Microbiologically active Mannich bases derived from 1,2,4-triazoles. the effect of C-5 substituent on antibacterial activity

Article abstract of DOI:10.1007/s00044-012-0248-y

Our research proved that chemical character of the C-5 substituent significantly determines the antibacterial activity of the Mannich bases derived from 4,5-disubstituted 1,2,4-triazole-3-thiones. This activity was considerably increased by an introduction of a chlorine atom to the phenyl ring. The obtained compounds were particularly active against opportunistic bacteria (Bacillus subtilis and Bacillus cereus). The antibacterial activity of some Mannich bases was similar or higher than the activity of commonly used antibiotics such as ampicillin and cefuroxime.

Full text of DOI:10.1007/s00044-012-0248-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2531–2537
DOI 10.1007/s00044-012-0248-y
ORIGINAL RESEARCH
Microbiologically active Mannich bases derived from
1,2,4-triazoles. The effect of C-5 substituent on antibacterial
activity
•
Tomasz Plech Monika Wujec Magdalena Majewska
•
•
•
Urszula Kosikowska Anna Malm
Received: 17 March 2012 / Accepted: 14 September 2012 / Published online: 29 September 2012
Ó The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract Our research proved that chemical character of
the C-5 substituent significantly determines the antibacte-
rial activity of the Mannich bases derived from 4,5-
disubstituted 1,2,4-triazole-3-thiones. This activity was
considerably increased by an introduction of a chlorine
atom to the phenyl ring. The obtained compounds were
particularly active against opportunistic bacteria (Bacillus
subtilis and Bacillus cereus). The antibacterial activity of
some Mannich bases was similar or higher than the activity
of commonly used antibiotics such as ampicillin and
cefuroxime.
appearance of the so-called super resistant NDM-1 gene
(Yong et al., 2009; Rolain et al., 2010) which spreads via
DNA segments called plasmids. In the view of growing
bacterial drug-resistance, the search of chemical substances
which can efficiently treat infections caused by this type of
bacteria seems to be necessary.
The Mannich reaction is known to be very useful for
the synthesis of antibacterial compounds. This reaction
makes it possible to introduce amine fragment into the
different chemical scaffolds which can increase the
affinity of the obtained molecule toward appropriate
molecular target. 1,2,4-Triazole-3-thione derivatives
known for their antibacterial activity (Turan-Zitouni et al.,
2005; Eswaran et al., 2009; Shafiee et al., 2002) were
used by many researchers as substrates for the Mannich
reaction. The obtained aminomethyl derivatives included
both compounds which acted stronger than their N2-un-
substituted predecessors (Isloor et al., 2009; Ashok et al.,
2007; Bayrak et al., 2009a), as well as significantly less
active compounds (Bayrak et al., 2009b; Almajan et al.,
2009). In our previous studies we proved that the presence
of the 4-bromophenyl moiety in the N-4 position benefited
the antibacterial activity of 4,5-disubstituted 1,2,4-tria-
zole-3-thione derivatives (Plech et al., 2011a, b). Further
research also indicated that the activity of this type of
Mannich bases decreases with the increased volume of
substituent in the N2 position (Plech et al., 2011b). The
goal of current research was to analyze the impact of the
substituent in the C-5 position on the antibacterial activity
of obtained compounds. First of all, it has been decided to
examine if, and to what degree, the strength of the new
derivatives’ activity changes after introducing a chlorine
atom to the phenyl ring. Also, the disparities in the
activity of appropriate ortho-, meta-, and para- derivatives
were analyzed.
Keywords Aminomethylation Á Five-membered ring Á
Opportunistic bacteria Á MRSA
Introduction
Excessive and uncontrolled intake of antibiotics resulted in
a selection of bacterial strains resistant to commonly used
drugs. Recently, the world has been focused on the
Study results were presented in part at the Eighth Multidisciplinary
Conference on Drug Research, Rawa Mazowiecka, Poland (June,
2012).
T. Plech (&) Á M. Wujec Á M. Majewska
Department of Organic Chemistry, Faculty of Pharmacy,
Medical University, Chodzki 4A, 20-093 Lublin, Poland
e-mail: tomasz.plech@umlub.pl
U. Kosikowska Á A. Malm
Department of Pharmaceutical Microbiology, Faculty of
Pharmacy, Medical University, Chodzki 1, 20-093 Lublin,
Poland
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Med Chem Res (2013) 22:2531–2537
Results and discussion
Chemistry
existence of the mentioned compounds as the C=S
tautomers.
