Sulfonamide-1,2,4-triazole derivatives as antifungal and antibacterial agents: Synthesis, biological evaluation, lipophilicity, and conformational studies

Article abstract of DOI:10.1016/j.bmc.2007.10.082

A series of 10 new 5-[2-(substituted sulfamoyl)-4,5-dimethoxy-benzyl]-4aryl-s-triazole-3-thiones were synthesized and evaluated for in vitro antifungal and antibacterial activity. All compounds tested showed significant antifungal activity against all the micromycetes, compared to the commercial fungicide bifonazole. Differences in their activity depend on the substitution of different reactive groups. More specifically, best antifungal activity among synthetic analogues was shown with N-dimethylsulfamoyl group. All the compounds tested against bacteria showed the same activity as the commercial agent streptomycin, except for Enterobacter cloacce and Salmonella species. Chloramphenicol showed lower bactericidal effect than the synthetic compounds. Furthermore, it is apparent that different compounds reacted in different ways against bacteria. Gram (-) bacteria seem to be more sensitive to these compounds than Gram (+) species. An effort was made to correlate the above-mentioned differences in activity with lipophilicity studies. Furthermore, molecular modeling was used to obtain the main conformational features of this class of molecules for future structure-activity relationship studies.

Full text of DOI:10.1016/j.bmc.2007.10.082

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1150–1161
Sulfonamide-1,2,4-triazole derivatives as antifungal
and antibacterial agents: Synthesis, biological
evaluation, lipophilicity, and conformational studies
Iraj Rahavi Ezabadi,a Charalabos Camoutsis,a,* Panagiotis Zoumpoulakis,b
c
d
d
d
´
´
´
Athina Geronikaki, Marina Sokovic, Jasmina Glamocˇilija and Ana Ciric
aSchool of Health Sciences, Department of Pharmacy, Laboratory of Pharmaceutical Chemistry, University of Patras, Patras, Greece
bLaboratory of Molecular Analysis, Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation,
48 Vas. Constantinou Avenue, 11635 Athens, Greece
cSchool of Pharmacy, Department of Pharmaceutical Chemistry of Aristotelian, University of Thessaloniki, Thessaloniki, Greece
dDepartment of Plant Physiology, Mycological Laboratory, Institute of Biological Research, Bulevar Despota Stefana 142,
11000 Belgrade, Serbia
Received 13 July 2007; accepted 23 October 2007
Available online 28 November 2007
Abstract—A series of 10 new 5-[2-(substituted sulfamoyl)-4,5-dimethoxy-benzyl]-4aryl-s-triazole-3-thiones were synthesized and
evaluated for in vitro antifungal and antibacterial activity. All compounds tested showed significant antifungal activity against
all the micromycetes, compared to the commercial fungicide bifonazole. Differences in their activity depend on the substitution
of different reactive groups. More specifically, best antifungal activity among synthetic analogues was shown with N-dimethylsulfa-
moyl group. All the compounds tested against bacteria showed the same activity as the commercial agent streptomycin, except for
Enterobacter cloacce and Salmonella species. Chloramphenicol showed lower bactericidal effect than the synthetic compounds. Fur-
thermore, it is apparent that different compounds reacted in different ways against bacteria. Gram (À) bacteria seem to be more
sensitive to these compounds than Gram (+) species. An effort was made to correlate the above-mentioned differences in activity
with lipophilicity studies. Furthermore, molecular modeling was used to obtain the main conformational features of this class of
molecules for future structure–activity relationship studies.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
makes it necessary to continue the search for new anti-
microbial substances.
Systemic fungal infections are important problems in
phytopathology and especially in medicine, the care of
patient’s immunosuppressed by infectious diseases, che-
motherapy, or age. Infections caused by fungal species
are common in immunocompromised patients and carry
significant treatment costs and mortality.1 Standard sys-
temic antibiotic therapy alone is frequently unsatisfac-
tory in certain circumstances. Also, more attention is
being focused on addressing the problem of multidrug-
resistant bacteria and the staggering costs and conse-
quences resulting from this. The emerging resistance of
microorganisms to some synthetic antimicrobial agents
The aim of this paper is to investigate the antibacterial
and antifungal activity of various triazole derivatives
against food poisoning, plant, animal, and human
pathogens. Triazole moiety may be considered as a bio-
isostere of imidazole, which is a part of the azole group
of antifungal drugs (i.e., fluconazole). The biological
activities of various triazole derivatives have been exten-
sively studied. It is known from the literature that the s-
triazole moiety has great versatility in fusing to various
ring systems and possesses a broad spectrum of biolog-
ical activities. Among the most important effects, tria-
zole derivatives have been reported to exhibit
antibacterial,2–16 antifungal,2–4,17–20 and antimycobacte-
rial21–25 properties.
Keywords: Triazoles; Antifungal; Antibacterial; Lipophilicity; Confor-
mational properties.
*
Prompted by these observations and in continuation of
our search for bioactive molecules, we designed the syn-
Corresponding author. Tel.: +30 2107273869; fax: +30
2107273872; e-mail: pzoump@eie.gr
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.082

I. R. Ezabadi et al. / Bioorg. Med. Chem. 16 (2008) 1150–1161
1151
thesis of a series of novel sulfonamide-s-triazole. In par-
ticular, we emphasized in the strategy of combining two
chemically different but pharmacologically compatible
molecules (the sulfonamide nucleus and the s-triazole
nucleus) in one frame, in order to study their antifungal
and antibacterial activities.
We found that II reacts readily with secondary amines in
anhydrous benzene to give excellent yields of the corre-
sponding sulfonamides (IIIb–e). In the contrary to the
preparation of the above sulfonamides, the N-di-
methyl-sulfonamide IIa was prepared by treatment of
sulfonyl chloride II with 3 equiv of a 40% aqueous solu-
tion of dimethylamine in chloroform at 0 ꢁC.27 The
latter were converted to the desired 2-(N-substituted sul-
famoyl)-4,5-dimethoxy-phenylacetylhydrazides IV(a–e)
by treatment with hydrazine hydrate in xylol. The hith-
erto unknown 1-[2-(N-substituted sulfamoyl)-4,5-dime-
2. Chemistry
The synthetic pathway followed for the preparation of
the title compounds was accomplished as shown in
Scheme 1.
thoxy-phenylacetyl]-4-aryl-thiosemicarbazides
(Va–j)
were synthesized by condensing of acid hydrazides
IVa–e with suitable aryl isothiocyanates.28 These thiose-
micarbazides were cyclized into their corresponding 5-
[2-(N-substituted sulfamoyl)-4,5-dimethoxy-benzyl]-4-
aryl-triazole-3-thiones (VIa–j) in the presence of aque-
ous 2 N sodium hydroxide solution.28
Since the para position to one of the methoxy groups in
ethyl 3,4-dimethoxyphenylacetate (I) is activated, direct
chlorosulfonation at low temperature afforded ethyl-[2-
(chlorosulfonyl)-4,5-dimethoxy-phenyl]acetate (II).26
O
O
H3CO
C
O CH2CH3
H3CO
C
O
CH2CH3
ClSO2OH
H3CO
H3CO
SO2
Cl
II
I
O
R
HN
H3CO
H3CO
H3CO
H3CO
C
O
CH2CH3
CONHNH2
R
H2NNH2, H2O
SO2
N
SO2
N
IV
R
R
III
R
R
S
R1
N C S
H3CO
NaOH
CONHNHCNH
R1
H3CO
SO2
N
V
R
R
N
NH
H3CO
S
N
SO2
H3CO
N
R
R
VI
R1
CH3
R
R
CH2CH3
CH2CH3
N
,
N
=
N
,
N
N
N
O
,
,
CH3
= H, Cl
R1
Scheme 1.

