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

Article abstract of DOI:10.1248/cpb.58.160

A series of thirteen new thiadiazole compounds were synthesized and evaluated for in vitro antifungal and antibacterial activity. All compound tested showed significant antifungal activity against all the micromycetes, compared to the commercial fungicide

Full text of DOI:10.1248/cpb.58.160

160
Chem. Pharm. Bull. 58(2) 160—167 (2010)
Vol. 58, No. 2
Sulfonamide-1,2,4-thiadiazole Derivatives as Antifungal and Antibacterial
Agents: Synthesis, Biological Evaluation, Lipophilicity, and
Conformational Studies
Charalabos CAMOUTSIS, ,a Athina GERONIKAKI,b Ana CIRIC,c Marina SOKOVIC´,c
*
d
Panagiotis ZOUMPOULAKIS,d and Maria ZERVOU
a School of Health Sciences, Department of Pharmacy, Laboratory of Pharmaceutical Chemistry, University of Patras;
Patras 26500, Greece: b School of Pharmacy, Department of Pharmaceutical Chemistry of Aristotelian, University of
Thessaloniki; Thessaloniki 54124, Greece: c Department of Plant Physiology, Mycological Laboratory, Institute of
Biological Research; Bulevar Despota Stefana 142, 11000 Belgrade, Serbia: and d Laboratory of Molecular Analysis,
Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation; 48 Vas. Constantinou Avenue,
11635 Athens, Greece. Received May 6, 2009; accepted November 26, 2009; published online November 30, 2009
A series of thirteen new thiadiazole compounds were synthesized and evaluated for in vitro antifungal and
antibacterial activity. All compound tested showed significant antifungal activity against all the micromycetes,
compared to the commercial fungicide bifonazole. Differences in their activity depend on the substitution of dif-
ferent reactive groups. More specifically, best antifungal activity was shown for the synthetic analogue with
methylpiperazine reactive group. Furthermore, it is apparent that different compounds reacted on different ways
against bacteria. An effort was made to correlate the above mentioned differences in activity with lipophilicity
studies. Furthermore, NMR and molecular modelling were used to obtain the main conformational features of a
potent analogue, for future in silico studies.
Key words thiadiazole; antifungal; antibacterial; bifonazole; ketoconazole
Bacterial infections have increased dramatically in recent tivities.
years. Bacteria have been the cause of some of the most
Chemistry The synthetic pathway followed for the
deadly diseases and widespread epidemics in human civiliza- preparation of the title compounds was accomplished as
tion.1) Moreover, the widespread use and misuse of antibi- shown in Chart 1.
otics has caused bacterial resistance. Some of these resistant
Starting from ethyl(2-chlorosulfonyl-4,5-dimethoxy-
strains, such as vancomycin-resistant enterococci (VRE) and phenyl)acetate (1)17,18) by reaction with secondary aliphatic
multidrug resistant Staphylococcus aureus (MRSA), are ca- amines in anhydrous benzene the corresponding sulfon-
pable of surviving the effects of most, if not all, antibiotics amides (2a—d) were obtained. The latter were converted
currently in use.2—6) With the increase in resistance of bacte- to the desired 2-(N-substituted sulfamoyl)-4,5-dimethoxy-
ria to antibiotic treatment, attention was given on developing phenylacetylhydrazides (3a—d) by treatment with hydrazine
novel approaches to antimicrobial therapy.7—13)
hydrate in xylol. The hitherto unknown 1-[2-(N-substituted
We have previously reported the significant antifungal ac- sulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-aryl-thiosemicar-
tivity of a series of sulfonamide-1,2,4-triazole derivatives bazides (4a—m) were obtained upon the reaction of acid
against a series of micromycetes, compared to the commer- hydrazides (3a—d) with suitable aryl isothiocyanates.4) Cy-
cial fungicide bifonazole. These compounds have also shown clization of 4a—m with concentrated sulfuric acid in cold re-
a comparable bactericidal effect to that of streptomycin but sulted to the formation of N-{5-[2-(N-substituted sulfamoyl)-
better activity than chlorampenicol respectively against vari- 4,5-dimethoxy benzyl]-1,3,4-thiadiazole-2-yl}-N-arylamines
ous bacteria.14)
(5a—m) respectively.
Thiadiazoles belong to the wider category of imidazole
and triazole synthetic antifungal drugs which are designed to Results and Discussion
inhibit the enzyme cytochrome P450 14a-demethylase and
Biological Evaluation and Lipophilicity Studies The
inhibit the conversion of lanosterol to ergosterol, which is re- results of antibacterial and antifungal activity of compounds
quired in fungal cell membrane synthesis.
5a—m against a panel of selected Gram positive, Gram neg-
1,3,4-Thiadiazoles are known to possess antibacterial and ative bacteria and fungi are presented in Tables 1 and 2 in
antifungal properties similar to those of well known comparison with those of the reference drugs ampicillin and
sulphonamide drugs.15) Thus, the 1,3,4-thiadiazoles exhibit a streptomycin, bifonazole and ketoconazole respectively.
broad spectrum of biological activities possibly due to the
Results of antibacterial activity of compounds (Table 1)
presence of the toxophoric –N–C–S moiety.16) Prompted by show that the minimum inhibitory concentration (MIC) of
these observations and in continuation of our search for compounds varies in the range of 0.92—4.6ꢀ10ꢁ2 mmol/ml,
bioactive molecules, we designed the synthesis of a series of while minimum bactericidal concentration (MBC) varies
novel sulfonamide-1,3,4-thiadiazoles, emphasizing, in partic- between 1.75—9.20ꢀ10ꢁ2 mmol/ml. Compounds 5a, 5b, 5c
ular, on the strategy of combining two chemically different and 5g showed the lowest antibacterial activity among the
but pharmacologically compatible molecules (the sulfon- tested compounds with 5a being the worst, with MIC of
amide nucleus and the five member heterocycle) in one 1.15—4.60ꢀ10ꢁ2 mmol/ml and MBC of 1.75—9.20ꢀ10ꢁ2
frame, in order to study their antibacterial and antifungal ac- mmol/ml. Moderate activity was observed for compounds
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: pzoump@otenet.gr

February 2010
161
5d—f, 5i, 5j and 5l. Compounds 5h, 5k and 5m showed the
higher antibacterial activity with 5h ranked as the best
among them with MIC varying in the interval 0.95—
1.89ꢀ10ꢁ2 mmol/ml and bactericidal effect between 1.89—
3.78ꢀ10ꢁ2 mmol/ml. It should be noted that less active com-
pounds have lower ClogP values which indicates that the in-
crease in activity goes in parallel with their lipophilicity ex-
pressed as the calculated ClogP values.
The most sensitive bacterial species on these compounds
is Bacillus cereus, while Listeria monocytogenes is the most
resistant species. It can be seen that all the compounds tested
for antibacterial activity showed much better effect than com-
mercial antibiotics, streptomycin and ampicillin.
In general, compounds tested are more active against
Gram positive bacteria than Gram negative while compound
5k exhibited the best activity against Gram positive bacteria
(B. cereus, Micrococcus flavus) with MIC 0.92—1.84ꢀ10ꢁ2
mmol/ml and the same good activity for Gram negative bac-
teria such as Pseudomonas aeruginosa (ATCC 27853) and
Salmonella typhimurium (ATCC 13311). Almost the same
behaviour was observed for compound 5h which exhibited
the best activity against Gram positive bacteria (Bacillus
cereus and Staphylococcus aureus (ATCC 6538) as well as
against Gram negative bacteria Pseudomonas aeruginosa
(ATCC 27853) and Salmonella typhimurium (ATCC 13311).
As regards the relationships between the structure and the
detected antibacterial activity, it was observed that pyrroli-
dine derivatives are mostly endowed with higher activity with
respect to piperidine, methylpiperazine and dimethylamino
derivatives. Moreover the inhibitory effect appears to be de-
pendent on the substitution at the benzene ring. Thus the in-
troduction of CF3 substituent as well as a chloro atom in the
para-position of the benzene ring respectively, improved an-
tibacterial activity in respect to compound 5a.
Chart 1. Synthetic Pathway for the Preparation of the Thiadiazole Deriva-
tives
Results of antifungal activity of the tested compounds are
Table 1. Antibacterial Activity of Compounds Tested by Microdilution Method (MIC and MBC in mmolꢀ10ꢁ2
)
5a
mic
mbc
5b
mic
mbc
5c
mic
mbc
5d
mic
mbc
5e
mic
mbc
5f
mic
mbc
5g
mic
mbc
5h
mic
mbc
5i
mic
mbc
5j
mic
mbc
5k
mic
mbc
5l
mic
mbc
5m
mic
mbc
Str
mic
mbc
Amp
mic
mbc
Bacteria
Bacillus cereus
2.30
4.60
1.07
2.13
1.05
2.09
0.99
1.99
1.09
2.17
2.02
4.04
1.98
3.96
0.95
1.89
0.98
1.97
1.93
3.86
0.92
1.84
1.91
3.82
1.79
1.79
4.3
8.6
24.8
37.2
Micrococcus
flavus
1.15
1.75
2.13
4.26
2.09
8.36
0.99
1.99
1.09
2.17
2.02
4.04
1.98
3.96
1.89
3.78
1.97
3.94
1.93
3.86
0.92
1.84
0.96
1.91
1.79
3.58
8.6
17.2
24.8
37.2
Staphylococcus 4.60
aureus 9.20
2.13
4.26
4.18
8.36
1.99
3.98
2.17
4.34
2.02
4.04
1.98
3.96
0.95
1.89
1.97
3.94
3.86
7.72
1.84
3.68
3.82
7.64
1.79
3.58
17.2
34.4
24.8
37.2
Escherichia coli 4.60
4.26
8.52
2.09
8.36
3.98
7.96
2.17
4.34
1.52
4.04
1.98
3.96
1.89
3.78
1.97
3.94
1.93
3.86
1.84
3.68
1.91
7.64
1.79
1.79
17.2
34.4
37.2
49.2
4.60
Pseudomonas
aeruginosa
4.60
4.60
4.26
8.52
1.57
4.18
1.99
3.98
2.17
4.34
2.02
4.04
1.98
3.96
0.95
1.89
0.98
1.97
0.97
1.93
0.92
1.84
1.91
7.64
1.79
3.58
17.2
34.4
74.4
124.0
Proteus
mirabilis
4.60
4.60
2.13
4.26
4.18
8.36
1.99
7.96
2.17
4.34
2.02
4.04
3.96
7.92
1.89
3.78
1.97
7.87
3.86
7.72
3.68
7.36
3.82
7.64
1.79
3.58
17.2
34.4
37.2
49.2
Salmonella
typhimurium
4.60
9.20
2.13
4.26
1.05
2.09
0.99
1.99
1.09
2.17
1.01
2.02
3.96
7.92
0.95
1.89
0.98
1.97
0.97
1.93
0.92
1.84
0.96
3.82
1.79
3.58
17.2
34.4
24.8
49.2
Listeria
monocytogenes 9.20
4.60
8.52
8.52
4.18
8.36
3.98
7.96
2.17
4.34
4.04
4.04
1.98
3.96
1.89
1.89
3.94
7.88
1.93
7.72
1.84
7.36
1.91
7.64
1.79
3.58
25.8
51.6
37.2
74.4
ClogP 3.34
4.08
3.15
4.27
3.98
4.71
3.78
4.91
5.27
4.34
5.47
4.46
4.66

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Vol. 58, No. 2
Table 2. Antifungal Activity of Compounds Tested by Microdilution Method (MIC and MFC in mmolꢀ10ꢁ2
)
5a
mic
mfc
5b
mic
mfc
5c
mic
mfc
5d
mic
mfc
5e
mic
mfc
5f
mic
mfc
5g
mic
mfc
5h
mic
mfc
5i
mic
mfc
5j
mic
mfc
5k
mic
mfc
5l
mic
mfc
5m
mic
mfc
Bif
mic
mfc
Ket
mic
mfc
Fungi
Penicillium
funiculosum
1.15
2.30
1.07
2.13
1.05
2.09
0.99
1.99
2.17
4.34
1.01
2.02
0.99
1.98
1.89
3.78
1.97
3.94
1.93
3.86
0.92
1.84
1.91
3.82
0.89
1.79
64.0
80.0
38.0
95.0
Penicillium
ochrochloron
2.30
4.60
2.13
4.26
1.09
4.18
0.99
3.98
2.17
4.34
2.02
4.04
1.98
3.96
1.89
3.78
1.97
3.94
1.93
3.86
1.84
3.68
1.91
3.82
1.79
3.58
48.0
64.0
380.0
380.0
Trichoderma
viride
1.15
2.30
1.07
2.13
1.05
2.09
0.99
1.99
1.09
2.17
1.01
2.02
0.99
1.98
0.95
1.89
0.98
1.97
0.97
1.93
0.92
1.84
0.96
1.91
0.89
1.79
64.0
80.0
475.0
570.0
Aspergillus
fumigatus
1.15
4.60
1.07
4.26
2.09
4.18
1.99
3.98
2.17
4.34
2.02
4.04
0.99
1.98
1.89
3.78
1.97
3.94
1.93
3.86
0.92
3.68
0.96
3.82
0.89
1.79
48.0
64.0
38.0
95.0
Aspergillus
niger
2.30
4.60
1.07
4.26
2.09
4.18
1.99
3.98
2.17
4.34
2.02
4.04
1.98
3.96
1.89
3.78
1.97
3.94
1.93
3.86
1.84
3.68
1.91
3.82
1.79
3.58
48.0
64.0
38.0
95.0
Aspergillus
flavus
2.30
4.60
1.07
4.26
2.09
4.18
1.99
3.98
2.17
4.34
2.02
4.04
1.98
3.96
1.89
3.78
0.98
1.97
1.93
3.86
1.84
3.68
1.91
3.82
1.79
3.58
48.0
64.0
285.0
380.0
Aspergillus
versicolor
1.15
2.30
1.07
4.26
2.09
4.18
0.99
3.98
2.17
4.34
2.02
4.04
1.98
3.96
1.89
3.78
0.98
1.97
1.93
3.86
0.92
1.84
3.82
3.82
1.79
3.58
32.0
64.0
38.0
95.0
Fulvia fulvum
1.15
2.30
2.13
8.52
1.05
2.09
0.99
1.99
1.09
2.17
1.01
2.02
0.99
1.98
1.89
7.56
0.98
1.97
0.97
1.93
0.92
1.84
0.96
1.91
0.89
1.79
32.0
64.0
38.0
95.0
Table 3. Observed ROE Constraints, Common in All Studied Compounds
presented in Table 2. As in the case of the antibacterial activ-
ity, compounds showed strong antifungal potential. MIC
varies between 0.89—2.30ꢀ10ꢁ2 mmol/ml and minimal fun-
gicidal concentration (MFC) in the interval 1.79—8.52ꢀ
10ꢁ2 mmol/ml. The lowest antifungal activity was observed
for compounds 5e, 5f, 5h, 5j and 5l. Among them, com-
pound 5e possessed the lowest antifungal potential, by in-
hibiting fungal growth at 1.09—2.17ꢀ10ꢁ2 mmol/ml and
showing fungicidal effect at 2.17—4.34ꢀ10ꢁ2 mmol/ml.
Compounds 5a—d, 5g and 5i were ranked in the middle of
the activities score, according to their antifungal potential.
Compounds 5k and 5m, showed the best activity against all
Distance constraints
5m structure
12–14
9–13
9–17/21
6–17/21
6–12
the fungi, with 5m exhibiting the highest antifungal potential troscopy (DQF-COSY) and rotating frame Overhauser en-
(MIC 0.89—1.79ꢀ10ꢁ2 mmol/ml and MFC 1.79—3.58ꢀ hancement spectroscopy (ROESY) spectra. It was observed
10ꢁ2 mmol/ml). The majority of compounds showed the best that all the tested compounds had common ROEs which re-
activity against Trichoderma viride while Fulvia fulvum is flect similar conformational properties (Table 3). This result
the most resistant species. Fungi were in general more sensi- indicates that the conformation of the molecules’ scaffold is
tive than bacterial species.
not influenced by the performed substitutions.
In conclusion, according to obtained results, compound 5h
In Fig. 1, we present the ROESY spectrum for the 5m ana-
showed the best antibacterial activity while compounds 5k logue indicating the critical ROE correlations. Resonance
and 5m showed the best antifungal activity. peaks assignment is indicated on the 1D projection. The ob-
Conformational Analysis Studies As a first step for fu- served ROE between H6 and the aromatic proton at 7.15 ppm
ture in silico docking and pharmacophore alignment studies, clearly attributed this singlet peak to H12. Furthermore, the
we performed a conformational study using NMR and mo- aromatic protons H9 and H12 showed ROE correlations with
lecular modelling. Here we demonstrate the conformational the methoxy protons H13 and H14 respectively leading to the
properties of the most active compounds 5m, 5h and of the unequivocal assignment of those proton resonances. ROE be-
lowest activity compound 5a. The solvent used in NMR tween H23 and the doublet at 7.73 ppm (not shown) led to
analysis is DMSO-d6 and not D2O because of little solubility the assignment of the more deshielded doublet to the
of tested compounds in aqueous media. Selection of the sol- H25/H29 while the peak attributed to H26/28 was confirmed
vent though is not inadequate since drug’s bioactive confor- by COSY correlation with the neighbouring nuclei. The ob-
mation is finally determined from the environment of the ac- served ROE signals between both H6 and H9 with H17, H21
tive site of the target protein.
are indicative of the spatial proximity between the methylene
Structure elucidation for each of the above mentioned group and the dimethoxy benzyl ring with the methylpiper-
compounds was performed following standard procedures azine moiety. Interestingly, no ROE signal was observed be-
using homonuclear double quantum filtered correlation spec- tween the benzene protons (H25—29) and the rest of the

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163
Fig. 1. ROESY Spectrum of 5m Acquired at 600 MHz
Distance constraints are indicated in red circles.
molecule and this was valid for all of the tested compounds.
In order to identify low energy conformations consistent
with the observed ROE constrains, we performed molecular
modelling studies for the 5a, 5h and 5m. The most important
conformational features for all the analogues are related to
the dihedral angles (t1—t8) which are presented in Fig. 2.
The 3D models of the studied molecules following their opti-
mization were subjected to Monte Carlo conformational
search. The produced low energy conformations for each
analogue were clustered according to the dihedral angles val-
ues and the lowest energy members of the families were fur-
ther investigated for their consistency with the ROE data.
Here we present the results for the 5m analogue stating that
similar configurations were observed for the rest of the com-
pounds. Three representative favourable conformations of
5m namely 5m_a, 5m_b and 5m_c are displayed in Fig. 3.
Conformer 5m_a forms a cluster between the thiadiazole
system and methylpiperazine group while 5m_b adopts an
“open” conformation, differing in the orientation of methyl-
piperazine which is now closer to the aromatic ring. Both
5m_a and 5m_b conformers support the experimentally ob-
served proximity of the methylpiperazine (H17, H21) with
the methylene group H6 and the H9 proton of the aromatic
ring. On the other hand, 5m_c conformer brings in spatial
proximity the methylpiperazine group and the benzene ring
which is inconsistent with the ROE data. Moreover, the ab-
sence of any ROE signal between the benzene ring (H25—
29) and the rest of the molecule features indicates its distal
positioning with its conformational flexibility depending on
Fig. 2. Critical Torsion Angles of the Thiadiazole Analogues for Their
Conformational Properties
X and Y indicate the substitution groups for all compounds.
Fig. 3. Representative Low Energy Conformers of 5m
t7 and t8 dihedrals. Finally, the thiadiazole system may adopt the H23 with the thiadiazole plane (t7ꢂ0° or 180°). Further-
different orientations bringing the sulphur atom or the dia- more, H23 seems to be preferably directed towards the sul-
zole moiety in geometrically opposite directions.
phur instead of the nitrogen atom.
In order to further examine the conformational relation-
Concerning the benzene ring flexibility, no specific limita-
ship between the thiadiazole and the benzene rings, we per- tions are observed since t8 dihedral energetically favours
formed systematic search through t7 and t8 dihedrals. The several values around ꢃ35°, ꢃ145°.
isoenergetic map (Fig. 4) confirmed a coplanar position of
Systematic search was also performed for t3 and t4 dihe-

164
Vol. 58, No. 2
dral angles to examine the relative orientation of the methoxy gal properties of 1,3,4-thiadiazoles, similar to those of well
groups. Results showed that energetically low conformations known sulphonamide drugs, we presented a series of novel
are achieved when adopting opposite directions, t3ꢂ0° and sulfonamide-1,3,4-thiadiazoles emphasizing, in particular, on
t4ꢂ180° and vice versa with a margin of ꢃ60°. All these the strategy of combining two chemically different but phar-
orientations are accepted as they are in accordance with ROE macologically compatible molecules (the sulfonamide nu-
data.
cleus and the five member heterocycle) in one frame. All the
As already stated, detailed conformational search of the compounds showed very strong antibacterial and antifungal
studied compounds, revealed similar conformational features activity against all the species tested. Compounds 5h, 5k and
for 5m (most active antifungal and good antibacterial), 5h 5m showed better antibacterial potential with 5h ranking as
(most active antibacterial and least active antifungal) and 5a the more active while compounds 5k and 5m, showed better
(least active antibacterial). In an effort to explain the differ- activity against all the fungi, with 5m showing the highest
ent activity profile, we calculated the lipophilicity maps for antifungal potential.
the derived favorable conformations of the three compounds
As regards the relationships between the structure and the
(Fig. 5). All molecules showed distinct lipophilic and hy- detected antibacterial activity, it was observed that in general
drophilic areas giving a more amphiphilic character. This pyrrolidine derivatives are mostly endowed with higher activ-
characteristic has been proved crucial for lateral diffusion ity with respect to piperidine, methylpiperazine and dimethyl-
through lipid bilayers.19,20) Interestingly, 5a has significantly amino derivatives. Moreover the inhibitory effect appears to
lower lipophilic area (ca. 20 Å2) compared to 5h (ca. 66 Å2) be dependent on the substitution at the benzene ring. Thus
which may partially explain their difference in antibacterial the introduction of CF3 substituent as well as a chloro atom
activity.
These results will be used for further in silico studies, pro- proved antibacterial activity in respect to compound 5a.
viding information on their pharmacophore models. It has to Conformational studies of more and less potent analogues
in the para-position of the benzene ring respectively, im-
be noticed that fruitful discussion of quantitative structure– with NMR and molecular modelling techniques provided
activity relationship (QSAR) is difficult, based on such nar- data for further pharmacophore generation and in silico
row-range activity data.
docking studies on CYP450. All studied analogues provided
similar low energy conformations consistent with ROE ex-
perimental data which indicates that the conformation of the
Conclusion
Prompted by the well established antibacterial and antifun- molecules’ scaffold is not influenced by the performed sub-
stitutions. The lipoplilic surface of the resulting conformers
show an amphoteric character, important for the interaction
of the molecules with membrane bilayers while supports that
greater lipophilic surface is related to antibacterial activity
increase.
Experimental
General Experimental Considerations Melting points were taken in
glass capillary tubes on a Haake Bucher apparatus and are uncorrected. IR
spectra were recorded on a FT-IR Jasco spectrophotometer in solid phase
KBr. All proton NMR spectra were determined with a Varian 300 MHz spec-
trometer using deuterated dimethylsulfoxide (DMSO-d6) and are reported in
d (ppm) units. Thin layer chromatography (TLC) was performed in E.
Merck precoated silica gel plates. Visualization was obtained by exposure 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 experimental
error (ꢃ0.4% of the calculated values).
General Procedure for the Preparation of the Ethyl-[2-(N-substituted
sulfamoyl)-4,5-dimethoxy-phenyl]acetate (2) To a flask containing 0.01
Fig. 4. Isoenergetic Map from Simultaneous Grid Scan of t7 and t8 Dihe-
mol of ethyl-(2-chloro-sulfonyl-4,5-dimethoxy-phenyl) acetate (1) 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 re-
duced pressure and ice-water was added to yield the corresponding sulfon-
drals
Energy minimum values are achieved when t7 is at 0, 180, 360°. This indicates a
coplanar orientation of NH and thiadiazole group.
Fig. 5. Selected Low Energy Conformers of 5m, 5h, and 5a in Accordance with ROE Experimental Data and Their Lipophilicity Maps Showing Am-
phiphilic Character for the Selected Molecules
Light gray represents hydrophilic and dark gray represents hydrophobic areas.

February 2010
165
amides. The title compounds prepared are reported below.
(s, 1H, NH), 10.23 (s, 1H, NH). Anal. Calcd for C21H25N5O7S2: C, 48.18; H,
Ethyl-[2-(N,N-dimethyl sulfamoyl)-4,5-dimethoxy-phenyl]acetate (2a): 4.78; N, 13.38. Found: C, 48.26; H, 4.68; N, 13.25.
mp 108—110 °C (methanol) (ref. 14; 109—110 °C).
1-[2-(1-Pyrrolidinesulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-(p-trifluo-
Ethyl-[2-(1-pyrrolidine sulfamoyl)-4,5-dimethoxy-phenyl]acetate (2b): romethyl-phenyl)-thiosemicarbazide (4h): Yield: 78%. mp 199—200 °C
mp 107—108 °C (ethylacetate) (ref. 14; 107—108 °C).
Ethyl-[2-(1-piperidine sulfamoyl)-4,5-dimethoxy-phenyl]acetate (2c): mp 1325 (S–Oantisym), 1124 (S–Osym). 1H-NMR (DMSO-d6) d (ppm): 1.72—1.76
100—101 °C (methanol) (ref. 17; 100—101 °C). (m, 4H, pyrrolidine), 3.13—3.17 (m, 4H, pyrrolidine), 3.79 (s, 3H, CH3O),
Ethyl-[2-(4-methyl-piperazine sulfamoyl)-4,5-dimethoxy-phenyl]acetate 3.81 (s, 3H, CH3O), 3.92 (s, 2H, CH2), 7.08 (s, 1H, ArH), 7.24 (s, 1H, ArH),
(2d): Yield: 64%. mp 119—120 °C (ethylacetate). IR cmꢁ1: 1734 (COO),
7.69 (d, 2H, ArH, Jꢂ8.7 Hz), 7.80 (d, 2H, ArH, Jꢂ8.4 Hz), 9.46 (bs, 1H,
1329 (S–Oantisym), 1142 (S–Osym). Anal. Calcd for C17H26N2O6S: C, 52.85; H, NH), 9.98 (s, 1H, NH), 10.20 (s, 1H, NH). Anal. Calcd for C22H25F3N4O5S2:
(methanol). IR cmꢁ1: 3396, 3327, 3219 (3NH), 1684 (CONH), 1518 (CꢂS),
6.73; N,7.25. Found: C, 52.91; H, 6.68; N, 7.31.
C, 48.35; H, 4.57; N, 10.25. Found: C, 48.22; H, 4.66; N, 10.33.
General Procedure for the Preparation of the 2-(N-Substituted sul-
1-[2-(1-Piperidinesulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-( p-
famoyl)-4,5-dimethoxy-phenylacetylhydrazides (3) To a flask containing chlorophenyl)-thiosemicarbazide (4i): mp 211—213 °C (methanol–di-
0.01 mol of ethyl-[2-(N-substituted sulfamoyl)-4,5-dimethoxy-phenyl]ac- chloromethane) (ref. 14; 212—213 °C).
etate in 20 ml of xylol was added 0.02 mol of 80% hydrazine hydrate. The
1-[2-(1-Piperidinesulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-(p-nitro-
mixture was refluxed for 20 h. The solvent was then removed under reduced phenyl)-thiosemicarbazide (4j): Yield: 56%. mp 197—198 °C (methanol).
pressure and the solid residue after crystallization from the appropriate sol-
vent was collected by filtration.
The following compounds were prepared by an analogous procedure.
IR cmꢁ1: 3333, 3298, 3086 (3NH), 1670 (CONH), 1514 (CꢂS), 1332
(S–Oantisym), 1145 (S–Osym). 1H-NMR (DMSO-d6) d (ppm): ꢁ1.40—1.49
(m, 6H, piperidine), 2.99 (s, 4H, piperidine), 3.80 (s, 6H, 2CH3O), 3.90 (s,
2-(N,N-Dimethyl sulfamoyl)-4,5-dimethoxy-phenylacetylhydrazide (3a): 2H, CH2), 7.08 (s, 1H, ArH), 7.20 (s, 1H, ArH), 7.93 (d, 2H, ArH, Jꢂ
mp 147—149 °C (ethanol) (ref. 14; 147—148 °C). 6.97 Hz), 8.20 (d, 2H, ArH, Jꢂ7.92 Hz), 9.58 (s, 1H, NH), 10.17 (s, 1H,
2-(1-Pyrrolidine sulfamoyl)-4,5-dimethoxy-phenylacetylhydrazide (3b): NH), 10.23 (s, 1H, NH). Anal. Calcd for C22H27N5O7S2: C, 49.16; H, 5.02;
mp 139—141 °C (ethanol) (ref. 14; 140—141 °C).
N, 13.03. Found: C, 49.23; H, 5.15; N, 12.97.
2-(1-Piperidine sulfamoyl)-4,5-dimethoxy-phenylacetylhydrazide (3c):
mp 134—136 °C (ethanol) (ref. 14; 136—137 °C).
1-[2-(1-Piperidinesulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-(p-trifluo-
rophenyl)-thiosemicarbazide (4k): Yield: 60%. mp 202—203 °C (methanol).
2-(4-Methyl-piperazine sulfamoyl)-4,5-dimethoxy-phenylacetylhydrazide IR cmꢁ1: 3306, 3078 (3NH), 1687 (CONH), 1514 (CꢂS), 1325 (S–Oantisym),
(3d): Yield: 90%, mp 127—129 °C (powder ethanol–n-hexane), IR cmꢁ1
:
1145 (S–Osym). H-NMR (DMSO-d6) d (ppm): 1.40—1.49 (m, 6H, piperi-
1
3275, 3090 (NH, NH2), 1672 (CONH), 1320 (S–Oantisym), 1135 (S–Osym). dine), 2.99 (s, 4H, piperidine), 3.79 (s, 3H, CH3O), 3.80 (s, 3H, CH3O), 3.88
Anal. Calcd for C15H24N4O5S: C, 48.38; H, 6.45; N,15.05. Found: C, 48.44; (s, 2H, CH2), 7.08 (s, 1H, ArH), 7.19 (s, 1H, ArH), 7.69 (d, 2H, ArH,
H, 6.50; N, 15.09.
Jꢂ7.56 Hz), 7.79 (d, 2H, ArH, Jꢂ7.58 Hz), 9.42 (bs, 1H, NH), 9.98 (s, 1H,
NH), 10.20 (s, 1H, NH). Anal. Calcd for C23H27F3N4O5S2: C, 49.28; H, 4.82;
General Procedrure for the Preparation of the 1-[2-(N-Substituted
sulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-aryl-thiosemicarbazides (4) N, 10.00. Found: C, 49.39; H, 4.72; N, 10.12.
Equimolar quantities of hydrazide (1 mmol) and aryl isothiocyanate
(1 mmol) in 3 ml of absolute ethanol were refluxed on a stream bath for 1 h.
1-[2-(4-Methylpiperazine sulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-(p-
chlorophenyl)-thiosemicarbazide (4l): Yield: 84%. mp 177—179 °C (ethanol
The resulting solid was filtered and recrystallized from the appropriate sol- powder). IR cmꢁ1: 3306, 3180 (3NH), 1697 (CONH), 1518 (CꢂS), 1325
vent.
The following compounds were prepared by an analogous procedure.
(S–Oantisym), 1141 (S–Osym). 1H-NMR (DMSO-d6) d (ppm): 2.15 (s, 3H,
N–CH3), 2.34 (s, 4H, piperazine), 3.01 (s, 4H, piperazine), 3.80 (s, 3H,
1-[2-(N,N-Dimethyl sulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-phenyl- CH3O), 3.82 (s, 3H, CH3O), 3.88 (s, 2H, CH2), 7.09 (s, 1H, ArH), 7.20 (s,
thiosemicarbazide (4a): mp 218—220 °C (methanol–dichloromethane) (ref.
14; 219—221 °C).
1H, ArH), 7.38 (d, 2H, ArH, Jꢂ8.9 Hz), 7.53 (d, 2H, ArH, Jꢂ8.9 Hz), 9.30
(bs, 1H, NH), 9.85 (s, 1H, NH), 10.16 (s, 1H, NH). Anal. Calcd for
1-[2-(N,N-Dimethyl sulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-(p- C22H28ClN5O5S2: C, 48.75; H, 5.17; N, 12.92. Found: C, 48.82; H, 5.09; N,
chlorophenyl)-thiosemicarbazide (4b): mp 197—198 °C (methanol–di-
chloromethane) (ref. 14; 196—198 °C).
12.85.
1-[2-(4-Methylpiperazine sulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-(p-
1-[2-(N,N-Dimethyl sulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-(p-nitro-
trifluoromethylphenyl)-thiosemicarbazide (4m): Yield: 87%. mp 148—
phenyl)-thiosemicarbazide (4c): Yield: 76%. mp 207—208 °C (meth- 151 °C (methanol powder). IR cmꢁ1: 3304, 3190 (3NH), 1697 (CONH),
anol–chloroform). IR cmꢁ1: 3350, 3312, 3177 (3NH), 1685 (CONH), 1516 1518 (CꢂS), 1325 (S–Oantisym), 1141 (S–Osym). 1H-NMR (DMSO-d6) d
(CꢂS), 1338 (S–Oantisym), 1142 (S–Osym). 1H-NMR (DMSO-d6) d (ppm):
(ppm): 2.15 (s, 3H, N–CH3), 2.36 (s, 4H, piperazine), 3.02 (s, 4H, piper-
2.66 (s, 6H, N(CH3)2, 3.80 (s, 3H, CH3O), 3.82 (s, 3H, CH3O), 3.91 (s, 2H, azine), 3.80 (s, 3H, CH3O), 3.82 (s, 3H, CH3O), 3.91 (s, 2H, CH2), 7.10 (s,
CH2), 7.09 (s, 1H, ArH), 7.20 (s, 1H, ArH), 7.92 (d, 2H, ArH, Jꢂ8.75
Hz), 8.20 (d, 2H, ArH, Jꢂ8.95 Hz), 9.61 (s, 1H, NH), 10.15 (s, 1H, NH),
1H, ArH), 7.21 (s, 1H, ArH), 7.70 (d, 2H, ArH, Jꢂ8.4 Hz), 7.81 (d, 2H,
ArH, Jꢂ8.5 Hz), 9.49 (s, 1H, NH), 10.02 (bs, 1H, NH), 10.22 (s, 1H, NH).
10.22 (s, 1H, NH). Anal. Calcd for C19H23N5O7S2: C, 45.87; H, 4.62; N, Anal. Calcd for C23H28F3N5O5S2: C, 48.00; H, 4.87; N, 12.17. Found: C,
14.08. Found: C, 45.83; H, 4.67; N, 14.01.
48.11; H, 4.81; N, 12.25.
1-[2-(N,N-Dimethyl sulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-(p-trifluo-
romethylphenyl)-thiosemicarbazide (4d): Yield: 69%. mp 196—197 °C
General Procedure for the Preparation of N-{5-[2-(N-Substituted sul-
famoyl)-4,5-dimethoxy-benzyl]-1,3,4-thiadiazol-2-yl}-N-arylamines (5)
(methanol). IR cmꢁ1: 3377, 3329, 3188 (3NH), 1684 (CONH), 1525 (CꢂS), A mixture of the 1-[2-(N-substituted sulfamoyl)-4,5-dimethoxy-phenyl-
1323 (S–Oantisym), 1126 (S–Osym). 1H-NMR (DMSO-d6) d (ppm): 2.66 (s,
6H, N(CH3)2), 3.80 (s, 3H, CH3O), 3.82 (s, 3H, CH3O), 3.90 (s, 2H, CH2), acid (5 ml) was stirred for 15 min. The resulting solution was then allowed to
7.09 (s, 1H, C6), 7.20 (s, 1H, C3), 7.68 (d, 2H, ArH, Jꢂ8.4 Hz), 7.79 (d, 2H, reach ambient temperature, left stirring for 30 min and poured cautiously
acetyl]-4-aryl-thiosemicarbazide (1 mmol) in cold concentrated sulphuric
ArH, Jꢂ8.7 Hz), 9.47 (bs, 1H, NH), 9.98 (s, 1H, NH), 10.18 (s, 1H, NH). into ice cold water. The reaction mixture was made alkaline to pH 8 with
Anal. Calcd for C20H23F3N4O5S2: C, 46.15; H, 4.42; N, 10.77. Found: C, aqueous ammonia and the precipitated product was filtered, washed with
46.09; H, 4.47; N, 10.81.
water and recrystallized from the appropriate solvent. The following com-
1-[2-(1-Pyrrolidinesulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-phenyl- pounds were prepared by an analogous procedure.
thiosemicarbazide (4e): mp 188—190 °C (ethanol–dichloromethane) (ref.
14; 189—191 °C).
1-[2-(1-Pyrrolidinesulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-( p-
chlorophenyl)-thiosemicarbazide (4f): mp 212—213 °C (ethanol) (ref. 14;
212—214 °C).
N-{5-[2-(N,N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-1,3,4-thiadia-
zol-2-yl}-N-phenylamine (5a): Yield: 69%. mp 227—228 °C (methanol–
chloroform). IR cmꢁ1: 3285 (NH), 1329 (S–Oantisym), 1138 (S–Osym). 1H-
NMR (DMSO-d6) d (ppm): 2.67 (s, 6H, 2NCH3), 3.83 (s, 3H, CH3O), 3.84
(s, 3H, CH3O), 4.56 (s, 2H, CH2), 6.97 (t, 1H, ArH, Jꢂ 7.32—7.34 Hz),
7.14 (s, 1H, ArH), 7.26 (s, 1H, ArH), 7.32 (t, 2H, ArH, Jꢂ7.55–8.22 Hz),
7.56 (d, 2H, ArH, Jꢂ7.88 Hz), 10.17 (s, 1H, NH). Anal. Calcd for
1-[2-(1-Pyrrolidinesulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-(p-nitro-
phenyl)-thiosemicarbazide (4g): Yield: 74%. mp 200—201 °C (methanol).
IR cmꢁ1: 3333, 3196, 3090 (3NH), 1670 (CONH), 1514 (CꢂS), 1325 C19H22N4O4S2: C, 52.53; H, 5.07; N, 12.90. Found: C, 52.45; H, 5.15; N,
1
(S–Oantisym), 1145 (S–Osym). H-NMR (DMSO-d6) d (ppm): 1.72—1.77 (m,
4H, pyrrolidine), 3.13—3.18 (m, 4H, pyrrolidine), 3.79 (s, 3H, CH3O), 3.81
(s, 3H, CH3O), 3.93 (s, 2H, CH2), 7.08 (s, 1H, ArH), 7.24 (s, 1H, ArH), 7.92
12.81.
N-{5-[2-(N,N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-1,3,4-thiadia-
zol-2-yl}-N-(p-chlorophenyl)amine (5b): Yield: 85%. mp 264—265 °C
(d, 2H, ArH, Jꢂ8.7 Hz), 8.20 (d, 2H, ArH, Jꢂ9 Hz), 9.60 (s, 1H, NH), 10.16 (methanol–chloroform). IR cmꢁ1: 3262 (NH), 1329 (S–Oantisym), 1136

166
Vol. 58, No. 2
1
13.45.
(S–Osym). H-NMR (DMSO-d6) d (ppm): 2.61 (s, 6H, 2NCH3), 3.80 (s, 3H,
CH3O), 3.81 (s, 3H, CH3O), 4.54 (s, 2H, CH2), 7.12 (s, 1H, ArH), 7.24 (s,
1H, ArH), 7.34 (d, 2H, ArH, Jꢂ8.50 Hz), 7.58 (d, 2H, ArH, Jꢂ8.43 Hz),
10.28 (s, 1H, NH). Anal. Calcd for C19H21ClN4O4S2: C, 48.66; H, 4.48; N,
11.95. Found: C, 48.75; H, 4.43; N, 12.03.
N-{5-[2-(1-Piperidinesulfamoyl)-4,5-dimethoxy-benzyl]-1,3,4-thiadiazol-
2-yl}-N-(p-trifluoromethylphenyl)amine (5k): Yield: 80%. mp 253—254 °C
(methanol–dichloromethane). IR cmꢁ1: 3268 (NH), 1331 (S–Oantisym), 1142
(S–Osym). 1H-NMR (DMSO-d6) d (ppm): 1,41 (m, 2H, piperidine), 1,51 (m,
4H, piperidine), 3,00 (m, 4H, piperidine), 3.83 (s, 3H, CH3O), 3.84 (s, 3H,
CH3O), 4.59 (s, 2H, CH2), 7.15 (s, 1H, ArH), 7.27 (s, 1H, ArH), 7.68 (d, 2H,
ArH, Jꢂ8,77 Hz), 7,78 (d, 2H, ArH, Jꢂ8,66 Hz), 10.62 (s, 1H, NH). Anal.
Calcd for C23H25F3N4O4S2: C, 50.92; H, 4.61; N, 10.33. Found: C, 50.98; H,
4.70; N, 10.37.
N-{5-[2-(4-Methylpiperazinesulfamoyl)-4,5-dimethoxy-benzyl]-1,3,4-
thiadiazol-2-yl}-N-(p-chlorophenyl)amine (5l): Yield: 67%. mp 263—
264 °C (methanol–chloroform). IR cmꢁ1: 3262 (NH), 1342 (S–Oantisym),
1138 (S–Osym). 1H-NMR (DMSO-d6) d (ppm): 2,08 (s, 3H, N–CH3), 2.26
(m, 4H, piperazine), 2.96 (m, 4H, piperazine), 3.81 (s, 3H, CH3O), 3.82 (s,
3H, CH3O), 4.54 (s, 2H, CH2), 7.14 (s, 1H, ArH), 7.26 (s, 1H, ArH), 7.34 (d,
2H, ArH, Jꢂ8,78 Hz), 7,59 (d, 2H, ArH, Jꢂ8,77 Hz), 10.31 (s, 1H, NH).
Anal. Calcd for C22H26ClN5O4S2: C, 50.43; H, 4.96; N, 13.37. Found: C,
50.48; H, 4.91; N, 13.41.
N-{5-[2-(4-Methylpiperazinesulfamoyl)-4,5-dimethoxy-benzyl]-1,3,4-
thiadiazol-2-yl}-N-(p-trifluoromethylphenyl)amine (5m): Yield: 67%. mp
248—249 °C (methanol–dichloromethane). IR cmꢁ1: 3271 (NH), 1329
(S–Oantisym), 1136 (S–Osym). 1H-NMR (DMSO-d6) d (ppm): 2,08 (s, 3H,
N–CH3), 2.27 (m, 4H, piperazine), 2.97 (m, 4H, piperazine), 3.83 (s, 6H,
2CH3O), 4.58 (s, 2H, CH2), 7.17 (s, 1H, ArH), 7.28 (s, 1H, ArH), 7.67 (d,
2H, ArH, Jꢂ8,56 Hz), 7,77 (d, 2H, ArH, Jꢂ8,54 Hz), 10.62 (s, 1H, NH).
Anal. Calcd for C23H26F3N5O4S2: C, 49.55; H, 4.66; N, 12.56. Found: C,
49.61; H, 4.61; N, 12.50.
Pharmacology. Test for Antibacterial Activity The following Gram-
negative bacteria were used: Escherichia coli (ATCC 35210), Pseudomonas
aeruginosa (ATCC 27853), Salmonella typhimurium (ATCC 13311), Pro-
teus mirabilis (human isolate) as well as Gram-positive: Listeria monocyto-
genes (NCTC 7973), Bacillus cereus (clinical isolate), Micrococcus flavus
(ATCC 10240), and Staphylococcus aureus (ATCC 6538). The organisms
were obtained from the Mycological Laboratory, Department of Plant Physi-
ology, Institute for Biological Research “Sinisˇa Stankovic´,” Belgrade, Ser-
N-{5-[2-(N,N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-1,3,4-thiadia-
zol-2-yl}-N-(p-nitrophenyl)amine (5c): Yield: 89%. mp 268—269 °C
(methanol–dichloromethane). IR cmꢁ1: 3263 (NH), 1329 (S–Oantisym), 1136
1
(S–Osym). H-NMR (DMSO-d6) d (ppm): 2.64 (s, 6H, 2NCH3), 3.81 (s, 6H,
2CH3O), 4.59 (s, 2H, CH2), 7.14 (s, 1H, ArH), 7.24 (s, 1H, ArH), 7.77 (d,
2H, ArH, Jꢂ8.70 Hz), 8.21 (d, 2H, ArH, Jꢂ8.70 Hz), 10.90 (s, 1H, NH).
Anal. Calcd for C19H21N5O6S2: C, 47.60; H, 4.38; N, 14.61. Found: C, 47.68;
H, 4.32; N, 14.55.
N-{5-[2-(N,N-Dimethyl sulfamoyl)-4,5-dimethoxy-benzyl]-1,3,4-thiadia-
zol-2-yl}-N-(p-trifluoromethylphenyl)amine (5d): Yield: 73%. mp 253—
254 °C (methanol–dichloromethane). IR cmꢁ1
: 3263 (NH), 1329 (S–
Oantisym), 1138 (S–Osym). 1H-NMR (DMSO-d6) d (ppm): 2.65 (s, 6H,
2NCH3), 3.81 (s, 3H, CH3O), 3.82 (s, 3H, CH3O), 4.57 (s, 2H, CH2), 7.13 (s,
1H, ArH), 7.24 (s, 1H, ArH), 7.65 (d, 2H, ArH, Jꢂ8.58 Hz), 7.75 (d, 2H,
ArH, Jꢂ8.57 Hz), 10.61 (s, 1H, NH). Anal. Calcd for C20H21F3N4O4S2: C,
47.80; H, 4.18; N, 11.15. Found: C, 47.75; H, 4.27; N, 11.07.
N-{5-[2-(1-Pyrrolidinesulfamoyl)-4,5-dimethoxy-benzyl]-1,3,4-thiadia-
zol-2-yl}-N-phenylamine (5e): Yield: 57%. mp 242—243 °C (methanol–
chloroform). IR cmꢁ1: 3292 (NH), 1333 (S–Oantisym), 1142 (S–Osym). 1H-
NMR (DMSO-d6) d (ppm): 1.68—1.73 (m, 4H, pyrrolidine), 3.09—3.14
(m, 4H, pyrrolidine), 3.80 (s, 3H, CH3O), 3.81 (s, 3H, CH3O), 4.56 (s, 2H,
CH2), 6.94 (t, 1H, ArH, Jꢂ7.27 Hz), 7.12 (s, 1H, ArH), 7.29 (s, 1H, ArH),
7.29 (t, 2H, ArH, Jꢂ7.14—8.71 Hz), 7.53 (d, 2H, ArH, Jꢂ7.83 Hz), 10.14
(s, 1H, NH). Anal. Calcd for C21H24N4O4S2: C, 54.78; H, 5.21; N, 12.17.
Found: C, 54.87; H, 5.28; N, 12.20.
N-{5-[2-(1-Pyrrolidinesulfamoyl)-4,5-dimethoxy-benzyl]-1,3,4-thiadia-
zol-2-yl}-N-(p-chlorophenyl)amine (5f): Yield: 75%. mp 275—277 °C
(methanol–chloroform). IR cmꢁ1: 3267 (NH), 1331 (S–Oantisym), 1141
1
(S–Osym). H-NMR (DMSO-d6) d (ppm): 1.68—1.73 (m, 4H, pyrrolidine),
3.09—3.13 (m, 4H, pyrrolidine), 3.80 (s, 3H, CH3O), 3.81 (s, 3H, CH3O),
4.56 (s, 2H, CH2), 7.12 (s, 1H, ArH), 7.29 (s, 1H, ArH), 7.34 (d, 2H, ArH,
Jꢂ9Hz), 7.58 (d, 2H, ArH, Jꢂ9 Hz), 10.29 (s, 1H, NH). Anal. Calcd for
C21H23ClN4O4S2: C, 50.96; H, 4.65; N, 11.32. Found: C, 50.91; H, 4.71; N,
11.37.
bia.
21—23)
The antibacterial assay was carried out by microdilution method
in
order to determine the antibacterial activity of compounds tested against the
human pathogenic bacteria.
N-{5-[2-(1-Pyrrolidinesulfamoyl)-4,5-dimethoxy-benzyl]-1,3,4-thiadia-
zol-2-yl}-N-(p-nitrophenyl)amine (5g): Yield: 89%. mp 285—286 °C
(methanol–chloroform). IR cmꢁ1: 3262 (NH), 1332 (S–Oantisym), 1140
The bacterial suspensions were adjusted with sterile saline to a concentra-
tion of 1.0ꢀ105 colony forming unit (CFU)/ml. The inocula were prepared
daily and stored at ꢄ4 °C until use. Dilutions of the inocula were cultured on
solid medium to verify the absence of contamination and to check the valid-
ity of the inoculum.
1
(S–Osym). H-NMR (DMSO-d6) d (ppm): 1.66—1.73 (m, 4H, pyrrolidine),
3.09—3.14 (m, 4H, pyrrolidine), 3.80 (s, 3H, CH3O), 3.81 (s, 3H, CH3O),
4.61 (s, 2H, CH2), 7.15 (s, 1H, ArH), 7.29 (s, 1H, ArH), 7.76 (d, 2H, ArH,
Jꢂ9.3Hz), 8.22 (d, 2H, ArH, Jꢂ8.22 Hz), 10.94 (s, 1H, NH). Anal. Calcd
for C21H23N5O6S2: C, 49.90; H, 4.55; N, 13.86. Found: C, 49.93; H, 4.61; N,
13.89.
Microdilution Test The minimum inhibitory and bactericidal concen-
trations (MICs and MBCs) were determined using 96-well microtitre plates.
The bacterial suspension was adjusted with sterile saline to a concentration
of 1.0ꢀ105 cfu/ml. Compounds to be investigated were dissolved in broth
LB medium (100 ml) with bacterial inoculum (1.0ꢀ104 cfu per well) to
achieve the wanted concentrations (1 mg/ml). The microplates were incu-
bated for 24 h at 48 °C. The lowest concentrations without visible growth (at
the binocular microscope) were defined as concentrations that completely in-
hibited bacterial growth (MICs). The MBCs were determined by serial sub-
cultivation of 2 ml into microtitre plates containing 100 ml of broth per well
and further incubation for 72 h. The lowest concentration with no visible
growth was defined as the MBC, indicating 99.5% killing of the original in-
oculum. The optical density of each well was measured at a wavelength of
655 nm by Microplate manager 4.0 (Bio-Rad Laboratories) and compared
with a blank and the positive control. Streptomycin and Ampicillin were
used as a positive control (1 mg/ml DMSO). Two replicates were done for
each compound.
Test for Antifungal Activity For the antifungal bioassays, Aspergillus
flavus (ATCC 9643), Aspergillus fumigatus (plant isolate), Aspergillus niger
(ATCC 6275), Aspergillus versicolor (ATCC 11730)), Fulvia fulvum (TK
5318), Penicillium funiculosum (ATCC 36839), Penicillium ochrochloron
(ATCC 9112) and Trichoderma viride (IAM 5061) were used.
The organisms were obtained from the Mycological Laboratory, Depart-
ment of Plant Physiology, Institute for Biological Research “Sinisˇa
Stankovic´,” Belgrade, Serbia.
N-{5-[2-(1-Pyrrolidinesulfamoyl)-4,5-dimethoxy-benzyl]-1,3,4-thiadia-
zol-2-yl}-N-(p-trifluoromethylphenyl)amine (5h): Yield: 90%. mp 260—
261 °C (methanol–chloroform). IR cmꢁ1: 3265 (NH), 1329 (S–Oantisym),
1
1142 (S–Osym). H-NMR (DMSO-d6) d (ppm): 1.68—1.73 (m, 4H, pyrroli-
dine), 3.09—3.14 (m, 4H, pyrrolidine), 3.80 (s, 3H, CH3O), 3.81 (s, 3H,
CH3O), 4.59 (s, 2H, CH2), 7.14 (s, 1H, ArH), 7.29 (s, 1H, ArH), 7.65 (d, 2H,
ArH, Jꢂ8.7 Hz), 7.74 (d, 2H, ArH, Jꢂ8.4 Hz), 10.56 (s, 1H, NH). Anal.
Calcd for C22H23F3N4O4S2: C, 50.00; H, 4.35; N, 10.60. Found: C, 50.04; H,
4.29; N, 10.64.
N-{5-[2-(1-Piperidinesulfamoyl)-4,5-dimethoxy-benzyl]-1,3,4-thiadiazol-
2-yl}-N-(p-chlorolphenyl)amine (5i): Yield: 86%. mp 258—259 °C
(methanol–chloroform). IR cmꢁ1: 3262 (NH), 1333 (S–Oantisym), 1142
(S–Osym). 1H-NMR (DMSO-d6) d (ppm): 1.37—1.39 (m, 2H, piperidine),
1.45—1.48 (m, 4H, piperidine), 2.95—2.97 (m, 4H, piperidine), 3.80 (s, 3H,
CH3O), 3.81 (s, 3H, CH3O), 4.53 (s, 2H, CH2), 7.10 (s, 1H, ArH), 7.24 (s,
1H, ArH), 7.35 (d, 2H, ArH, Jꢂ4.5 Hz), 7.57 (d, 2H, ArH, Jꢂ4.5 Hz), 10.29
(s, 1H, NH). Anal. Calcd for C22H25ClN4O4S2: C, 51.91; H, 4.91; N, 11.01.
Found: C, 51.96; H, 4.85; N, 10.97.
