Biological evaluation and molecular docking of novel 1,3,4-thiadiazole-resorcinol conjugates as multifunctional cholinesterases inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104617

Two series of novel 1,3,4-thiadiazole-resorcinol conjugates were efficiently synthesized and evaluated as cholinesterases inhibitors. N-Butyl- and N-chlorophenyl-5-amino-1,3,4-thiadiazol-2-yl)benzene-1,3-diols were identified as the most promising compoun

Full text of DOI:10.1016/j.bioorg.2020.104617

Bioorganic Chemistry 107 (2021) 104617
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Biological evaluation and molecular docking of novel 1,3,4-thiadiazole-re-
sorcinol conjugates as multifunctional cholinesterases inhibitors
,
d
*
b
c
c
Alicja Skrzypeka, Joanna Matysiaka , Monika Karpinska , Kamila Czarnecka , Paweł Kręcisz ,
´
d
e
d
c
´
Dorota Stary , Jędrzej Kukułowicz , Beata Paw , Marek Bajda , Paweł Szymanski ,
Andrzej Niewiadomya
a Department of Chemistry, University of Life Sciences in Lublin, Akademicka 15, 20-950 Lublin, Poland
b Łukasiewicz Research Network – Institute of Industrial Organic Chemistry, Annopol 6, 03-236 Warsaw, Poland
c Department of Pharmaceutical Chemistry, Drug Analyses and Radiopharmacy, Faculty of Pharmacy, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland
d Department of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Cracow, Poland
e Department of Medicinal Chemistry, Medical University of Lublin, Jaczewskiego 4, 20-090 Lublin, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two series of novel 1,3,4-thiadiazole-resorcinol conjugates were efficiently synthesized and evaluated as cho-
linesterases inhibitors. N-Butyl- and N-chlorophenyl-5-amino-1,3,4-thiadiazol-2-yl)benzene-1,3-diols were
identified as the most promising compounds of low nanomolar activity against AChE (IC50 = 29–76 nM) and
moderate activity against BuChE. The inhibition mechanism studies proved that the compounds are mixed type
inhibitors. The docking simulations showed great affinity of the compounds for both enzymes. The modelled
amine derivatives exhibited a similar arrangement in the catalytic anionic site of AChE similar to that of tacrine.
1,3,4-Thiadiazole
Acetylcholinesterase
Butyrylcholinesterase
Cytotoxicity
Inhibitor
Molecular docking
Beta amyloid
The thiadiazole ring interacted with Trp84 and the phenyl groups created π-π stacking interactions with the
residue – Phe330. The compounds showed better inhibition of the in vitro self-induced Aβ (1–42) aggregation
than that compared with curcumin as well as antioxidant properties similar to those of quercetin. They exhibited
metal ion chelating properties, acceptable cytotoxicity in vitro and favourable ADMET profile determined in
silico.
Antioxidant
1. Introduction
diseases treatment and AChE is a goal molecular target in the drug
design [6,7].
Nowadays the issue of neurodegenerative diseases is one of the most
troublesome effects of people’s longer life and aging. That includes such
disorders as Parkinson’s (PD) and Alzheimer’s (AD) diseases as well as
senile dementia [1,2]. The causes of AD, one of the most common
neurodegenerative diseases are not clear, although several factors are
known to influence its development. Among them there are oxidative
stress, deposition of β-amyloid, collecting of neurofibrillary tangles,
biometal dyshomeostasis, inflammation processes as well as decreased
acetylcholine level caused by damage in the cholinergic receptor signal
transduction [3–5].
Some researchers postulated that formation and aggregation of the
β-amyloid (Aβ) peptide plays a major role in AD development. The
neurotoxic peptide Aβ is formed from the amyloid precursor protein
(APP) by β- and γ-secretases in the process of enzymatic cleavage. It
possesses a tendency towards aggregation, particularly that containing
42-amino acid residues, which leads to cognitive and mental disorders
caused by neuritis, oxidative stress or loss of neurons, Therefore the
approach to reduce the level of Aβ is considered as rational for the anti-
Alzheimer’s drugs design [8]. The current AD treatment approaches
show that AChE can also interact directly with β-amyloid increasing its
deposition into insoluble plaques. This can be important in the amyloid
fibril formation due to the presence of the PAS (the peripheral anionic
site). The PAS stimulates aggregation and formation of AChE-β-amyloid
complexes which are more neurotoxic than β-amyloid alone [9].
The cholinergic hypothesis is the basis of the main therapeutic
approach in the treatment of AD. The cholinergic function deterioration
leads to cognitive decline and memory loss. Acetylcholinesterase (AChE)
inhibitors are applied as relevant medicines in the neurodegenerative
* Corresponding author.
E-mail address: joanna.matysiak@up.lublin.pl (J. Matysiak).
https://doi.org/10.1016/j.bioorg.2020.104617
Received 23 July 2020; Received in revised form 24 November 2020; Accepted 28 December 2020
Available online 5 January 2021
0045-2068/© 2020 Published by Elsevier Inc.

A. Skrzypek et al.
Bioorganic Chemistry 107 (2021) 104617
Nitrogen and sulfur containing heterocycles are commonly employed
in the design of AChE and / or butyrylcholinesterase (BuChE) inhibitors
including the 1,3,4-thiadiazole ring, although this group of compounds
is poorly explored in this area. As the AChE and / or BuChE inhibitors
benzyl [10], carboxamide [11], carbothioamide [12], urea [13] and
glycosyl derivatives [14] were discussed. A few analogues are active
below 1 µM (IC50). Recently the drug-1,3,4-thiadiazole conjugates have
been presented as novel prominent mixed-type AChE inhibitors (IC50
about 18 nM) and free radical scavengers [15]. The other researchers
studied the triazolo-thiadiazole hybrids [16–18] or the compounds
including the isomeric 1,2,4-thiadiazole ring [19,20].
analogues. Using the hydrazides in the reaction there were obtained
other 2,5-disubstituted 1,3,4-thiadiazoles (11–19) which are presented
in Fig. 1 [21,28]. The analogues of STB applied in the synthesis included
-Et or -Cl substituent in the resorcinol moiety. The final products were
prepared with the yields in the range of 69–83%. The analytical data of
new compounds (1H NMR, 13 C NMR, MS, elemental analyses) confirm
their structure and they are consistent with those obtained for other
analogues (Supplementary materials) [25].
