Synthesis and biological evaluation of some new N1-[4-(4- Chlorophenylsulfonyl)benzoyl]-N4-(aryl)-thiosemicarbazides and products of their cyclization

Article abstract of DOI:10.1002/hc.21095

In the present study, new 1,2,4-triazoles, 1,3,4-thiadiazoles, and acylthiosemicarbaz-ides derived from 4-(4-chlorophenylsulfonyl)benzoic acid hydrazide were synthesized and screened for their antimicrobial and analgesic activities. Acylthiosemicarbazides 2-4 were synthesized by a reaction of 4-(4-chlorophenyl-sulfonyl)benzoic acid hydrazide 1 with different arylisothiocyanates.4,5-Disubstituted-2,4-dihydro-3H-1,2,4-triazol-3-thiones 5-7 and 2,5-disubstituted-1,3,4-thiadiazoles 8-10 were obtained by dehydrative cyclization of corresponding acylthiosemicarbazide derivatives 2-4 in basic media (8% aqueous sodium hydroxide) and in acidic media (sulfuric acid or phosphorous oxychloride), respectively. The structures of the newly synthesized compounds have been confirmed on the basis of elemental analysis and spectral studies (IR, ¹H NMR, ¹³C NMR, MS). Their antimicrobial activities against some bacteria and yeasts were investigated. The analgesic activity of all compounds was performed with two pharmacological tests: the writhing test induced with acetic acid and hot-plate test. The results showed that triazole 7 had the best antimicrobial activity against Bacillus cereus. In the chemical stimulus test, triazoles 6 and 7 were the most active compounds whereas in the hot-plate test thiadiazoles 9 and 10 exhibited the highest analgesic activity.

Full text of DOI:10.1002/hc.21095

Heteroatom Chemistry
Volume 24, Number 4, 2013
Synthesis and Biological Evaluation of Some
New N1-[4-(4-Chlorophenylsulfonyl)benzoyl]-
N4-(aryl)-thiosemicarbazides and Products of
Their Cyclization
Stefania-Felicia Barbuceanu,1 Gabriela Bancescu,2
Gabriel Saramet,3 Florica Barbuceanu,4 Constantin Draghici,5
Flavian Stefan Radulescu,6 Aura Ionescu,7 and Simona Negres7
1Organic Chemistry Department, Faculty of Pharmacy, 020956, Bucharest, Romania
2Microbiology Department, Faculty of Dentistry, 050051, Bucharest, Romania
3Pharmaceutical Techniques Department, Faculty of Pharmacy, 020956, Bucharest, Romania
4Institute for Diagnosis and Animal Health, 050557, Bucharest, Romania
5C. D. Nenitescu Institute of Organic Chemistry, Romanian Academy, 060023, Bucharest,
Romania
6Pharmaceutical Industry Department, Faculty of Pharmacy, 020956, Bucharest, Romania
7Pharmacology and Clinical Pharmacy Department, Faculty of Pharmacy, 020956, Bucharest,
Romania
Received 8 December 2012; revised 3 April 2013
disubstituted-1,3,4-thiadiazoles 8–10 were obtained
ABSTRACT: In the present study, new 1,2,4-
by dehydrative cyclization of corresponding acylth-
triazoles, 1,3,4-thiadiazoles, and acylthiosemicarbaz-
iosemicarbazide derivatives 2–4 in basic media (8%
ides derived from 4-(4-chlorophenylsulfonyl)benzoic
aqueous sodium hydroxide) and in acidic media (sul-
acid hydrazide were synthesized and screened for their
furic acid or phosphorous oxychloride), respectively.
antimicrobial and analgesic activities. Acylthiosemi-
The structures of the newly synthesized compounds
carbazides 2–4 were synthesized by a reaction of
have been confirmed on the basis of elemental analy-
4-(4-chlorophenyl-sulfonyl)benzoic acid hydrazide 1
sis and spectral studies (IR, 1H NMR, 13C NMR, MS).
with different arylisothiocyanates.4,5-Disubstituted-
Their antimicrobial activities against some bacteria
2,4-dihydro-3H-1,2,4-triazol-3-thiones 5–7 and 2,5-
and yeasts were investigated. The analgesic activity of
all compounds was performed with two pharmacolog-
ical tests: the writhing test induced with acetic acid
and hot-plate test. The results showed that triazole
Correspondence to: Stefania-Felicia Barbuceanu; e-mail:
sbarbuceanu@gmail.com.
7 had the best antimicrobial activity against Bacil-
lus cereus. In the chemical stimulus test, triazoles
6 and 7 were the most active compounds whereas
in the hot-plate test thiadiazoles 9 and 10 exhibited
Contract grant sponsor: Sectoral Operational Programme Hu-
man Resources Development, European Social Fund.
Contract grant sponsor: Government of Romania.
Contract grant number: POSDRU/89/1.5/S/64109.
ꢀC
2013 Wiley Periodicals, Inc.
309

310 Barbuceanu et al.
ꢀC
the highest analgesic activity. 2013 Wiley Periodi-
cals, Inc. Heteroatom Chem. 24:309–321, 2013; View
this article online at wileyonlinelibrary.com. DOI
10.1002/hc.21095
are several well-known drugs, for example, acetazo-
lamide (diuretic), sulfamethizole, and cefazolin (an-
tibacterial) that contain the 1,3,4-thiadiazole hetero-
cycle [12–14].
In addition, the literature survey indicates that a
large number of 1,2,4-triazole derivatives are known
to exhibit various pharmacological properties in-
cluding antibacterial [15–17], antifungal [15–18],
analgesic [19–21], anti-inflammatory [19–24], and
anticancer [25, 26]. It was reported that biological
activities of triazole are due to the presence of the
NH-C(S) NH function in a molecule, and the
change in activity depends on the nature of the sub-
stituents [27].
On the other hand, many 1,3,4-thiadiazole
derivatives are also associated with different phar-
macological activities, viz. antibacterial [15, 16,
28], antifungal [15, 16, 28], analgesic [19, 21], anti-
inflammatory [19–22], antitumoral [29, 30]. The lit-
erature reveals that broad spectrum of biological ac-
tivities of 1,3,4-thiadiazole is probably due to the
INTRODUCTION
The serious medical problem of bacterial and fun-
gal resistance and the rate at which it develops has
led to increasing levels of attention in classical an-
tibiotics. Owing to the development of antimicro-
bial resistance, which has increased in this century,
there is a great need to synthesize new antimicro-
bial agents, which have to be more selective, potent,
and less toxic compared to the drugs used in clinical
treatment.
On the other hand, nonsteroidal anti-
inflammatory drugs, which are widely used for
reducing pain and swelling associated with inflam-
mation, represent an area of continuous and ever
growing development.
Sulfone derivatives are an important class of
compounds since they have biological and pharma-
cological properties, such as antibacterial and antivi-
ral properties [1–3]. On the other hand, the sulfone
moiety is usually incorporated as an active part in
many analgesic anti-inflammatory molecules avail-
able as drugs [4]. Dapsone, a prototype of sulfones,
has antibacterial and anti-inflammatory activity, be-
ing used as a primary drug on leprosy and is also
used in several inflammatory dermatitis [5,6].
Heterocyclic compounds also play an important
role in the design and discovery of new pharmaco-
logically active compounds.
presence of toxophoric N
C S moiety [28].
Moreover, acylthiosemicarbazides, the key inter-
mediates used in the synthesis of triazoles and thia-
diazoles, are biologically versatile compounds dis-
playing a variety of biological effects, which include
antibacterial, antifungal, and analgesic properties
[31–34].
In the design of new drugs, the combination of
different pharmacophore frames may lead to com-
pounds with interesting biological profiles. On the
basis of this premise, we aimed to obtain new deriva-
tives with potential biological activity by association
of these pharmacophore groups (a diphenylsulfone
fragment and 1,2,4-triazole or 1,3,4-thiadiazole ring)
into a unique molecule.
The use of acylthiosemicarbazides in organic
synthesis has become a classical strategy for the
synthesis of several heterocycles. Among the het-
erocyclic compounds, which are obtained from
acylthiosemicarbazides are 1,2,4-triazoles and 1,3,4-
thiadiazoles, which are known for their biological
activity.
The 1,2,4-triazole and 1,3,4-thiadiazole nucleus
have been incorporated into a wide variety of ther-
apeutically important agents. 1,2,4-Triazoles have
revolutionized the prevention and treatment of in-
vasive fungal infections. Drugs such as flucona-
zole, itraconazole, terconazole, voriconazole, and
posaconazole including in their molecule 1,2,4-
triazole moiety have been used for the treatment of
fungal infections [7–9]. Letrozole and anastrozole
(antitumoral) [9, 10], ribavirin (antiviral), and riza-
triptan (antimigraine) [10,11] are other examples of
drugs containing this heterocycle. Moreover, there
We have recently reported [35] some
heterocyclic
analogues
carrying
5-(4-(4-X-
phenylsulfonyl)phenyl)- moiety (where X = H,
Br) and other groups (4-trifluoromethoxyphenyl
and 3,4,5-trimethoxyphenyl) in their molecules
as potential antimicrobial agents. Some of them
have promising biological properties. These find-
ings prompted us to extend our studies on the
development of novel antimicrobial agents.
Taking into account the literature data and en-
couraged by the research in the domain of potential
biological active heterocyclic compounds contain-
ing sulfur and nitrogen atoms [36–39], in this report
we describe the design, chemical synthesis, charac-
terization, and biological evaluation of some new
1,2,4-triazoles, 1,3,4-thiadiazoles, and their acylth-
iosemicarbazides (wherein the sulfone group is 5-(4-
(4-chlorophenylsulfonyl)phenyl)- moiety) as antimi-
crobial and analgesic agents.
Heteroatom Chemistry DOI 10.1002/hc

Some New N1-[4-(4-Chlorophenylsulfonyl)benzoyl]-N4-(aryl)-thiosemicarbazides and Their Cyclization Products 311
Ar
NH
O
H
N
C
C
S
HN
C
NH
NH2
a
O
Cl
S
O
O
1
Cl
S
O
O
2–4
d
c
b
X
SH
Ar
H
N
S
N
N
Ar
Ar
H
N
N
O
N
O
H
Ar
N
N
N
N
N
N
S
S
Cl
S
Cl
S
O
O
5–7
Cl
S
O
Cl
S
O
O
O
9,10
8
2 5 8
3 6 9
4 7 10
, , : Ar = 2-F-C6H4-; , , : Ar = 4-F3CO-C6H4-; , , : Ar = 3,4,5-(H3CO)3-C6H2-
SCHEME 1 Synthesis of target compounds 2–10. Reagents and conditions: (a) ArNCS, C2H5OH, reflux; (b) NaOH 8%, reflux;
(c) H2SO4 0◦C; (d) POCl3, reflux.
RESULTS AND DISCUSSION
Chemistry
cyclodehydration of acylthiosemicarbazides 3 and 4
in the presence of concentrated sulfuric acid, at 0◦C.
An attempt to cyclize acylthiosemicarbazide 2 with
sulfuric acid, in the same condition, did not yield
the expected 1,3,4-thiadiazole 8. This compound
was successfully obtained by cyclization of this
thiosemicarbazide via heating with phosphorus
oxychloride.
The synthesis pathway leading to the title com-
pounds is shown in Scheme 1.
The precursor used for the synthesis of new com-
pounds, 4-(4-chlorophenylsulfonyl)-benzoic acid hy-
drazide 1, was already described in the literature
[40].
The reaction of 4-(4-chlorophenylsulfonyl)-
benzoic acid hydrazide 1 with appropriate aryliso-
thiocyanates (2-fluorophenyl-, 4-trifluoromethoxy-
phenyl-, and 3,4,5-trimethoxyphenyl isothiocyanate)
gave N1-[4-(4-chlorophenylsulfonyl)benzoyl]-N4-(Ar)-
thiosemicarbazides 2–4. Heterocyclic compounds
from 1,2,4-triazole-3-thione class, 5-(4-(4-chlorop-
henylsulfonyl)phenyl)-4-(Ar)-2H-1,2,4-triazol-3(4H)-
thiones 5–7, were prepared by cyclodehydration
of the corresponding acylthiosemicarbazides 2–4
with a sodium hydroxide solution, under reflux
conditions. Alternatively, the thiosemicarbazides
2–4 were cyclized into the heterocyclic compounds
from 1,3,4-thiadiazole class 8–10, in acidic media.
The physical and spectral data of newly synthe-
sized compounds 2–10 are in agreement with the
proposed structures.
The infrared spectra of acylthiosemicarbazides
2–4 confirmed that nucleophilic addition of 4-
(4-chlorophenylsulfonyl)-benzoic acid hydrazide 1
to corresponding arylisothiocyanates took place
through the presence of new absorption band char-
acteristic to C S stretching vibration at 1210–
1245 cm−1. The absorption bands due to the
vibration of νC O and νNH from acylthiosemi-
carbazides appeared in the region 1677–1684 and
3145–3327 cm−1, respectively [20, 30, 41, 42]. In-
tramolecular cyclization of acylthiosemicarbazides
to heterocyclic compounds from triazole and thiadi-
azole class was confirmed in IR spectra of 5–10 by
the disappearance of the carbonyl absorption band,
The
5-(4-(4-chlorophenylsulfonyl)phenyl)-N-(Ar)-
1,3,4-thiadiazol-2-amines 9 and 10 were prepared by
Heteroatom Chemistry DOI 10.1002/hc

312 Barbuceanu et al.
which was present in acyclic intermediates at 1677–
[19,29,43]. The C(3) carbon resonated at ∼169 ppm
in the 13C NMR spectra of 1,2,4-triazole 5–7, as-
signed for C S, confirming a thione form of the tri-
azole ring [19,27,35].
The signals corresponding to protons and
carbons from 4-(4-chlorophenylsulfonyl)phenyl, 2-
fluorophenyl, 4-trifluoromethoxyphenyl, and 3,4,5-
trimethoxyphenyl moieties appeared in 1H and
13CNMR spectra of new compounds 2–10 at the ex-
pected chemical shifts [35–39,46].
Further evidence for the formation of these new
compounds 2–4 was obtained by recording its mass
spectra. All new synthesized compounds showed in
the mass spectra molecular ions corresponding to
their molecular formula. Because of the presence
of the chlorine atom in their molecule, their mass
spectra show the characteristic peaks corresponding
to isotopic distribution (35Cl and 37Cl isotopes). The
important common fragments are presented in the
Experimental.
1684 cm−1
.
The IR spectra of heterocyclic compounds 5–
10 showed a single vibration band corresponding
to the νNH group, which appeared in the region
3276–3428 cm−1 for triazole and 3243–3357 for thia-
diazole [16, 18, 41, 42]. Although 1,2,4-triazoles may
exist in thione–thiol tautomeric forms, our inves-
tigations show that in the case of compounds 5–7
the thione dominates in the solid state and in so-
lution, as indicated by the IR and NMR data. The
IR spectra of triazoles 5–7 displayed a C S stretch-
ing band about 1217–1240 cm−1 and no absorption
attributed to SH was observed in the region 2500–
2690 cm−1 [16,18,20,27,42]. A new absorption band
that appeared in IR spectra of compounds 5–10 in
the 1596–1628 cm−1 region indicated the presence of
C N from triazole and thiadiazole rings [16,20,43].
The SO2 stretching vibrations appeared in these
compounds in the 1157–1331 cm−1 region. The
absorption bands associated with other functional
groups appeared in the expected regions.
Biological Evaluation
The NMR spectra of compounds 2–10 con-
tain two subspectra characteristic of the 5-(4-(4-
chlorophenylsulfonyl)phenyl)- and of the triazole/
thiadiazole/thiosemicarbazide moiety. All com-
pounds were also identified by the homo- and het-
eronuclear spectra.
Antimicrobial Activity Evaluation. The in vitro
antimicrobial activity of newly compounds 2–10
was tested, by the broth microdilution method,
against the following reference bacterial strains:
Escherichia coli ATCC 25922, Enterobacter cloacae
ATCC 49141, Acinetobacter baumannii ATCC 19606,
Pseudomonas aeruginosa ATCC 27853, Staphylococ-
cus aureus ATCC 25923, Bacillus cereus ATCC 13061,
and the following reference yeasts strains: Candida
albicans ATCC 90028, C. parapsilosis ATCC 22019, C.
glabrata ATCC 15126, and C. tropicalis ATCC 13803.
For quality control, E. coli ATCC 25922 and
P. aeruginosa ATCC 27853 were tested against
amikacin, whereas fluconazole against C. parapsilo-
sis ATCC 22019 using the same broth microdilution
method.
1
In the H NMR spectra of compounds 2–4, the
protons linked to nitrogen atoms appeared as sin-
1
glets at δ 9.64–10.80 ppm [19, 28, 42, 44]. In the H
NMR spectra of compounds 5–7, the singlet signal
of the NH group from the 1,2,4-triazole-3-thione
derivatives was observed in the 14.26–14.46 ppm
range [18, 41]. In the case of compounds contain-
ing the 1,3,4-thiadiazole moiety (8–10), the 1H NMR
spectra displayed a singlet signal, which resonated
in region 10.52–10.89 ppm, assigned to secondary
amine NH protons [20,28,30].
The 13C NMR spectra of compounds 2–4 have
confirmed a reaction between hydrazide and isothio-
cyanates through the presence of a new signal, which
is the most deshielded, corresponding to C S car-
bon, occurring at δ 180.63–182.17 ppm [42, 43]. In
addition, the C O signal was observed at δ 164.28–
164.63 ppm [43]. The 13C NMR of compounds 5–
10 displayed no signals belonging to C O and C S
groups from thiosemicarbazide. Instead of these,
new signals derived from triazole and thiadiazole
rings appeared at the expected chemical shifts: The
C(3)/C(5) heterocyclic carbon from 1,2,4-triazole
ring resonated at 168.96–169.43 ppm/148.94–149.16
ppm [19, 30, 45], and the C(2)/C(5) heterocyclic
carbon from the 1,3,4-thiadiazole ring appeared
at 164.84–165.46/155.45–156.84 ppm, respectively
The results regarding the investigation of the an-
tibacterial and antifungal activity of newly synthe-
sized compounds are indicated in Table 1.
The analysis of the results obtained through the
determination of the antibacterial activity against
the Gram-positive bacteria (Table 1) showed that
all compounds have better action against B. cereus.
The best antimicrobial activity was shown by tri-
azole 7 with the lowest minimum inhibitory con-
centration (MIC) value (MIC = 8 μg/mL) followed
by thiosemicarbazide 4 (MIC = 16 μg/mL), which
contained in their molecule the trimethoxyphenyl
fragment.
Results obtained against S. aureus showed that
except for thiosemicarbazides 2 and 4, which had a
better activity (with a MIC value of 128 μg/mL), the
Heteroatom Chemistry DOI 10.1002/hc

Some New N1-[4-(4-Chlorophenylsulfonyl)benzoyl]-N4-(aryl)-thiosemicarbazides and Their Cyclization Products 313
TABLE 1 Antimicrobial Activities of Compounds 2–10 as MIC Values (μg/mL)
Gram-Positive
Bacteriaa
Gram-Negative Bacteriab
Yeastsc
Compound
Sa
Bc
Ec
Ecl
Ab
Pa
Ca
Cp
Cg
Ct
2
128
512
128
256
512
256
512
512
512
–
128
32
16
32
32
8
128
64
32
–
128
128
128
128
128
128
128
128
128
2
128
128
128
128
128
128
128
128
128
–
64
32
32
64
32
32
32
32
32
–
64
64
64
128
64
64
128
64
64
2
64
64
64
128
128
64
64
64
64
–
64
64
64
64
64
64
64
64
64
–
64
64
64
64
64
64
64
64
64
–
128
64
64
64
64
64
128
64
64
–
3
4
5
6
7
8
9
10
Control (AM)d
Control (FL)
–
–
–
–
–
–
2
–
–
aSa (S. aureus ATCC 25923); Bc (B. cereus ATCC 13061).
bEc (E. coli ATCC 25922); Ecl (Enterobacter cloacae ATCC 49141); Ab (A. baumannii ATCC 19606); Pa (P. aeruginosa ATCC 27853).
cCa (C. albicans ATCC 90028); Cp (C. parapsilosis ATCC 22019); Cg (C. glabrata ATCC 15126); Ct (tropicalis ATCC 13803).
dControl: AM = amikacin; FL = fluconazole.
other ones presented the poorest antibacterial activ-
ity (MIC = 256 or 512 μg/mL) against this strain from
all the reference strains investigated in this study.
Against Gram-negative bacteria, all compounds
showed the same MIC value (of 128 μg/mL) on both
bacteria belonging to the family of Enterobacteri-
aceae (the reference strains of E. coli and E. cloacae).
The newly synthesized compounds revealed a
much better action (MIC = 32 μg/mL) against A.
baumannii, except for thiosemicarbazide 2 and tria-
zole 5, which have in their molecule 2-fluorophenyl
fragment (MIC = 64 μg/mL).
tion 2 of amino-thiadiazole is beneficial in the case
of B. cereus (compounds 4, 7, and 10).
Some of these results are similar to those previ-
ously obtained in the case of compounds with similar
chemical structures [35].
In any case, the antimicrobial activity of the
compounds with this substituent was not less, but
at least equal to activity in which the substituent
was the 2-fluorophenyl- or 4-trifluoromethoxyphenyl
fragment.
The presence of fluorine (with the strong elec-
tron attracting effect, –I) directly linked to the aro-
matic ring, on the same reference strains, leads gen-
erally to lower antimicrobial activity, except for the
cases where all the compounds have the same value
for MIC.
Another conclusion from the analysis of the re-
sults obtained by especially testing antibacterial and
antifungal activities is that in most cases, cycliza-
tion of thiosemicarbazides to triazoles or thiadia-
zoles, except activity against B. cereus, did not result
in significant changes.
Referring to the correlation between molecular
descriptors of the new compounds synthesized and
their antimicrobial activity (Table 2), it is noted that
the compounds containing the trimethoxyphenyl
fragment show the smallest value of octanol–water
partition coefficient (log P) and that in some cases
this coefficient coincided with the lowest MIC value
(B. cereus). In all cases, log P of the compounds
that contained the trimethoxyphenyl fragment was
lower than log P of compounds that contained the
fluorophenyl or trifluoromethoxyphenyl fragment.
However, in the case of compounds containing
A better antibacterial activity was recorded
against P. aeruginosa, all compounds indicated the
same MIC value (64 μg/mL), except for triazole 5 and
thiadiazole 8, which contained the 2-fluorophenyl
fragment (MIC = 128 μg/mL).
Regarding the antifungal activity, all the tested
compounds developed the same MIC (64 μg/mL)
against the four reference strains of Candida, ex-
cept for triazoles 5 and 6 (with 2-fluorophenyl and
4-trifluoromethoxyphenyl fragments) against C. al-
bicans and compounds 2 and 8 (with 2-fluorophenyl
fragment) against C. tropicalis, which showed a
weaker activity (MIC = 128 μg/mL).
The MIC values obtained for amikacin and
fluconazole against the tested microbial reference
strains are indicated in Table 1.
A qualitative assessment of the influence of
structure on antimicrobial activity reveals that
the presence of the 3,4,5-trimethoxy substituent
(electron-donating group, +M) on the aromatic ring
linked to the N-4 nitrogen atom from thiosemicar-
bazides and triazoles or to the NH group from posi-
Heteroatom Chemistry DOI 10.1002/hc

314 Barbuceanu et al.
TABLE 2 Calculated QSAR Parameters of the Compounds 2–10
Compounds Log P MV
SAS
PSA
μ
αG
αM
MinPA MaxPA
MR
DE
HBD HBA NFB
2
3.80 343
3.84 378
550.55 87.30 4.89 46.40 45.89 56.82 112.45 119.678 98.68
609.45 96.53 4.53 48.77 48.09 67.19 123.53 122.532 104.82
3
3
3
1
1
1
1
1
1
6
7
9
3
4
6
5
6
8
7
9
10
2
4
5
4
6
7
3
4
3.33 423.5 687.51 114.99 4.89 54.43 53.51 74.12 130.50 138.852 114.82
4.25 315
4.45 350
4.04 395.5 652.67 95.44 4.21 52.79 51.95 69.48 121.99 134.228 125.59
4.74 315
4.86 350
5
515.30 67.75 3.94 44.77 44.33 58.76 104.48 115.055 110.88
575.12 76.98 3.77 47.15 46.52 67.91 115.33 117.909 116.77
6
7
8
525.71 71.95 4.55 45.01 42.71 52.17 115.42 123.004 113.79
585.48 81.18 4.55 47.39 44.97 54.7 121.87 125.858 121.51
9
10
4.28 395.5 663.44 99.64 4.78 53.05 50.34 63.36 131.41 142.177 131.21
3
2
2
˚
˚
Abbreviations: MV, volume (cm /mol); SAS, solvent accessible surface area (A ); PSA, polar surface area (A ); μ, dipole moment; αG,
3
3
2
˚
˚
˚
polarizability, Glen’s method (A ); αM, polarizability, Miller’s method (A ); MinPA, minimal projection area (A ); MaxPA, maximal projection area
(A ); MR, molecular refractivity; DE, Dreiding energy (kcal/mol); HBD, the number of hydrogen bond donors; HBA, the number of hydrogen
2
˚
bond acceptors; NFB, the number of freely rotatable bonds.
TABLE 3 Analgesic Effect intensity (%) against the Control
Group of Tested Compounds 2–10, in the Chemical Stimulus
Test
fluorine, although the log P value is lower than that
with the OCF3 substituent, antimicrobial activity
was lower.
Owing to the high values of log P, between 3.33
and 4.86 (Table 2), it is reasonable to assume that the
low water solubility is the limiting factor of their ab-
sorption and penetration across the lipid interfaces.
Nevertheless, the high lipophilicity is likely to deter-
mine a rapid distribution to lipidic compartments.
When both log P and polar surface area (PSA) are
considered, the current approaches seem to indicate
Effect(%)/
Control
t-Student
p/Control
Compound
Control
Mean SEM
30.90 0.64
–
–
ASA
2
8.0 0.68
21.30 1.415
24.00 2.517
16.20 1.172
27.00 1.619
14.60 1.984
15.20 1.925
23.40 1.384
20.80 1.482
19.60 1.439
74.11
31.06
22.33
47.57
12.62
52.72
50.80
24.27
32.68
36.56
<0.0001
<0.0001
0.0160
<0.0001
0.0380
<0.0001
<0.0001
0.0001
<0.0001
<0.0001
3
4
5
a low permeability across blood–brain barrier, since
6
2
˚
optimal PSA is lower than 60–70 A [47–51].
7
8
The results obtained show that a direct relation-
ship between log P and antimicrobial activity can-
not be established in the case of new synthesized
compounds.
9
10
Pharmacological Activity Evaluation. In the
pharmacological study, we investigated the anal-
gesic activity of both thiosemicarbazide intermedi-
ates 2–4 and corresponding triazoles 5–7 and thia-
diazoles 8–10.
parable to that of acetylsalicylic acid (ASA; Tables 3
and 4).
The analgesic activity, in the chemical stimulus
test, was the most intense for triazole 6 (52.72%),
which contained the trifluoromethoxyphenyl frag-
ment in a molecule and triazole 7 (50.80%) that has
the trimethoxyphenyl fragment, but lower than that
of ASA (74.11%). Of all the compounds studied, the
lowest analgesic effect was observed in triazole 5
(12.62%) that contained in its molecule the fluorine
atom (Table 3 and Fig. 1).
In the thiadiazoles class, compound 10, which
contains the trimethoxyphenyl fragment in the
molecule, showed better activity (36.56%) followed
by 9 that contained the trifuoromethoxyphenyl frag-
ment (32.68%) and than by compound 8 with the
fluorophenyl substituent (24.27%).
First, acute toxicity of newly synthesized com-
pounds was tested. Experimental data highlighted
the fact that compounds investigated, administered
in 1000 mg/kg-body weight (bw) dose, do not alter
motor or sexual behavior nor the exterior aspects
of treated animals. Body weight increased as did
for the untreated (control) animals, housed in nor-
mal laboratory conditions. The data supported the
hypothesis that newly synthesized compounds are
low-toxicity substances according to Hodge–Sterner
scale of chemical classification based on LD50 [52].
The testing for analgesic activity was performed
with two pharmacological tests: the writhing test in-
duced with acetic acid [53] and hot-plate test [54].
The results indicated that, in both tests of anal-
gesia, some compounds have analgesic action com-
Moreover, in a class of thiosemicarbazides, the
most intense activity was shown by compound 4
(47.57%) with the trimethoxyphenyl substituent.
Heteroatom Chemistry DOI 10.1002/hc

Some New N1-[4-(4-Chlorophenylsulfonyl)benzoyl]-N4-(aryl)-thiosemicarbazides and Their Cyclization Products 315
TABLE 4 Analgesic Effect Intensity (%) for the Tested Compounds 2–10 against the Control Group, in the Hot-Plate Test
Basal Lick T (s)
Mean SEM
Lick T after 1 h (s)
Mean SEM
Effect%/
Immediate
p/Control/
Group
Effect%/
Control
Compound
p/Basal/Group
Control
7.930
7.700
8.070
8.450
7.730
7.740
8.000
7.390
8.880
8.580
8.900
0.4395
0.4946
0.5237
0.5780
0.9048
0.8042
0.7501
0.5178
0.4914
0.6868
0.6725
8.330
15.51
10.34
12.02
10.80
14.99
14.09
9.50
0.4573
1.087
1.017
1.131
0.7722
1.713
0.9966
0.6188
1.190
1.582
1.513
0.5362 ns
<0.0001
0.0627 ns
<0.0001
0.0188
0.0012
0.0001
0.0053
0.0560 ns
0.0008
5.044136
101.4286
28.12887
42.24852
39.71539
93.66925
76
34.64141
29.61712
81.23543
78.53933
–
–
ASA
2
<0.0001
0,0883 ns
0,0073
0,0254
0,0014
<0.0001
0,0496
0,0226
0,0004
0,0001
86.19448
24.12965
44.29772
29.65186
79.95198
69.02761
19.44778
38.17527
86.67467
90.7563
3
4
5
6
7
8
11.51
15.55
15.89
9
10
0.0005
Statistical significance of results (t Student), against the immediate measurement of each group.
FIGURE 1 Analgesic effect intensity (%) against the control group for the tested compounds 2–10 and for reference substance
(acetylsalicylic acid), in the chemical stimulus test (***p < 0.001).
In the thermal stimulus test (hot plate), a statis-
tically significant analgesic effect was documented
against the control group for the compounds 3–
7, 9, 10 (Table 4 and Fig. 2). The most intense
analgesic action in this test was shown by thiadi-
azole 10 (90.75%), which has in the molecule the
trimethoxyphenyl substituent followed by thiadia-
zole 9 with trifluoromethoxy fragment (86.67%),
higher than of ASA (86.19%).
As shown in Table 4 and Fig. 2, although triazole
7 had good analgesic activity in the writhing test in-
duced with acetic acid (50.8%; Table 3, Fig. 1), it had
the weakest effect in the hot-plate test (19.44%). In
the class of acylthiosemicarbazides, the analgesic ef-
fect intensity variation as follows: trifluoromethoxy
> trimethoxy > fluorine, whereas in the case of tria-
zoles this variation is fluorine > trifluoromethoxy >
trimethoxy.
CONCLUSIONS
To summarize, a new series of 1,2,4-triazole, 1,3,4-
thiadiazole, and acylthiosemicarbazide derivatives
were successfully synthesized. All these new com-
1
pounds were confirmed by IR, H NMR, 13C NMR,
mass spectra, as well as elemental analysis.
All compounds synthesized were screened for
their antimicrobial and analgesic activities. Their
Heteroatom Chemistry DOI 10.1002/hc

316 Barbuceanu et al.
FIGURE 2 Analgesic effect intensity (%) against the control group for the tested compounds 2–10 and for reference substance
(acetylsalicylic acid), in the thermal stimulus test (*p < 0.05; **p < 0.01; ***p < 0.001).
antimicrobial activities were evaluated against ref-
erence bacterial and yeasts strains. The analgesic ac-
tivity was performed with the writhing test induced
with acetic acid and the hot-plate test.
apparatus and are uncorrected. Elemental analyses
(the percentage of C, H, N) for all compounds
were performed using a Costech ECS 4010 micro-
dosimeter. Nuclear magnetic resonance (1H NMR
and 13C NMR) spectra were recorded on a Varian
Gemini 300 BB instrument working at 300 MHz for
The results of antimicrobial data show that,
among the synthesized compounds, triazole 7,
which contains the trimethoxyphenyl fragment, was
the most active (MIC = 8 μg/mL) against B. cereus.
The analgesic data indicated that, in the chemi-
cal stimulus test, triazoles 6 (which contains the
trifluoromethoxyphenyl fragment) and 7 (with the
trimethoxyphenyl fragment) were the most active
compounds. In the hot-plate test, the thiadiazoles 10
(which contains trimethoxyphenyl fragment) and 9
(with the trifluoromethoxyphenyl fragment) exhib-
ited the highest analgesic activity and were more ac-
tive than acetylsalicylic acid.
1
a H and 75 MHz for 13C, using dimethylsulfoxide
(DMSO-d6) as a solvent. The chemical shifts are
expressed in δ (ppm) relative to trimethylsilane
as an internal reference. Splitting patterns are as
follows: s = singlet, d = doublet, dd = double
doublet; t = triplet, q = quartet, m = multiplet,
and b = broad peak). Infrared spectra (IR) were
obtained with a Vertex 70 Bruker spectropho-
tometer using potassium bromide pellets, and
the results are expressed in wave number (cm−1).
Mass spectra were obtained on a triple quadrupole
mass spectrometer Varian 1200 L/MS/MS with
electrospray interface (ESI) coupled with a Varian
ProStar 240 SDM pump. Sample solution (2 μg/mL
in methanol/0.1% ammonia in water 10/1, v/v) was
introduced in the ESI interface by direct infusion,
at a flow rate of 20 μL/min. The instrument was
operated in positive ions mode. Fragmentation of
the protonated molecular ion was performed by
collision with argon at 1.5 mTorr. Ultraviolet (UV)
spectra were recorded on a Specord 40 Analytik
Jena spectrometer in methanol at approximatively
2.5 × 10−5 mol/L.
To understand the structure–biological activity
relationship and the mechanisms involved in the
antimicrobial and analgesic properties, it is nec-
essary to synthesize new compounds with similar
structures.
EXPERIMENTAL
Chemistry
All chemical and solvents were of analytical reagent
grade. Melting points were recorded on a Boetius
Heteroatom Chemistry DOI 10.1002/hc

Some New N1-[4-(4-Chlorophenylsulfonyl)benzoyl]-N4-(aryl)-thiosemicarbazides and Their Cyclization Products 317
2.79; N, 7.88; ESI-MS, m/z (%): 530/532 [M + H]+;
General Procedure for the Synthesis of
353/355 (24/11) [M – F3COC6H4NH]+; 311/313 (100)
[ClC6H4SO2C6H4CONHNH3]+; 279/281 (10/11)
[ClC6H4SO2C6H4CO]+.
N1-[4-(4-Chlorophenylsulfonyl)Benzoyl]-N4-
(Ar)-Thiosemicarbazides 2–4
4-(4-Chlorophenylsulfonyl)benzoic acid hydrazide 1
(5 mmol) was refluxed for 10 h in 25 mL of
ethanol with the addition of stoechiometric quantity
(5 mmol) of appropriate isothiocyanate. The mix-
ture was then cooled, precipitate was filtered off,
washed with cold ethanol, dried, and recrystallized
from ethanol.
N1-[4-(4-Chlorophenylsulfonyl)benzoyl]-N4-(3,4,
5-trimethoxyphenyl)-thiosemicarbazide
4. Yield
91.1%; mp = 183–185◦C; IR (KBr, ν, cm−1): 3301,
3150, 3145 (NH), 3090, 3002 (CHaryl), 2839 (CH3),
1677 (C O), 1575, 1507, 1459 (C C), 1300, 1158
(SO2), 1236 (C S+ C
O C), 1128 (C O C), 766
(C Cl); 1H NMR (DMSO-d6) δ : 3.63 (s, 6H, 3-OCH3,
5-OCH3), 3.73 (s, 3H, 4-OCH3), 6.82 (s, 2H, ArH),
7.71 (d, J = 8.8 Hz, 2H, ArH), 8.01 (d, J = 8.8 Hz,
2H; ArH), 8.10 (d, J = 8.8 Hz, 2H, ArH), 8.13 (d, J =
8.8 Hz, 2H, ArH), 9.80 (bs, 2H; 2NH), 10.80 (bs, 1H,
NH) ppm; 13C NMR (DMSO-d6) δ: 55.93 (3-OCH3,
5-OCH3), 60.15 (4-OCH3), 103.39, 127.59, 129.35,
129.58, 134.77; 134.87, 137.48, 139.11, 139.27,
139.47, 143.30, 152.27, 164.63 (C O); 180.63(C S)
ppm; UV (CH3OH, λmax, nm (log ε)): 252.0 (4.40),
360.4 (3.31); Anal. calcd for C23H22ClN3O6S2 (536.02
g/mol): C, 51.53; H, 4.14; N, 7.84. Found. C, 51.61; H,
4.09; N, 7.79; ESI-MS, m/z (%): 536/538 [M + H]+;
352/354 (2) [ClC6H4SO2C6H4CON=NC=S + H]+;
311/313 (100) [ClC6H4SO2C6H4CONHNH3]+; 226
(41) [(CH3O)3C6H2NHC=S]+; 184 (11) [(CH3O)3
C6H2NH3]+.
N1-[4-(4-Chlorophenylsulfonyl)Benzoyl]-N4-(2-
Fluorophenyl)-Thiosemicarbazide 2. Yield 91.2%;
mp = 186–188◦C; IR (KBr, ν, cm−1): 3318, 3170
(NH), 3083, 3052, 3019 (CHaryl), 1684 (C O), 1596,
1543, 1524, 1480 (C C), 1313, 1297, 1157 (SO2),
1264 (C F), 1245 (C S), 762 (C Cl); 1H NMR
(DMSO-d6) δ: 7.10–7.35 (m, 4H, ArH), 7.71 (d, J =
8.5 Hz, 2H, ArH), 8.02 (d, J = 8.5 Hz, 2H, ArH),
8.11 (d, J = 8.8 Hz, 2H; ArH), 8.15 (d, J = 8.8 Hz,
2H, ArH), 9.64 (s, 1H, NH), 9.96 (s, 1H, NH), 10.87
(bs, 1H, NH) ppm; 13C NMR (DMSO-d6) δ: 115.70
(d, J2
= 19.2 Hz); 123.94 (d, J4
= 2.9 Hz);
C-F
C-F
127.03 (d, J2
= 11.3 Hz); 127.44; 127.46 (d; J3
C-F
C-F
= 8.3 Hz); 128.19; 129.38 (d; J3
= 7.2 Hz); 129.48;
C-F
130.00; 137.22; 139.14; 139.40; 143.25; 157.34 (d;
J1
= 246.8 Hz); 164.58 (C O); 182.17 (C S) ppm;
C-F
UV (CH3OH, λmax, nm (log ε)): 251.1 (4.44), 357.7
(3.47); Anal. calcd for C20H15ClFN3O3S2 (463.93
g/mol): C, 51.78; H, 3.26; N, 9.06. Found: C, 51.72;
H, 3.19; N, 9.11; ESI-MS, m/z (%): 464/466 [M +
H]+; 353/355 (85/90) [M – FC6H4NH]+; 311/313
(100) [ClC6H4SO2C6H4CONHNH3]+; 279/281 (11/9)
[ClC6H4SO2C6H4CO]+; 154 (3) [FC6H4NHCS]+; 111
(10) [FC6H4NH2]+.
General Procedure for Synthesis of 5-(4-(4-
Chlorophenylsulfonyl)phenyl)-4-(Ar)-2H-1,2,4-
triazol-3(4H)-thiones 5–7
Appropriate acylthiosemicarbazide (2 mmol) was re-
fluxed in 30 mL of 8% NaOH aqueous solution for
2 h. The hot mixture was filtered, cooled, and acid-
ified with diluted hydrochloric acid (pH ∼ 5) to the
precipitation of 1,2,4-triazole. The crude product
was filtered off, washed with water, dried, and puri-
fied by recrystallization from chloroform/petroleum
ether (1:2, v/v).
N1-[4-(4-Chlorophenylsulfonyl)benzoyl]-N4-(4-
trifluoromethoxyphenyl)-thiosemicarbazide 3. Yield
91.3%; mp = 180–182◦C; IR (KBr; ν, cm−1): 3327,
3308, 3194 (NH), 3095, 3072, 3016 (CHaryl), 1683
(C O), 1576, 1543, 1525, 1480 (C C), 1323, 1290,
1158 (SO2), 1264 (C
O
C), 1219 (C F), 1210
5-(4-(4-Chlorophenylsulfonyl)phenyl)-4-(2-fluor-
1
(C S), 758 (C Cl); H NMR (DMSO-d6) δ: 7.32 (d,
J = 8.5 Hz, 2H, ArH), 7.59 (bd, J = 8.5 Hz, 2H, ArH),
7.72 (d, J = 8.8 Hz, 2H, ArH), 8.01 (d, J = 8.8 Hz,
2H, ArH), 8.11 (d, J = 9.0 Hz, 2H, ArH), 8.15 (d, J =
9.0 Hz, 2H, ArH), 9.91 (bs, 2H, NH), 10.60 (bs, 1H,
ophenyl)-2H-1,2,4-triazol-3(4H)-thione
5. Yield
83.2%; mp = 231–233◦C; IR (KBr, ν, cm−1): 3276
(NH), 3092, 3078 (CHaryl), 1596 (C N), 1580, 1508,
1471 (C C), 1331, 1288, 1159 (SO2), 1260 (C F),
1240 (C S), 770 (C Cl); 1H NMR (DMSO-d6) δ:
7.38 (td, J = 9.2 Hz, J = 1.5 Hz, 1H, ArH), 7.45 (m,
1H, Ar-H), 7.55–7.66 (m, 2H, ArH), 7.57 (d, J = 8.8
Hz; 2H, ArH), 7.69 (d, J = 8.8 Hz, 2H, ArH), 7.88
(d, J = 8.8 Hz, 2H, ArH), 7.99 (d, J = 8.8 Hz, 2H,
ArH), 14.46 (s, 1H, NH) ppm; 13C NMR (DMSO-d6)
NH) ppm; 13C NMR (DMSO-d6) δ: 120.00 (q; JC-F
=
255.0 Hz; OCF3), 120.55, 127.36, 127.46, 129.09,
129.18, 129.86, 137.30, 138.29, 139.03, 139.37,
143.18, 144.99, 164.28 (C O), 181.07 (C S) ppm; UV
(CH3OH, λmax, nm (log ε)): 251.1 (4.51), 361.2 (3.52);
Anal. calcd for C21H15ClF3N3O4S2 (529.94 g/mol):
C, 47.59; H, 2.85; N, 7.93. Found: C, 47.62; H,
δ: 116.81 (d, J2
= 18.9 Hz); 121.65 (d, J3
=
C-F
C-F
12.3 Hz), 125.56 (d, J4
= 3.4 Hz), 127.98, 128.75,
C-F
Heteroatom Chemistry DOI 10.1002/hc

318 Barbuceanu et al.
[(OCH3)3C6H2 + H]+, 153 (21) [(OCH3)3C6H2
+
129.57, 130.04, 130.14 (d, J2
= 12.6 Hz), 130.15,
C-F
H-CH3]+.
132.62 (d, J3
= 12.3 Hz), 139.02, 139.26, 142.30,
C-F
148.94 (C-5 triazolic ring), 156.76 (d, J1
= 249.3
C-F
Hz), 169.43 (C-3 triazolic ring) ppm; UV (CH3OH,
λmax, nm, (log ε)): 255.5 (4.54), 325.1 (4.04); Anal.
calcd for C20H13ClFN3O2S2 (445.92 g/mol): C, 53.87;
H, 2.94; N, 9.42. Found: C, 53.92; H, 2.89; N, 9.36;
ESI-MS, m/z (%): 446/448 [M + H]+; 271 (100)
[M + H – ClC6H4SO2]+.
General Procedure for the Synthesis of
5-(4-(4-chlorophenylsulfonyl)phenyl)-N-(Ar)-
1,3,4-thiadiazol-2-amines 8–10
Synthesis
of
5-(4-(4-Chlorophenylsulfonyl)
phenyl)-N-(2-fluorophenyl)-1,3,4-thiadiazol-2-amine
8. A mixture of the acylthiosemicarbazide
(2 mmol) and phosphorous oxychloride (10 mL)
was refluxed for 4 h. After phosphorous oxychloride
evaporation under reduced pressure, the obtained
residue was added in a beaker with ice when a
precipitate was obtained. To the precipitate, a
diluted solution of sodium hydrogen carbonate was
added until pH ∼ 8 and the crude product was
filtered off, washed with water, dried and purified
by recrystallized from chloroform/petroleum ether
(1:2, v/v).
2
5-(4-(4-Chlorophenylsulfonyl)phenyl)-4-(4-triflu-
oromethoxyphenyl)-2H-1,2,4-triazol-3(4H)-thione 6.
Yield 80.2%; mp = 251–253◦C; IR (KBr, ν, cm−1):
3428 (NH), 3091, 3021 (CHaryl), 1600 (C N),
1580, 1512, 1476 (C C), 1330, 1161 (SO2), 1261
1
(C
O
C), 1230 (C-F), 1217 (C S), 769 (C Cl); H
NMR (DMSO-d6, δ, ppm, J, Hz): 7.51 (d, J = 9.8 Hz,
2H, ArH); 7.52 (d, J = 8.8 Hz, 2H, ArH); 7.56 (d, J =
9.8 Hz, 2H, ArH); 7.68 (d, J = 8.5 Hz, 2H, ArH); 7.92
(d, J = 8.8 Hz, 2H, ArH); 7.95 (d, J = 8.5 Hz, 2H,
ArH); 14.32 (s; 1H; NH) ppm; 13C NMR (DMSO-d6)
δ: 119.93 (q, JC-F = 256.6 Hz, 4-OCF3), 121.83,
127.70, 129.53, 129.56, 130.10, 130.56, 130.65,
130.96, 139.14, 139.23, 142.02, 148.65, 148.98 (C-5
triazolic ring), 168.96 (C-3 triazolic ring) ppm; UV
(CH3OH, λmax, nm (log ε)): 254.6 (4.42), 322.5 (3.88);
Anal. calcd for C21H13ClF3N3O3S2 (511.92 g/mol): C,
49.27; H, 2.56; N, 8.21. Found: C, 49.21; H, 2.51; N,
8.15; ESI-MS, m/z (%): 512/514 [M + H]+; 337 (100)
[M + H – ClC6H4SO2]+; 201 (9) [F3COC6H4NCN]+;
177 (25) [F3COC6H4NH2]+.
Synthesis
of
5-(4-(4-Chlorophenylsulfonyl)
phenyl)-N-(Ar)-1,3,4-thiadiazol-2-amines 9 and 10.
To 30 mL of concentrated sulfuric acid, the corre-
sponding acylthiosemicarbazide 3,4 (2 mmol) was
added and stirred at 0◦C for 3 h and then 3 h at room
temperature. To the reaction mixture, on ice bath,
a dilute ammonium hydroxide solution was added
until neutralization and the obtained solid product
was filtered out, washed with water, dried, and puri-
fied by recrystallization from chloroform/petroleum
ether (1:2, v/v).
5-(4-(4-Chlorophenylsulfonyl)phenyl)-N-(2-fluor-
ophenyl)-1,3,4-thiadiazol-2-amine 8. Yield 88.0%;
mp = 218–220◦C; IR (KBr, ν, cm−1): 3357 (NH),
3088, 3060 (CHaryl), 1623 (C N), 1596, 1576, 1548,
1494, 1477 (C C), 1323, 1293, 1157 (SO2), 1255
(C F), 764 (C Cl); 1H NMR (DMSO-d6) δ: 7.05–
7.38 (m, 3H, Ar-H); 7.72 (d, J = 8.5 Hz, 2H, Ar-H);
8.01 (d, J = 8.5 Hz, 2H, Ar-H); 8.09 (s, 4H, Ar-H);
8.38 (bt, 8.5 Hz, 1H, Ar-H); 10.52 (bs, 1H, NH) ppm;
5-(4-(4-Chlorophenylsulfonyl)phenyl)-4-(3,4,5-
trimethoxyphenyl)-2H-1,2,4-triazol-3(4H)-thione 7.
Yield 89.8%; mp = 258–260◦C; IR (KBr, ν, cm−1):
3420 (NH), 3087, 3033 (CHaryl), 2985, 2922, 2839
(CH3), 1601 (C N), 1545, 1506, 1469 (C C), 1322,
1279, 1160 (SO2), 1236 (C S + C
O C), 1129
(C
O
C), 768 (C Cl); 1H NMR (DMSO-d6) δ: 3.60
(s, 6H, 3-OCH3, 5-OCH3); 3.69 (s, 3H, 4-OCH3);
6.74 (s, 2H, ArH); 7.60 (d, J = 8.6 Hz, 2H, ArH);
7.68 (d, J = 8.6 Hz, 2H, ArH); 7.95 (d, J = 8.6
Hz, 2H, ArH); 7.97 (d, J = 8.6 Hz, 2H, ArH);
14.26 (s, 1H, NH) ppm; 13C NMR (DMSO-d6,) δ:
56.25 (3-OCH3; 5-OCH3); 60.24 (4-OCH3); 106.75;
127.69; 129.30; 129.53; 129.68; 130.05; 130.90;
141.85; 138.11; 139.25; 139.32; 153.13; 149.14 (C-5
triazolic ring); 168.99 (C-3 triazolic ring) ppm; UV
(CH3OH, λmax, nm, (log ε)): 252.0 (4.30), 317.2
(3.75); Anal. calcd for C23H20ClN3O5S2 (518.01
g/mol): C, 53.33; H, 3.89; N, 8.11. Found: C, 53.25,
H, 3.95, N, 8.06; ESI-MS, m/z (%): 518/520 [M
+ H]+; 351/353 (15) [M + H – C6H2(OCH3)3]+,
210 (10) [(OCH3)3C6H2NH(CH2)2]+, 168 (100)
13C NMR (DMSO-d6) δ: 115.38 (d, J2
= 18.8 Hz);
C-F
122.14 (d, J3
= 8.2 Hz); 123.62 (d, J3
= 7.2 Hz);
C-F
C-F
124.75 (d, J4
= 3.0 Hz); 127.81; 127.87; 128.01
C-F
(d; J2
= 11.5 Hz); 129.37; 130.03; 134.98; 139.08;
C-F
139.54; 141.37; 152.06 (d, J1
= 243.8 Hz); 156.84
C-F
(C-5 thiadiazole ring); 165.21 (C-2 thidiazole ring)
ppm; UV (CH3OH, λmax, nm (log ε)): 256.4 (4.21);
341.9 (4.16); Anal. calcd for C20H13ClFN3O2S2
(445.92 g/mol): C, 53.87; H, 2.94; N, 9.42. Found: C,
53.92; H, 2.89; N, 9.36; ESI-MS, m/z (%): 446/448 [M
+ H]+; 271 (81) [M + H – ClC6H4SO2]+; 110 (100)
[FC6H4NH]+.
5-(4-(4-Chlorophenylsulfonyl)phenyl)-N-(4-triflu-
oromethoxyphenyl)-1,3,4-thiadiazol-2-amine 9. Yield
Heteroatom Chemistry DOI 10.1002/hc

Some New N1-[4-(4-Chlorophenylsulfonyl)benzoyl]-N4-(aryl)-thiosemicarbazides and Their Cyclization Products 319
87.7%; mp = 230–232◦C; IR (KBr, ν, cm−1): 3243
were performed for each compound (at the concen-
tration of 2048 μg/mL) using the dimethyl sulfoxide
as the solvent. The microplates were filled in with
50 μL of Mu¨ller–Hinton broth when testing the com-
pounds for their antibacterial action and with 50 μL
of Sabouraud broth when testing for their antifun-
gal action. For each compound, a series of twofold
dilutions was performed in both Mu˝ller–Hinton and
Sabouraud broth from 1:2 to 1:1024.
(NH), 3090, 3061, 3018 (CHaryl), 1613 (C N), 1557,
1555, 1510, 1480 (C C), 1323, 1157 (SO2), 1263
(C
O
C), 1222 (C F); 764 (C Cl); 1H NMR
(DMSO-d6,) δ: 7.38 (d, J = 9.1 Hz, 2H, Ar-H), 7.71
(d, J = 8.5 Hz, 2H, Ar-H); 7.77 (d, J = 9.1 Hz, 2H,
Ar-H); 8.00 (d, J = 8.5 Hz, 2H, Ar-H), 8.09 (s, 4H,
Ar-H), 10.89 (s, 1H, NH) ppm; 13C NMR (DMSO-
d6) δ: 118.92, 120.16 (q, JC-F = 253.4 Hz, OCF3),
122.10, 127.92, 128.44, 129.43, 130.03, 133.09,
134.89, 139.29, 139.54, 141.40, 142.84, 156.23 (C-5
thiadiazole ring), 164.81 (C-2 thiadiazole ring) ppm;
UV (CH3OH, λmax, nm (log ε)): 261.7 (4.28); 346.3
(4.32); Anal. calcd for C21H13ClF3N3O3S2 (511.92
g/mol): C, 49.27; H, 2.56; N, 8.21. Found: C, 49.35;
H, 2.52; N, 8.14; (ESI-MS) m/z (%): 512/514 [M +
H]+; 337 (29) [M + H – ClC6H4SO2]+; 176 (100)
[F3COC6H4NH]+.
The inoculum for each reference bacterial strain
was prepared by suspending five colonies from a
24-h culture on Columbia Blood Agar (BioMe´rieux,
France) in 5 mL of Mu¨ller–Hinton broth and ad-
justing the turbidity at 0.5 McFarland. The bacterial
suspension was diluted afterward 1:100 in Mu¨ller–
Hinton broth, and aliquots of 50 μL were inoculated
within 15 min in all wells containing the compounds
(including the growth control well), except for the
sterility control well, which was filled in only with
compound-free Mu¨ller–Hinton broth (100 μL).
The inoculum for each reference yeast strain was
prepared by suspending five colonies from a 24 h
culture on Sabouraud Dextrose Agar in 5 mL of
sterile distilled water and adjusting the turbidity at
0.5 McFarland. Afterward, the microbial suspension
was diluted in distilled water for obtaining a work-
ing inoculum of 1–5 × 105 CFU/mL. All the wells
(including the growth control one) already contain-
ing the compounds diluted in Sabouraud broth were
inoculated with 50 μL of the yeast inoculum within
15’, except for the sterility control well, which was
filled in only with compound-free Sabouraud broth
(100 μL).
The inoculated microplates were sealed with
sterile adhesive sheets, covered with the proper lid
and incubated at 37◦C for 20 h in the case of testing
against the bacterial strains and 24 h in the case of
testing against the yeasts strains (or 48 h for C. para-
psilosis). After the incubation period, the microbial
growth in the wells was examined by the naked eye
and MIC was considered the lowest concentration of
the respective compound, which inhibited the visible
microbial growth.
5-(4-(4-Chlorophenylsulfonyl)phenyl)-N-(3,4,5-
trimethoxyphenyl)-1,3,4-thiadiazol-2-amine 10. Yield
87.1%; mp. = 297–299◦C; IR (KBr, ν, cm−1): 3266
(NH), 3086, 3065 (CHaryl), 2940, 2835 (CH3), 1629
(νC = N), 1581, 1513m, 1468 (νC C), 1327, 1286,
1157 (SO2), 1244, 1133 (C
O C); 764 (C Cl);
1H NMR (DMSO-d6) δ : 3.62 (s, 6H, 3- OCH3,
5-OCH3); 3.78 (s, 3H, 4-OCH3); 7.00 (s, 2H, Ar-H);
7.71 (d, J = 8.8 Hz, 2H, Ar-H); 8.01 (d, J = 8.8
Hz, 2H, Ar-H); 8.07 (s, 4H, Ar-H); 10.71 (s, 1H,
NH); ppm; 13C NMR (DMSO-d6) δ: 55.87 (3-OCH3;
5- OCH3); 60.23 (4-OCH3); 95.90; 127.85; 128.64;
129.56; 133.09; 135.52; 136.50; 139.16; 139.60;
141.29; 142.08; 153.19; 155.45 (C-5 thiadiazole
ring); 165.46 (C-2 thiadiazole ring) ppm; UV
(CH3OH, λmax, nm (log ε)): 229.9 (4.19); 273.1
(4.18); 361.2 (4.16); Anal. calcd for C23H20ClN3O5S2
(518.01 g/mol): C, 53.33; H, 3.89; N, 8.11. Found: C,
53.40; H, 3.84; N, 8.07; (ESI-MS) m/z (%): 518/520
[M + H]+; 502/504 (1) [M + H – CH4]+; 486/488
(163) [M + H– 2CH4]+; 470/472 (4) [M + H– 3CH4]+.
Biological Activity
Antimicrobial Activity. The antimicrobial activ-
ities of all compounds synthesized were investigated
against some bacteria (S. aureus ATCC 25923, B.
cereus ATCC 13061, E. coli ATCC 25922, Enterobacter
cloacae ATCC 49141, A. baumannii ATCC 19606, and
P. aeruginosa ATCC 27853) and yeasts (C. albicans
ATCC 90028, C. parapsilosis ATCC 22019, C. glabrata
ATCC 15126, and Candida tropicalis ATCC 13803).
The broth microdilution method was used for
the investigation of the antimicrobial activity of
the nine compounds in 96 flat-bottomed wells
microplates (Nunc, Denmark). The stock solutions
The antimicrobial activity of the compounds was
investigated in duplicate against all the reference
strains. In addition, as control, E. coli ATCC 25922
and P. aeruginosa ATCC 27853 were tested against
amikacin, while C. parapsilosis ATCC 22019 was
tested against fluconazole using the broth microdi-
lution method [55,56].
Pharmacological Activity
Acute Toxicity. For acute toxicity, 11 groups of
white, male, NMRI strain mice were formed (10
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