Synthesis of novel sulfonamide-1,2,4-triazoles, 1,3,4-thiadiazoles and 1,3,4-oxadiazoles, as potential antibacterial and antifungal agents. Biological evaluation and conformational analysis studies.

Abstract

The significant antifungal activity of a series of sulfonamide-1,2,4-triazole and 1,3,4-thiazole derivatives against a series of micromycetes, compared to the commercial fungicide bifonazole has been reported. These compounds have also shown a comparable bactericidal effect to that of streptomycin and better activity than chloramphenicol against various bacteria. In view of the potential biological activity of members of the 1,2,4-triazole, 1,3,4-thiadiazole and 1,3,4-oxadiazole ring systems and in continuation of our search for bioactive molecules, we designed the synthesis of a series of novel sulfonamide-1,2,4-triazoles, -1,3,4-thiadiazoles and -1,3,4-oxadiazoles emphasizing, in particular, on the strategy of combining two chemically different but pharmacologically compatible molecules (the sulfomamide nucleus and the five member) heterocycles in one frame. Synthesized compounds were tested in vitro for antibacterial and antifungal activity and some analogues exhibited very promising results especially as antifungal agents. In order to explain structure-activity relationships, conformational analysis was performed for active and less active analogues using NMR spectroscopy and molecular modeling techniques. Furthermore, molecular properties which can be further used as descriptors for SAR studies, were predicted for the synthesized analogues. In general, antifungal activity seems to depend more on the triazol-3-thione moiety rather than the different length of the alkyl chain substitutions.

Full text

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Synthesis of novel sulfonamide-1,2,4-triazoles, 1,3,4-thiadiazoles and
1,3,4-oxadiazoles, as potential antibacterial and antifungal agents. Biological
evaluation and conformational analysis studies
P. Zoumpoulakisa,⇑, Ch. Camoutsisb, G. Pairasb, M. Sokovic ´c, J. Glamoc ˇlijac, C. Potamitisa, A. Pitsasb
aLaboratory of Molecular Analysis, Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., 11635 Athens, Greece
bLaboratory of Pharmaceutical Chemistry, Department of Pharmacy, University of Patras, 26500 Patras, Greece
cDepartment of Plant Physiology, Mycological Laboratory, Institute of Biological Research, University of Belgrade, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia
a r t i c l ei n f o
Article history:
Received 21 September 2011
Revised 14 December 2011
Accepted 17 December 2011
Available online 30 December 2011
Keywords:
Antifungal
Antibacterial
Sulfonamides
Thiadiazoles
Triazoles
Oxadiazoles
Conformational analysis
a b s t r a c t
The significant antifungal activity of a series of sulfonamide-1,2,4-triazole and 1,3,4-thiazole derivatives
against a series of micromycetes, compared to the commercial fungicide bifonazole has been reported.
These compounds have also shown a comparable bactericidal effect to that of streptomycin and better
activity than chloramphenicol against various bacteria.
In view of the potential biological activity of members of the 1,2,4-triazole, 1,3,4-thiadiazole and 1,3,4-
oxadiazole ring systems and in continuation of our search for bioactive molecules, we designed the syn-
thesis of a series of novel sulfonamide-1,2,4-triazoles, -1,3,4-thiadiazoles and -1,3,4-oxadiazoles empha-
sizing, in particular, on the strategy of combining two chemically different but pharmacologically
compatible molecules (the sulfomamide nucleus and the five member) heterocycles in one frame. Syn-
thesized compounds were tested in vitro for antibacterial and antifungal activity and some analogues
exhibited very promising results especially as antifungal agents.
In order to explain structure–activity relationships, conformational analysis was performed for active
and less active analogues using NMR spectroscopy and molecular modeling techniques. Furthermore,
molecular properties which can be further used as descriptors for SAR studies, were predicted for the syn-
thesized analogues. In general, antifungal activity seems to depend more on the triazol-3-thione moiety
rather than the different length of the alkyl chain substitutions.
? 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Bacterial infection such as food poisoning, rheumatic, salmonel-
losis and diarrhea are caused by multidrug-resistant Gram-positive
and Gram-negative pathogens. Principal players among these
problematic organisms are isolates of methicillin-resistant Staphy-
lococcus aureus, Staphylococcus pyogenes, Salmonella typhimurium
and Escherichia coli.1
In contrast to the large number of antibacterial antibiotics,
there are very few antifungal agents that can be used for life-
threatening fungal infections. These drugs are amphotericin B,2
5-fluorocytosine, azoles (such as fluconazole and itraconazole2)
and echinocandins (such as caspofungin and micafungin).3Because
of their high therapeutic index, azoles are first-line drugs for the
treatment of invasive fungal infections. Unfortunately, the broad
use of azoles has led to development of severe resistance,4,5which
significantly reduced the efficacy of them. This situation has led to
an ongoing search for new azoles. Several novel triazole antifungal
agents such as voriconazole,6posaconazole,7ravuconazole8and
albaconazole,9are marketed or are at the late stages of clinical
trials.
In general, azoles have been the leading agents used to treat
fungal infections of plants, animals and humans. These drugs act
by competitive inhibition of the lanosterol 14a-demethylase
(CYP51),10which is the key enzyme in sterol biosynthesis of fungi.
Selective inhibition of CYP51 would cause depletion of ergosterol
and accumulation of lanosterol and other 14-methyl sterols result-
ing in the growth inhibition of fungal cells.11Being an essential en-
zyme for the fungal life cycle, CYP51 is the target for azole
antifungals. Several in silico docking studies have been performed
to explore the mechanism of action of azole antifungals.12–18These
studies have been performed on homologous 3D models of CYP51
based on the crystal structures of known prokaryotic P450. The
majority of these studies indicate that apart from the coordination
of the heme iron from the azole ring nitrogen atom, crucial inter-
actions for azole binding into the putative active site of CYP51
are hydrophobic interactions including p–p stacking.13,18
0968-0896/$ - see front matter ? 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.031
⇑Corresponding author. Tel.: +30 210 7273869; fax: +30 210 7273872.
E-mail address: pzoump@eie.gr (P. Zoumpoulakis).
Bioorganic & Medicinal Chemistry 20 (2012) 1569–1583
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc

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Triazole antifungals which act predominantly by inhibition of
lanosterol to ergosterol through the cytochrome P450, are com-
pounds containing 1,2,4-triazole and 1,2,4-triazolone ring.19More-
over, our group has previously reported the significant antifungal
activity of a series of sulfonamide-1,2,4-triazole and 1,3,4-thiazole
derivatives against a series of micromycetes, compared to the com-
mercial fungicide bifonazole. These compounds have exerted sim-
ilar bactericidal effect to that of streptomycin and higher activity
than chloramphenicol against various bacteria.20
Prompted by these observations and in continuation of search
for bioactive molecules, we designed and synthesized a series of
novel sulfonamide-1,2,4-triazoles, sulfonamide-1,3,4-thiadiazoles
and sulfonamide-1,3,4-oxadiazoles. On our design we emphasized
on the strategy of combining two chemically different but pharma-
cologically compatible molecules, the sulfomamide nucleus and
the five member heterocycles in one frame.20–28Furthermore, the
designed molecules contain different aliphatic side chains (methyl,
ethyl, propyl, isopropyl, butyl and tert-butyl), aiming to increase
the hydrophobic interactions and to determine the optimum chain
length for increased activity.
2. Materials and methods
2.1. Synthesis
Melting points were taken in glass capillary tubes on a Haake
Bucher apparatus. IR spectra were recorded on a FT-IR Jasco spec-
trophotometer in solid phase KBr. All proton NMR spectra were re-
corded on a Varian 300 MHz spectrometer using deuterated
dimethylsulfoxide (DMSO-d6) and are reported in d units (ppm)
relative to tetramethylsilane (TMS). Thin layer chromatography
(TLC) was performed in E.Merck precoated silica gel plates (Kiesel-
gel 60F254). Visualization was obtained by exposure to iodine va-
pors and/or under UV light (254 nm). The elemental analysis (C,
H, N) of synthesized compounds are within the range of experi-
mental error (±0.4% of the calculated values). Accurate mass spec-
trometricdata were obtained
spectrometer (Thermo Scientific). Ionization was performed with
heated electrospray source operated in the positive mode (HESI+).
Full scan mass spectral data were acquired from m/z 100 to 1000 at
a resolving power of 60,000. The molecular formulas of the com-
pounds were confirmed by analysis with a mass spectrometer that
provided accurate mass data. In all cases, the protonated molecules
([M+H]+) and sodiated adducts ([M+Na]+) were detected with mass
error less than 1.5 ppm.
using LTQOrbitrap Velos™
2.1.1. General procedure for the preparation of the 1-[2-(N-dim
ethylsulfamoyl)-4,5-dimethoxy-phenylacetyl]-4-alkyl-thiosemi
carbazide/semicarbazide (2)
Equimolar quantities of hydrazide (1 mmol) and alkyl isothio-
cyanate (1 mmol) in 5 ml of absolute ethanol were refluxed on a
steam bath for 1 h. The formed precipitate was filtered and recrys-
tallized from the appropriate solvent. The following compounds
were prepared by an analogous procedure.
2.1.1.1.
tyl]-4-methyl-thiosemicarbazide (2a).
211 ?C (ethanol). IR cm?1: 3300 (NH), 1700 (CONH), 1560, 1520
(C@S), 1325 (S-Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 2.66 (s, 6H, N(CH3)2), 2.87 (d,
J = 4.35 Hz, 3H, CH3), 3.81 (s, 3H, CH3O), 3.83 (s, 3H, CH3O), 3.85
(s, 2H, CH2), 7.03 (s, 1H, ArH), 7.21 (s, 1H, ArH), 7.63 (m, 1H, NH),
9.32 (s, 1H, NH), 9.86 (s, 1H, NH).
Anal. Calcd for C14H22N4O5S2: C, 43.07; H, 5.64; N, 14,35. Found:
C, 43.11; H, 5.67; N, 14.31.
1-[2-(N-Dimethylsulfamoyl)4,5-dimethoxy-phenylace-
Yield: 95%. Mp 210–
2.1.1.2. 1-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-phenylace-
tyl]-4-ethyl-thiosemicarbazide (2b).
193 ?C (ethanol). IR cm?1: 3305, 3195 (NH), 1700 (CONH), 1550,
1520 (C@S), 1325 (S-Oantisym), 1150 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 1.06 (t, J = 7.19 Hz, 3H, CH3), 2.66 (s,
6H, N(CH3)2), 3.44 (pentaplet, J = 6.88 Hz, 2H, CH2), 3.81 (s, 3H,
CH3O), 3.83 (s, 3H, CH3O), 3.83 (s, 2H, CH2), 7.05 (s, 1H, ArH), 7.20
(s, 1H, ArH), 7.65 (m, 1H, NH), 9.27 (s, 1H, NH), 9.86 (s, 1H, NH).
Anal. Calcd for C15H24N4O5S2: C, 44.51; H, 5.60; N, 13.06. Found:
C, 44.55; H, 5.62; N, 13.10.
Yield: 90%. Mp 191–
2.1.1.3. 1-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-phenylace-
tyl]-4-propyl-thiosemicarbazide (2c).
236 ?C (ethanol). IR cm?1: 3325, 3180 (NH), 1720 (CONH), 1605
(C@N), 1555, 1520 (C@S), 1325 (S-Oantisym), 1145 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 0.80 (t, J = 7.42 Hz, 3H, CH3), 1.49
(hexaplet, J = 7.07 Hz, 2H, CH2), 2.64 (s, 6H, N(CH3)2), 3.34 (m,
2H, CH2), 3.79 (s, 3H, CH3O), 3.81 (s, 3H, CH3O), 3.81 (m, 2H,
CH2), 7.04 (s, 1H, ArH), 7.18 (s, 1H, ArH), 7.65 (s, 1H, NH), 9.27 (s,
1H, NH), 9.87 (s, 1H, NH).
Anal. Calcd for C16H26N4O5S2: C, 45.93; H, 6.22; N, 13.39. Found:
C, 45.90; H, 6.27; N, 13.36.
Yield: 87%. Mp 235–
2.1.1.4. 1-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-phenylace-
tyl]-4-isopropyl- thiosemicarbazide (2d).
214–216 ?C (ethanol). IR cm?1: 3335, 3255 (NH), 1690 (C0NH),
1550, 1510 (C@S), 1330 (S-Oantisym), 1140(S-Osym).
1H NMR (DMSO-d6) d(ppm): 1.07 (d, J = 6.59 Hz, 6H, 2CH3), 2.66
(s, 6H, N(CH3)2), 3.77 (s, 2H, CH2), 3.80 (s, 3H, CH3O), 3.82 (s, 3H,
CH3O), 4.37 (m, 1H, CH), 7.08 (s, 1H, ArH), 7.16 (s, 1H, ArH), 7.27
(m, 1H, NH), 9.21 (s, 1H, NH), 9.85 (s, 1H, NH).
Anal. Calcd for C16H26N4O5S2: C, 45.93; H, 6.22; N, 13.39. Found:
C, 45.97; H, 6.18; N, 13.42.
Yield: 74%. Mp
2.1.1.5. 1-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-phenylace-
tyl]-4-butyl-thiosemicarbazide (2e).
211 ?C (ethanol). IR cm?1: 3335, 3255 (NH), 1690 (C0NH), 1550,
1510 (C@S), 1330 (S-Oantisym), 1140(S-Osym).
1H NMR (DMSO-d6) d(ppm): 0.84 (t, J = 6.78 Hz, 3H, CH3), 1.22
(hexaplet, J = 7.22 Hz, 2H, CH2), 1.45 (pentaplet, J = 6.51 Hz, 2H,
CH2), 2.64 (s, 6H, N(CH3)2), 3.37 (m, 2H, CH2), 3.81 (s, 3H, CH3O),
3.81 (s, 3H, CH3O), 3.81 (m, 2H, CH2), 7.04 (s, 1H, ArH), 7.18 (s,
1H, ArH), 7.60 (s, 1H, NH), 9.25 (s, 1H, NH), 9.86 (s, 1H, NH).
Anal. Calcd for C17H28N4O5S2: C, 47.22; H, 6.48; N, 12.96. Found:
C, 47.26; H, 6.44; N, 13.00.
Yield: 89%. Mp 210–
2.1.1.6. 1-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-phenylace-
tyl]-4-tert-butyl-thiosemicarbazide
187–188 ?C (ethanol). IR cm?1: 3355, 3315, 3240 (NH), 1670
(C0NH), 1550, 1520 (C@S), 1335 (S-Oantisym), 1150 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 1.42 (s, 9H, 3CH3), 2.65 (s, 6H,
N(CH3)2), 3.75 (s, 2H, CH2), 3.80 (s, 3H, CH3O), 3.82 (s, 3H, CH3O),
6.89 (s, 1H, NH), 7.08 (s, 1H, ArH), 7.15 (s, 1H, ArH), 9.04 (s, 1H,
NH), 9.83 (s, 1H, NH).
Anal. Calcd for C17H28N4O5S2: C, 47.22; H, 6.48; N, 12.96. Found:
C, 47.27; H, 6.52; N, 12.89.
(2f).
Yield: 74%.Mp
2.1.1.7. 1-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-phenylace-
tyl]-4-ethyl-semicarbazide (2g).
(ethanol). IR cm?1: 3350, 3270 (NH), 1685 (C0NH), 1665 (NHCO
NH), 1325 (S-Oantisym), 1145 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 0.97 (t, J = 7.20 Hz, 3H, CH3), 2.64
(s, 6H, N(CH3)2), 3.00 (m, 2H, CH2), 3.79 (s, 3H, CH3O), 3.80 (s,
2H, CH2), 3.82 (s, 3H, CH3O), 6.22 (m, 1H, NH), 7.08 (s, 1H, ArH),
7.19 (s, 1H, ArH), 7.74 (s, 1H, NH), 9.59 (s, 1H, NH).
Anal. Calcd for C15H24N4O6S: C, 46.39; H, 6.18; N, 14.43. Found:
C, 46.45; H, 6.23; N, 14.38.
Yield: 74%. Mp 133–135 ?C
1570
P. Zoumpoulakis et al./Bioorg. Med. Chem. 20 (2012) 1569–1583