2.2. AChE and BuChE inhibition evaluation
It is known that the oxidative stress has a significant impact on the
pathogenesis of neurodegenerative diseases [12]. Polyphenols are
commonly used as antioxidant agents. Therefore this is justified to
create thiadiazole-resorcinol conjugates as cholinesterases (ChEs) in-
hibitors with the additional antioxidant properties. The previous studies
carried out on resorcinol substituted 1,3,4-thiadiazoles and benzimid-
azoles revealed nanomolar AChE and moderate BuChE inhibition in vitro
[21–23]. The benzimidazole-resorcinols also showed the ability to
inhibit aggregation of Aβ-amyloid at low concentrations and a signifi-
cant antioxidant potential. The molecular modeling studies exhibited
that, among others, the hydroxyl groups and heterocyclic ring partici-
pate in the crucial interactions with AChE [22].
All presented compounds were evaluated by the in vitro assay as the
AChE and BuChE inhibitors. For the SAR elucidation some previously
described analogues (3, 8, 10) were also subjected to the study [25]. The
potency of compounds was assessed using the modified Ellman’s tech-
nique and it was shown as the half of maximal compound concentration,
IC50 [29]. Tacrine, neostigmine and donepezil commonly applied in the
AD therapy drugs were studied for comparison. The results are presented
in Table 1. In all the analyses p-value below the assumed degree of
significance was obtained, which indicates that the differences between
the compounds are statistically significant (p < 0.001).
As follows from the data the potency of compounds is varied but the
majority of them exhibit good to remarkable inhibitory activities,
particularly towards AChE. The IC50 values of the most potent analogues
against AChE and BuChE are ranged from 0.029 to 0.085 µM and from
3.154 to 24.711 µM, respectively. The compounds show greater activity
towards AChE than BuChE except for compound 15. Some of them are
highly selective for AChE with respect to BuChE, particularly compound
5 (the selectivity is higher than 6579). Some compounds show no sig-
nificant inhibitory activity against the studied enzymes (IC50 < 500 µM).
Under our experimental conditions most of the active thiadiazoles are
characterized by the activity similar to that of neostygmine and slightly
smaller than that of donepezil used as reference drugs. A variety in the
structure of the hydrophobic gorges of active center of both enzymes is
responsible for differences in the inhibitory potency [30].
As follows from the other studies carried out on the 1,3,4-thiadia-
zole-resorcinol conjugates some compounds are characterized by a
relatively low cytotoxicity against normal cells. One of them, wildly
explored 2,5-substitutes 1,3,4-thiadiazole (FABT) exhibited even neu-
roprotective properties [24]. The behavioural studies carried out on
mice proved analgesic, anxiolytic as well as anticonvulsant activities
[25,26].
Taking into account the therapeutic potential of that class of com-
pounds, novel 2,5-disubstituted 1,3,4-thiadiazoles were prepared and
their biological properties as potential AD drugs were examined. The
studies include evaluation of cholinesterases activities, determination of
the inhibition mechanism and molecular docking. The estimation of in
vitro self-induced Aβ (1–42) aggregation inhibition, antioxidant poten-
tial and metal chelating ability were also made. For broader assessment
of the biological potential of compounds, their cytotoxicity to normal
cells and the ADMET profile in silico were determined.
The structure–activity elucidation shows that thiadiazoles with the
–NH– group are more potent than their analogues without it, but the
amine group is not necessary for the inhibitory potency against the
AChE. Butylamine derivative (8) is the most active against both en-
zymes. Whereas N,N-disubstituted aminothiadiazole 10 is inactive
against them. The prominent activity is also observed for some halo-
genphenylaminothiadiazoles (2, 4–6, 8) as well as for naphthen-2-
ylthiadiazole (16). Among the active isomeric chlorophenylamine de-
rivatives (4, 6, 7) the strongest effect is observed for ortho- isomer 4. The
para- isomer is the least active. The compounds with the phenyl ring
possessing extended fluoroalkoxy substituents (13–15) or the de-
rivatives with the furan ring (18–19) show weak potency or are inactive.
The previous studies of 5-substitutted 2-(2,4-dihydroxyphenyl)-1,3,4-
thiadiazoles also indicate that the alkyl substituent (without –NH–
group) can be favourable for the compounds potency against AChE [23].
The analogues with the additional Cl substituent on the 2,4-dihy-
droxyphenyl moiety show a smaller activity against AChE compared
with the unsubstituted ones (8 and 9; 11 and 12; 18 and 19). A smaller
activity is also observed for the chlorine analogues compared to the
ethyl ones (2 and 3; 4 and 5; 16 and 17; 18 and 19). This effect is even
more evident against BuChE. All analogues including a chlorine atom in
the benzenediol ring moiety are inactive. The results obtained so far do
not indicate explicitly the influence on the activity of the ethyl (Et)
substituent.
