Synthesis and anticandidal activity of new triazolothiadiazine derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.09.020

New triazolothiadiazine derivatives were synthesized via the ring closure reaction of 4-amino-5-substituted-2,4-dihydro-3H-1,2,4-triazol-3-thiones with phenacyl bromides. The compounds were tested in vitro against various Candida species and compared with

Full text of DOI:10.1016/j.ejmech.2011.09.020

European Journal of Medicinal Chemistry 46 (2011) 5562e5566
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticandidal activity of new triazolothiadiazine derivatives
,
a
a
Mehlika Dilek Altıntopa, Zafer Asım Kaplancıklıa , Gülhan Turan-Zitouni , Ahmet Özdemir ,
*
b
c
d
_
Gökalp Is¸ can , Güls¸ en Akalın , S¸ afak Ulusoylar Yıldırım
a Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskis¸ehir, Turkey
b Anadolu University, Faculty of Pharmacy, Department of Pharmacognosy, 26470 Eskis¸ehir, Turkey
c Anadolu University, Faculty of Pharmacy, Department of Biochemistry, 26470 Eskis¸ehir, Turkey
d Anadolu University, Faculty of Pharmacy, Department of Pharmacology, 26470 Eskis¸ehir, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2011
Received in revised form
9 August 2011
Accepted 15 September 2011
Available online 22 September 2011
New triazolothiadiazine derivatives were synthesized via the ring closure reaction of 4-amino-5-
substituted-2,4-dihydro-3H-1,2,4-triazol-3-thiones with phenacyl bromides. The compounds were
tested in vitro against various Candida species and compared with ketoconazole. Among these
compounds, the compound bearing cyclohexyl moiety and p-chlorophenyl substituent on tri-
azolothiadiazine ring (2i) was found to be the most potent derivative against Candida albicans (ATCC
90028). It is clear that there is a positive correlation between anticandidal activity and two functional
moieties, namely cycloaliphatic group and p-chlorophenyl substituent on triazolothiadiazine ring. The
compounds were also investigated for their cytotoxic effects using MTT assay. Compound 2a exhibited
the highest cytotoxic activity, whereas compound 2f possessed the lowest cytotoxic activity against NIH/
3T3 cells.
Keywords:
Triazole
Triazolothiadiazine
Anticandidal activity
Cytotoxicity
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
azoles via decreased accumulation of the drug resulting from
enhanced efflux, interference of their action on lanosterol 14
a-
Candida species have emerged as the most common cause of
systemic fungal infections worldwide over the last two decades. A
large number of studies have attempted to assess risk factors for
candidaemia. The most frequently implicated risk factors include
treatment with broad-spectrum antibiotics, use of central venous
catheters and implantable prosthetic devices, parenteral nutrition,
prolonged intensive care unit stay, hemodialysis and immunosup-
pression (including HIV infection, neutropenia, use of glucocorti-
costeroids, chemotherapeutic agents, and immunomodulators)
[1e7].
demethylase, alterations in other enzymes of the biosynthetic
pathway of ergosterol, and decreased permeability of the fungal
membrane to the drug. As a consequence of this situation, medic-
inal chemists have focused on the development of new effective
anticandidal agents [3].
Among triazole derivatives, 1,2,4-triazoline-3-thiones and their
fused heterocyclic derivatives including triazolothiadiazines have
received considerable attention due to their biological importance
[10e12]. Some studies have confirmed that triazolothiadiazine
derivatives possess anticandidal activity [13e17].
Azoles, especially triazole antifungal agents, play a leading role
in the treatment of systemic fungal infections owing to their broad
spectrum and improved safety profile. The prominent drugs
bearing triazole ring are fluconazole, itraconazole, voriconazole,
and posaconazole, all of which are widely used antifungal drugs for
the treatment of systemic fungal infections [8,9].
In continuation of our previous work on the synthesis and
antimicrobial evaluation of triazolothiadiazine derivatives [15],
herein we described the discovery of new triazolothiadiazine
derivatives, which were tested in vitro against various Candida
species and investigated for their cytotoxic effects.