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2.1.1.8. 1-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-phenylace-
tyl]-4-propyl-semicarbazide (2h).
(ethanol). IR cm?1: 3350 (NH), 1685 (C0NH), 1660 (NHCONH),
1330 (S-Oantisym), 1145(S-Osym).
1H NMR (DMSO-d6) d(ppm): 0.80 (t, J = 7.30 Hz, 3H, CH3), 1.60
(hexaplet, J = 6.90 Hz, 2H, CH2), 2.62 (s, 6H, N(CH3)2), 2.94 (t,
J = 6.40 Hz, 2H, CH2), 3.79 (s, 3H, CH3O), 3.81 (s, 3H, CH3O), 3.80
(s, 2H, CH2), 6.23 (m, 1H, NH), 7.08 (s, 1H, ArH), 7.21 (s, 1H, ArH),
7.72 (s, 1H, NH), 9.59 (s, 1H, NH).
Anal. Calcd for C16H26N4O6S: C, 47.76; H, 6.46; N, 13.93. Found:
C, 47.81; H, 6.41; N, 13.88.
Yield: 70%. Mp 124–126 ?C
2.1.1.9. 1-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-phenylace-
tyl]-4-isopropyl-semicarbazide (2i).
179 ?C (ethanol). IR cm?1: 3405, 3370, 3215, 3090 (NH), 1695
(CONH), 1670 (NHCONH), 1330 (S-Oantisym), 1140(S-Osym).
1H NMR (DMSO-d6) d(ppm): 1.00 (d, J = 6.50 Hz, 6H, 2CH3), 2.64
(s, 6H, N(CH3)2), 3.68 (m, 1H, CH), 3.77 (s, 2H, CH2), 3.79 (s, 3H,
CH3O), 3.82 (s, 3H, CH3O), 6.01 (m,1H, NH), 7.09 (s, 1H, ArH),
7.18 (s, 1H, ArH), 7.65 (s, 1H, NH), 9.59 (s, 1H, NH).
Anal. Calcd for C16H26N4O6S: C, 47.76; H, 6.46; N, 13.93. Found:
C, 47.71; H, 6.49; N, 13.97.
Yield: 76%. Mp 178–
2.1.1.10.
acetyl]-4-butyl-semicarbazide (2j).
130 ?C (ethanol). IR cm?1: 3355 (NH), 1685 (C0NH), 1660
(NHCONH), 1330 (S-Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 0.83 (t, J = 7.0 Hz, 3H, CH3), 1.22
(m, 2H, CH2), 1.31 (m, 2H, CH2), 2.64 (s, 6H, N(CH3)2), 2.95 (m,
2H, CH2), 3.71 (s, 2H, CH2), 3.79 (s, 3H, CH3O), 3.80 (s, 3H, CH3O),
6.22 (m, 1H, NH), 7.08 (s, 1H, ArH), 7.20 (s, 1H, ArH), 7.72 (s, 1H,
NH), 9.59 (s, 1H, NH).
Anal. Calcd for C17H28N4O6S: C, 49.03; H, 6.73; N, 13.46. Found:
C, 49.07; H, 6.69; N, 13.51.
1-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-phenyl-
Yield: 77%. Mp 128–
2.1.1.11.1-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-phenylace-
tyl]-4-tert-butyl-semicarbazide (2k).
127 ?C (ethanol). IR cm?1: 3530, 3350 (NH), 1685 (C0NH), 1665
(NHCONH), 1325 (S-Oantisym), 1145 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 1.21 (s, 9H, 3CH3), 2.63 (s, 6H,
N(CH3)2), 3.76 (s, 2H, CH2), 3.81 (s, 3H, CH3O), 3.83 (s, 3H, CH3O),
5.90 (s, 1H, NH), 7.13 (s, 1H, ArH), 7.19 (s, 1H, ArH), 7.50 (s, 1H,
NH), 9.58 (s, 1H, NH).
Anal. Calcd for C17H28N4O6S: C, 49.03; H, 6.73; N, 13.46. Found:
C, 49.01; H, 6.76; N, 13.50.
Yield: 81%. Mp 125–
2.1.2. General procedure for the preparation of the 5-[2-(N-
dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-4-alkyl-s-triazole-
3-thiones/3-ones (3)
A suspension of thiosemicarbazide/semicarbazide (1 mmol) in
sodium hydroxide solution (5%, 5 ml) was refluxed for 1 h. The
reaction mixture was allowed to cool and then adjusted to pH 6
with 10% hydrochloric acid. The formed precipitate was filtered,
washed with water, dried and recrystallized from the appropriate
solvent.
The following compounds were prepared by an analogous
procedure.
2.1.2.1.
methyl-s-triazole-3-thione (3a).
(methanol). IR cm?1: 3100, 3050 (NH), 1600 (C@N), 1575, 1520
(C@S), 1330 (S-Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 2.59 (s, 6H, N(CH3)2), 3.41 (s, 3H,
NCH3), 3.77 (s, 3H, CH3O), 3.81 (s, 3H, CH3O), 4.28 (s, 2H, CH2),
7.02 (s, 1H, ArH), 7.22 (s, 1H, ArH), 13.44 (s, 1H, NH).
5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-4-
Yield: 76%. Mp 194–196 ?C
Anal. Calcd for C14H20N4O4S2: C, 45.16; H, 5.37; N, 15.05. Found:
C, 45.21; H, 5.41; N, 15.00.
2.1.2.2.
ethyl-s-triazole-3-thione (3b).
(methanol). IR cm?1: 3100, 3050 (NH), 1600 (C@N), 1570, 1510
(C@S), 1330 (S-Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 1.18 (t, J = 7.12 Hz, 3H, CH3), 2.61
(s, 6H, N(CH3)2), 3.79 (s, 3H, CH3O), 3.83 (s, 3H, CH3O), 3.98 (q,
J = 7.05 Hz, 2H, NCH2), 4.35 (s, 2H, CH2), 7.07 (s, 1H, ArH), 7.24 (s,
1H, ArH), 13.43 (s, 1H, NH).
Anal. Calcd for C16H22N4O4S2: C, 46.63; H, 5.70; N, 14.50. Found:
C, 46.58; H, 5.73; N, 14.54.
MS (HESI+). (m/z) 387.1159 (100%, [M+H]+), 409.0979 (20%),
[M+Na]+).
5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-4-
Yield: 86%. Mp 193–194 ?C
2.1.2.3.
propyl-s-triazole-3-thione (3c).
(methanol). IR cm?1: 3100, 3050 (NH), 1605 (C@N), 1580, 1510
(C@S), 1330 (S-Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 0.87 (t, J = 7.45 Hz, 3H, CH3), 1.60
(hexaplet, J = 7.73 Hz, 2H, CH2), 2.59 (s, 6H, N(CH3)2), 3.77 (s, 3H,
CH3O), 3.81 (s, 3H, CH3O), 3.87 (t, J = 7.88 Hz, 2H, NCH2), 4.33 (s,
2H, CH2), 7.05 (s, 1H, ArH), 7.22 (s, 1H, ArH), 13.43 (s, 1H, NH).
Anal. Calcd for C16H24N4O4S2: C, 48.00; H, 6.00; N, 14.00. Found:
C, 48.07; H, 5.96; N, 14.03.
5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-4-
Yield: 96%. Mp 189–190 ?C
2.1.2.4.
isopropyl-s-triazole-3-thione (3d).
230 ?C (methanol–chloroform). IR cm?1: 3295, 3050 (NH), 1600
(C@N), 1560, 1520 (C@S), 1330 (S-Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 1.46 (d, J = 7.02 Hz, 6H, 2CH3), 2.59
(s, 6H, N(CH3)2), 3.78 (s, 3H, CH3O), 3.81 (s, 3H, CH3O), 4.38 (s, 2H,
CH2), 4.91 (m, 1H, NCH), 7.07 (s, 1H, ArH), 7.21 (s, 1H, ArH), 13.38
(s, 1H, NH).
Anal. Calcd for C16H24N4O4S2: C, 48.00; H, 6.00; N, 14.00. Found:
C, 47.96; H, 6.04; N, 14.05.
MS (HESI+). (m/z) 401.1315 (100%, [M+H]+), 423.1135 (20%),
[M+Na]+).
5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-4-
Yield: 78%. Mp 228–
2.1.2.5.
butyl-s-triazole-3-thione. (3e).
(methanol). IR cm?1: 3100, 3050 (NH), 1605 (C@N), 1575, 1510
(C@S), 1330 (S-Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 0.87 (t, J = 7.38 Hz, 3H, CH3), 1.28
(hexaplet, J = 7.35 Hz, 2H, CH2), 1.54 (m, 2H, CH2), 2.59 (s, 6H,
N(CH3)2), 3.77 (s, 3H, CH3O), 3.81 (s, 3H, CH3O), 3.91 (t,
J = 7.51 Hz, 2H, NCH2), 4.33 (s, 2H, CH2), 7.05 (s, 1H, ArH), 7.22 (s,
1H, ArH), 13.41 (s, 1H, NH).
Anal. Calcd for C17H26N4O4S2: C, 49.27; H, 6.28; N, 13.52. Found:
C, 49.33; H, 6.23; N, 13.55.
5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-4-
Yield: 59%. Mp 190–191 ?C
2.1.2.6.
tert-butyl-s-triazole-3-thione (3f).
196 ?C (methanol–chloroform). IR cm?1: 3355, 3320 (NH), 1600
(C@N), 1550, 1520 (C@S), 1335 (S-Oantisym), 1150 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 1.84 (s, 9H, 3CH3), 2.59 (s, 6H,
N(CH3)2), 3.77 (s, 3H, CH3O), 3.82 (s, 3H, CH3O), 4.48 (s, 2H, CH2),
6.91 (s, 1H, ArH), 7.23 (s, 1H, ArH), 13.34 (s, 1H, NH).
Anal. Calcd for C17H26N4O4S2: C, 49.27; H, 6.28; N, 13.52. Found:
C, 49.23; H, 6.33; N, 13.48.
5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-4-
Yield: 53%. Mp 194–
2.1.2.7.
ethyl-s-triazole-3-one
(methanol). IR cm?1: 3365, 3170 (NH), 1695 (NHCO), 1600
(C@N), 1330 (S-Oantisym), 1140 (S-Osym).
5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-4-
(3g).
Yield: 65%.Mp192–193 ?C
P. Zoumpoulakis et al./Bioorg. Med. Chem. 20 (2012) 1569–1583
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1H NMR (DMSO-d6) d(ppm): 1.05 (t, J = 7.11 Hz, 3H, CH3), 2.62
(s, 6H, N(CH3)2), 3.52 (q, J = 7.08 Hz, 2H, NCH2), 3.77 (s, 3H,
CH3O), 3.81 (s, 3H, CH3O), 4.19 (s, 2H, CH2), 6.96 (s, 1H, ArH),
7.24 (s, 1H, ArH), 11.37 (s, 1H, NH).
Anal. Calcd for C15H22N4O5S:C, 48.64; H, 5.94; N, 15.13. Found:
C, 48.59; H, 5.99; N, 15.09.
2.1.2.8.
propyl-s-triazole-3-one (3h).
(methanol). IR cm?1: 3170 (NH), 1710 (NHCO), 1600 (C@N), 1335
(S-Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 0.80 (t, J = 7.51 Hz, 3H, CH3), 1.46
(hexaplet, J = 7.45 Hz, 2H, CH2), 2.61 (s, 6H, N(CH3)2), 3.45 (t,
J = 7.32 Hz, 2H, NCH2), 3.76 (s, 3H, CH3O), 3.81 (s, 3H, CH3O), 4.19
(s, 2H, CH2), 6.96 (s, 1H, ArH), 7.23 (s, 1H, ArH), 11.37 (s, 1H, NH).
Anal. Calcd for C16H24N4O5S:C, 50.00; H, 6.25; N, 14.58. Found:
C, 50.04; H, 6.31; N, 14.54.
5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-4-
Yield: 26%. Mp 158–159 ?C
2.1.2.9.
isopropyl-s-triazole-3-one (3i).
(methanol). IR cm?1: 3165 (NH), 1690 (NHCO), 1600 (C@N), 1330
(S-Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 1.30 (d, J = 6.83 Hz, 6H, 2CH3), 2.62
(s, 6H, N(CH3)2), 3.76 (s, 3H, CH3O), 3.81 (s, 3H, CH3O), 4.08 (m, 1H,
NCH), 4.18 (s, 2H, CH2), 6.91 (s, 1H, ArH), 7.24 (s, 1H, ArH), 11.30 (s,
1H, NH).
Anal. Calcd for C16H24N4O5S:C, 50.00; H, 6.25; N, 14.58. Found:
C, 49.95; H, 6.30; N, 14.63.
5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-4-
Yield: 40%. Mp 223–225 ?C
2.1.2.10. 5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-4-
butyl-s-triazole-3-one (3j).
thanol). IR cm?1: 3175 (NH), 1715 (C@O), 1600 (C@N), 1335 (S-
Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 0.82 (t, J = 7.34 Hz, 3H, CH3), 1.20
(hexaplet, J = 7.61 Hz, 2H, CH2), 1.38 (m, 2H, CH2), 2.61 (s, 6H,
N(CH3)2), 3.48 (t, J = 7.36 Hz, 2H, NCH2), 3.76 (s, 3H, CH3O), 3.80
(s, 3H, CH3O), 4.19 (s, 2H, CH2), 6.96 (s, 1H, ArH), 7.23 (s, 1H,
ArH), 11.37 (s, 1H, NH).
Anal. Calcd for C17H26N4O5S:C, 51.25; H, 5.32; N, 14.07. Found:
C, 51.31; H, 5.25; N, 14.01.
Yield: 28%. Mp 167–169 ?C (me
2.1.2.11. 5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-4-
tert-butyl-s-triazole-3-one (3k).
(methanol). IR cm?1: 3540, 3350 (NH), 1685, 1665 (C@O), 1605
(C@N), 1325 (S-Oantisym), 1145 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 1.21 (s, 9H, 3CH3), 2.64 (s, 6H,
N(CH3)2), 3.76 (s, 2H, CH2), 3.79 (s, 3H, CH3O), 3.83 (s, 3H, CH3O),
5.91 (s, 1H, NH), 7.14 (s, 1H, ArH), 7.19 (s, 1H, ArH), 7.51 (s, 1H,
NH), 9.59 (s, 1H, NH).
Anal. Calcd for C17H26N4O5S: C, 51.25; H, 5.32; N, 14.07. Found:
C, 51.20; H, 5.37; N, 14.11.
Yield: 63%. Mp 137–138 ?C
2.1.2.12. General procedure for the preparation of N-{5-[2-(N-
dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-1,3,4-thiadiazol-2-
yl}-N-alkylamines (4).
A mixture of the 1-[2-(N-sulfamoyl)-
4,5-dimethoxy-phenylacetyl]-4-thiosemicarbazide
cold concentrated sulphuric acid (5 ml) was stirred for 15 min.
The resulting solution was then allowed to reach ambient temper-
ature, left stirring for 30 min and poured cautiously into ice cold
water. The reaction mixture was alkalified to pH 8 with aqueous
ammonia and the precipitated product was filtered, washed with
water and recrystallized from the appropriate solvent.
The following compounds were prepared by an analogous
procedure.
(1 mmol)in
2.1.2.13.
zyl]-1,3,4-thiadiazol-2-yl}-N-methylamine (4a).
Mp 190–191 ?C (methanol–dichloromethane). IR cm?1: 3280
(NH), 1600 (C@N), 1330 (S-Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 2.62 (s, 6H, N(CH3)2), 2.78 (d,
J = 4.77 Hz, 3H, CH3), 3.78 (s, 3H, CH3O), 3.80 (s, 3H, CH3O), 4.44
(s, 2H, CH2), 7.04 (s, 1H, ArH), 7.21 (s, 1H, ArH), 7.43 (q,
J = 4.77 Hz, 1H, NH).
Anal. Calcd for C14H20N4O4S2:C, 45.16; H, 5.37; N, 15.05. Found:
C, 45.12; H, 5.30; N, 15.09.
N-{5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-ben-
Yield: 91%.
2.1.2.14.
zyl]-1,3,4-thiadiazol-2-yl}-N-ethylamine
Mp 193–194 ?C (methanol–chloroform). IR cm?1: 3335 (NH),
1600 (C@N), 1330 (S-Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 1.10 (t, J = 7.19 Hz, 3H, CH3), 2.62
(s, 6H, N(CH3)2), 3.18 (m, 2H, CH2), 3.78 (s, 3H, CH3O), 3.80 (s,
3H, CH3O), 3.98 (q, J = 7.05 Hz, 2H, NCH2), 4.43 (s, 2H, CH2), 7.04
(s, 1H, ArH), 7.21 (s, 1H, ArH), 7.49 (m, 1H, NH).
Anal. Calcd for C15H22N4O4S2:C, 46.63; H, 5.70; N, 14.50. Found:
C, 46.66; H, 5.66; N, 14.46.
N-{5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-ben-
(4b).
Yield:82%.
2.1.2.15.
zyl]-1,3,4-thiadiazol-2-yl}-N-propylamine (4c).
Mp 170–171 ?C (methanol–chloroform). IR cm?1: 3340 (NH),
1600 (C@N), 1325 (S-Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 0.85 (t, J = 7.46 Hz, 3H, CH3), 1.49
(hexaplet, J = 7.18 Hz, 2H, CH2), 2.62 (s, 6H, N(CH3)2), 3.11 (q,
J = 5.75 Hz, 2H, CH2), 3.78 (s, 3H, CH3O), 3.80 (s, 3H, CH3O), 4.43
(s, 2H, CH2), 7.05 (s, 1H, ArH), 7.21 (s, 1H, ArH), 7.52 (t,
J = 5.37 Hz, 1H, NH).
Anal. Calcd for C16H24N4O4S2: C, 48.00; H, 6.00; N, 14.00. Found:
C, 47.97; H, 6.05; N, 14.05.
N-{5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-ben-
Yield: 70%.
2.1.2.16.
zyl]-1,3,4-thiadiazol-2-yl}-N-isopropylamine
63%. Mp 192–193 ?C (methanol). IR cm?1: 3335 (NH), 1600
(C@N), 1325 (S-Oantisym), 1135 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 1.11 (d, J = 6.43 Hz, 6H, 2CH3), 2.62
(s, 6H, N(CH3)2), 3.69 (m, 1H, CH), 3.79 (s, 3H, CH3O), 3.81 (s, 3H,
CH3O), 4.43 (s, 2H, CH2), 7.06 (s, 1H, ArH), 7.22 (s, 1H, ArH), 7.40
(d, J = 7.16 Hz, 1H, NH).
Anal. Calcd for C16H24N4O4S2:C, 48.00; H, 6.00; N, 14.00. Found:
C, 48.05; H, 5.94; N, 13.97.
N-{5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-ben-
(4d).
Yield:
2.1.2.17.
zyl]-1,3,4-thiadiazol-2-yl}-N-butylamine
Mp 174–175 ?C (methanol–chloroform). IR cm?1: 3340 (NH),
1600 (C@N), 1330 (S-Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 0.85 (t, J = 7.23 Hz, 3H, CH3), 1.27
(hexaplet, J = 7.63 Hz, 2H, CH2), 1.47 (pentaplet, J = 7.76 Hz, 2H,
CH2), 2.62 (s, 6H, N(CH3)2), 3.15 (tetraplet, J = 5.74 Hz, 2H, CH2),
3.78 (s, 3H, CH3O), 3.80 (s, 3H, CH3O), 4.43 (s, 2H, CH2), 7.05 (s,
1H, ArH), 7.21 (s, 1H, ArH), 7.50 (t, J = 5.47 Hz, 1H, NH).
Anal. Calcd for C17H26N4O4S2:C, 49.27; H, 6.28; N, 13.52. Found:
C, 49.22; H, 6.32; N, 13.49.
MS (HESI+). (m/z) 415.1473 (100%, [M+H]+), 437.1292 (20%),
[M+Na]+).
N-{5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-ben-
(4e).
Yield:53%.
2.1.3. General procedure for the preparation of N-{5-[2-(N-
dimethylsulfa-moyl)-4,5-dimethoxy-benzyl]-1,3,4-oxadiazol-2-
yl}-N-alkylamines (5)
To a stirred, cooled (0–5 ?C) solution of the respective thiosem-
icarbazide derivative (1 mmol) in ethanol (6 ml) 2 N sodium
hydroxide was added until the solution acquired pH 9. Iodine in
potassium iodide solution (5%) was then added dropwise with stir-
1572
P. Zoumpoulakis et al./Bioorg. Med. Chem. 20 (2012) 1569–1583

Page 5

ring at room temperature until the yellow colour of iodine per-
sisted. The solvent was removed under reduced pressure and the
residue was crystallized from the proper solvent to give the corre-
sponding oxadiazole derivative.
The following compounds were prepared by an analogous
procedure.
2.1.3.1. N-{5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-
1,3,4-oxadiazol-2-yl}-N-methylamine (5a).
201–202 ?C (ethanol–hexane). IR cm?1: 3400 (NH), 1600 (C@N),
1330 (S-Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 2.62 (s, 6H, N(CH3)2), 3.16 (s, 3H,
CH3), 3.67 (s, 3H, CH3O), 3.79 (s, 3H, CH3O), 4.20 (s, 2H, CH2),
6.73 (s, 1H, ArH), 7.25 (s, 1H, ArH).
Anal. Calcd for C14H20N4O4S5:C, 47.19; H, 3.61; N, 15.73. Found:
C, 47.24; H, 5.64; N, 15.69.
Yield: 60%. Mp
2.1.3.2. N-{5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-
1,3,4-oxadiazol-2-yl}-N-ethylamine
183–185 ?C (ethanol). IR cm?1: 3445 (NH), 1600 (C@N), 1330 (S-
Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 1.06 (t, J = 7.12 Hz, 3H, CH3), 2.60
(s, 6H, N(CH3)2), 3.74 (s, 3H, CH3O), 3.80 (s, 3H, CH3O), 3.86 (q,
J = 7.14 Hz, 2H, CH2), 4.29 (s, 2H, CH2), 6.96 (s, 1H, ArH), 7.23 (s,
1H, ArH).
Anal. Calcd for C15H22N4O5S:C, 48.64; H, 5.95; N, 15.13. Found:
C, 48.60; H, 5.91; N, 15.17.
(5b).
Yield:81%.Mp
2.1.3.3. N-{5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-
1,3,4-oxadiazol-2-yl}-N-propylamine (5c).
177–179 ?C (ethanol). IR cm?1: 3365 (NH), 1605 (C@N), 1330 (S-
Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 0. 37 (t, J = 7.41 Hz, 3H, CH3), 1.38
(hexaplet, J = 7.57 Hz, 2H, CH2), 2.62 (s, 6H, N(CH3)2), 3.64 (m, 2H,
CH2),3.67 (s, 3H, CH3O), 3.79 (s, 3H, CH3O), 4.24 (s, 2H, CH2), 6.82
(s, 1H, ArH), 7.24 (s, 1H, ArH).
Anal. Calcd for C16H24N4O5S:C, 50.00; H, 6.25; N, 14.58. Found:
C, 50.02; H, 6.21; N, 15.01.
Yield: 78%. Mp
2.1.3.4. N-{5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-
1,3,4-oxadiazol-2-yl}-N-butylamine (5d).
128–129 ?C (ethanol–n-hexane). IR cm?1: 3450 (NH), 1600
(C@N), 1335 (S-Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 0.78 (t, J = 7.20 Hz, 3H, CH3), 1.17
(hexaplet, J = 7.31 Hz, 2H, CH2), 1.33 (m, 2H, CH2), 2.61 (s, 6H,
N(CH3)2), 3.68 (s, 3H, CH3O), 3.70 (m, 2H, CH2), 3.79 (s, 3H,
CH3O), 4.24 (s, 2H, CH2), 6.83 (s, 1H, ArH), 7.24 (s, 1H, ArH).
Anal. Calcd for C17H26N4O5S:C, 51.25; H, 6.53; N, 14.07. Found:
C, 51.29; H, 6.49; N, 14.10.
Yield:75%.Mp
2.1.3.5. N-{5-[2-(N-Dimethylsulfamoyl)-4,5-dimethoxy-benzyl]-
1,3,4-oxadiazol-2-yl}-N-tert-butylamine
Mp 221–224 ?C (ethanol–n-hexane). IR cm?1: 3420 (NH), 1600
(C@N), 1330 (S-Oantisym), 1140 (S-Osym).
1H NMR (DMSO-d6) d(ppm): 1.84 (s, 9H, 3CH3), 2.59 (s, 6H,
N(CH3)2), 3.77 (s, 3H, CH3O), 3.82 (s, 3H, CH3O), 4.48 (s, 2H, CH2),
6.91 (s, 1H, ArH), 7.23 (s, 1H, ArH).
Anal. Calcd for C17H26N4O5S:C, 51.25; H, 6.53; N, 14.07. Found:
C, 51.21; H, 6.56; N, 14.03.
(5e).
Yield:95%.
2.2. Pharmacology
2.2.1. Antibacterial activity
The following Gram-negative bacteria were used: Escherichia
coli (ATCC 35210), Pseudomons aeruginosa (ATCC 27853), Salmo-
nella typhimurium (ATCC 13311), Enterobacter cloacae (human iso-
late) and the following Gram-positive bacteria: Bacillus cereus
(clinical isolate), Micrococcus flavus (ATCC 10240), and Listeria mon-
ocytogenes (NCTC 7973), Staphylococcus aureus (ATCC 6538). The
organisms were obtained from the Mycological Laboratory,
Department of Plant Physiology, Institute for Biological Research
‘Siniša Stankovic ´’, Belgrade, Serbia.
The antibacterial activity of tested compounds against human
pathogenic bacteria was determined using the microdilution
method.29
The bacterial suspensions were adjusted with sterile saline to a
concentration of 1.0 ? 105CFU/ml. The inocula were prepared dai-
ly and stored at +4 ?C until use. Dilutions of the inocula were cul-
tured on solid medium to verify the absence of contamination and
to check the validity of the inoculum.
2.2.2. Microdilution test
The minimum inhibitory and bactericidal concentrations (MICs
and MBCs) were determined using 96-well microtitre plates. The
bacterialsuspensionwasadjustedwithsterilesalinetoaconcentra-
tion of 1.0 ? 105cfu/ml. Compounds to be investigated were dis-
solved in broth LB medium (100 ll) with bacterial inoculum
(1.0 ? 104cfu per well) to achieve the wanted concentrations
(1 mg/ml). The microplates were incubated for 24 h at 48 ?C. The
lowest concentrations without visible growth (at the binocular
microscope) were defined as concentrations that completely inhib-
ited bacterial growth (MICs). The MBCs were determined by serial
sub-cultivation of 2 ll into microtitre plates containing 100 ll of
brothperwellandfurtherincubationfor72 h.Thelowestconcentra-
tionwithnovisiblegrowthwasdefinedastheMBC,indicating99.5%
killing of the original inoculum. The optical density of eachwell 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.Streptomycinandampicillinwereusedasapositivecontrol
(1 mg/ml DMSO). Two replicates were done for each compound.
2.2.3. Antifungal activity
For the antifungal bioassays, following fungi were used: Asper-
gillus flavus (ATCC 9643), Aspergillus fumigatus (human isolate),
Aspergillus niger (ATCC 6275), Aspergillus versicolor (ATCC 11730),
Aspergillus ochraceus (ATCC 12066), Penicillium funiculosum (ATCC
36839), Penicillium ochrochloron (ATCC 9112) and Trichoderma vir-
ide (IAM 5061).
The micromycetes were maintained on malt agar and the cul-
tures stored at 4 ?C and sub-cultured once a month.1In order to
investigate the antifungal activity of the extracts, a modified mic-
rodilution technique was used.29The fungal spores were washed
from the surface of agar plates with sterile 0.85% saline containing
0.1% Tween 80 (v/v). The spore suspension was adjusted with ster-
ile saline to a concentration of approximately 1.0 ? 105in a final
volume of 100 ll per well. The inocula were stored at 4 ?C for fur-
ther 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 compounds 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.
The fungicidal concentrations (MFCs) were determined by serial
subcultivation of a 2 ll into microtitre plates containing 100 ll of
broth per well and further incubation 5 days at 28 ?C. The lowest
concentration with no visible growth was defined as MFC indicat-
ing 99.5% killing of the original inoculum. DMSO was used as a neg-
ative control, commercial fungicides, bifonazole and ketoconazole,
were used as positive controls (1–5000 lg/ml).
P. Zoumpoulakis et al./Bioorg. Med. Chem. 20 (2012) 1569–1583
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Page 6

2.3. NMR spectroscopy
DMSO-d6 and ultra precision NMR tubes (Norell 509-UP-7,
5 mm) were used for the NMR experiments. Compounds were dis-
solved in DMSO-d6to a final concentration of 10 mM and a series of
experiments were performed using Varian 600 MHz spectrometer
at 300 K temperature. All data are collected using pulse sequences
and phase-cycling routines provided in the Varian libraries. The1H
spectral width was set to 8500 Hz at 600 MHz. Typically the 2D
NOESY spectrum was acquired with 4096 data points in t2dimen-
sion, 64 scans, 256 increments in t1 dimension, 150 ms mixing
time and a relaxation delay of 1 s. Data processing including
apodization with cosine square Bell function, Fourier transforma-
tion and phasing, were performed using Varian VNMR software.
Intramolecular distances were calculated from cross-peak volumes
in NOESY while a tolerance of ±10% of the calculated value was ap-
plied to produce the upper and the lower limit distance
constraints.
2.4. Molecular modeling
Computer calculations were performed using Schrodinger Suite
2011 molecular modeling package. More specifically, Molecular
CH3O
CH3O
CONHNH2
SO2
N
CH3
CH3
R-N=C=X
X=O, S
CH3O
CH3O
CONHNHCNHR
SO2
N
CH3
CH3
O
CH3O
CH3O SO2
N
CH3
CH3
N
NH
N
X
R
X=O, S
5% NaOH
HCl
CH3O
CH3O
SO2
N
CH3
CH3
S
NN
N
H
R
H2SO4
0 oC
I2/NaOH
CH3O
CH3O SO2
N
CH3
CH3
O
N
N
N
H
R
R: -CH3, -CH2CH3, -CH2CH2CH3, -CH
-CH2CH2CH2CH3, -C
CH3
CH3
CH3
CH3
CH3
21
3
4
5
Scheme 1. Synthetic pathway for the preparation of the title compounds.
Table 1
Chemical structures of synthesized molecules
O
O
SO2
N
H3C CH3
N
NH
N
X
R
H3C
H3C
SO2
N
H3CCH3
O
O
H3C
H3C
S
N
N
N
H
R
SO2
N
H3CCH3
O
O
H3C
H3C
O
N
N
N
H
R
X = S X = O
–CH3
–CH2CH3
–CH2CH2CH3
3a
3b
3c
—
3g
3h
4a
4b
4c
5a
5b
5c
CH
CH3
CH3
3d
3i
4d
—
–CH2CH2CH2CH3
3e
3j
4e
5d
CCH3
CH3
CH3
3f
3k
—
5e
1574
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Table 2
Antibacterial activity of compounds 3a–ktested by microdilution method (MIC and MBC in lmol)
Bacteria Activity
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
StrAmp
Staphylococcus aureus
MIC
MBC
0.27
0.54
0.26
0.52
0.50
0.50
0.38
0.38
0.24
0.48
0.24
0.42
0.27
0.54
0.26
0.39
0.39
0.39
0.25
0.50
0.39
0.39
0.04
0.09
0.25
0.37
Bacillus cereus
MIC
MBC
0.27
0.54
0.52
0.52
0.25
0.50
0.25
0.50
0.24
0.48
0.24
0.48
0.27
0.81
0.26
0.65
0.26
0.78
0.25
0.76
0.25
0.78
0.09
0.17
0.25
0.37
Micrococcus flavus
MIC
MBC
0.27
0.54
0.26
0.52
0.25
0.50
0.63
0.63
0.24
0.48
0.24
0.42
0.40
0.40
0.65
0.65
0.39
0.39
0.63
0.63
0.39
0.39
0.17
0.34
0.25
0.37
Listeria monocytogenes
MIC
MBC
0.27
0.27
0.26
0.52
0.25
0.25
0.63
0.63
0.24
0.24
0.24
0.42
0.40
0.54
0.65
0.78
0.39
0.39
0.38
0.38
0.39
0.39
0.17
0.34
0.37
0.49
Pseudomonas aeruginosa
MIC
MBC
0.54
0.54
0.26
0.26
0.50
0.50
0.63
0.63
0.48
0.48
0.24
0.42
0.40
0.54
0.65
0.78
0.39
0.39
0.38
0.38
0.39
0.39
0.17
0.34
0.74
1.24
Enterobacter cloacae
MIC
MBC
0.27
0.27
0.26
0.52
0.25
0.25
0.63
0.63
0.24
0.24
0.24
0.42
0.54
0.54
0.65
0.78
0.39
0.39
0.50
0.76
0.25
0.25
0.17
0.34
0.37
0.49
Salmonella typhimurium
MIC
MBC
0.27
0.27
0.26
0.26
0.50
0.50
0.63
0.63
0.24
0.24
0.24
0.42
0.40
0.54
0.65
0.65
0.39
0.39
0.50
0.76
0.25
0.25
0.17
0.34
0.25
0.49
Escherichia coli
MIC
MBC
0.54
0.54
0.26
0.52
0.50
0.50
0.63
0.63
0.48
0.48
0.24
0.42
0.27
0.27
0.65
0.65
0.39
0.39
0.13
0.25
0.13
0.25
0.26
0.52
0.37
0.74
Table 3
Antifungal activity of compounds 3a–ktested by microdilution method (MIC and MFC in lmol)
FungiActivity
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
Bif Keto
Penicillium funiculosum
MIC
MFC
0.14
0.27
0.13
0.26
0.13
0.25
0.13
0.25
0.12
0.24
0.12
0.24
0.14
0.27
0.13
0.26
0.26
0.26
0.13
0.25
0.13
0.25
0.64
0.8
0.38
0.95
Penicillium ochrochloron
MIC
MFC
0.14
0.27
0.13
0.26
0.13
0.25
0.13
0.38
0.12
0.48
0.12
0.36
0.14
0.27
0.13
0.39
0.13
0.39
0.13
0.38
0.25
0.38
0.48
0.64
3.8
3.8
Trichoderma viride
MIC
MFC
0.07
0.14
0.07
0.13
0.06
0.13
0.01
0.03
0.06
0.12
0.24
0.24
0.14
0.27
0.07
0.13
0.03
0.07
0.03
0.06
0.03
0.25
0.64
0.8
4.75
5.7
Aspergillus fumigatus
MIC
MFC
0.14
0.27
0.13
0.26
0.13
0.25
0.13
0.25
0.12
0.24
0.02
0.12
0.14
0.27
0.13
0.26
0.03
0.13
0.25
0.25
0.25
0.38
0.48
0.64
0.38
0.95
Aspergillus niger
MIC
MFC
0.07
0.14
0.07
0.13
0.06
0.13
0.13
0.38
0.06
0.12
0.24
0.36
0.27
0.45
0.13
0.26
0.26
0.39
0.13
0.38
0.25
0.38
0.48
0.64
0.38
0.95
Aspergillus flavus
MIC
MFC
0.07
0.14
0.07
0.26
0.06
0.13
0.13
0.38
0.06
0.24
0.24
0.36
0.27
0.45
0.13
0.26
0.26
0.39
0.13
0.38
0.25
0.38
0.48
0.64
2.85
3.8
Aspergillus versicolor
MIC
MFC
0.14
0.27
0.13
0.26
0.25
0.5
0.13
0.38
0.24
0.48
0.24
0.36
0.14
0.45
0.13
0.39
0.13
0.39
0.13
0.38
0.25
0.38
0.32
0.64
0.38
0.95
Fulvia fyulvum
MIC
MFC
0.07
0.14
0.07
0.07
0.06
0.13
0.25
0.25
0.12
0.24
0.24
0.24
0.27
0.27
0.26
0.26
0.26
0.26
0.25
0.25
0.25
0.25
0.32
0.64
0.38
0.95
Table 4
Minimal inhibitory and bactericidal concentration (MIC and MBC (lmol/ml)) of tested compounds, 4a–e
BacteriaActivity
4a
4b
4c
4d
4e
StreptomycinAmpicillin
Staphylococcus aureus
MIC
MBC
0.27
0.54
0.52
0.52
0.5
0.5
0.38
0.46
0.48
0.48
0.04
0.09
0.25
0.37
Bacillus cereus
MIC
MBC
0.27
0.27
0.26
0.26
0.25
0.5
0.25
0.5
0.24
0.24
0.09
0.17
0.25
0.37
Micrococcus flavus
MIC
MBC
0.27
0.54
0.26
0.52
0.25
0.5
0.38
0.38
0.24
0.48
0.17
0.34
0.25
0.37
Listeria monocytogenes
MIC
MBC
0.27
0.54
0.52
0.52
0.5
0.5
0.38
0.46
0.24
0.48
0.17
0.34
0.37
0.49
Pseudomonas aeruginosa
MIC
MBC
0.27
0.27
0.26
0.26
0.5
0.5
0.25
0.38
0.48
0.48
0.17
0.34
0.74
1.24
Enterobacter cloacae
MIC
MBC
0.27
0.27
0.52
0.52
0.5
0.5
0.46
0.46
0.24
0.24
0.17
0.34
0.37
0.49
Salmonella typhimurium
MIC
MBC
0.27
0.27
0.26
0.26
0.25
0.5
0.25
0.5
0.24
0.24
0.17
0.34
0.25
0.49
Escherichia coli
MIC
MBC
0.27
0.27
0.26
0.26
0.25
0.25
0.38
0.46
0.48
0.48
0.26
0.52
0.37
0.74
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Mechanics calculations were performed under Macromodel30
module of Schrodinger Suite 2011, using the OPLS 2005 force field.
Compounds were first minimized with TNCG (Truncated Newton
Conjugate Gradient) algorithm using 1000 iterations and an energy
tolerance of 0.01 kcal/mol?1Å?1, to reach a local minimum. The
dielectric constant (e) was set to 47 during minimization, simulat-
ing the DMSO environment of the NMR solvent.
To generate random conformers, the 3D models of the studied
molecules following their optimization were subjected to Confor-
mational Search (Macromodel) using the Mixed torsional/Low-
mode sampling. This method uses a combination of the random
changes in torsion angles and/or molecular position from the tor-
sional sampling (MCMM) method, together with the low-mode
steps from the LMOD method, which is highly efficient and has
the advantage that ring structures and variable torsion angles do
not need to be specified.
Epik was used for the tautomers generation of compound 3fby
employing protonation and tautomerization state adjustment con-
sistent with a specified pH range. The tautomerization facility of
Epik relies on a database of tautomeric templates. Tautomers in
the database are assigned probabilities to assist in focusing on
the most highly populated tautomeric forms.31–33
QikProp predicts physically significant descriptors and phar-
maceutically relevant properties of organic molecules. It rapidly
analyses atom types and charges, rotor counts, and the sample
molecule’s volume and surface area. QikProp then uses this infor-
mation, along with the physical descriptors calculated using algo-
rithms, which mimic the full Monte Carlo simulations and
produce comparable results with experimentally determined
properties, in regression equations. This procedure results in an
accurate prediction of a molecule’s pharmacologically relevant
properties.34
The calculations of the hydrophilic and hygrophobic surfaces
were performed by the Hydrophobic/philic Surfaces panel of
Maestro.35
3. Results and discussion
3.1. Chemistry
The synthetic pathway followed for the preparation of the title
compounds was accomplished as shown in Scheme 1. All synthe-
sized molecules are presented in Table 1.
Table 5
Minimal inhibitory and fungicidal concentration (MIC and MFC lmol/ml) of tested compounds 4a–e
FungiActivity
4a
4b
4c
4d
4e
BifonazoleKetoconazole
Penicillium funiculosum
MIC
MFC
0.14
0.27
0.13
0.26
0.13
0.25
0.25
0.5
0.12
0.24
0.64
0.8
0.38
0.95
Penicillium ochrochloron
MIC
MFC
0.14
0.27
0.13
0.26
0.13
0.25
0.5
0.75
0.12
0.24
0.48
0.64
3.8
3.8
Trichoderma viride
MIC
MFC
0.07
0.13
0.07
0.13
0.06
0.13
0.13
0.25
0.06
0.12
0.64
0.8
4.75
5.7
Aspergillus fumigatus
MIC
MFC
0.14
0.27
0.13
0.26
0.13
0.25
0.13
0.25
0.12
0.24
0.48
0.64
0.38
0.95
Aspergillus niger
MIC
MFC
0.07
0.14
0.13
0.26
0.06
0.13
0.25
0.75
0.06
0.12
0.48
0.64
0.38
0.95
Aspergillus flavus
MIC
MFC
0.14
0.27
0.13
0.26
0.13
0.25
0.25
0.75
0.12
0.24
0.48
0.64
2.85
3.8
Aspergillus versicolor
MIC
MFC
0.27
0.54
0.26
0.52
0.25
0.5
0.25
0.75
0.12
0.24
0.32
0.64
0.38
0.95
Fulvia fyulvum
MIC
MFC
0.07
0.07
0.07
0.07
0.06
0.13
0.13
0.25
0.06
0.12
0.32
0.64
0.38
0.95
Table 6
Antibacterial activity of compounds 5a–etested by microdilution method (MIC and MBC in lmol)
BacteriaActivity
5a
5b
5c
5d
5e
StrAmp
Staphylococcus aureus
MIC
MBC
0.28
0.28
0.14
0.27
0.26
0.26
0.25
0.38
0.25
0.5
0.04
0.09
0.25
0.37
Bacillus cereus
MIC
MBC
0.28
0.28
0.14
0.27
0.26
0.26
0.25
0.25
0.39
0.39
0.09
0.17
0.25
0.37
Micrococcus flavus
MIC
MBC
0.56
0.84
0.27
0.54
0.13
0.26
0.25
0.5
0.25
0.5
0.17
0.34
0.25
0.37
Listeria monocytogenes
MIC
MBC
0.42
0.42
0.27
0.54
0.13
0.26
0.25
0.75
0.25
0.39
0.17
0.34
0.37
0.49
Pseudomonas aeruginosa
MIC
MBC
0.42
0.56
0.27
0.54
0.26
0.26
0.13
0.25
0.39
0.39
0.17
0.34
0.74
1.24
Enterobacter cloacae
MIC
MBC
0.7
0.84
0.67
0.67
0.63
0.75
0.63
0.75
0.63
0.63
0.17
0.34
0.37
0.49
Salmonella typhimurium
MIC
MBC
0.42
0.42
0.14
0.27
0.13
0.26
0.13
0.25
0.13
0.25
0.17
0.34
0.25
0.49
Escherichia coli
MIC
MBC
0.28
0.42
0.14
0.27
0.63
0.63
0.63
0.63
0.39
0.39
0.26
0.52
0.37
0.74
1576
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3.2. Pharmacology
3.2.1. Triazoles group
Results of antibacterial activity of the triazole compounds 3a–k
are presented in Table 2. All the compounds showed antibacterial
activity with MIC in range 0.13–0.65 lmol/ml and MBC of 0.24–
0.76 lmol/ml. The best antibacterial activity was obtained for
compound 3fwith MIC and MBC of 0.24–0.48 lmol/ml while the
lowest antibacterial activity was achieved for compound 3gwith
MIC and MBC of 0.27–0.81 lmol/ml. It can be seen that differences
between MIC and MBC of these compounds are very small. Strep-
tomycin showed MIC in range of 0.04–0.26 lmol/ml and MBC of
0.09–0.52 lmol/ml. Ampicillin showed inhibitory effect at 0.25–
0.74 lmol/ml and bactericidal at 0.37–1.24 lmol/ml. Compounds
3a, 3cand 3eshowed better antibacterial activity than streptomy-
cin against Listeria monocytogenes. Compound 3balso possessed
better activity than streptomycin but only against Pseudomonas
aeruginosa. Enterobacter cloacae and Salmonella typhimurium were
more sensitive to compounds 3a, 3eand 3kthan to streptomycin.
Escherichia coli was the most sensitive bacteria to the tested com-
pounds, especially to 3f, 3iand 3k, where the MIC and MBC were
lower than for streptomycin.
Compounds 3a, 3b, 3c, 3e, 3f, 3iand 3kpossessed better antibac-
terial activity than ampicillin against Enterobacter cloacae and Sal-
monella typhimurium. All compounds tested showed stronger
antibacterial potential than ampicillin against Escherichia coli and
Pseudomonas aeruginosa. Compounds 3a, 3c, 3e, 3f, 3i, 3jand 3kre-
acted with higher antibacterial activity than ampicillin against Lis-
teria monocytogenes.
From the obtained results it can be noticed that several com-
pounds possessed better antibacterial activity than streptomycin
and ampicillin.
Results of antifungal activity are presented in Table 3. It can be
seen that all the compounds showed very good antifungal activity
with MIC of 0.01–0.27 lmol/ml and MFC 0.06–0.50 lmol/ml better
than the commercial antifungal agents, bifonazole (MIC 0.32–
0.64 lmol/ml, MFC 0.64–0.80 lmol/ml) and ketoconazole (MIC
0.38–4.75 lmol/ml, MFC 0.95–5.70 lmol/ml). Triazoles, exhibited
much better antifungal activity than these mycotics, in some cases
( A. niger, A. flavus andT. viride) this activity was 10–70 times higher.
Compound 3dshowed the best antifungal activity among all the
others with lowest MIC (0.01–0.25 lmol/ml) and MFC (0.03–
0.38 lmol/ml). The most sensitive fungi to all tested compounds
were Trichoderma viride, while Aspergillus versicolor was the most
resistant.
3.2.2. Thiadiazoles group
The results of antibacterial activity of thiadiazole compounds 4
a–eare presented in Table 4. The range of antibacterial activity was
in 0.24–0.54 lmol/ml (MIC and MBC). All compounds tested pos-
sessed higher antibacterial activity than streptomycin against
Escherichia coli. Pseudomonas aeruginosa and Escherichia coli were
more sensitive to all the compounds than to ampicillin. Compound
4eshowed the best antibacterial activity with MIC and MBC of
0.24–0.48 lmol/ml. Compounds 4a,4band 4eshowed better bacte-
ricidal activity than streptomycin against Escherichia coli and Sal-
monellatyphimurium
andbetter
ampicillin against Bacillus cereus and Salmonella typhimurium. Bet-
ter bactericidal potential than ampicillin against Enterobacter cloa-
cae was achieved also for compounds 4a,4dand 4e,while 4dand 4e
showed better activity than ampicillin only against Listeria
monocytogenes.
The results of antifungal activity are presented in Table 5. The
tested compounds showed very good antifungal activity with
MIC of 0.06–0.50 lmol/ml and MFC 0.07–0.75 lmol/ml. All the
compounds showed higher antifungal potential than ketoconazol
and almost all showed higher potential than bifonazole (MIC,
MFC), except compound 4dwhich exhibited slightly lower poten-
tial than bifonazole against Penicillium ochrochloron, Aspegillus ni-
ger, Aspergillus versicolor and A. flavus.
The best antifungal potential could be seen for compound 4e
MIC (0.06–0.12 lmol/ml) and MFC (0.12–0.24 lmol/ml). The most
sensitive fungus on all tested compounds was Fulvia fulvum, while
Aspergillus versicolor was the most resistant species. In conclusion,
thiadiazoles exhibited much better antifungal activity than used
mycotics, in some cases (A. niger, T. viride andF. fulvum) this activity
was 8–10 times higher.
bactericidalactivitythan
3.2.3. Oxadiazoles group
The results of antibacterial activity of the oxadiazoles 5a–eare
presented in Table 6. Antibacterial activity was achieved at 0.13–
0.70 lmol/ml (MIC) and 0.25–0.84 lmol/ml (MBC). All compounds
showed almost the same activity with small differences. The high-
est inhibition and bactericidal potential was observed for com-
pound 5b with MIC of 0.14–0.67 lmol/ml and MBC of 0.27–
0.67 lmol/ml. Compounds 5dand 5cshowed better antibacterial
activity than streptomycin against Salmonella typhimurium, while
compound 5cwas also more effective than streptomycin against
Listeria moonocytognes and Micrococcus flavus. Compounds 5b,5c
and 5dpossessed higher antibacterial activity than streptomycin
against Salmonella typhimurium. Ampicillin was less effective than
Table 7
Antifungal activity of compounds 5a–etested by microdilution method (MIC and MFC in lmol)
Fungi Activity
5a
5b
5c
5d
5e
Bif Keto
Penicillium funiculosum
MIC
MFC
0.14
0.28
0.14
0.27
0.26
0.78
0.25
0.5
0.13
0.25
0.64
0.8
0.38
0.95
Penicillium ochrochloron
MIC
MFC
0.14
0.45
0.14
0.27
0.13
0.26
0.25
0.75
0.13
0.38
0.48
0.64
3.8
3.8
Trichoderma viride
MIC
MFC
0.03
0.14
0.03
0.03
0.07
0.13
0.13
0.13
0.06
0.13
0.64
0.8
4.75
5.7
Aspergillus fumigatus
MIC
MFC
0.14
0.28
0.27
0.27
0.13
0.26
0.13
0.25
0.13
0.25
0.48
0.64
0.38
0.95
Aspergillus niger
MIC
MFC
0.14
0.45
0.14
0.14
0.13
0.39
0.25
0.75
0.25
0.38
0.48
0.64
0.38
0.95
Aspergillus flavus
MIC
MFC
0.14
0.45
0.14
0.4
0.13
0.39
0.25
0.75
0.13
0.25
0.48
0.64
2.85
3.8
Aspergillus versicolor
MIC
MFC
0.14
0.45
0.14
0.4
0.26
0.39
0.25
0.75
0.13
0.38
0.32
0.64
0.38
0.95
Fulvia fyulvum
MIC
MFC
0.28
0.45
0.27
0.27
0.13
0.26
0.13
0.25
0.25
0.25
0.32
0.64
0.38
0.95
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all the compounds tested against Pseudomonas aeruginosa and
Salmonella typhimurium. More specifically, compound 5c showed
higher antibacterial activity than ampicillin against Micrococcus
flavus, Bacilluscereus, Listeriamonocytogenes, Pseudomonas aeruginosa
and Salmonella typhimurium, while 5d suppressed Bacillus cereus,