2. Results and discussion
2.1. Chemistry
The enzymatic studies carried out so far showed that in the group of
4-(1,3,4-thiadiazol-2-yl)benzene-1,3-diol derivatives particularly active
against cholinesterases are those containing 5-halogenphenyl or 5-alkyl
substituent on heterocyclic ring (IC about 60 nM) [21,23]. Molecular
modeling studies exhibited that the considered 2,5-disubstituted 1,3,4-
thiadiazoles (especially diaryl derivatives), due to their relatively high
symmetry, oriented differently toward the AChE binding site, which
makes it difficult to use the results to design new analogues. However,
they indicated a very important function of -OH groups in the in-
teractions with the enzymes. Carrying on the research in this area, N-
alkyl/aryl and N,N-dialkyl substituted amino-1,3,4-thiadiazole-
resorcinols (1–10) as the first group of compounds, were designed for
evaluation. This is justified by the fact that many molecules that act as
AChE inhibitors have an amine group [27]. The other series includes
phenyl derivatives with the extended fluoroalkoxy substituents (11–15),
the compounds with naphthalene (16–17) and furan (18–19) rings. In
search for the optimal substitution of the benzenediol moiety the Cl
atom was added, which was pointed out as favorable. For comparison
the Et substituent of other electronic properties was considered.
2.3. Kinetic studies
To define the mechanism of enzyme AChE inhibition, the kinetic
study was carried out using the most active thiadiazoles (8 and 16). The
type of inhibition was interpreted using the Lineweaver-Burk plots
(Fig. 2). These plots present the mutual velocity compared to the
N-substituted 2-amino-1,3,4-thiadiazoles (1–10) were afforded by
treatment of commercially available 4-substituted-3-thiosemicarbazides
with sulfinylbis[(2,4-dihydroxyphenyl)methanethione] (STB) or its
2

A. Skrzypek et al.
Bioorganic Chemistry 107 (2021) 104617
Fig. 1. Synthesis scheme and structure of compounds 1–19.
negative reciprocal substrate concentrations in the range of 0.05 µM
gradually decreased by 0.01 µM for various concentrations (0.015 µM,
0.03 µM, 0.06 µM) of inhibitors and without an inhibitor. The test
showed the decreased KM and Vmax values with higher inhibitor con-
centrations for compound 8. In the case of compound 16, the studies
showed an increase in Km and a decrease in Vmax with higher inhibitor
concentrations (Table 2). This indicates that the compound can be
combined with the free enzyme or the enzyme-substrate complex. The
potential inhibitors can be combined with the enzyme by non-covalent
interactions such as hydrogen or ionic bonds as well as hydrophobic
ones. It is sometimes free from any chemical binding with the enzyme.
Such interactions occur immediately and can be easily reversed.
Therefore the enzyme - inhibitor complex is rapidly separated. This
proves that the reversible inhibitors exhibit definitely a smaller risk of
side effects than the irreversible ones.
potency is observed for compound 16 with the naphthyl substituent. The
activity of monochlorophenylamine (4, 5, 6) derivatives is at the similar
level, however, the compound with the best properties proved to be 5
with the 2-chlorophenylamine substituent and the additional chlorine
atom in the benzenodiol moiety. Compound (5) at 3 µM shows 37.64 ±
3.21% of inhibition whereas at the concentrations of 13–100 µM it is
always above 55%. All of them presented the aggregation inhibition
values above 20% (in a concentration 25–100 µM). Taking into account
the activity of compounds against AChE and the degree of aggregation
inhibition, compound 4 appears to be the most interesting one (IC50
=
31 nM).
2.5. Antioxidant activity
To evaluate the effectiveness of antioxidants efficiency of com-
pounds as radical scavengers versus the stable free radicals the 2,2-
diphenylpicrylhydrazyl (DPPH) was used. The results of this test for
the studied thiadiazoles are shown in Table 1. They are expressed as IC50
which is the concentration of the antioxidant eliminating 50% of DPPH
radicals [32]. All considered thiadiazoles are characterized by a signif-
icant antioxidant potential at IC50 < 0.195 µM. Of them compounds 4, 5,
16 showed the highest scavenging capacity (IC50 in the range of
0.022–0.036 µM), which was more than or equal to that of the quercetin
(IC50 0.036 ± 0.02 µM). The most active derivatives include compounds
of different structures of high activity against AChE: 2-chlorophenyla-
mino (4, 5), butylamino (8) and naphthyl (16) derivatives. This phe-
nomenon can be explained by the fact that the polarity of antioxidants is
proportional to their DPPH radical scavenging potency. The existence of
two or more hydroxyl groups on the phenyl ring can increase the adical-
scavenging activity by allowing more hydrogen atoms of the phenolic
hydroxyl groups to be donated to stabilize the free radicals [33]. The
analyses p-value below the assumed degree of significance was ob-
tained, which indicates that the differences between the compounds are
statistically significant (p < 0.001).
2.4. Inhibition of self-mediated Aβ42 aggregation
One of the hallmarks of AD patient brains is the misfolding and ag-
gregation of specific proteins that accumulate extracellularly (e.g. senile
plaques, mostly composed of beta-amyloid peptide (Aβ) aggregates) and
interfere with relevant brain biological functions [9]. To evaluate the
anti-amyloidogenic effects of the new compounds, the experiments
based on the in vitro tests with thioflavin-T (ThT) were conducted. The
selected compounds of the highest activity (4, 5, 6, 8, 16) against AChE
were included in the studies. The data are presented in Fig. 3 and
Table 3. They show that tested compounds 4, 5, 6 and 16 induced a
decrease of the ThT fluorescence associated with the Aβ fibril binding,
thus indicating inhibition of the Aβ(1–42) self-aggregation process
[8,31]. The overwhelming majority of the tested compounds show
better inhibitory parameters compared to curcumin at the tested con-
centrations. One tested compound 8 of the highest activity against
cholinesterases (with the butylamine substituent) did not exhibit an
amyloid-beta inhibitory activity at any used concentration. The lowest
3

A. Skrzypek et al.
Bioorganic Chemistry 107 (2021) 104617
efficient for complex production. Our previous study described two
models of ion complexing of very similar compounds, where the com-
pound:ion ratios were equal to 1:1 or 2:1 [35]. The current results may
be due to the ion complexity of one molecule of compound and a col-
ligand or two molecules of compound depending on the concentration of
the compound in the mixture.