N-{5-[2-(1-Piperidinesulfamoyl)-4,5-dimethoxy-benzyl]-1,3,4-thiadiazol-
2-yl}-N-(p-nitrolphenyl)amine (5j): Yield: 74%. mp 273—274 °C
(methanol–dichloromethane). IR cmꢁ1: 3262 (NH), 1337 (S–Oantisym), 1142
(S–Osym). 1H-NMR (DMSO-d6) d (ppm): 1,42 (m, 2H, piperidine), 1,51 (m,
4H, piperidine), 3,01 (m, 4H, piperidine), 3.84 (s, 6H, 2CH3O), 4.61 (s, 2H,
CH2), 7.16 (s, 1H, ArH), 7.27 (s, 1H, ArH), 7.79 (d, 2H, ArH, Jꢂ8,89 Hz),
8,25 (d, 1H, ArH, Jꢂ8,86 Hz), 10.96 (s, 1H, NH). Anal. Calcd for
C22H25N5O6S2: C, 50.86; H, 4.81; N, 13.48. Found: C, 50.91; H, 4.85; N,
The micromycetes were maintained on malt agar and the cultures stored at
4 °C and sub-cultured once a month24) in order to investigate the antifungal
activity of the extracts, a modified microdilution technique was used.21—23)
The fungal spores were washed from the surface of agar plates with sterile

February 2010
167
0.85% saline containing 0.1% Tween 80 (v/v). The spore suspension was ad- References
justed with sterile saline to a concentration of approximately 1.0ꢀ105 in a
final volume of 100 ml per well. The inocula were stored at 4 °C for further
use. Dilutions of the inocula were cultured on solid malt agar to verify the
absence of contamination and to check the validity of the inoculum.
Minimum inhibitory concentration (MIC) determinations were performed
by a serial dilution technique using 96-well microtiter plates. The com-
pounds investigated were dissolved in DMSO (1 mg/ml) and added in broth
Malt medium with inoculum. The microplates were incubated for 72 h at
28 °C, respectively. The lowest concentrations without visible growth (at the
binocular microscope) were defined as MICs.
1) Todar’s Online Textbook of Bacteriology. http://www.textbookofbacte-
riology.net/.
2) Hooper D. C., Clin. Infect. Dis., 33, S157—S160 (2001).
3) Galimand M., Courvalin P., Lambert T., Antimicrob. Agents
Chemother., 47, 2565—2571 (2003).
4) Tenover F. C., Clin. Infect. Dis., 33, S108—S115 (2001).
5) Leclercq R., Courvalin P., Antimicrob. Agents Chemother., 46, 2727—
2734 (2002).
6) Nagai K., Davies T. A., Jacobs M. R., Appelbaum P. C., Antimicrob.
Agents Chemother., 46, 1273—1280 (2002).
The fungicidal concentrations (MFCs) were determined by serial subculti-
vation of a 2 ml into microtiter plates containing 100 ml of broth per well and
further incubation 72 h at 28 °C. The lowest concentration with no visible
growth was defined as MFC indicating 99.5% killing of the original inocu-
lum. DMSO was used as a negative control, commercial fungicides, bifona-
zole and ketoconazole, were used as positive controls (1—3000 mg/ml).
NMR Spectroscopy DMSO-d6 and ultra precision NMR tubes Wilmad
7) Fung-Tomc J. C., Clark J., Minassian B., Pucci M., Tsai Y. H., Gradel-
ski E., Lamb L., Medina I., Huczko E., Kolek B., Chaniewski S., Fer-
raro C., Washo T., Bonner D. P., Antimicrob. Agents Chemother., 46,
971—976 (2002).
8) Chopra I., Curr. Opin. Pharmacol., 1, 464—469 (2001).
9) Nagano R., Shibata K., Adachi Y., Imamura H., Hashizume T., Mori-
shima H., Antimicrob. Agents Chemother., 44, 489—495 (2000).
535—5 mm (SPINTEC ROTOTEC) were used for the NMR experiments. 10) Capobianco J. O., Cao Z., Shortridge V. D., Ma Z., Flamm R. K.,
The compounds were dissolved in DMSO-d6 to a final concentration of
around 10 mM and a series of experiments were performed using Varian
600MHz spectrometer at 300 K. All data are collected using pulse sequences
Zhong P., Antimicrob. Agents Chemother., 44, 1562—1567 (2000).
11) Choudhry A. E., Mandichak T. L., Broskey J. P., Egolf R. W., Kinsland
C., Begley T. P., Seefeld M. A., Ku T. W., Brown J. R., Zalacain M.,
Ratnam K., Antimicrob. Agents Chemother., 47, 2051—2055 (2003).
1
and phase-cycling routines provided in the Varian libraries. The H spectral
width was set to 8500 Hz at 600 MHz. The offset compensated ROESY ex- 12) Colca J. R., McDonal W. G., Waldon D. J., Thomasco L. M., Gadwood
periments were performed using a mixing time of 150 ms in the phase-sensi-
tive mode, a relaxation delay of 1 s and 4 kHz spin-locking field strength.
The performance of a series of nuclear Overhauser effect spectroscopy
(NOESY) experiments using mixing times of 100 ms, 150 ms, 300 ms,
500 ms and 1 s revealed that the mixing time of 150 ms could ensure the op-
eration at the initial linear part of the NOE buildup curve. Experimental data
were processed using VNMR routines. Chemical shifts (d) are reported in
ppm while spectra were referenced by the standard experimental setup.
Molecular Modeling Conformational analyses studies and lipophilicity
maps calculations were performed using MacroModel 9.5 (Schrödinger Inc.)
R. C., Lund E. T., Cavey G. S., Mathews W. R., Adams L. D., Cecil E.
T., Pearson J. D., Bock J. H., Mott J. E., Shinabarger D. L., Xiong L.,
Mankin A. S., J. Biol. Chem., 278, 21972—21979 (2003).
13) Oliva B., Miller K., Caggiano, N., O’Neill A. J., Cuny G. D., Hoemann
M. Z., Hauske J. R., Chopra I., Antimicrob. Agents Chemother., 47,
458—466 (2003).
14) Ezabadi I. R., Camoutsis Ch., Zoumpoulakis P., Geronikaki A.,
Sokovic´ M., Clamocilija J., Ciric A., Bioorg. Med. Chem., 16, 1150—
1161 (2008).
15) Sandström J., Adv. Heterocycl. Chem., 9, 165—209 (1968).
s/w. The OPLS5 force field was applied for the potential energy calculations 16) Mohsen A., Omar M. E., Wafa A., J. Heterocycl. Chem., 23, 1339—
and all studies were run using a dielectric constant eꢂ45 to simulate DMSO 1341 (1986).
environment. Monte Carlo search was performed using MCMM/LMOD 17) Catsoulacos P., J. Heterocycl. Chem., 8, 947—950 (1971).
routine. PRCG algorithm was used for energy optimization with 0.005 con-
vergence threshold and 5000 steps. Consequently, the minimized conformers
18) Kothari P. J., Kishore V., Stenberg V. I., Parmar S. S., J. Heterocycl.
Chem., 15, 1101—1104 (1978).
are representatives of the local minima of the potential energy surface. The 19) Theodoropoulou E., Marsh D., Biochim. Biophys. Acta, 1461, 135—
derived conformers were clustered by torsional RMSD in 5 families, the 146 (1999).
lowest energy members of which were chosen for further analysis. Grid scan 20) Zoumpoulakis P., Daliani I., Zervou M., Kyrikou I., Siapi E., Lam-
search was performed using Maestro Dihedral Scan. The initial structures
were subjected to a systematic variation around the specific torsion angles
prinidis G., Mikros E., Mavromoustakos T., Chem. Phys. Lipids, 125,
13—25 (2003).
from 0° to 360° applying a torsion step of 10°. The derived conformations 21) Daouk K. D., Dagher M. S., Sattout J. E., J. Food Protect., 58, 1147—
were optimized by applying PRCG algorithm with an energy gradient toler- 1149 (1995).
ance of 0.001 Kcal/mol Å as convergence criterion. Visualization of the 3D 22) Espinel-Ingroff A., J. Clin. Microbiol., 39, 1360—1367 (2001).
structures has been enabled by the use of Accelrys DS Visualizer s/w.
23) Hanel H., Raether W., Mycoses, 31, 148—154 (1988).
24) Booth C., “Methods in Microbiology,” Vol. IV, ed. by Norris J. R., Rib-
bons D. W., Academic Press, London, New York, 1971, pp. 49—94.