Does dehydrocyclization of 4-benzoylthiosemicarbazides in acetic acid lead to s-triazoles or thiadiazoles?

Article abstract of DOI:10.1007/s11224-012-0065-4

Ever since the recognition of strong pharmaceutical activities of triazoles and thiadiazoles, these scaffolds have been the subject of vigorous studies. One of the best strategies for synthesis of these azoles is dehydrocyclization of 1,4-disubstituted thiosemicarbazides, which leads to s-triazoles in alkaline media, whereas in strong acidic media 1,3,4-thiadiazoles are formed. However, the literature is riddled with contradictory communications regarding the nature of the products of such reactions under mild acidic conditions. As these compounds are not amenable to X-ray analysis, we have resorted to NMR and theoretical modelling to resolve this discrepancy. In this article, we present arguments indicating that dehydrocyclization of 4-benzoylthiosemicarbazides in glacial acetic acid leads to thiadiazole derivatives. These structural findings are augmented by studies of bioactivity of a few members of the studied class of compounds.

Full text of DOI:10.1007/s11224-012-0065-4

Struct Chem
DOI 10.1007/s11224-012-0065-4
ORIGINAL RESEARCH
Does dehydrocyclization of 4-benzoylthiosemicarbazides
in acetic acid lead to s-triazoles or thiadiazoles?
•
Agata Siwek Paweł Sta˛czek Urszula Kosikowska
•
•
•
Anna Malm Piotr Paneth Stefan Jankowski
•
Received: 1 May 2011 / Accepted: 5 October 2011
Ó Springer Science+Business Media, LLC 2012
Abstract Ever since the recognition of strong pharma-
ceutical activities of triazoles and thiadiazoles, these scaf-
folds have been the subject of vigorous studies. One of the
best strategies for synthesis of these azoles is dehydro-
cyclization of 1,4-disubstituted thiosemicarbazides, which
leads to s-triazoles in alkaline media, whereas in strong
acidic media 1,3,4-thiadiazoles are formed. However, the
literature is riddled with contradictory communications
regarding the nature of the products of such reactions under
mild acidic conditions. As these compounds are not ame-
nable to X-ray analysis, we have resorted to NMR and
theoretical modelling to resolve this discrepancy. In this
article, we present arguments indicating that dehydrocyc-
lization of 4-benzoylthiosemicarbazides in glacial acetic
acid leads to thiadiazole derivatives. These structural
findings are augmented by studies of bioactivity of a few
members of the studied class of compounds.
Keywords Thiadiazole ꢀ Triazole ꢀ NMR ꢀ DFT
Introduction
The triazole and thiadiazole core structures represent
pharmacophores that have been widely exploited in
medicinal chemistry. The triazole- and thiadiazole-based
compounds have been reported as antimicrobials [1–21],
antivirals [22–24], antitumors [6, 10, 25–31], anticonvul-
sants [11, 32, 33] antidepressants [34–36], analgesic [8,
37–41] and anti-inflammatory [8, 9, 37, 38, 42–46] agents.