The Mannich reaction was carried out in mild conditions;
it was quick (30 min) and efficient (yields: 76–87 %). The
structure and purity of the products (10–21) was confirmed
using 1H-NMR, 13C-NMR (for compound 20), and IR
spectra as well as elemental analysis. The 1H-NMR spectra
showed characteristic signals which indicated the presence
of aminomethyl fragment. Two protons of the N2–CH2–
group resonated as a singlet in the range of 5.22–5.34 ppm,
while the signals of the amine residues were visible at
1.20–3.76 ppm. In addition to this, peaks characteristic for
para-substituted phenyl rings were visible in the area typ-
ical for aromatic protons. The IR spectra also confirmed the
suggested structure of the Mannich bases (10–21).
Scheme 1 shows subsequent stages of the synthesis. The
substrates for the syntheses included commercially available
hydrazides (1–3). Appropriate thiosemicarbazide deriva-
tives (4–6) were obtained from the reaction of the hydrazides
(1–3) with 4-bromophenyl isothiocyanate using the method
described earlier (Plech et al., 2011a). The reaction carried
out in the anhydrous ethanol medium lasted 5 min. Spectral
and physicochemical properties of the derivatives 4–6 were
given elsewhere (Li et al., 2001; Oruc¸ et al., 2004). The
cyclization of compounds 4–6 in the presence of sodium
hydroxide resulted in the formation of 4-(4-bromophenyl)-5-
substituted-2,4-dihydro-3H-1,2,4-triazole-3-thiones (7–9).
Generally, the 1,2,4-triazole-3-thione derivatives may exist
in two tautomeric forms: thione (C=S) and thiole (C–SH).
Our earlier studies showed that the thione tautomer is ener-
getically favored (Wujec et al., 2007). The IR spectra
of compounds 7–9 showed the absorption bands at
3,437–3,411 cm-1 and 1,331–1,328 cm-1, indicating the
Antibacterial screening
The antibacterial activity of compounds 10–21 was deter-
mined for Gram-positive and Gram-negative bacteria. The
growth of Gram-negative bacteria (Escherichia coli ATCC
25922, Klebsiella pneumoniae ATCC 13883, Proteus
mirabilis ATCC 12453, and Pseudomonas aeruginosa
ATCC 9027) was not inhibited by any of the compounds.
Therefore, Table 1 shows the Mannich bases activity only
1
presence of NH and C=S groups, respectively. In the H-
NMR spectra, NH proton resonated as a singlet at *14 ppm.
Crystallographic data (unpublished results) also confirm the
Scheme 1 Synthetic route to
target compounds 10–21.
O
NCS
O
Reagents and conditions:
a EtOH, reflux, 5 min; b 2 %
NaOH, reflux, 2 h; c HCHO,
amine, EtOH, 30 min
a
NH
NH
NH
NH
+
NH2
S
R1
Br
R1
1-3
4-6
Br
b
R2
H
N
N
N
N
c
S
S
N
N
R1
R1
10-21
7-9
Br
Br
R1 = H, 2-Cl, 4-Cl
CH3
CH3
N
R2 =
N
N
N
O
,
,
,
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Med Chem Res (2013) 22:2531–2537
2533
Fig. 1 Chemical structures of
compounds 22–25
R
S
CH
CH
3
3
N
N
R =
R =
23.
25.
N
N
R =
R =
22.
24.
