Synthesis and in vitro antifungal and cytotoxicity evaluation of tlnazolo-4H-1,2,4-triazoles and 1,2,3-thiadiazolo-4H-1,2,4-triazoles

Article abstract of DOI:10.1002/1521-4184(200010)333:10<347::AID-ARDP347>3.0.CO;2-6

The increasing clinical importance of drag-resistant fungal pathogens has lent additional urgency to microbiological and antifungal research. Various thiazolo(or 1,2,3-thiadiazolo)thiosemicarbazides (2a-2e), 3-thiono-1,4-dihydrotriazolothiazoles-(or 1,2,3-thiadiazoles) (3a-3e), their related substituted thio-4H-1,2,4-triazoles (4a-4p) and sulfones (5a-5o) were synthesized. Most of the compounds tested for antifungal activity exhibited significant effects against Cryptococcus neoofrmans and Sacchromyces cerevisiae at MIC ranges of 0.53 to 12.5μg/mL, whereas their activities were moderate against Candida albicans and weak against Aspergillus fumigatus. At 10 ppm concentration, all compounds showed low toxicity on brine shrimps (higher than 80% survival), except compounds 4c and 2c. At 100 ppm concentration most of the compounds showed toxicity except compounds 2b, 2e, 3c, 3d, 3e, and 4e. Compounds 4b, 4c, and 4h showed in vitro cytotoxicity against Kbalb cell lines and compounds 4c and 4g against 143B cell lines at 0.1 mM concentration.

Full text of DOI:10.1002/1521-4184(200010)333:10<347::AID-ARDP347>3.0.CO;2-6

Thiazolo- and 1,2,3-Thiadiazolo-4H-1,2,4-triazoles
347
Synthesis and in Vitro Antifungal and Cytotoxicity Evaluation of
Thiazolo-4H-1,2,4-triazoles and 1,2,3-Thiadiazolo-4H-1,2,4-triazoles
a)
b)
a)
a)
c)
Amir Reza Jalilian , Saeed Sattari , Maria Bineshmarvasti , Abbas Shafiee , and Mohsen Daneshtalab *
a) Department of Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box:14155-6451, Tehran, Iran
b) Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2N8, Canada
c) School of Pharmacy, Memorial University of New Foundland, St. John’s, New Foundland A1B 3V6, Canada
Key Words: 1,2,4-4H-Triazoles; thiazoles; 1,2,3-thiadiazoles; brine shrimp toxicity; in vitro cytotoxicity; antifungal activity
substituted triazole analogues have been reported to exhibit
Summary
excellent efficacy in in vivo animal models of candidiasis and
[5]
aspergillosis . Several small molecule triazole-, thiadiaz-
The increasing clinical importance of drug-resistant fungal patho-
gens has lent additional urgency to microbiological and antifungal
research. Various thiazolo(or 1,2,3-thiadiazolo)thiosemicarbaz-
ides (2a–2e), 3-thiono-1,4-dihydrotriazolothiazoles-(or1,2,3-thia-
diazoles) (3a–3e), their related substituted thio-4H-1,2,4-triazoles
(4a–4p) and sulfones (5a–5o) were synthesized. Most of the
compounds tested for antifungal activity exhibited significant ef-
fects against Cryptococcus neoofrmans and Sacchromyces cere-
visiae at MIC ranges of 0.53 to 12.5µg/mL, whereas their activities
were moderate against Candida albicans and weak against Asper-
gillus fumigatus. At 10 ppm concentration, all compounds showed
low toxicity on brine shrimps (higher than 80% survival), except
compounds 4c and 2c. At 100 ppm concentration most of the
compounds showed toxicity except compounds 2b, 2e, 3c, 3d, 3e,
and 4e. Compounds 4b, 4c, and 4h showed in vitro cytotoxicity
against Kbalb cell lines and compounds 4c and 4g against 143B
cell lines at 0.1 mM concentration.
ole-, and oxadiazole-based heterocycles have also been re-
ported to possess potential bioactivity, including anti-
[6–14]
microbial, anticonvulsant, antiallergy, etc.
. In continu-
ation of our current research on the synthesis and biological
evaluation of small bioactive heterocyclic molecules a new
series of thiazolo-1H-1,2,4-triazole and 1,2,3-thiadiazolo-
4H-1,2,4-triazoles were synthesized and their antifungal and
cytotoxic properties were evaluated.
