
10
15
20
5
4
a–50 mM
4
4
a–100 mM
a–150 mM
4
b–50 mM
4
4
b–100 mM
b–150 mM
4
d–50 mM
4
4
d–100 mM
d–150 mM
5
c–50 mM
5
5
c–100 mM
c–150 mM
5
e–50 mM
5
5
e–100 mM
e–150 mM
6
a–50 mM
6
6
a–100 mM
a–150 mM
10
10
5
10
25
5
6
b–50 mM
6
6
b–100 mM
b–150 mM
5
5
5
5
10
6
e–50 mM
5
5
10
6
6
e–100 mM
e–150 mM
10
15
0
0
5
5
5
5
0
L929, Murine aneuploid fibrosarcoma cell line; GMK, Green monkey kidney cell line; A549, Adenocarcinomic human alveolar basal epithelial cells;
HeLa, Human cervical cancer cells; TOV-112D, Human ovarian cancer cell line; T47D, Human ductal breast epithelial tumor cell line.

DOI: 10.3109/14756366.2013.855926
Mannich bases bearing 1,2,4-triazole moiety
9
Table 3. The influence of synthesized compounds 1–6 on the growth of Gram-positive and Gram-negative bacteria of on the basis of MIC (mg/ml).
Compound
Sa6538
Sa25923 (MSSA) Sa43300 Se12228 Bs6633 Bc10876 Ml10240 Ec25922 Kp13883 Pm12453 Pa27853
1
1
1
2
2
3
3
3
4
4
4
4
5
5
6
6
6
a
b
c
a
c
a
b
e
a
b
d
e
c
e
a
b
e
62.5
62.5
31.25
125
62.5
500
500
500
500
500
62.5
62.5
31.25
62.5
62.5
31.25
125
62.5
250
250
500
125
250
500
500
125
250
250
31.25
62.5
125
250
250
500
500
500
1000
500
125
nd
125
125
250
125
125
250
500
500
500
1000
1000
250
125
nd
62.5
62,5
62.5
125
125
250
500
500
125
125
125
500
31.25
31.25
31.25
31.25
62.5
125
250
500
62.5
62.5
125
250
125
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
125
125
500
500
500
500
500
125
500
125
nd
nd
nd
nd
nd
1000
1000
nd
1000
41000
nd
nd
nd
nd
500
500
500
500
1000
500
500
nd
1000
1000
nd
1000
41000
nd
nd
nd
nd
1000
1000
nd
1000
41000
nd
nd
nd
nd
1000
41000
125
62.5
nd
500
500
500
nd
250
500
500
500
62.5
nd
500
500
1000
500
500
500
500
500
500
500
500
500
500
500
500
1000
1000
1000
500
250
500
500
500
500
1000
1000
1000
nd, not determined; Sa6538, Staphylococcus aureus ATCC 6538; Sa25923, Staphylococcus aureus ATCC 25923; Sa43300, Staphylococcus aureus
ATCC 43300; Se12228, Staphylococcus epidermidis ATCC 12228; Bs6633, Bacillus subtilis ATCC 6633; Bc10876, Bacillus cereus ATCC 10876;
Ml10240, Micrococcus luteus ATCC 10240; Ec25922, Escherichia coli ATCC 25922; Pm12453, Proteus mirabilis ATCC 12453; Pa27853,
Pseudomonas aeruginosa ATCC 27853.
concentration of 100 and 150 mM also caused small decreases (10 opportunistic (e.g. S. epidermidis, M. luteus, B. subtilis or B.
and 20%, respectively) in cell viability versus control.
cereus) Gram-positive bacteria and may be of value for searching
We have noted, that T47D cell line was the most susceptible new derivatives showing better antimicrobial activity.
cancer cell line, among the others, on compounds’ influencing.
The slight cytotoxic activity against T47D cell line in the set of
checked compounds (except compound 6b) were found also for Conclusions
compounds number 5e and 6a. In the concentration of 50, 100 and
1
In the current study, we synthesized and characterized by H
NMR, C NMR and elemental analyses a new series of Mannich
base derivatives containing 1,2,4-triazole moiety.
Twelve of obtained compounds were analyzed for their in vitro
antiproliferative acitivity against six cancer cell lines. Our study
revealed that synthesized compounds 5e, 6a, 6e possess minor
antiproliferative activity and some further studies in this direction
need to be done.
1
50 mM, these compounds showed 15–25% growth inhibition
1
3
against breast tumor cell line after 24, 48 and 72 h of incubation.
The other compounds that affected on this cell line, but to a much
lesser extent, were compound number 4b, 4d, 5c, 6a and 6e.
No visual changes in cell viability against TOV-112D cell line,
L929 and GMK cell line during tested compound treatment, were
not observed. Our study revealed that some of our synthesized
compounds possess minor antiproliferative activity.
All the newly prepared compounds were also screened for their
in vitro antimicrobial activity by agar dilution technique against
Microbiology
1
1 reference strains of Gram-positive and Gram-negative bacteria.
On the basis of the preliminary results obtained by agar plate Our antimicrobial study revealed that compounds 1a, 1b, 1c, 2a,
method, it was shown that some of the newly synthesized 2c and 5c had good or moderate activity against the reference
compounds listed in Table 3 have shown a potential activity Gram-positive bacteria and the compounds 3a, 3b, 4a, 4b, 6a, 6b
against reference strains mainly of Gram-positive bacteria. On the and 6e had mild or moderate inhibitory effect on the Gram-
basis of MIC estimation some compounds was found to possess negative bacteria growth. Obtained compounds may be regarded
good, moderate or mild bioactivity against Gram-positive bacter- as precursor compounds for searching new derivatives showing
ial species with MIC ranging from 31.25 to 500 mg/ml (Table 3). better antimicrobial activity against pathogenic or opportunistic
The highest activity possessed compounds 1a, 1b, 1c, 2a, 2c and bacteria. Nowadays due to several serious problems such as
5
c (MIC ¼ 31.25–250 mg/ml). M. luteus ATCC 102740 was the growing drug resistance of bacteria or undesirable side effects of
most sensitive to the selected derivatives (MIC ¼ 31.25–250 mg/ drugs it is very important to find new substances with antimicro-
ml). In our experiments, MIC of cefuroxime was 0.24–1.95 mg/ml bial activity.
for Staphylococcus species and 0.49–31.25 mg/ml for the other
Gram-positive bacteria.
Among the tested compounds, only seven (3a, 3b, 4a, 4b, 6a,
Declaration of interest
6
b and 6e) had mild or moderate inhibitory effect on the Gram- The project was partially supported by the Research Grant for Young
negative bacteria growth with MIC values ranging from 250 to Scientists (MNmb25). The authors declare no conflicts of interest. The
authors alone are responsible for the content and writing of this article.
ꢃ1000 mg/ml.
In general, our obtained thiosemicarbazide derivatives showed
better activity as compared to cyclic compounds, what is
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