N
O
N
N
Cl
Br
Table 1 In vitro antibacterial screening of compounds 10–25 (MICs and MBCs are given in lg ml-1
)
Compounds S. aureus ATCC
25923
S. aureus
(MRSA)
S. epidermidis ATCC B. subtilis ATCC
6633
B. cereus ATCC
10876
M. luteus ATCC
10240
12228
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
10
1,000
1,000
1,000
1,000
62.5
*
1,000
1,000
1,000
1,000
125
125
500
250
62.5
62.5
1,000
125
–
*
1,000
1,000
1,000
1,000
62.5
31.25
1,000
125
*
1,000
500
*
1,000
1,000
1,000
1,000
62.5
62.5
1,000
125
*
1,000
500
*
11
*
*
*
1,000
*
*
1,000
1,000
1,000
250
1,000
*
12
*
*
*
1,000
1,000
31.25
15.63
250
*
500
13
*
*
*
*
*
250
14
*
*
*
62.5
125
*
*
62.5
62.5
500
15
31.25
500
500
*
500
*
500
*
16
*
*
17
125
500
250
500
*
500
250
1,000
*
500
125
250
500
125
*
*
62.5
15.63
7.81
1,000
62.5
31.25
62.5
31.25
31.25
–
250
62.5
250
*
18
31.25
31.25
1,000
62.5
31.25
31.25
1,000
62.5
62.5
250
250
31.25
62.5
1,000
125
31.25
31.25
1,000
62.5
62.5
62.5
62.5
62.5
62.5
31.25
–
250
19
1,000
*
20
*
*
21
1,000
*
*
1,000
125
500
500
1,000
1,000
–
*
*
22a
23b
24a
25a
Ampicillin
31.25
31.25
31.25
31.25
–
–
*
*
*
*
–
–
–
62.5
62.5
62.5
62.5
–
*
500
*
*
–
–
*
*
–
–
62.5
125
1,000
*
*
–
–
*
–
–
–
500
–
–
–
–
–
Cefuroxime 0.49
Vancomycin –
–
–
–
0.24
–
15.63
–
–
0.98
–
–
–
0.98
3.91
–
–
– not determined, * MIC or MBC values higher than 1,000 lg ml-1
a
Data derived from Plech et al. (2011b)
b
Data derived from Plech et al. (2011a)
for five investigated Gram-positive bacterial strains. The
activity toward the pathogenic Staphylococcus aureus
strains was moderate. Minimum concentrations which
inhibited the growth of S. aureus ATCC 25923 ranged to
31.25 lg ml-1 (15, 18, 19), and the most active toward
methicillin-resistant (MRSA) strain were derivatives with
diethylaminomethyl (18) and pyrrolidinylmethyl (19) sub-
stituents. In both cases, the MIC values equaled
62.5 lg ml-1. Opportunistic (relatively pathogenic) bac-
teria was by far more sensitive to the newly obtained
compounds. In the case of Bacillus cereus ATCC 10876,
the activity of three derivatives (14, 15, 21) was similar to
the activity of ampicillin, and the activity of another two
derivatives (18, 19) was twice as strong. Moreover, the
antibacterial activity of the compound with the N2-pyrro-
lidinylmethyl fragment (15) toward Bacillus subtilis ATCC
6633 was as strong as cefuroxime’s; as far as Micrococcus
luteus ATCC 10240 is concerned, the most active com-
pound was the derivative of 4-(4-bromophenyl)-5-(4-
chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione with
pyrrolidinylmethyl substituent (19, MIC = 7.81 lg ml-1).
Based on the MBC/MIC ratio it might be concluded that
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Med Chem Res (2013) 22:2531–2537
the obtained compounds do show bactericidal (MBC/
MIC B4) or bacteriostatic (MBC/MIC [4) activity. This
may suggest a presence of more than one mechanism of
action for these derivatives.
Experimental
General comments
In order to analyze the impact of the type of substituent
in the C-5 position on the antibacterial activity, derivatives
including phenyl (10–13), 2-chlorophenyl (14–17), and
4-chlorophenyl (18–21) rings were obtained. In order to
ensure more comprehensive analysis, the discussion also
considered the compounds with 3-chlorophenyl fragment
(22–25) (Fig. 1)—synthesized and described by us recently
(Plech et al., 2011a, b). Regardless of the type of amino-
methyl substituent in the N2 position, none of the C5-
phenyl derivatives showed antibacterial activity which
would be worth noticing. The activity of the obtained
Mannich bases was significantly increased only after an
electron-withdrawing chlorine atom had been introduced to
the phenyl ring. Interesting conclusions can be drawn when
comparing the activity of appropriate ortho-, meta-, and
para- analogs. Balanced activity toward all analyzed
Gram-positive bacteria was characteristic for compounds
with 3-chlorophenyl fragment, regardless of the type of
substituent in the N2 position. While the activity of ortho-
and para- analogs depended largely on the type of ami-
nomethyl fragment. It is worth mentioning that compounds
with pentatomic (apart from hydrogen atoms) amine sub-
stituent in the N2 position (pyrrolidine and diethylamine)
were microbiologically more active than appropriate
compounds with six-membered substituents (piperidine
and morpholine). Also, the disparity between the activities
of piperidinyl and morpholinyl derivatives shows that the
oxygen atom in the morpholine molecule is important for
the binding with a potential molecular target. This is
probably caused by the fact that the oxygen atom can
participate in the formation of hydrogen bonds in the drug-
target site.