Chemistry
1,2,3-Thiadiazolo-1,2,4-triazoles, bearing alkyl or aryl sub-
stituent on the triazole ring were synthesized fortheir possible
antifungal effect. In order to prepare different thiosemicar-
bazides in alkaline aqueous phase, isothiocyanates were al-
lowed to react with various hydrazides. The more sterically
hindered isothiocyanates afforded less chemical yields
Introduction
Since 1980s, the risk of opportunistic fungal infections has (R = 1-butyl: 85%, R = 2-naphthyl: 76% and R = 1-
1
1
1
been greatly increased due to the increasing number of im- adamantyl: 60%). The substitution on the thiosemicarbazide
munocompromised patients such as those infected with played an important role in cyclization step. Namely, the
HIV-1, organ-transplant patients, and those undergoing can- semicarbazides with larger substituents showed lower reac-
[1]
cer chemotherapy . This rising incidence of fungal infec- tion yield, compared to the analogues with smaller substi-
tions, along with the emergence of fungal resistance to tuents (R = 1-butyl: 85%, R = 2-naphthyl: 71% and
1
1
[2]
conventionally-utilized azole antifungals , and severe tox-
R = 1-adamantyl: 62%). All cyclizations were accom-
1
[3]
icity of amphotericin B , has added considerable urgency to plished in aqueous media, except adamantyl derivatives, due
the pursuit of safe and effective therapies in the past decade. to their low solubility in water. In these cases, DMF was
In this respect, the search for new antifungal agents which are added to improve the solubility. In thioalkylation reactions,
fungicidal, have a broad-spectrum of activity, and have fewer the solubility of thionotriazole salt in aqueous media played
side effects has become critical. Traditionally, small mole- an important role in the completion of reaction time (1-butyl:
cules have been a reliable source for discovering novel bio- 2 h, 2-naphthyl: 4 h, and 1-adamantyl: 20 h). To improve
logically active compounds. Compared to their natural solubility, ethanol was added to the alkaline mixture. In most
counterparts with complex structures, these molecules are cases, we observed only the formation of thioalkyl(or benzyl)
easily synthesized and their smooth structural optimization derivative, except the reaction between 4-(1-butyl)-3-thiono-
would usually lead to a feasible candidate compound. 5-(1,2,3-thiadiazol-5-yl)-1,2,4-triazole and 4-methoxyben-
Through utilization of combinatorial chemistry large libraries zyl chloride. In this case, both thioalkyl and N -alkylated
1
of small molecules have been generated and screened for triazole were isolated. Oxidation reaction of substituted thio
specific biological activities. We have previously reported derivatives using MCPBA, was sensitive to water and quality
the synthesis and antimicrobial activity of a series of of the MCPBA used. Thus, a yield of 60–80% was obtained
substituted-thiazolo-1,3,4-thiadiazole, -1,3,4-oxadiazole, by recrystallization of MCPBA and using anhydrous dichlo-
[4]
and -1,2,4-triazole derivatives . Alkylthio- or alkylsulfonyl- romethane.
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0365-6233/00/1010/0347 $17.50 +.50/0

348
Daneshtalab and co-workers
Table 1. Minimum inhibitory concentration (MIC) (µg/mL) of different
compounds against selected fungal speciesa.
——————————————————————————————
No.
Candida
albicans
Saccharo- Crypto-
myces coccus
cerevisiae neo-
Asper-
gillus
fum-
ATCC
14053
PLM
454
formans
PLM 589
igatus
PLM 112b
——————————————————————————————
2b
12.5
12.5
25
6.25
6.25
3.12
6.25
6.25
6.25
6.25
6.25
3.12
1.06
6.25
6.25
6.25
6.25
3.12
3.12
25
25
25
25
25
25
25
25
25
25
25
25
25
25
2c
2d
12.5
25
3.12
2e
12.5
12.5
12.5
25
3a
3b
3.12
6.25
3d
3e
25
12.5
4b
12.5
12.5
25
6.25
0.53
4c
4e
12.5
4d
25
25
25
25
4l
25
5f
25
Amphotericin B
Fluconazole
0.26
0.26
0.13
25
0.26
0.53
0.26
3.25
——————————————————————————————
a
Microbroth dilution method according to NCCLS was used to determine
b
the MICs.
MICs observed at the concentration, at which, compounds
remain soluble in media (25 µg/mL).
Scheme 1. Synthesis of the substituted 1,2-4H-triazoles. a: NaOH, C2H5OH,
H2O, R1N=C=S; b: NaOH, H2O, reflux; c: R2X, NaOH, C2H5OH; d:
mCPBA; CH2Cl2; RT.
Table 2. Kbalb and 143B cell lines survival (%) in the presence of different
concentrations of some compounds.
——————————————————————————————
Results and Discussion
No.a
Kbalbb
(1 mM)
Kbalb
(0.1 mM)
143B
143B
(0.1 mM)
In order to obtain novel chemotherapeutic heterocyclic
cores, we synthesized new thiazolo- and 1,2,3-thiadiazolo-
1,2,4-triazoles that showed antifungal activities on some
pathogenic fungal organisms. Some of the compounds also
showed cytotoxic activity against 143B and Kbalb cancer cell
cultures in vitro. Compounds 2b, 2c, 3a, 3b, and specially 4c
(4c, MIC = 0.53µg/mL compared to MIC = 0.53µg/mL for
fluconazole and MIC = 0.26µg/mL for Amphotricin B)
showed significant inhibitory effect on Cryptococcus neofor-
mans, whereas, most compounds showed moderate effects on
Candida albicans. The detailed data are tabulated in Table 1.
Compounds 4b, 4g, and 4c showed a significant cytotoxic
activity against cell cultures (34% Kbalb cell survival for 4c
at 1 mM, 43% for 4b and 52% for 4g) and (33% for 4c at
0.1 mM, 53% for 4b, 30% for 4g in 143B cell line). At 1 mM
concentration the thioalkyl compounds, 4c, 4b, and 4h
showed significant cytotoxicity against Kbalb cell line. In
143B cell line, only 4c and 4g showed significant toxicity at
0.1 mM concentration, while 4b and 4i showed moderate
toxicity. The detailed data are tabulated in Table 2.
