Convenient Synthesis and Biological Activity of Mono and Diacyl 2,5-Dimercapto-1,3,4-thiadiazole Derivatives

Article abstract of DOI:10.1002/jhet.2942

New derivatives of 2,5-dimercapto-1,3,4-thiadiazole substituted both at one or two exocyclic sulfur atoms with a series of aroyl or ethoxycarbonyl groups were synthesized in reactions of 2,5-dimercapto-1,3,4-thiadiazole salts with appropriate acid chlorides or ethyl chloroformate in mild conditions. The products were characterized by spectroscopy (¹H NMR, ¹³C NMR, IR, and HRMS). Some from the synthesized compounds were screened in vitro and in vivo for antibacterial and antifungal activities against a panel of reference strains of microorganisms. The study revealed that ethyl S-(5-mercapto-1,3,4-thiadiazol-2-yl) carbonothioate seems to be the most active and versatile compound against Gram-positive bacteria, Gram-negative bacteria, and plant pathogenic fungi.

Full text of DOI:10.1002/jhet.2942

Month 2017
Convenient Synthesis and Biological Activity of Mono and Diacyl 2,5-
Dimercapto-1,3,4-thiadiazole Derivatives
Karolina Jasiak,a Agnieszka Kudelko, Monika Wróblowska, Anna Biernasiuk, Anna Malm, and Maria Krawczyk
a
a
b
b
c
*
aDepartment of Chemical Organic Technology and Petrochemistry, The Silesian University of Technology, Krzywoustego
4, PL 44100 Gliwice, Poland
bDepartment of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University, Chodźki 1, PL 20093 Lublin,
Poland
cInstitute of Industrial Organic Chemistry, Annopol 6, PL 03236 Warsaw, Poland
*
E-mail: agnieszka.kudelko@polsl.pl
Received March 9, 2017
DOI 10.1002/jhet.2942
Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com).
New derivatives of 2,5-dimercapto-1,3,4-thiadiazole substituted both at one or two exocyclic sulfur atoms
with a series of aroyl or ethoxycarbonyl groups were synthesized in reactions of 2,5-dimercapto-1,3,4-
thiadiazole salts with appropriate acid chlorides or ethyl chloroformate in mild conditions. The products were
characterized by spectroscopy (1H NMR, 13C NMR, IR, and HRMS). Some from the synthesized
compounds were screened in vitro and in vivo for antibacterial and antifungal activities against a panel of
reference strains of microorganisms. The study revealed that ethyl S-(5-mercapto-1,3,4-thiadiazol-2-yl)
carbonothioate seems to be the most active and versatile compound against Gram-positive bacteria,
Gram-negative bacteria, and plant pathogenic fungi.
J. Heterocyclic Chem., 00, 00 (2017).
INTRODUCTION
N,N0-diacylhydrazines which treated with diphosphorus
pentasulfide [35–37] or Lawesson’s reagent [38–40]
undergo cyclization forming the desired heterocyclic
arrangement. Other sources describe also exchange of
oxygen atom in 1,3,4-oxadiazoles to sulfur using thiourea
[41–43], the cyclization of bithioureas [44,45], or
thiosemicarbazides with another compounds containing a
carbonyl group [46–48].
One of the representatives of thiadiazole family is 2,5-
dimercaptothiadiazole (DMTD 1), prepared efficiently in
a simple manner from easy accessible reagents: carbon
disulfide and hydrazine hydrate [49]. This compound
played a role of the leading unit in our study on mono-
and diacylation reactions of heterothiols by means of
aromatic acid chlorides. Bearing in mind a versatile
nature and broad range of biological interactions of
thiadiazole derivatives, we have decided to synthesize a
series of heterocyclic molecules containing this moiety.
All the synthesized compounds were screened in vitro
and in vivo for their antimicrobial activity against
different fungal and bacterial pathogenic strains in order
to find new agents for the treatment of microbial
infection. Therefore, the main purpose of these studies is
to investigate the antimicrobial potential of the obtained
1,3,4-thiadiazoles and identify compounds with a wide
spectrum of activity, which could find an application in
agriculture and medicine.
Thiadiazoles belong to the group of five-membered
heterocyclic arrangements containing one sulfur and two
nitrogen atoms in the structure and were first described in
literature in 1882 by Fischer [1–4]. Among several
thiadiazole isomers, 1,3,4-thiadiazoles are the objects of the
highest interest and intensive study [5,6]. Such
arrangements exhibit a broad spectrum of biological activity
and are used widely in medicine due to their precious
antibacterial [7,8], antifungal [9,10], antituberculosis
[11,12], anti-inflammatory [13,14], anticonvulsant [15,16],
anticancer [17,18], and antiparasitic [19,20] activities. 1,3,4-
Thiadiazoles also serve as adenosine/histamine receptor
antagonist and antidepressant agents [21,22]. It is worth
mentioning that some compounds of this group such as
cefazolin [23]—a thiadiazole analogue of antibacterial
cephalosporanic acid, acetazolamide, and metazolamide
[24]—anhydrase inhibitors of the sulphonamide nature,
belong to commercial drugs and are successfully applied in
treatment (Scheme 1). Thiadiazoles are also used in
agriculture as herbicides, fungicides, insecticides, and plant-
growth regulators [25–30], and in material industry. The
latter area comprises lubricants, corrosion inhibitors,
thermoplastic resins, and dyes [31–34].
The most commonly reported methodology for
the synthesis of this group of compounds uses
© 2017 Wiley Periodicals, Inc.

K. Jasiak, A. Kudelko, M. Wróblowska, A. Biernasiuk, A. Malm, and M. Krawczyk
Vol 000
Scheme 1. Structure of the commercially available drug including 1,3,4-
thiadiazole unit.
product
derivative 4.