An excellent strategy for the synthesis of these azoles is the
dehydrocyclization of 1,4-disubstituted thiosemicarbaz-
ides. It is generally accepted that the cyclization of these
compounds results in the formation of s-triazoles in an
alkaline medium, whereas in an acidic medium 1,3,4-thi-
adiazoles are formed. Results from our laboratory, how-
ever, show that this is not generally true, and for some
substituents, s-triazoles are obtained both under alkaline
and acidic conditions. This leads to confusion regarding the
actual structure of the bioactive compounds. For example,
there is some disagreement in the literature regarding the
identity of the product of 4-benzoylthiosemicarbazides
dehydrocyclization in acetic acid. Some authors have
reported the formation of s-triazoles [47, 48], while others
claim to have obtained thiadiazoles [49–54] as illustrated in
the Scheme 1. As this class of compounds exhibits sig-
nificant antibacterial activity, it is important to unequivo-
cally identify the structure of this bioactive compound. The
uncertainty described above prompted us to study the
A. Siwek (&)
Department of Organic Chemistry, Faculty of Pharmacy,
Medical University, Chodzki 4a, 20-093 Lublin, Poland
e-mail: agata.siwek@am.lublin.pl
P. Sta˛czek
Department of Genetics of Microorganisms, University of Lodz,
Banacha 12/16, 90-237 Lodz, Poland
U. Kosikowska ꢀ A. Malm
Department of Pharmaceutical Microbiology, Faculty of
Pharmacy, Medical University, Chodzki 1, 20-093 Lublin,
Poland
P. Paneth
Institute of Applied Radiation Chemistry, Technical University
of Lodz, Zeromskiego 116, 90-924 Lodz, Poland
S. Jankowski
Institute of Organic Chemistry, Technical University of Lodz,
Zeromskiego 116, 90-924 Lodz, Poland
123

Struct Chem
NMR spectra of 2-benzoylamino-5-(pyrrol-2-yl)-1,3,4-
thiadiazole (2)
R
1H, 13C and 15N NMR spectra were recorded at 300 K in
DMSO-d6 on Bruker Avance II Plus spectrometer at
1
700.21, 176.09 and 70.96 MHz, respectively. H and 13C
chemical shifts were calibrated on solvent signals at 2.49
and 39.7 ppm, respectively. Liquid ammonia was the ref-
erence compound for 15N chemical shifts. The signal
1
assignments were based on analyses of H and 13C 1D
NMR, 1H-1H COSY, 1H-13C HSQC and HMBC, and
1H-15N HSQC spectra. As presented in the discussion
based on the obtained spectra, the compound was identified
as 2-benzoylamino-5-(pyrrol-2-yl)-1,3,4-thiadiazole (2)
with the following signal assignments:
R
R
1H NMR d (DMSO-d6) 6.21 (dt, 1 H, J 2.4 and 3.6 Hz,
C0-4), 6.73 (m, 1H, C0-3), 6.98 (m, 1 H, C0-5), 7.56 (t, 2H, J
7.77 Hz, Ph3,5), 7.66 (t, 1 H, Ph4), 8.12 (dd, 2 H, J 8.5,
1.3 Hz, Ph2,6), 11.97 (br s, 1 H, N-2), 12.99 (br s, 1 H,
N-1); and 13C NMR d (DMSO-d6) 109.9 (C-4 pyrrol),
111.4 (C-3 pyrrol), 122.1 (C-5 pyrrol), 122.3, 128.5 (Ph2,6),
128.8 (Ph3,5), 131.7 (Ph1), 133.1 (Ph4), 155.7 (C-2 pyrrol
or thiadiazol), 157.2 (C-2 pyrrol or thiadiazol), 165.1
(carbonyl).
Scheme 1 Proposed [47–54] the direction of dehydrocyclization of
4-benzoylthiosemicarbazides in acetic acid
structure of the product of the 4-benzoylthiosemicarbazide
dehydrocyclization (Scheme 1).
In this article, we present results of structural studies on
dehydrocyclization of 4-benzoyl-1-(pyrrol-2-ylcarbonyl)-
thiosemicarbazide (1, R = pyrrol-2-yl), which allowed us
to confirm that the product of this reaction is 2-benzoyla-
mino-5-(pyrrol-2-yl)-1,3,4-thiadiazole (2). Further studies
compare bioactivity of its more complicated analogue,
2-benzoylamino-5-(4-methyl-imidazol-5-yl)-1,3,4-thiadiazole
(3, R = 4-methyl-imidazol-5-yl), with its acyclic (‘linear’)
precursor, 4-benzoyl-1-(4-methyl-imidazol-5-ylcarbonyl)-
thiosemicarbazide (4), which shows excellent activity against
Gram-positive bacteria, as well as inhibitory activity towards
topoisomerase IV (topo IV) [55].
Synthesis of 4-benzoyl-1-(pyrrol-2-ylcarbonyl)-
thiosemicarbazide (1)
The compound (1) was obtained as described previously
for the synthesis of (4) [55].
