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Original article
Synthesis and biological evaluation of 1,3,4-thiadiazole
analogues as novel AChE and BuChE inhibitors
Alicja Skrzypek
a
, Joanna Matysiak
a
,
*
, Andrzej Niewiadomy
a
,
b
,
Marek Bajda
c
,Pawe1Szyma
nski
d
a
Department of Chemistry, University of Life Sciences, Akademicka 15, 20-950 Lublin, Poland
b
Institute of Industrial Organic Chemistry, Annopol 6, 03-236 Warszawa, Poland
c
Department of Physicochemical Drug Analysis, Chair of Pharmaceutical Chemistry, Jagiellonian University Medical College, Medyczna 9,
30-688 Krakow, Poland
d
Department of Pharmaceutical Chemistry and Drug Analyses, Medical University, Muszy
nskiego 1, 90-151 Łód
z, Poland
article info
Article history:
Received 8 September 2012
Received in revised form
19 November 2012
Accepted 22 December 2012
Available online 11 January 2013
Keywords:
1,3,4-Thiadiazole
Acetylcholinesterase
Butyrylcholinesterase
Inhibitor
Molecular docking
In silico pharmacokinetics
abstract
In this paper a series of new 1,3,4-thiadiazole derivatives has been designed, synthesized and evaluated
as the acetyl- and butyrylcholinesterase inhibitors. Some analogues showed promising inhibition of both
enzymes in vitro in the nM range. Generally, inhibitory potency of compounds was stronger against AChE
than BuChE, and one of them was 1154-fold more active inhibiting AChE (IC
50
¼0.17
m
M) than BuChE.
The kinetic studies showed that one of the most active analogues 8(IC
50
¼0.09
m
M, AChE) acted as
a non-competitive AChE inhibitor and was characterized by the high selectivity index (300). The other
derivative (1) exhibited a mixed-type of AChE inhibition. Docking simulations enabled the detection of
key binding interactions of the compounds with AChE and revealed that they occupied mainly the
catalytic active site. The scoring function for the novel compounds was similar or higher than for the
reference inhibitor. Additionally, based on Lipinski and other filters, the drug-likeness of compounds was
assessed. They revealed that the compounds possess properties which can suggest the favourable
pharmacokinetics in the human body after oral admission.
Ó2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
Alzheimer’s disease (AD) is a progressive degeneration of the
central nervous system, characterised by a loss of memory and
reduced ability to perform basic everyday activities of daily living.
Based on the cholinergic hypothesis, the mainstays of current
pharmacotherapy of AD are drugs aimed at increasing the levels of
acetylcholine (ACh) through the inhibition of cholinesterases
(ChEs) [1e3]. The studies have shown that AChE performs sec-
ondary noncholinergic functions. It colocalizes with the
b
-amyloid
peptide (A
b
) deposit present in the brain of Alzheimer’s patients. It
is postulated that AChE binds to A
b
and induces a conformational
transition from A
b
into its amyloidogenic form in vitro [4,5].
Two types of the ChE enzymes are found in the nervous system
eacetylcholinesterase (AChE, E.C. 3.1.1.7) and butyrylcholinesterase
(BuChE, E.C. 3.1.1.8). Both enzymes are able to hydrolyse ACh, but
AChE has a 10
13
efold higher hydrolytic ACh activity than BuChE
under the same conditions. In the normal brain AChE predominates
over the BuChE activity but it is reported that BuChE has a key role
that can partly compensate for the action of AChE.
Cholinesterase inhibitors have been approved as efficacious
treatment to reduce the symptoms of early medium stage of AD.
Several antiacetylcholinesteraseagents such as donepezil [6], tacrine
[7], galantamine [8], and ensaculin [9] have shown to induce modest
improvementin memory and cognitive functions. Unfortunately,the
potential effectiveness offered by these inhibitors is often limited by
the appearance of central and peripheral side effects. For example,
clinical studies have shown that tacrine has hepatotoxic liability
[10,11]. A large diversity of multi-target-directed AChE inhibitors of
tacrine and nimodipine hybrids has been also evaluated [12,13].
Recent research aims at new types of compounds as potential
AChE and BuChE inhibitors. To synthesize more metabolically sta-
ble cholinomimetic ligands, it points out to the possibility of
replacing the ester group with five-membered rings like thiadia-
zoles, triazoles, tetrazoles as well as oxadiazoles. Some scientists
have studied widely 1,3,4-thiadiazole derivatives as potential drugs
to treat AD [14,15]. The thiadiazole ring can act as the “hydrogen
binding domain”,“two-electron donor system”as well as it enables
to create
p
e
p
stocking interactions.
*Corresponding author. Tel.: þ48 81 4456816; fax: þ48 81 5333549.
E-mail address: joanna.matysiak@up.lublin.pl (J. Matysiak).
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
0223-5234/$ esee front matter Ó2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.060
European Journal of Medicinal Chemistry 62 (2013) 311e319
3-(Thiadiazolyl)pyridine 1-oxide derivatives possessing the
antioxidant and muscarinic receptor binding properties were
reported as potential acetylcholinesterase inhibitors [16]. Sarkandi
et al. described 1-benzyl-4-[2-(5-phenyl-1,3,4-thiadiazole-2-yl)
aminoethyl]piperidines [17]. Recently, the thiadiazolidin-3,5-dione
(TDZD) analogues have been reported as the first non-ATP com-
petitive inhibitors of glycogen synthase kinase 3
b
(GSK-3
b
). This is
one of the most attractive molecular targets for the development of
effective inhibitors of AD treatment [18].
Recently 1,3,4-thiadiazole based compounds have been obtained
and tested by our team [19]. Some of them acted as strong AChE and
BuChE inhibitors with the IC
50
values in the nM ranges. The other
derivatives were highly selective towards AChE, exhibiting selec-
tivity ratios of ca. 950. Behavioural studies performed on mice
revealed considerable analgesic activity and anxiolytic effect of some
4-(1,3,4-thiadiazol-2-yl)benzene-1,3-diols. At the same time none of
the studied derivatives displayed any neurotoxic activity and their
LD
50
values were in the range of 1000e2500 mg/kg of body weight
(unpublishedresults). These studies can suggest that the compounds
under consideration can cross the bloodebrain-barrier. Additionally,
cytotoxic studies performed on normal and cancer cells confirmed
relatively low toxicity of (1,3,4-thiadiazol-2-yl)benzene-1,3-diol set.
One of them showed even neuoroprotective properties [20].This
encouraged us to explore (1,3,4-thiadiazol-2-yl)benzene-1,3-diols as
potential AD drugs.
The synthesis and biological evaluation of a new series of
4-(5-phenyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol derivatives
as the AChE and BuChE inhibitors are discussed in the paper. To
gain more insights into the molecular determinants responsible
for the observed ability to inhibit the AChE activity, a modelling
study was undertaken through molecular docking of the most
active compounds. Recently the attention has been paid to
optimization of the pharmacokinetics of the substance at a very
early stage of its research as a potential drug. Therefore, based
on the molecule structure, the number of descriptors necessary
for its estimation was determined. Lipinski and other drug-
likeness filterswereusedtopredictpharmacokineticsofthe
obtained compounds [21e24].
2. Chemistry
5-Phenyl(modified)-(1,3,4-thiadiazol-2-yl)benzene-1,3-diols
were formed by the reaction of the commercially available hydra-
zides with sulfinylbis[(2,4-dihydroxyphenyl)methanethione] (STB)
in methanol under reflux (3e4 h) as presented in Scheme 1. The
yields of processes were in the range of 64e84%. The starting re-
agent STB was prepared from 2,4-dihydroxybenzenecarbodithioic
acid and SOCl
2
in ethoxyethane [25]. In the reaction the electro-
philic substrate STB also acts as an endogeneous cyclising reagent.
Purity of the obtained compounds was checked by the reversed-
phase (RP-18) HPLC chromatography using the MeOHeH
2
O mix-
ture as a mobile phase. The structures of the obtained compounds
are shown in Table 1. Their analytical and spectroscopic data are in
accordance with the predicted structures.
In the
1
H NMR spectra the resonance signals of hydroxyl groups
protons are usually registered as broad singlets in the range ca. 11.1
and 10.1 ppm. The aromatic protons of the benzenediol substituent
are detected as two doublets at 8.1 ppm (J¼8.7 Hz) and 6.6e6.5 ppm
(J¼2.3 Hz) corresponding to HeC(5) and HeC(2) respectively. The
third proton appears as doublets of doublets in the range of 6.5 to
6.4 ppm (J¼8.7 and 2.3 Hz) of HeC(6). The
13
C NMR spectrum
shows characteristic signals of aryl-substituted 1,3,4-thiadiazole
ring carbon atoms in the range of 166e160 ppm [26].
The IR spectra show two strong bands in the region about
3480e3140 and 1632e1620 cm
1
, corresponding to
n
(OeH) and
n
(C]N) respectively [26]. In the mass spectra (EI), the molecular ion
peaks M
þ
shown that the predicted compound has formed.
3. Biological results and discussion
3.1. Inhibition studies of AChE and BuChE
All considered compounds have been assessed as the AChE and
BuChE inhibitors. Their inhibitory potency was described as the half
of maximal inhibitory concentration, IC
50
. The modified Ellman’s
method has been used in the evaluation [27]. Two drugs: neo-
stigmine and donepezil were used as the standards (Table 1). The
results are summarized in Table 1. They clearly show that most of
the designed compounds exhibit good to moderate inhibitory ac-
tivities. The IC
50
values for AChE are ranged from 128.42 to 0.06
m
M
and for BuChE from >500 to 0.29
m
M. At the same time all
Table 1
Chemical structures, in vitro inhibition (IC
50
,
m
M) and selectivity of the studied
compounds on AChE and BuChE.
N
N
S
HO
OH
R1
R3
R2
R4
No. R
1
R
2
R
3
R
4
IC
50
for
AChE
a
[
m
M]
IC
50
for
BuChE
b
[
m
M]
Selectivity
for AChE
c
1. H Me H H 0.16 0.01 17.02 0.40 106.4
2. F H H H 1.07 0.02 >500 >467
3. Cl H H H 1.55 0.02 196.24 7.00 126.6
4. Br H H H 0.06 0.003 0.29 0.04 4.8
5. Cl H Cl H 128.42 7.13 >500 >3.9
6. Cl H H Cl 112.01 43.42 19.06 0.92 0.17
7. H Cl H Cl 45.26 0.50 >500 >11.1
8. H H CF
3
H 0.09 0.004 26.49 1.22 294.3
9. OMe H H H 26.70 0.21 212.38 6.13 7.9
10. H OMe H H 1.87 0.11 27.32 0.30 25.5
11. H H OMe H 2.18 0.20 96.34 0.31 44.2
12. H OMe OMe H 19.34 0.52 >500 >25.9
13. OMe H H OMe 1.56 0.08 63.28 0.05 40.5
14. H OMe OMe OMe 0.17 0.01 196.21 7.22 1154.2
15. OH H OH H 1.89 0.04 26.74 0.90 14.2
16. H OH OH H 0.19 0.05 2.16 0.20 11.4
17. H OMe OH H 19.21 1.00 112.41 2.33 5.9
18. H NH
2
H H 1.73 0.23 84.32 1.00 48.7
19. NO
2
H H H 2.12 0.08 57.32 0.74 27.0
20. H NO
2
H H 0.67 0.04 32.14 0.81 47.9
21. H Me NO
2
H 20.86 0.91 147.62 4.31 7.1
Neostigmine 0.05 0.007 0.07 0.009 1.4
Donepezil 0.02 0.008 7.52 0.20 376
a
IC
50
: 50% inhibitory concentration (means SD of three independent experi-
ments) of AChE.
b
IC
50
: 50% inhibitory concentration (means SD of three experiments) of BuChE.
c
Selectivity for AChE ¼IC
50
(BuChE)/IC
50
(AChE).
S
S
O
SOH
OH
OH
HO
NH
O
NH
2
+
N
N
S
HO
OH
R
R
MeOH, 3-4 h
Scheme 1. Synthesis scheme of modified 4-(5-phenyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diols.
A. Skrzypek et al. / European Journal of Medicinal Chemistry 62 (2013) 311e319312
synthesized compounds are significantly more active towards AChE
than BuChE, with the exception for compound 6. Some of the de-
rivatives proved to be highly selective for AChE with respect to
BuChE (1,3,8,14).
To find the influence of the type of substitution of the aryl ring
on the potency of compounds the structureeactivity analyses have
been performed. To obtain better results some previously described
compounds were included in the biological screening. The results
show that compound 4with the Br substituent at the ortho-posi-
tion exhibits the most promising activity with the IC
50
values of
0.06 and 0.29
m
M against AChE and BuChE respectively. Compounds
2and 3with F or Cl atom in the same position are slightly weaker
inhibitors. The activity of these analogues changes in the following
order: Br >Cl >F. A similar trend against BuChE is observed,
however, activity changes are much larger. The presence of OMe or
NO
2
substituents at C(2) of the phenyl ring decreased potency of
the compounds against both enzymes (9,19).
Placement of the Me group in the meta-position of the phenyl
ring (compound 1) resulted favourably (IC
50
¼0.16
m
M), suggesting
the importance of the hydrophobic character for the interaction
with enzyme. Compounds 10 and 18 bearing OMe or NH
2
groups
(
p
<0) in the same position are weaker inhibitors. High potency of
the compound with the strong electron-withdrawing NO
2
sub-
stituent for AChE was also observed (20). In the para-position the
presence of the hydrophobic electron-withdrawing CF
3
substituent
is strongly preferable to the hydrophilic electron donating OMe
substituent.
Analysing the compounds with the OMe substituents (9e14), it
can be found that the analogue with three MeO groups in 3, 4, 5
positions (compound 14) shows higher potency compared to less
substituted ones 9e13. Different electronic effects of the OMe
groups depending on their position, hydrophobic character of the
flexible group as well as the presence of O atoms as the potential
proton acceptors in the hydrogen bond formation could enable
access to the enzyme-binding site.
Interposition of 3-Me and 4-NO
2
substituents on the phenyl ring
(compound 21) led to a significant decrease in activity
(IC
50
¼20.86
m
M) as compared with 1. Also compounds with two Cl
atoms (5,6,7) are weaker inhibitors of both enzymes. In the case of
analogue 6the inversion of the affinity is observed (Table 1). Sim-
ilarly to tacrine it has a small selectivity for AChE over BuChE [28].
It is interesting that compounds 8and 14 are much weaker in-
hibitors against BuChE compared with AChE, indicating that these
compounds can serve as selective inhibition agents for AChE. Par-
ticularly 14 is 1154-fold more active inhibiting AChE not BuChE,
being slightly more selective than donepezil and neostigmine.
3.2. Molecular modelling
Molecular modelling studies were performed to find a possible
binding mode of the most active compounds (4,8,14) with AChE.
The structure of enzyme was obtained from the Protein Data Bank
[29]. The 1ACJ complex was selected for docking according to the
validation process. All compounds were bound mainly with the
catalytic active site. Interactions with a peripheral anionic sitewere
reduced.
The derivatives were located between the residues Trp84 and
Phe330. The central part of each molecule ethe thiadiazole ring
was parallel to the indole moiety of Trp84, creating
p
e
p
stacking
interactions (distance 3.1e3.2
A for each compound). The two hy-
droxyl substituents in compound 4created the following hydrogen
bonds: the first one in position 4 with the amine group of Gly117
(2.7
A) and with the hydroxyl substituent of Tyr130 (2.5
A), the
second one in position 2 formed H-bond with Glu199 (2.3
A)
(Fig. 1). The bromine atom was directed to the conserved water
molecule (WAT643), thus phenyl ring could form
p
e
p
stacking and
p
eCH interactions with the aromatic side chains of Phe330 and
Trp432. The interaction CHeoxygen atom from the hydroxyl sub-
stituent in Tyr334 (2.2
A) was also observed in the case of this ring.
Compound 8due to the lack of bromine atom and introduction
of CF
3
was slightly shifted (Fig. 2). It caused a few changes in
binding. The OH group in position 4 created H-bond with the OH
substituent of Tyr130 (3.2
A) and ]C(]O) from Gly117 (2.2
A), the
second hydroxyl substituent still formed the hydrogen bond with
Glu199 (2.9
A). The thiadiazole moiety was rotated and nitrogen
atoms were directed towards the conserved water (WAT 643)
(3.1 and 3.5
A). Phenyl substituted by the CF
3
group formed two
kinds of interactions:
p
e
p
with Phe330 (3.6
A), CHeoxygen atom
from OH Tyr334 (2.8
A). Trifluoromethyl could create
hydrophobic interactions with Trp432.
For compound 14 (Fig. 3) the thiadiazole moiety was placed in
the same way as in the case of compound 8but the outermost
phenyl rings were replaced with each other. Trimethoxyphenyl was
orientated towards Ser122 and Gly123. The oxygen atom from the
3-CH
3
O group created the hydrogen bond with OH Ser122 (3.5
A)
and oxygen from 4-CH
3
O with NH of Gly123 (2.6
A). The HOeC(2)
substituent from the second phenyl ring formed H-bond with the
carbonyl group of His440 (2.6
A), 4-hydroxy group interacted with
p
electrons of Trp432 (3.5
A). The phenyl ring was engaged in
p
e
p
stacking with Phe330 (3.6
A) and CHeOH interactions with Tyr334
Fig. 1. Binding mode of compound 4with AChE.
Fig. 2. Binding mode of compound 8with AChE.
A. Skrzypek et al. / European Journal of Medicinal Chemistry 62 (2013) 311e319 313
(2.5
A). These differences in binding of compounds 4,8and 14 could
be the reason for differences in activity.
The scoring function for the novel compounds is similar or
higher than for the reference inhibitor etacrine (GoldScore: 67.95;
IC
50
180 nM) [30]. Two most active compounds 4and 8obtained
76.48 and 69.17 (GoldScore) and are more potent than the reference
(IC
50
60 nM and 90 nM, respectively).
3.3. Kinetic studies
The linear LineweavereBurk equation which is a double recip-
rocal form of the MichaeliseMenten one was converted to evaluate
the type of inhibition. The graphical analysis of steady-state inhi-
bition data for representative compounds 1and 8is shown in
Fig. 4A and B, respectively. In Fig. 4A the graph shows that the
mechanism of AChE inhibition of compound 1is of the mixed-type.
In Fig. 4B the lines crossing the xaxis in the same point indicate
unchanged K
M
and decrease V
max
with the increasing inhibitor
concentrations. This is a typical trend of non-competitive inhibition
which is similar to that of donepezil [31].
Docking simulation showed that compounds bound mainly
with the catalytic active site of AChE creating with them a lot of
interactions significant for activity. The studies revealed that the
compounds interact in a different way depending on the type of
aryl ring modification. Only 1,3,4-thiadiazole ring occupies the
same space but its orientation and type of interactions are also
different. This can explain significant differences in the activity,
selectivity as well as different types of inhibitions of the studied
analogues.
3.4. Virtual screening of pharmacokinetic properties
To predict some aspects of the pharmacokinetics of the obtained
compounds, their physicochemical and topological properties were
calculated. Table 2 presents the octanolewater partition co-
efficients expressed as Clog Pand Mlog P(Moriguchi log P),
a number of H-bond donors (HBD), a number of H-bond acceptors
(HBA), a number of rotatable bonds (RBC), and the polar surface
area (tPSA).
The descriptors obtained in silico were compared with the filters
for prediction of solubility and permeability of drug candidates after
the oral admission. The results show that the compounds under
consideration obey the Lipinski rule of 5: their MW 500, HBD 5,
HBA 10 and Clog P5(Table 2)[21,22]. The molecular weight
MW of compounds is found between 284 and 360 Da, their Clog Pis
in the range of 1.49e3.97, they possess 2-4 H-bond donors and 4-7
H-bond acceptors. All properties are of medium value. According to
Lipinski a compound that fails the alert will likely to be poorly
bioavailable because of poor absorption or permeation [21].
The derivatives also meet the Oprea’s criterions, which addi-
tionally includes a number of ring 5 and MLog Pin the range 2.0
to 4.5. The compounds possess 2 rings and their Mlog Pvalues are
in the range of 0.64e2,71 [23].Afilter of two properties is proposed
by Veber: a number of HBA and HBD 12 (tPSA 140
A
2
),
RBC 10. It is postulated that limited molecular flexibility,
expressed as the number of rotatable bonds (RBC), and low polar
Fig. 3. Binding mode of compound 14 with AChE.
A
-5
0
5
10
15
20
25
30
-12 -8 -4 0 4 8 12
[S]
-1
mM
-1
V
-1
min/ A
no inhibitor
0.12 µM
0.17 µM
B
-5
0
5
10
15
20
25
-12 -8 -4 0 4 8 12
[
S
]
-1
mM
-1
V
-1
min/ A
no inhibitor
0.10 µM
0.14 µM
Fig. 4. Steady state inhibition of 1(A) and 8(B) against AChE. The plot A shows
mixed-type inhibition and the plot B non-competitive inhibition.
Table 2
Molecular descriptors
a
in silico of (1,3,4-thiadiazol-2-yl)benzene-1,3-diols.
No. HBD HBA Clog PMlog PtPSA [
A
2
] RBC
1. 2 4 3.04 2.46 65.18 2
2. 2 4 2.65 2.33 65.18 2
3. 2 4 3.01 2.46 65.18 2
4. 2 4 3.11 2.59 65.18 2
5. 2 4 3.72 2.71 65.18 2
6. 2 4 3.72 2.71 65.18 2
7. 2 4 3.97 2.71 65.18 2
8. 2 4 3.43 2.83 65.18 3
9. 2 5 2.04 1.67 74.41 3
10. 2 5 2.60 1.67 74.41 3
11. 2 5 2.60 1.67 74.41 3
12. 2 6 2.32 1.15 83.64 4
13. 2 6 2.11 1.15 83.64 4
14. 2 7 2.04 0.65 92.87 5
15. 4 6 1.49 0.64 105.64 2
16. 4 6 1.86 0.64 105.64 2
17. 3 6 1.99 0.90 96.64 3
18. 4 5 1.59 1.42 91.20 2
19. 2 6 2.29 1.98 116.99 3
20. 2 6 2.29 1.98 116.99 3
21. 2 6 2.71 2.24 116.99 3
a
Clog Pethe octanolewater partition coefficient, Mlog PeMoriguchi log P, HBD
ea number of H-bond donors, HBA ea number of H-bond acceptors, tPSA ethe
polar surface area, RBC ea number of rotatable bonds.
A. Skrzypek et al. / European Journal of Medicinal Chemistry 62 (2013) 311e319314
surface are important predictors of oral bioavailability, indepen-
dent of molecular weight [24].
The results show that the synthesized compounds would have
favourable pharmacokinetics: solubility and permeability after the
oral admission as drug candidates. They are drug likeliness inde-
pendent of the criterion used.
4. Conclusions
In summary, a series of new 1,3,4-thiadiazole analogues has
been synthesized and evaluated as the AChE and BuChE inhibitors.
Compounds 4and 8(IC
50
¼0.06 and 0.09
m
M respectively) are the
most potent ones in the series. At the same time they show strong
affinity for BuChE but compound 14 is 1154-fold more active
inhibiting AChE (IC
50
¼0.17
m
M) than BuChE. It is slightly more
selective than donepezil and neostigmine. That compound can
serve as a selective inhibition agent for AChE over BuChE. The ki-
netic studies suggest that in a series of the investigated compounds,
the inhibition mechanisms can be various.
Docking simulation showed that the compounds are bound
mainly with the catalytic active site of AChE. The scoring function
for the novel compounds is similar or higher than for the reference
inhibitor. Modelled derivatives 4,8and 14 create a lot of in-
teractions with the catalytic active site of AChE which confirms
their high inhibitory potency. However, the compounds locate in
different ways depending on the type of aryl ring modification.
Particularly a molecule of compound 14 docs otherwise, probably
due to a large volume of trimetoxyphenyl substituent. This can
cause a poor fit and week interactions with the BuChE residues
and its low inhibition. It can explain very high selectivity of 14
towards AChE.
The analysis of adequate descriptors revealed that the com-
pounds possess properties which can suggest favourable pharma-
cokinetics in the human body after the oral admission. They are
drug likeliness independent of the criterion used.
5. Experimental section
5.1. Chemistry
The IR spectra were measured with a PerkineElmer FT-IR 1725X
spectrophotometer (in KBr) or a Varian 670-IR FT-IR spectrometer
(ATR). The spectra were made in the range of 600e4000 cm
1
.
1
H
NMR and
13
C NMR spectra were recorded in DMSO-d
6
a Varian
Mercury 400 or a Bruker DRX 500 instrument. Chemical shifts
(
d
, ppm) were described in relation to tetramethylsilane (TMS).
The spectra MS (EI, 70 eV) were recorded using the apparatus
AMD-604. Elemental analyses (C, H, N) were performed using
a PerkineElmer 2400 instrument and were found to be in good
agreement (0.4%) with the calculated values. The melting point
(mp) was determined using a Büchi B-540 (Flawil, Switzerland)
melting point apparatus.
The purity of the compounds was examined by HPLC Knauer
(Berlin, Germany) with a dual pump, a 20-
m
L simple injection valve
and a UVevisible detector (330 nm). The Hypersil Gold C18(1.9
m
m,
100 2.1 mm) column was used as the stationary phase. The
mobile phase included different contents of MeOH and acetate
buffer (pH 4, 20 nM) as the aqueous phase. The flow rate was
0.5 mL min
1
at room temperature. The retention time of an
unretained solute (t
o
) was determined by the injection of a small
amount of acetone dissolved in water. The log kvalues for 70% of
methanol (v/v) in the mobile phase are presented. The log kvalues
were calculated as log k¼log(t
R
t
o
)/t
o
, where: t
R
¼the retention
time of a solute, t
o
¼the retention time of an unretained solute.
5.2. Synthesis of compounds
5.2.1. A general procedure for the synthesis of compounds
A mixture of the corresponding hydrazide (0.01 mol) and STB
(0.0075 mol) in MeOH (50 mL) was refluxed for 3 h. The hot mix-
ture was filtered. Water (50 mL) was added to the filtrate and the
filtrate was left at room temperature (48 h) (compounds 1,9e11,14
and 18) or the filtrate was concentrated (compounds 3,5e8,12,13,
16,19 ,21). The obtained solid was recrystallized from MeOH/H
2
O.
5.2.2. 4-[5-(3-Methylphenyl)-1,3,4-thiadiazol-2-yl]benzene-1,3-
diol (1)
Yield: 74%; pale yellow needles; HPLC: (C-18): log k¼0.308;
m.p.: 234e236
C; anal. calc. for C
15
H
12
N
2
O
2
S (284.06): C, 63.36;
H, 4.25; N, 9.85; found: C, 63.42; H, 4.23; N, 9.89;
1
H NMR
(400 MHz, DMSO-d
6
, ppm)
d
: 11.14 (s, 1H, HOeC(3)), 10.10 (s, 1H,
HOeC(3)), 8.08 (d, J¼8.73 Hz, 1H, HOeC(5)), 7.83 (m, 2H, HOe
C(2
0
,4
0
)), 7.45 (t, J¼7.55 Hz, 1H, HeC(5
0
)), 7.36 (m, 1H, HeC(6
0
)),
6.53 (d, J¼2.18 Hz, 1H, HOeC(2)), 6.48 (dd, J¼8.74 and
2.36 Hz, 1H, HOeC(6)), 2.41 (s, 3H, CH
3
);
13
C NMR (125 MHz,
DMSO-d
6
, ppm)
d
: 166.2, 162.6, 161.3, 156.3, 138.8, 131.3, 130.1,
129.2, 128.9, 127.7, 124.4, 108.4, 108.3, 102.3, 20.8 (CH
3
); IR (KBr,
cm
1
): 3138 (OH), 1632 (C]N), 1598, 1528, 1471, 1443, 1318 (C]C),
1281, 1262, 1214, 1175 (CeO), 1141, 1095 (HeAr), 1041 (N]CeSe
C]N), 986, 966, 877, 846, 778 (HeAr), 684, 665 (CeSeC), 596,
522; EI-MS (m/z, %): 284 (M
þ
, 100), 168 (4), 167 (38), 153 (6), 150
(3), 149 (15), 142 (4), 135 (13), 119 (5), 118 (4), 117 (5), 116 (4), 107
(5), 91 (9), 80 (3), 65 (4).
5.2.3. 4-[5-(2-Chlorophenyl)-1,3,4-thiadiazol-2-yl]benzene-1,3-
diol (3)
Yield: 68%; yellow needles; HPLC (C-18): log k¼0.175; m.p.:
268e269
C; anal. calc. for C
14
H
9
ClN
2
O
2
S (304.75): C, 55.18; H, 2.98;
N, 9.19; found: C, 55.06; H, 2.99; N, 9.21;
1
H NMR (500 MHz, DMSO-
d
6
, ppm)
d
: 11.17 (s, 1H, HOeC(3)), 10.14 (br.s., 1H, HOeC(1)), 8.17
(m, 1H, HeC(3
0
)), 8.14 (d, J¼8.70 Hz, 1H, HeC(5)), 7.69 (m, 1H, He
C(6
0
)), 7.55 (m, 2H, HeC(4
0
,5
0
)), 6.56 (d, J¼2.32 Hz, 1H, HeC(2)),
6.51 (dd, J¼8.70 and 2.33 Hz, 1H, HeC(6));
13
C NMR (125 MHz,
DMSO-d
6
, ppm)
d
: 164.2, 161.8,161.4,156.4, 131.8, 131.3,130.9, 130.6,
129.0, 128.9, 127.8, 108.4, 108.1, 102.4; IR (KBr, cm
1
): 3186 (OH),
1629 (C]N),1598 (C]C), 1522 (C]C), 1473 (C]C), 1417, 1314,1230,
1183 (CeO),1068 (N]CeSeC]N), 1042, 995, 982, 965, 845 (HeAr),
798, 757, 733, 714, 681 (CeSeC), 648, 632; EI-MS (m/z, %): 304 (M
þ
,
100), 171 (6), 169 (19), 168 (7), 167 (78), 157 (8), 156 (5), 155 (24), 153
(12), 140 (4), 139 (10), 138 (8), 137 (14), 136 (3), 135 (18), 134 (10),
125 (5), 111 (15), 108 (10), 107 (19), 106 (8), 102 (13), 97 (8), 95 (5),
90 (4), 84 (5), 80 (13), 79 (7), 76 (6), 75 (15), 69 (17), 66 (5), 65 (7), 63
(11), 62 (6), 53 (6), 52 (24), 51 (18), 50 (10), 45 (7), 39 (22), 38 (5).
5.2.4. 4-[5-(2,4-Dichlorophenyl)-1,3,4-thiadiazol-2-yl]benzene-1,3-
diol (5)
Yield: 67%; orange needles; HPLC (C-18): log k¼0.533; m.p.:
272e273
C; anal. calc. for C
14
H
8
Cl
2
N
2
O
2
S (339.20): C, 49.57; H,
2.38; N, 8.26; found: C, 49.66; H, 2.36; N, 8.23;
1
H NMR (400 MHz,
DMSO-d
6
, ppm)
d
: 11.21 (s, 1H, HOeC(3)), 10.15 (s, 1H, HOeC(1)),
8.22 (dd, J¼8.56 and 0.28 Hz, 1H, HeC(5
0
)), 8.14 (d, J¼8.71 Hz,
1H, HeC(5)), 7.89 (dd, J¼2.11 and 0.27 Hz, 1H, HeC(3
0
)), 7.65 (dd,
J¼8.57 and 2.11 Hz, 1H, HeC(6
0
)), 6.54 (d, J¼2.20 Hz, 1H, HeC(2)),
6.41 (dd, J¼8.71 and 2.29 Hz, 1H, HeC(6));
13
C NMR (125 MHz,
DMSO-d
6
, ppm)
d
: 164.3, 161.5, 160.0, 156.4, 135.6, 132.2, 132.0,
130.0, 128.9, 128.4, 128.1, 108.5, 108.1, 102.3; IR (KBr, cm
1
): 3350,
3196 (OH), 1630 (C]N), 1595 (C]N), 1524 (C]N), 1478 (C]N),
1411, 1341, 1315, 1246, 1246, 1177 (CeO), 1142, 1110 (CeCl), 1067
(N]CeSeC]N), 994, 966, 947, 815 (HeAr), 745, 684, 668 (CeSeC),
648, 614; EI-MS (m/z, %): 338 (M
þ
, 100), 205 (7), 203 (10), 191 (5),
A. Skrzypek et al. / European Journal of Medicinal Chemistry 62 (2013) 311e319 315
189 (7), 171 (5), 169 (6), 168 (7), 167 (65), 153 (8), 135 (8), 119 (6), 107
(7), 80 (4), 69 (5), 52 (6), 39 (4).
5.2.5. 4-[5-(2,5-Dichlorophenyl)-1,3,4-thiadiazol-2-yl]benzene-1,3-
diol (6)
Yield: 69%; orange needles; HPLC (C-18): log k¼0.530; m.p.:
250e252
C; anal. calc. for C
14
H
8
Cl
2
N
2
O
2
S (339.20): C, 49.57; H, 2.38;
N, 8.26; found: C, 49.62; H, 2.39; N, 8.30;
1
H NMR (500 MHz, DMSO-
d
6
,ppm)
d
: 11.18 (s, 1H, HOeC(3)), 10.11 (s, 1H, HOeC(1)), 8.20 (d,
J¼2.61 Hz, 1H, HeC(6
0
)), 8.13 (d, J¼8.67 Hz, 1H, HeC(5)), 7.74 (d,
J¼8.65 Hz,1H, HeC(3
0
)), 7.64 (dd, J¼8.64 and 2.63 Hz, 1H, HeC(4
0
)),
6.52 (d, J¼2.29 Hz, 1H, HeC(2)), 6.47 (dd, J¼8.69 and 2.31 Hz, 1H,
HeC(6));
13
C NMR (125 MHz, DMSO-d
6
,ppm)
d
: 164.5, 161.5,160.58,
156.5,132.8,132.3, 131.3, 130.7, 130.0,129,9, 128.9,108.5, 108.1, 102.3;
IR (KBr, cm
1
): 3372 (OH), 3065 (C
Ar
eH), 1621 (C]N), 1590 (C]C),
1519 (C]C), 1473 (C]C), 1402,1339,1314,1291, 1259, 1218, 1184 (Ce
O), 1141, 1105 (CeCl), 1062 (N]CeSeC]N), 1015, 986, 970, 881, 844
(HeAr), 825, 763, 742, 721, 683 (CeSeC), 658, 638; EI-MS (m/z,%):
338 (M
þ
, 10 0), 205(8), 203 (12), 191 (7), 189 (11),173 (8),171(11),169
(7), 168 (10),167 (77), 154 (4), 153 (12), 136 (5), 135 (13), 133 (5), 124
(4),119(10), 112 (5), 109 (4), 108(6), 107 (11), 100 (4), 97 (4), 80 (8), 69
(9), 52 (10), 51 (6), 39 (7).
5.2.6. 4-[5-(3,5-Dichlorophenyl)-1,3,4-thiadiazol-2-yl]benzene-1,3-
diol (7)
Yield: 66%; orange needles; HPLC (C-18): log k¼0.781; m.p.:
273e275
C; anal. calc. for C
14
H
8
Cl
2
N
2
O
2
S (339.20): C, 49.57; H,
2.38; N, 8.26; found: C, 49.65; H, 2.40; N, 8.31;
1
H NMR (500 MHz,
DMSO-d
6
, ppm)
d
: 11.21 (s, 1H, HOeC(1)), 10.12 (s, 1H, HOeC(3)),
8.11 (d, J¼8.71 Hz, 1H, HeC(5)), 8.02 (d, J¼1.93 Hz, 2H, HeC(2
0
,
6
0
)), 7.78 (t, J¼1.89 Hz, 1H, HeC(4
0
)), 6.53 (d, J¼2.31 Hz, 1H, He
C(2)), 6.48 (dd, J¼8.70 and 2.31 Hz, 1H, HeC(6));
13
C NMR
(125 MHz, DMSO-d
6
, ppm)
d
: 163.7, 163.3, 161.6, 156.5, 135.0 (2C),
133.4, 129.7, 128.9, 135.5 (2C), 108.5, 108.1, 102.3; IR (ATR, cm
1
):
3200 (OH), 3077 (HeAr), 2917 (CeH), 2850 (CeH), 1626 (C]N),
160 6 (C]C), 1590 (C]C), 1567 (C]C), 1523 (C]C), 1486, 1417, 1330,
1278, 1261, 1193 ( CeO), 1124, 1110 (CeCl), 1060 (N]CeSeC]N),
988, 872, 855, 816 (HeAr), 765, 673 (CeSeC); EI-MS (m/z, %): 338
(M
þ
, 100), 205 (13), 203 (19), 191 (7), 189 (9), 173 (7), 171 (10), 170
(4), 169 (7), 168 (10), 167 (74), 153 (13), 145 (5), 135 (18),133 (4), 119
(11), 112 (5), 109 (5), 108 (7), 107 (13), 106 (5), 97 (5), 80 (9), 79 (4),
69 (9), 52 (12), 51 (6), 39 (17), 38 (6).
5.2.7. 4-[5-(4-Trifluoromethylphenyl)-1,3,4-thiadiazol-2-yl]
benzene-1,3-diol (8)
Yield: 64%; orange needles; HPLC (C-18): log k¼0.494; m.p.:
252e254
C; anal. calc. for C
15
H
9
F
3
N
2
O
2
S (338.30): C, 53.25; H, 2.68;
N, 8.28; found: C, 53.30; H, 2.65; N, 8.30;
1
H NMR (500 MHz, DMSO-
d
6
, ppm)
d
: 11.20 (s, 1H, HOeC(1)), 10.11 (s, 1H, HOeC(3)), 9.23 (d,
J¼8.11 Hz, 2H, HeC(3
0
,5
0
)), 8.10 (d, J¼8.69 Hz, 1H, HeC(5)), 7.90 (d,
J¼8.32 Hz, 2H, HeC(2
0
,6
0
)), 6.54 (d, J¼2.30 Hz, 1H, HeC(2)), 6.46
(dd, J¼8.70 and 2.31 Hz, 1H, HeC(6));
13
C NMR (125 MHz, DMSO-
d
6
, ppm)
d
: 164.1, 163.4, 161.6, 156.5, 134.0, 129.0, 128.9, 128.0, 125.1
(CF
3
), 126.3, 126.2, 125.0, 108.4, 108.2, 102.3; IR (KBr, cm
1
): 3452,
3202 (OH), 1631 (C]N), 1600, 1523, 1473 (C]C), 1413, 1320 (CF
3
),
1235, 1170 (CeO), 1141 (C
AR
eCF
3
), 1115, 1067 (N]CeSeC]N), 1012,
996, 985, 964, 843, 829, 799, 671 (CeSeC); EI-MS (m/z, %): 338 (M
þ
,
100), 319 (9), 309 (4), 203 (20), 189 (14), 171 (4), 169 (4),167 (47),153
(13), 152 (6), 145 (12), 139 (8), 135 (15), 121 (7), 119 (9), 112 (5), 108
(7), 107 (14), 106 (5), 95 (7), 80 (90), 69 (11), 51 (8), 39 (11).
5.2.8. 4-(5-(2-Methoxyphenyl)-1,3,4-thiadiazol-2-yl)benzene-1,3-
diol (9)
Yield: 70%; yellow needles; HPLC (C-18): log k¼0.301; m.p.:
218e220
C; anal. calc. for C
15
H
12
N
2
O
3
S (300.33): C, 59.99; H, 4.03;
N, 9.33; found: C, 59.91; H, 4.01; N, 9.36;
1
H NMR (500 MHz, DMSO-
d
6
, ppm)
d
: 10.22 (s, 1H, HOeC(3)), 9.20 (s, 1H, HOeC(1)), 7.52 (dd,
J¼7.53 and 1.71 Hz, 1H, HeC(6
0
)), 7.24 (d, J¼8.64 Hz, 1H, HeC(5)),
6.75 (m, 1H, HeC(4
0
)), 6.49 (m, 1H, HeC(5
0
)), 6.37 (m, 1H, HeC(3
0
)),
5.69 (d, J¼2.32 Hz, 1H, HeC(2)), 5.66 (dd, J¼8.64 and 2.34 Hz, 1H,
HeC(6)), 3.21 (s, 3H, OCH
3
);
13
C NMR (125 MHz, DMSO-d
6
, ppm)
d
:
164.4, 161.0, 160.2, 156.4, 155.5,131.9, 129.1, 127.6, 121.1,118.9, 112.4,
108.5,108.3, 102.5, 56.1 (OCH
3
); IR (KBr, cm
1
): 3410 (OH), 2935 (Ce
H), 2846 (AreOCH
3
), 1628 (C]N), 1601 (C]C), 1519 (C]C), 1468
(C]C), 1428, 1320, 1257 (CeOeC), 1170 (CeO), 1136, 1059 (N]Ce
SeC]N), 1019, 990, 967, 848, 799, 754 (HeAr), 693 (CeSeC), 658,
632, 601, 527; EI-MS (m/z, %): 300 (M
þ
, 24), 284 (7), 256 (73), 167
(7), 166 (6), 165 (9), 153 (12), 149 (15), 148 (7), 142 (5), 137 (45), 136
(21), 135 (100), 132 (6), 78 (5), 77 (29), 71 (5), 69 (9), 64 (7), 63 (9),
57 (5), 53 (5), 52 (7), 51 (11), 50 (6), 43 (5), 41 (5), 39 (11), 36 (5).
5.2.9. 4-(5-(3-Methoxyphenyl)-1,3,4-thiadiazol-2-yl)benzene-1,3-
diol (10)
Yield: 78%; orange needles; HPLC (C-18): log k¼0.242; m.p.:
145 e147
C; anal. calc. for C
15
H
12
N
2
O
3
S (300.33): C, 59.99; H, 4.03;
N, 9.33; found: C, 59.94; H, 4.01; N, 9.38;
1
H NMR (500 MHz, DMSO-
d
6
, ppm)
d
: 11.14 (s, 1H, HOeC(3)), 10.10 (s, 1H, HOeC(1)), 8.09 (d,
J¼8.67 Hz, 1H, HeC(5)), 7.58 (m, 1H, HeC(4
0
)), 7.54 (m, 1H, He
C(2
0
)), 7.45 (t, J¼8.04 Hz, 1H, HeC(5
0
)), 7.13 (m, 1H, HeC(6
0
)),
6.55 (d, J¼2.37 Hz, 1H, HeC(2)), 6.50 (dd, J¼8.68 and 2.33 Hz,
1H, HeC(6)), 3.87 (s, 3H, OCH
3
);
13
C NMR (125 MHz, DMSO-d
6
,
ppm)
d
: 165.0, 163.0, 161.4, 159.7, 156.5, 131.5, 130.6, 129.0, 119.9,
116.6, 111.9, 108.4, 108.3, 102.5, 55.4 (OCH
3
); IR (KBr, cm
1
): 3626,
3128 (OH), 2834 (AreOCH
3
), 1604 (C]N), 1580 (C]C), 1455 (C]C),
1408, 1382, 1295, 1270 (CeOeC), 1218, 1198, 1173 (CeO), 1107 (Are
H), 1016 (N]CeSeC]N), 976, 885, 873, 807, 792, 774 (AreH), 682
(CeSeC), 644; EI-MS (m/z, %): 300 (M
þ
, 100), 299 (4), 271 (4), 168
(4), 167 (40), 165 (11), 153 (6),151 (10), 150 (5), 136 (6),135 (13), 134
(5), 133 (11), 122 (6), 119 (6), 112 (6), 108 (8), 107 (8), 103 (5), 90 (4),
80 (5), 77 (8), 69 (5), 64 (4), 63 (6), 52 (8), 51 (5), 39 (7).
5.2.10. 4-(5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl)benzene-1,3-
diol (11 )
Yield: 72%; pale orange needles; HPLC (C-18): log k¼0.193;
m.p.: 218e220
C; anal. calc. for C
15
H
12
N
2
O
3
S (300.33): C, 59.99; H,
4.03; N, 9.33; found: C, 60.06; H, 4.01; N, 9.29;
1
H NMR (500 MHz,
DMSO-d
6
, ppm)
d
: 11.07 (s, 1H, HOeC(3)), 10.06 (s, 1H, HOeC(1)),
8.05 (d, J¼8.69 Hz, 1H, HeC(5)), 7.95 (m, 2H, HeC(2
0
,6
0
)), 7.11
(m, 2H, HeC(3
0
,5
0
)), 6.53 (d, J¼2.34 Hz, 1H, HeC(2)), 6.48 (dd,
J¼8.66 and 2.34 Hz, 1H, HeC(6)), 3.84 (s, 3H, OCH
3
);
13
C NMR
(125 MHz, DMSO-d
6
, ppm)
d
: 165.8, 162.1, 161.2, 161.1, 156.2, 128.9,
128.8 (2C), 122.7,114.7 (2C), 108.4, 108.3,102.4, 55.4 (OCH
3
); IR (KBr,
cm
1
): 3848, 3110 (OH), 2837 (CeOCH
3
), 2360, 1606 (C]N), 1521
(C]C), 1455 (C]C), 1417, 1309, 1257 (CeOeC), 1178 (C
Ar
eO), 1137,
1173 (CeO), 1029 (N]CeSeC]N), 981, 963, 885, 834, 801 (AreH),
744, 683, 669 (CeSeC), 641; EI-MS (m/z, %): 300 (M
þ
, 100), 284 (10),
168 (4), 167 (42), 165 (13), 160 (5), 153 (10), 151 (18), 150 (23), 137
(7), 136 (5), 135 (21), 134 (12), 133 (14), 122 (8), 120 (5), 119 (7), 108
(6), 107 (8), 90 (5), 80 (5), 77 (5), 69 (5), 63 (6).
5.2.11. 4-(5-(3,4-Dimethoxyphenyl)-1,3,4-thiadiazol-2-yl)benzene-
1,3-diol (12)
Yield: 73%; yellow plaques; HPLC (C-18): log k¼0.012; m.p.:
214e216
C;anal.calc.forC
16
H
14
N
2
O
4
S (330.36): C, 58.17; H, 4.27;
N, 8.48; found: C, 58.15; H, 4.29; N, 8.50;
1
H NMR (400 MHz,
DMSO-d
6
,ppm)
d
: 11.07 (s, 1H, HOeC(1)), 10.07 (s, 1H, HOeC(3)),
8.04 (d, J¼8.56 Hz, 1H, HeC(5)), 7.56 (d, J¼2.02 Hz, 1H, He
C(2
0
)), 7.53 (dd, J¼8.23 and 2.02 Hz, 1H, HeC(6
0
)), 7.11 (d,
J¼8.56 Hz, 1H, HeC(5
0
)), 6.51 (d, J¼2.35 Hz, 1H, HeC(2)), 6.47
(dd, J¼8.73 and 2.35 Hz, 1H, HeC(6)), 3.88 (s, 3H, CH
3
), 3.84 (s,
A. Skrzypek et al. / European Journal of Medicinal Chemistry 62 (2013) 311e319316
3H, CH
3
);
13
C NMR (125 MHz, DMSO-d
6
, ppm)
d
: 166.0, 162.3,
161.1, 156.3, 150.9, 149.1, 128.9, 122.8, 120.8, 112.0, 109.7, 108.4,
108.3, 102.4, 55.7 (OCH
3
), 55.6 (OCH
3
); IR (KBr, cm
1
): 3478, 3151
(OH), 2943 (AreH), 2840 (C
Ar
eOCH
3
), 1627 (C]N), 1597 (C]C),
1522 (C]C), 1454 (C]C), 1432 (C]C), 1269 (CeOeC), 1246, 1178
(CeO), 1128, 1095 (N]CeSeC]N), 1020, 985, 967, 859, 805, 756
(AreH), 679, 643 (CeSeC); EI-MS (m/z, %): 330 (M
þ
, 100), 329 (5),
315 (12), 301 (4), 287 (9), 181 (7),180 (5), 167 (14), 165 (7), 164 (5),
163 (14), 153 (8), 152 (4), 148 (6), 137 (4), 136 (4), 135 (6), 120 (7),
107 (4), 94 (5), 65 (4), 39 (4).
5.2.12. 4-(5-(2,5-Dimethoxyphenyl)-1,3,4-thiadiazol-2-yl)benzene-
1,3-diol (13)
Yield: 79%; pale yellow plaques; HPLC (C-18): log k¼0.102;
m.p.: 248e249
C; anal. calc. for C
16
H
14
N
2
O
4
S (330.36): C, 58.17; H,
4.27; N, 8.48; found: C, 58.23; H, 4.29; N, 11.41;
1
H NMR (500 MHz,
DMSO-d
6
, ppm)
d
: 11.03 (s, 1H, HOeC(3)), 10.03 (s, 1H, HOeC(1)),
8.05 (d, J¼8.64 Hz, 1H, HeC(5)), 7.87 (d, J¼3.16 Hz, 1H, He
C(6
0
)), 7.25 (d, J¼9.12, 1H, HeC(3
0
)), 7.11 (dd, J¼9.07 and
3.19 Hz, 1H, HeC(4
0
)), 6.50 (d, J¼2.29 Hz, 1H, HeC(2)), 6.47 (dd,
J¼8.64 and 2.32 Hz, 1H, HeC(6)), 3.97 (s, 3H, OCH
3
), 3.82 (s, 3H,
OCH
3
);
13
C NMR (125 MHz, DMSO-d
6
, ppm)
d
: 164.5, 161.0, 160.0,
156.4,153.3, 149.9, 129.1, 119.4, 117.9,114.0,111.3, 108.5,108.3, 102.4,
56.6, 55.6; IR (KBr, cm
1
): 3434 (OH), 3001 (AreH), 2938 (AreH),
2836 (CH
3
), 1629 (C]N), 1598 (C]C), 1503 (C]C), 1465 (C]C),
1427 (C]C), 1402, 1338, 1289 (CeOeC), 1224, 1182 (CeO), 1129,
1047 ( N]CeSeC]N), 1018(AreH), 970, 876, 846, 805, 728, 691,
669 (CeSeC), 644, 616; EI-MS (m/z, %): 330 (M
þ
, 100), 329 (16), 301
(15), 287 (9), 271 (7), 196 (10), 195 (49), 194 (18), 180 (13), 179 (7),
178 (11), 168 (7), 167 (46), 166 (17), 165 (14), 163 (13), 162 (23), 162
(23), 153 (28), 152 (9), 150 (9), 149 (16), 148 (27), 137 (8), 136 (17),
135 (22), 134 (21), 120 (11), 107 (9), 79 (8), 69 (7).
5.2.13. 4-(5-(3,4,5-Trimethoxyphenyl)-1,3,4-thiadiazol-2-yl)
benzene-1,3-diol (14)
Yield: 71%; yellow needles; HPLC (C-18): log k¼0.022; m.p.:
202e204
C; anal. calc. for C
17
H
16
N
2
O
5
S (360.38): C, 56.66; H, 4.47;
N, 7.77; found: C, 56.74; H, 4.45; N, 7.80;
1
H NMR (500 MHz, DMSO-
d
6
, ppm)
d
: 11.05 (s, 1H, HOeC(1)), 10.02 (s, 1H, HOeC(3)), 8.08 (d,
J¼8.60 Hz, 1H, HeC(5)), 7.85 (d, J¼3.19 Hz, 2H, HeC(2
0
,6
0
)), 6.50 (d,
J¼2.29 Hz, 1H, HeC(2)), 6.48 (dd, J¼8.63 and 2.30 Hz, 1H, HeC(6)),
3.90 (s, 6H, OCH
3
), 3.82 (s, 3H, OCH
3
);
13
C NMR (125 MHz, DMSO-d
6
,
ppm)
d
: 166.0, 162.7, 161.2, 156.3, 156.3, 153.4, 139.5, 128.9, 125.7,
108.4, 108.3, 104.6 (2C), 102.3, 60.2 (OCH
3
), 56.1 (OCH
3
, 2C); IR (KBr,
cm
1
): 3408 (OH), 2934 (AreH), 2836 (CH
3
), 1628 (C]N), 1592 (C]
C), 1517 (C]C), 1467 (C]C), 1414, 1332, 1248 (CeOeC), 1172 (CeO),
1130, 1045 (N]CeSeC]N), 1002, 968, 925, 845, 804, 767, 737, 681,
650 (CeSeC), 600; EI-MS (m/z, %): 360 (M
þ
, 100), 347 (4), 346 (11),
345 (6), 331 (4), 317 (10), 314 (5), 302 (7), 193 (4), 180 (4), 178 (10),
167 (7), 153 (14), 152 (4), 150 (12), 135 (9), 120 (4), 118 (4).
5.2.14. 4-(5-(3,4-Dihydroxyphenyl)-1,3,4-thiadiazol-2-yl)benzene-
1,3-diol (16)
Yield: 82%; yellow plaques; HPLC (C-18): log k¼0.293; m.p.:
296e298
C; anal. calc. for C
14
H
10
N
2
O
4
S (302.31): C, 55.62; H, 3.33; N,
9.27; found: C, 55.70; H, 3.32; N, 9.31;
1
HNMR(500MHz,DMSO-d
6
,
ppm)
d
:11.03(s,1H,HOeC(3)), 10.02 (s, 1H, HOeC(1)), 9.55 (br.s., 2H,
HOeC(3
0
,4
0
)), 8.00 (d, J¼8.68 Hz, 1H, HeC(5)), 7.44 (d, J¼2.14 Hz, 1H,
HeC(2
0
)), 7.28 (dd, J¼8.2 and 2.21 Hz, 1H, HeC(6
0
)), 6.88 (d,
J¼8.19 Hz, 1H, HeC(5
0
)), 6.51 (d, J¼2.29 Hz, 1H, HeC(2)), 6.47 (dd,
J¼8.66 and 2.3 Hz, 1H, HeC(6));
13
CNMR(125MHz,DMSO-d
6
, ppm)
d
: 166.4, 161.8, 161.0, 156.2,148.2, 145.8, 128.9, 121.4, 119.5, 116.2, 113.9,
108.4, 108.3, 102.4; IR (KBr, cm
1
): 3392 (OH), 1631 (C]N), 1530 (C]
C),1440 (C]C),1335,1292, 1228,1185 (CeO ), 1141, 1020 (N ]CeSeC]
N), 987, 969, 886, 884 (AreH), 805, 772, 745, 728, 686 (CeSeC), 644;
EI-MS (m/z, %): 302 (M
þ
, 100), 169 (6), 168 (9), 167 (67), 153 (39), 138
(5), 137 (5), 136 (10),135 (34), 121 (11),119 (8), 112 (5), 108 (7), 107 (13),
106 (10), 97 (7), 89 (6), 81 (7), 80 (10), 79 (9), 75 (7), 69 (13), 65 (7), 63
(17), 62 (7), 55 (6), 53 (7), 52 (21), 51 (18), 50 (7), 39 (17), 38 (6).
5.2.15. 4-(5-(3-Aminophenyl)-1,3,4-thiadiazol-2-yl)benzene-1,3-
diol (18)
Yield: 69%; yellow needles; HPLC (C-18): log k¼0.107; m.p.:
160 e162
C; anal. calc. for C
14
H
11
N
3
O
2
S (285.32): C, 58.93; H, 3.89;
N, 14.73; found: C, 58.98; H, 3.91; N, 14.68;
1
H NMR (500 MHz,
DMSO-d
6
, ppm)
d
: 11.14 (s, 1H, HOeC(3)), 10.07 (s, 1H, HOeC(1)),
8.08 (d, J¼8.68 Hz, 1H, C(5)), 7.88 (m, 2H, HeAr), 7.61 (m, 1H,
HeAr), 6.53 (d, J¼2.34 Hz, 1H, C(2)), 6.47 (dd, J¼8.70 and 2.33 Hz,
1H, C(6)), 6.38 (s, 2H, NH
2
), 6.36 (d, 1H, HeAr);
13
C NMR (125 MHz,
DMSO-d
6
, ppm)
d
: 165.5, 161.4, 156.2, 147.5, 133.9, 130.2, 128.9,
120.8, 118.8, 112.0, 109.7, 108.5, 107.9, 102.3; IR (KBr, cm
1
): 3433
(OH, NH), 1622 (C]N), 1506 (C]C), 1464 (C]C), 1341, 1234 (CeOe
C), 1171 (CeO), 978, 892, 843, 801, 744, 682 (CeSeC), 649; EI-MS
(m/z, %): 28 (5), 258 (4), 256 (10), 192 (7), 160 (11), 149 (4), 128
(9), 110 (7), 96 (6), 91 (6), 81 (4), 80 (7), 77 (5), 76 (5), 73 (4), 69 (4),
66 (10), 64 (100), 60 (6), 57 (7), 55 (8), 51 (7), 48 (12), 45 (8), 44 (80),
43 (14), 41 (13), 40 (46), 39 (12), 38 (6), 36 (15), 34 (4).
5.2.16. 4-(5-(2-Nitrophenyl)-1,3,4-thiadiazol-2-yl)benzene-1,3-
diol (19)
Yield: 64%; orange needles; HPLC (C-18): log k¼0.360; m.p.:
282e284
C; anal. calc. for C
14
H
9
N
3
O
4
S (315.30): C, 53.33; H, 2.88; N,
13.33; found: C, 53.41; H, 2.90; N, 13.29;
1
H NMR (500 MHz, DMSO-
d
6
,ppm)
d
:11.30(s,1H,HOeC(3)), 10.18 (s, 1H, HOeC(1)), 8.14 (d,
J¼8.80 Hz, 1H, HeC(5)), 8.09 (dd, J¼7.80 and 1.28 Hz, 1H, HeC(3
0
)),
7.97 (dd, J¼7.52 and 1.46 Hz, 1H, HeC(6
0
)), 7.85 (td, J¼7.52 and
1.46 Hz, 1H, HeC(5
0
)), 7.79 (td, J¼7.70 and 1.47 Hz, 1H, HeC(4
0
)), 6.55
(d, J¼2.20 Hz, 1H, HeC(2)), 6.48 (dd, J¼8.70 and 2.33 Hz, 1H, He
C(6));
13
C NMR (125 MHz, DMSO-d
6
,ppm)
d
: 164 .1, 161. 6, 161.1,
156.5 , 148.5, 133.1, 132. 0, 131.6, 128 .9, 124. 6, 123.4 , 108.5, 10 8.1, 102. 3;
IR (KBr, cm
1
): 3443, 3143 (OH), 3039 (AreH), 1606 (C]N), 1529,
146 6 (C ]C), 1411, 1368,1350,1317,1291 (NO
2
), 1216 (C eO) , 1109 , 104 2
(N]CeSeC]N), 984, 852, 812, 771, 745, 719, 684 (CeSeC), 648; EI-
MS (m/z,%):315(M
þ
, 49), 155 (5), 154 (9), 153 (100), 150 (3), 135 (7),
134 (12), 108 (4), 107 (4), 104 (9), 97 (4), 90 (4), 76 (4), 69 (3).
5.2.17. 4-(5-(3-Methyl-4-nitrophenyl)-1,3,4-thiadiazol-2-yl)
benzene-1,3-diol (21)
Yield: 84%; yellow needles; HPLC (C-18): log k¼0.193; m.p.:
295e296
C; anal. calc. for C
15
H
11
N
3
O
4
S (329.33): C, 54.71; H, 3.37;
N, 12.76; found: C, 54.62; H, 3.39; N, 12.81;
1
H NMR (500 MHz,
DMSO-d
6
, ppm)
d
: 11.22 (s, 1H, HOeC(1)), 10.13 (s, 1H, HOeC(3)),
8.15 (m, 2H, HeAr), 8.12 (d, J¼8.71 Hz, 1H, HeC(5)), 8.10 (m, 1H,
HeAr), 6.54 (d, J¼2.3 Hz, 1H, C(2)), 6.48 (dd, J¼8.72 and 2.34 Hz,
1H, C(6)), 2.63 (s, 3H, CH
3
);
13
C NMR (125 MHz, DMSO-d
6
, ppm)
d
:
164.0, 163.7, 161.6, 156.6, 149.5, 134.3, 134.2, 131.2, 128.9, 125.6,
125.5, 108.5, 108.2, 102.3, 19.5 (CH
3
); IR (ATR, cm
1
): 3358 (OH),
2947 (CH), 2835 (CH), 1633 (C]N), 1558 (C]C), 1507 (C]C), 1449,
1424,1346, 1323, 1284,1214,1185 (CeO), 1112, 1023, 865; EI-MS (m/
z, %): 329 (M
þ
, 100), 299 (9), 284 (5), 283 (8), 167 (37),153 (20),148
(10), 136 (5), 135 (14), 134 (7), 132 (9), 122 (7), 121 (9), 119 (7), 116
(10),108 (7), 107 (10),106 (5),104 (10), 97 (6), 90 (8), 89 (13), 80 (7),
78 (5), 77 (9), 69 (9), 65 (5), 63 (11), 52 (13), 51 (10), 45 (6), 39 (13).
Compounds: 4-[5-(2-fluorophenyl)-1,3,4-thiadiazol-2-yl]benzene-
1,3 - d iol (2), 4-[5-(2-bromophenyl)-1,3,4-thiadiazol-2-yl]benzene-1,3-
diol (4), 4-[5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazol-2-yl]benzene-
1,3 - d iol (15 ), 4-[5-(4-hydroxy-3-methoxyphenyl)-1,3,4-thiadiazol-2-
yl]benzene-1,3-diol (17 ) and 4-[5-(3-nitrophenyl)-1,3,4-thiadiazol-2-
yl]benzene-1,3-diol (20) were prepared according to the procedure
already described [26,32,33].
A. Skrzypek et al. / European Journal of Medicinal Chemistry 62 (2013) 311e319 317
5.3. Biological assay
5.3.1. In vitro AChE and BuChE inhibition assay
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from the electric eel),
butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum),
acetylthiocholine iodide (ATCh), butylthiocholine iodide (BTCh),
5,5
0
-dithiobis-(2-nitrobenzoic acid) (DTNB), neostigmine bromide
and donepezil hydrochloride monohydrate were purchased from
SigmaeAldrich (Steinheim, Germany). The inhibitory activities
against AChE and BuChE of the prepared compounds were per-
formed by means of the method previously developed by Ellman
et al. [27], using donepezil and neostigmine as the reference
compounds. This is based on the reaction of released thiocholine to
give a coloured product with a chromogenic reagent. Seven dif-
ferent concentrations of the synthesized compounds in the range
10
3
e10
9
M were measured at 412 nm. All the assays were under
0.1 M KH
2
PO
4
/K
2
HPO
4
buffer (pH ¼8) using a Varian Cary 50
Spectrophotometer. Enzyme solutions were prepared to give
2 units mL
1
in 2 mL aliquots.
The assay medium contained phosphate buffer, pH 8.0 (1 mL),
50
m
L of 0.01 M DTNB, 10
m
L of enzyme, 50
m
L of acetylthiocholine
iodide (ATCh) and 50
m
L of the test compound solution. ATCh was
added to the assay medium after 10 min of incubation time. The
activity was determined by measuring the increase in absorbanceat
412 nm in the 1 min intervals at 37
C. For determining the blank
value, additionally 50
m
L buffer replaced the enzymesolution. In vitro
the BuChE assay uses a similar method to that described above.
Each concentration was analysed in triplicate. The 50% inhib-
itory concentration (IC
50
) was calculated from a doseeresponse
curve obtained by plotting the percentage of inhibition versus the
log concentration with the use of GraFit 4.09 software [34]. The
results were expressed as the mean standard deviation (SD).
5.3.2. Molecular modelling studies
The inhibitor structures were created and prepared by Corina on-
line (Molecular Networks) and Sybyl 8.0 (Tripos). Types of atoms
were checked, hydrogen atoms were added and then Gasteiger-
Marsili charges were assigned. Ligands were docked to acetylcholi-
nesterase from the 1ACJ crystal complex. Before docking with
GoldSuite (CCDC) the protein was prepared. All histidine residues
were protonated at Nε, the hydrogen atoms were added, a few water
molecules (616, 634, 643) were retained, the others and ligands were
removed and the binding site was defined as all amino acid residues
within 12
A from tacrine. A standard set of genetic algorithm with
the population size 100, the number of operations 100,000 and
clustering with a tolerance of 1
A were applied. As a result, 10 ligand
poses, sorted by the GoldScore function value were obtained. The
results were visualized by PyMOL 0.99 (DeLano ScientificLLC).
5.3.3. Kinetic studies of AChE inhibition
Kinetic characterization of AChE was performed using a reported
method [35]. Different concentrations of AChE and inhibitors (1and 8)
were mixed in the assay buffer (pH 8.0), containing 350
m
M of DTNB,
0.035 unit mL
1
AChE and 550
m
M ATCh. The test compounds were
added to the assay solution and pre-incubated with the enzyme at
37
C for 15 min, followed by the addition of substrate. Determination
of kinetic characterization of the AChE-catalysed hydrolysis of ATCh
was made spectrometrically at 412 nm. A parallel control experiment
was carried out without the test compound in the mixture.
5.4. Computational methods
The log Pvalues and tPSA were calculated using the ChemDraw
Ultra 10.0 [36] and Virtual Computational Chemistry Laboratory
services [37]. The polar surface area (tPSA) was calculated by the
atom-based method [38].
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2012.12.
060. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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