3 is usually accompanied with diacylated
With the optimized reaction conditions in hand, the key
DMTD 1 was treated with a range of aromatic acid
chlorides (2a–f) to give a series of S-monosubstituted (5a–f)
and S,S0-disubstituted (6a–f) derivatives of DMTD. The
yields of 5-mercapto-1,3,4-thiadiazol-2-yl benzothioates
(5a–f) and 1,3,4-thiadiazole-2,5-diyl dibenzothioates (6a–f)
are listed in Table 1. Comparing the two series of DMTD
derivatives, it has to be noticed that the yields of S,S0-
dibenzoyl derivatives (4, 6a–f, 40–72%) are lower than
their monosubstituted counterparts (3, 5a–f, 61–83%).
This might be attributed to the formation of side product,
small amounts of monoacyl derivative, during the
synthesis of disubstituted arrangement. The structure of
new products was confirmed by means of elemental
analysis, 1H, 13C NMR spectroscopy, high resolution mass
spectrometry, and IR spectrophotometry. Products of both
groups are crystallic solids, hardly soluble in many
solvents. It was noticed that melting points of diacyl
derivatives (6a–f, Table 1) are generally lower in contrast
to their monoacylated counterparts (5a–f, Table 1), which
may be caused by the existence of hydrogen bonding in
the latter group. The results of elemental analysis remain
in a good agreement with the calculated values. In the
1H NMR spectra of 5-mercapto-1,3,4-thiadiazol-2-yl
alkanethioates (5a–f), one can observe the characteristic
peak of the proton adjacent to sulfur atom at the position 5
of the ring or to the corresponding tautomeric NH form,
which appears as a broad singlet in the range from 10.23
to 12.70 ppm. Such signal disappears in the series of
disubstituted 1,3,4-thiadiazole-2,5-diyl dialkanethioates
(6a–f). The presence of carbonyl groups introduced during
the acylation at the mercapto function of the starting
DMTD 1 may be traced by means of 13C NMR
spectroscopy, where the characteristic C═O peak appears
in the range between 184 and 192 ppm.
RESULTS AND DISCUSSION
2,5-Dimercapto-1,3,4-thiadiazole (DMTD 1), prepared
from hydrazine hydrate and carbon disulfide according to
the methodology presented in literature [20], ethyl
chloroformate, and the selected aromatic acid chlorides
(2a–f), derived from commercially available acids and
thionyl chloride SOCl2, were the key reagents in our
synthesis (Scheme 2). First trials of S-acylation, aiming at
searching for the best conditions, were conducted by
means of DMTD
1 and ethyl chloroformate in
chloroform as a solvent, and in the presence of triethyl
amine (TEA), as the agent responsible for binding of
acidic hydrogen from the mercapto group.
The reactions proceeded smoothly at low temperatures
(~0°C) in a relatively short time (1 h for 3 and 5 h for 4),
and the type of product depended mainly on the molar
ratio of DMTD and acylation agent. Monoacyl derivative
of DMTD 3 was obtained in high yield using equimolar
amount of ethyl chloroformate, while its diacyl derivative
4 needed long reaction time and a slight excess of ethyl
chloroformate (2.2 equiv; Table 1). It was also found that
the better yield of thiadiazole 3 might be achieved when
the solution of acylation agent is gradually introduced
into DMTD 1–TEA mixture. Otherwise, monoacylation
Scheme 2. Synthesis of 5-mercapto-1,3,4-thiadiazol-2-yl benzothioates (5a–f) and 1,3,4-thiadiazole-2,5-diyl dibenzothioates (6a–f). Reagents and
conditions: (i) NaOH, H2SO4, reflux; (ii) SOCl2, toluene, reflux, 2–8 h; (i') TEA, 2-butanol, rt; (ii') TEA, CHCl3, rt.
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Synthesis and Biological Activity of 2,5-Dimercapto-1,3,4-thiadiazole Derivatives
Table 1
Products of the reaction of DMTD 1 with ethyl chloroformate and aromatic acid chlorides.
Monosubstituted DMTD 3, 5a–f
Disubstituted DMTD 4, 6a–f
Product
Yield, %
Mp, °C
Product
Yield, %
Mp, °C
3
83
78
82
73
61
65
74
101–103
227–229
217–219
173–175
210–212
191–193
188–190
4
72
68
82–84
5a (R1 = H)
6a (R1 = H)
192–194
214–216
116–118
138–140
171–173
168–170
5b (R1 = 4-CH3O)
5c (R1 = 3-NO2)
5d (R1 = 4-NO2)
5e (R1 = 3-Cl)
5f (R1 = 4-Cl)
6b (R1 = 4-CH3O)
6c (R1 = 3-NO2)
6d (R1 = 4-NO2)
6e (R1 = 3-Cl)
71
57
52
40
54
6f (R1 = 4-Cl)
The complementary IR analysis also indicates to the
presence of the carbonyl group in both series of
monosubstituted (3, 5a–f) and disubstituted (4, 6a–f)
derivatives of DMTD forming a strong band in the range
from 1650 to 1690 cmꢀ1. In order to confirm the
bacteria. The minimum concentrations, which inhibited
the growth of these microorganisms, ranged from
62.5 μg/mL to 125 μg/mL (MBC = 62.5–250 μg/mL).
The reference bacteria belonging to Bacillus spp.
were the most susceptible to this substance
(MIC = MBC = 62.5 μg/mL). The compound 3 indicated
also moderate or mild activity towards some of Gram-
negative bacteria (MIC = MBC = 250–1000 μg/mL), and
also moderate antifungal activity against Candida
albicans spp. ATTC with MIC = 250–500 μg/mL and
MFC = 500 μg/mL. It is worth mentioning that all
reference Gram-positive bacteria were also sensitive
structure of new products,
a high-resolution mass
spectrometric analysis was also performed. The measured
mass of the [M + H]+ ion is consistent with the expected
mass for all compounds.
Some from the synthesized monosubstituted derivatives
of DMTD 1 such as 3, 5a, 5b, 5d, and 5f were screened
in vitro for antibacterial and antifungal activities using the
broth microdilution method according to European
Committee on Antimicrobial Susceptibility Testing
(EUCAST) [50] and Clinical and Laboratory Standards
Institute guidelines [51] against a panel of reference and
clinical or saprophytic strains of microorganisms,
including Gram-positive bacteria, Gram-negative bacteria,
and fungi. The microorganisms belonging to ATCC
originate from American Type Culture Collection,
routinely used for the evaluation of antimicrobials. The
inhibition of microbial growth was assessed by
comparison with a control culture prepared without any
sample tested [52]. Ciprofloxacin (Cip) or fluconazole
(Flu) were used as reference antibacterial or antifungal
compounds, respectively.