(1 mM)c
——————————————————————————————
2c
3c
3a
4c
4b
2d
4h
4g
4k
4i
69
68
73
34
43
50
47
52
51
65
85
82
77
42
56
69
82
63
100
89
80
74
100
33
53
62
87
30
68
37
82
86
100
47
60
100
100
45
78
59
——————————————————————————————
a
Tests were carried out in duplicate and variability of results between
b
duplicate runs was no more than one dilution. Kirsten virus-transformed
murine sarcoma cell line. c Human osteosarcoma.
Arch. Pharm. Pharm. Med. Chem. 333, 347–354 (2000)

Thiazolo- and 1,2,3-Thiadiazolo-4H-1,2,4-triazoles
349
Table 3. Survival percentage of shrimps in the presence of different concen-
trations (100 and 10 ppm) of compounds.
in their lipophilic characteristics), while contributes to a
modest increase in the toxicity profiles of the compounds
(4b–k). The activity of all the compounds against Aspergillus
fumigatus was negligible, while some of the compounds
exhibited a modest activity against Candida albicans. Most
of the antifungal active compounds, specifically 4c, have
shown significant activity against Cryptococcus neoformans,
indicating a mode of action different from the presently
marketed antifungal agents.
——————————————————————————————
No.
100 ppm
10 ppm
No.
100 ppm
10 ppm
——————————————————————————————
2b
2c
2d
2e
3a
3c
3d
3e
4b
93
0
100
63
4c
4e
4g
4h
4i
23
90
0
53
96
73
86
40
80
93
82
60
100
100
90
86
30
70
0
96
Acknowledgments
100
82
We gratefully acknowledge Professor Leonard I. Wiebe for his unfailing
support and the technical assistance of Mrs. Panteha Khalili for performing
cell line toxicity tests.
96
4k
100
96
Sea Water 100
100
100
0
DMSO
Taxol
100
0
86
Experimental
——————————————————————————————
Materials
Toxicity test on brine shrimps showed that these com-
pounds have low toxicity. There was no correlation between
brine shrimp and cell cytotoxicity with most of compounds
except compounds 4h, 4g, and 4c. The compounds 2c and 4c
were the most toxic to the shrimps even at 10 ppm concentra-
tion. The minority of the compounds demonstrated consider-
able toxicity against brine shrimps. The detailed data are
tabulated in Table 3.
Taking all the above biological data in consideration, it can
be concluded that the acyclic compounds (2b–e), and their
cyclic counterparts (3b–e), while exhibiting significant anti-
fungal effect against pathogenic Candida and Cryptococcus
species, show minimal toxicity on Kbalb cells and brine
shrimp. Alkylation of the mercapto group, in general, im-
proves the antifungal properties (probably due to the increase
Most chemicals were purchased from Aldrich Chemical Company, Mil-
waukee, WI. Thiazolyl blue (MTT) was purchased from Sigma. Ethyl
4-methyl-1,2,3-thiadiazole-5-carboxylate was prepared according to a pre-
viously reported procedure[15]. 4-Methylthiazole-5-carboxylic acid hydraz-
ide was prepared according to literature[16]. All chemicalswere recrystallized
or redistilled before use. Flash chromatography was performed on Silica gel
60, class 70–230, 0.063–0.2 mm mesh, Rose Scientific. Two cell lines were
used in this study, the Kbalb (a Kirsten virus-transformed murine sarcoma
cell line, ATCC) and 143B cell lines, a human osteosarcoma cell line,
obtained from ATCC. Agar dilution method according NCCLS was used to
determine the antifungal potency of the compounds1). Brine shrimp test was
performed for in vitro toxicity evaluation of the compounds according to the
–——
1) The fungal strains used were Candidiae albicans ATCC 14053, Crypto-
coccus neoformans PLM589, Saccharomyces cerevisiae PLM 454, and
Aspergillus niger PLM 1140.
Table 4. Physical constants for compounds (2a) to (2e)a).
—————————————————————————————————————————————————————————————–
No.
R1
Time
(h)
Yield
(%)
Mp
Method
1H NMR (DMSO-d6)
(°C)b)
—————————————————————————————————————————————————————————————–
2a
2-naphthyl
3
76
165–167
A
8.5 (bs, 1H, NH), 7.94 (bs, 2H, NH), 7.35–7.71 (m, 7H, aromatic),
2.87 (s, 3H, CH3), 2.75 (s, 3H, CH3)
2b
1-butyl
2
85
215–220
A
9.2 (bs, 1H, NH), 8.5 (bs, H, NH2), 5.2 (s, 1H, NH), 3.21–3.11
(t, 2H, CH2), 2.06 (s, 3H, CH3), 1.94 (s, 3H, CH3), 1.56 (m, 2H, CH2),
1.38 (m, 2H, CH2), 0.91 (t, 3H, CH3).
2c
2d
2e
1-adamantyl 24
2-naphthyl
1-adamantyl 24
90
79
86
204–206
220–223
164–166
A
A
A
8.25 (s, 1H, NH), 7.4 (s, 1H, NH), 4.43 (s, 1H, NH), 2.15 (d, 6H, CH3),
2.07 (bs, 8H, adamantyl), 1.64 (s, 7H, adamantyl).