All reagents and solvents were purchased from Alfa Aesar
(Ward Hill, USA) and Merck Co. (Darmstadt, Germany).
Melting points were determined using Fisher-Johns appa-
ratus (Fisher Scientific, Schwerte, Germany) and are
uncorrected. The 1H-NMR and 13C-NMR spectra were
recorded on a Bruker Avance spectrometer (Bruker Bio-
Spin GmbH, Rheinstetten, Germany) using TMS as an
internal standard. The IR spectra (KBr) were obtained on a
Perkin-Elmer 1725X FTIR spectrophotometer. Elemental
analyses were performed on an AMZ 851 CHX analyzer
´
(PG, Gdansk, Poland) and the results were within 0.2 %
of the theoretical value. All the compounds were purified
by flash chromatography (PuriFlash 430evo, Interchim,
USA).
Synthesis of thiosemicarbazide derivatives (4–6)
Three derivatives of thiosemicarbazide: 1-benzoyl-4-(4-
bromophenyl)thiosemicarbazide (4), 4-(4-bromophenyl)-
1-[(2-chlorophenyl)carbonyl]thiosemicarbazide (5), and
4-(4-bromophenyl)-1-[(4-chlorophenyl)carbonyl]thiosem-
icarbazide (6) were synthesized according to the proce-
dure described earlier (Plech et al., 2011a). Their spectral
and physicochemical properties were consistent with
(Li et al., 2001; Oruc¸ et al., 2004).
Synthesis of 1,2,4-triazole derivatives (7–9)
Appropriate thiosemicarbazides (4–6) were dissolved in
2 % solution of NaOH. Next, the resulting solution was
heated under reflux for 2 h. After cooling, the reaction
mixture was neutralized with HCl. The precipitated product
was filtered off, washed with distilled water, and recrys-
tallized from EtOH.
Conclusions
4-(4-Bromophenyl)-5-phenyl-2,4-dihydro-3H-1,2,4-
triazole-3-thione (7)
Our research showed that chemical character of the C-5
substituent significantly determines the antibacterial
activity of the N2-aminomethyl derivatives of the 1,2,4-
triazole. This activity can be considerably increased by an
introduction of an electron-withdrawing chlorine atom to
the phenyl ring in the C-5 position. In addition to this, the
number of atoms which form the aminomethyl substituent
seems to be important. The activity of the obtained Man-
nich bases was particularly strong toward opportunistic
bacteria. The antibacterial activity of some compounds was
similar or higher than the activity of commonly used
antibiotics such as ampicillin and cefuroxime.
Yield: 87 %, CAS Registry Number: 162221-97-8.
4-(4-Bromophenyl)-5-(2-chlorophenyl)-2,4-dihydro-3H-
1,2,4-triazole-3-thione (8)
Yield: 83 %, m.p. 282–284 °C, 1H-NMR (250 MHz)
(DMSO-d6) d (ppm): 7.08–7.76 (m, 8H, Ar–H), 14.03 (s,
1H, NH, exch. D2O). IR (KBr, m, cm-1): 3411, 3045, 1531,
1331. Anal. Calc. for C14H9BrClN3S (%): C 45.86, H 2.47,
N 11.46. Found: C 45.99, H 2.35, N 11.43.
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Med Chem Res (2013) 22:2531–2537
2535
4-(4-Bromophenyl)-5-(4-chlorophenyl)-2,4-dihydro-3H-
1,2,4-triazole-3-thione (9)
4-(4-Bromophenyl)-2-(morpholin-4-ylmethyl)-5-phenyl-
2,4-dihydro-3H-1,2,4-triazole-3-thione (13)
Yield: 82 %, CAS Registry Number: 537017-82-6.
Yield: 83 %, m.p. 146–147 °C, 1H-NMR (250 MHz)
(CDCl3) d (ppm): 2.95 (t, 4H, 2 9 CH2, J = 4.26 Hz),
3.76 (t, 4H, 2 9 CH2, J = 4.26 Hz), 5.26 (s, 2H, CH2),
7.18 (d, 2H, Ar–H, J = 8.80 Hz), 7.24–7.35 (m, 5H, Ar–
H), 7.62 (d, 2H, Ar–H, J = 8.81 Hz). IR (KBr, m, cm-1):
3074, 3021, 2961, 2831, 1574, 1512, 1328, 786. Anal. Calc.
for C19H19BrN4OS (%): C 52.90, H 4.44, N 12.99. Found:
C 52.98, H 4.56, N 13.05.
General procedure for the synthesis of Mannich bases
(10–21)
10 mmol of the 1,2,4-triazole derivative (7–9) was dis-
solved (with heating) in 20 ml of anhydrous ethanol and
then equimolar amounts of appropriate secondary amine
(diethylamine, pyrrolidine, piperidine, and morpholine)
and formaldehyde solution (37 %) were added. The
obtained mixture was stirred at room temperature for
30 min. Next, 5 ml of distilled water was added, the pre-
cipitate was filtered off, washed with distilled water, and
recrystallized from ethanol.
4-(4-Bromophenyl)-5-(2-chlorophenyl)-2-
[(diethylamino)methyl]-2,4-dihydro-3H-1,2,4-triazole-3-
thione (14)
Yield: 79 %, m.p. 172–173 °C, 1H-NMR (250 MHz)
(CDCl3) d (ppm): 1.20 (t, 6H, 2 9 CH3, J = 7.55 Hz),
2.90 (q, 4H, 2 9 CH2, J = 7.55 Hz), 5.30 (s, 2H, CH2),
7.17 (d, 2H, Ar–H, J = 8.89 Hz), 7.22–7.32 (m, 4H, Ar–
H), 7.62 (d, 2H, Ar–H, J = 8.90 Hz). IR (KBr, m, cm-1):
3030, 2986, 2832, 1603, 1541, 1341, 813. Anal. Calc. for
C19H20BrClN4S (%): C 50.51, H 4.46, N 12.40. Found: C
50.41, H 4.38, N 12.29.
4-(4-Bromophenyl)-2-[(diethylamino)methyl]-5-phenyl-
2,4-dihydro-3H-1,2,4-triazole-3-thione (10)
Yield: 78 %, m.p. 118–120 °C, 1H-NMR (250 MHz)
(CDCl3) d (ppm): 1.20 (t, 6H, 2 9 CH3, J = 7.17 Hz),
2.90 (q, 4H, 2 9 CH2, J = 7.18 Hz), 5.32 (s, 2H, CH2),
7.18 (d, 2H, Ar–H, J = 8.69 Hz), 7.25–7.34 (m, 5H, Ar–
H), 7.61 (d, 2H, Ar–H, J = 8.70 Hz). IR (KBr, m, cm-1):
3065, 2931, 2796, 1612, 1520, 1331, 799. Anal. Calc. for
C19H21BrN4S (%): C 54.68, H 5.07, N 13.42. Found: C
54.60, H 5.02, N 13.53.
4-(4-Bromophenyl)-5-(2-chlorophenyl)-2-(pyrrolidin-1-
ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (15)
Yield: 84 %, m.p. 143–145 °C, 1H-NMR (250 MHz)
(CDCl3) d (ppm): 1.76–1.83 (m, 4H, 2 9 CH2), 2.96 (t,
4H, 2 9 CH2, J = 6.40 Hz), 5.32 (s, 2H, CH2), 7.17 (d,
2H, Ar–H, J = 8.75 Hz), 7.22–7.30 (m, 4H, Ar–H), 7.63
(d, 2H, Ar–H, J = 8.75 Hz). IR (KBr, m, cm-1): 3099,
2956, 2825, 1589, 1530, 1327, 802. Anal. Calc. for
C19H18BrClN4S (%): C 50.73, H 4.03, N 12.46. Found: C
50.66, H 4.12, N 12.45.
4-(4-Bromophenyl)-5-phenyl-2-(pyrrolidin-1-ylmethyl)-
2,4-dihydro-3H-1,2,4-triazole-3-thione (11)
Yield: 82 %, m.p. 142–143 °C, 1H-NMR (250 MHz)
(CDCl3) d (ppm): 1.75–1.83 (m, 4H, 2 9 CH2), 2.99 (t,
4H, 2 9 CH2, J = 6.43 Hz), 5.34 (s, 2H, CH2), 7.19 (d,
2H, Ar–H, J = 8.86 Hz), 7.25–7.33 (m, 5H, Ar–H), 7.61
(d, 2H, Ar–H, J = 8.84 Hz). IR (KBr, m, cm-1): 3084,
3008, 2915, 2868, 1584, 1513, 1323, 806. Anal. Calc. for
C19H19BrN4S (%): C 54.94, H 4.61, N 13.49. Found: C
55.05, H 4.50, 13.50.
4-(4-Bromophenyl)-5-(2-chlorophenyl)-2-(piperidin-1-
ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (16)
Yield: 80 %, m.p. 180–181 °C, 1H-NMR (250 MHz)
(CDCl3) d (ppm): 1.36–1.69 (m, 6H, 3 9 CH2), 2.85 (t, 4H,
2 9 CH2, J = 5.40 Hz), 5.22 (s, 2H, CH2), 7.18 (d, 2H, Ar–
H, J = 8.71 Hz), 7.23–7.34 (m, 4H, Ar–H), 7.63 (d, 2H, Ar–
H, J = 8.70 Hz). IR (KBr, m, cm-1): 3062, 2985, 2800,
1594, 1526, 1342, 784. Anal. Calc. for C20H20BrClN4S (%):
C 51.79, H 4.35, N 12.08. Found: C 51.90, H 4.35, N 12.00.
4-(4-Bromophenyl)-5-phenyl-2-(piperidin-1-ylmethyl)-
2,4-dihydro-3H-1,2,4-triazole-3-thione (12)
Yield: 77 %, m.p. 122–123 °C, 1H-NMR (250 MHz)
(CDCl3) d (ppm): 1.44–1.68 (m, 6H, 3 9 CH2), 2.87 (t,
4H, 2 9 CH2, J = 5.40 Hz), 5.25 (s, 2H, CH2), 7.19 (d,
2H, Ar–H, J = 8.90 Hz), 7.24–7.35 (m, 5H, Ar–H), 7.61
(d, 2H, Ar–H, J = 8.90 Hz). IR (KBr, m, cm-1): 3110,
2918, 2785, 1603, 1519, 1342, 808. Anal. Calc. for
C20H21BrN4S (%): C 55.94, H 4.93, N 13.05. Found: C
56.00, H 4.90, N 13.17.
4-(4-Bromophenyl)-5-(2-chlorophenyl)-2-(morpholin-4-
ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (17)
Yield: 76 %, m.p. 174–175 °C, 1H-NMR (250 MHz)
(CDCl3) d (ppm): 2.91 (t, 4H, 2 9 CH2, J = 4.75 Hz),
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Med Chem Res (2013) 22:2531–2537
3.72 (t, 4H, 2 9 CH2, J = 4.75 Hz), 5.23 (s, 2H, CH2),
7.17 (d, 2H, Ar–H, J = 8.81 Hz), 7.23–7.34 (m, 4H, Ar–
H), 7.64 (d, 2H, Ar–H, J = 8.81 Hz). IR (KBr, m, cm-1):
3037, 2903, 2785, 1600, 1521, 1328, 806. Anal. Calc. for
C19H18BrClN4OS (%): C 48.99, H 3.90, N 12.03. Found: C
49.11, H 3.84, N 12.17.
2H, Ar–H, J = 8.70 Hz). IR (KBr, m, cm-1): 3074, 3033,
2951, 2856, 1603, 1541, 1318, 798. Anal. Calc. for
C19H18BrClN4OS (%): C 48.99, H 3.90, N 12.03. Found: C
49.10, H 3.97, N 12.00.
Antibacterial screening
4-(4-Bromophenyl)-5-(4-chlorophenyl)-2-
[(diethylamino)methyl]-2,4-dihydro-3H-1,2,4-triazole-
3-thione (18)
Tested microorganism: S. aureus ATCC 25923, S. aureus
Microbank 14001 (MRSA), Staphylococcus epidermidis
ATCC 12228, B. subtilis ATCC 6633, B. cereus ATCC
10876, M. luteus ATCC 10240, E. coli ATCC 25922, K.
pneumoniae ATCC 13883, P. mirabilis ATCC 12453, and
P. aeruginosa ATCC 9027. Preliminary antibacterial in
vitro potency of the tested compounds was screened using
the agar dilution method on the basis of the growth inhibition
on the Mueller–Hinton agar to which the tested compounds
at concentration 1,000 lg ml-1 were added. The plates were
poured on the day of testing. 10 ll of each bacterial sus-
pension was put onto Mueller–Hinton agar containing the
tested compounds; medium without the compounds was
used as a control. The plates were incubated at 37 °C for
18 h. Then the in vitro antibacterial activity of the com-
pounds with inhibitory effect was determined by broth
microdilution method. Ampicillin, cefuroxime, and vanco-
mycin were used as control antimicrobial agents. The
microbial suspensions were prepared in sterile saline
with an optical density of 0.5 McFarland standard—
150 9 106 CFU ml-1 (CFU—colony forming unit). All
stock solutions of the tested compounds were dissolved in
DMSO. Mueller–Hinton broth was used with a series of
twofold dilutions of the tested substances in the range of
final concentrations from 3.91 to 1,000 lg ml-1. Minimum
inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) are given in lg ml-1 (CLSI 2008).
Yield: 82 %, m.p. 175–176 °C, 1H-NMR (250 MHz)
(CDCl3) d (ppm): 1.20 (t, 6H, 2 9 CH3, J = 7.24 Hz),
2.90 (q, 4H, 2 9 CH2, J = 7.24 Hz), 5.30 (s, 2H, CH2),
7.17 (d, 2H, Ar–H, J = 8.63 Hz), 7.22–7.33 (m, 4H, Ar–
H), 7.62 (d, 2H, Ar–H, J = 8.63 Hz). IR (KBr, m, cm-1):
3088, 3009, 2917, 2826, 1589, 1526, 1319, 778. Anal. Calc.
for C19H20BrClN4S (%): C 50.51, H 4.46, N 12.40. Found:
C 50.43, H 4.52, N 12.41.
4-(4-Bromophenyl)-5-(4-chlorophenyl)-2-(pyrrolidin-1-
ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (19)
Yield: 87 %, m.p. 143–144 °C, 1H-NMR (250 MHz)
(CDCl3) d (ppm): 1.76–1.84 (m, 4H, 2 9 CH2), 2.97 (t,
4H, 2 9 CH2, J = 6.10 Hz), 5.32 (s, 2H, CH2), 7.18 (d,
2H, Ar–H, J = 8.76 Hz), 7.23–7.34 (m, 4H, Ar–H), 7.64
(d, 2H, Ar–H, J = 8.76 Hz). IR (KBr, m, cm-1): 3040,
2921, 2787, 1609, 1541, 1333, 783. Anal. Calc. for
C19H18BrClN4S (%): C 50.73, H 4.03, N 12.46. Found: C
50.62, H 3.92, N 12.52.
4-(4-Bromophenyl)-5-(4-chlorophenyl)-2-(piperidin-1-
ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (20)
Yield: 76 %, m.p. 180–182 °C, 1H-NMR (250 MHz)
(CDCl3) d (ppm): 1.36–1.68 (m, 6H, 3 9 CH2), 2.85 (t,
4H, 2 9 CH2, J = 5.62 Hz), 5.22 (s, 2H, CH2), 7.18 (d,
2H, Ar–H, J = 8.74 Hz), 7.23-7.31 (m, 4H, Ar–H), 7.63
(d, 2H, Ar–H, J = 8.72 Hz). 13C-NMR (90 MHz) (CDCl3)
d (ppm): 23.81, 25.91, 51.82, 71.09, 123.64, 124.10,
129.11, 129.87, 130.02, 133.27, 134.45, 137.27, 148.18,
170.64. IR (KBr, m, cm-1): 3085, 2882, 2790, 1600, 1531,
1323, 809. Anal. Calc. for C20H20BrClN4S (%): C 51.79, H
4.35, N 12.08. Found: C 51.86, H 4.32, N 12.18.
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