The results of antimicrobial screening revealed, that
two among the studied monosubstituted DMTD
derivatives: 5a, bearing the benzoyl group at the
mercapto substituent and 5d with 4-nitrobenzoyl group,
showed no inhibitory effect on the growth of all
reference strains of bacteria and fungi (Table 2). The
other compounds (3, 5b, 5f) exhibited a different
to
compounds
containing
4-methoxybenzoyl
substituent 5b and 4-chlorobenzoyl substituent 5f
(MIC = 250–1000 μg/mL and MBC = 250–1000 μg/mL).
The remaining 5a and 5d showed lower activity against
some of the tested Gram-positive and Gram-negative
bacteria with MIC
=
500–1000 μg/mL, mainly
1000 μg/mL (MBC ≥ 1000 μg/mL) or had no inhibitory
effect on the growth of reference microorganisms. The
study also revealed that these derivatives were generally
inactive against the examined fungi.
We have also studied influence of some synthesized
monosubstituted DMTD derivatives (3, 4, 5a–f) on the
selected plant pathogenic fungi (Table 3).
The biological experiments (in vitro and in vivo) were
carried out according to European Plant Protection
Organization (EPPO) Standards [53] with the use of
the following strains of fungi: Botrytis cinerea,
Phythophtora cactorum, Rhizoctonia solani, Phoma
betae, Fusarium culmorum, Fusarium oxysporum,
Alternaria alternate, Blumeria graminis, Phytophtora
infestans and mold fungi: Penicillium ochrochloron,
which play the key role in biodeterioration of
technical materials (attacks plastic and textiles). The
fungicidal activity of the tested compounds was
expressed as percentage of mycelial growth
inhibition of fungi (in vitro assays) or percentage of
wheat leaf area infected (in vivo assays) with respect to
the control. Table 3 presents the results of both
antifungal tests.
bacteriostatic
or
bactericidal
activity
against
tested microorganisms (MIC
=
62.5–1000 μg/mL,
MBC = 125–2000 μg/mL) or were just inactive against
these bacteria. The widest spectrum of antimicrobial
activity against both of bacteria and fungi exhibited ethyl
S-(5-mercapto-1,3,4-thiadiazol-2-yl) carbonothioate (3).
This compound showed good activity with bactericidal
effect (MBC/MIC
=
1–2) towards Gram-positive
Journal of Heterocyclic Chemistry
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K. Jasiak, A. Kudelko, M. Wróblowska, A. Biernasiuk, A. Malm, and M. Krawczyk
Vol 000
Table 2
The activity data expressed as MIC (MBC) [μg/mL] against the reference strains of bacteria and fungi for the selected monosubstituted DMTD derivatives
(3, 5a, 5b, 5d, 5f).
MIC (MBC or MFC) [μg/mL] of the tested compounds
Species
3
5a
5b
5d
5f
Positive control
Gram-positive bacteria
Cipa
Staphylococcus aureus
ATCC 25923
Staphylococcus aureus
ATCC 6538
Staphylococcus aureus
ATCC 43300
Staphylococcus epidermidis
ATCC 12228
Micrococcus luteus
ATCC 10240
Bacillus subtilis
ATCC 6633
62.5 (125)
125 (250)
125 (125)
125 (125)
62.5 (125)
62.5 (62.5)
62.5 (62.5)
2000 (2000)
500 (2000)
1000 (2000)
1000 (1000)
1000 (2000)
500 (000)
1000 (>2000)
1000 (>2000)
1000 (>2000)
1000 (>2000)
1000 (>2000)
1000 (>2000)
2000 (>2000)
500 (1000)
1000 (1000)
1000 (1000)
500 (1000)
500 (1000)
250 (250)
0.488
2000 (2000)
1000 (2000)
2000 (2000)
1000 (1000)
1000 (2000)
500 (1000)
0.244
0.244
0.122
0.976
0.031
0.061
250 (500)
Bacillus cereus
500 (500)
500 (500)
ATCC 10876
Gram-negative bacteria
Cipa
Bordetella bronchiseptica
ATCC 4617
Escherichia coli
ATCC25922
Klebsiella pneumoniae
ATCC 13883
Proteus mirabilis
ATCC 12453
Salmonella typhimurium
ATCC1 4028
Pseudomonas aeruginosa
ATCC 9027
250 (250)
1000 (1000)
2000 (2000)
500 (500)
1000 (2000)
2000 (2000)
2000 (>2000)
2000 (2000)
—
2000 (2000)
1000 (>2000)
1000 (2000)
0.976
2000 (>2000)
2000 (>2000)
2000 (2000)
0.004
0.122
0.030
0.061
0.488
—
—
—
2000 (>2000)
2000 (>2000)
2000 (2000)
1000 (1000)
2000 (>2000)
—
—
—
—
—
—
—
Fungi
Flub
Candida albicans
ATCC 2091
Candida albicans
ATCC 10231
Candida parapsilosis
ATCC 22019
500 (500)
250 (500)
2000 (>2000)
2000 (2000)
2000 (2000)
2000 (>2000)
2000 (>2000)
2000 (>2000)
2000 (>2000)
2000 (2000)
2000 (2000)
2000 (2000)
0.244
—
0.976
1.953
1000 (1000)
2000 (>2000)
aThe standard antibiotic for Gram-positive and Gram-negative bacteria—ciprofloxacin (Cip) used as positive control.
bThe standard antibiotic for fungi—fluconazole (Flu) used as positive control.
cBioactivity ranges: no bioactivity when MIC >1000 μg/mL; mild bioactivity for MIC 501–1000 μg/mL; moderate bioactivity for MIC 126–500 μg/mL;
good bioactivity for MIC 26–125 μg/mL; strong bioactivity for MIC 10–25 μg/mL; very strong bioactivity when MIC <10 μg/mL; ATCC—American
Type Culture Collection.