3
9.30 (bs, 1H, NH), 8.69 (bs, 1H, NH), 7.29–8.09 (m, 7H, aromatic),
2.09 (s, 3H, CH3).
9.35 (bs, 1H, NH), 8.60 (bs, 1H, NH), 5.6 (bs, 1H, NH), 2.11 (s, 3H, CH3),
2.07 (bs, 8H, adamantyl), 1.64 (s, 7H, adamantyl).
—————————————————————————————————————————————————————————————–
a All the compounds were crystallized in ethanol-water mixture. b All the compounds showed decomposition at their melting points.
Arch. Pharm. Pharm. Med. Chem. 333, 347–354 (2000)

350
Daneshtalab and co-workers
1
method of McLaughlin and Meyer [17,18]. The H NMR spectra were ob-
tained on a Varian Unity Plus (400 MHz) instrument, with tetramethylsilane
as the internal standard (0.00 ppm). The solvents used were CHCl3-d1 and
DMSO-d6. Infrared spectra were taken on a Perkin-Elmer 781 (KBr disks).
Thin layer chromatography (TLC) was performed on silica gel polymer-
backed (F 1500/LS 254, 20 × 20 cm, TLC Ready Foil, Schleicher & Schuell).
Purification of compounds was carried out with different mobile phase
systems, I: ethyl acetate:hexane (3:1, v/v) and II: ethyl acetate:dichlo-
romethane (1:4, v/v). ELISA reader used for cytotoxicity evaluation of
compounds was a Molecular Device. Melting points were determined on a
Reichert-Jung hot stage microscope and are uncorrected. Elemental mi-
croanalyses were within ± 0.4% of theoretical values for C, H, F, and N.
Method B: 5-Azolyl-4-substituted-2,4-dihydro-1,2,4-triazole-3-thione
(3a,3e)
Cyclization of different thiosemicarbazides to 3-thiono-1,2,4-triazoles was
performed with a modified method reported previously[19]. A mixture of
thiosemi-carbazides (2a, 2b, and 2d) (8 mmole), sodium bicarbonate (10 g,
0.12 mole), and water (100 mL) was refluxed at 120–140 °C for times given
in Table 5. The mixture was then cooled to room temperature and acidified
(pH 5) by the addition of hydrochloric acid solution (1 M), while cooling in
an ice bath. The mixture was filtered and the residue was crystallized in a
60% ethanol to give different 1,2,4-triazole derivatives, as colorless crystals
(Table 5).
Method C: 4-(1-Adamantyl)-5-azolyl-2,4-dihydro-1,2,4-triazole-3-thione
(3c and 3e)
4-Methyl-1,2,3-thiadiazole-5-carboxylic Acid Hydrazide (1b)
To a stirred mixture of ethyl 4-methyl-1,2,3-thiadiazole-5-carboxylate[15]
(8.6 g, 0.05 mole) in ethanol (100 mL), hydrazine hydrate (100 mL) was
added dropwise. The reaction mixture was warmed to 50 °C for 2 h with
vigorous stirring. The reaction was checked with TLC until completion. The
mixture was concentrated at reduced pressure to give a yellow solid which
was crystallized in ethanol to give 6.7 g of 1b as yellow needles: 85%.
mp = 115–118 °C; 1H NMR (DMSO-d6): ppm 10.8 (bs, 1H, NH), 9.45 (s,
2H, NH2), 2.10 (s, 3H, CH3). FT-IR (KBr) ν 3352 (NH), 1670 (C=O), 1560
(N=N).
A mixture of 2c or 2e (8 mmole), sodium bicarbonate (10 g, 0.12 mole),
dimethylformamide (DMF) (80 mL) and water (30 mL) were refluxed at
140 °C at different times. The mixture was then cooled to room temperature
and filtered. The precipitate was separated and the filtrate was acidified
(pH 5) by the addition of hydrochloric acid solution (1 M) in an ice bath. The
mixture was filtered and the residue was mixed with initial precipitate. The
mixed precipitate was crystallized in ethyl acetate to give the product as
colorless crystals (Table 5).
Method D: 5-Azolyl-4-substituted-3-methylthio-4H-1,2,4-triazole (4a–4e)
Alkylation of 3-thiono compounds was performed with a modification of
the previously reported method[19]. A mixture of 3a–3e (0.5 mmole) in
ethanol (5 mL) and sodium hydroxide (0.02 g, 0.5 mmole) was stirred until
a homogeneous mixture was formed. Iodomethane (30µL, 0.5 mmole) was
then added to above mixture in one portion and the reaction was accom-
plished by stirring at room temperature. The reaction mixture was checked
by TLC until the completion using solvent system I. Water (5 mL) was then
added to the mixture. In case of precipitate forming, the mixture was filtered,
and the precipitate was washed with water (3 × 10 mL). If no precipitate was
formed, the mixture was extracted with chloroform (3 × 20 mL) and the
organic layer was dried over sodium sulfate. The solvent was removed under
vacuum and the residue was crystallized in ethyl acetate:hexane mixture or
Method A: 1-(Azolyl-5-carboxyl)-4-substituted-thiosemicarbazide (2a–2c)
Different thiosemicarbazides (2a–2e) were prepared according to the
method reported previously[19]. A sodium hydroxide solution (10 mL, 1 M)
was added dropwise to a stirred mixture of 1b (1.71 g, 0.01 mole) and ethanol
(10 mL). The stirring was continued until a transparent wine-colored solution
was obtained, the related isothiocyanate (0.01 mole) was then added to above
solution in one portion and the mixture was stirred at times given in Table 4.