The widespread use of these agents has led to the development
of resistance in recent years. Several researchers have extensively
studied the mechanisms of resistance to azoles in Candida species.
At the molecular level, Candida species can develop resistance to
2. Chemistry
The compounds possessing triazoline-3-thione structure
(1aeb) were previously synthesized via the one-pot reaction of
thiocarbohydrazide with appropriate propionic acids under
solvent-free conditions [18].
New triazolothiadiazine derivatives (2aej) were obtained by the
ring closure reaction of 1,2,4-triazoline-3-thiones (1aeb) with
* Corresponding author. Tel.: þ90 222 3350580/3776; fax: þ90 222 3350750.
E-mail address: zakaplan@anadolu.edu.tr (Z.A. Kaplancıklı).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.020

M.D. Altıntop et al. / European Journal of Medicinal Chemistry 46 (2011) 5562e5566
5563
In the 1H-NMR spectra of compounds 2fej, the cyclohexyl
protons and the methylene protons directly attached to cyclo-
hexane ring were observed at 0.84e1.77 ppm. The methylene
protons directly attached to triazolothiadiazine appeared at
2.87e2.97 ppm as a triplet peak. All other aromatic and aliphatic
protons were observed at expected regions.
In the 13C-NMR spectra of all compounds (2aej), the peaks
belonging to triazolothiadiazine carbons except C-7 appeared at
140e160 ppm. All other aromatic and aliphatic carbons were
observed at expected regions.
In the mass spectra of all compounds (2aej), M þ 1 peaks were
consistent with their molecular weights. All compounds gave
satisfactory elemental analysis.
All compounds were tested in vitro against Candida species and
compared with ketoconazole (Table 2). Among these compounds,
compounds 2a, 2b, 2f, 2g and 2j exhibit the highest inhibitory
activity against Candida albicans (Clinical Isolate). This outcome
confirms that phenyl and p-nitrophenyl groups on tri-
azolothiadiazine ring may have a considerable influence on anti-
fungal activity against C. albicans (Clinical Isolate). Although
compounds 2e and 2j possess 4-methylphenyl moiety on tri-
azolothiadiazine ring, they show different levels of anticandidal
activity. The former bearing 4-hydroxyphenyl group exhibits the
lowest antifungal activity, whereas the latter bearing cyclohexyl
moiety exhibits the highest antifungal activity against C. albicans
(Clinical Isolate). This could result from increased lipophilicity
associated with cyclohexyl group.
The compound bearing cyclohexyl moiety and p-chlorophenyl
substituent on triazolothiadiazine ring (2i) was found to be the
most potent derivative against C. albicans (ATCC 90028), Candida
parapsilosis, C. albicans (NRRL Y-12983), Candida glabrata (Clinical
Isolate, Anadolu University, Faculty of Science). Among Candida
species, C. albicans (ATCC 90028) is the most susceptible fungus to
compound 2i. Compound 2i exhibits the inhibitory activity against
C. albicans (ATCC 90028) with a MIC value of 0.03 mg/mL, whereas
ketoconazole exhibits the inhibitory activity with a MIC value of
0.04 mg/mL. It is apparent that there is a positive correlation
between anticandidal activity and two functional moieties, namely
cycloaliphatic group and p-chlorophenyl substituent on tri-
Scheme 1. The synthesis of the compounds (2aej).
phenacyl bromides [19]. These reactions are summarized in
Scheme 1 and some properties of the compounds are given in
Table 1.
3. Results and discussion
The structures of all compounds (2aej) were confirmed by 1H-
NMR, 13C-NMR, mass spectral data and elemental analysis.
In the 1H-NMR spectra of the previously synthesized
compounds 1aeb, we observed NH2, NH protons at 5e6 ppm and
13e14 ppm, respectively [18].
In the 1H-NMR spectra of all compounds (2aej), the absence of
the peaks corresponding to NH2 and NH protons of compounds
1aeb clearly confirmed the formation of triazolothiadiazine ring.