Most of the tested compounds showed week to moderate
antifungal activity against plant pathogens. The best results
were observed for one compound: ethyl S-(5-mercapto-
1,3,4-thiadiazol-2-yl) carbonothioate (3) which caused the
complete growth inhibition of five strains at the
concentration of 200 mg/L. It is worth noting, that this
compound was still active even after lowering the
concentration to 100 mg/L. The most sensitive species to
the compound 3 was Phytophtora cactorum for which at
the concentration of 100 mg/L the mycelial growth was
not observed up to 7 days on PDA medium. In addition,
disubstituted DMTD derivative 4 showed moderate
antifungal effect in the in vivo test against wheat powdery
mildew on winter wheat seedlings.
CONCLUSION
Thus, we have elaborated the easy and efficient
methodology for the preparation of new S-monoacylated
and symmetrically substituted S,S0-diacylated 2,5-
dimercapto-1,3,4-thiadiazole derivatives basing on 2,5-
dimercapto-1,3,4-thiadiazole salts and aromatic acid
chlorides or chloroformates. It was shown experimentally
that compounds of these groups exhibit antibacterial and
antifungal activities. Among them in particular ethyl S-(5-
mercapto-1,3,4-thiadiazol-2-yl), carbonothioate seems to
be the most active and versatile compound against Gram-
positive bacteria, Gram-negative bacteria, and plant
pathogenic fungi.
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Synthesis and Biological Activity of 2,5-Dimercapto-1,3,4-thiadiazole Derivatives
Table 3
Antifungal activity of monosubstituted DMTD derivatives (3, 4, 5a–f) against various strains of plant pathogenic fungi—percentage of mycelial growth
inhibition and percentage of wheat leaf area infected.
Species
3
3
4
5a
5b
5c
5d
5e
5f
In vitro C [mg/L]
Alternaria alternata
Botrytis cinerea
Fusarium culmorum
Phytophtora cactorum
Rhizoctonia solani
Phytophtora infestans
Fusarium oxysporum
Penicillium ochrochloron
In vivo C [mg/L]
200
48
100
100
100
60
100
100
33.3
100
—
80
29
100
56
15
19
—
—
—
200
44
80
46.6
18.5
56
36
56
37
1000
44.4
200
42
60
16.6
0
0
0
12
0
200
0
28
13.3
0
0
0
0
200
0
20
16.6
0
0
0
7.6
0
200
4
28
16.6
0
0
0
0
200
14
30
16.6
0
0
0
0
200
0
16
16.6
0
0
0
0
0
0
0
1000
0
1000
16
1000
1000
30.8
1000
12.3
1000
11.1
1000
Blumeria graminis
4.9
7.4
7.4
EXPERIMENTAL
4-Nitrobenzoyl chloride (2d). Yield 70%, yellow solid,
mp 71–73°C (mp 72–73°C [56]).
3-Chlorobenzoyl chloride (2e).
liquid, BP 100–105°C (15 mmHg) [BP 103–104°C
(14 mmHg) [57]].
Yield 74%, colorless
All solvents and reagents were purchased from
commercial sources and used without further purification.
Melting points were determined on a Stuart SMP3
1
melting point apparatus without corrections. H and 13C
4-Chlorobenzoyl chloride (2f). Yield 90%, colorless liquid,
BP 105–110°C (15 mmHg) [BP 107–108°C (17 mmHg) [58]].
Synthesis of ethyl S-(5-mercapto-1,3,4-thiadiazol-2-yl)
carbonothioate (3). The solution of ethyl chloroformate
NMR spectra were recorded on an Agilent 400-NMR
spectrometer using DMSO-d6, CDCl3 or CD3CN as the
solvent and TMS as the internal standard. Elemental
analyses were performed with a VarioEL analyzer. FT-IR
spectra were recorded between 4000 and 650 cmꢀ1 on an
(2.1 mL, 0.022 mol) in 10 mL of 2-butanol was added
dropwise to a cooled (0°C) solution of DMTD 1 (3.00 g,
0.020 mol) and TEA (2.8 mL, 0.020 mol) in 30 mL of 2-
butanol. The mixture was agitated at 0°C for 3 h. Then,
20 mL of water was introduced to wash the organic layer,
and the aqueous layer was discarded. The organic layer
was washed with 20% H2SO4 solution (20 mL), again
with water (20 mL) and dried over anhydrous MgSO4.
The solvent was evaporated, and the crude yellow
FT-IR Nicolet 6700 apparatus with
a Smart iTR
accessory. High-resolution mass spectra were obtained by
means of a Waters ACQUITY UPLC/Xevo G2QT
instrument. Thin-layer chromatography was performed on
silica gel 60 F254 (Merck) TLC plates using
benzene/EtOAc (3:1 v/v) as the mobile phase.
2,5-Dimercapto-1,3,4-thiadiazole (DMTD, 1).
was
product 3 was crystallized from toluene.
Ethyl S-(5-mercapto-1,3,4-thiadiazol-2-yl) carbono-thioate
(3). Yield 83%, gray solid, mp 101–103°C, IR (ATR) ν,
synthesized based on the methodology described in
literature from commercially available hydrazine hydrate
and carbon disulfide yielding 87% of white solid, mp
163–165°C (mp 164–170°C [20]).
2965, 2883, 2846, 2553, 2162 (S―H), 1721 (C═O),
1497, 1463, 1441, 1328, 1267, 1165, 1123, 1110, 1063,
1
1004, 844, 772, 663 cmꢀ1. H NMR (400 MHz, CDCl3,
Carboxylic acid chlorides (2a–f).
were prepared by
ppm) δ: 1.36 (3H, t, J = 6.8 Hz, CH3); 4.41 (2H, q,
J = 6.8 Hz, OCH2); 11.86 (1H, br s, SH). 13C NMR
(100.6 MHz, CDCl3, ppm) δ: 14.1 (CH3); 66.4 (OCH2);
161.6; 164.4; 190.4 (CO). Anal. Calcd. for C5H6N2O2S3:
C, 27.01; H, 2.72; N, 12.60. Found: C, 27.12; H, 2.78;
refluxing the selected carboxylic acids (0.10 mol) with
thionyl chloride (15 mL) in dry toluene (100 mL) until
the acids were fully consumed (TLC, 2–8 h). The crude
acid chlorides obtained in high yields (2a–f, 68–92%)
were purified by distillation under reduced pressure or
crystallization and used in the subsequent reactions with
DMTD 1.