The reaction was cooled to room temperature, filtered, and the filtrate was
acidified (pH 5) by the addition of hydrochloric acid solution (1 M). The
precipitate was filtered and washed with water and dried to give the related
thiosemicarbazides (2a–2e) (Table 4).
Table 5. Physical constants for compounds 3a to 3e.
————————————————————————————————————————————————————————––————–
No.
R1
Time
(h)
Yield
(%)
Mp
(°C)
Method
1H NMR (DMSO-d6)
—————————————————————————————————————————————————————————————–
3a
3b
3c
2-naphthyl 12
71
85
62
75–77a
B
B
C
8.62 (bs, 1H, NH), 7.31–8.14 (m, 7H, aromatic), 2.09 (s, 3H, CH3),
1.98 (s, 3H, CH3).
1-butyl
7
215–217
240–242a
8.29 (bs, 1H, NH), 3.19 (t, 2H, CH2), 2.08 (s, 3H, CH3), 1.89 (s, 3H, CH3),
1.65 (m, 2H, CH2), 1.41(m, 2H, CH2), 0.97 (t, 3H, CH3).
1-adamantyl 48
3.11 (bs, 1H, NH), 2.55 (s, 3H, CH3), 2.53 (s, 3H, CH3), 1.56–2.13
(m, 15H, adamantyl).
3d
3e
2-naphthyl
5
73
55
185–188
230–233a
C
C
8.79 (bs, 1H, NH), 7.29–8.21 (m, 7H, aromatic), 2.13 (s, 3H, CH3).
1-adamantyl 4
9.25 (bs, 1H, NH), 2.11 (bs, 4H, adamantyl), 2.01 (s, 3H, CH3), 1.99
(s, 5H, adamantyl), 1.68 (s, 6H, adamantyl).
—————————————————————————————————————————————————————————————————————–
a These compounds were decomposed at their melting points.
Arch. Pharm. Pharm. Med. Chem. 333, 347–354 (2000)

Thiazolo- and 1,2,3-Thiadiazolo-4H-1,2,4-triazoles
351
Table 6. Physical constants for compounds (4a) to (4e):
—————————————————————————————————————————————————————————————–
No.
R1
Time
(h)
Yield
(%)
Mp
(°C)
Method
1H NMR (CHCl3-d1)
—————————————————————————————————————————————————————————————–
4a
2-naphthyl
4
2
67
72
47–50a
D
7.5–8.32 (m, 7H, aromatic), 2.64 (s, 3H, SCH3), 2.61 (s, 3H, CH3),
2.59 (s, 3H, CH3).
3.19 (t, 2H, CH2), 2.41 (s, 3H, CH3), 2.06 (s, 3H, CH3), 1.99 (s, 3H, CH3),
1.55 (m, 2H, CH2), 1.38 (m, 2H, CH2), 0.92 (t, 3H, CH3).
4b
1-butyl
oil
D
4c
4d
4e
1-adamantyl 20
2-naphthyl
1-adamantyl 18
50
70
48
154–157
62–64b
132–134
D
D
D
3.04 (s, 3H, SCH3), 2.68 (s, 6H, CH3), 1.62–2.18 (m, 15H, adamantyl).
7.31–8.35 (m, 7H, aromatic), 2.7 (s, 3H, SCH3), 2.09 (s, 3H, CH3).
2.63 (s, 3H, SCH3), 2.10 (bs, 5H, adamantyl), 2.03 (s, 3H, CH3),
1.92 (bs, 6H, adamantyl), 1.69 (s, 4H, adamantyl)
3
—————————————————————————————————————————————————————————————–
a Purified by column chromatography using solvent system I; b decomposed at their melting points.
Table 7. Physical constants for compounds (4f) to (4p) c.
————————————————————————————————————————————————————————————–—
No.
R1
X
Time
(h)
Yield
(%)
Mp
(°C)
Method
1H NMR (CHCl3-d1)
—————————————————————————————————————————————————————————————–
4f
2-naphthyl OCH3
4
86
39–41a
E
7.43–8.37 (m, 1H, aromatic), 4.35 (s, 2H, CH2), 3.82
(s, 3H, CH3), 2.08 (s, 3H, CH3), 2.00 (s, 3H, CH3)
7.47–6.82 (q, 4H, aromatic), 4.31 (s, 2H, CH2), 3.77
(s, 3H, CH3), 3.18 (m, 2H, CH2), 2.06 (s, 3H, CH3), 2.01
(s, 3H, CH3), 1.54 (m, 2H, CH2), 1.38 (m, 2H, CH2), 0.95
(t, 3H, CH3).