Table 1
In vitro inhibitory concentration of the compounds against AChE and BuChE
(IC50
,
μ
M), selectivity and the radical scavenging effect IC50(DPPH)
.
No.
AChEa IC50
BuChEb IC50
Selectivity for
DPPHd IC50
[
μ
M]*
[μ
M]*
AChEc
[µM]**
1.
0.537 ±
0.00
> 500
> 931.1
129.6
> 6.2
591.7
> 6578.9
1599.3
560.6
59.7
0.147 ±
0.006
2.
0.085 ±
0.007
11.018 ±
0.529
0.049 ±
0.015
2.7. Cytotoxicity of compounds
3.
80.670 ±
0.791
> 500
0.172 ±
0.005
Compounds 4 and 8 of the highest AChE inhibitory activity were
further evaluated for their cytotoxic effect to human hepatic stellate
cells by the MTT cytotoxicity test. The cells were treated with various
concentrations of compounds for 48 h and their viability was deter-
mined by MTT cell proliferation assay. Compound 4 was analysed in the
concentration range of 25–70 µM and compound 8 in the range of
10–250 µM. The results showed that compound 4 at the concentration of
51.62 ± 2.15 µM (IC50) and compound 8 at the concentration of 153.85
± 1.96 µM (IC50) inhibited about 50% of the cell viability as compared to
control cells. Examined earlier curcumin by Shy et al. shows IC50 con-
centration similar to those of compounds 4 and 8 (concentration in the
range of 50–150 µM) [36]. These results demonstrate that new com-
pounds are not cytotoxic in the range of concentrations that were
effective towards AChE and BuChE in in vitro studies.
4.
0.031 ±
0.004
18.344 ±
0.072
0.036 ±
0.006
5.
0.076 ±
0.003
> 500
0.025 ±
0.005
6.
0.061 ±
0.008
97.560 ±
0.526
0.095 ±
0.006
7.
0.202 ±
0.004
113.240 ±
0.287
0.118 ±
0.006
8.
0.029 ±
0.005
1.731 ±
0.013
0.042 ±
0.012
9.
0.318 ±
0.011
> 500
> 1572.3
–
0.147 ±
0.014
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
> 500
> 500
0.083 ±
0.008
0.043 ±
0.004
12.006 ±
0.196
279.2
> 277.0
570.2
–
0.054 ±
0.013
1.805 ±
0.009
> 500
0.195 ±
0.004
2.8. ADMET analysis
0.197 ±
0.001
112.333 ±
0.317
0.091 ±
0.009
Compounds 4, 5, 6, 8 and 16 were analyzed using the ACD/Percepta
software to obtain their estimated values of pharmacokinetic and
mutagenic properties (Table 5) [37,38]. They exhibited moderate
permeation through Blood Brain Barrier (BBB) and satisfactory log PS
values (ꢀ 1.3 to ꢀ 1.8), indicating rapid penetration into the brain tissue.
All compounds, except 5, showed acceptable log BB values (ꢀ 0.07 to
0.25), indicating sufficient brain penetration for central nervous system
activity. Derivative 5 showed moderate brain penetration (log BB =
ꢀ 0.3). Therefore, this analogs has the weakest BBB permeation prop-
erties in our group of compounds. All compounds have a very high
percentage of oral bioavailability at the 50 mg dose dependent on log P
(%F-log P) and sufficient volume of distribution to penetrate into the
intracellular fluid. They showed a very high cumulative percentage of
binding to human plasma proteins (%PPB), which indicates potentially
high effective doses in clinical applications and a very high risk of
occurrence of side effects when used in combination with other drugs.
Neither of our compounds has a significant probability of the positive
Ames test result. The detailed results are presented in Table 5.
> 500
> 500
0.121 ±
0.006
80.740 ±
0.150
3.154 ±
0.022
0.04
0.069 ±
0.007
0.074 ±
0.004
3.028 ±
0.023
40.9
0.022 ±
0.006
3.071 ±
0.012
> 500
> 162.8
110.9
> 992.1
1.2
0.117 ±
0.005
0.217 ±
0.003
24.073 ±
0.764
0.081 ±
0.004
0.504 ±
0.005
> 500
0.137 ±
0.003
neostygmine
donepezil
tacrine
0.054 ±
0.005
0.067 ±
0.005
7.420 ±
0.100
0.016 ±
0.012
–
–
0.019 ±
0.004
390.5
0.19
–
0.084 ±
0.050
quercetin
–
–
0.036 ±
0.003
a
IC50 – 50% inhibitory concentration (means ± SEM of three independent
experiments) of AChE (* p < 0.001; Kruskal-Wallis test).
b
2.9. Molecular modeling studies
IC50 – 50% inhibitory concentration (means ± SEM of three independent
experiments) of BuChE (* p < 0.001; Kruskal-Wallis test).
c
d
selectivity for AChE = IC50(BuChE)/IC50(AChE).
The in silico studies on the selected 1,3,4-thiadiazole derivatives
allowed us to present their binding mode within the enzymes and to
explain differences in their activity due to various substituents in the 2-
(2,4-dihydroxyphenyl)-thiadiazole system. The modelled compounds of
different activity: 5, 8, 14 and 16 were docked into the active sites of
AChE (PDB code: 1ACJ) and BuChE (PDB code: 4BDS) according to the
validated docking procedure [39]. All analyzed derivatives were
unionized under physiological conditions based on the predicted pKa
values.
IC50: 50% inhibitory concentration (means ± SD of three experiments) (**
p
< 0.001; one-way ANOVA test).
2.6. Metal ion chelating
Metal ion chelating ability is a very desirable property for multi-
functional anti-AD agents. The current state of knowledge indicates that
the excess of Cu2+, Fe3+ and Zn2+ ions in the CNS plays an important
role in the amyloid β aggregation process and its neurotoxicity [34].