1152
I. R. Ezabadi et al. / Bioorg. Med. Chem. 16 (2008) 1150–1161
It is interesting to note that compounds VIa–j were pres-
ent in the solid state in the C@S form as indicated by
their IR and H NMR spectra. In the IR spectra, the
pounds. Compounds VIa and VIb showed the best anti-
fungal activity against micromycetes investigated, while
VIi and VIj possessed the lowest antifungal effect. These
better effects for compounds VIa and VIb could be ex-
plained as a consequence of the presence of an N-
(CH3)2 group in their structure, and in the contrary
compounds VIi and VIj do not possess this reactive
group in their structure. However, all the compounds
exhibited much higher antifungal activity than commer-
cial fungicide bifonazole which is the imidazole-type
fungicide.
1
presence of two absorption maxima at 1342 and
1320 cmÀ1, characteristic of the C@S group in this type
of compounds, and the lack of absorption bands at
around 2500–2600 cmÀ1 for the S–H stretching support
the thione form of VIa–j.29 In addition N–H stretching
due to s-triazoline-3-thiones was detected within the
1
3015–3300 cmÀ1 region. Furthermore in the H NMR
spectra, the broad signal between 13.38 and 13.70 ppm
due to triazole–NH indicates that the above compounds
exist in the C@S form.
Antibacterial activity of compounds tested is presented in
Table 2. Compounds VIa, VIb, VIc, VIb, VIe, VIf, and
VIg showed inhibitory effect at 100.0 lg/ml against Esch-
erichia coli, the same MIC as antibiotic streptomycin, but
much better than chloramphenicol (MIC at 250.0 lg/ml).
Compounds VIi and VIj showed greater MIC at 150.0 lg/
ml against E. coli. The highest inhibitory effect against
Salmonella typhimurium can be seen for compounds
VIe, VIf, VIh, and VIj with MIC at 100.0 lg/ml, these
compounds showed lower activity than streptomycin
but much better than chloramphenicol. Compound VIj
exhibited inhibitory effect at 100.0 lg/ml against Entero-
bacter cloacce, better than streptomycin but lower than
chloramphenicol. The rest of the compounds showed
MIC at 150.0 lg/ml (Fig. 3). The values for minimal bac-
tericidal concentrations of compounds tested are the same
for all of them (150.0 lg/ml), except against E. cloacce
where the MBC is lower (100.0 lg/ml) and same as for
streptomycin but higher than that for chloramphenicol.
All the compounds tested showed the same bactericidal
activity as streptomycin, except for E. cloacce and Salmo-
nella species. Chloramphenicol showed lower bactericidal
effect than compounds tested. Demirayak et al.32 and
Ulusoy et al.33 showed in their results that triazole deri-
vates possessed antibacterial and antifungal activities
with MIC of 6.25–250 lg/ml. The most resistant species
was E. coli, while the most sensitive was Salmonella aur-
eus. Our results showed that E. coli was the most suscep-
tible species to triazole derivates tested in this work.
3. Results and discussion
3.1. Biological evaluation
Antifungal activity of compounds tested is presented in
Table 1. All the compounds tested showed significant
antifungal activity against all the micromycetes. It can
be seen that compounds VIa and VIb showed the best
inhibitory activity with lowest MIC (50.0 lg/ml) against
mycotoxic species Aspergillus flavus and Trichoderma
viride. Compounds VIc and VId also possessed great
inhibitory effect with MIC of 50.0 lg/ml against T. vir-
ide. Compounds VIg, VIh, VIi, and VIj exhibited the
lowest inhibitory activity with MIC of 100.0–150.0 lg/
ml, but still greater than commercial fungicide bifonaz-
ole which showed MIC of 150.0–200.0 lg/ml (Fig. 1).
Among the compounds tested, VIa and VIb showed
the highest fungicidal potential with MFC of 100.0 lg/
ml against A. flavus, Penicillium funiculosum, and T. vir-
ide. Compounds VIc and VId also possessed great fungi-
cidal effect against T. viride with MFC of 100.0 lg/ml.
All the other compounds showed fungicidal effect at
150.0 lg/ml which is lower than MFC for bifonazole
(200.0-250.0 lg/ml) (Fig. 2). It is interesting that all the
compounds exhibited the best antifungal activity against
T. viride. Previous results of antifungal activities of both
natural and synthetic antifungal agents against numer-
ous fungal strains showed that T. viride is the most resis-
tant species tested.30,31
It is obvious that different compounds reacted in differ-
ent ways against bacteria. Gram (À) bacteria seem to be
more sensitive in these compounds than Gram (+) spe-
cies. Chloramphenicol and streptomycin also showed
differences in their activities (Figs. 3 and 4).
It can be concluded that there is a connection between
antifungal activity and chemical structure of these com-
Table 1. Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs-lg/ml)
Micromycetes
VIa Mic VIb Mic VIc Mic VId Mic VIe Mic VIf Mic VIg Mic VIh Mic VIi Mic VIj Mic Bifonazole
Mic (Mfc)
(Mfc)
(Mfc)
(Mfc)
(Mfc)
(Mfc)
(Mfc)
(Mfc)
(Mfc)
(Mfc)
(Mfc)
A. niger
100
150
100
150
100
150
50
100
150
100
150
100
150
50
100
150
100
150
100
150
100
150
100
150
50
100
150
100
150
100
150
100
150
100
150
50
100
150
100
150
100
150
100
150
100
150
100
150
100
150
100
150
100
150
100
150
100
150
100
150
150
150
100
150
100
150
100
150
100
150
100
150
150
150
100
150
100
150
100
150
100
150
100
150
150
150
100
150
100
150
100
150
100
150
150
150
150
150
100
150
100
150
100
150
100
150
150
150
200
250
150
200
150
200
150
200
200
250
200
250
A. ochraceus
A. versicolor
A. flavus
100
P. funiculosum 100
100
100
100
50
100
50
T. viride
100
100
100
100

I. R. Ezabadi et al. / Bioorg. Med. Chem. 16 (2008) 1150–1161
1153
μg/ml
200
180
160
140
120
100
80
Column
1
2
3
4
5
6
7
8
Compound
VIa
VIb
VIc
VId
VIe
VIf
VIg
VIh
9
10
11
VIi
VIj
Bifonazole
60
40
20
0
A. niger
A. ochraceus
A. versicolor
A. flavus
P. funiculosum
T. viride
Figure 1. Minimal inhibitory concentrations (lg/ml) of compounds tested and bifonazole against micromycetes. The table on the right provides the
correlation between columns and compounds.
μg/ml
250
200
Column
1
2
Compound
VIa
150
100
50
VIb
3
4
5
6
7
8
9
10
11
VIc
VId
VIe
VIf
VIg
VIh
VIi
VIj
Bifonazole
0
A. niger
A. ochraceus
A. versicolor
A. flavus
P. funiculosum
T. viride
Figure 2. Minimal fungicidal concentrations (lg/ml) of compounds tested and bifonazole against micromycetes.