7.32–6.84 (q, 4H, aromatic), 4.62 (s, 2H, CH2), 3.82
(s, 3H, CH3), 3.64 (m, 2H, CH2), 2.01 (s, 3H, CH3), 1.89
(s, 3H, CH3), 1.65 (m, 2H, CH2), 1.42 (m, 2H, CH2), 0.98
(t, 3H, CH3).
4g
1-butyl
1-butyl
OCH3
OCH3
2
42
oilb
E
4h
4i
2
31
55
oilb
E
E
1-adamantyl OCH3
12
215–218
7.28–6.86 (q, 4H, aromatic), 4.46 (s, 2H, CH2), 3.82
(s, 3H, OCH3), 2.32 (d, 6H, adamantyl), 2.14 (s, 4H,
adamantyl), 2.01 (s, 3H, CH3), 1.86 (s, 3H, CH3),
1.71 (s, 5H, adamantyl).
4j
2-naphthyl CF3
3
3
54
84
137–139
oil
E
E
7.41–8.36 (m, 1H, aromatic), 4.31 (s, 2H, CH2), 2.06
(s, 3H, CH3), 2.03 (s, 3H, CH3).
4k
1-butyl
CF3
7.59–7.43 (q, 4H, aromatic), 4.39 (s, 2H, CH2), 3.58
(m, 2H, CH2), 2.01 (s, 3H, CH3), 1.88 (s, 3H, CH3), 1.63
(m, 2H, CH2), 1.43 (m, 2H, CH2), 0.95 (t, 3H, CH3).
7.63–7.47 (q, 4H, aromatic), 4.41 (s, 2H, CH2), 2.14–1.93
(m, 14H, CH3 & adamantyl), 1.64 (s, 7H, adamantyl);
7.44–8.36 (m, 1H, aromatic), 4.34 (s, 2H, CH2), 3.84
(s, 3H, CH3), 2.23 (s, 3H, CH3).
7.53–7.98 (q, 4H, aromatic), 4.65 (s, 3H, CH3), 3.88
(s, 2H, CH2), 2.21 (s, 3H, CH3), 2.14 (bs, 5H, adamantyl),
2.05 (s, 4H, adamantyl), 1.67 (s, 6H, adamantyl).
7.41–8.34 (m, 1H, aromatic), 4.32 (s, 2H, CH2), 3.81
(s, 3H, CH3), 2.19 (s, 3H, CH3).
4l
1-adamantyl CF3
2-naphthyl OCH3
1-adamantyl OCH3
10
3
73
91
86
108–111
oilb
E
E
E
4m
4n
18
121–124a
4o
4p
2-naphthyl CF3
1-adamantyl CF3
1.5
16
90
73
66–71
E
E
201–204a
7.54–7.96 (q, 4H, aromatic), 3.86 (s, 2H, CH2), 2.21
(s, 3H, CH3), 2.12 (bs, 5H, adamantyl), 2.01 (s, 4H,
adamantyl), 1.64 (s, 6H, adamantyl).
—————————————————————————————————————————————————————————————–
a These compounds were decomposed at their melting points. b These compounds were repurified by column chromatography using solvent system I, but
yielded no crystals. c Compound (3b) afforded two different products (4g, 4h) in reaction with 4-methoxybenzyl bromide.
Arch. Pharm. Pharm. Med. Chem. 333, 347–354 (2000)

352
Daneshtalab and co-workers
Table 8. Physical constants for compounds (5a) to (5o).
—————————————————————————————————————————————————————————————–
No.
R1
R2
Time
(h)
Yield
(%)
Mp
(°C)
Method 1H NMR (CHCl3-d1)
—————————————————————————————————————————————————————————————–
5a
2-naphthyl
CH3
3
2
76
81
86–88
oil
F
7.5–8.32 (m, 7H, aromatic), 3.76 (s, 3H, CH3),
2.64 (s, 3H, SCH3), 2.61 (s, 3H, CH3), 2.59
(s, 3H, CH3).
5b
1-butyl
CH3
F
3, 72 (s, 3H, CH3), 3.19 (t, 2H, CH2), 2.41
(s, 3H, CH3), 2.06 (s, 3H, CH3), 1.99 (s, 3H, CH3), 1.55
(m, 2H, CH2), 1.38 (m, 2H, CH2), 0.92 (t, 3H, CH3).
5c
1-adamantyl
2-naphthyl
CH3
8
68
76
186–189
oil
F
F
3.68 (s, 3H, CH3), 3.04 (s, 3H, SCH3), 2.68
(s, 6H, CH3), 1.62–2.18 (m, 15H, adamantyl).
5d
CH2C6H4OCH3
2.5
7.43–8.37 (m, 1H, aromatic), 4.35 (s, 2H, CH2),
3.82 (s, 3H, CH3), 3.72 (s, 3H, CH3), 2.08 (s, 3H,
CH3), 2.00 (s, 3H, CH3).
5e
5f
1-butyl
CH2C6H4OCH3
CH2C6H4OCH3
1.5
4
80
58
246–248
155–158
F
F
7.47–6.82 (q, 4H, aromatic), 4.31 (s, 2H, CH2), 3.77
(s, 3H, CH3), 3.71 (s, 3H, CH3), 3.18 (m, 2H, CH2),
2.06 (s, 3H, CH3), 2.01 (s, 3H, CH3), 1.54 (m, 2H,
CH2), 1.38 (m, 2H, CH2), 0.95 (t, 3H, CH3).
1-adamantyl
7.28–6.86 (q, 4H, aromatic), 4.46 (s, 2H, CH2),
3.82 (s, 3H, OCH3), 3.76 (s, 3H, CH3), 2.32 (d, 6H,
adamantyl), 2.14 (s, 4H, adamantyl), 2.01 (s, 3H,
CH3), 1.86 (s, 3H, CH3), 1.71 (s, 5H, adamantyl).
5g
5h
2-naphthyl
1-butyl
CH2C6H4CF3
CH2C6H4CF3
1.5
69
75
oil
F
F
7.41–8.36 (m, 1H, aromatic), 4.31 (s, 2H, CH2),
3.74 (s, 3H, CH3), 2.06 (s, 3H, CH3), 2.03 (s, 3H, CH3).
0.75
131–135
7.59–7.43 (q, 4H, aromatic), 4.39 (s, 2H, CH2),
3.71 (s, 3H, CH3), 3.58 (m, 2H, CH2), 2.01 (s, 3H,
CH3), 1.88 (s, 3H, CH3), 1.63 (m, 2H, CH2), 1.43
(m, 2H, CH2), 0.95 (t, 3H, CH3).
5i
1-adamantyl
CH2C6H4CF3
3
66
93–95
F
7.63–7.47 (q, 4H, aromatic), 4.41 (s, 2H, CH2),
3.79 (s, 3H, CH3), 2.14–1.93 (m, 14H, CH3 &
adamantyl), 1.64 (s, 7H, adamantyl).
5j
2-naphthyl
CH3
CH3
2.5
4.5
40
30
167–169
186–189
F
F
7.31–8.35 (m, 7H, aromatic), 3.69 (s, 3H, CH3),
2.7 (s, 3H, SCH3), 2.09 (s, 3H, CH3).
5k
1-adamantyl
3, 73 (s, 3H, CH3), 2.63 (s, 3H, SCH3), 2.10 (bs, 5H,
adamantyl), 2.03 (s, 3H, CH3), 1.92 (bs, 6H,
adamantyl), 1.69 (s, 4H, adamantyl).
5l
2-naphthyl
CH2C6H4OCH3
CH2C6H4OCH3
5
8
48
85
80–83
F
F
7.44–8.36 (m, 1H, aromatic), 4.34 (s, 2H, CH2),
3.84 (s, 3H, CH3), 3.77 (s, 3H, CH3), 2.23 (s, 3H, CH3).
5m
1-adamantyl
136–138
7.47–7.61 (q, 4H, aromatic), 5.11 (s, 2H, CH2),
4.08 (s, 3H, OCH3), 1.91–2.14 (m, 18H, CH3 &
adamantyl).
5n
5o
2-naphthyl
CH2C6H4CF3
CH2C6H4CF3
0.5
3
44
45
89–91
F
F
7.41–8.34 (m, 1H, aromatic), 4.32 (s, 2H, CH2),
3.81 (s, 3H, CH3), 3.74 (s, 3H, CH3), 2.19 (s, 3H, CH3).
1-adamantyl
243–245
7.54–7.96 (q, 4H, aromatic), 3.86 (s, 2H, CH2),
3.70 (s, 3H, CH3), 2.21 (s, 3H, CH3), 2.12 (bs, 5H,
adamantyl), 2.01 (s, 4H, adamantyl), 1.64 (s, 6H,
adamantyl).
—————————————————————————————————————————————————————————————–
All compounds were purified by flash chromatography using solvent system II.
Arch. Pharm. Pharm. Med. Chem. 333, 347–354 (2000)

Thiazolo- and 1,2,3-Thiadiazolo-4H-1,2,4-triazoles
353
purified by flash chromatography using solvent system I, to give 4a–4e
(Table 6).
MTT working solution was added to each well and the plates were incubated
at 37 °C in an incubator for 4 h. At the end of the 4 h, the media was carefully
aspirated from the wells, leaving approximately 20 µL in each well. Hundred
and fifty microlitres of DMSO was then added to each well and the plates
were shaken slowly on a shaker for up to 15 min to dissolve the formazan
crystals. The plates were then read at 540 nm on a scanning multiwell ELISA
reader. Table 2 summarizes the estimates of surviving cell percentage of each
cell line after exposure to the highest (1 mM) and second highest concentra-
tions (0.1 mM) of each of the investigated drugs.
Method E: 5-Azolyl-4-substituted-3-(4-substitutedbenzyl)thio-4H-1,2,4-
triazole (4f–4p)
A mixture of (3a–3e) (0.5 mmole) in ethanol (5 mL) and potassium hy-
droxide (28 mg, 0.5 mmole) was stirred for 0.5 h. 4-Methoxybenzyl chloride
(78 mg, 0.5 mmole) or 4-trifluoromethylbenzyl bromide (120 mg,
0.5 mmole) was added to above mixture in one portion and the reaction was
stirred at room temperature. The reaction progression was checked by TLC.