In the 1H-NMR spectra of all compounds (2aej), the signal due
to the eSeCH2e protons of triazolothiadiazine ring gave rise to
a singlet peak at 4.3e4.45 ppm.
azolothiadiazine ring. It can be attributed to þ
p effect of chloro
In the 1H-NMR spectra of compounds 2aee, the methylene
protons directly attached to phenol and triazolothiadiazine rings
were observed at 2.90e2.94 ppm and 3.08e3.12 ppm, respectively.
The protons of phenol ring were observed in the region
6.62e7.01 ppm. The OeH proton of phenol appeared at 9.20 as
a singlet peak.
substituent and cyclohexyl group.
The compound possessing 4-hydroxyphenyl moiety and phenyl
substituent on triazolothiadiazine ring (2a) is the most effective
derivative against C. glabrata (Clinical Isolate, Osmangazi Univer-
sity, Faculty of Medicine).
The biological results indicate that Candida tropicalis is more
susceptible to compounds 2a, 2b and 2i.
Compound 2h is the most potent derivative against Candida
krusei. It is obvious that cyclohexyl moiety and 4-fluorophenyl
group on triazolothiadiazine ring have an important impact on
antifungal activity against C. krusei.
All compounds were also evaluated for their cytotoxic proper-
ties using MTT assay. The biological study indicated that compound
2a possessed the highest cytotoxicity, whereas compound 2f
exhibited the lowest cytotoxicity against NIH/3T3 cells among the
title compounds (Table 3).
Table 1
Some properties of the compounds (2aej).
Compound R
R0 Yield
(%)
M.p. (ꢀC) Molecular
formula
Molecular
weight
2a
2b
2c
2d
2e
4-(OH)
C6H5
4-(OH)
C6H5
4-(OH)
C6H5
4-(OH)
C6H5
4-(OH)
C6H5
H
75
NO2 80
65
220
C18H16N4OS
336
381
354
244e245 C18H15N5O3S
206e211 C18H15FN4OS
F
Cl 65
CH3 60
274e275 C18H15ClN4OS 370.5
4. Conclusion
234e236 C19H18N4OS
350
In conclusion, we focused on the synthesis of new tri-
azolothiadiazine derivatives, which were tested in vitro against
various Candida species.
Generally the compounds bearing cyclohexyl moiety (2fej) are
more effective than the compounds bearing 4-hydroxyphenyl
2f
2g
2h
2i
C6H11
C6H11
C6H11
C6H11
C6H11
H
70
NO2 75
65
168e170 C18H22N4S
153e156 C18H21N5O2S
183e185 C18H21FN4S
201e202 C18H21ClN4S
326
371
344
360.5
340
F
Cl 65
CH3 55
2j
253
C19H24N4S

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M.D. Altıntop et al. / European Journal of Medicinal Chemistry 46 (2011) 5562e5566
Table 2
Anticandidal activities of the compounds (2aej) as MIC values (mg/mL).
2a
2ba
2c
2d
2e
2f
2ga
2h
2i
2j
Ketoconazole
A
B
C
D
E
F
0.09
0.08
0.04
0.09
0.37
0.18
0.37
0.18
0.09
0.09
0.09
0.09
0.18
0.04
0.75
0.09
0.18
1.5
1.5
1.5
0.18
1.5
0.75
0.75
0.75
0.75
0.75
0.37
1.5
0.75
0.75
0.75
1.5
0.37
0.37
1.5
0.09
0.09
0.09
0.18
0.18
0.37
0.18
0.09
0.09
0.09
0.09
0.18
0.18
0.18
0.09
0.09
0.37
0.09
0.37
0.75
0.09
0.37
0.09
0.09
0.18
0.04
0.09
0.09
0.37
0.02
0.04
0.04
0.09
0.18
0.09
0.75
0.18
0.37
0.09
0.18
0.03
0.03
0.015
0.003
0.003
0.007
0.015
0.007
G
H
1.5
0.37
0.75
0.75
Ketoconazole: 16 mg/1 mL
A: C. albicans (Clinical Isolate, Osmangazi University, Faculty of Medicine, Eskis¸ ehir, Turkey), B: C. albicans (ATCC 90028), C: C. glabrata (Clinical Isolate, Osmangazi University,
Faculty of Medicine, Eskis¸ ehir, Turkey), D: C. tropicalis (NRRL Y-12968), E: C. krusei (NRRL Y-7179), F: C. parapsilosis (NRRL Y-12696), G: C. albicans (NRRL Y-12983), H:
C. glabrata (Clinical Isolate, Anadolu University, Faculty of Science, Department of Biology, Eskis¸ ehir, Turkey).
a
The compounds were solved in 12 mg/2 mL DMSO, other derivatives were solved in 24 mg/2 mL DMSO.
moiety (2aee) against Candida species. It can be attributed to
increased lipophilicity associated with cyclohexyl group.
Compound 2i is the most effective compound against C. albicans
(ATCC 90028), C. parapsilosis, C. albicans (NRRL Y-12983), Candida
glabrata (Clinical Isolate, Anadolu University, Faculty of Science).
Among Candida species, C. albicans (ATCC 90028) is the most
susceptible fungus to compound 2i. Compound 2i exhibits the
inhibitory activity against C. albicans (ATCC 90028) with a MIC
value of 0.03 mg/mL, whereas ketoconazole exhibits the inhibitory
activity with an MIC value of 0.04 mg/mL. This outcome confirms
that cycloaliphatic group and p-chlorophenyl substituent on tri-
azolothiadiazine ring may have a considerable influence on anti-
candidal activity.
and tetramethylsilane was used as an internal standard. Mass
spectra were recorded on a VG Quattro Mass spectrometer (Agi-
lent, Minnesota, USA). Elemental analyses were performed on
a Perkin Elmer EAL 240 elemental analyser (Perkin Elmer, Nor-
walk, CT, USA).
5.1.1. General procedure for the synthesis of the compounds
5.1.1.1. 4-Amino-5-(2-cyclohexyl/(4-hydroxyphenyl)ethyl)-2,4-dihydro-
3H-1,2,4-triazol-3-thione (1aeb). A mixture of 3-cyclohexyl/(4-
hydroxyphenyl)propionic acid (0,1 mol) and thiocarbohydrazide
(0,1 mol) was heated in an oil bath at 160e170 ꢀC for 2 h. It was then
dispersed with hot water and collected by filtration. The product was
crystallized from ethanol.
The cytotoxic effects of the compounds were also investigated
and compound 2a possessed the highest cytotoxicity, whereas
compound 2f exhibited the lowest cytotoxicity against NIH/3T3
cells.
5.1.1.2. 3-[2-Cyclohexyl/(4-hydroxyphenyl)ethyl]-6-aryl-7H-1,2,4-triaz-
olo[3,4-b]-1,3,4-thiadiazines (2aej). A solution of triazole (0.005 mol)
and appropriate phenacyl bromide (0.005 mol) in absolute ethanol
(30 mL) was heated under reflux for 1 h, cooled to room temperature
and then neutralized with ammonium hydroxide. The product thus
obtained was recrystallized from ethanol.
5. Experimental
5.1.1.2.1. 3-[2-(4-Hydroxyphenyl)ethyl]-6-phenyl-7H-1,2,4-triazolo
[3,4-b]-1,3,4-thiadiazine (2a). 1H-NMR (400 MHz, DMSO-d6):
2.90e2.94 (2H, t, phenol-CH2), 3.08e3.12 (2H, t, CH2-triazole), 4.32
(2H, s, S-CH2), 6.62e6.65 (2H, d, J ¼ 8.44 Hz, phenol-H3, H5),
6.99e7.01 (2H, d J ¼ 8.44 Hz, phenol-H2, H6), 7.63e7.66 (3H, m,
phenyl H3, H4, H5), 7.98e8.00 (2H, dd, J ¼ 1.55, J ¼ 1.50, phenyl H2,
H6), 9.20 (1H, s, OeH).