Pseudomonas aeruginosa and Salmonella typhimurium better than
ampicillin. Listeria monocytogenes was more sensitive to compounds
5cand 5ethan to ampicillin. Compound 5bshowed better activity
than ampicillin against Listeria monocytogenes, Staphylococcus aureus,
Pseudomonasaeruginosa, Salmonellatyphimurium, Escherichia coli and
Bacillus cereus.
Results of antifungal activity are presented in Table 7. Inhibitory
activity of compounds was in range of 0.03–0.28 lmol/ml and fun-
gicidal in range of 0.03–0.75 lmol/ml. The tested compounds
showed higher antifungal potential than bifonazole, except com-
pound 5dwhich exhibited slightly lower potential against Penicil-
lium ochrochloron, Aspegillus niger, A. versicolor and A. flavus.
Moreover, oxadiazoles possessed much better antifungal potential
than ketokonazol. The best antifungal potential was achieved for
compound 5b with MIC 0.03–0.27 lmol/ml and MFC 0.03–
0.40 lmol/ml. Compounds 5a, 5band 5eshowed almost the same
activity with small differences in MIC, while compounds 5cand
5dpossessed similar antifungal potential. The most sensitive fun-
gus on all tested compounds was Trichoderma viride, while Asper-
gillus versicolor and Penicillium species were the most resistant.
3.3. SAR—structure activity relationship
From the biological results, it becomes clear that different sub-
stituents on triazole, thiadiazole and oxadiazole scaffolds have a
noticeable effect on antibacterial and antifungal activities. In gen-
eral, studied molecules exhibited much better antifungal than anti-
bacterial activity.
3.3.1. Antibacterial activity
The antimicrobial activity of all compounds is promising. It
seems that the presence of the aliphatic chain increases lipophilic-
ity which is mandatory for the increased activity. More specifically,
the activity is strongly depended on the number of carbon atoms of
the chain length. In general oxadiazole analogues with propyl (5c)
and butyl (5d) chains exhibited the best MIC activity over most of
the studied bacteria.
3.3.2. Antifungal activity
All the tested compounds exhibited much better antifungal
activity than ketokonazol and bifonazole against all fungi species
being 8 to 10-fold higher in cases of A. niger, T. viride, A. flavus
and F. fulvum. Among all groups of tested compounds, triazole-3-
thiones, in general, exhibited the best antifungal potency over all
fungal species.
3.3.2.1. Penicillium funiculosum and Penicillium ochrochlo-
ron.
Triazole-3-thiones analogues exhibited the best MIC
activity against these species especially those with butyl (3e) and
tert butyl (3f) substitutions. The thiadiazole analogue with butyl
chain substitution (4e) also exhibited equipotent activity.
3.3.2.2. Trichoderma viride.
ited by triazole-3-thione with isopropyl substitution (3d). In gen-
eral, triazole-3-ones with longer chains (isopropyl, butyl, tert
butyl) and oxadiazoles with methyl or ethyl substitutions showed
remarkable inhibition.
The best MIC activity was exhib-
3.3.2.3. Aspergillus fumigates.
tyl substitution (3f) and triazole-3-one with isopropyl chain (3i)
exhibited the best activity against this specie.
Triazole-3-thione with tert bu-
Table 8
Predicted properties for the synthesized analogues
Compd
Dipole
FOSA
SASA
FISA
PISA
WPSA
Volume
HBd
Hba
Dip^2/V
QPlogPC16
QPlogPoct
QPlogPw
QPlogPo/w
QPlogS
CIQPlogS
IP (eV)
EA (eV)
PSA
3⁄HBd ? QPlogPo/w
3a
8.85
347.19
566.06
100.93
43.33
74.61
1053.98
1
9.5
0.074
9.90
18.20
11.59
1.63
?2.59
?3.66
8.43
1.02
90.50
1.37
3b
10.94
398.13
620.73
115.93
36.94
69.72
1130.80
1
9.5
0.106
10.53
19.37
11.56
1.93
?3.30
?3.93
8.31
1.01
92.44
1.07
3c
13.07
431.90
655.81
115.89
38.41
69.61
1193.70
1
9.5
0.143
11.11
20.47
11.43
2.31
?3.72
?4.21
8.29
1.01
92.33
0.69
3d
11.51
428.21
603.51
89.23
24.70
61.37
1163.64
1
9.5
0.114
10.58
19.86
11.25
2.30
?2.98
?4.21
8.13
1.05
89.97
0.7
3e
9.85
424.29
608.36
91.69
30.35
62.03
1185.88
1
9.5
0.082
10.85
19.28
10.87
2.44
?2.74
?4.49
8.24
1.09
90.47
0.56
3f
10.29
449.34
614.25
81.26
25.94
57.72
1192.99
1
9.5
0.089
10.76
19.88
11.23
2.53
?3.14
?4.49
8.03
0.99
83.76
0.47
3g
3.59
382.77
574.58
147.66
44.14
0.00
1074.05
1
9
0.012
9.88
16.97
11.19
1.34
?2.42
?3.82
9.25
0.77
109.32
1.66
3h
7.73
438.51
624.23
152.05
33.28
0.38
1146.92
1
9
0.052
10.42
18.16
11.05
1.72
?3.07
?4.09
9.15
0.95
113.38
1.28
3i
9.46
434.23
587.91
128.48
24.91
0.29
1131.22
1
9
0.079
10.16
18.56
10.96
1.81
?2.64
?4.09
8.82
0.88
108.27
1.19
3j
6.46
460.10
639.31
137.77
40.62
0.82
1190.30
1
9
0.035
10.77
18.26
10.82
2.10
?3.16
?4.37
9.13
0.83
106.98
0.9
3k
8.65
478.64
620.48
114.35
27.49
0.00
1177.02
1
9
0.064
10.42
18.87
10.96
2.18
?3.17
?4.37
8.81
0.76
103.63
0.82
4a
5.85
425.45
617.69
120.17
49.08
22.99
1103.10
1
8.5
0.031
10.04
17.22
10.66
1.98
?3.41
?4.05
8.66
0.63
90.19
1.02
4b
7.61
410.68
595.64
123.31
50.28
11.37
1112.64
1
8.5
0.052
10.15
17.48
10.41
2.00
?2.83
?4.33
8.99
0.77
89.41
1
4c
2.38
462.68
648.03
117.45
46.31
21.59
1186.42
1
8.5
0.005
10.77
17.50
10.25
2.50
?3.59
?4.61
8.90
0.66
89.75
0.5
4d
5.23
502.38
670.46
117.15
43.31
7.62
1210.41
1
8.5
0.023
10.88
18.20
10.50
2.58
?4.07
?4.61
8.63
0.77
90.69
0.42
4e
6.15
512.77
705.50
120.00
47.61
25.13
1272.23
1
8.5
0.030
11.65
18.73
10.20
3.01
?4.42
?4.89
8.68
0.66
90.67
?0.01
5a
9.77
407.30
589.95
123.96
58.19
0.50
1062.84
1
9
0.090
9.63
17.73
11.16
1.44
?2.67
?3.54
8.98
0.75
100.12
1.56
5b
10.04
425.53
600.75
116.33
58.32
0.57
1097.01
1
9
0.092
9.90
17.96
10.92
1.70
?2.69
?3.82
8.94
0.77
96.89
1.3
5c
1.37
460.04
633.77
124.56
47.65
1.52
1163.24
1
9
0.002
10.48
17.37
10.76
2.03
?3.07
?4.09
8.87
0.73
100.51
0.97
5d
3.39
488.34
665.08
124.43
51.80
0.51
1222.44
1
9
0.009
11.05
18.03
10.64
2.39
?3.43
?4.37
8.97
0.67
99.71
0.61
5e
1.99
466.32
626.54
112.13
47.36
0.73
1183.71
1
9
0.003
10.60
17.95
10.88
2.25
?3.11
?4.37
8.83
0.74
97.22
0.75
1578
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3.3.2.4. Aspergillus niger.
zole-3-thiones and thiadiazoles with propyl and butyl chain sub-
stitutions (3c, 3e, 4c, 4e).
The best MIC activity show tria-
3.3.2.5. Aspergillus flavus.
with linear alkyl chains (methyl, ethyl, propyl, butyl) exhibited
very good activity.
All triazole-3-thiones substituted
3.3.2.6. Aspergillus versicolor.
and triazole-3-ones showed very good activity. From the thiadia-
zole analogues, 4eexhibited the best activity.
Almost all triazole-3-thiones
3.3.2.7. Fulvia fyulvum.
exhibited the best activity against this specie.
Thiadiazoles and triazole-3-thiones
3.4. Prediction of molecular properties
In order to proceed with prediction of physically significant
descriptors and pharmaceutically relevant properties of the
synthesized molecules, QikProp (Maestro) as a fast and accurate
prediction software was used. The produced values can be used
as descriptors for QSAR and in silico screening techniques. The fol-
lowing properties34were selected, calculated and presented in Ta-
ble 8.
(a) Computed dipole moment of the molecule (Dipole); (b) total
solvent accessible surface area (SASA) in square angstroms using a
probe with a 1.4 Å radius, (c) hydrophobic component of the SASA
(FOSA); (d) hydrophilic component of the SASA (FISA); (e) p com-
ponent of the SASA (PISA); (f) weakly polar component of the SASA
(halogens, P, and S) (WPSA); (g) total solvent-accessible volume in
cubic angstroms using a probe with a 1.4 Å radius (volume); (h)
estimated average number of hydrogen bonds (taken over a num-
ber of configurations) that would be donated by the solute to water
molecules in an aqueous solution (HBd); (i) estimated average
number of hydrogen bonds (taken over a number of configura-
tions) that would be accepted by the solute from water molecules
in an aqueous solution (HBa); (j) square of the dipole moment di-
vided by the molecular volume (dip^2/V), a relevant parameter for
Figure 1. NOESY NMR spectrum for compound 4eindicating the critical NOE signals.
Table 9
Critical interproton distances, calculated from ROE signals
Distance constrains ±10% (Å)
4e Structure
9–18
9–13
12–14
12–6
6–17
2.55–3.11
2.03–2.49
2.05–2.50
2.04–2.49
2.65–3.24
4e structure
N
2
SO2
N
H3C
1
CH3
7
O
O
H3C
H3C
13
O
N
N
H
CH3
23
6
22
9
21
12
14
19
20
81
7
8
10
11
5
P. Zoumpoulakis et al./Bioorg. Med. Chem. 20 (2012) 1569–1583
1579