N, 12.62. HRMS (ES): 222.9660 [M + H]+.
Synthesis of S,S0-(1,3,4-thiadiazole-2,5-diyl) dicarbono-thioate
(4).
The solution of ethyl chloroformate (4.2 mL,
Benzoyl chloride (2a). Yield 92%, colorless liquid, BP
195–198°C (BP 195–197°C [54]).
0.044 mol) in 40 mL of chloroform was added dropwise
to a cooled (0°C) solution of DMTD 1 (3.00 g,
0.020 mol) and TEA (5.6 mL, 0.040 mol) in 40 mL of
chloroform. The mixture was agitated at 0°C for 5 h.
Then, 30 mL of water was introduced to wash the
organic layer, and the aqueous layer was discarded. The
organic layer was washed with 20% H2SO4 solution
4-Methoxybenzoyl chloride (2b). Yield 85%, colorless
liquid, BP 140–145°C (25 mmHg) [BP 142°C (25 mmHg)
[55]].
3-Nitrobenzoyl chloride (2c). Yield 68%, yellow solid,
mp 32–34°C (mp 32–33°C [56]).
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K. Jasiak, A. Kudelko, M. Wróblowska, A. Biernasiuk, A. Malm, and M. Krawczyk
Vol 000
(30 mL), again with water (30 mL) and dried over
anhydrous MgSO4. The solvent was evaporated, and the
crude yellow product 4 was purified by means of column
chromatography (Al2O3, benzene/EtOAc, 1:5 v/v).
Diethyl S,S0-(1,3,4-thiadiazole-2,5-diyl) dicarbono-thioate
(4). Yield 72%, yellow solid, mp 82–84°C (mp 86°C
C6H4); 12.70 (1H, br s, SH). 13C NMR (100.6 MHz,
CD3CN, ppm) δ: 118.3; 123.3; 130.3; 132.1; 134.4;
146.8 (C6H4); 161.7; 166.3; 186.2 (CO). Anal. Calcd.
for C9H5N3O3S3: C, 36.11; H, 1.68; N, 14.04. Found:
C, 36.09; H, 1.66; N, 14.03. HRMS (ES): 299.9563
[M + H]+.
[59]), IR (ATR) ν, 2960, 2885, 2846, 2551, 2162, 1721
(C═O), 1471, 1457, 1447, 1389, 1375, 1331, 1181, 1150,
S-(5-Mercapto-1,3,4-thiadiazol-2-yl)
(5d).
4-nitrobenzo-thioate
Yield 61%, yellow solid, mp 210–212°C, IR
1
1117, 1010, 1073, 1003, 864, 805, 663 cmꢀ1. H NMR
(ATR) ν, 3043, 2954, 2869, 2872, 2558 (S―H), 2167,
1671 (C═O), 1607, 1527, 1471, 1405, 1346, 1322, 1260,
(400 MHz, CDCl3, ppm) δ: 1,38 (6H, t, J = 7.2 Hz,
CH3); 4.42 (4H, q, J = 7.2 Hz, OCH2). 13C NMR
(100.6 MHz, CDCl3, ppm) δ: 14.2 (CH3); 66.1 (OCH2);
161.4; 165.2; 183.5; 183.7 (CO). Anal. Calcd. for
C8H10N2O4S3: C, 32.64; H, 3.42; N, 9.52. Found: C,
32.60; H, 3.40; N, 9.55. HRMS (ES): 294.9878 [M + H]+.
General procedure for the synthesis of S-(5-mercapto-1,3,4-
1
1198, 1182, 1112, 1052, 912, 861, 714 cmꢀ1. H NMR
(400 MHz, DMSO-d6, ppm) δ: 8.16 (2H, d, J = 8.0 Hz,
C6H4); 8.32 (2H, d, J = 8.0 Hz, C6H4); 10.26 (1H, br s,
SH). 13C NMR (100.6 MHz, DMSO-d6, ppm) δ: 123.7;
130.6; 136.4; 149.9 (C6H4); 162.0; 165.8; 186.0 (CO).
Anal. Calcd. for C9H5N3O3S3: C, 36.11; H, 1.68; N,
14.04. Found: C, 36.15; H, 1.70; N, 14.07. HRMS (ES):
thiadiazol-2-yl) benzothioates 5a–f.
In 20 mL of
299.9569 [M + H]+.
S-(5-Mercapto-1,3,4-thiadiazol-2-yl) 3-chlorobenzo-thioate
(5e). Yield 65%, white solid, mp 191–193°C, IR (ATR)
chloroform, 1.50 g (0.010 mol) of DMTD 1 and 1.4 mL
(0.010 mol) of TEA were dissolved. The resulted clear
orange solution was added dropwise to a stirring solution
of 0.010 mol of acyl chloride R1C6H4COCl (2) in
10 mL of chloroform and agitated at room temperature
for the next 24 h. The crude precipitated product was
washed with water (2 × 25 mL), dried on air, and
crystallized from the appropriate solvent (i-PrOH, hexane,
ν, 3059, 2957, 2874, 2841, 2543 (S―H), 2162, 1683
(C═O), 1586, 1570, 1488, 1418, 1268, 1195, 1123, 1061,
945, 786, 723 cmꢀ1 1H NMR (400 MHz, DMSO-d6,
.
ppm) δ: 7.86 (1H, s, C6H4); 7.92 (1H, d, J = 8.0 Hz,
C6H4); 8.08 (1H, d, J = 8.0 Hz, C6H4); 8.12 (1H, t,
J = 8.0 Hz, C6H4); 11.62 (1H, br s, SH). 13C NMR
(100.6 MHz, DMSO-d6, ppm) δ: 127.9; 128.8; 130.6;
132.6; 133.3; 137.6 (C6H4); 161.7; 165.9; 184.2 (CO).