5.1. Chemistry
All reagents were purchased from commercial suppliers and
were used without further purification. Melting points (m.p.) were
determined on an Electrothermal 9100 melting point apparatus
(Weiss-Gallenkamp, Loughborough, UK) and are uncorrected.
Proton nuclear magnetic resonance (1H-NMR) spectra were
recorded on Bruker 400 MHz spectrometer (Bruker, Billerica, MA,
USA). Carbon nuclear magnetic resonance (13C-NMR) spectra were
recorded on Bruker 300 MHz spectrometer (Bruker, Billerica, MA,
USA). Chemical shifts were expressed in parts per million (ppm)
13C-NMR (300 MHz, DMSO-d6): 22.91 (CH2), 26.35 (CH2), 31.63
(CH2), 115.08 (2CH), 127.39 (2CH), 129.01 (2CH), 129.24 (2CH),
130.48 (CH), 131.80 (C), 133.55 (C), 140.04 (C), 152.38 (C), 154.63 (C),
155.65 (C).
For C18H16N4OS, calculated: C, 64.26; H, 4.79; N, 16.65; found: C,
64.25; H, 4.80; N, 16.65.
Table 3
In vitro cytotoxicity of the compounds (2aej).
MS (FAB) [M þ 1]þ: m/z 337.
Compound
IC50
(
m
g/mL)a
5.1.1.2.2. 3-[2-(4-Hydroxyphenyl)ethyl]-6-(4-nitrophenyl)-7H-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine (2b). 1H-NMR (400 MHz,
DMSO-d6): 2.90e2.94 (2H, t, phenol-CH2), 3.08e3.12 (2H, t, CH2-
triazole), 4.40 (2H, s, S-CH2), 6.62e6.65 (2H, d, J ¼ 8.43 Hz, phenol-
H3, H5), 6.99e7.01 (2H, d, J ¼ 8.42 Hz, phenol-H2, H6), 8.23e8.26
(2H, d, J ¼ 8.94 Hz, p-nitrophenyl H2, H6), 8.39e8.41 (2H, d,
J ¼ 8.96 Hz, p-nitrophenyl H3, H5), 9.20 (1H, s, OeH).
13C-NMR (300 MHz, DMSO-d6): 22.98 (CH2), 26.35 (CH2), 31.63
(CH2), 115.08 (2CH), 124.07 (2CH), 128.73 (2CH), 129.24 (2CH),
133.55 (C), 139.52 (C), 139.87 (C), 149.08 (C), 152.93 (C), 154.01 (C),
155.65 (C).
2a
2b
2c
2d
2e
2f
2g
2h
2i
31.6 þ 2.8
306.7 þ 27.5
366.7 þ 28.9
86.7 þ 10.4
400 þ 100
433 þ 28.8
210 þ 50
106 þ 28.8
103 þ 32.14
198 þ 41.9
2j
a
Cytotoxicity of the compounds to mouse fibroblast
(NIH/3T3) cell line. Incubation for 24 h. IC50 is the drug
concentration required to inhibit 50% of the cell growth.
For C18H15N5O3S, calculated: C, 56.68; H, 3.96; N, 18.36; found:
C, 56.69; H, 3.96; N, 18.35.
The values represent mean
triplicate determinations.
ꢁ
standard deviation of
MS (FAB) [M þ 1]þ: m/z 382.

M.D. Altıntop et al. / European Journal of Medicinal Chemistry 46 (2011) 5562e5566
5565
5.1.1.2.3. 3-[2-(4-Hydroxyphenyl)ethyl]-6-(4-fluorophenyl)-7H-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine (2c). 1H-NMR (400 MHz,
DMSO-d6): 2.90e2.94 (2H, t, phenol-CH2), 3.08e3.12 (2H, t, CH2-
triazole), 4.36 (2H, s, S-CH2), 6.62e6.65 (2H, d, J ¼ 8.45 Hz, phenol-
H3, H5), 6.99e7.01 (2H, d J ¼ 8.45 Hz, phenol-H2, H6), 7.42e7.45 (2H,
m, p-fluorophenyl H3, H5), 8.05e8.10 (2H, dd, J ¼ 5.38, J ¼ 5.41, p-
fluorophenyl H2, H6), 9.20 (1H, s, OeH).