Yield: (2.68 g, 93 %). Mp: 203-5 °C. IR (m, cm-1) 3298
(NH), 1672 (C=O), 1621, 1489, 886, 753, 707 (Ar–H). 1H-
NMR (300 MHz, DMSO-d6) dH 6.15–6.18 (m, 1H, CH),
6.97–6.99 (m, 2H, 2 9 CH), 7.51–7.57 (m, 2H, 2 9 CH),
7.64–7.70 (m, 1H, CH), 7.97–8.03 (m, 2H, 2 9 CH),
10.63, 11.71, 12.33 (3s, 3H, 4 9 NH). Anal. Calc. for
C13H12N4O2S: C, 54.15; H, 4.19; N, 19.43. Found: C,
54.55; H, 3.87; N, 19.30.
Experimental
Chemistry
All commercial reactants and solvents with the highest
purity were purchased from either Sigma-Aldrich or Lan-
caster and used without further purification. Melting points
were determined on a Fischer–Johns block and were
uncorrected. Elemental analysis was determined by an
AMZ-CHX elemental analyser (are within 0.4 % of the
theoretical values). IR spectra (m, cm-1) were recorded in
Synthesis of 2-benzoylamino-5-(pyrrol-2-yl/4-methyl-
imidazol-5-yl)-1,3,4-thiadiazole (2) and (3)
A solution of (1) or (4) (0.01 mol) in glacial acetic acid
(30 mL) was refluxed, and progress of the reaction was
monitored by thin layer chromatography. After 5 h, the
reaction was completed, and the reaction mixture was kept
for 12 h at room temperature. The precipitate was filtered,
dried and crystallized from ethanol.
1
KBr using a Specord IR-75 spectrophotometer. H-NMR
spectra (d, ppm) of (1) and (3) were recorded on a Bruker
Avance 300 in DMSO-d6 with TMS as internal standard.
Analytical thin layer chromatography (TLC) was per-
formed with Merc 60F254 silica gel plates and visualized by
UV irradiation (254 nm).
2-Benzoylamino-5-(pyrrol-2-yl)-1,3,4-thiadiazole (2)
Yield: (2.16 g, 80 %). Mp: 276–8 °C. IR (m, cm-1) 3458
(NH), 3002, 1581, 1537, 1468, 905, 735, 696 (Ar–H), 1671
(C=O), 1600 (C=N). Anal. Calc. for C13H10N4OS: C,
123

Struct Chem
57.76; H, 3.73; N, 20.73. Found: C, 58.10; H, 4.11; N,
20.54.
were used to spread the microbial suspensions (0.5
McFarland inoculum diluted 1:100 in Mueller–Hinton
broth) onto the medium surface, and then the compound
solution at a concentration of 5,000 lg/mL was introduced
into wells (80 lL per well separately). The wells (8-mm
diameter) were made on the agar with a sterile cork borer.
The plates were preincubated at room temperature for
1.5 h, to allow the diffusion of solution into the medium,
and were then incubated at 37 °C for 18 h.
2-Benzoylamino-5-(4-methyl-imidazol-5-yl)-1,3,4-thia-
diazole (3)
Yield: (1.17 g, 41 %). Mp: 256–8 °C. IR (m, cm-1) 3229
(NH), 1666 (C=O), 1610, 1579, 1511, 1488, 766, 711 (Ar–
H), 2924, 2853, 1451, 1353 (Aliph.), 1622 (C=N). 1H-
NMR (300 MHz, DMSO-d6) dH 2.56 (s, 3H, CH3),
6.54–7.69 (m, 3H, 3 9 CH), 7.70 (s, 1H, CH), 8.11–8.14
(m, 2H, 2 9 CH), 12.45 (s, 1H, NH), 12.91 (s, 1H, NH).
Anal. Calc. for C13H11N5OS: C, 54.72; H, 3.89; N, 24.55.
Found: C, 54.47; H, 3.74; N, 24.85.
The agar dilution method
The agar dilution method was used to determine MICs for
(1). For the determination, the compound was dissolved in
DMSO. Concentrations of the agent tested in solid medium
ranged from 6.25 to 400 lg/mL. The final inoculum of all
organisms studied was 104 colony-forming units per mL
(CFU/mL). Minimal inhibitory concentrations were read
after 18 h (for bacteria) of incubation at 35 °C. Cipro-
floxacin (5 lg per disc) was used as the control antimi-
crobials. The MIC results were repeated three times and are
illustrated in Table 1.
Antimicrobial assay
Microorganisms used in this study were as follows:
S. aureus ATCC 25923, S. aureus ATCC 6538, S. epide-
rmidis ATCC 12228, M. luteus ATCC 10240, B. subtilis
ATCC 6633, B. cereus ATCC 10876 and B. cereus ATCC
11778 (as representative examples of Gram-positive bac-
teria); and E. coli ATCC 25922, E. coli ATCC 10538,
E. coli NCTC 8196, P. vulgaris NCTC 4635, K. pneumo-
niae ATCC 13883, P. aeruginosa ATCC 15442, P. aeru-
ginosa NCTC 6749, P. aeruginosa ATCC 27853,
P. aeruginosa ATCC 9027, P. mirabilis ATCC 12453 and
B. bronchiseptica ATCC 4617 (as representative examples
of Gram-negative bacteria).
The broth microdilution method
The broth microdilution method was used to determine
MICs for (3). This method was performed in 96-well
microplates with the Mueller–Hinton broth containing
from 1.95 to 1,000 lg/mL of (1). 20 lL of each bacterial
0.5 McFarland suspension was added to the Mueller–Hin-
ton broth per each well; total volume was 200 lL. After
Preliminary antibacterial potencies of (1) and (3) against
a panel of Gram-positive and Gram-negative bacteria were
screened on the basis of growth inhibition zones (GIZ) by
means of the agar well diffusion method. Then, the MICs
were determined by the agar dilution method (for (1)) or
the broth microdilution technique (for (3)). The minimum
inhibitory concentration (MIC) was defined as the lowest
concentration of the compound, preventing growth of the
tested microorganism. In both the methods, the recom-
mended Mueller–Hinton medium was used—agar and
broth, respectively [56, 57].
incubation (37 °C for 18 h), the optical density (OD600
)
measurements were determined for bacterial cultures in the
presence and in the absence of tested compounds. Cefur-
oxim at concentrations of 0.06 to 500 lg/mL was used as
control antimicrobials. The MIC results were repeated
three times and are illustrated in Table 1.
Inhibition of bacterial type IIA topoisomerases
The disc diffusion method
Supercoiling assay
The disc diffusion method was used to determine pre-
liminary activity of (1). For this method, sterile filter paper
discs (9-mm diameter) were dripped with the compound
solution to load 400 lg of one compound per disc. Dry
discs were placed on the surface of Mueller–Hinton II agar
medium. The diameter of the growth inhibition zone was
read after 18 h of incubation at 35 °C.
The assays were performed using S. aureus Gyrase Super-
coiling Assay Kits (Inspiralis). In brief, supercoiled
pBR322 plasmid DNA (0.5 lg) was incubated with 1 unit
of gyrase, in the dedicated supercoiling assay buffer sup-
plied by the manufacturer, in the presence of varying con-
centrations of the test compounds. Reactions were carried
out at 37 °C for 1 h and then terminated by the addition of
equal volume of 29 STOP Buffer (40 % sucrose, 100 mM
Tris–Cl pH 7.5, 1 mM EDTA, and 0.5 mg/ml bromophenol
blue) and chloroform/iso-amyl alcohol. Samples were vor-
texed, centrifuged and run through a 15 cm 1 % agarose gel
The agar well diffusion method
The agar well diffusion method was used to determine
preliminary activity of (3). For this method, sterile swabs
123

Struct Chem
Table 1 In vitro antimicrobial
activities of (1), (3),(4) and
reference antibiotics expressed
as the growth inhibition zone
(GIZ, mm) and minimal
inhibitory concentration
(MIC, lg/mL)
Compound
S. aureus
S. epidermidis
M. luteus
B. subtilis
B. cereus
GIZ MIC
GIZ
MIC
GIZ
MIC
GIZ MIC
GIZ MIC
(1)
(3)
18a,b 50a,b
20c
19c
50c
62.5c
15d
44d
25d
31.25d 50e
20e
25e
14f
8g
25f
[1,000g
50a
8a
34a
18b
125a
500b
500a
1,000b
0.49a
0.98b
0.5a
62.5e
(4)
24c
3.91c
0.98c
0.5c
50d
0.98d
0.98d
2d
21e
7.81e
8g
62.5g
31.25g
1f
Cefuroxim
Ciprofloxacin
0.98e
a
Strains ATCC: 25923, 6538,
b
\0.125e
d
e
f
c12228, 10240, 6633, 11778,
0.5b
g10876
in TAE buffer (40 mM Tris–acetate, 2 mM EDTA) for 3 h
at 50 V. Gels were stained with ethidium bromide and
visualized under UV light.
Discussion
Recently, a series of products of dehydrocyclization of
4-benzoylthiosemicarbazides in glacial acetic acid were
obtained in our laboratory with the aim of comparing their
bioactivity with those of their acyclic 4-benzoylthiose-
micarbazide precursors. All the compounds were charac-
Decatenation assay
The assay was performed using S. aureus topoisomerase IV
decatenation kits (Inspiralis). Interlinked kDNA substrate
(0.5 lg) was incubated with 1 unit of topoisomerase IV
(Inspiralis), in the dedicated decatenation assay buffer
supplied by the manufacturer, in the presence of varying
concentrations of the test compounds. Reactions were
carried out at 37 °C for 1 h and then terminated by the
addition of equal volume of 29 STOP Buffer (40 %
sucrose, 100 mM Tris–Cl pH 7.5, 1 mM EDTA and
0.5 mg/ml bromophenol blue) and chloroform/iso-amyl
alcohol. Samples were vortexed, centrifuged and run
through a 15 cm 1 % agarose gel in TAE buffer for 1.5 h at
80 V. Gels were stained with ethidium bromide and visu-
alized under UV light. The concentrations of the inhibitor
that prevented 50 % of the kinetoplast DNA from being
converted into decatenated minicircles (IC50 values) were
determined by plotting the results obtained from the
densytometric analyses of the gel images by means of
Quantity One software (BioRad).
1
terized by H NMR spectra, which clearly confirmed the
process of aromatization of the ‘linear’ substrates. How-
ever, based on the broad resonance of the NH group at
about 13 ppm, we could not confirm whether the s-triazole
or thiadiazole derivative was formed. Typically, the 1H
NMR spectra of s-triazoles display a sharp NH resonance
signal at about 14 ppm, while for thiadiazoles, a broad
signal in the range 10–12 ppm is observed. The NH signals
at about 13 ppm, for products of aromatization of 4-ben-
zoylthiosemicarbazides in glacial acetic acid, are halfway
between these typical values, and thus, are ascribed in the
literature either to s-triazole or thiadiazole moieties.
Unfortunately, structures of these compounds cannot be
confirmed based on X-ray analysis because none of them
are prone to crystallization. Thus, in order to establish the
actual structure, 2D NMR experiments (as detailed in
the ‘‘Experimental’’ section) were carried out based on the
example of dehydrocyclization of (1).
1H-15N HSQC spectrum shows two correlations of NH
protons to attached nitrogen atoms: 12.99–130.5 and
11.97–155.5 ppm. In the COSY spectrum (Fig. 1a), the
signal at 11.97 ppm shows correlations with other protons
and is the entering point for assignment of protons at 6.21,
6.73 and 6.98 ppm as protons of the pyrrole ring. Thus, the
second spin system in the COSY spectra at 7.56, 7.66 and
8.12 ppm corresponds to the phenyl ring protons. Signal at
8.12 ppm was assigned as ortho protons in phenyl ring.
The second NH atom is connected with carbonyl carbon in
the benzamide fragment (left) or to thiocarbonyl group
within triazole ring. In HMBC spectrum (Fig. 1b), signal at
12.99 ppm correlates with the carbon atom at 165.1 ppm.
Correlation with the same atom was detected for ortho
Theoretical calculations
Structures of alternative products of dehydrocyclization of
(1) (s-triazole and thiadiazole) were optimized by means of
the B3PW91 functional expressed in the 6-31?G(d,p) basis
set [58, 59], as implemented in Gaussian09 [60]. Contin-
uum solvent model IEFPCM with parameters for DMSO
was used in all calculations. Theoretical NMR shifts were
then obtained for the optimized structures by means of the
B3LYP functional [61] with 6-311?G(2d,p) basis set.
Default thresholds were used in all calculations. It has been
shown that reoptimization by means of the B3LYP func-
tional led to same conclusions.
123

Struct Chem
1
1
Fig. 1 a H-1H COSY and b H-13C HMBC NMR spectra used in elucidation of the structure of the product of dehydrocyclization of (1)
phenyl protons at 8.12 ppm. From this, it can be concluded
that the product of the dehydrocyclization of (1) is the
thiadiazole (2).
Figure 2 shows all 13C signals and relates them to the
corresponding carbon atoms.
We have recently documented for the first time inhibi-
tory properties of 4-benzoylthiosemicarbazides against
bacterial topoisomerase IV [55]. Among tested compounds
4-benzoyl-1-(indol-2-ylcarbonyl)-thiosemicarbazide (5, R =
indol-2-yl) was found to be the best inhibitor of topo IV
with IC50 = 14 lM. A decrease in this inhibitory activity
(IC50 = 90 lM) was observed when indole moiety was
replaced with imidazole in (4). 1,4-Dibenzoylthiosemi-
carbazide (6, R = phenyl), with a considerably different
structure from both (4) and (5), was inactive as inhibitor of
both DNA gyrase and topoisomerase IV. Based on the DFT
and docking studies it was proposed that two factors: (i) the
structure of the thiosemicarbazide and (ii) substituent at
N1 nitrogen atom are the key functionalities required
for inhibition of topo IV rather than H-bond interaction
between NH–NH–C(=S)–NH core and the enzyme. In
order to expand these initial findings with further details on
the structure–activity relationship for this chemical class,
(3), the cyclic product of (4), was synthesized and its
biological activity evaluated. It was found that (3) exhibits
no inhibitory activity against topo IV. Based on these
findings, we can conclude that both substituents as well as
the NH–NH–C(S)–NH core are important for interactions
of thiosemicarbazide-based compounds with topo IV.
Interestingly, inhibitory activities of 4-benzoyltiosemic-
arbazides and their cyclic derivatives do not parallel their
antibacterial activities. The comparison of these activities
for compounds (1) and (3) with the corresponding literature
data on (4) is given in Table 1 in the Experimental section.
The assignment was augmented by the results of theo-
retical modeling of 13C and 15N spectra. The three signals
with the highest chemical shifts and two signals with the
lowest chemical shifts in theoretical 13C spectra of both
alternative products clearly support the thiadiazole struc-
ture as evidenced by the data illustrated in Table 2. Also
the difference between NH signals in 15N spectra calcu-
lated for triazole (40.1 ppm) and thiadiazole (20.8 ppm),
when compared with the experimental value of 24.5 ppm,
supports this conclusion. The unambiguous assignment of
some carbon signals, which is experimentally impossible
due to lack of long-range couplings, was achieved in the
theoretical manner. The selected DFT functional, B3PW91
[62–64], has been shown previously to be adequate for
calculating properties of compounds of this class [65–68].
Table 2 Comparison of selected 13C NMR signals (ppm), pre-
dicted theoretically at the IEFPCM(DMSO)/B3LYP/6-311?G(2d,p)//
IEFPCM(DMSO)/B3PW91/6-31?G(d,p) for alternative structures
with the experimental values
Thiadiazole
Experiment
Triazole
166.3
160.8
159.1
111.7
111.6
164.9
157.0
155.6
111.2
109.7
170.6
168.6
152.1
118.6
112.2
123

Struct Chem
Fig. 2 13C NMR spectrum
predicted theoretically for (2).
Atom numbering at the top
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Conclusion
In this contribution, arguments indicating that dehydro-
cyclization of 4-benzoylthiosemicarbazides in glacial ace-
tic acid leads to thiadiazole derivatives were presented.
These structural findings were augmented with studies on
bioactivities of a few members of the studied class of
compounds. They further evidence that thiosemicarbazide
derivatives have at least two different targets correspond-
ing to their antibacterial activities.
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