The solvent was removed from the reaction under vacuum, and water (5 mL)
was added to the mixture. In case of precipitate forming, the mixture was
filtered, and the precipitate was washed with water (3 × 10 mL). If no
precipitate was formed the mixture was extracted with chloroform (3 ×
20 mL) and the organic layer was dried over sodium sulfate. The solvent was
removed under vacuum and the residue was crystallized or purified by flash
chromatography using solvent system II to give (4f–4p) (Table 7).
Brine Shrimp Test
Brine shrimp toxicity test was determined on different compounds by a
modification of the previously reported methods[17,18]. Two different con-
centrations (10 and 100 ppm) of test compounds were prepared by dissolving
in DMSO as the solvent. Taxol was used as positive toxic standard. Seawater
was prepared by dissolving commercially available sea salt (3.8 g) into
tap-water (1 L). Brine shrimps hatched in seawater media at room tempera-
ture in 48 h as an air flow was bubbling through the media. Ten shrimps,
seawater (5 mL) and different amounts of each test compound, were put in
a test tube. Three blank samples were prepared containing, DMSO (50 µL in
5 mL seawater), Taxol (10 ppm in DMSO) and of seawater (5 mL) and a
blank sample of seawater alone (5 mL). All test tubes were left at room
temperature for 24 h and the survived brine shrimps were counted and
reported as survival percentage (Table 3).
Procedure F: 5-Azolyl-4-substituted-3-(substituted)sulfonyl-4H-1,2,4-tria-
zole (5a–5o)
Oxidation of thioalkyl(or benzyl) compounds was performed with a modi-
fied method previously reported[19]. MCPBA (0.6 g, 3.5 mmole) was added
in one portion, to a stirring mixture of (4a–4p) (1 mmole) in anhydrous
dichloromethane (15 mL) at 0–5°C. The stirring was continued at different
times and checked by TLC. After completion of reaction, the mixture was
filtered, and the filtrate was concentrated in vacuum. The residue was
dissolved in ethyl acetate (20 mL) and washed by saturated sodium bicar-
bonate solution (20 mL). The organic layer was washed with water (3 ×
10 mL), saturated saline solution (20 mL), and dried over sodium sulfate.
The organic layer was then concentrated in vacuum to give an oil which was
purified by flash chromatography to give 5a–5o (Table 8).
References and Notes
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Mitscher et al. (1972) with minor modifications[20,21]. The test was done in
duplicates, and the positive antifungal results were read based on no growth
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to measure the minimum inhibitory concentration (MIC) values [22]. Namely,
the fungal organisms, taken from SDA plates, were suspended in normal
saline to obtain T% = 75–77% at 530 nm, which is equal to 106 CFU/mL.
The fungal suspension was diluted 1000 times in the medium and 100µL
aliquots were added to each well. Compounds were dissolved in DMSO to
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medium (SD broth) in 96 microwell plates, leaving 100 µL in each well. The
plates were incubated at 35 °C for 24–96 h.
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formazan product, by the cancer cell mitochondrial enzyme succinate dehy-
drogenase[23]. The reduction of tetrazolium salt to the formazan, which is
proportional to the number of living cells present, can only take place in the
living cells and indicates the toxic effects of the compounds. The cells were
suspended in complete medium [Dulbecco’s Modified Eagle’s Medium
(DMEM) + fetal bovine serum 10% and antibiotic 1%] and diluted to a
concentration of approximately 8 × 103 cells/mL. The cells were then plated
in 100µL volumes (800 cells) into 96-well microplates and incubated for 24 h
at 37 °C in 5% CO2. The compounds were dissolved in DMEM and added
to the cells in 100µL volumes to produce a range of final concentrations of
the drugs (between 10–3 to 10–7 M). The control wells were filled with 100µL
of medium, without any drugs. The blank rows contained 200 µL of medium
alone (no cells). The plates were incubated for 3 days in an incubator at 37 °C
and 5% CO2. MTT working solution was prepared freshly on the day of the
assay by 1:5 dilution of the MTT stock solution in pre-warmed DMEM. MTT
stock solution is prepared by dissolving MTT in phosphate buffer solution
(PBS) at 5 mg/mL. On the day of the assay, 50 µL of the freshly prepared
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[16] W. R . Boon, J. Chem. Soc. 1945, 601–603.
Arch. Pharm. Pharm. Med. Chem. 333, 347–354 (2000)

354
Daneshtalab and co-workers
[17] B. N. Meyer, N. R. Ferrigni, J. E. Putnam, L. B. Jacobson, D. E. Nichols,
[21] L. Mitscher, R. Lue, M. S. Bathala, W. Wu, J. L Beal, Lloydia. 1972,
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[18] J. L. McLaughlin, Vol.6 in the Methods of plant Biochemistry, 1st ed.;
Hostettmann, K., Academic Press: London, 1991; pp 1–32.
[22] National Committee for Clinical Laboratory Standards (1992). Refer-
ence method for broth dilution antifungal susceptibility testing for
yeasts. Proposed standards, Document M27-P. National Committee for
Clinical Laboratory Standards, Villanova, PA.
[19] J. M. Kane, M. A. Staeger, C. R. Dalton, F. P. Miller, M. W. Dudley,
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Received: July 5, 2000 [FP504]
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Arch. Pharm. Pharm. Med. Chem. 333, 347–354 (2000)