tives of 3-r-4-phenyl-∆2-1,2,4-triazoline-5

tives of 3-r-4-phenyl-∆2-1,2,4-triazoline-5

Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 62 No. 6 pp. 443ñ449, 2005
ISSN 0001-6837
Polish Pharmaceutical Society
DRUG SYNTHESIS
SYNTHESIS AND BIOLOGICAL ACTION OF 1-AMINOMETHYL DERIVATIVES OF 3-R-4-PHENYL-∆2-1,2,4-TRIAZOLINE-5-THIONE
MONIKA PITUCHA1, MONIKA WUJEC1, MARIA DOBOSZ1,
URSZULA KOSIKOWSKA2 and ANNA MALM2
Department of Organic Chemistry, Faculty of Pharmacy, Medical University,
6 Staszica Str., 20-081 Lublin, Poland*
2
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University,
1 Chodüki Str, 20-093 Lublin, Poland
1
Abstract: 4-Phenyl-1,2,4-triazoline-5-thione [I] and 4-phenyl-3-(pyridin-3-yl)-1,2,4-triazoline-5-thione [II]
were subjected to the reaction of aminomethylation with piperazine derivatives. Compounds [III] and [IX]
were screened for their antibacterial activities.
Keywords: synthesis, 1-aminomethyl-3-R-4-phenyl-∆2-1,2,4-triazoline-5-thione, spectral analysis, biological
activity.
Some derivatives of 1,2,4-triazoline-5-thione
have been received and tested for antibacterial (1-6)
and antifungal (7-11) activity. Based on this fact we
attempted to obtain new derivatives of 1-aminomethyl3-R-4-phenyl- ∆2-1,2,4-triazoline-5-thione which has
not been described in the literature yet.
One of the method of synthesis of derivatives
with 1,2,4-triazoline-5-thione system is the cyclization of acyl derivatives of thiosemicarbazide in alkaline media (12-15). By this method 4-phenyl-1,2,4triazoline-5-thione [I] and 4-phenyl-3-(pyridin-3yl)-1,2,4-triazoline-5-thione [II] were obtained. The
thiosemicarbazide of formic and nicotinic acid were
cyclized with an aqueous 2% solution of sodium
hydroxide (16). In the nucleophilic substitution
reactions these compounds can give both S- or Nderivatives, but their characteristic reactions were
only those of N-derivatives (17). Compounds [I]
and [II] were subjected to the reaction with piperazine derivatives and transformed into the corresponding 1-aminomethyl-3-R-4-phenyl-∆2-1,2,4-triazoline-5-thione derivatives. The aminomethylation
reactions were carried out in ethanolic solution, in
the presence of formalin, using N-phenyl-piperazine, 1-(4-methoxyphenyl)piperazine, 1-(2-fluorophenyl)piperazine, 1-(4-fluoro-phenyl)piperazine,
1-(3-chlorophenyl)piperazine and 1-(4-pyridyl)
piperazine (Scheme 1).
The obtained compounds were screened for
their antibacterial activity.
EXPERIMENTAL
Chemistry
Melting points were determined in a FisherJohns block without corrections. IR spectra were
recorded in KBr using Specord IR-75 spectrophotometer. The 1H NMR spectra were recorded on a
Brucker Avance 300 in DMSO-d6 with TMS as
internal standard. Chemicals were purchased from
Merck Co. or Lancaster and used without purification.
1-aminomethyl-4-phenyl-1,2,4-triazoline-5thione derivatives [III-VI, VIII ] and 1-aminomethyl-4-phenyl-3-(pyridin-3-yl)-1,2,4-triazoline5-thione derivatives [IX, X, XII-XIV]
Compound [I] or [II] (0.01 mole) was dissolved in 10 cm3 of anhydrous ethanol. Then 0.8 g
(0.01 mole) of 37% formaldehyde aqueous solution
and 0.02 mole of appropriate amine were added. The
mixture was refluxed for 6 h (2 h for VIII, 10 h for
XIII, XIV). After cooling the precipitate was filtered off, dried and crystallized from ethanol.
1-[(3-chlorophenylpiperazinyl)methyl]-4-phenyl1,2,4-triazoline-5-thione [VII] and 1-[(2-fluorophenylpiperazinyl)methyl]-4-phenyl-3-(pyrid3-yl)-1,2,4-triazoline-5-thione [XI]
Compound [I] or [II] (0.01 mole) was dissolved in 15 cm3 of anhydrous ethanol. Then 0.8 g
* Corresponding author: e-mail ñ [email protected]
443
444
MONIKA PITUCHA et al.
(0.01 mole) of 37% formaldehyde aqueous solution
and 0.01 mole of appropriate amine were added. The
mixture was heated while boiling for 6-9 h under
reflux. Then the solvent was distilled off and the
residue were washed with hot heptane to yield pure
produducts.
Microbiology
Compounds III and IX were assayed for their
antibacterial activity in vitro against 8 strains of aer-
obic bacteria (Gram- positive ñ Staphylococcus
aureus, Staphylococcus epidermidis, Enterococcus
faecalis and Gram-negative ñ Escherichia coli,
Klebsiella pneumoniae, Proteus vulgaris, Enterobacter aerogenes and Pseudomonas aeruginosa).
The tested microorganisms were originally clinical
isolates from the collection of the Department of
Pharmaceutical Microbiology of Medical University
of Lublin. In addition, two reference strains of bacteria ñ Staphylococcus aureus ATCC 25923 and
Table 1. Physical and analytical data for compounds III-VIII
No. Rí
III C6H5
Formula
Yield %
Molecular
O
M.p.
C
Weight
Analysis
Calculated/Found
%C
%H
%N
H NMR- δ (ppm)
IR (cm-1)
1
KBr
DMSO ñ d6
C19H21N5S
351.47
70
148-150
64.92 6.02 19.92 3133 CH arom.
64.9 5.9 19.9 2949,1432 CH aliph.
1600 C=N
1497 C-N
2.69-2.99 (m,4H,2CH2)
3.12 3.73 (m,4H,2CH2)
5.17 (s, 2H,CH2)
6.77-7.65 (m,10H,2 arom. benz.)
8.83 (s,1H,CH)
IV 4-OCH3C6H4 C13H16N5S
381.5
71
130-132
62.97 6.07 18.36 3103 CH arom.
62.9 6.0 16.3 2938, 1428 CH aliph.
1594 C=N
1498 C-N
2.86-2.93 (m,4H,2CH2)
3.43 (s,3H,CH3)
3.65-3.74 (m,4H,2CH2)
5.16 (s, 2H,CH2)
6.85-6.97 (m,4H, arom. benz.)
7.49-7.69 (m. 5H, arom. benz.)
8.84 (s,1H,CH)
V 2-FC6H4
C19H20N5SF 69
369.46
84-86
61.76 5.46 18.95 3092 CH arom.
61.7 5.5 19.0 2940, 1430 CH aliph.
1587 C=N
1486 C-N
2.87-2.88 (m,4H,2CH2)
2.92-2.94 (m,4H,2CH2)
5.33 (s, 2H,CH2)
6.93-7.14 (m. 5H, arom. benz.)
7.48-7.68 (m. 4H, arom. benz.)
8.67 (s,1H,CH)
VI 4-FC6H4
C19H20N5SF 69
369.46
133-134
61.76 5.46 18.95 3135 CH arom.
61.8 5.4 18.9 2947, 1426 CH aliph.
1594 C=N
1511 C-N
2.87-2.96 (m,4H,2CH2)
3.00-3.09 (m,4H,2CH2)
5.17 (s, 2H,CH2)
6.90-7.06 (m,4H, arom. benz.)
7.48-7.67 (m,5H, arom. benz.)
8.82 (s,1H,CH)
VII 3-ClC6H4
C19H20N5SCl 72
385.91
96-97
59.13 5.22 18.15 3087 CH arom.
59.1 5.3 18.2 2948, 1428 CH aliph.
1582 C=N
1498 C-N
2.52-2.80 (m,4H,2CH2)
2.97-3.18 (m,4H,2CH2)
5.17 (s, 2H,CH2)
6.75-7.29 (m,4H, arom. benz.)
7.48-7.67 (m,4H, arom. benz.)
8.70 (s,1H,CH)
VIII C5H4N
C18H20N6S
352.46
61.34 5.72 23.84 3033 CH arom.
61.4 5.7 23.9 2956, 1436 CH aliph.
1593 C=N
1516 C-N
2.83-2.86 (m,4H,2CH2)
3.32-3.46 (m,4H,2CH2)
5.18 (s, 2H,CH2)
6.80-6.83 (d,2H, arom. pyrid.)
7.49-7.65 (m,5H, arom. benz.)
8.12-8.14 (d,2H, arom.pyrid.)
8.77 (s,1H,CH)
71
92-93
Synthesis and biological action of 1-aminomethyl derivatives...
Figure 1. Antibacterial activity of the compound III assessed by broth dilution method.
445
446
MONIKA PITUCHA et al.
Figure 2. Antibacterial activity of the compound IX assessed by broth dilution method.
447
Synthesis and biological action of 1-aminomethyl derivatives...
Scheme 1:
I: R = H II: R = 3-pyridyl
III
IV
V
VI
VII
VIII
R
Rí
H
H
H
H
H
H
C6H5
4-CH3OC6H4
2-FC6H4
4-FC6H4
3-ClC6H4
C5H4N
Escherichia coli ATCC 35218 were included in the
studies.
The minimal inhibitory concentration (MIC),
defined as the lowest concentration of the compound
preventing the visible growth, was estimated for
newly synthesized compounds by the dilution
method using Mueller-Hinton agar.
The compounds were dissolved in a mixture of
dimethylsulfoxide (DMSO) and 95% ethanol (1:1).
The final concentration of each solvent in the medium had no influence on the growth of tested
microorganisms. Microbial suspensions (optical
density of 0.5 McFarland standard, 0.025 mL) were
put onto Petri dishes with 20 mL agar medium containing several concentrations of the tested compounds (500, 250, 125, 62.5, 31.25 mg L-1). The
media were incubated for 18 h at 35OC. The medium
without assayed compounds added served as a control. Antibacterial activity of both compounds
against some species of bacteria was also assayed by
the dilution method, using Mueller-Hinton broth
containing several concentrations of the tested compounds (500, 250 or 125 mg L-1), using optical density (OD600) measurements. Incubation conditions
were the same as in case of the agar dilution method.
RESULTS AND DISCUSSION
Chemistry
Thiosemicarbazide derivatives were obtained
according to the method described in (16). These
compounds were cyclized in 2% NaOH and 4-
X
X
XI
XII
XIII
XIV
R
Rí
3-pyridyl
3-pyridyl
3-pyridyl
3-pyridyl
3-pyridyl
3-pyridyl
C6H5
4-CH3OC6H4
2-FC6H4
4-FC6H4
3-ClC6H4
C5H4N
phenyl-1,2,4-triazoline-5-thione [I] and 4-phenyl-3(pyrid-3-yl)-1,2,4-triazoline-5-thione [II] derivatives were obtained. These compounds reacted with
amines: N-phenylpiperazine, 1-(4-methoxyphenyl)
piperazine, 1-(2-fluorophenyl)piperazine, 1-(4-fluorophenyl)piperazine, 1-(3-chlorophenyl)piperazine
and 1-(4-pyridyl)piperazine. Taking into account the
possibility of thiol-thione isomerism we proved that
all compounds exist in the thione form. In the substitution reactions 1-aminomethyl-4-phenyl-1,2,4triazoline-5-thione [III-VIII ] and 1-aminomethyl4-phenyl-3-(pyrid-3-yl)-1,2,4-triazoline-5-thione
[IX-XIV ] were obtained.
The structure of new compounds was confirmed
by elemental analysis, as well as the IR and 1H NMR
spectra. The signal of protons characteristic for the
ñNH-C=S group found in the substrates was not
observed in the 1H NMR spectra of new compounds.
The detailed physico-chemical data of the new
obtained compounds [III-VIII] and [IX-XIV] are
presented in Table 1 and Table 2, respectively.
The detailed physicochemical data of compounds [IX-XIV] are presented in Table 2.
Microbiology
According to the results from Table 3, the
newly synthesized compounds showed inhibitory
activity on growth of all tested species of aerobic
bacteria but only at high MIC values ranging from
≥500 ñ 250 mg L-1.
As shown in Figs. 1 and 2, both compounds, at
concentrations below MIC values had only slight
448
MONIKA PITUCHA et al.
Table 2. Physical and analytical data for compounds IX-XIV
No.
R
Formula
Yield %
Molecular
M.p.OC
Weight
Analysis
Calculated/Found
%C
C24H24N6S
428.56
%H
%N
H NMR- δ (ppm)
IR (cm-1)
1
KBr
DMSO ñ d6
IX
C6H5
72
155-157
67.26 5.64 19.61
67.3 5.6 19.6
3065 CH arom.
2969, 1427 CH aliph.
1597 C=N
1499 C-N
2.98-3.07 (m, 4H, CH2)
3.15-3.18 (m, 4H, 2CH2)
5.20 (s, 2H, CH2)
6.36-7.53 (m, 10H, 2 arom. benz.)
8.53-8.60 (m, 4H, arom. pyrid.)
X
4-OCH3C6H4 C25H26N6OS 75
458.58
165-166
65.48 5.71 18.32
65.5 5.7 18.3
3050 CH arom.
2942, 1432 CH aliph.
1594 C=N
1470 C-N
2.52-2.69 (m, 4H, 2CH2)
2.98 (s, 3H, CH3)
3.66-3.77 (m, 2H, CH2)
5.26 (s, 2H, CH2)
7.38-7.54 (m, 4H, arom. benz.)
7.64-7.74 (m, 5H, arom. benz.)
8.49-8.62 (m, 4H, arom. pyrid.)
XI
2-FC6H4
C24H23N6SF 74
446.55
170-171
64.55 5.19 18.82
64.6 5.2 18.7
3052 CH arom.
2950, 1451 CH aliph.
1587 C=N
1480 C-N
2.49-2.71 (m, 4H, 2CH2)
2.99-3.03 (m, 4H, 2CH2)
5.21 (s, 2H, CH2)
6.94-7.15 (m, 9H, 2 arom. benz.)
7.44-8.52 (m, 4H, arom. pyrid.)
XII
4-FC6H4
C24H23N6SF 72
446.55
171-172
64.55 5.19 18.82
64.6 5.2 18.7
3044 CH arom.
2965, 1418 CH aliph.
1597 C=N
1455 C-N
2.52-2.71 (m, 4H, 2CH2)
2.98-3.11 (m, 4H, 2CH2)
5.27 (s, 2H,CH2)
6.92-7.71 (m, 9H, 2 arom. benz.)
8.52-8.61 (m, 4H, arom. pyrid.)
XIII
3-ClC6H4
C24H23N6SCl 70
463.00
162-163
62.26 5.01 18.15
62.2 5.2 18.2
3065 CH arom.
2949, 1417 CH aliph.
1591 C=N
1451 C-N
2.52-2.68 (m, 4H, 2CH2)
2.97-3.46 (m, 4H, 2CH2)
5.27 (s, 2H, CH2)
6.77-7.12 (m, 4H, arom. benz.)
7.38-7.71 (m, 4H, arom. benz.)
8.51-8.61 (m, 4H, arom. pyrid.)
XIV
4-C5H4N
C23H23N7S
429.54
64.31 5.39 22.82
64.3 5.4 22.8
3035 CH arom.
2940, 1482 CH aliph.
1597 C=N
1452 C-N
2.93-2.96 (m, 4H, 2CH2)
3.29-3.48 (m, 4H, 2CH2)
5.28 (s, 2H, CH2)
6.83 (d, 2H, arom. pyrid.)
7.40-7.53 (m, 5H, arom. benz.)
8.13 (d, 2H,a rom. pyrid.)
8.50-8.60 (m, 4H, arom. pyrid.)
71
100-101
inhibitory effect on growth of Gram-positive bacteria, except Staphylococcus epidermidis. The growth
of this species was inhibited by compound IX in a
concentration-dependent manner.
In contrast, both compounds at concentrations
below MIC values showed concentration-dependent
inhibitory effect on growth of Gram-negative bacteria (Figs. 1 and 2). This inhibitory effect was
stronger in case of compound III compared to that
of compound IX. The observed difference may be
probably due to the larger size of molecule of compound IX and hence its lower permeability across
the surface structures (i.e. cell wall, cytoplasmic
membrane) of bacteria.
Our preliminary data suggest that although the
newly synthesized compounds had no significant
antibacterial activity in vitro, compound III appears
to be a promising precursor of compounds with the
increased inhibitory activity especially against
Gram-negative bacteria.
449
Synthesis and biological action of 1-aminomethyl derivatives...
Table 3. Antibacterial activity of the compounds III and IX, assessed by agar dilution method.
Species
Concentration (mg L-1)
Compound III
Compound IX
Gram-positive
bacteria
Staphylococcus aureus
Staphylococcus aureus ATCC25923
Staphylococcus epidermidis
Enterococcus faecalis
500
500
500
500
>500
>500
250
>500
Gram-negative
bacteria
Escherichia coli
Escherichia coli ATCC35218
Klebsiella pneumoniae
Proteus vulgaris
Enterobacter aerogenes
Pseudomonas aeruginosa
500
500
500
500
500
500
>500
>500
>500
500
>500
>500
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Received: 12.07.2005