Therefore the most active compounds were tested for chelating ability of
those ions and the compound: ion ratios in the obtained complex mol-
ecules were estimated. The pH impact on the UV spectra was also
examined. The test did not show any significant influence of the
observed pH changes in the pH range used during the test on the UV
spectra for the compounds and ionic mixtures. The results are collected
in Table 4. They show that the tested compounds complex Cu2+ and Fe3+
ions. The results of compound:ion ratio determination are ambiguous for
compounds 6 and 8. The compound:ion ratios 2:1 and 1:1 were equally
The top-ranked poses of the tested 1,3,4-thiadiazole derivatives
within the acetylcholinesterase active site are presented in Fig. 4 and S1
(Supplementary materials). Compounds 5 and 8 showed a similar
arrangement: the thiadiazole rings interacted with Trp84 from the cat-
alytic anionic site while their phenyl groups created π-π stacking in-
teractions with the anionic site residue – Phe330. The phenylthiadiazole
system of these compounds mimicked the binding mode of tacrine in the
active site of enzyme. Similarly to tacrine compound 8 is also a mixed-
type, reversible inhibitor of cholinesterase [40] (details, Supplementary
materials).
4

A. Skrzypek et al.
Bioorganic Chemistry 107 (2021) 104617
Fig. 2. Lineweaver-Burk reciprocal plots for the AChE inhibition by compounds 8 and 16.
The ADMET prediction analysis indicated that the tested compounds
have sufficient BBB permeability to the central nervous system activity,
satisfactory pharmacokinetic profile and low probability of mutagenic
properties. Furthermore, the selected compounds are not very toxic to
normal cells. Therefore this group of compounds seems to be more
promising as far as the searching for drugs for AD treatment is
concerned.
Table 2
The KM and Vmax values at different compounds 8 and 16 concentrations towards
AChE.
Concentration (µM)
KM (µM)
Vmax (A/min)
Compound 8
0.0
555.55
227.30
152.44
113.12
714.29
175.43
95.24
0.015
0.03
The molecular docking studies of alkyl(aryl)amine derivatives to
AChE showed that they mimicked the binding mode of tacrine and
similar to it they are a mixed-type, reversible inhibitor. Considering the
previously obtained results for the resorcinol-1,3,4-thiadiazole conju-
gates, it should be assumed that the NH group is not necessary for the
activity against AChE, although the compounds from this group seem to
be more promising which has been confirmed by the molecular docking
studies. The presence of the extended alkoxy substituents on the phenyl
ring is not conducive to activity, which has been also shown by the
molecular docking studies. The additional Cl atom in the 2,4-dihydrox-
yphenyl moiety is unfavorable for the inhibitory potency against both
cholinesterases. The observation results will be explored for designing
and synthesizing of new more effective cholinesterase inhibitors based
on the 1,3,4-thiadiazole-resorcinol unit.
0.06
44.05
Compound 16
0.0
76.73
54.64
48.51
43.66
40.16
0.015
0.03
96.98
126.72
148.51
0.06
Considering the interactions of investigated compounds with BuChE
we could observe that compounds 8 and 16 were able to create spcific
hydrogen bond interactions with the enzyme, although they bound to
the active site in a different manner.
The most active derivative 8 with IC50 = 1.73 µM interacted with the
BuChE binding pocket as shown in Fig. 5. The alkyl substituent created
hydrophobic interactions with amino acids such as Trp82, Leu125 and
Tyr114. Moreover, one nitrogen atom of thiadiazole interacted with
Ser198 of catalytic triad through the H-bond. The hydroxyl groups in
positions 2 and 4 of the phenyl substituent created hydrogen bonds with
the oxyanion site residues: Ala199, Gly117 and Leu286 from the acyl
pocket (details, Supplementary materials).
4. Experimental part
4.1. Analytical studies
The purity of compounds was monitored by reverse-phase high-
performance liquid chromatography RP-18/MeOH/H2O (Eurosil Bio-
3. Conclusions
select C 18, 5 μm, 300 × 4.6 mm; Knauer, Berlin, Germany). Melting
point (m.p.) was assigned by a BÜCHI B-540 (Flawil, Switzerland)
melting point apparatus and it was uncorrected. The elemental analysis
was made using Perkin-Elmer 2400. The contribution of C, H and N was
within 0.5% of the theoretical value. The MS spectra (EI, 70 eV) were
registered by means of the apparatus AMD-604. The 1D NMR spectra
(1H, 13C and 19F NMR) were made in DMSO‑d6 using a Bruker DRX 500
(Bruker Daltonics, Inc. Billerica, MA, USA). The chemical shifts (δ, ppm)
were presented in relation to tetramethylsilane (TMS) and the coupling
constants (J) were expressed in Hz.
The new 1,3,4-thiadiazle-resorcinol conjugates show advantageous
biological properties. The halogenophenylamine and alkylamine de-
rivatives seem to be of specific interest, although the type of N-substi-
tution of 2-amino-1,3,5-thiadizole ring has a significant effect on the
spectrum of biological potency. Butylamine derivative (8) of the highest
activity against both enzymes is not an inhibitor of the amyloid Aβ(1–42)
aggregation. Whereas chlorophenylaminothiadiazoles (4, 5, 6), being a
strong inhibitor of AChE, show a significant antioxidant potential and
inhibit the amyloid beta aggregation at a low concentration better than
the reference agents. The compounds show metal ion chelating ability
that is a very desirable property for the multifunctional anti-AD agents.
The excess of Cu2+, Fe3+ and Zn2+ ions in the CNS favours an amyloid β
aggregation process, intensifies oxidative stress and neurotoxicity.
4.2. General procedure for the synthesis of compounds
The corresponding 4-substituted 3-thiosemicarbazide or hydrazide
5

A. Skrzypek et al.
Bioorganic Chemistry 107 (2021) 104617
Fig. 3. Results of the amyloid beta aggregation inhibition for all tested compounds and the reference as the mean ± SD of triplicate of three independent
experiments.
Table 3
Table 5
The results of the amyloid beta aggregation inhibition for the selected thiadia-
zoles and curcumin of triplicate in three independent experiments. The data are
shown as means ± SD, n = 9.
ADMET prediction results.
No.
log PS
log BB
%PPB a
Vd [L/kg] b
Ames test c
%F-log P d
4
ꢀ 1.3
ꢀ 1.7
ꢀ 1.3
ꢀ 1.8
ꢀ 1.3
0.08
97.94 l
99.20 l
98.61 l
91.95 l
98.81 l
2.9
0.7
3.1
2.0
3.4
0.20
0.11
0.16
0.34
0.20
94.70
95.10
82.30
99.20
86.50
Compound
Concentrations of compound (μM)
5
ꢀ 0.30
0.21
6
3
13
25
50
100
8
ꢀ 0.07
0.25
4
37.56 ±
2.65
42.28 ±
2.38
55.77 ±
5.86
56.42 ±
3.90
56.42 ±
5.09
16
a
b
c
%PPB - cumulative percentage of bonding to human plasma proteins.
Vd - volume of distribution.
5
37.64 ±
3.21
55.55 ±
5.71
67.42 ±
5.91
55.32 ±
4.51
57.14 ±
5.06
6
23.69 ±
2.15
35.26 ±
1.65
46.15 ±
5.39
36.03 ±
3.18
56.34 ±
3.19
Ames test - probability of positive Ames test result.
%F-log P - oral bioavailability at 50 mg dose dependent on log P.
d
16
7.87 ±
0.39
15.79 ±
0.88
22.36 ±
1.39
32.40 ±
1.03
42.27 ±
1.69
4.2.1. 4-Ethyl-6-(5-(3-fluorophenylamino)-1,3,4-thiadiazol-2-yl)benzene-
1,3-diol (1)
Curcumin
8.62 ±
0.56
15.25 ±
0.75
22.74 ±
2.34
27.37 ±
3.60
30.40 ±
2.81
Yield: 84%; m.p.: 253–254 ◦C; EI MS (m/z, %): 332.1 (21), 331.1
(M+, 100), 317.1 (18), 316.1 (92), 168.0 (12), 148.1 (13), 69.3 (12); 1H
NMR (500 MHz, DMSO‑d6) δ: 10.66 (s, 1 H, HO-C(3)), 10.47 (s, 1 H, HO-
Table 4
′
–
–
C
C(3)), 9.86 (s, 1 H, HO-C(1), 7.71 (s, 1H, H- C(2 )), 7.69 (s, 1H, H-
Results of metal ion chelating examination – estimated compound:ion ratio for
′
′
–
complex molecule.
No.
(5)), 7.36 (m, 1H, H- C(4 )), 7.30 (d, J = 8.0 Hz, 1H, C(6 )-H), 6.80 (t, J
′
–
= 7.54 Hz, 1H, H- C(5 )), 6.51 (s, 1H, C(2)-H), 2.53 (q, J = 7.4 Hz, 2H,
Metal ions
Et), 1.44 (t, J = 7.4 Hz, 3H, Et); 13C NMR (125 MHz, DMSO‑d6, δ): 163.6,
163.0 (d, 1J (19F,13C) = 241.8 Hz, C’(3)) 158.5, 155.5, 154.0, 142.0 (d,
3J (19F,13C) = 11.8 Hz, C’(1)), 130.0 (d, 3J (19F,13C) = 9.8 Hz, C’(5)),
[Cu2+] from Cu
[Zn2+] from Zn
(COOCH3)2
[Fe3+] from
FeCl3
(COOCH3)2
4
5
6
8
42-Cu
4-Zn
4-Fe
5-Fe
6-Fe
8-Fe2
127.6, 122.9, 113.5 (d, 4J (19F,13C) = 2.7 Hz, C’(6)), 108.4, 108.0 (d, 2J
52-Cu
5-Zn
4
(
19F,13C) = 21.4 Hz, C’(4)), 104.5 (d, J (19F,13C) = 26.8 Hz, C’(2)),
62-Cu and/or 6-Cu
82-Cu and/or 8-Cu
No complex
No complex
102.7, 22.6, 14.8 ppm. Anal. calcd for C16H14FN3O2S: C, 57.99; H, 4.26;
N, 12.68. Found: C, 57.65; H, 4.24; N, 12.62%.
(15 mmol) and sulfinylbis[(2,4-dihydroxyphenyl)methanethione] (15
mmol) were dissolved in in methanol (75 mL) placed in a round bottom
flask. A mixture was heated to reflux (3–4 h) and then the hot mixture
was filtered. To separate the product, the obtained filtrate was left at
room temperature or was concentrated. Finally, the compounds were
crystallized from the MeOH or MeOH/H2O solution.
4.2.2. 4-(5-(2-Chlorophenylamino)-1,3,4-thiadiazol-2-yl)-6-ethylbenzene-
1,3-diol (4)
Yield: 80%; m.p.: 265–267 ◦C; EI MS (m/z, %): 349.1 (38), 348.1
(20), 347.1 (M+, 100), 333.1 (16), 332.1 (87), 312.30 (30), 184 (12),
152 (10), 149 (23), 148.6 (15), 148.1 (17), 122.1 (9), 69.3 (16); 1H NMR
(500 MHz, DMSO‑d6) δ: 10.63 (s, 1H, HO-C(3)), 9.82 (s, 1H, HO-C(1),
′
–
9.64 (s, 1H, NH), 8.31 (d, J = 8.2 Hz, 1H, H- C(6 )), 7.67 (s, 1H, H-
6

A. Skrzypek et al.
Bioorganic Chemistry 107 (2021) 104617
Fig. 4. Binding mode of compound 8 within the active site of acetylcholinesterase.
Fig. 5. Binding mode of compound 8 within the active site of butyrylcholinesterase.
′
–
–
–
C
C(5)), 7.49 (dd, J = 8.0 and 1.2 Hz, 1H, H- C(3 )), 7.37 (m, 1H, H-
150.1 (15), 184.0 (35), 149.1 (150), 75.3 (11), 169.3 (11); 1H NMR
′
′
–
(5 )), 7.07 (m, 1H, H- C(4 )), 6.50 (s, 1H, C(2)-H), 2.51 (m, 2H, Et),
1.13 (t, J = 7.5 Hz, 3H, Et); 13C NMR (125 MHz, DMSO‑d6, δ): 164.5,
158.4, 156.5, 153.9, 137.8, 130.1, 128.3, 127.7, 124.2, 123.0, 122.9,
121.9, 105.5, 102.7, 22.6, 14.8 ppm. Anal. calcd for C16H14ClN3O2S: C,
56.25; H, 4.06; N, 12.08. Found: C, 56.02; H, 4.04; N, 12.12%.
(500 MHz, DMSO‑d6) δ: 11.17 (s, 1 H, HO-C(3)), 10.74 (s, 1 H, HO-C(1),
′
–
9.75 (s, 1H, NH), 8.29 (dd, J = 8.1 and 1.1 Hz, 1 H, H- C(6 )), 7.94 (s,
1H, C(5)-H), 7.50 (dd, J = 8.0 and 1.2 Hz, 1H, C(3′)-H), 7.38 (td, J = 8.0
and 1.4 Hz, 1H, C(5′)-H), 7.09 (td, J = 8.1 and 1.4 Hz, 1H, C(4′)-H), 6.75
(s, 1H, C(2)-H); 13C NMR (150 MHz, DMSO‑d6) δ: 165.3, 156.0, 154.2,
154.5, 137.8, 130.0, 128.3, 127.6, 124.5, 123.4, 122.2, 112.1, 110.0,
104.1 ppm. Anal. calcd for C14H9Cl2N3O2S: C, 47.47; H, 2.56; N, 11.86.
Found: C, 47.67; H, 4.05; N, 11.91%.
4.2.3. 4-Chloro-6-(5-(2-chlorophenylamino)-1,3,4-thiadiazol-2-yl)
benzene-1,3-diol (5)
Yield: 83%; m.p.: 234–235 ◦C; EI MS (m/z, %): 355.0 (71), 354.0
(21), 353.0 (M+, 100), 320.0 (35), 319.0 (20), 318 (51), 187.0 (15),
186.0 (13), 184.0 (35), 170.0 (11), 169.0 (15), 159.0 (13), 152.0 (13),
7

A. Skrzypek et al.
Bioorganic Chemistry 107 (2021) 104617
4.2.4. 4-(5-(4-Chlorophenylamino)-1,3,4-thiadiazol-2-yl)-6-ethylbenzene-
1,3-diol (7)
N, 6.03%.-
Yield: 81%; m.p.: 261 ◦C; EI MS (m/z, %): 349.1 (54), 348.1 (34),
347.1 (M+, 100), 334.0 (36), 332.0 (17), 184.0 (14), 166.0 (14), 152.0
(11), 148.1 (15), 69.3 (13); 1H NMR (500 MHz, DMSO‑d6) δ: 10.63 (s, 1
–
4.2.9. 4-(5-(2,5-Bis(2,2,2-trifluoroethoxy)phenyl)-1,3,4-thiadiazol-2-yl)-
6-ethylbenzene-1,3-diol (14)
Yield: 72%; m.p.: 236–238 ◦C; EI MS (m/z, %): 494.2 (M+, 100),
479.2 (99), 201.0 (22), 215.9 (14), 148.0 (15); 1H NMR (500 MHz,
H, HO-C(3)), 10.74 (s, 1 H, HO-C(1), 9.83 (s, 1H, NH), 7.68 (m, 3H, H
C
(6′)), 7.38 (d, 2H, C(5)-H), 6.48 (s, 1H, C(2)-H), 2.49 (m, 2H, Et), 1.13 (t,
J = 7.4 Hz, 3H, Et); 13C NMR (150 MHz, DMSO‑d6) δ: 163.7, 158.5,
1554, 153.9, 140.2, 129.3 (2C), 127.6, 125.3, 122.7, 119.1 (2), 108.4,
102.6, 22.5, 14.8 ppm. Anal. calcd for C16H14ClN3O2S: C, 55.25; H, 4.06;
N, 12.08. Found: C, 55.45; H, 4.08; N, 12.12%.
DMSO‑d6) δ: 7.98 (m, 2H, H- C(Ar)), 7.38 (d, J = 9.2 Hz, 1H, H-
–
–
C
–
–
(Ar)), 7.32 (m, 1H, H- C(Ar)), 6.65 (s, 1H, H- C(2)), 5.01 (q, J = 9.0
Hz, 2H, CH2), 4.85 (q, J = 9.0 Hz, 2H, CH2), 2.54 (m, J = 7.9 Hz, 2H,
CH2), 1.66 (t, J = 7.8 Hz, 3H, CH3) (OH and NH protons are invisible);
13C NMR (125 MHz, DMSO‑d6) δ: 165.2, 159.9, 159.4, 155.0, 152.4,
148.7, 127.8, 124.0 (q, 1J (19F,13C) = 276 Hz), 123.5 (q, 1J (19F,13C) =
2
4.2.5. 4-(5-(Butylamino)-1,3,4-thiadiazol-2-yl)-6-chlorobenzene-1,3-diol
(9)
256 Hz), 123.0, 120.7, 119.0, 115.6, 114.1, 108.2, 102.4, 65.9 (q, J
(
19F,13C) = 30 Hz, 2C, CH2), 22.6 (CH2), 14.7 (CH3) ppm. Anal. calcd for
Yield: 68%; m.p.: 222–223 ◦C; EI MS (m/z, %): 301.0 (48), 300.1
(22), 299.1 (M+, 100), 270.0 (23), 266.1 (25), 258.0 (23), 257.0 (49),
256.0 (49), 245.0 (36), 243.0 (85), 186.9 (35), 171.6 (36), 169 (74),
130.1 (33), 245.0 (36), 97.2 (20), 88.2 (16), 57.5 (16); 1H NMR (500
MHz, DMSO‑d6) δ: 10.96 (s, 1 H, HO-C(3)), 10.56 (s, 1 H, HO-C(1)), 7.75
(s, 1H, C(5)-H), 7.69 (s, 1 H, NH), 6.63 (s, 1H, C(2)-H), 3.27 (t, J = 6.8
Hz, 2H, CH2), 1.58 (m, J = 7.0 Hz, 2H, CH2), 1.35 (m, J = 7.2 Hz, 2H,
CH2), 0.90 (t, J = 7.3 Hz, 3H, CH3) ppm.; 13C NMR (150 MHz, DMSO‑d6)
