Synthesis and in vitro antifungal and cytotoxicity evaluation of substituted 4,5-dihydronaphtho[1,2-d][1,2,3]thia(or selena)diazoles.

Article abstract of DOI:10.1016/S0014-827X(02)00029-0

Unsubstituted 4,5-dihydronaphtho[1,2-d][1,2,3]thia (or selena)diazoles (2a, 2b), prepared from the semicarbazone (1a), were nitrated using fuming nitric acid at 0 degrees C to yield various mono-nitrated dihydronaphthalenes (3a-3e). Related sulfamoyl deri

Full text of DOI:10.1016/S0014-827X(02)00029-0

Il Farmaco 58 (2003) 63Á68
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Synthesis and in vitro antifungal and cytotoxicity evaluation of
substituted 4,5-dihydronaphtho[1,2-d][1,2,3]thia(or selena)diazoles
A.R. Jalilian a, S. Sattari b, M. Bineshmarvasti a, M. Daneshtalab b, Abbas Shafiee c,*
a Cyclotron Department, Nuclear Research Center for Agriculture and Medicine, P.O.Box 31585-4395, Karaj, Iran
b Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alta., Canada T6G 2N8
c Department of Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran, Iran
Received 30 July 2002; accepted 6 November 2002
Abstract
Unsubstituted 4,5-dihydronaphtho[1,2-d][1,2,3]thia (or selena)diazoles (2a, 2b), prepared from the semicarbazone (1a), were
nitrated using fuming nitric acid at 0 8C to yield various mono-nitrated dihydronaphthalenes (3aÁ3e). Related sulfamoyl derivatives
/
(4a, 4b) were prepared using chlorosulfonic acid, followed by the addition of ammonia solution. Synthesis of 6,9-dimethoxy-4,5-
dihydronaphtho[1,2-d][1,2,3]thiadiazole derivative (2c) was performed using 5,8-dimethoxy-a-tetralone semicarbazone (1b) and
thionylchloride at low temperature. At 10 ppm concentration, all compounds showed low toxicity (higher than 80% survival) on
brine shrimps, while at 100 ppm concentration compounds 2d, 3d, and 4b exhibited toxicity (less than 60% survival). Compounds 3a,
3e, and especially 4a showed significant antifungal activity against Cryptococcus neoformans. Compound 4a, while being the most
active antifungal agent in this series, possessed low toxicity.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Antifungal activity; Cytotoxicity; 1,2,3-Thiadiazole; 1,2,3-Selenadiazole
1. Introduction
emergence of fungal resistance to azoles has made the
pursuit of safe and effective therapies in the past decade
[3,4]. In this respect, search for new antifungal agents,
which are fungicidal, have a broad-spectrum of activity,
and have fewer side effects has become critical. Small
heterocyclic molecules with potential phamacophoric
properties have attracted the attention of scientific
communities as feasible alternatives to conventional
drugs. Their synthetic diversity, easy to manipulate
skeletons and economical feasibility has made these
molecules the top runners in drug discovery endeavors.
In this context, the syntheses and antifungal activity of a
series of 1,2,3-selenadiazole, 1,2,3-thiadiazole, 1,2,4-
triazolo[4,3-a]pyrimidine, and other relevant azoloazine
derivatives has been reported [5]. Also, the syntheses and
antibacterial/antifungal activity of a series of arylsulfo-
nyl-1,2,3-selenadiazoles was previously reported [6]. We
have recently reported the syntheses and antifungal
activity of a series of thiazolo-4H-1,2,4-triazoles and
1,2,3-thiadiazolo-4H-1,2,4-triazole derivatives [7]. Based
on the above reports and in continuation of our research
on the syntheses of biologically-active small heterocyclic
The substantial increase in the incidence of fungal
infections during the past two decades has been sug-
gested as the major cause of morbidity and mortality
among immunocompromized patients [1]. Aggressive
immunosuppression, HIV-1 infection, cancer, myelo-
toxic therapeutic regimens, and organ transplantation
have opened the door to pathogenic fungal organisms.
Disseminated candidiasis, pulmonary aspergillosis, and
infections caused by emerging opportunistic fungi are
the most common of the serious mycoses [2]. Ampho-
tericin B has been the gold standard in antifungal
therapy for the past half a century, and newer chemi-
cally modified azoles, such as fluconazole and itracona-
zole, have provided the clinicians with favorable
therapeutic options. Despite remarkable efficacy of
both classes of compounds in the treatment of invasive
mycoses, severe toxic reactions to amphotericin B and
* Correspondence and reprints.
E-mail address: ashafiee@ams.ac.ir (A. Shafiee).
´
0014-827X/02/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 0 1 4 - 8 2 7 X ( 0 2 ) 0 0 0 2 9 - 0

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A.R. Jalilian et al. / Il Farmaco 58 (2003) 63Á68
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Table 1
Physical constants for compounds 3a to 3e
a
b
c
Number
Position
7
X
S
Yield (%) M.p. (8C) Rf
1H NMR (CDCl3)
3a
55
30
173Á
160Á
142Á
167Á
155Á
/
176
163
144
169
157
0.5
8.42 (d, 1H, H9, J8,9ꢂ
(d, 1H, H6, J6,8 3 Hz), 3.36 (t, 2H, CH2), 3.24 (t, 2H, CH2)
0.35 9.09 (d, 1H, H9, J7,9 2.4 Hz), 8.18 (dd, 1H, H7, J7,9ꢂ2.4 Hz, J6,7ꢂ
7.48 (d, 1H, H6, J6,7ꢂ6.7), 3.35 (t, 2H, CH2), 7.23 (t, 2H, CH2)
0.70 8.58 (dd, 1H, H9, J8,9ꢂ7.6 Hz, J7,9ꢂ2.24 Hz), 7.88 (dd, 1H, H7, J7,8ꢂ
J7,9ꢂ2.24), 7.52 (dd, 1H, H8, J8,9ꢂ7.6 Hz, J7, 8ꢂ8.2 Hz), 3.44Á3.29 (m, 4H, CH2)
0.45 8.42 (d, 1H, H9, J8,9ꢂ8.5 Hz), 8.27 (dd, 1H, H8, J8,6ꢂ2.1 Hz, J9,8ꢂ8.5 Hz),
8.20 (d, 1H, H6, J6,8ꢂ2.1 Hz), 3.38 (t, 2H, CH2), 3.25 (t, 2H,CH2)
9.10 (d, 1H, H9, J9,7 2.1Hz), 8.17 (dd, 1H, H7, J7,6ꢂ7.1 Hz, J7,9
7.48 (d, 1H, H6, J6,7ꢂ7.1 Hz), 3.39 (t, 2H, CH2), 3.22 (t, 2H, CH2)
/
9.4 Hz), 8.27 (dd, H8, J6,8ꢂ
/
3 Hz, J8,9ꢂ
/
9.4 Hz), 8.21
6.7 Hz),
8.2 Hz,
ꢂ/
3b
3c
3d
3e
8
6
7
8
S
/
ꢂ
/
/
/
/
Se 21
Se 35
Se 41
/
/
/
/
/
/
/
/
/
/
/
/
/
/
0.2
ꢂ
/
/
ꢂ/2.1 Hz),
/
a
All compounds were crystallized from ethyl acetate:hexane (65:35) mixture.
Crystals decomposed at their melting points.
Mobile phase: ethyl acetate: hexane (40:60).
b
c
molecules, the syntheses and anitfungal evaluation of
the title compounds are reported.
a Varian Unity Plus (300 MHz) instrument, with
tetramethylsilane as the internal standard. Infrared
spectra were taken on a PerkinÁ
/
Elmer 781 (KBr disks).
0.4% of theore-
tical values for C, H and N. Thin layer chromatography
Elemental microanalyses were within 9
/
2. Chemistry
(TLC) was performed on silica gel polymer-backed (F
1500/LS 254, 20ꢀ
and Schuell). Flash chromatography was performed
using silica gel 60, class 70Á230, 0.063Á0.2 mm mesh,
Rose Scientific.
/
20 cm, TLC Ready Foil, Schleicher
Reaction of thionyl chloride with the 1a according to
the procedure reported previously gave compound 2a
[8]. Compound 2b was synthesized by the reaction of
selenium dioxide with 1a in acetic acid [9,10]. Com-
pound 2c was obtained through the reaction of semi-
carbazone 1b with thionyl chloride at low temperature.
Compound 2d was synthesized according to the proce-
/
/
3.1.1. 4,5-Dihydronaphtho[1,2-d][1,2,3]-thiadiazole
(2a)
dure reported previously [11]. Compounds 3aÁ3e were
/
This compound was prepared in 46% yield from the
relevant semicarbazone according to literature [8].
synthesized through the nitration of compounds 2a and
2b. Reaction of compounds 2a or 2b with chlorosulfonic
acid followed by ammonia gave compounds 4a or 4b,
respectively.
3.1.2. 6,9-Dimethoxy-4,5-dihydronaphtho[1,2-
d][1,2,3]thiadiazole (2c)
To a recrystallized portion of 5,8-dimethoxy-a-tetra-
lone semicarbazone [12] (1b) (2.63 g, 0.01 mol) in a
3. Experimental
cooled flask in an ice-salt bath at ꢁ5 8C, distilled
/
thionyl chloride (10 ml) was added through a dropping
funnel. After addition, the flask was kept at above
temperature and the mixture was stirred vigorously for
24 h until a homogenous yellow suspension was formed.
Chloroform (30 ml) was then added to the mixture. The
final mixture was added dropwise to a beaker containing
3.1. Chemistry
Melting points were determined on a ReichertÁJung
/
hot stage microscope and are uncorrected. 1H NMR
spectra were obtained on a Brucker FT-80 (80 MHz) or

A.R. Jalilian et al. / Il Farmaco 58 (2003) 63Á
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65
Table 2
Minimum inhibitory concentration (MIC) (mg/ml) of different compounds on some fungal species
a
Number
Candidia albicans
Saccharomyces cerevisiae
Cryptococcus neoformans
Aspergilus fumigatus
2b
2c
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
0.14
0.26
12.5
25
25
12.5
12.5
12.5
3.12
12.5
12.5
25
25
25
2d
3a
25
25
25
0.53
12.5
3.12
12.5
3.12
3.12
3.12
0.06
25
3b
3c
25
25
3d
3e
25
25
3.12
0.53
12.5
0.13
0.53
12.5
4a
4b
25
25
Amphotricin B
Fluconazole
DMSO
0.14
3.25
25
3.12
a
All compounds were soluble in media and sterility of media was checked carefully.
saturated sodium bicarbonate solution (30 ml). After
neutralization, the mixture was decanted and the
Table 3
Survival percentage of shrimps in presence of different concentrations
(100 and 10 ppm) of test compounds
organic layer was washed with water (3ꢀ10 ml). The
/
organic layer was then dried over sodium sulfate and
filtered. The filtrate was evaporated at reduced pressure
at 40 8C and the residue was immediately purified by
flash column chromatography with a gradient of ethyl
acetate: hexane mixture (from 10:90 to 90:10) to give 1.5
Number
10 ppm
100 ppm
2c
2d
97
81
80
23
77
60
77
50
80
70
57
63
100
100
0
3a
3b
97
97
45 8C. 1H NMR (CDCl3) d 7.03
7.8 Hz), 4.17 (s, 3H, OCH3),
7.8 Hz),
3c
3d
81
81
g (55%) of 2a; m.p.: 43Á
(ABq, 2H, aromatic, J7,8
4.05 (s, 3H, OCH3), 3.19 (t, 2H, CH2, J4,5
3.04 (t, 2H, CH2, J4,5ꢂ7.8 Hz); UV(CH3OH) lmax 254
nm (log oꢂ3.5).
/
ꢂ
/
3e
4a
100
100
93
ꢂ
/
/
4b
5b
93
/
Sea water
DMSO
Taxol
100
100
0
3.1.3. 4,5-Dihydronaphtho[1,2-d][1,2,3]selenadiazole
(2b)
3.1.5. 8-Sulfamoyl-4,5-dihydronaphtho[1,2-
d][1,2,3]thiadiazole (4a)
This compound was prepared according to the
literature [10].
Chlorosulfonic acid (25 ml) was added to 2a (188 mg,
1 mmol) with stirring in an ice bath. After stirring for 10
min, the mixture was warmed at 60Á
/
80 8C for 2.5Á3 h.
/
3.1.4. Nitration of 4,5-dihydronaphtho[1,2-
d][1,2,3]thia(or selena)diazoles
Ethyl acetate (25 ml) was added to the reaction mixture
and the mixture was added to ice-water (50 ml). The
organic layer was decanted. It was washed with water
Fuming nitric acid (5 ml) was added dropwise to 2a or
2b (1 mmol) with stirring in an ice bath during 10 min.
After stirring for 1 h, the mixture was warmed to room
temperature (r.t.) and the reaction mixture was added to
ice-water (50 ml). The resulting mixture was then
(3ꢀ
was filtered and the solvent evaporated under reduced
pressure to give a yellowÁbrown oil. To the latter
/10 ml) and dried over sodium sulfate. The mixture
/
residue, a mixture of water (25 ml), methanol (25 ml)
and ammonia solution (25 ml, 25% v/v) was added. The
reaction mixture was stirred at r.t. for 0.5 h, then
successively extracted with chloroform (3ꢀ10 ml) and
/
the organic layer was dried over sodium sulfate. After
filteration the solvent was evaporated at reduced
pressure to give a yellow solid which was purified by
flash chromatography using ethyl acetate:hexane (from
25:75 to 100%) as eluent to yield different mono-nitrated
compounds (Table 1).
warmed at 60Á80 8C for 0.5 h. The mixture was
/
evaporated at reduced pressure to remove methanol,
then cooled to r.t. and acidified (pH 5) by the addition
of concentrated hydrochloric acid. The resulting mixture
was extracted with ethyl acetate. The organic layer was

66
A.R. Jalilian et al. / Il Farmaco 58 (2003) 63Á68
/
Scheme 1. Reagents and condition a, SOCl2; b, SeO2, AcOH; c, HNO3 (fuming) 5Á0 8C; d, HSO3Cl, NH3.
/
dried (sodium sulfate), filtered and evaporated at
reduced pressure to give a yellow residue which was
crystallized from ethyl acetate:hexane (3:1) to give 144
1H, H9, J7,9ꢂ
/
1.86 Hz), 7.74Á
/
7.82 (dd, H7, J6,7ꢂ
/
7.9
Hz, J7,9 1.86 Hz), 7.56 (d, 1H, H6, J7,6ꢂ
ꢂ
/
/
7.9 Hz), 7.40
(bs, 2H, NH2), 3.40 (t, 2H, CH2), 3.12 (t, 2H, CH2).
3.2. Biological in vitro tests
mg (54%) of 4a; m.p. 225Á
d 8.55 (d, 1H, H9, J7,9ꢂ
J6,7ꢂ7.9 Hz, J7,9 1.86 Hz), 7.59 (d, 1H, H6, J7,6ꢂ
/
227 8C, 1H NMR (DMSO-d6)
1.86 Hz), 7.77Á7.84 (dd, H7,
7.9
/
/
/
ꢂ
/
/
Hz), 7.46 (bs, 2H, NH2), 3.43 (t, 2H, CH2), 3.16 (t, 2H,
CH2).
3.2.1. Antifungal tests
3.1.6. 8-Sulfamoyl-4,5-dihydronaphtho[1,2-
d][1,2,3]selenadiazole (4b)
This compound was prepared similar to 4a in 39%
3.2.1.1. Agar dilution method. The method used for this
test was adopted from Muanza et al. (1994) and
Mitscher et al. (1972) with little modifications [13,14].
The test was done in duplicates and, the positive
yield; m.p. 201Á
203 8C.1H NMR (DMSO-d6) d 8.54 (d,
/

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67
antifungal results were read based on no growth
compared with solvent control.
On brine shrimp toxicity test only 2d showed a strong
toxicity at 100 ppm, while the rest of the compounds
exhibited slight toxicity. The minority of the compounds
demonstrated considerable toxicity against brine
shrimps. The detailed data are tabulated in Table 3.
3.2.1.2. Microbroth dilution method. Recommendations
of NCCLS (1992) were mostly used to measure the
minimum inhibitory concentration (MIC) values. The
fungal organisms, taken from SDA plates, were sus-
pended in normal saline to obtain Tꢂ
/
75Á77% at 530
/
5. Conclusion
nm, which was equal to 106 CFU/ml. The fungal
suspension was diluted 1000-times in the medium and
100 ml aliquots were added to each well. Compounds
were dissolved in DMSO to make a concentration of 250
mg/ml, leaving 100 ml in each well. The 96-well plates
In order to obtain new chemotherapeutic heterocyclic
cores, we have synthesized substituted-naphtho[1,2-
d][1,2,3]thia(or selena)diazoles that showed antifungal
activities against some pathogenic fungal organisms, in
vitro. The toxicity test on brine shrimps demonstrated
that most of the compounds have low toxicity. Com-
pound 4a exhibited significant antifungal activity
against Cryptococcus neoformans (4a, MIC: 0.53 mg/
ml, compared with 0.53 mg/ml for fluconazole and 0.13
mg/ml for amphotericin B), while other compounds,
were incubated at 35 8C for 24Á96 h (Table 2).
/
3.2.1.3. Brine shrimp test. Brine shrimp toxicity test was
determined on different compounds by a modification of
the previously reported methods [15,16]. Two different
concentrations (10 and 100 ppm) of test compounds
were prepared by dissolving in DMSO as the solvent.
Seawater was prepared by dissolving commercially
available sea salt (3.8 g) into tap water (1 l). Brine
shrimps hatched in seawater media at r.t. for 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. Two blank samples
were prepared containing, DMSO (50 ml in 5 ml sea
water) and of sea water (5 ml). Taxol (10 ppm in
DMSO) was used as the positive standard. All test tubes
were left at r.t. for 24 h and the survived brine shrimps
were counted and reported as survival percentage (Table
3).
such as 3a and 3e (MICꢂ3.12 mg/ml) showed moderate
/
effects (Scheme 1).
Acknowledgements
We gratefully acknowledge Professor L.I. Wiebe
(Faculty of Pharmacy and Pharmaceutical Sciences,
University of Alberta, Edmonton, Alberta, Canada)
for his support.
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4. Results and discussion
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/