Anal. Calcd. for C9H5ClN2OS3: C, 37.43; H, 1.75; N,
9.70. Found: C, 37.46; H, 1.73; N, 9.68. HRMS (ES):
or acetone).
S-(5-Mercapto-1,3,4-thiadiazol-2-yl) benzothioate (5a). Yield
78%, white solid, mp 227–229°C, IR (ATR) ν, 3084, 2965,
2890, 2847, 2553 (S―H), 2164, 1667 (C═O), 1607, 1578,
1489, 1446, 1431, 1269, 1210, 1179, 1126, 1064, 903, 767,
722, 677 cmꢀ1. 1H NMR (400 MHz, CDCl3, ppm) δ: 7.25–
7.55 (5H, m, Ph); 12.11 (1H, br s, SH). 13C NMR
(100.6 MHz, CDCl3, ppm) δ: 127.9; 129.3; 134.9; 135.1
(Ph); 160.3; 166.1; 185.5 (CO). Anal. Calcd. for
C9H6N2OS3: C, 42.50; H, 2.38; N, 11.01. Found: C, 42.53;
H, 2.39; N, 11.03. HRMS (ES): 254.9714 [M + H]+.
288.9329 [M + H]+.
S-(5-Mercapto-1,3,4-thiadiazol-2-yl) 4-chlorobenzo-thioate
(5f). Yield 74%, white solid, mp 188–190°C, IR (ATR)
ν, 3056, 2953, 2875, 2843, 2496 (S―H), 2162, 1673
(C═O), 1584, 1569, 1485, 1434, 1399, 1261, 1200, 1128,
1110, 890, 832, 724 cmꢀ1
.
1H NMR (400 MHz,
DMSO-d6, ppm) δ: 7.57 (2H, d, J = 8.0 Hz, C6H4); 7.95
(2H, d, J = 8.0 Hz, C6H4); 10.23 (1H, br s, SH). 13C
NMR (100.6 MHz, DMSO-d6, ppm) δ: 128.7; 129.6;
131.1; 137.7 (C6H4); 162.8; 166.4; 186.3 (CO). Anal.
Calcd. for C9H5ClN2OS3: C, 37.43; H, 1.75; N, 9.70.
Found: C, 37.46; H, 1.77; N, 9.72. HRMS (ES):
S-(5-Mercapto-1,3,4-thiadiazol-2-yl) 4-methoxybenzo-thioate
(5b).
Yield 82%, yellow solid, mp 217–219°C, IR
(ATR) ν, 3044, 2952, 2866, 2843, 2766, 2551 (S―H),
2162, 1652 (C═O), 1596, 1569, 1496, 1424, 1263, 1224,
1
1172, 1118, 1058, 1018, 894, 840, 724 cmꢀ1. H NMR
(400 MHz, DMSO-d6, ppm) δ: 3.81 (3H, s, OCH3); 7.00
(2H, d, J = 8.0 Hz, C6H4); 7.87 (2H, d, J = 8.0 Hz,
C6H4), 12.62 (1H, br s, SH). 13C NMR (100.6 MHz,
DMSO-d6, ppm) δ: 55.4 (OCH3); 113.8; 122.9; 128.6;
166.9 (C6H4); 162.8; 165.3; 190.9 (CO). Anal. Calcd. for
C10H8N2O2S3: C, 42.24; H, 2.84; N, 9.85. Found: C,
42.28; H, 2.85; N, 9.87. HRMS (ES): 284.9826 [M + H]+.
288.9326 [M + H]+.
General procedure for the synthesis of S,S0-(1,3,4-tiadiazole-
2,5-diyl) bis(R1-substituted benzothioates 6a–f. In 20 mL of
chloroform, 1.50 g (0.010 mol) of DMTD 1 and 2.8 mL
(0.020 mol) of TEA were dissolved. The resulted solution
was added dropwise to a stirring solution of 0.022 mol of
acyl chloride R1C6H4COCl (2) in 10 mL of chloroform
and agitated at room temperature for 24 h. The crude
precipitated product was washed with water (25 mL), 5%
NaHCO3 aqueous solution (25 mL), and again with water
(25 mL). The solid was dried on air and crystallized or
washed with the appropriate solvent (i-PrOH, hexane or
acetone).
S-(5-Mercapto-1,3,4-thiadiazol-2-yl)
(5c).
3-nitrobenzo-thioate
Yield 73%, yellow solid, mp 173–175°C, IR
(ATR) ν, 3041, 2952, 2867, 2831, 2556 (S―H), 2165,
1688 (C═O), 1610, 1530, 1495, 1473, 1433, 1344, 1322,
1
1264, 1207, 1130, 1063, 935, 856, 720 cmꢀ1. H NMR
(400 MHz, CD3CN, ppm) δ: 7.83 (1H, t, J = 8.0 Hz,
C6H4); 8.44 (2H, d, J = 8.0 Hz, C6H4); 8.69 (1H, s,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Synthesis and Biological Activity of 2,5-Dimercapto-1,3,4-thiadiazole Derivatives
S,S0-(1,3,4-Thiadiazole-2,5-diyl) dibenzothioate (6a).
Yield
Found: C, 44.96; H, 1.91; N, 6.57. HRMS (ES):
426.9199 [M + H]+.
bis(4-chlorobenzo-thioate)
(6f). Yield 54%, white solid, mp 168–170°C, IR (ATR)
68%, white solid, mp 192–194°C (mp 185°C [60]), IR
(ATR) ν, 3047, 2165, 1668 (C═O), 1591, 1578, 1490,
S,S0-(1,3,4-Tiadiazole-2,5-diyl)
1444, 1276, 1207, 1175, 1069, 898, 770, 682 cmꢀ1. H
1
ν, 3086, 2163, 1673 (C═O), 1584, 1570, 1485, 1399,
NMR (400 MHz, DMSO-d6, ppm) δ: 7.56–7.68 (6H, m,
2 × Ph); 7.82 (4H, m, 2 × Ph). 13C NMR (100.6 MHz,
DMSO-d6, ppm) δ: 128.1; 129.2; 134.7; 135.0 (Ph); 161.2;
165.9; 185.5; 187.1 (CO). Anal. Calcd. for C16H10N2O2S3:
C, 53.61; H, 2.81; N, 7.81. Found: C, 53.59; H, 2.80; N,
1372, 1275, 1198, 1174, 1070, 893, 830, 738, 716 cmꢀ1
.