The susceptibility of a microorganism to some agents
depends, first of all, on the properties of the agents
and the microorganism itself. It is known that Gram
(+) bacteria are more susceptible to antimicrobial agents
and Gram (À) bacteria are less sensitive. Fungi are more
susceptible than bacteria in general.34 These observa-
tions are confirmed with our results. The results ob-
tained clearly demonstrate that compounds tested
present a great potential for medical procedures and
for food, cosmetics, and pharmaceutical industries.
correlate antibacterial and antifungal activities of the
synthesized compounds with lipophilicity was unsuc-
cessful. It is obvious from the obtained results (Table
3) that lipophilicity does not affect so much the antibac-
terial/antifungal activities of synthesized compounds.
Probably the structural characteristics of the synthesized
molecules are more important for this kind of activity.
3.3. Molecular modeling studies
As a first step for future structure–activity relationship
studies, we performed conformational analysis using
molecular modeling. In this paper, we demonstrate the
conformational properties of two of the synthesized
compounds. For comparison reasons we selected VIa
as one of the most active and VIe from the less active
molecules.
3.2. Lipophilicity studies
The examined compounds have the same skeleton with
variation only in the amino moiety N(R)2 and the sub-
stituent in the 4-position of aromatic ring substituted
in the C1 position with triazole cycle. The attempt to

1154
I. R. Ezabadi et al. / Bioorg. Med. Chem. 16 (2008) 1150–1161
Table 2. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) in lg/ml of compounds tested in micro-
dilution method
Bacteria
VIa
Mic
VIb
Mic
VIc
Mic
VId
Mic
VIe
Mic
VIf
Mic
VIg
Mic
VIh
Mic
VIi
Mic
VIj
Mic
Streptomycin Chloramphenicol
Mic (Mfc)
Mic (Mfc)
(Mfc) (Mfc) (Mfc) (Mfc) (Mfc) (Mfc) (Mfc) (Mfc) (Mfc) (Mfc)
E. coli
100
150
150
150
150
150
150
150
100
150
150
150
150
150
150
150
150
150
150
150
100
100
100
150
150
150
150
150
150
150
150
150
150
150
100
100
100
150
150
150
150
150
150
150
150
150
150
150
100
100
100
150
150
150
150
150
150
150
100
150
150
150
100
100
100
150
150
150
150
150
150
150
100
150
150
150
100
100
100
150
150
150
150
150
150
150
150
150
150
150
100
100
100
150
150
150
150
150
150
150
100
150
150
150
100
100
150
150
150
150
150
150
150
150
150
150
150
150
100
100
150
150
150
150
150
150
150
150
150
150
150
150
100
100
100
150
100
150
100
150
100
150
50
250
250
150
200
150
200
100
100
150
200
150
200
150
200
B. cereus
M. flavus
S. epidermidis
S. typhimurium 100
150
100
50
S. enteritidis
E. cloacce
150
150
100
100
100
50
100
μg/ml
300
250
200
150
100
50
Column
1
2
3
4
5
6
7
8
Compound
VIa
VIb
VIc
VId
VIe
VIf
VIg
VIh
VIi
9
10
11
12
VIj
Str
Chlo
0
E. coli
B. cereus
M. flavus
S. epidermidis
Sal. typhimurium
Sal. enteritidis
Ent. cloacce
Figure 3. Minimal inhibitory concentrations (lg/ml) of compounds tested and antibiotics against bacteria.
300 μg/ml
Column
Compound
VIa
250
200
150
100
50
1
2
3
4
5
6
7
8
9
VIb
VIc
VId
VIe
VIf
VIg
VIh
VIi
10
11
12
VIj
Str
Chlo
0
E. coli
B. cereus
M. flavus
S. epidermidis
Sal. typhimurium
Sal. enteritidis
Ent. cloa
Figure 4. Minimal bactericidal concentrations (lg/ml) of compounds tested and streptomycin against bacteria.
3.3.1. Conformational analysis of VIa and VIe. The most
important conformational properties of both com-
pounds are related to the dihedral angles s1–s7 which
are presented in Figure 5.
The two molecules were designed and subjected to en-
ergy minimization procedures using first and second or-
der algorithms. The last were input as initial
conformations for the generation of low energy con-

I. R. Ezabadi et al. / Bioorg. Med. Chem. 16 (2008) 1150–1161
Table 3. Calculated lipophilicity of tested compounds
1155
Compound
N(R)2
R1
AlogP
IAlogP
ClogP
Cosmo logP
mlogP
Kowwin
XlogP
Average
VIa
VIb
VIc
VId
N(Me)2
N(Me)2
N(Et)2
N(Et)2
H
2.59
3.30
3.32
3.96
2.31
2.96
2.49
2.65
3.687
4.40
4.25
4.55
5.58
5.88
1.72
2.40
2.48
3.15
3.56
4.20
4.54
5.18
2.39
3.02
3.24
3.86
2.93
3.55
3.77
4.31
Cl
H
4.745
5.458
Cl
VIe
VIf
VIg
VIh
VIi
VIj
H
3.08
3.68
2.10
2.82
2.78
3.37
3.43
3.83
3.16
2.73
2.55
3.69
4.880
5.593
3.587
4.297
4.321
5.034
5.53
5.84
4.28
4.58
4.81
510
2.63
3.31
1.57
2.25
2.13
2.11
4.92
5.56
3.17
3.81
4.42
5.07
3.26
3.88
1.99
2.62
2.90
3.52
3.96
4.53
2.98
3.30
3.42
3.98
N
N
Cl
H
N
N
N
N
O
Cl
H
O
Cl
N
NH
N
NH
τ2
τ2
τ3
H3C
H3C
O
O
τ3
τ6
H3C
O
O
τ6
N
τ1
S
N
τ4
SO2
τ1
τ4
SO2
τ7
τ5
N
τ7
H3C
τ5
N
H3C
CH3
Figure 5. Chemical structures of VIa (left) and VIe (right).
formers using Random Sampling algorithm. The con-
formers are produced by random modifications of the
dihedral angles followed by minimization procedure.
Consequently, the minimized conformers are representa-
tives of the potential energy surface.
in Figure 7 and their dihedral values are shown in Table
5. VIe_1 and VIe_2 favor a cluster between the two phe-
nyl rings as in case of VIa. Furthermore, these two con-
formers have a mirror image relationship and an energy
equal to 37.47 kcal/mol. VIe_3 (38.65 kcal/mol) and
VIe_4 (40.40 kcal/mol) differ in s1, s2, and s3 dihedral
angles and favor a cluster between the phenyl ring at-
tached to the triazole system and the pyridine ring. Grid
Scan analysis on s2 and s3 has proved that also VIe_3
and VIe_4 can have enantiomeric conformations
which were not produced with simple use of Random
Sampling method. A more detailed conformational
analysis study of all series of synthesized molecules is
still in progress.
Compund VIa produced 1000 low energy conformers
which were grouped into eight different classes accord-
ing to their atom coordinates with an RMSD value
equal to 2.38. The four lowest energy conformers are
displayed in Figure 6 and their dihedral values are
shown in Table 4. Differences among VIa conformers
are mainly due to s2 and s3 dihedrals resulting in two
main classes. The first one includes VIa_1 and VIa_2
and favors a cluster between the two phenyl rings. The
second includes VIa_3 and VIa_4 and extends the two
phenyl systems away from each other. Interestingly,
the four low energy conformers have a mirror image
relationship forming two pairs of enantiomers. This is
also apparent from the identical energy values of each
pair (IVa_1 and IVa_2 have an energy of 26.85 kcal/
mol, and IVa_3 and IVa_4 have an energy of
27.34 kcal/mol).