For C18H21N5O2S, calculated: C, 58.20; H, 5.70; N,18.85; found: C,
58.20; H, 5.69; N, 18.85.
MS (FAB) [M þ 1]þ: m/z 372.
5.1.1.2.8. 3-[2-Cyclohexylethyl]-6-(4-fluorophenyl)-7H-1,2,4-triaz-
olo[3,4-b]-1,3,4-thiadiazine (2h). 1H-NMR (400 MHz, DMSO-d6):
0.90e0.93 (2H, m, cyclohexyl-H), 1.15e1.30 (4H, m, cyclohexyl-H),
1.59e1.77 (7H, m, cyclohexyl-H, cyclohexyl-CH2), 2.87e2.90 (2H, t,
CH2-triazole), 4.40 (2H, s, S-CH2), 7.43e7.47 (2H, m, p-fluorophenyl
H2, H6), 8.07e8.10 (2H, m, p-fluorophenyl H3, H5).
13C-NMR (300 MHz, DMSO-d6): 21.41 (CH2), 22.98 (CH2), 25.68
(2CH2), 26.06 (CH2), 32.43 (2CH2), 33.88 (CH2), 36.37 (CH), 129.00
(2CH), 129.26 (2CH), 130.44 (C), 131.80 (C), 140.04 (C), 152.99 (C),
153.56 (C).
13C-NMR (300 MHz, DMSO-d6): 22.75 (CH2), 26.33 (CH2), 31.68
(CH2), 115.07 (2CH), 129.00 (2CH), 129.19 (2CH), 129.26 (2CH),
130.44 (C), 132.36 (C), 136.63 (C), 139.93 (C), 152.99 (C), 153.56 (C),
155.66 (C).
For C18H15FN4OS, calculated: C, 61.00; H, 4.27; N,15.81; found: C,
61.00; H, 4.28; N, 15.80.
MS (FAB) [M þ 1]þ: m/z 355.
For C18H21FN4S, calculated: C, 62.76; H, 6.15; N, 16.27; found: C,
62.75; H, 6.15; N, 16.25.
5.1.1.2.4. 3-[2-(4-Hydroxyphenyl)ethyl]-6-(4-chlorophenyl)-7H-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine (2d). 1H-NMR (400 MHz,
DMSO-d6): 2.90e2.94 (2H, t, phenol-CH2), 3.08e3.12 (2H, t, CH2-
triazole), 4.32 (2H, s, S-CH2), 6.62e6.65 (2H, d, J ¼ 8.45 Hz, phenol-
H3, H5), 6.99e7.01 (2H, d, J ¼ 8.46 Hz, phenol-H2, H6), 7.63e7.66
(2H, d, J ¼ 8.72 Hz, p-chlorophenyl H3, H5), 7.98e8.00 (2H, d,
J ¼ 8.69 Hz, p-chlorophenyl H2, H6), 9.20 (1H, s, OeH).
13C-NMR (300 MHz, DMSO-d6): 22.75 (CH2), 26.33 (CH2), 31.68
(CH2), 115.07 (2CH), 129.00 (2CH), 129.19 (2CH), 129.26 (2CH), 130.44
(C),132.36 (C),136.63 (C),139.93 (C),152.99 (C),153.56 (C),155.66 (C).
For C18H15ClN4OS, calculated: C, 58.30; H, 4.08; N, 15.11; found:
C, 58.32; H, 4.08; N, 15.10.
MS (FAB) [M þ 1]þ: m/z 345.