1H NMR (400 MHz, DMSO-d6, ppm) δ: 7.65 (4H, d,
J = 8.0 Hz, 2 × C6H4); 8.02 (4H, d, J = 8.0 Hz,
2 × C6H4). 13C NMR (100.6 MHz, DMSO-d6, ppm) δ:
128.9; 129.3; 131.1; 137.7 (C6H4); 162.3; 165.4; 187.1;
187.5 (CO). Anal. Calcd. for C16H8Cl2N2O2S3: C, 44.97;
H, 1.89; N, 6.56. Found: C, 44.99; H, 1.87; N, 6.54.
HRMS (ES): 426.9193 [M + H]+.
7.83. HRMS (ES): 358.9979 [M + H]+.
S,S0-(1,3,4-Tiadiazole-2,5-diyl) bis(4-methoxybenzo-thioate)
(6b). Yield 71%, white solid, mp 214–216°C, IR (ATR)
ν, 3101, 2841, 2163, 1664 (C═O), 1597, 1574, 1512,
1456, 1278, 1261, 1223, 1168, 1117, 1072, 1016, 903,
Microbiology. In vitro antimicrobial screening with the
use of American Type Culture Collection ATCC.
1
830, 792 cmꢀ1. H NMR (400 MHz, DMSO-d6, ppm) δ:
3.81 (6H, s, 2 × OCH3); 7.16 (4H, d, J = 8.0 Hz,
2 × C6H4); 7.90 (4H, d, J = 8.0 Hz, 2 × C6H4). 13C
NMR (100.6 MHz, DMSO-d6, ppm) δ: 55.4 (OCH3);
113.9; 123.9; 128.7; 167.0 (C6H4); 162.7; 165.5; 189.6;
190.4 (CO). Anal. Calcd. for C18H14N2O4S3: C, 51.66;
H, 3.37; N, 6.69. Found: C, 51.68; H, 3.40; N, 6.71.
HRMS (ES): 419.0191 [M + H]+.
bis(3-nitrobenzothioate)
Yield 57%, yellow solid, mp 116–118°C, IR
(ATR) ν, 3090, 2164, 1691 (C═O), 1617, 1526, 1478,
Compounds 3, 5a, 5b, 5d, and 5f were screened in vitro
for antibacterial and antifungal activities using the broth
microdilution method according to European Committee
on Antimicrobial Susceptibility Testing (EUCAST) [50]
and Clinical and Laboratory Standards Institute
guidelines [51] against a panel of reference and clinical
or saprophytic strains of microorganisms, including
Gram-positive bacteria (Staphylococcus aureus ATCC
S,S0-(1,3,4-Tiadiazole-2,5-diyl)
(6c).
25923,
Staphylococcus
aureus
ATCC
43300,
Staphylococcus aureus ATCC 6538, Staphylococcus
epidermidis ATCC 12228, Bacillus subtilis ATCC 6633,
Bacillus cereus ATCC 10876, Micrococcus luteus ATCC
10240), Gram-negative bacteria (Escherichia coli ATCC
25922, Klebsiellapneumoniae ATCC 13883, Proteus
mirabilis ATCC 12453, Bordetella bronchiseptica ATCC
1442, 1401, 1348, 1324, 1265, 1192, 1133, 1071, 921,
821, 777, 716 cmꢀ1 1H NMR (400 MHz, DMSO-d6,
.
ppm) δ: 7.85 (2H, t, J = 8.0 Hz, 2 × C6H4); 8.30 (2H, d,
J = 8.0 Hz, 2 × C6H4); 8.56–8.62 (4H, m, 2 × C6H4). 13C
NMR (100.6 MHz, DMSO-d6, ppm) δ: 119.8; 121.1;
130.5; 133.9; 134.2; 147.1 (Ph); 162.3; 165.9; 187.5;
188.0 (CO). Anal. Calcd. for C16H8N4O6S3: C, 42.85; H,
1.80; N, 12.49. Found: C, 42.81; H, 1.82; N, 12.49.
HRMS (ES): 448.9682 [M + H]+.
4617,
Salmonella
typhimurium
ATCC
14028,
Pseudomonas aeruginosa ATCC 9027), and fungi
(Candida albicans ATCC 2091, Candida albicans ATCC
10231, Candida parapsilosis ATCC 22019). The
microorganisms belonging to ATCC originate from
American Type Culture Collection and are usually used
for the evaluation of antimicrobials. Microbial cultures
were subcultured on nutrient agar or Sabouraud agar at
35°C for 18–24 h or 30°C for 24–48 h for bacteria and
fungi, respectively. The surface of adequate agar (for
bacteria: Mueller-Hinton agar and for fungi: RPMI 1640
with MOPS) was inoculated with the suspension of the
analyzed species. Microbial suspensions were prepared in
sterile saline (0.85% NaCl) with an optical density of
S,S0-(1,3,4-Tiadiazole-2,5-diyl)
(6d).
bis(4-nitrobenzothioate)
Yield 52%, yellow solid, mp 138–140°C, IR
(ATR) ν, 3101, 2165, 1670 (C═O), 1607, 1526, 1472,
1410, 1346, 1322, 1261, 1197, 1182, 1115, 1052, 902,
1
846, 714 cmꢀ1. H NMR (400 MHz, DMSO-d6, ppm) δ:
8.16–8.36 (8H, m, 2 × C6H4). 13C NMR (100.6 MHz,
DMSO-d6, ppm) δ: 123.8; 130.5; 134.9; 137.7 (C6H4);
162.9; 165.9; 187.4; 187.9 (CO). Anal. Calcd. for
C16H8N4O6S3: C, 42.85; H, 1.80; N, 12.49. Found: C,
42.86; H, 1.81; N, 12.47. HRMS (ES): 448.9677 [M + H]+.