Page 12

Figure 2. Representative low energy conformers of 4e.
Figure 3. (a) Low energy conformers of compounds 4e, 3f, 5b, consistent with NOE data; (b) hydrophobic (brown) and hydrophilic (blue) surface area distribution.
1580
P. Zoumpoulakis et al./Bioorg. Med. Chem. 20 (2012) 1569–1583

Page 13

the energy of solvation of a dipole of volume V; (k) hexadecane/gas
partition coefficient (QPlogPC16); (l) octanol/gas partition coeffi-
cient (QPlogPoct); (m) water/gas partition coefficient (QPlogPw);
(n) octanol/water partition coefficient (QPlogPo/w); (o) aqueous
solubility in mol dm?3(QPlogS); (p) conformation-independent
predicted aqueous solubility (CIQPlogS); (q) PM3 calculated ioniza-
tion potential (IP(ev)); (r) PM3 calculated electron affinity
(EA(eV)); (s) van der Waals surface area of polar nitrogen and oxy-
gen atoms (PSA).
The empirical equation 3⁄HBd ? QPlogPo/w, was also calcu-
lated, which combines descriptors for both polarity and lipophilic-
ity, and could serve as a predictor for compounds’ bioavailability.36
A value above 6 is correlated to very poor bioavailability. All of the
tested compounds showed considerable lower values (Table 8).
The obtained results show that molecules which exhibited good
antifungal activities against all tested species, are characterized by
high dipole moments, increased number of hydrogen bond accep-
tors, high dip^2/V values leading to high energy of solvation, in-
creased polar contributions to the total solvent accessible surface
area, high water/gas partition coefficients, as well as increased
electron affinity and low ionization potential values. Interestingly,
the class of triazole-3-thiones is following the trend described
above.
3.5. Conformational analysis
Conformational analysis was performed using NMR and molec-
ular modeling techniques to one representative compound from
each category, exhibiting different activities. From the triazoles,
3fwas chosen among the best antifungal and antibacterial agents.
Compound 4efrom the thiadiazole group was selected showing
good activity against tested bacteria and fungi but less compared
to 3f. Finally, for comparison reasons, 5bwas selected from the oxa-
diazole group, because it exhibited lower activity relatively to the
3fand 4e.
Here we demonstrate the conformational analysis of the thiadi-
azole 4e. The solvent used in NMR analysis is DMSO-d6and not D2O
because of little solubility of tested compounds in aqueous media.
Selection of the solvent though is not inadequate since drug’s bio-
active conformation is finally determined from the environment of
the active site of the target protein.
Structure elucidation for each of the above mentioned com-
pounds was performed following standard procedures using
homonuclear gCOSY and NOESY spectra.
In Figure 1, we present the NOESY spectrum for the 4eanalogue
indicating the NOE correlations. Resonance peaks assignment is
indicated on the 1D projection. The triplet peak at 0.85 ppm is
attributed to H23. From COSY correlation of the neighbouring nu-
clei, peaks at 1.28, 1.47, 3.16 and 7.47 ppm are attributed to H22,
H21, H20 and H19 correspondingly. The single peak at 4.43 ppm,
integrated for two protons is attributed to H6. The observed NOE
between H6 and the aromatic proton at 7.05 ppm clearly attrib-
uted this singlet peak to H12. Furthermore, NOE correlation of
H12 with the single peak at 3.78 ppm, leads to the unequivocal
assignment of those proton resonances to the methoxy protons
H14. Thus, aromatic H9 and the second methoxy group H13 are as-
signed to 7.21 and 3.80 ppm correspondingly. Finally, the single
peak at 2.62 ppm is integrated for 6 protons and is assigned for
H17 and H18.
Critical interproton distances for the conformation of 4ein solu-
tion are calculated from NOE signal volumes and are presented in
Table 9. Signals between H12 with H14 and H9 with H13 define the
orientation of the methoxy groups towards the aromatic ring. Sig-
nal between H9 with H18 and H6 with H17 define the orientation
of the dimethyl sulfamoyl group. Finally, signals between protons
of the alkyl chain were not taken into consideration due its high
mobility in solution.
In order to identify low energy conformations, consistent with
the experimentally observed constrains, we performed molecular
modeling studies for the 4e. The 3D models of the studied mole-
cules following their optimization were subjected conformational
search after examining the existence of possible tautomers. The
produced low energy conformations for each analogue were clus-
tered according to their heavy atoms and the lowest energy mem-
bers of the families were further investigated for their consistency
with the NOE data.
Four representative favourable conformations of 4enamely 4e_1,
4e_2, 4e_3and 4e_4, are displayed in Figure 2. 4e_1and 4e_3adopt a
more extended conformation without forming any kind of clusters
between functional groups as in the case of 4e_2and 4e_4. From the
presented conformers, 4e_1is in accordance with the critical NOE
data of Table 9.
The same procedure was followed for compounds 3fand 5b.
Low energy conformers, consistent with NOE data are shown in
Figure 3a together with 4e_1. Compound 3fis found in two tauto-
meric forms (3f–aand 3f–b) at pH 7 ± 2 (Fig. 4) as calculated using
Epik module of Schrodinger software. Nevertheless, the presence
of a broad single peak at ?13 ppm in the NMR spectrum proves
the existence of 3fin the ‘a’ form. Interestingly, the compound 3f
exhibits much higher potential energy relative to 4eand 5b, which
is attributed to the increased bend energy of the triazole-3-thione
ring due to the steric interactions of the tert-butyl group.
Proposed conformations consistent with NOE data were used to
generate the hydrophobic (brown) and hydrophilic surfaces (blue)
Figure 4. Tautomeric forms of compound 3f(3f–aand 3f–b) at pH 7 ± 2.
P. Zoumpoulakis et al./Bioorg. Med. Chem. 20 (2012) 1569–1583
1581

Page 14

of the selected compounds (Fig. 3b). Results show that all mole-
cules have an amphoteric character, important for antifungal activ-
ity, with higher hydrophilic and lower hydrophobic areas.
As a next step, we recalculated the molecular properties of com-
pounds 3f(both tautomers), 4eand 5b, this time using their low en-
ergyconformationssatisfying
Differences are expected to occur compared to Table 8, only to
properties which depend on the molecules’ conformation. Results
are presented in Table 10 and indicate that higher WPSA, Qplog-
Poct and lower FISA, PISA and IP values may be related to higher
antifungal activity to most of the tested species.
theexperimental NOEdata.
4. Conclusions
The synthesis of a series of 21 novel sulfonamide-1,2,4-tria-
zoles, sulfonamide-1,3,4-thiadiazoles and sulfonamide-1,3,4-oxa-
diazoles is presented emphasizing, on the strategy of combining
two chemically different but pharmacologically compatible mole-
cules (the sulfomamide nucleus and the five member) heterocycles
in one frame. Synthesized compounds were tested in vitro for anti-
bacterial and antifungal activity. Results indicate that increase of
the length of the aliphatic chain, increases lipophilicity which is
mandatory for antibacterial activity. More specifically, oxadiazole
analogues with propyl (5c) and butyl (5d) chains exhibited the best
MIC activity over most of the studied bacteria. Although the title
compounds did not exhibit significantly higher antifungal activity
than previous synthesized by our group triazole and thiadiazole
analogues, all of them exhibited much better antifungal activity
than commercial ketokonazol and bifonazol. In some cases of fungi
(i.e., A. niger, T. viride, A. flavus and F. fulvum) this activity was 8–10
times higher. More specifically, triazole-3-thiones exhibited the
best activities over all fungal species. Prediction of the molecular
properties of synthesized molecules showed that triazole-3-thi-
ones share by high dipole moment, increased number of hydrogen
bond acceptors, high energy of solvation, increased polar contribu-
tions to the total solvent accessible surface area, high water/gas
partition coefficients, as well as increased electron affinity and
low ionization potential values.
In general, larger alkyl groups (butyl, tert-butyl, isopropyl) gave
better results against fungi. Nevertheless, main differences in the
antifungalactivityseemtodependmoreonthetriazol-3-thionecore
rather than the different length of the alkyl chain substitutions.
Acknowledgments
The research leading to these results has received funding from
the European Union’s Seventh Framework Programme (FP7-REG-
POT-2009-1) under grant agreement No. 245866.
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Table 10
Predicted properties for 3f(including its two tautomers a and b), 4eand 5banalogues
Compd
Dipole
FOSA
SASA
FISA
PISA
WPSA
Volume
HBd
HBa
Dip^2/V
QPlogPC16
QPlogPoct
QPlogPw
QPlogPo/w
QPlogS
CIQPlogS
IP (eV)
EA (eV)
3f_a
11.74
424.82
620.92
102.86
30.89
62.354
1181.45
1
9.5
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11.423
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?3.269
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63.51
1181.6
0.8
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0.0897
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18.471
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?5.084
8.65
0.60
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10.088
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?4.503
?4.89
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5b
9.926
397.84
569.25
131.95
39.46
0
1073.34
1
9
0.0918
9.762
17.718
10.82
1.449
?2.168
?3.815
8.98
0.75
1582
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