5.1.1.2.9. 3-[2-Cyclohexylethyl]-6-(4-chlorophenyl)e7H-1,2,4-tria-
zolo[3,4-b]-1,3,4-thiadiazine (2i). 1H-NMR (400 MHz, DMSO-d6):
0.90e0.95 (2H, m, cyclohexyl-H), 1.05e1.31 (4H, m, cyclohexyl-H),
1.59e1.77 (7H, m, cyclohexyl-H, cyclohexyl-CH2), 2.90e2.94 (2H, t,
CH2-triazole), 4.44 (2H, s, S-CH2), 7.67e7.69 (2H, d, J ¼ 8.55 Hz, p-
chlorophenyl H2, H6), 8.04e8.06 (2H, d, J ¼ 8.55 Hz, p-chlorophenyl
H3, H5).
13C-NMR (300 MHz, DMSO-d6): 21.41 (CH2), 22.98 (CH2), 25.68
(2CH2), 26.06 (CH2), 32.43 (2CH2), 33.88 (CH2), 36.37 (CH), 129.00
(2CH), 129.26 (2CH), 130.44 (C), 131.80 (C), 140.04 (C), 152.99 (C),
153.56 (C).
MS (FAB) [M þ 1]þ: m/z 371.
5.1.1.2.5. 3-[2-(4-Hydroxyphenyl)ethyl]-6-(4-methylphenyl)-7H-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine (2e). 1H-NMR (400 MHz,
DMSO-d6): 2.40 (3H, s, CH3), 2.92e2.95 (2H, t, phenol-CH2),
3.09e3.12 (2H, t, CH2-triazole), 4.33 (2H, s, S-CH2), 6.64e6.66 (2H, d,
J ¼ 8.41 Hz, phenol-H3, H5), 7.01e7.03 (2H, d, J ¼ 8.42 Hz, phenol-H2,
H6), 7.38e7.40 (2H, d, J ¼ 8.15 Hz, p-methylphenyl H2, H6), 7.89e7.91
(2H, d, J ¼ 8.27 Hz, p-methylphenyl H3, H5), 9.19 (1H, s, OeH).
13C-NMR (300 MHz, DMSO-d6): 21.00 (CH3), 23.00 (CH2), 27.00
(CH2), 32.00 (CH2), 115.50 (2CH), 128.00 (2CH), 129.90 (2CH), 130.90
(2CH), 131.00 (C), 132.36 (C), 140.60 (C), 142.90 (C), 153.50 (C),
155.00 (C), 156.20 (C).
For C18H21ClN4S, calculated: C, 59.90; H, 5.86; N, 15.52; found: C,
59.90; H, 5.85; N, 15.50.
MS (FAB) [M þ 1]þ: m/z 361.
5.1.1.2.10. 3-[2-Cyclohexylethyl]-6-(4-methylphenyl)-7H-1,2,4-
triazolo[3,4-b]-1,3,4-thiadiazine (2j). 1H-NMR (400 MHz, DMSO-
d6): 0.90e0.95 (2H, m, cyclohexyl-H), 1.05e1.31 (4H, m, cyclohexyl-
H), 1.61e1.77 (7H, m, cyclohexyl-H, cyclohexyl-CH2), 2.40 (3H, s,
CH3), 2.94e2.97 (2H, t, CH2-triazole), 4.45 (2H, s, S-CH2), 7.40e7.42
(2H, d, J ¼ 8.13 Hz, p-methylphenyl H2, H6), 7.94e7.96 (2H, d,
J ¼ 8.27 Hz, p-methylphenyl H3, H5).
13C-NMR (300 MHz, DMSO-d6): 20.97 (CH3), 21.41 (CH2), 22.98
(CH2), 25.68 (2CH2), 26.06 (CH2), 32.43 (2CH2), 33.88 (CH2), 36.37
(CH), 127.39 (2CH), 129.01 (2CH), 130.48 (C), 131.80 (C), 140.04 (C),
152.38 (C), 154.63 (C).
For C19H18N4OS, calculated: C, 65.12; H, 5.18; N, 15.99; found: C,
65.10; H, 5.18; N, 16.00.
MS (FAB) [M þ 1]þ: m/z 351.
5.1.1.2.6. 3-[2-Cyclohexylethyl]-6-phenyl-7H-1,2,4-triazolo[3,4-
b]-1,3,4-thiadiazine (2f). 1H-NMR (400 MHz, DMSO-d6): 0.84e0.95
(2H, m, cyclohexyl-H), 1.05e1.31 (4H, m, cyclohexyl-H), 1.60e1.77
(7H, m, cyclohexyl-H, cyclohexyl-CH2), 2.87e2.91 (2H, t, CH2-tri-
azole), 4.41 (2H, s, S-CH2), 7.57e7.62 (3H, m, phenyl H3, H4, H5),
8.00e8.02 (2H, m, phenyl H2, H6).
For C19H24N4S, calculated: C, 67.02; H, 7.10; N, 16.45; found: C,
67.00; H, 7.10; N, 16.46.
MS (FAB) [M þ 1]þ: m/z 341.
5.2. Microbiology
13C-NMR (300 MHz, DMSO-d6): 21.41 (CH2), 22.98 (CH2), 25.68
(2CH2), 26.06 (CH2), 32.43 (2CH2), 33.88 (CH2), 36.37 (CH), 127.39
(2CH), 129.01 (2CH), 130.48 (CH), 131.80 (C), 140.04 (C), 152.38 (C),
154.63 (C).
5.2.1. Anticandidal evaluation
A
modified microbroth dilution method was carried out
according to the procedure and a previous work [20,21]. Ketoco-
nazole was used as a reference drug.
For C18H22N4S, calculated: C, 66.22; H, 6.79; N, 17.16; found: C,
66.20; H, 6.80; N, 17.15.
The compounds (2aej) were tested in vitro against C. albicans
(Clinical isolate, Osmangazi University, Faculty of Medicine, Eski-
s¸ ehir, Turkey), C. albicans (ATCC 90028), C. glabrata (Clinical isolate,
Osmangazi University, Faculty of Medicine, Eskis¸ ehir, Turkey),
C. tropicalis (NRRL Y-12968), C. krusei (NRRL Y-7179), C. parapsilosis
(NRRL Y-12696), C. albicans (NRRL Y-12983), C. glabrata (Clinical
Isolate, Anadolu University, Faculty of Science, Department of
Biology, Eskis¸ ehir, Turkey).
MS (FAB) [M þ 1]þ: m/z 327.
5.1.1.2.7. 3-[2-Cyclohexylethyl]-6-(4-nitrophenyl)-7H-1,2,4-triazolo
[3,4-b]-1,3,4-thiadiazine (2g). 1H-NMR (400 MHz, DMSO-d6):
0.90e0.93 (2H, m, cyclohexyl-H), 1.04e1.30 (4H, m, cyclohexyl-H),
1.59e1.77 (7H, m, cyclohexyl-H, cyclohexyl-CH2), 2.87e2.91 (2H, t,
CH2-triazole), 4.46 (2H, s, S-CH2), 8.23e8.26 (2H, d, J ¼ 8.95 Hz, p-
nitrophenyl H2, H6), 8.39e8.41 (2H, d, J ¼ 8.93 Hz, p-nitrophenyl H3,
H5).
5.3. Toxicity
13C-NMR (300 MHz, DMSO-d6): 21.41 (CH2), 22.98 (CH2), 25.68
(2CH2), 26.06 (CH2), 32.43 (2CH2), 33.88 (CH2), 36.37 (CH), 124.07
(2CH), 128.73 (2CH), 133.55 (C), 139.52 (C), 139.87 (C), 152.93 (C),
154.01 (C).
The level of cellular MTT (Sigma) reduction was quantified as
previously described in the literature with small modifications
[22,23].

5566
M.D. Altıntop et al. / European Journal of Medicinal Chemistry 46 (2011) 5562e5566
5.3.1. Cell culture and drug treatment
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NIH/3T3 cells were obtained from the American Type Culture
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made with fresh culture medium (the concentration of DMSO in the
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