Synthesis and antimicrobial activity of thiosemicarbazides, s-triazoles and their Mannich bases bearing 3-chlorophenyl moiety

Article abstract of DOI:10.1016/j.ejmech.2010.11.010

A fast and efficient synthesis of some 1,4-disubstituted thiosemicarbazide derivatives is described. The reaction of 3-chlorobenzoic acid hydrazide with various aryl isothiocyanates gave thiosemicarbazide derivatives (1-11) in good yield. The cyclization of compounds (1-11) in the presence of 2% NaOH resulted in the formation of compounds (12-22) containing the 1,2,4-triazole ring. A series of new Mannich bases (23-33) related to the structure of 1,2,4-triazole has been also synthesized. All of these compounds were tested for their in vitro antibacterial activity against the reference strains of aerobic bacteria - 6 Gram-positive and 3 Gram-negative ones; 12 Staphylococcus aureus clinical isolates were also examined. An attempt was made to clarify the influence of the nature/position of substituents on antibacterial activity of compounds described.

Full text of DOI:10.1016/j.ejmech.2010.11.010

European Journal of Medicinal Chemistry 46 (2011) 241e248
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antimicrobial activity of thiosemicarbazides, s-triazoles and their
Mannich bases bearing 3-chlorophenyl moiety
,
a
a
b
b
Tomasz Plech a , Monika Wujec , Agata Siwek , Urszula Kosikowska , Anna Malm
*
a Department of Organic Chemistry, Faculty of Pharmacy, Medical University, Staszica 6, 20-081 Lublin, Poland
b Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University, Chodzki 1, 20-093 Lublin, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A fast and efficient synthesis of some 1,4-disubstituted thiosemicarbazide derivatives is described. The
reaction of 3-chlorobenzoic acid hydrazide with various aryl isothiocyanates gave thiosemicarbazide
derivatives (1e11) in good yield. The cyclization of compounds (1e11) in the presence of 2% NaOH
resulted in the formation of compounds (12e22) containing the 1,2,4-triazole ring. A series of new
Mannich bases (23e33) related to the structure of 1,2,4-triazole has been also synthesized. All of these
compounds were tested for their in vitro antibacterial activity against the reference strains of aerobic
bacteria - 6 Gram-positive and 3 Gram-negative ones; 12 Staphylococcus aureus clinical isolates were also
examined. An attempt was made to clarify the influence of the nature/position of substituents on
antibacterial activity of compounds described.
Received 5 August 2010
Received in revised form
5 November 2010
Accepted 5 November 2010
Available online 3 December 2010
Keywords:
s-Triazoles
Mannich bases
Minimal inhibitory concentration (MIC)
Minimal bactericidal concentration (MBC)
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
reaction we obtained a series of new derivatives containing a pyr-
rolidin-1-ylmethyl substituent coupled with a 1,2,4-triazole ring.
Since the introduction of the first antibiotic (penicillin, 1942)
into medical practice, to date, there has been an ongoing “race”
between scientists creating new drugs and pathogenic bacteria.
This specific “arms race” causes that thousands of potentially active
chemicals are synthesized in laboratories around the world every
The choice of pyrrolidine as one of the substrates in the Mannich
reaction was dictated by its positive impact on antimicrobial
activity in other groups of drugs [11].
Another issue taken up in this article is the modification of
synthesis of 1,4-disubstituted derivatives of thiosemicarbazide
allowing for maximum shortening of the synthesis duration and
obtaining high reaction efficiency. The synthesis methods adapted
by other researchers include, among others: (i) synthesis in dime-
thylacetamide (DMA) environment [12], (ii) solvent-free synthesis
(solid-state synthesis) [13], (iii) synthesis in dimethylformamide
(DMF)/ethanol mixture [1], and (iv) synthesis in ethanol environ-
ment [14e17]. The use of DMA as a solvent required 24-h heating of
the reaction mixture, whilst the reaction yield reached 88%.
Solvent-free synthesis lasted 12 h and as a result appropriate
1,4-disubstituted thiosemicarbazides with a yield of 85e95% were
obtained. The lowest yield (36e63%) was obtained for the reaction
of hydrazides with isothiocyanates in DMF/EtOH mixture (reaction
time: 4 h). In turn, the said synthesis was carried out in ethanol
environment most frequently. Reaction mixture was heated in this
case for 2 h (66,5e82,5% yield), 3 h (74e96%), 4 h (85e90%). Since
the fact of the sensitivity of isothiocyanates to water is well known,
we used only anhydrous ethanol (contrary to the previously cited
authors) in the experiments the results of which are presented in
this article.
day.
A literature survey reveals that 1,4-disubstituted thio-
semicarbazides as well as 1,2,4-triazoles [1e3] are known as
promising antimicrobial agents. Compounds of the said structure
often exhibit higher activity than standard antibiotics: penicillin
G [4], ampicillin [1], gentamicin [5]. On the other hand, many
authors reveal that the introduction of halogen atoms into the
pharmacophore structure can, in many cases, be beneficial for
antimicrobial activity [6,7]. With this in mind, we have synthesized
a number of compounds in which the nature of the halogen atom
seems to influence their biological action. Moreover, according to
some authors, the improvement of activity of 1,2,4-triazoles can be
obtained through the introduction of substituents in N-2 nitrogen
position of a 1,2,4-triazole ring [8e10]. The aminomethylation
reaction is most commonly used for this purpose and it results in
the creation of the so-called Mannich bases. Using the Mannich
* Corresponding author. Tel.: þ48 081 532 05 19; fax: þ48 081 532 45 46.
E-mail address: tomasz.plech@umlub.pl (T. Plech).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.010

242
T. Plech et al. / European Journal of Medicinal Chemistry 46 (2011) 241e248
2. Results and discussion
studies aim to explain the differences in the length of reaction time
depending on the used dissolvent (or the lack of such an agent). The
results shall be presented shortly.
2.1. Chemistry
Alkaline cyclization of compounds (1e11) using 2% solution of
sodium hydroxide afforded the corresponding 5-(3-chlorophenyl)-
The synthesis pathway leading to the title compounds is
depicted in Scheme 1. In order to select the optimum synthesis
method, various experimental parameters, including solvents
(ethanol, dimethylacetamide, the lack of solvent) and temperature
conditions were examined (data not shown). 3-Chlorobenzoic acid
hydrazide (commercially available) was added to appropriate aryl
isothiocyanates, and 1-[(3-chlorophenyl)carbonyl]-4-substituted
thiosemicarbazides (1e11) were obtained with a short reaction
time (from 2 to 5 min, details in the Experimental section). The
reaction medium, in all cases, was anhydrous ethanol and the
syntheses were performed at boiling point temperature. Good to
excellent yields (up to 96% in the case of compound 6) were
obtained.