S,S0-(1,3,4-Tiadiazole-2,5-diyl)
(6e).
bis(3-chlorobenzo-thioate)
Yield 40%, yellow solid, mp 171–173°C, IR
McFarland
standard
scale
0.5—approximately
(ATR) ν, 3071, 2164, 1690 (C═O), 1597, 1575, 1475,
1.5 × 108 CFU (colony forming units)/mL for bacteria
and 0.5 McFarland standard scale—approximately
5 × 105 CFU/mL for fungi. Each examined compound
was dissolved in 1 mL of DMSO. Both bacterial and
fungal suspensions were placed on Petri dishes with solid
media containing 2 mg/mL of the tested compound and
incubated at 37°C for 24 h and 30°C for 48 h for bacteria
and fungi, respectively. The inhibition of microbial
growth was assessed by comparison with a control
1417, 1301, 1261, 1231, 1172, 1138, 1035, 897, 850,
1
748, 719 cmꢀ1. H NMR (400 MHz, DMSO-d6, ppm) δ:
6.49 (2H, s, 2 × C6H4); 6.65 (2H, t, J = 8.0 Hz,
2 × C6H4); 6.71 (2H, d, J = 8.0 Hz, 2 × C6H4); 7.31 (2H,
d, J = 8.0 Hz, 2 × C6H4). 13C NMR (100.6 MHz,
DMSO-d6, ppm) δ: 125.8; 127.7; 128.2; 130.3; 130.5;
134.4 (C6H4); 162.9; 165.8; 191.6; 192.0 (CO). Anal.
Calcd. for C16H8Cl2N2O2S3: C, 44.97; H, 1.89; N, 6.56.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

K. Jasiak, A. Kudelko, M. Wróblowska, A. Biernasiuk, A. Malm, and M. Krawczyk
Vol 000
culture prepared without any sample tested. Ciprofloxacin
or fluconazole was used as a reference antibacterial or
antifungal compound, respectively. Minimal inhibitory
concentration (MIC) for the examined compounds was
evaluated by the microdilution broth method [52], using
the twofold dilutions in Mueller-Hinton broth (for
bacteria) and RPMI 1640 broth with MOPS (for fungi)
prepared in 96-well polystyrene plates. Final
concentrations of the compounds ranged from 1000 to
0.488 μg/mL. Microbial suspensions were prepared in
sterile saline (0.85% NaCl) with an optical density of 0.5
McFarland standard. Next, 2 μL of each bacterial or
fungal suspension was added per each well containing
200-μL broth and various concentrations of the examined
compounds. After incubation (37°C for 24 h for bacteria
and 30°C for 48 h for fungi), the MIC was assessed by
spectrophotometric method as the lowest concentration of
the compound exhibiting complete bacterial or fungal
growth inhibition. Appropriate DMSO, growth and sterile
controls were carried out. The medium with no tested
substances was used as control. The minimal bactericidal
concentration (MBC) or minimal fungicidal concentration
(MFC), presenting the lowest concentration of the
compound required to kill a particular bacteria or fungi,
was determined by removing 20 μL of the culture used
for MIC determinations from each well and spotting onto
appropriate agar medium. The plates were incubated for
37°C for 24 h and 30°C for 48 h for bacteria and fungi,
respectively. The lowest concentration of the compound
with no visible growth observed was chosen as a MBC
or MFC concentration. Each experiment was repeated
three times. Bioactivity ranges were defined as follows:
no bioactivity when MIC >1000 μg/mL; mild bioactivity
for MIC 501–1000 μg/mL; moderate bioactivity for MIC
126–500 μg/mL; good bioactivity for MIC 26–
125 μg/mL; strong bioactivity for MIC 10–25 μg/mL;
very strong bioactivity when MIC <10 μg/mL. The
MBC/MIC or MFC/MIC ratios were calculated in order
to determine bactericidal/fungicidal (MBC/MIC ≤4,
MFC/MIC ≤4) or bacteriostatic/fungistatic (MBC/MIC
>4, MFC/MIC >4) effect of the tested compounds.
synthesized compounds at the concentration of 200 mg/L
was infected with agar discs with thin mycelium of fungi
taken from the margin of young vigorously growing 7-
day-old culture. Linear growth of each colony was
determined after incubation for 7 days at 25 2°C. The
fungicidal activity of the tested compounds was
expressed as
a percentage inhibition of mycelium
compared to the control combination. Percentage
inhibition was calculated as (1 ꢀ A / B) × 100%, where
A represents a colony diameter in Petri dishes with tested
compounds and B is the mean colony diameter in control
dishes without compounds. Each measurement consisted
of at least three replicates.
In vivo fungicidal bioassay.
Wheat plants (Triticum
aestivum, L.) winter cultivar Kobra were grown under
normal glasshouse propagation conditions (temperature,
20–25°C; lighting, 14-h photoperiod of daylight
supplemented by lamps 400 W) when plants had two
expanded leaves. Seedlings were sprayed with acetone
solution of the tested compounds at the concentration of
1000 mg/L containing 0.0125% Tween-20 as surfactant.
Two hours after spraying, they were inoculated with
powdery mildew (B. graminis) using dry inoculums from
diseased wheat plants. After inoculation, plants were
maintained in the growth chamber for approximately
8 days (at 20°C, 65% relative humidity and under 12 h
dark/12 h light with 200 L × mol/m2 × s photon flux
density supplied by high output white fluorescent tubes).
Macroscopic assessment of the percentage area covered
by powdery mildew on the inoculated leaf was scored
according to assessment keys presented in the EPPO
Standards [53]. Disease scores were converted into
relative values, expressed as a percentage of the reading
on the control, and efficacy was calculated according to
the Abbott’s formula.
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet