Synthesis and antibacterial activity of some novel N2-hydroxymethyl and N2-aminomethyl derivatives of 4-aryl-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4- triazole-3-thione

Article abstract of DOI:10.1002/hc.20737

In this investigation, several novel N2-hydroxymethyl and N2-aminomethyl derivatives of 5-(3-chlorophenyl)-4-(4-methylphenyl)-2,4-dihydro- 3H-1,2,4-triazole-3-thione and 4-(4-bromophenyl)- 5-(3-chlorophenyl)-2,4- dihydro-3H-1,2,4-triazole-3- thione were prepared. All synthesized compounds were screened for their antibacterial activity against six Gram-positive and four Gram-negative bacterial strains.

Full text of DOI:10.1002/hc.20737

Heteroatom Chemistry
Volume 22, Number 6, 2011
Synthesis and Antibacterial Activity of Some
Novel N2-Hydroxymethyl and N2-Aminomethyl
Derivatives of 4-aryl-5-(3-Chlorophenyl)-2,4-
Dihydro-3H-1,2,4-Triazole-3-Thione
Tomasz Plech,1 Monika Wujec,1 Barbara Kapron´,1
Urszula Kosikowska,2 and Anna Malm2
1Department of Organic Chemistry, Faculty of Pharmacy, Medical University,
Chodzki 4A, 20-093 Lublin, Poland
2Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University,
Chodzki 1, 20-093 Lublin, Poland
Received 30 November 2010; revised 29 April 2011
ity of aminomethyl (strictly: pyrrolidin-1-ylmethyl)
ABSTRACT: In this investigation, several novel N2-
derivatives of 4-aryl-5-(3-chlorophenyl)-2,4-dihydro-
hydroxymethyl and N2-aminomethyl derivatives of
3H-1,2,4-triazole-3-thione. Some authors report that
5-(3-chlorophenyl)-4-(4-methylphenyl)-2,4-dihydro-
the aminomethylation of 1,2,4-triazoles permits
3H-1,2,4-triazole-3-thione and 4-(4-bromophenyl)-
them to obtain more active compounds [13–15], but
5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-
the results of studies by other researchers do not con-
thione were prepared. All synthesized compounds
firm this thesis [16–18]. Using the results obtained
were screened for their antibacterial activity
by us it has been discovered that, for the described
against six Gram-positive and four Gram-negative
compounds, the Mannich reaction gave more active
ꢀ
C
bacterial strains.
2011 Wiley Periodicals, Inc. Het-
derivatives when in the N-4 position a phenyl ring
with electron-donating substituents (CH3, OCH3)
was present. On the other hand, for the 1,2,4-triazole
derivatives with electron-withdrawing substituents
in the phenyl ring (in the N-4 position), no increased
antibacterial activity was found after transforming
these compounds into respective Mannich bases.
As in the cited work, only pyrrolidin-1-ylmethyl
derivatives of 4-aryl-5-(3-chlorophenyl)-2,4-dihydro-
3H-1,2,4-triazole-3-thione were described; it seemed
useful to check how the antibacterial activity of
the compounds will change after the introduc-
tion of different substituents in the N-2 position.
Figure 1 shows the parent compounds (A, B) that
were used as the substrates in the Mannich reaction.
The choice of these compounds resulted from their
completely different microbiological and physico-
chemical properties (A—microbiologically inactive
eroatom Chem 22:737–743, 2011; View this article online
at wileyonlinelibrary.com. DOI 10.1002/hc.20737
INTRODUCTION
The triazole ring is considered to be an im-
portant element of many pharmacophore struc-
tures. It determines antifungal [1,2], antibacterial
[3,4], antiviral [5], antiepileptic [6], anticancer [7–
9], anxiolytic [10], and antimigraine [11] activi-
ties, and others. In our previous study [12] we
described, among others, the antibacterial activ-
Correspondence to: Tomasz Plech; e-mail: tomasz.plech@
umlub.pl.
c
ꢀ
2011 Wiley Periodicals, Inc.
737

738 Plech et al.
Hydroxymethyl derivatives (1, 2) were obtained
from the reaction of compounds (A, B, respectively)
with formaldehyde while the compounds (3–14)
were synthesized using the so-called Mannich re-
action. Its mechanism was extensively described by
Almajan et al. [18]. For all the described substances
the reaction time was 1 h, while the reaction yields
were from 74 to 90% (Table 1). In the 1H NMR
spectra, the signal of the two protons of the CH2
group (bound to the N-2 nitrogen atom) was vis-
ible in the range of 5.19–5.74 ppm. For the com-
pounds (1, 2), the signal of the hydroxyl group
proton was registered at 7.09 ppm and 7.14 ppm,
respectively. The chemical shifts of the aromatic
protons were in the range of 6.85–7.76 ppm. Proof
that the reaction is completed can also be found
in the disappearance of the peak of the proton
bound to the N-2 nitrogen atom in the initial com-
pounds (A and B), observed earlier at 14.01 ppm
and 14.24 ppm, respectively. In the IR spectra, char-
acteristic bands are visible: for the C S group in
the range of 1317–1348 cm−1, for aromatic rings
in the range of 3009–3112 cm−1, and for aliphatic
C-H bonds—the vibrations in the range of 2809–
2991 cm−1. Furthermore, the N2-hydroxymethyl
derivatives (1, 2) have characteristic wide bands of
stretching vibrations at 3437 cm−1 and at 3401 cm−1,
respectively.
FIGURE 1 Structure of the parent compounds (A—inactive,
MIC >1000 μg/mL), (B—active, MIC = 15.63–31.25 μg/mL).
compound with electron-donating substituent in
the para position of the phenyl ring; B—
microbiologically active compound with electron-
withdrawing substituent in this position).
RESULTS AND DISCUSSION
Chemistry
The parent compounds (A, B) were synthesized ac-
cording to the method reported in [12]. The syn-
thesis of compounds (1–14) has been carried out as
depicted in Fig. 2.
FIGURE 2 Synthesis of compounds 1–14. Reagents and conditions: (a) HCOH, EtOH, rt, 1 h; (b) HCOH, amine, EtOH, rt,
1 h.
Heteroatom Chemistry DOI 10.1002/hc

Synthesis and Antibacterial Activity of Some Novel N2-Hydroxymethyl and N2-Aminomethyl Derivatives 739
TABLE 1 Physical Data of the Compounds 1–14
No.
R1
R2
M.p. (◦C)
Yield (%)
M.w
1
2
3
4
5
–CH3
–Br
–CH3
–Br
–
–
142–144
130–131
112–114
96–98
86
74
75
76
83
331.82
396.69
386.94
451.81
398.95
-N(C2H5)2
–N(C2H5)2
–CH3
118–120
6
7
–Br
–CH3
106–108
146–148
77
79
463.82
400.92
8
9
–Br
–CH3
138–140
120–122
82
83
465.79
476.04
10
11
–Br
78–80
80
90
540.90
494.03
–CH3
150–152
12
13
–Br
88–90
88
90
558.90
713.74
–CH3
252–254
14
–Br
212–214
79
843.48
against Gram-negative bacteria were insignificant
(MIC ≥500), which is why in Table 2 only the values
of MIC (and/or MBC) for the strains of Gram-positive
bacteria are presented.
Microbiological Part
The determination of the antibacterial activity of the
newly synthesized compounds was made with the
use of the broth microdilution method. The micro-
biological studies were performed for six strains of
Gram-positive and 4 strains of Gram-negative bac-
teria. The activity was expressed as the minimum
concentration that inhibits the growth of the bac-
teria (MIC—minimal inhibitory concentration); for
some substances the values of minimal bacterici-
dal concentration (MBC) were also determined. Ce-
furoxime and ampicillin were used as reference an-
tibiotics. The values of MIC (and/or MBC) are given
in Table 2. The activities of the substances (1–14)
The N2-substituted derivatives of 4-(4-bromo-
phenyl)-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (B) completely inhibited the
growth of the bacteria at the concentration of
31.25–125◦C μg/mL, with the only exception being
the inactive compound (14). The most promising
activity was that of the N2-hydroxymethyl derivative
(2), which acted against B. cereus ATCC 10876 twice
as strongly as ampicillin. Moreover, four other
derivatives (4, 6, 8, 10) of the initial compound (B)
demonstrated an antibacterial activity equal to that
Heteroatom Chemistry DOI 10.1002/hc

740 Plech et al.
TABLE 2 In vitro Antibacterial Screening of the Compounds 1–14 (MIC and MBC Values Are Given in μg/mL)
Microorganisms
S. aureus
ATCC 25923
S. aureus
ATCC 6538
S. epidermidis
ATCC 12228
B. subtilis
ATCC 6633
B. cereus
ATCC 10876
M. luteus
ATCC 10240
Compounds
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
Aa
>1000
31.25
62.5
31.25
62.5
31.25 >1000
62.5 >1000
31.25 >1000
125 >1000
31.25 >1000
250
62.5
125
nd
nd
nd
nd
>1000
>1000
31.25
125
62.5
62.5
62.5
125
62.5
125
125
250
125
250
125
nd
nd
nd
>1000
31.25
125
62.5
125
62.5
125
62.5
125
125
250
125
250
nd
nd
nd
>1000
15.63
62.5
31.25
125
62.5
125
62.5
125
62.5
250
62.5
62.5
125
>1000
>1000
nd
nd
nd
nd
>1000
15.63
62.5
31.25
62.5
62.5
125
62.5
125
62.5
250
62.5
125
125
>1000
>1000
62.5
31.25
nd
nd
nd
nd
>1000
>1000 31.25
>1000
1000
>1000
>1000 31.25
nd
nd
nd
nd
nd
nd
nd
nd
>1000
31.25
125
62.5
62.5
nd
nd
nd
Ba
1
2
nd
nd
nd
nd
3
>1000
>1000
1000
>1000
>1000
>1000
nd
nd
nd
nd
nd
>1000
>1000
>1000
>1000
>1000
>1000
nd
nd
nd
nd
nd
500
500
500
1000
1000
1000
nd
nd
nd
nd
nd
1000
500
>1000
4
5
62.5
31.25 >1000
62.5
6
7
>1000
500
nd
nd
nd
nd
nd
nd
nd
8
9
nd
nd
nd
nd
nd
nd
nd
nd
250
62.5
250
10
11
12
125
125
125
13
>1000
>1000
nd
>1000
>1000
nd
>1000
>1000
nd
>1000
>1000
nd
14
nd
nd
nd
nd
nd
nd
nd
nd
nd
Ampicillin
Cefuroxime
0.49
0.98
0.24
15.63
0.98
nd
nd—not determined. a—data derived from [12].
of ampicillin against this strain. Unfortunately, none
of the introduced substituents caused an intensifica-
tion of the action of the initial compound (B). On the
other hand, some very interesting results have been
obtained for the derivatives of the antibacterially
inactive 5-(3-chlorophenyl)-4-(4-methylphenyl)-2,4-
dihydro-3H-1,2,4-triazole-3-thione (A). With the
exception of compound (13), which was inactive,
the remaining N2-substituted derivatives showed
the activity on the level of concentrations 62.5–
250 μg/mL. The N2-hydroxymethyl derivative (1)
and the N2-(diethylamino)methyl derivative (3)
affected the B. cereus ATCC 10876 strain as strongly
as the reference antibiotic (ampicillin). When com-
pared to the inactive initial compound (A), it is a
significant, positive change. Interesting conclusions
are also provided by the comparison of the MIC
and MBC values for the tested substances. A high
value of the MBC/MIC ratio suggests that these
compounds display a bacteriostatic mechanism of
action.
25923, a significant dependence structure/activity is
visible. The introduction of new substituents into
the active compound (B) does not improve its ac-
tivity in any way. The parent compound (B) and its
derivatives (2, 4, 6, 8) are characterized by the same
MIC values. Only after the introduction of large-
volume substituents (10, 12, 14) does the efficiency
of these derivatives decrease gradually. It could be
supposed that a substituent in the N-2 position could
be a steric hindrance, which impedes (10, 12) or
totally prevents (14) the binding with the potential
drug-target sites. Another situation is observed in
the case of substance (A), initially inactive, which
after the transformation into N2-substituted deriva-
tives starts to act as a significant antibacterial agent.
In summary, it is possible to propose the following
hypotheses: (1) a substituent in the N-4 position de-
termines the antibacterial activity to a much larger
extent than a substituent in the N-2 position; (2)
the presence of an electron-withdrawing substituent
(-Br) at the phenyl ring is important—probably be-
cause the electron characteristic of this substituent
determines the possibility of binding the molecule
with the potential drug-target site; (3) the interac-
tion of the 4-bromophenyl moiety with the active
site is strong enough for the influence of the pres-
ence of a substituent in the N-2 position to be un-
noticeable (but only until this substituent starts to
be a steric hindrance, impeding the binding of the
Despite a large number of scientific reports on
the antibacterial activity of the derivatives of 1,2,4-
triazole, the mechanism of action of these sub-
stances has not been found yet. Knowing this mech-
anism would help in fighting an increasing amount
of common drug-resistant strains of pathogen bacte-
ria, such as S. aureus. For most of the studied bacte-
rial strains, and particularly for the S. aureus ATCC
Heteroatom Chemistry DOI 10.1002/hc

Synthesis and Antibacterial Activity of Some Novel N2-Hydroxymethyl and N2-Aminomethyl Derivatives 741
whole molecule); and (4) the influence of the N2-
General Procedure for the Synthesis of N2-
aminomethyl Derivatives (3–14). 10 mmol of the
starting compound (A or B) was dissolved in 20 mL
of anhydrous ethanol and then equimolar amounts
of appropriate secondary amine and formaldehyde
(37%) were added to this solution. In order to
obtain the compounds (13) and (14), a double ex-
cess of the triazole derivative (A or B, respectively),
in relation to the quantity of piperazine, was used.
The obtained mixture was stirred at room temper-
ature for 1 h. Next, distilled water was added and
the precipitate formed was filtered off, washed sev-
eral times with distilled water, and crystallized from
ethanol.
5-(3-Chlorophenyl)-2-[(diethylamino)methyl]-4-
(4-methylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-
thione (3) 1H NMR (250 MHz) (CDCl3) δ (ppm):
1.24 (t, 6H, 2 × CH3, J = 7.2 Hz), 2.46 (s, 3H, CH3),
2.94 (q, 4H, 2 × CH2, J = 7.1 Hz), 5.35 (s, 2H, CH2),
7.11–7.54 (m, 8H, Ar-H). IR (KBr, ν, cm−1): 3042,
3020, 2991, 2906, 1601, 1524, 1491, 1319, 701. Anal.
Calc. for C20H23ClN4S (%): C 62.08, H 5.99, N 14.48.
Found: C 62.00, H 6.11, N 14.32.
substituted fragment becomes visible in the case
of substances with electron-donating substituents
bound to the phenyl ring in the N-4 position of the
1,2,4-triazole. Due to the relatively small number of
compounds, the proposed hypotheses are only pre-
liminary and need to be completed. This will be done
successively.
EXPERIMENTAL
Chemistry
General Comments. All reagents were pur-
chased from Lancaster and Merck Co. and used
without further purification. Melting points were de-
termined by using Fischer-Johns apparatus (Sanyo,
1
Japan) and are uncorrected. The H NMR spectra
were recorded on a Bruker Avance 250 MHz in-
strument using DMSO-d6 or CDCl3 as solvents and
TMS as an internal standard. Chemical shifts are ex-
pressed as δ (ppm). The IR spectra were recorded
in KBr discs using a Perkin-Elmer 1725X FTIR
spectrometer. The purity of the compounds was
checked by TLC on plates precoated with silica
gel Si 60 F254, produced by Merck Co. (Darmstadt,
Germany). The spots were detected by the exposure
to UV-lamp at λ = 254 nm. Elemental analyses were
performed on AMZ 851 CHX analyzer and the results
were within 0.4% of the theoretical value.
4-(4-Bromophenyl)-5-(3-chlorophenyl)-2-[(dieth-
ylamino)methyl]-2,4-dihydro-3H-1,2,4-triazole-3-
thione (4) 1H NMR (250 MHz) (CDCl3) δ (ppm):
1.23 (t, 6H, 2 × CH3, J = 7.1 Hz), 2.93 (q, 4H, 2
× CH2, J = 7.1 Hz), 5.34 (s, 2H, CH2), 7.06–7.68
(m, 8H, Ar-H). IR (KBr, ν, cm−1): 3050, 3026, 2967,
2843, 1574, 1539, 1492, 1412, 1330, 809. Anal. Calc.
for C19H20BrClN4S (%): C 50.51, H 4.46, N 12.40.
Found: C 50.56, H 4.38, N 12.43.
General Procedure for the Synthesis of N2-
hydroxymethyl Derivatives (1, 2). A mixture of
10 mmol of compounds (A) or (B) and formalde-
hyde (37%, 15 mmol) in ethanol medium (20 ml)
was stirred at room temperature for 1 h. The ob-
tained products were filtered off, dried, and crystal-
lized from ethanol.
5-(3-Chlorophenyl)-4-(4-methylphenyl)-2-(piperi-
din-1-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione
(5) H NMR (250 MHz) (CDCl3) δ (ppm): 1.41–1.50
1
(m, 2H, CH2), 1.61–1.69 (m, 4H, 2 × CH2), 2.46 (s,
3H, CH3), 2.90 (t, 4H, 2 × CH2, J = 5.4 Hz), 5.27
(s, 2H, CH2), 7.12–7.50 (m, 8H, Ar-H). IR (KBr, ν,
cm−1): 3067, 2983, 2871, 1590, 1520, 1482, 1341,
714. Anal. Calc. for C21H23ClN4S (%): C 63.22, H
5.81, N 14.04. Found: C 63.36, H 5.88, N 14.11.
4-(4-Bromophenyl)-5-(3-chlorophenyl)-2-(piperi-
din-1-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione
(6) Yield 77%, m.p. 106–108◦C. 1H NMR (250 MHz)
(CDCl3) δ (ppm): 1.46–1.50 (m, 2H, CH2), 1.61–1.69
(m, 4H, 2 × CH2), 2.89 (t, 4H, 2 × CH2, J = 5.4 Hz),
5.26 (s, 2H, CH2), 7.07–7.72 (m, 8H, Ar-H). IR (KBr,
ν, cm−1): 3061, 2982, 2860, 1591, 1541, 1417, 1329,
764. Anal. Calc. for C20H20BrClN4S (%): C 51.79, H
4.35, N 12.08. Found: C 51.85, H 4.21, N 12.18.
5-(3-Chlorophenyl)-4-(4-methylphenyl)-2-(morpho-
lin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione
5-(3-Chlorophenyl)-2-(hydroxymethyl)-4-(4-methy-
lphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione
(1)
1H NMR (250 MHz) (CDCl3) δ (ppm): 2.43 (s, 3H,
CH3), 5.74 (s, 2H, CH2), 7.09 (s, 1H, OH), 7.18–7.46
(m, 8H, Ar-H). IR (KBr, ν, cm−1): 3437, 3091, 2962,
2903, 1588, 1519, 1481, 1333, 709. Anal. Calc. for
C16H14ClN3OS (%): C 57.91, H 4.25, N 12.66. Found:
C 58.02, H 4.19, N 12.75.
4-(4-Bromophenyl)-5-(3-chlorophenyl)-2-(hydro-
xymethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2)
1H NMR (250 MHz) (DMSO-d6) δ (ppm): 5.62 (d,
2H, CH2, J = 7.6 Hz), 7.14 (t, 1H, OH, J = 7.3 Hz),
7.28–7.81 (m, 8H, Ar-H). IR (KBr, ν, cm−1): 3401,
3055, 2972, 2899, 1583, 1529, 1491, 1333, 714. Anal.
Calc. for C15H11BrClN3OS (%): C 45.42, H 2.79, N
10.59. Found: C 45.30, H 2.97, N 10.41.
1
(7) H NMR (250 MHz) (CDCl3) δ (ppm): 2.46 (s,
3H, CH3), 2.96 (t, 4H, 2 × CH2, J = 4.7 Hz), 3.77
(t, 4H, 2 × CH2, J = 4.6 Hz), 5.28 (s, 2H, CH2),
Heteroatom Chemistry DOI 10.1002/hc

742 Plech et al.
7.11–7.56 (m, 8H, Ar-H). IR (KBr, ν, cm−1): 3051,
2980, 2899, 1583, 1513, 1478, 1331, 777. Anal. Calc.
for C20H21ClN4OS (%): C 59.91, H 5.28, N 13.97.
Found: C 59.82, H 5.20, N 13.80.
(KBr, ν, cm−1): 3112, 3046, 2961, 2915, 2862, 1594,
1505, 1466, 1348, 753. Anal. Calc. for C36H34Cl2N8S2
(%): C 60.58, H 4.80, N 15.70. Found: C 60.66, H
5.01, N 15.83.
4-(4-Bromophenyl)-5-(3-chlorophenyl)-2-(morpho-
lin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione
(8) H NMR (250 MHz) (CDCl3) δ (ppm): 2.95 (t,
4-(4-Bromophenyl)-2-{[4-[[4-(4-bromophenyl)-3-
(3-chlorophenyl)-5-thioxo-1,2,4-triazol-1-yl]methyl]
piperazin-1-yl]methyl}-5-(3-chlorophenyl)-1,2,4-tria-
1
1
4H, 2 × CH2, J = 4.6 Hz), 3.77 (t, 4H, 2 × CH2,
J = 4.6 Hz), 5.27 (s, 2H, CH2), 7.05–7.73 (m, 8H,
Ar-H). IR (KBr, ν, cm−1): 3052, 2935, 2909, 2851,
1575, 1552, 1489, 1436, 1327, 774. Anal. Calc. for
C19H18BrClN4OS (%): C 48.99, H 3.90, N 12.03.
Found: C 48.82, H 3.95, N 12.00.
5-(3-Chlorophenyl)-4-(4-methylphenyl)-2-[(4-phe-
nylpiperazin-1-yl)methyl]-2,4-dihydro-3H-1,2,4-tria-
zole-3-thione (9) 1H NMR (250 MHz) (CDCl3) δ
(ppm): 2.46 (s, 3H, CH3), 3.15–3.28 (m, 8H, 4 ×
CH2), 5.29 (s, 2H, CH2), 7.18–7.50 (m, 13H, Ar-H).
IR (KBr, ν, cm−1): 3052, 3009, 2927, 2838, 1598,
1527, 1478, 1345, 801. Anal. Calc. for C26H26ClN5S
(%): C 65.60, H 5.51, N 14.71. Found: C 65.50, H
5.63, N 14.85.
4-(4-Bromophenyl)-5-(3-chlorophenyl)-2-[(4-phe-
nylpiperazin-1-yl)methyl]-2,4-dihydro-3H-1,2,4-tria-
zole-3-thione (10) 1H NMR (250 MHz) (CDCl3) δ
(ppm): 3.10–3.16 (m, 4H, 2 × CH2), 3.23–3.30 (m,
4H, 2 × CH2), 5.37 (s, 2H, CH2), 6.85–7.72 (m,
13H, Ar-H). IR (KBr, ν, cm−1): 3061, 3039, 2985,
2809, 1591, 1540, 1477, 1348, 740. Anal. Calc. for
C25H23BrClN5S (%): C 55.51, H 4.29, N 12.95. Found:
C 55.56, H 4.31, N 12.91.
zole-3-thione (14) H NMR (250 MHz) (DMSO-d6)
δ (ppm): 2.89 (br. s, 8H, 4 × CH2), 5.19 (s, 4H, 2 ×
CH2), 7.33–7.76 (m, 16H, Ar-H). IR (KBr, ν, cm−1):
3078, 3015, 2909, 2861, 2833, 1603, 1525, 1477,
1319, 750. Anal. Calc. for C34H28Br2Cl2N8S2 (%): C
48.41, H 3.35, N 13.28. Found: C 48.31, H 3.28, N
13.34.
Microbiology
The antimicrobial activity of the compounds was
tested on the Gram-positive strains (Staphylo-
coccus aureus ATCC 25923, Staphylococcus au-
reus ATCC 6538, Staphylococcus epidermidis ATCC
12228, Bacillus subtilis ATCC 6633, Bacillus cereus
ATCC 10876, Micrococcus luteus ATCC 10240) and
on the Gram-negative strains (Escherichia coli ATCC
25922, Klebsiella pneumoniae ATCC 13883, Pro-
teus mirabilis ATCC 12453, Pseudomonas aerugi-
nosa ATCC 9027). Ampicillin and cefuroxime, an-
tibiotics widely used in the treatment of infectious
diseases, were used as control antimicrobial agents.
MIC, defined as the lowest concentration of com-
pound at which there was no visible growth of
tested microorganisms, was achieved by a broth mi-
crodilution method, according to the CLSI recom-
mendation [19]. Briefly, microbial suspensions were
prepared in sterile saline with an optical density
of 0.5 McFarland standard −150 × 106 CFU/mL
(CFU—colony forming unit). All stock solutions of
the tested compounds were dissolved in dimethyl
sulfoxide. Mueller–Hinton broth was used with a se-
ries of two-fold dilutions of the tested substances
in the range of final concentrations from 3.91 to
1000 μg/mL. MBC was determined by a broth mi-
crodilution technique by plating out the contents of
wells (20 μL) that showed no visible growth of bacte-
ria, onto Mueller-Hinton agar plates and incubating
at 35◦C for 18 h. MBC was defined as the lowest
concentration of compound that resulted in >99.9%
reduction in CFU of the initial inoculum. All the tests
were repeated three times and the results were aver-
aged.
5-(3-Chlorophenyl)-2-{[4-(4-fluorophenyl)pipera-
zin-1-yl]methyl}-4-(4-methylphenyl)-2,4-dihydro-
1
3H-1,2,4-triazole-3-thione (11) H-NMR (250 MHz)
(CDCl3) δ (ppm): 2.46 (s, 3H, CH3), 3.19 (br. s, 8H, 4 ×
CH2), 5.38 (s, 2H, CH2), 7.21–7.51 (m, 12H, Ar-H).
IR (KBr, ν, cm−1): 3069, 2941, 2861, 1584, 1531,
1510, 1470, 1317, 698. Anal. Calc. for C26H25ClFN5S
(%): C 63.21, H 5.10, N 14.18. Found: C 63.32, H
5.00, N 14.08.
4-(4-Bromophenyl)-5-(3-chlorophenyl)-2-{[4-(4-
fluorophenyl)piperazin-1-yl]methyl}-2,4-dihydro-3H-
1,2,4-triazole-3-thione (12) 1H NMR (250 MHz)
(CDCl3) δ (ppm): 3.13–3.19 (m, 8H, 4 × CH2), 5.37
(s, 2H, CH2), 6.93–7.70 (m, 12H, Ar-H). IR (KBr, ν,
cm−1): 3071, 3051, 2977, 2843, 1588, 1532, 1485,
1332, 763. Anal. Calc. for C25H22BrClFN5S (%): C
53.73, H 3.97, N 12.53. Found: C 53.79, H 4.08, N
12.44.
5-(3-Chlorophenyl)-2-{[4-[[3-(3-chlorophenyl)-4-
(4-methylphenyl)-5-thioxo-1,2,4-triazol-1-yl]methyl]
piperazin-1-yl]methyl}-4-(4-methylphenyl)-1,2,4-tria-
zole-3-thione (13) 1H NMR (250 MHz) (CDCl3) δ
(ppm): 2.46 (s, 6H, 2 × CH3), 3.03 (s, 8H, 4 × CH2),
5.29 (s, 4H, 2 × CH2), 7.10–7.48 (m, 16H, Ar-H). IR
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