The test results indicate that the method of synthesis used is
definitely quicker and more efficient than the solid-state synthesis
method [13,18e20], and even faster than reactions executed in
similar conditions described by other authors [8,14e17]. Taking into
consideration time and efficiency, this method of the synthesis of
thiosemicarbazides may be treated as competitive to the micro-
wave-assisted method. The currently executed computational
4-substituted-2,4-dihydro-3H-1,2,4-triazole-3-thiones
(12e22).
The structures and purity of these compounds were characterized
using the 1H NMR, IR and MS methods together with elemental
analysis. The 1H NMR spectra of the compounds showed the signals
of aromatic protons in the range of 6.89e8.01 ppm. For the thio-
semicarbazides, the signals of the proton linked to N1-N2 and N3
nitrogens were shown at 9.25e10.79 and 8.14e9.87 ppm, respec-
tively. In the obtained 1,2,4-triazoles, the signal of the NH proton
was observed in the range of 12.42e14.31 ppm. It is important to
note that all synthesized 1,2,4-triazoles are present in thione form
(eC¼S) as indicated by their IR (presence of absorption maxima at
1328e1360 cmꢀ1 characteristic for the C]S group) and NMR
spectra (absence of a signal of the proton of the SH group).
Mannich bases (23e33) were synthesized in the reaction of
suitable 4,5-disubstituted derivatives of 1,2,4-triazolino-3-thione
(12e22) with pyrrolidine and formaldehyde (37%), in ethanol (96%)
environment. The occurrence of the aminomethylation reaction is
shown by the loss of signal for the proton at the N-2 nitrogen atom
of the 1,2,4-triazole ring. Moreover, in 1H NMR spectra a sharp
O
O
NH
NH
NCS
NH2
NH
NH
a
S
R
R
Cl
Cl
1-11
b
N
S
N
NH
N
N
N
S
N
c
Cl
Cl
R
R
12-22
23-33
R
R
H
2-I
4-Cl
4-F
1, 12, 23
2, 13, 24
3, 14, 25
4, 15, 26
5, 16, 27
6, 17, 28
7, 18, 29
8, 19, 30
9, 20, 31
10, 21, 32
11, 22, 33
4-CH3
4-OCH3
4-Br
2,5-diF
2,6-diF
2-Br
4-I
Scheme 1. Reagents and conditions: (a) EtOH, 2e5 min; (b) 2% NaOH, 2 h; (c) HCOH, pyrrolidine, EtOH, 1 h.

T. Plech et al. / European Journal of Medicinal Chemistry 46 (2011) 241e248
243
Table 1
Screening for antimicrobial activity of the compounds.
Microorganisms/MIC (mg/mL)
Compounds
S. aureus
S. aureus
S. epidermidis
B. subtilis
B. cereus
M. luteus
ATCC 25923
ATCC 6538
ATCC 12228
ATCC 6633
ATCC 10876
ATCC 10240
1
2
125
62.5
125
62.5
125
125
125
125
a
a
a
a
3
62.5
125
125
125
125
62.5
4
5
6
7
15.63
62.5
31.25
31.25
15.63
31.25
31.25
31.25
15.63
125
31.25
31.25
31.25
125
31.25
15.63
15.63
62.5
15.63
31.25
15.63
62.5
31.25
31.25
8
9
31.25
31.25
31.25
31.25
62.5
31.25
a
a
a
a
a
a
10
11
12
13
62.5
62.5
125
125
250
125
62.5
125
250
250
250
250
a
a
a
a
a
125
a
a
a
a
a
a
a
a
a
a
a
a
14
15
16
17
18
31.25
31.25
31.25
125
15.63
1000
15.63
1000
31.25
a
a
a
a
a
250
500
250
31.25
250
a
a
a
a
a
19
20
21
22
125
125
125
125
125
125
a
a
a
a
a
a
a
a
250
250
6
1000
500
3
1000
250
62.5
1000
125
6
250
2
250
3
Ampicillin
a
- MIC value higher than 1000 mg/mL.
singlet corresponding to the methylene group, linking the triazole
ring with the pyrrolidine ring, occurs in the range of 5.25e5.38
ppm. The signal of the four protons of pyrrolidine deriving from the
fragment e(CH2)2Ne becomes evident in the form of a triplet at
2.90e3.01 ppm. The signal of the remaining four protons of pyr-
rolidine is shown as a multiplet (1.69e1.86 ppm). In turn, in the IR
spectra, absorption bands characteristic for fragments: C]S, C]N
against the reference strains of Gram-negative and Gram-positive
bacterial species (Table 1, Table 2).
Among the Gram-positive species, the most sensitive to all of
the assayed compounds were Staphylococcus aureus ATCC 25923
(MIC from 15.63 to >1000
from 15.63 to >1000 g/mL). It was found that the most effective
against Gram-positive species was compound (4) (MIC ¼ 15.63 g/
mL; for Bacillus subtilis ATCC 6633 MIC ¼ 31.25 g/mL). Compounds
(6) and (7) (MIC ¼ 15.63e31.25 g/mL) as well as compound (8)
g/mL) were also characterized by good activity
mg/mL) and S. aureus ATCC 6538 (MIC
m
m
and eCH2e, are visible in the ranges of: 1321e1338 cmꢀ1
,
m
1581e1599 cmꢀ1 and 2833e2971 cmꢀ1, respectively.
m
Among the above-mentioned compounds there are both newly-
synthesized compounds as well as earlier known derivatives (details
in the Experimental section). An evaluation of their antimicrobial
activities was performed and is described here for the first time.
(MIC ¼ 31.25e62.5
m
against the tested Gram-positive bacteria. Among the investigated
triazoles, the most active was compound (15) (MIC ¼ 15.63e31.25
mg/mL). As can be seen from Table 1, compounds (4), (6), (15)
appeared to be four-fold more effective against Bacillus cereus ATCC
10876 than ampicillin in the same in vitro test. Compounds (1e22)
were found to be inactive against Gram-negative bacteria.
2.2. Antimicrobial evaluation
The novel Mannich bases (23e33) had differential activity
against Gram-positive bacteria, with MIC values ranged from 31.25
The in vitro antimicrobial activity of the synthesized compounds
(1e33) was evaluated using the broth microdilution method
Table 2
Screening for antimicrobial activity of the compounds.
Species
Sa 25923
MIC
Sa 6538
MIC
Se 12228
MIC MBC
125
Bs 6638
MIC
Bc 10876
Ml 10240
MIC
Ec 25922
Kp 13883
Pm 12453
Compounds
MBC
MBC
MBC
MIC
MBC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
23
24
25
26
27
28
29
30
31
32
33
Amp.
125
31.25
125
31.25
250
125
500
125
125
125
125
2
500
125
62.5
250
62.5
250
250
500
1000
125
31.25
250
500
1000
500
125
125
125
125
62.5
125
62.5
500
125
500
1000
125
125
125
6
1000
500
500
250
500
500
a
a
a
a
a
a
a
a
a
125
250
250
500
250
500
1000
125
125
125
6
500
500
1000
1000
250
500
250
500
500
a
a
a
a
a
a
a
a
a
62.5
500
62.5
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
1000
1000
125
250
125
125
125
125
3
1000
125
500
125
250
125
125
62.5
1000
a
a
a
a
500
a
a
a
125
250
500
500
nd
1000
500
1000
1000
nd
1000
500
1000
1000
nd
1000
500
500
500
7.81
1000
500
500
250
1000
62.5
1000
125
125
125
3
250
500
500
nd
250
1000
500
nd
500
500
500
500
1000
0.5
1000
a
a
a
1000
nd
1000
nd
1000
nd
1000
nd
Abbreviations: Sa25923 - Staphylococcus aureus ATCC 25923, Sa6538 - Staphylococcus aureus ATCC 6538, Se12228 - Staphylococcus epidermidis ATCC 12228, Bs6638 - Bacillus
subtilis ATCC 6638, Bc10876 - Bacillus cereus ATCC 10876, Ml10240 - Micrococcus luteus ATCC 10240, Ec25922 - Escherichia coli ATCC 25922, Kp13883 - Klebsiella pneumoniae
ATCC 13883, Pm12453 - Proteus mirabilis ATCC 12453, Amp. e ampicillin, nd e not determined.
a
MIC or MBC value higher than 1000 mg/mL.

244
T. Plech et al. / European Journal of Medicinal Chemistry 46 (2011) 241e248
to >1000
m
g/mL. On the basis of MBC/MIC ratio, the tested
(m), energy of the highest occupied molecular orbital (EHOMO),
compounds are considered to have bactericidal (MBC/MIC ꢁ 4) or
bacteriostatic (MBC/MIC > 4) effect on the tested strains [21,22]. As
opposed to compounds (1e22), some of the synthesized Mannich
bases were also active towards Gram-negative bacteria. The MIC
values for compounds (23), (25), (31e33) were in the range of
energy of the lowest unoccupied molecular orbital (ELUMO) were
determined. Compounds (1), (12), (23), containing an unsub-
stituted phenyl ring, demonstrate weak activity against Gram-
positive bacteria. Among other compounds there were derivatives
with electron-donating or electron-withdrawing substituents
connected to the phenyl ring. The introduction of the halogen atom
in general increased antibacterial activity. Moreover, the advanta-
geous position for the electron-withdrawing substituents is the
para position in the phenyl ring. It is clearly visible in the case of the
corresponding bromine and iodine derivatives. Para isomers of
these compounds are characterized by higher antibacterial activity
than their ortho isomers. Unexpectedly, compounds (9) and (20),
containing a 4-fluorophenyl fragment, were deprived of any
activity directed towards the tested microorganisms. Furthermore,
it should be noted that the compounds (1e22) were not active
against Gram-negative bacteria. Next, comparing the activity of
1,2,4-triazoles (12e22) with their respective N-2-(pyrrolidin-1-
ylmethyl) derivatives (23e33), it is clear that the substitution of
hydrogen at nitrogen N-2 causes a significant increase in antibac-
terial activity mainly for those compounds, which had the electron-
donating substituents (CH3, OCH3) at the phenyl ring. For example,
compound (13), which was inactive against all tested bacterial
strains, after moving it in the appropriate Mannich base (24),
acquires relatively substantial activity. Moreover, the introduction
of the substituent in N-2 position extends the spectrum of activity
of certain compounds also on the Gram-negative bacteria.
According to the data listed in Table S.1 (in the Supplementary data
section), it can be hypothesize that lipophilicity (logP) has no
decisive influence on antibacterial activity for these compounds.
Therefore, it is necessary to think that the structural or electronic
parameters may have a greater impact on the activity. Investigation
250e1000
mg/mL. The MBC values were in the similar range,
suggesting bactericidal activity.
Moreover, the most active (against the reference strains)
compounds (4e8, 10, 15, 24, 26) were examined against twelve
clinical isolates of S. aureus. It was found that most of the tested
compounds inhibited the growth of these bacterial isolates with
MIC ranging from 15.63 to 31.25
value was 15.63 g/mL. Compounds (6), (7) and (8) had the highest
activity, with MIC ¼ 15.63 g/mL for all of the tested clinical isolates
of S. aureus. Only derivatives (15), (24) and (26) showed MIC values
higher than 31.25 g/ml. The difference of sensitivity of referential
mg/mL (Table 3); the mode MIC
m
m
m
strains and clinical strains on the studied substances is clearly
noticeable. For example, compound (4) inhibited the growth of
referential strains (S. aureus ATCC 25923, S. aureus ATCC 6538) in
the concentration of 15.63
majority of clinical strains (8 out of 12) took place in a concentra-
tion that was twice as high (31.25 g/mL). A contrary situation is
mg/mL, while stopping the growth of the
m
observed in the case of iodine and chlorine derivatives of thio-
semicarbazide (6,7,8). The mentioned compounds were character-
ized by the values of MIC ¼ 31.25
and S. aureus ATCC 6538, while the concentration inhibiting the
growth of all twelve clinical strains was twice as low (15.63 g/mL).
mg/mL for S. aureus ATCC 25923
m
From the microbiological point of view, the difference written
above in the sensitivity of both types of strains of S. aureus is easy to
explain. A similar situation is described by Lopes et al. [23], who
investigated different enterococci strains. Clinical strains, which
were used in the studies, were isolated from patients at various
ages, treated due to various diseases, treated by various drugs.
Therefore, there was a great probability of a mutation taking place
in the bacterial genome, which caused a different sensitivity to the
studied substances. Also due to this, conclusions concerning
structure-activity dependencies were formulated on the basis of
results obtained for referential strains.
of the dependency between the dipole moments (m) and antimi-
crobial activity for analogs with bromo and iodo substitution (4-7,
15-18, 26e29) revealed that 4-bromo (4, 15, 26) and 4-iodo (6, 17,
28) analogs, with lower dipole moments than their 2-bromo (5, 16,
27) and 2-iodo (7, 18, 29) isomers, were more biologically active.
3. Conclusions
Results obtained in this work clearly indicate that among the
described compounds, thiosemicarbazide derivatives exhibit much
stronger antibacterial activity compared to related 1,2,4-triazoles
and their Mannich bases. Only compound (15) (MIC ¼ 15.63e31.25
We describe a fast (several minute long), facile and efficient
method for the preparation of 1,4-disubstituted thiosemicarbazide
derivatives. A variety of novel s-triazoles and their Mannich bases
were prepared as well. Some derivatives showed promising anti-
microbial activity, especially against Gram-positive bacteria.
Compounds (4), (6), (15) appeared to be four-fold more effective
against B. cereus ATCC 10876 than ampicillin. We think that our
results should be of value for further studies, especially for
searching new derivatives showing better antimicrobial activity
against both opportunistic (e.g., Bacillus spp., Staphylococcus
epidermidis or Escherichia coli and Proteus mirabilis) or pathogenic
(e.g., S. aureus, Klebsiella pneumoniae) bacteria.
m
g/mL), based on the 1,2,4-triazole scaffold, displays activity similar
to that of the initial thiosemicarbazide. The antibacterial activity of
the title compounds distinctly depends on the nature of substitu-
ents and their location. In order to analyze the relationship: nature/
position of the substituentethe activity of compounds (1e33), the
values of the following parameters: surface area (SA), volume (V),
hydration energy (HE), logarithm of the partition coefficient (logP),
molecular refractivity (MR), polarizability (a), dipole moment
Table 3
4. Experimental
Antibacterial activity of the tested compounds against twelve clinical isolates of
Staphylococcus aureus.
4.1. Chemistry
Compound
MIC (mg/mL)
4.1.1. General comments
15.63
31.25
All reagents were purchased from Lancaster and Merck Co. and
used without further purification. Melting points were determined
by using Fischer-Johns apparatus (Sanyo, Japan) and are uncor-
rected. The 1H NMR spectra were recorded on a Bruker Avance 250
MHz instrument using DMSO-d6 or CDCl3 as solvents and TMS as an
Number (%) of isolates
4
5
6
7
8
10
4 (33.3)
7 (58.3)
12 (100)
12 (100)
12 (100)
0 (0)
8 (66.7)
5 (41.7)
0 (0)
0 (0)
0 (0)
internal standard. Chemical shifts are expressed as
d (ppm). The IR
12 (100)
spectra were recorded in KBr discs using a PerkineElmer 1725X

T. Plech et al. / European Journal of Medicinal Chemistry 46 (2011) 241e248
245
FTIR spectrometer. The mass spectra were obtained on a Finnigan
Trace DSQ spectrometer operating at 70 eV. 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
(10), 50 (7). Anal. Calc. for C14H10ClF2N3OS (%): C 49.20, H 2.95,
N 12.30. Found: C 49.12, H 2.87, N 12.32.
1-[(3-Chlorophenyl)carbonyl]-4-(2,6-difluorophenyl)thiosemicar-
bazide (11) Yield 92%, m.p. 152e154 ꢃC, 1H NMR (250 MHz) (DMSO-
were detected by exposure to UV-lamp at
analyses were performed on AMZ 851 CHX analyser and the results
were within ꢂ0.2% of the theoretical value.
l
¼ 254 nm. Elemental
d6) d (ppm): 7.10e7.17 (m, 2H, AreH), 7.32e7.40 (m, 1H, AreH),
7.49e7.57 (m, 1H, AreH), 7.62e7.67 (m, 1H, AreH), 7.85e7.90 (m,
1H, AreH), 8.00 (s, 1H, AreH), 9.46, 10.08, 10.79 (3s, 3H, 3NH, exch.
D2O). IR (KBr, n
, cmꢀ1): 3324 (NH), 3017 (CHarom.), 1688 (C]O),
4.1.2. General procedure for the synthesis of 1-[(3-chlorophenyl)
carbonyl]-4-substituted-thiosemicarbazides (1e11)
1353 (C]S). EI-MS m/z (%): 341 (Mþ, 3), 212 (12.5), 171 (42.3), 139
(100), 129 (61.5), 111 (30.7), 75 (21), 63 (9.6), 50 (6.7). Anal. Calc. for
C14H10ClF2N3OS (%): C 49.20, H 2.95, N 12.30. Found: C 49.12, H
2.87, N 12.32.
A solution of 0.01 mol of 3-chlorobenzoic acid hydrazide and
equimolar amount of appropriate isothiocyanate in 25 ml of
anhydrous EtOH was heated under reflux for 2 min (for compounds
2, 6, 8e10) or for 5 min (in the case of compounds 1, 3e5, 7, 11).
Next, the solution was cooled and the solid formed was filtered off,
washed with diethyl ether, dried, and crystallized from EtOH.
1-[(3-Chlorophenyl)carbonyl]-4-phenylthiosemicarbazide (1) Yield:
81%, spectral and physicochemical data consistent with [24].
1-[(3-Chlorophenyl)carbonyl]-4-(4-methylphenyl)thiosemicarba-
zide (2) Yield 93%, CAS Registry Number: 891082-71-6.
4.1.3. General procedure for the synthesis of 5-(3-chlorophenyl)-4-
substituted-2,4-dihydro-3H-1,2,4-triazole-3-thiones (12e22)
Suitable substituted thiosemicarbazides (1e11) (0.01 mol) were
dissolved in 2% solution of sodium hydroxide and the resulting
solution was heated under reflux for 2 h. After cooling, the mixture
was neutralized with 3M hydrochloric acid. The precipitate formed
was filtered and washed several times with distilled water. The
compounds were crystallized from EtOH.
5-(3-Chlorophenyl)-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thi-
one (12) Yield: 87%, spectral and other physicochemical data
consistent with [26].
5-(3-Chlorophenyl)-4-(4-methylphenyl)-2,4-dihydro-3H-1,2,4-tri-
azole-3-thione (13) Yield 73%, CAS Registry Number: 893725-08-1.
5-(3-Chlorophenyl)-4-(4-methoxyphenyl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (14) Yield 89%, CAS Registry Number:
714214-94-5.
1-[(3-Chlorophenyl)carbonyl]-4-(4-methoxyphenyl)thiosemicarba-
zide (3) Yield 84%, CAS Registry Number: 905262-18-2.
4-(4-Bromophenyl)-1-[(3-chlorophenyl)carbonyl]thiosemicarbazide
(4) Yield 88%, spectral and physicochemical data consistent with [25].
4-(2-Bromophenyl)-1-[(3-chlorophenyl)carbonyl]thio-
semicarbazide (5) Yield 86%, m.p. 150e152 ꢃC, 1H NMR (250 MHz)
(DMSO-d6)
d (ppm): 7.15e7.23 (m, 1H, AreH), 7.33e7.42 (m, 2H,
AreH), 7.49e7.68 (m, 3H, AreH), 7.85e7.90 (m, 1H, AreH), 8.00
(s, 1H, AreH), 9.68, 9.85, 10.72 (3s, 3H, 3NH, exch. D2O). IR (KBr,
n
, cmꢀ1): 3359 (NH), 3027 (CHarom.), 1671 (C]O), 1338 (C]S). EI-
4-(4-Bromophenyl)-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-tri-
MS m/z (%): 384 (Mþ, 0.1), 213 (50), 170 (19.2), 139 (100), 111 (52.9),
90 (13.5), 75 (47.1), 63 (11.5), 50 (20.2). Anal. Calc. for
C14H11BrClN3OS (%): C 43.71, H 2.88, N 10.92. Found: C 43.87, H 2.71,
N 10.99.
azole-3-thione (15) Yield 95%, m.p. 224e226 ꢃC, 1H NMR (250 MHz)
(DMSO-d6)
d
(ppm): 7.22e7.25 (m, 1H, AreH), 7.36 (dd, 2H, J ¼ 8.70
Hz, 1.90 Hz, AreH, p-substituted ring), 7.39e7.45 (m, 2H, AreH),
7.51e7.55 (m, 1H, AreH), 7.72 (dd, 2H, J ¼ 8.73 Hz, 1.90 Hz, AreH,
1-[(3-Chlorophenyl)carbonyl]-4-(4-iodophenyl)thiosemicarbazide
p-substituted ring), 14.24 (s, 1H, NH, exch. D2O). IR (KBr, n
, cmꢀ1):
(6) Yield 96%, m.p. 180e182 ꢃC, 1H NMR (250 MHz) (DMSO-d6)
3437 (NH), 3062 (CHarom.), 1578 (C]N), 1328 (C]S). EI-MS m/z (%):
370 ([Mþ4]þ, 27), 368 ([Mþ2]þ, 94), 366 (Mþ, 100), 308 (10.5), 285
(9.6), 227 (13.5),169 (15.5), 152 (44.2), 143 (25), 137 (38.4), 125 (75),
111 (26), 90 (42.3), 75 (60.5), 63 (30), 50 (23). Anal. Calc. for
C14H9BrClN3S (%): C 45.86, H 2.47, N 11.46. Found: C 45.72, H 2.42,
N 11.50.
d
(ppm): 7.27e7.32 (m, 2H, AreH), 7.50e7.57 (m, 1H, AreH),
7.63e7.68 (m, 3H, AreH), 7.84e7.89 (m, 1H, AreH), 7.98 (s, 1H,
AreH), 9.84 (s, 2H, 2NH, exch. D2O), 10.67 (s, 1H, NH, exch. D2O). IR
(KBr, n
, cmꢀ1): 3320 (NH), 3023 (CHarom.), 1669 (C]O), 1345 (C]S).
EI-MS m/z (%): 431 (Mþ, 0.13), 261 (93.2), 219 (27.8), 212 (12.5), 170
(19), 139 (100), 111 (50), 90 (24), 75 (36.2), 50 (27.5). Anal. Calc. for
C14H11ClIN3OS (%): C 38.95, H 2.57, N 9.73. Found: C 38.87, H 2.52, N
9.83.
4-(2-Bromophenyl)-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-tri-
azole-3-thione (16) Yield 85%, m.p. 228e230 ꢃC, 1H NMR (250 MHz)
(DMSO-d6)
d (ppm): 7.25e7.43 (m, 3H, AreH), 7.47e7.53 (m, 2H,
1-[(3-Chlorophenyl)carbonyl]-4-(2-iodophenyl)thiosemicarbazide
AreH), 7.56e7.64 (m, 1H, AreH), 7.71e7.82 (m, 2H, AreH), 14.31 (s,
(7) Yield 80%, m.p. 160e162 ꢃC, 1H NMR (250 MHz) (DMSO-d6)
d
1H, NH, exch. D2O). IR (KBr, n
, cmꢀ1): 3445 (NH), 3101 (CHarom.),
(ppm): 7.00e7.06 (m, 1H, AreH), 7.26e7.40 (m, 2H, AreH),
1577 (C]N), 1335 (C]S). EI-MS m/z (%): 367 ([Mþ1]þ, 4), 286
(65.4), 149 (100), 137 (8.6), 125 (11.5), 105 (13.5), 90 (8.6), 75 (13.5),
63 (5), 50 (5). Anal. Calc. for C14H9BrClN3S (%): C 45.86, H 2.47, N
11.46. Found: C 45.70, H 2.36, N 11.49.
7.49e7.56 (m, 1H, AreH), 7.62e7.67 (m, 1H, AreH), 7.83e7.91
(m, 2H, AreH), 8.02 (s, 1H, AreH), 9.67, 9.80, 10.71 (3s, 3H, 3NH,
exch. D2O). IR (KBr, n
, cmꢀ1): 3345 (NH), 3003 (CHarom.), 1670 (C]
O), 1337 (C]S). EI-MS m/z (%): 303 (9), 261 (68.2), 219 (5), 170
(15.3), 139 (100), 111 (52), 90 (15.4), 75 (28.8), 63 (10), 50 (18.2).
Anal. Calc. for C14H11ClIN3OS (%): C 38.95, H 2.57, N 9.73. Found:
C 38.81, H 2.50, N 9.83.
4-(4-Chlorophenyl)-1-[(3-chlorophenyl)carbonyl]thiosemicarbazide
(8) Yield 88%, spectral and physicochemical data consistent with [25].
1-[(3-Chlorophenyl)carbonyl]-4-(4-fluorophenyl)thiosemicarb-
azide (9) Yield 90%, CAS Registry Number: 316151-86-7.
1-[(3-Chlorophenyl)carbonyl]-4-(2,5-difluorophenyl)thiosemicar-
bazide (10) Yield 91%, m.p. 178e180 ꢃC, 1H NMR (250 MHz) (DMSO-
5-(3-Chlorophenyl)-4-(4-iodophenyl)-2,4-dihydro-3H-1,2,4-tri-
azole-3-thione (17) Yield 90%, m.p. 236e238 ꢃC, 1H NMR (250
MHz) (DMSO-d6)
d
(ppm): 7.19 (dd, 2H, J ¼ 8.49 Hz, 2.00 Hz,
AreH, p-substituted ring), 7.22e7.25 (m, 1H, AreH), 7.35e7.42
(m, 2H, AreH), 7.48e7.57 (m, 1H, AreH), 7.86 (dd, 2H, J ¼ 8.50
Hz, 1.95 Hz, AreH, p-substituted ring), 14.22 (s, 1H, NH, exch.
D2O). IR (KBr, n
, cmꢀ1): 3435 (NH), 3101 (CHarom.), 1578 (C]N),
1333 (C]S). EI-MS m/z (%): 415 ([Mþ2]þ, 32.1), 413 (Mþ, 100),
286 (9.6), 149 (21), 122 (11.5), 108 (18.2), 90 (25), 76 (34.6), 63
(18.2), 50 (16.3). Anal. Calc. for C14H9ClIN3S (%): C 40.65, H 2.19,
N 10.16. Found: C 40.62, H 2.22, N 10.20.
d6)
d (ppm): 7.08e7.22 (m, 2H, AreH), 7.28e7.37 (m, 1H, AreH),
7.49e7.70 (m, 2H, AreH), 7.83e8.00 (m, 2H, AreH), 9.70, 10.05,
5-(3-Chlorophenyl)-4-(2-iodophenyl)-2,4-dihydro-3H-1,2,4-triazole-
10.77 (3s, 3H, 3NH, exch. D2O). IR (KBr, n
, cmꢀ1): 3320 (NH), 3009
3-thione (18) Yield 65%, m.p. 260e261 ꢃC, 1H NMR (250 MHz)
(CHarom.), 1633 (C]O), 1352 (C]S). EI-MS m/z (%): 341 (Mþ, 0.5),
(DMSO-d6)
d (ppm): 7.25e7.43 (m, 4H, AreH), 7.48e7.69 (m, 3H,
212 (6), 171 (51), 152 (11), 139 (100), 129 (26), 111 (32), 75 (19), 63
AreH), 7.95e8.00 (m, 1H, AreH), 14.28 (s, 1H, NH, exch. D2O). IR

246
T. Plech et al. / European Journal of Medicinal Chemistry 46 (2011) 241e248
(KBr,
n
, cmꢀ1): 3434 (NH), 3099 (CHarom.), 1574 (C]N), 1334 (C]S).
(CHarom.), 2945, 2888 (CHaliph.), 1590 (C]N), 1338 (C]S). EI-MS m/z
(%): 400 (Mþ, 1.1), 317 (1.9), 138 (1.4), 84 (100), 55 (3), 42 (2.8). Anal.
calc. for C20H21ClN4OS (%): C 59.91, H 5.28, N 13.97. Found: C 59.98,
H 5.20, N 13.85.
4-(4-Bromophenyl)-5-(3-chlorophenyl)-2-(pyrrolidin-1-ylmethyl)-
2,4-dihydro-3H-1,2,4-triazole-3-thione (26) Yield 80%, m.p. 136e
EI-MS m/z (%): 413 (Mþ, 7.6), 286 (71), 149 (100), 122 (13.4), 105
(16.3), 90 (9.6), 76 (17.3), 63 (6.7), 50 (7.6). Anal. Calc. for
C14H9ClIN3S (%): C 40.65, H 2.19, N 10.16. Found: C 40.60, H 2.11,
N 10.23.
5-(3-Chlorophenyl)-4-(4-chlorophenyl)-2,4-dihydro-3H-1,2,4-tri-
azole-3-thione (19) Yield 85%, CAS Registry Number: 879074-80-3.
5-(3-Chlorophenyl)-4-(4-fluorophenyl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (20) Yield 90%, CAS Registry Number: 720667-
75-4.
138 ꢃC, 1H NMR (250 MHz) (CDCl3)
d
(ppm): 1.84e1.86 (m, 4H, 2ꢄ
CH2), 3.01 (t, 4H, 2ꢄ CH2, J ¼ 6.75 Hz), 5.37 (s, 2H, CH2), 7.05e7.10 (m,
1H, AreH), 7.21 (dd, 2H, J ¼ 8.78 Hz, 2.05 Hz, AreH, p-substituted
ring), 7.25e7.30 (m, 1H, AreH), 7.39e7.44 (m, 1H, AreH), 7.51 (t, 1H,
J ¼ 1.90 Hz, AreH), 7.67 (dd, 2H, J ¼ 8.80 Hz, 2.05 Hz, AreH, p-
5-(3-Chlorophenyl)-4-(2,5-difluorophenyl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (21) Yield 79%, m.p. 138e140 ꢃC, 1H NMR (250
substituted ring). IR (KBr,
n
, cmꢀ1): 3047 (CHarom.), 2961, 2876
MHz) (CDCl3)
AreH), 12.42 (s, 1H, NH, exch. D2O). IR (KBr,
d
(ppm): 7.16e7.28 (m, 5H, AreH), 7.39e7.46 (m, 2H,
, cmꢀ1): 3431 (NH),
(CHaliph.), 1584 (C]N), 1326 (C]S). EI-MS m/z (%): 450 ([Mþ1]þ,
0.3), 366 (2.1), 155 (1.5), 138 (1.3), 84 (100), 55 (3.8), 42 (5.1). Anal.
calc. for C19H18BrClN4S (%): C 50.73, H 4.03, N 12.46. Found: C 50.66,
H 4.18, N 12.41.
n
3092 (CHarom.), 1581 (C]N), 1360 (C]S). EI-MS m/z (%): 325
([Mþ2]þ, 32.3), 323 (Mþ, 100), 304 (67), 288 (7), 250 (11), 186 (12),
152 (37), 137 (22.3), 111 (21), 90 (13), 75 (27), 50 (9). Anal. Calc. for
C14H8ClF2N3S (%): C 51.94, H 2.49, N 12.98. Found: C 52.11, H 2.36,
N 13.13.
4-(2-Bromophenyl)-5-(3-chlorophenyl)-2-(pyrrolidin-1-ylmethyl)-
2,4-dihydro-3H-1,2,4-triazole-3-thione (27) Yield 86%, m.p. 172e
174 ꢃC, 1H NMR (250 MHz) (CDCl3)
d
(ppm): 1.69e1.77 (m, 4H, 2ꢄ
5-(3-Chlorophenyl)-4-(2,6-difluorophenyl)-2,4-dihydro-3H-1,2,4-
CH2), 2.95 (t, 4H, 2ꢄ CH2, J ¼ 6.60 Hz), 5.27 (s, 2H, CH2), 7.05e7.21
triazole-3-thione (22) Yield 77%, m.p. 158e160 ꢃC, 1H NMR (250
(m, 2H, AreH), 7.26e7.50 (m, 5H, AreH), 7.64e7.69 (m,1H, AreH). IR
MHz) (DMSO-d6)
d
(ppm): 7.25e7.28 (m, 1H, AreH), 7.36e7.48 (m,
(KBr, n
, cmꢀ1): 3053 (CHarom.), 2961, 2843 (CHaliph.), 1589 (C]N),
5H, AreH), 7.56e7.62 (m, 1H, AreH), 14.21 (s, 1H, NH, exch. D2O). IR
1321 (C]S). EI-MS m/z (%): 450 ([Mþ1]þ, 0.6), 286 (2.4), 149 (2.9),
84 (100), 55 (3.2), 42 (2.5). Anal. calc. for C19H18BrClN4S (%): C 50.73,
H 4.03, N 12.46. Found: C 50.81, H 4.10, N 12.49.
(KBr, n
, cmꢀ1): 3432 (NH), 3080 (CHarom.), 1570 (C]N), 1332 (C]S).
EI-MS m/z (%): 325 ([Mþ2]þ, 31.8), 323 (Mþ, 100), 304 (90), 250
(8.6), 167 (24), 152 (34.6), 137 (27.9), 125 (25.2), 90 (11.5), 75 (30),
63 (16), 50 (7). Anal. Calc. for C14H8ClF2N3S (%): C 51.94, H 2.49, N
12.98. Found: C 52.00, H 2.41, N 13.03.
5-(3-Chlorophenyl)-4-(4-iodophenyl)-2-(pyrrolidin-1-ylmethyl)-
2,4-dihydro-3H-1,2,4-triazole-3-thione (28) Yield 83%, m.p.
143e144 ꢃC, 1H NMR (250 MHz) (CDCl3)
d (ppm): 1.70e1.76 (m, 4H,
2ꢄ CH2), 2.90 (t, 4H, 2ꢄ CH2, J ¼ 6.60 Hz), 5.26 (s, 2H, CH2), 6.97 (dd,
2H, J ¼ 8.80 Hz, 2.10 Hz, AreH, p-substituted ring), 7.11e7.19 (m, 2H,
AreH), 7.28e7.34 (m, 1H, AreH), 7.41 (t, 1H, J ¼ 1.88 Hz, AreH), 7.71
(dd, 2H, J ¼ 8.80 Hz, 2.10 Hz, AreH, p-substituted ring). IR (KBr,
4.1.4. General procedure for the synthesis of Mannich bases
(23e33)
To a solution of corresponding compounds (12e22) (1 mmol) in
96% ethanol, pyrrolidine (1 mmol) and formaldehyde (37%, 0.2 mL)
were added and the mixture was stirred at room temperature for 1
h. Next, distilled water was added and the precipitate formed was
filtered off, washed several times with distilled water, and crys-
tallized from ethanol.
n
, cmꢀ1): 3062 (CHarom.), 2958, 2846 (CHaliph.), 1590 (C]N), 1329
(C]S). EI-MS m/z (%): 496 (Mþ, 0.6), 413 (3.2), 203 (1.9), 138 (2.5),
84 (100), 63 (1.2), 55 (6.7), 42 (9.6). Anal. calc. for C19H18ClIN4S (%):
C 45.94, H 3.65, N 11.28. Found: C 45.87, H 3.59, N 11.35.
5-(3-Chlorophenyl)-4-(2-iodophenyl)-2-(pyrrolidin-1-ylmethyl)-
2,4-dihydro-3H-1,2,4-triazole-3-thione (29) Yield 76%, m.p.
5-(3-Chlorophenyl)-4-phenyl-2-(pyrrolidin-1-ylmethyl)-2,4-dihy-
dro-3H-1,2,4-triazole-3-thione (23) Yield 85%, m.p. 102e104 ꢃC, 1H
166e168 ꢃC, 1H NMR (250 MHz) (CDCl3)
d (ppm): 1.70e1.77 (m, 4H,
NMR (250 MHz) (CDCl3)
d
(ppm): 1.70e1.77 (m, 4H, 2ꢄ CH2), 2.94 (t,
2ꢄ CH2), 2.94 (t, 4H, 2ꢄ CH2, J ¼ 6.60 Hz), 5.25 (s, 2H, CH2),
4H, 2ꢄ CH2, J ¼ 6.60 Hz), 5.28 (s, 2H, CH2), 6.99e7.14 (m, 2H, AreH),
7.06e7.22 (m, 3H, AreH), 7.26e7.53 (m, 4H, AreH), 7.88e7.93
7.18e7.30 (m, 3H, AreH), 7.33e7.36 (m,1H, AreH), 7.41e7.47 (m, 3H,
(m, 1H, AreH). IR (KBr, n
, cmꢀ1): 3054 (CHarom.), 2923, 2887 (CHa-
AreH). IR (KBr,
n
, cmꢀ1): 3045 (CHarom.), 2911, 2878 (CHaliph.), 1581
liph.),1585 (C]N),1323 (C]S). EI-MS m/z (%): 496 (Mþ, 0.9), 285 (4),
148 (11.5), 84 (100), 55 (4.2), 42 (4.7). Anal. calc. for C19H18ClIN4S
(%): C 45.94, H 3.65, N 11.28. Found: C 45.98, H 3.71, N 11.20.
4-(4-Chlorophenyl)-5-(3-chlorophenyl)-2-(pyrrolidin-1-ylmethyl)-
2,4-dihydro-3H-1,2,4-triazole-3-thione (30) Yield 77%, m.p. 138e
(C]N),1326 (C]S). EI-MS m/z (%): 370 (Mþ,1), 286 (3), 84 (100), 77
(4.8), 55 (3.8), 42 (4.8). Anal. calc. for C19H19ClN4S (%): C 61.53, H 5.16,
N 15.11. Found: C 61.55, H 5.21, N 15.05.
5-(3-Chlorophenyl)-4-(4-methylphenyl)-2-(pyrrolidin-1-ylmethyl)-
2,4-dihydro-3H-1,2,4-triazole-3-thione (24) Yield 72%, m.p. 104e
140 ꢃC, 1H NMR (250 MHz) (CDCl3)
d
(ppm): 1.70e1.76 (m, 4H, 2ꢄ
106 ꢃC, 1H NMR (250 MHz) (CDCl3)
d
(ppm): 1.81e1.86 (m, 4H, 2 ꢄ
CH2), 2.90 (t, 4H, 2ꢄ CH2, J ¼ 6.60 Hz), 5.26 (s, 2H, CH2), 6.95e7.01
(m,1H, AreH), 7.15 (dd, 2H, J ¼ 8.10 Hz, 2.00 Hz, AreH, p-substituted
ring), 7.19e7.21 (m, 1H, AreH), 7.28e7.33 (m, 1H, AreH), 7.40 (dd,
2H, J ¼ 8.10 Hz, 2.05 Hz, AreH, p-substituted ring), 7.41e7.44 (m, 1H,
CH2), 2.46 (s, 3H, CH3), 3.01 (t, 4H, 2ꢄ CH2, J ¼ 6.50 Hz), 5.38 (s, 2H,
CH2), 7.12e7.16 (m, 1H, AreH), 7.20 (dd, 2H, J ¼ 8.33 Hz, 2.00 Hz,
AreH, p-substituted ring), 7.30 (dd, 2H, J ¼ 8.30 Hz, 2.00 Hz, AreH,
p-substituted ring), 7.34e7.41 (m, 2H, AreH), 7.49 (t, 1H, J ¼ 1.90 Hz,
AreH). IR (KBr, n
, cmꢀ1): 3069 (CHarom.), 2954, 2874 (CHaliph.), 1589
AreH). IR (KBr,
n
, cmꢀ1): 3040 (CHarom.), 2964, 2833 (CHaliph.),1599
(C]N), 1333 (C]S). EI-MS m/z (%): 406 ([Mþ1]þ, 0.5), 320 (3.5), 138
(4.7), 125 (5.4), 111 (20), 84 (100), 55 (9.6), 42 (8.6). Anal. calc. for
C19H18Cl2N4S (%): C 56.30, H 4.48, N 13.82. Found: C 56.38, H 4.35, N
13.91.
(C]N), 1327 (C]S). EI-MS m/z (%): 384 (Mþ, 0.6), 300 (3.4), 163 (1),
138 (1.4), 84 (100), 55 (3.8), 42 (4.2). Anal. calc. for C20H21ClN4S (%): C
62.41, H 5.50, N 14.56. Found: C 62.58, H 5.61, N 14.49.
5-(3-Chlorophenyl)-4-(4-methoxyphenyl)-2-(pyrrolidin-1-ylme-
thyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (25) Yield 69%, m.p.
5-(3-Chlorophenyl)-4-(4-fluorophenyl)-2-(pyrrolidin-1-ylmethyl)-
2,4-dihydro-3H-1,2,4-triazole-3-thione (31) Yield 80%, m.p. 114e
102e104 ꢃC, 1H NMR (250 MHz) (CDCl3)
d
(ppm): 1.70e1.76 (m, 4H,
116 ꢃC, 1H NMR (250 MHz) (CDCl3)
d
(ppm): 1.70e1.77 (m, 4H, 2ꢄ
2ꢄ CH2), 2.98 (t, 4H, 2ꢄ CH2, J ¼ 6.60 Hz), 3.78 (s, 3H, OCH3), 5.27
(s, 2H, CH2), 6.92 (dd, 2H, J ¼ 8.80 Hz, 2.20 Hz, AreH, p-substituted
ring), 7.01e7.06 (m, 1H, AreH), 7.14 (dd, 2H, J ¼ 8.80 Hz, 2.30 Hz,
AreH, p-substituted ring), 7.19 (s, 1H, AreH), 7.25e7.30 (m, 1H,
CH2), 2.91 (t, 4H, 2ꢄ CH2, J ¼ 6.62 Hz), 5.27 (s, 2H, CH2), 6.98e7.04 (m,
1H, AreH), 7.14 (dd, 2H, J ¼ 8.05 Hz, 2.00 Hz, AreH, p-substituted
ring) 7.21 (dd, 2H, J ¼ 8.05 Hz, 1.90 Hz, AreH, p-substituted ring),
7.23e7.26 (m,1H, AreH), 7.28e7.33 (m,1H, AreH), 7.36 (t,1H, J ¼ 1.81
AreH), 7.40 (t, 1H, J ¼ 1.90 Hz, AreH). IR (KBr,
n
, cmꢀ1): 3056
Hz, AreH). IR (KBr, n
, cmꢀ1): 3046 (CHarom.), 2991, 2841 (CHaliph.),

T. Plech et al. / European Journal of Medicinal Chemistry 46 (2011) 241e248
247
1581 (C]N),1323 (C]S). EI-MS m/z (%): 388 (Mþ, 0.6), 304 (2.4),138
Appendix. Supplementary data
(1.5), 84 (100), 55 (3.8), 42 (4.8). Anal. calc. for C19H18ClFN4S (%): C
58.68, H 4.67, N 14.41. Found: C 58.71, H 4.58, N 14.40.
The supplementary data related to this article can be found
online at doi:10.1016/j.ejmech.2010.11.010.
5-(3-Chlorophenyl)-4-(2,5-difluorophenyl)-2-(pyrrolidin-1-ylme-
thyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (32) Yield 78%, m.p.
138e140 ꢃC, 1H NMR (250 MHz) (CDCl3)
d (ppm): 1.71e1.76 (m, 4H,
References
2ꢄ CH2), 2.90 (t, 4H, 2ꢄ CH2, J ¼ 6.60 Hz), 5.27 (s, 2H, CH2), 7.06e7.21
(m, 5H, AreH), 7.30e7.36 (m, 1H, AreH), 7.39 (t, 1H, J ¼ 1.82 Hz,
[1] U. Salgın-Göks¸ en, N. Gökhan-Kelekçi, Ö Göktas¸ , Y. Köysal, E. Kılıç, S¸ Is¸ ık,
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AreH). IR (KBr, n
, cmꢀ1): 3041 (CHarom.), 2968, 2845 (CHaliph.), 1583
(C]N),1327 (C]S). EI-MS m/z (%): 406 (Mþ, 0.8), 322 (1.2), 250 (1.6),
138 (1.5), 84 (100), 55 (3.6), 42 (5.1). Anal. calc. for C19H17ClF2N4S (%):
C 56.09, H 4.21, N 13.77. Found: C 56.15, H 4.31, N 13.72.
5-(3-Chlorophenyl)-4-(2,6-difluorophenyl)-2-(pyrrolidin-1-ylme-
thyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (33) Yield 70%, m.p.
94e96 ꢃC, 1H NMR (250 MHz) (CDCl3)
d (ppm): 1.70e1.77 (m, 4H,
2ꢄ CH2), 2.90 (t, 4H, 2ꢄ CH2, J ¼ 6.60 Hz), 5.28 (s, 2H, CH2),
6.98e7.07 (m, 2H, AreH), 7.12e7.22 (m, 2H, AreH), 7.30e7.36
(m, 1H, AreH), 7.39e7.51 (m, 2H, AreH). IR (KBr, n
, cmꢀ1): 3048
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(CHarom.), 2971, 2895 (CHaliph.), 1593 (C]N), 1324 (C]S). EI-MS m/z
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42 (4.7). Anal. calc. for C19H17ClF2N4S (%): C 56.09, H 4.21, N 13.77.
Found: C 56.10, H 4.27, N 13.79.
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4.2. Microbiology
The antimicrobial activity of the compounds was tested on the
Gram-positive strains (S. aureus ATCC 25923, S. aureus ATCC 6538,
S. epidermidis ATCC 12228, B. subtilis ATCC 6633, B. cereus ATCC
10876, Micrococcus luteus ATCC 10240) and on the Gram-negative
strains ( E. coli ATCC 25922, K. pneumoniae ATCC 13883, P. mirabilis
ATCC 12453). Besides, twelve clinical isolates of S. aureus from the
collection of the Department of Pharmaceutical Microbiology of
Medical University in Lublin were also used in our experiments.
Microbial suspensions were prepared in sterile saline (0.85% NaCl)
with an optical density of 0.5 McFarland standard e 150 ꢄ 106 CFU/
mL (CFU e colony forming units). All stock solutions of the tested
compounds were dissolved in dimethyl sulfoxide (DMSO). Muel-
lereHinton medium was used with a series of two-fold dilutions
of the tested substances in the range of final concentrations from
3.91 to 1000
mg/mL. It was dimethyl sulfoxide (DMSO) had no
antimicrobial activity against any of the tested microorganisms.
Ampicillin, antibiotic widely used in the treatment of infectious
diseases, was used as control antimicrobial agent.
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screened on the basis of MIC (minimal inhibitory concentration),
usually defined as the lowest concentration of compound at which
there was no visible growth of tested microorganisms. Determination
of the MIC values was achieved by a broth microdilution method,
according to CLSI recommendation [27]. MBC (minimal bactericidal
concentration), defined as the lowest concentration of compound
that resulted in >99.9% reduction in CFU of the initial inoculum, was
assessed only for Mannich bases (23e33). MBC was determined by
a broth microdilution technique by plating out the contents of wells
(20
mL), that showed no visible growth of bacteria, onto Muel-
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MBC values are given in mg/mL, according to CLSI reference [27].
4.3. Computational part
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The values of the following parameters: SA, V, HE, logP, MR, a, m,
EHOMO, ELUMO were calculated using semi-empirical AM1 method as
implemented in HyperChem [28]. In a first step, the molecular
geometries of all compounds were fully optimized in the gas phase
to gradients of 0.01 kcal/(mol A) and afterwards the molecular
descriptors were determined.

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