4. Experimental
4.1. General experimental considerations
Melting points were taken in glass capillary tubes on a
Haake Bucher apparatus and are uncorrected. IR spec-
tra were recorded on a FT-IR Jasco spectrophotometer
in solid phase KBr. All proton NMR spectra were deter-
mined with a Bruker AC300 spectrometer using deuter-
ated dimethylsulfoxide (DMSO-d6) and are reported in d
units (ppm) relative to tetramethylsilane (TMS) as an
internal standard. Thin layer chromatography (TLC)
Respectively, VIe produced 1000 low energy conformers
which were grouped into 8 different classes according to
their atom coordinates with an RMSD value equal to
2.68. The four lowest energy conformers are displayed

1156
I. R. Ezabadi et al. / Bioorg. Med. Chem. 16 (2008) 1150–1161
Figure 7. Low energy representative conformers of VIe.
Table 5. Dihedral angles for the low energy conformers of compound
VIe
Conformer
VIe_1
VIe_2
VIe_3
VIe_4
Dihedral
s1
s2
s3
s4
s5
s6
s7
À71.3
À56.8
À68.0
À94.8
52.7
À108.6
56.7
68.0
60.3
147.1
À86.0
119.4
170.7
179.5
À0.3
À93.6
74.5
À158.5
À105.8
À171.4
176.1
0.9
94.7
175.2
177.5
À1.4
Figure 6. Low energy representative conformers of VIa.
À177.7
1.3
Table 4. Dihedral angles for the low energy conformers of compound
VIa
fonamides. The title compounds prepared are reported
below.
Conformer
VIa_1
VIa_2
VIa_3
VIa_4
Dihedral
4.1.1.1. Ethyl-[2-(N-diethylsulfamoyl)-4,5-dimethoxy-
phenyl]acetate (IIIb). Mp 68–69 ꢁC (n-hexane-ethyl ace-
tate) (Ref. 35; 67–68 ꢁC).
s1
s2
s3
s4
s5
s6
s7
À65.9
À55.9
À67.3
À98.0
À171.8
À178.4
1.2
66.1
55.4
125.2
À158.3
96.3
À125.0
158.4
À96.1
À126.6
57.3
67.3
98.1
126.8
169.5
À179.2
À0.3
À53.7
178.3
À1.1
4.1.1.2. Ethyl-[2-(1-peperidinesulfamoyl)-4,5-dimethoxy-
phenyl]acetate (IIIc). Mp 101 ꢁC (methanol) (Ref. 26;
100–101 ꢁC).
179.4
0.3
4.1.1.3. Ethyl-[2-(4-morpholinesulfamoyl)-4,5-dimetho-
xy-phenyl]acetate (IIId). Mp 104–105 ꢁC (methanol)
(Ref. 26; 105–106 ꢁC).
was performed in E. Merck precoated silica gel plates
(Kieselgel 60F254). Visualization was obtained by expo-
sure to iodine vapors and/or under UV light (254 nm).
The elemental analyses (C,H,N) of all compounds were
performed by the Center of Instrumental Analysis of the
University of Patras and are within the range of experi-
mental error ( 0.4% of the calculated values).
4.1.1.4.
Ethyl-[2-(1-pyrrolidinesulfamoyl)-4,5-dime-
thoxy-phenyl]acetate (IIIe). Yield: 95%, Mp 107–108 ꢁC
(ethyl acetate). IR m cmÀ1: 1745 (COO), 1335 (S–Oanti-
sym), 1140 (S–Osym). Anal. calc for C16H23NO6S: C,
53.78; H, 6.44; N, 3.92. Found: C, 53.45; H, 6.55; N,
3.80.
4.1.1. General procedure for the preparation of the ethyl-
[2-(N-substituted sulfamoyl)-4,5-dimethoxy-phenyl]ace-
tate (III). To a flask containing 0.01 mol of ethyl-(2-
chlorosulfonyl-4,5-dimethoxy-phenyl)acetate in 30 ml
of anhydrous benzene was added 0.02 mol of aliphatic
amine. The mixture was heated under reflux for 2 h.
Then the solvent was evaporated under reduced pressure
and ice-water was added to yield the corresponding sul-
4.1.1.5. Ethyl-[2-(N-dimethylsulfamoyl)-4,5-dimethoxy-
a
phenyl]acetate (IIIa). To
solution of 3.225 g
(0.01 mol) of ethyl-[2-(chlorosulfonyl)-4,5-dimethoxy-
phenyl]acetate in 30 ml of chloroform was added
3.8 ml (0.034 mol) of a 40% aqueous solution of

I. R. Ezabadi et al. / Bioorg. Med. Chem. 16 (2008) 1150–1161
1157
dimethylamine. The reaction mixture was stirred for
3 h at 0ꢁC and then 30 ml of chloroform was added.
The organic layer was separated and washed with a
solution of dilute hydrochloric acid (30 ml) and water
(30 ml). The solvent was next dried and evaporated
and the resulting solid was recrystallized from ethyl
acetate to give 2.95 g (89%) of the N-dimethyllsulfona-
mide. Mp 109–110 ꢁC, IR m cmÀ1: 1735 (COO), 1330
(S–Oantisym), 1135 (S–Osym). Anal. calc for
C14H21NO6S: C, 50.75; H, 6.34; N, 4.23. Found: C,
51.03; H, 6.28; N, 4.42.
hydrazide (1 mmol) and aryl isothiocyanate (1 mmol)
in 3 ml of absolute ethanol were refluxed on a steam
bath for 1 h. The resulting solid was filtered and recrys-
tallized from the appropriate solvent.
The following compounds were prepared by an analo-
gous procedure.
4.1.3.1. 1-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-
phenylacetyl]-4-phenyl-thiosemicarbazide (Va). Yield:
85%. Mp 218–220 ꢁC (methanol–dichloromethane). IR
m cmÀ1: 3035, 3270, 3315 (3NH), 1620 (CONH), 1525
(C@S), 1325 (S–Oantisym), 1135 (S–Osym). 1H NMR
(DMSO-d6) d (ppm): 2.67 [s, 6H, N(CH3)2], 3.82 (d,
6H, 2CH3O, J = 5.58 Hz), 3.90 (s, 2H, CH2CO), 7.10
(s, 1H, C6), 7.12–7.18 (m, 1H, ArH), 7.21(s, 1H, C3),
7.30–7.35 (m, 2H, ArH), 7.50 (d, 2H, ArH,
J = 7.68 Hz), 9.29 (b, 1H, NH), 9.75 (s, 1H, NH),
10.13 (s, 1H, NH) Anal. calc for C19H24N4O5S2: C,
50.44; H, 5.30; N, 12.39. Found: C, 50.42; H, 5.15; N,
12.31.
4.1.2. General procedure for the preparation of the 2-(N-
substituted sulfamoyl)-4,5-dimethoxy-phenylacetylhyd-
razides (IV). To a flask containing 0.01 mol of ethyl-[2-
(N-substituted sulfamoyl)-4,5-dimethoxy-phenyl]acetate
in 20 ml of xylol was added 0.02 mol of 80% hydrazine
monohydrate. The mixture was refluxed for 20 h. The
solvent was then removed under reduced pressure and
the solid residue after crystallization from the appropri-
ate solvent was collected by filtration.
The following compounds were prepared by an analo-
gous procedure.
4.1.3.2. 1-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-
phenylacetyl]-4-(p-chlorophenyl)-thiosemicarbazide (Vb).
Yield: 82%. Mp 196–198 ꢁC (methanol–dichlorometh-
ane). IR m cmÀ1: 3170, 3320 (3NH), 1680 (CONH),
1520 (C@S), 1313 (S–Oantisym), 1155 (S–Osym). 1H
NMR (DMSO-d6) d (ppm): 2.67 [s, 6H, N(CH3)2],
3.82 (d, 6H, 2CH3O, J = 5.13 Hz), 3.90 (s, 2H,
CH2CO), 7.09 (s, 1H, C6), 7.21 (s, 1H, C3) 7.39 (d,
2H, ArH, J = 8.61 Hz), 7.53 (d, 2H, ArH,
J = 8.85 Hz) 9.36 (b, 1H, NH), 9.85 (br s, 1H, NH),
10.15 (s, 1H, NH) Anal. calc for C19H23ClN4O5S2: C,
46.86; H, 4.72; N, 11.51. Found: C, 46.75; H, 4.80;
N, 11.38.
4.1.2.1. 2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-phen-
ylacetylhydrazide (IVa). Yield: 78%. Mp 147–149 ꢁC
(ethanol). IR m cmÀ1: 3295, 3375 (NH, NH2), 1655,
1615 (CONH), 1370 (S–Oantisym), 1120 (S–Osym). Anal.
calc for C12H19N3O5S: C, 45.42; H, 5.99; N, 13.24.
Found: C, 45.31; H, 5.80; N, 13.11.
4.1.2.2. 2-(N-Diethylsulfamoyl)-4,5-dimethoxy-phen-
ylacetylhydrazide (IVb). Yield: 95%. Mp 160–161 ꢁC
(ethanol). IR m cmÀ1: 3180, 3290, 3375 (NH, NH2),
1660, 1615 (CONH), 1345 (S–Oantisym), 1120 (S–Osym).
Anal. calc for C14H23N3O5S: C, 48.69; H, 6.66; N,
12.17. Found: C, 48.81; H, 6.78; N, 12.05.
4.1.3.3. 1-[2-(N-Diethylsulfamoyl)-4,5-dimethoxy-
phenylacetyl]-4-phenyl-thiosemicarbazide (Vc). Yield:
81%. Mp 202–203 ꢁC (ethanol-dichloromethane). IR m
cmÀ1: 3185, 3335 (3NH), 1690 (CONH), 1525 (C@S),
1325 (S–Oantisym), 1140 (S–Osym). 1H NMR (DMSO-
d6) d (ppm): 0.96–1.05 (m, 6H, 2CH2CH3), 3.09–3.45
(m, 4H, 2CH2CH3), 3.82 (d, 6H, 2CH3O, J = 5.34 Hz),
3.89 (s, 2H, CH2CO), 7.10 (s, 1H, C6), 7.12–7.17 (m,
1H, ArH), 7.21 (s, 1H, C3), 7.30-7.35 (m, 3H, ArH),
7.49 (d, 2H, ArH, J = 12Hz), 9.29 (b, 1H, NH), 9.75
(s, 1H, NH), 10.13 (s, 1H, NH) Anal. calc for
C21H28N4O5S2: C, 52.50; H, 5.83; N, 11.66. Found: C,
52.38; H, 5.75; N, 11.53.
4.1.2.3. 2-(1-Piperidinesulfamoyl)-4,5-dimethoxy-phen-
ylacetylhydrazide (IVc). Yield: 82%. Mp 134–136 ꢁC
(ethanol). IR m cmÀ1: 3200, 3320 (NH, NH2), 1630
(CONH), 1330 (S–Oantisym), 1140 (S–Osym). Anal. calc
for C15H23N3O5S: C, 50.42; H, 6.44; N, 11.76. Found:
C, 50.58; H, 6.70; N, 11.50.
4.1.2.4. 2-(4-Morpholinesulfamoyl)-4,5-dimethoxy-phen-
ylacetylhydrazide (IVd). Yield: 62%. Mp 144–145 ꢁC (eth-
anol). IR m cmÀ1: 3230, 3390 (NH, NH2), 1640 (CONH),
1340 (S–Oantisym), 1155 (S–Osym). Anal. calc for
C14H21N3O6S: C, 46.79; H, 5.84; N, 11.70. Found: C,
46.58; H, 5.95; N, 11.63.
4.1.3.4. 1-[2-(N-Diethylsulfamoyl)-4,5-dimethoxy-phen-
ylacetyl]-4-(p-chlorophenyl)-thiosemicarbazide (Vd). Yield:
77%. Mp 199–200 ꢁC (methanol–dichloromethane). IR m
cmÀ1: 3280, 3320 (3NH), 1690 (CONH), 1535 (C@S),
1335 (S–Oantisym), 1140 (S–Osym). 1H NMR (DMSO-
d6) d (ppm): 0.98–1.05 (m, 6H, 2CH2CH3), 3.12-3.40
(m, 4H, 2CH2CH3), 3.82 (d, 6H, 2CH3O, J = 5.1 Hz),
3.90 (s, 2H, CH2CO), 7.09 (s, 1H, C6), 7.21 (s, 1H,
C3), 7.39 (d, 2H, ArH, J = 8.85 Hz), 7.53 (d, 2H, ArH,
J = 8.85 Hz), 9.35 (b, 1H, NH), 9.85 (s, 1H, NH),
10.15 (s, 1H, NH). Anal. calc for C21H27ClN4O5S2: C,
48.98; H, 5.24; N, 10.88. Found: C, 48.87; H, 5.30; N,
10.77.
4.1.2.5. 2-(1-Pyrrolidinesulfamoyl)-4,5-dimethoxy-phen-
ylacetylhydrazide (IVe). Yield: 81%. Mp 139–141 ꢁC
(ethanol). IR m cmÀ1: 3185, 3380, 3395 (NH, NH2),
1660, 1615 (CONH), 1320 (S–Oantisym), 1140 (S–Osym).
Anal. calc for C14H21N3O5S: C, 48.97; H, 6.12; N,
12.24. Found: C, 49.05; H, 6.38; N, 12.07.
4.1.3. General procedure for the preparation of the 1-[2-
(substituted sulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-
aryl-thiosemicarbazides (V). Equimolar quantities of

1158
I. R. Ezabadi et al. / Bioorg. Med. Chem. 16 (2008) 1150–1161
4.1.3.5. 1-[2-(1-Piperidinesulfamoyl)-4,5-dimethoxy-
(DMSO-d6) d (ppm): 1.74–1.78 (m, 4H, pyrrolidine),
3.13–3.18 (m, 4H, pyrrolidine), 3.82 (d, 6H, 2CH3O,
J = 4.47 Hz), 3.92 (s, 2H, CH2CO), 7.10 (s, 1H, C6),
7.12–7.17 (m, 1H, ArH), 7.25 (s, 1H, C3), 7.30–7.35
(m, 2H, ArH), 7.51 (d, 2H, ArH, J = 7.92 Hz), 9.29 (b,
1H, NH), 9.74 (s, 1H, ArH), 10.15 (s, 1H, NH). Anal.
calc for C21H26N4O5S2: C, 52.72; H, 5.44; N, 11.71.
Found: C, 52.65; H, 5.54; N, 11.80.
phenylacetyl]-4-phenyl-thiosemicarbazide (Ve). Yield:
66%. Mp 193–195 ꢁC (ethanol-dichloromethane). IR m
cmÀ1: 3180, 3335, 3400 (3NH), 1650 (CONH), 1525
(C@S), 1320 (S–Oantisym), 1135 (S–Osym). 1H NMR
(DMSO-d6) d (ppm): 1.42 (br s, 2H, piperidine), 1.51
(br s, 4H, piperidine), 3.01 (br s, 4H, piperidine), 3.82
(d, 6H, 2CH3O, J = 5.67 Hz), 3.89 (s, 2H, CO CH2),
7.11 (s, 1H, C6), 7.13–7.18 (m, 1H, ArH), 7.21 (s, 1H,
C3), 7.30–7.36 (m, 2H, ArH), 7.52 (d, 2H, ArH,
J = 7.8 Hz), 9.27 (br s, 1H, NH), 9.77 (s, 1H, NH),
10.16 (s, 1H, NH). Anal. calc for C22H28N4O5S2: C,
53.65; H, 5.69; N, 11.38. Found: C, 53.17; H, 5.65; N,
11.43.
4.1.3.10. 1-[2-(1-Pyrrolidinesulfamoyl)-4,5-dimethoxy-
phenylacetyl]-4-(p-chlorophenyl)-thiosemicarbazide (Vj).
Yield: 76%. Mp 212–214 ꢁC (ethanol). IR m cmÀ1
:
3070, 3275, 3315 (3NH), 1685 (CONH), 1520 (C@S),
1335 (S–Oantisym), 1155 (S–Osym). 1H NMR (DMSO-
d6) d (ppm): 1.74–1.78 (m, 4H, pyrrolidine), 3.14–3.18
(m, 4H, pyrrolidine), 3.82 (d, 6H, 2CH3O,
J = 4.47 Hz), 3.92 (s, 2H, CH2CO), 7.09 (s, 1H, C6),
7.25 (s, 1H, C3), 7.39 (d, 2H, ArH, J = 8.73 Hz), 7.54
(d, 2H, ArH, J = 8.97 Hz), 9.34 (b, 1H, NH), 9.85 (s,
1H, ArH), 10.17 (s, 1H, NH). Anal. calc for
C21H25ClN4O5S2: C, 49.17; H, 4.87; N, 10.92. Found:
C, 49.22; H, 5.05; N, 10.85.
4.1.3.6. [2-(1-Piperidinesulfamoyl)-4,5-dimethoxy-phen-
ylacetyl]-4-(p-chlorophenyl)-thiosemicarbazide (Vf). Yield:
72%. Mp 212–213 ꢁC (methanol–dichloromethane). IR
m cmÀ1: 3085, 3310, (3NH), 1690 (CONH), 1515
(C@S), 1315 (S–Oantisym), 1135 (S–Osym). 1H NMR
(DMSO-d6) d (ppm): 1.41–1.42 (m, 2H, piperidine),
1.51 (br s, 4H, piperidine), 2.99–3.00 (m, 4H, piperi-
dine), 3.82 (d, 6H, 2CH3O, J = 4.89 Hz), 3.89 (s, 2H,
COCH2), 7.09 (s, 1H, C6), 7.21 (s, 1H, C3), 7.39 (d,
2H, ArH, J = 8.61 Hz), 7.54 (d, 2H, ArH,
J = 8.61 Hz), 9.32 (br s, 1H, NH), 9.86 (s, 1H, NH),
10.17 (s, 1H, NH). Anal. calc for C22H27ClN4O5S2:
C, 50.14; H, 5.12; N, 10.63. Found: C, 50.20; H,
5.17; N, 10.55.
4.1.4. General procedure for the preparation of the
5-[2-(substituted
sulfamoyl)-4,5-dimethoxy-benzyl]-4-
aryl-s-triazole-3-thiones (VI). A suspension of thiosemi-
carbazide (1 mmol) in sodium hydroxide solution (5%,
5 ml) was heated under reflux for 1 h. The reaction
mixture was allowed to cool and then adjusted to pH
6 with 10% hydrochloric acid. The precipitate formed
was filtered, washed with water, dried, and recrystallized
from the appropriate solvent.
4.1.3.7.
[2-(4-Morpholinesulfamoyl)-4,5-dimethoxy-
phenylacetyl]-4-phenyl-thiosemicarbazide (Vg). Yield:
92%. Mp 213–214 ꢁC (methanol–dichloromethane). IR
m cmÀ1: 3070, 3255, 3315 (3NH), 1680 (CONH), 1530
(C@S), 1340 (S–Oantisym), 1120 (S–Osym). 1H NMR
(DMSO-d6) d (ppm): 2.87–2.99 (m, 4H, morpholine),
3.59–3.60 (m, 4H, morpholine), 3.83 (d, 6H, 2CH3O,
J = 5.1 Hz), 3.91 (s, 2H, CH2CO), 7.13 (s, 1H, C6),
7.15–7.18 (m, 1H, ArH), 7.22 (s, 1H, C3), 7.31–7.36
(m, 2H, ArH), 7.50 (d, 2H, ArH, J = 7.68 Hz), 9.29 (b,
1H, NH), 9.77 (br s, 1H, NH), 10.16 (s, 1H, NH). Anal.
calc for C21H26N4O6S2: C, 51.01; H, 5.26; N, 11.33.
Found: C, 51.09; H, 5.20; N, 11.41.
The following compounds were prepared by an analo-
gous procedure.
4.1.4.1. 5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-
benzyl]-4-phenyl-s-triazole-3-thione (VIa). Yield: 70%.
Mp 208–209 ꢁC (methanol). IR m cmÀ1: 3085, 3050
(NH), 1590, 1570, 1515 (C@N), 1335 (S–Oantisym),
1
1135 (S–Osym). H NMR (DMSO-d6) d (ppm): 2.49 [s,
6H, N(CH3)2], 3.82 (s, 6H, 2CH3O), 4.03 (s, 2H,
CH2CO), 7.10 (s, 1H, C6), 7.20 (s, 1H, C3), 7.43 (d,
2H, ArH, J = 7.32 Hz), 7.55–7.64 (m, 3H, ArH), 13.67
(s, 1H, NH). Anal. calc for C19H22N4O4S2: C, 52.53;
H, 5.07; N, 12.90. Found: C, 52.41; H, 5.15; N, 12.82.
4.1.3.8.
[2-(4-Morpholinesulfamoyl)-4,5-dimethoxy-
phenylacetyl]-4-(p-chlorophenyl)-thiosemicarbazide (Vh).
Yield: 71%. Mp 230–231 ꢁC (methanol–dichlorometh-
ane). IR m cmÀ1: 3095, 3280, 3315 (3NH), 1680
(CONH), 1530 (C@S), 1335 (S–Oantisym), 1155 (S–Osym).
1H NMR (DMSO-d6) d (ppm): 2.99 (m, 4H, morpho-
line), 3.59–3.60 (m, 4H, morpholine), 3.83 (d, 6H,
2CH3O, J = 5.1 Hz), 3.92 (s, 2H, CH2CO), 7.12 (s, 1H,
C6), 7.23 (s, 1H, C3), 7.40 (d, 2H, ArH, J = 8.61 Hz),
7.53 (d, 2H, ArH, J = 8.85 Hz), 9.35 (b, 1H, NH), 9.88
(br s, 1H, NH), 10.18 (s, 1H, NH). Anal. calc for
C21H25ClN4O6S2: C, 47.68; H, 4.73; N, 10.59. Found:
C, 47.75; H, 4.80; N, 10.51.
4.1.4.2. 5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-ben-
zyl]-4-(p-chlorophenyl)-s-triazole-3-thione (VIb). Yield:
58%. Mp 246–247 ꢁC (methanol). IR m cmÀ1: 3075,
3045 (NH), 1585, 1560, 1515 (C@N), 1325 (S–Oantisym),
1
1150 (S–Osym). H NMR (DMSO-d6) d (ppm): 2.49 [s,
6H, N(CH3)2], 3.80 (s, 6H, 2CH3O), 4.03 (s, 2H,
CH2CO), 7.04 (s, 1H, C6), 7.17 (s, 1H, C3), 7.43 (d,
2H, ArH, J = 8.55 Hz), 7.67 (d, 2H, ArH,
J = 8.55 Hz), 13.68 (b, 1H, NH). Anal. calc for
C19H21ClN4O4S2: C, 48.66; H, 4.48; N, 11.95. Found:
C, 48.72; H, 4.54; N, 11.88.
4.1.3.9.
[2-(1-Pyrrolidinesulfamoyl)-4,5-dimethoxy-
phenylacetyl]-4-phenyl-thiosemicarbazide (Vi). Yield:
80%. Mp 189–191 ꢁC (ethanol-dichloromethane). IR m
cmÀ1: 3200, 3335 3435 (3NH), 1675 (CONH), 1530
(C@S), 1320 (S–Oantisym), 1145 (S–Osym). 1H NMR
4.1.4.3. 5-[2-(N-Diethylsulfamoyl)-4,5-dimethoxy-ben-
zyl]-4-phenyl-s-triazole-3-thione (VIc). Yield: 75%. Mp
205–206 ꢁC (methanol–dichloromethane). IR m cmÀ1
:
3105, 3050 (NH), 1595, 1570, 1515 (C@N), 1330 (S–

I. R. Ezabadi et al. / Bioorg. Med. Chem. 16 (2008) 1150–1161
1159
Oantisym), 1140 (S–Osym). 1H NMR (DMSO-d6) d (ppm):
0.98–1.10 (m, 6H, 2CH2CH3), 3.11–3.43 [m, 4H,
2CH2CH3], 2.95 (s, 6H, 2CH3O), 3.16 (s, 2H, CH2CO),
6.23 (s, 1H, C6), 6.33 (s, 1H, C3), 6.56 (d, 2H, ArH,
J = 6.69 Hz), 6.68–6.77 (m, 3H, ArH), 12.79 (b, 1H,
NH). Anal. calc for C21H26N4O4S2: C, 54.54; H, 5.62;
N, 12.12. Found: C, 54.47; H, 5.56; N, 12.18.
(C@N), 1350 (S–Oantisym), 1140 (S–Osym). 1H NMR
(DMSO-d6) d (ppm): 2.75 (s, 4H, Morpholine), 3.45
(br s, 4H, Morpholine), 3.84 (s, 6H, 2CH3O), 4.07 (s,
2H, CH2CO), 7.08 (s, 1H, C6), 7.22 (s, 1H, C3), 7.43
(d, 2H, ArH, J = 7.2 Hz), 7.67 (d, 2H, ArH,
J = 6.3 Hz). Anal. calc for C21H23ClN4O5S2: C, 49.36;
H, 4.50; N, 10.97. Found: C, 49.41; H, 4.47; N, 10.92.
4.1.4.4. 5-[2-(N-Diethylsulfamoyl)-4,5-dimethoxy-ben-
zyl]-4-(p-chlorophenyl)-s-triazole-3-thione (VId). Yield:
69%. Mp 247–249 ꢁC (methanol–dichloromethane). IR
m cmÀ1: 3080, 3015 (NH), 1590, 1515 (C@N), 1330 (S–
Oantisym), 1145 (S–Osym). 1H NMR (DMSO-d6) d
(ppm): 0.98–1.10 (m, 6H, 2CH2CH3), 3.11–3.43 [m,
4H, 2CH2CH3], 3.80 (s, 6H, 2CH3O), 4.03 (s, 2H,
CH2CO), 7.05 (s, 1H, C6), 7.17 (s, 1H, C3), 7.44 (d,
2H, ArH, J = 8.55 Hz), 7.67 (d, 2H, ArH,
J = 8.55 Hz), 13.70 (b, 1H, NH). Anal. calc for
C21H25ClN4O4S2: C, 50.75; H, 5.03; N, 11.27. Found:
C, 50.68; H, 5.10; N, 11.21.
4.1.4.9. 5-[2-(1-Pyrrolidinesulfamoyl)-4,5-dimethoxy-
benzyl]-4-phenyl-s-triazole-3-thione (VIi). Yield: 70%.
Mp 241–242 ꢁC (methanol–dichloromethane). IR m
cmÀ1: 3085, 3020 (NH), 1510, 1500 (C@N), 1315 (S–
Oantisym), 1145 (S–Osym). 1H NMR (DMSO-d6) d
(ppm): 1.65 (m, 4H, Pyrrolidine), 2.92 (t, 4H, Pyrroli-
dine), 3.80 (s, 6H, 2CH3O), 4.04 (s, 2H, CH2CO), 7.07
(s, 1H, C6), 7.24 (s, 1H, C3), 7.41 (d, 2H, ArH,
J = 7.95 Hz), 7.57 (m, 3H, ArH), 13.64 (b, 1H, NH).
Anal. calc for C21H24N4O4S2: C, 54.78; H, 5.21; N,
12.17. Found: C, 54.82; H, 5.16; N, 12.21.
4.1.4.10. 5-[2-(1-Pyrrolidinesulfamoyl)-4,5-dimethoxy-
benzyl]-4-(p-chlorophenyl)-s-triazole-3-thione
4.1.4.5. 5-[2-(1-Piperidinesulfamoyl)-4,5-dimethoxy-
benzyl]-4-phenyl-s-triazole-3-thione (VIe). Yield: 44%.
Mp 225–226 ꢁC (methanol–dichloromethane). IR m
cmÀ1: 3090, 3025 (NH), 1590, 1560, 1520 (C@N), 1330
(S–Oantisym), 1140 (S–Osym). 1H NMR (DMSO-d6) d
(ppm): 1.32 (s, 6H, piperidine), 2.76 (s, 4H, piperidine),
3.80 (d, 6H, 2CH3O, J = 1.83 Hz), 4.01 (s, 2H, CH2CO),
7.07 (s, 1H, C6), 7.21 (s, 1H, C3), 7.40 (d, 2H, ArH,
J = 7.32 Hz), 7.53–7.59 (m, 3H, ArH), 13.70 (b, 1H,
NH). Anal. calc for C22H26N4O4S2: C, 55.69; H, 5.48;
N, 11.81. Found: C, 55.62; H, 5.52; N, 11.87.
(VIj).
Yield: 56%. Mp 232–233 ꢁC (methanol–dichlorometh-
ane). IR m cmÀ1: 3085, 3050 (NH), 1585, 1570, 1515
(C@N), 1340 (S–Oantisym), 1140 (S–Osym). 1H NMR
(DMSO-d6) d (ppm): 1.66 (m, 4H, Pyrrolidine), 2.93 (t,
4H, Pyrrolidine), 3.80 (s, 6H, 2CH3O), 4.06 (s, 2H,
CH2CO), 7.03 (s, 1H, C6), 7.23 (s, 1H, C3), 7.44 (d,
2H, ArH, J = 8.55 Hz), 7.67 (d, 2H, ArH,
J = 8.55 Hz), 13.69 (b, 1H, NH). Anal. calc for
C21H23ClN4O4S2: C, 50.96; H, 4.65; N, 11.32. Found:
C, 51.02; H, 4.59; N, 11.35.
4.1.4.6. 5-[2-(1-Piperidinesulfamoyl)-4,5-dimethoxy-ben-
zyl]-4-(p-chlorophenyl)-s-triazole-3-thione (VIf). Yield:
48%. Mp 248–249 ꢁC (methanol–dichloromethane). IR
m cmÀ1: 3085, 3040 (NH), 1595, 1570, 1525 (C@N),
1330 (S–Oantisym), 1140 (S–Osym). 1H NMR (DMSO-
d6) d (ppm): 1.33 (s, 6H, piperidine), 2.76 (s, 4H, piper-
idine), 3.80 (d, 6H, 2CH3O, J = 1.23 Hz), 4.03 (s, 2H,
CH2CO), 7.03 (s, 1H, C6), 7.20 (s, 1H, C3), 7.43 (d,
2H, ArH, J = 9.15 Hz), 7.67 (d, 2H, ArH,
J = 8.52 Hz), 13.69 (b, 1H, NH). Anal. calc for
C22H25ClN4O4S2: C, 51.91; H, 4.91; N, 11.01. Found:
C, 51.85; H, 4.98; N, 10.97.
4.2. Pharmacology
4.2.1. Test for antifungal activity
4.2.1.1. Microdilution test. In order to investigate the
antifungal activity of compounds we used the following
fungi: Aspergillus niger (ATCC 6275), Aspergillus och-
raceus (ATCC 12066), Aspergillus versicolor (ATCC
11730), A. flavus (ATCC 9643), P. funiculosum (ATCC
36839), and T. viride (IAM 5061).
The organisms were obtained from the Mycological
Laboratory, Department of Plant Physiology, Institute
ˇ ´
for Biological research ‘Sinisa Stankovic’, Belgrade,
4.1.4.7. 5-[2-(4-Morpholinesulfamoyl)-4,5-dimethoxy-
benzyl]-4-phenyl-s-triazole-3-thione (VIg). Yield: 60%.
Mp 241–242 ꢁC (methanol–dichloromethane). IR m
cmÀ1: 3105, 3070 (NH), 1590, 1575, 1515 (C@N), 1320
(S–Oantisym), 1145 (S–Osym). 1H NMR (DMSO-d6) d
(ppm): 2.76 (s, 4H, Morpholine), 3.43 (br s, 4H, Mor-
pholine), 3.81 (s, 6H, 2CH3O, J = 1.23 Hz), 4.04 (s,
2H, CH2CO), 7.11 (s, 1H, C6), 7.22 (s, 1H, C3), 7.41
(d, 2H, ArH, J = 10.32 Hz), 7.54–7.62 (m, 3H, ArH),
13.66 (b, 1H, NH). Anal. calc for C21H24N4O5S2: C,
52.94; H, 5.04; N, 11.76. Found: C, 53.00; H, 5.09; N,
11.71.
Serbia.
The micromycetes were maintained on malt agar (MA)
and the cultures were stored at +4 ꢁC and subcultured
once a month.36
In order to investigate the antifungal activity of com-
pounds the modified microdilution technique was
used.37,38 The fungal spores were washed from the sur-
face of agar plates with sterile 0.85% saline containing
0.1% Tween 80 (v/v). The spore suspension was adjusted
with sterile saline to a concentration of approximately
1.0 · 105 in a final volume of 100 ll per well. The inocula
were stored at +4 ꢁC for further use. Dilutions of the
inocula were cultured on solid MA to verify the absence
of contamination and to check the validity of the
inoculum.
4.1.4.8. 5-[2-(4-Morpholinesulfamoyl)-4,5-dimethoxy-
benzyl]-4-(p-chlorophenyl)-s-triazole-3-thione
(VIh).
Yield: 55%. Mp 246–247 ꢁC (methanol–dichlorometh-
ane). IR m cmÀ1: 3080, 3045 (NH), 1605, 1565, 1515

1160
I. R. Ezabadi et al. / Bioorg. Med. Chem. 16 (2008) 1150–1161
Minimum inhibitory concentration (MIC) determina-
tion was performed by a serial dilution technique using
96-well microtiter plates. Compounds investigated were
dissolved in DMSO and in concentrations of 50.0–
150.0 lg/ml added in broth malt medium with fungal
inoculum. The microplates were incubated for 72 h at
28 ꢁC. The lowest concentration without visible growth
(under the binocular microscope) was defined as MIC.
The minimum fungicidal concentrations (MFCs) were
determined by serial subcultivation of a 2 ll into micro-
titer plates containing 100 ll of broth per well and fur-
ther incubation for 72 h at 28 ꢁC. The lowest
concentration with no visible growth was defined as
the MFC, indicating = 99.5% killing of the original inoc-
ulum. The commercial fungicide bifonazole (imidazole-
type fungicide) was used as a positive control (100.0–
250.0 lg/ml).
ics O2. Molecular Mechanics calculations were carried
out using the CHARMm force field. Synthetic com-
pounds were first minimized with Steepest Descents,
Conjugate Gradients, and Newton–Raphson algorithms
using an energy tolerance of 0.01 kcal/molÀ1 AÀ1, to
reach a local minimum. The dielectric constant (e) was
set to 45 during minimization simulating the amphi-
philic environment. To generate random conformers, a
systematic search procedure (Random Sampling) was
applied. This method randomly changes all defined tor-
sion angles within a predefined angular window. The
range of the torsion angle varies during the search pro-
cedure and generates new conformations. CHARMm
energy minimization is performed for each randomly al-
tered conformation. In order to explore the preferred
torsion angles that correspond to the lowest energy con-
formers and energy barriers of the molecules under
study, stochastic search procedures (systematic grid
scan) were used. This method initiates a grid scan search
that generates conformations by varying specified tor-
sion angles over a grid of equally spaced values. Inter-
vals of 5ꢁ were applied for single bond rotation and
10ꢁ of two bond rotation. During these searches, the
predetermined torsion angle remained constant while
minimization using 1000 steps of conjugate gradient
algorithm was applied to ‘relax’ the whole molecule.
4.2.2. Tests for antibacterial activity. The following bac-
teria were used: E. coli (ATCC 35210), Bacillus subtilis
(ATCC 10907), Micrococcus flavus (ATCC 10240),
Staphylococcus epidermidis (ATCC 12228), S. typhimuri-
um (ATCC 13311), Salmonella enteritidis (ATCC
13076), and E. cloacce (human isolate).
The bacterial suspension was adjusted with sterile saline
to a concentration of 1.0 · 105 CFU mlÀ1. The inocula
were daily prepared and stored at +4 ꢁC until use. Dilu-
tions of the inocula were cultured on solid medium to
verify the absence of contamination and to check the
validity of the inoculum.
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