δ: 169.0, 155.1, 154.2, 152.3, 127.7, 111.7, 110.6, 104.2, 45.0, 31.2,
20.1, 14.1 ppm. Anal. calcd for C12H14ClN3O2S: C, 48.08; H, 4.71; N,
14.02 Found: C, 48.27; H, 4.73; N, 14.08%.
C20H16F6N2O4S: C, 48.59; H, 3.26; N, 5.67. Found: C, 48.73; H, 3.27; N,
5.69%.
4.2.10. 4-(5-((4-(Trifluoromethoxy)phenoxy)methyl)-1,3,4-thiadiazol-2-
yl)benzene-1,3-diol (15)
Yield: 82%; m.p.: 152–154 ◦C; EI MS (m/z, %): 384.1 (M+, 19), 208.1
(12), 207.1 (100), 153.1 (72); 1H NMR (500 MHz, DMSO‑d6) δ: 11.09 (s,
–
1 H, HO-C(3)), 10.08 (s, 1 H, HO-C(1), 8.04 (d, J = 9.1 Hz, 1H, H-
C
C
–
–
(5)), 7.34 (d, J = 9.7 Hz, 2H, H- C(Ar)), 7.20 (d, J = 9.6 Hz, 2H, H-
(Ar)), 6.50 (d, J = 2.5, 1H, C(2)-H), 6.43 (dd, 1H, J = 2.3 and 9.1 Hz, H-
C(6)), 5.67 (s, 2 H, CH2),13C NMR (150 MHz, DMSO‑d6) δ: 164.6,
–
164.5, 161.8, 156.8, 156.7, 142.8, 129.4, 123.1 (2C), 120.1 (q, 1J
4.2.6. 4-(5-(4-(trifluoromethoxy)phenyl)-1,3,4-thiadiazol-2-yl)benzene-
1,3-diol (11)
(
19F,13C) = 256 Hz, CF3), 116.8 (2C), 108.8, 108.6, 102.8, 65.9 ppm.
Anal. calcd for C16H11F3N2O4S: C, 50.00; H, 2.88; N, 7.29. Found: C,
50.25; H, 2.89; N, 7.31%.
Yield: 74%; m.p.: 226–228 ◦C; EI MS (m/z, %): 355.1 (18), 354.1
(M+, 100), 219.1 (11), 205.1 (10), 167.1 (16), 68.9 (11); 1H NMR (500
MHz, DMSO‑d6) δ: 11.25 (s, 1 H, HO-C(3)), 10.16 (s, 1 H, HO-C(1), 8.15
4.2.11. 4-Ethyl-6-(5-(naphthalen-2-yl)-1,3,4-thiadiazol-2-yl)benzene-1,3-
diol (16)
–
–
(d, J = 8.6 Hz, 2H, H C(Ar)), 8.10 (d, J = 8.7 Hz 1H, H C(5)), 7.55 (d,
J = 8.1 Hz, 2H, H- C(Ar)), 6.55 (d, J = 1.4 Hz, 1H, H- C(2)), 6.46 (dd,
J = 8.6 and 2.7 Hz, 1H,) ppm; 13C NMR (150 MHz, DMSO‑d6) δ: 165.1,
163.6, 161.8, 156.9, 150.1, 129.8, 129.7 (2C), 129.3, 122.3 (2C), 119.4
(q, 1J (19F,13C) = 261 Hz, CF3), 108.7, 108.8, 102.8 ppm. Anal. calcd for
Yield: 71%; m.p.: 213–214 ◦C; EI MS (m/z, %): 349.1 (23), 348.1
(M+, 90), 347.1 (10), 334.1 (19), 333.1 (80), 186.0 (17), 185.0 (100),
171.0 (13), 153.1 (25), 148.1 (16), 127.1 (15), 170.0 (11), 169.0 (15),
159.0 (13), 152.0 (13), 150.1 (15), 184.0 (35), 149.1 (150), 75.3 (11),
169.3 (11); 1H NMR (500 MHz, DMSO‑d6) δ: 10.93 (s, 1 H, HO-C(3)),
–
–
C
15H9F3N2O3S: C, 50.85; H, 2.56; N, 7.91. Found: C, 50.99; H, 2.57; N,
–
10.04 (s, 1 H, HO-C(1), 8.71 (d, 1H, J = 7.9 Hz, H- C(Ar)), 8.13 (d,
7.93%.
–
–
1H, J = 8.2 Hz, H- C(Ar)), 8.08 (d, 1H, J = 7.5 Hz, H- C(Ar)), 8.03 (s,
–
4.2.7. 4-Chloro-6-(5-(4-(trifluoromethoxy)phenyl)-1,3,4-thiadiazol-2-yl)
benzene-1,3-diol (12)
1H, C(5)-H), 7.95 (d, J = 7.0 Hz, 1H, H- C(Ar)), 7.66 (m, 3H, C(Ar)-H),
6.61 (s, 1H, C(2)-H), 2.56 (q, J = 7.4 Hz, 2H, t), 1.18 (t, J = 7.4 Hz, 3H,
Et); 13C NMR (150 MHz, DMSO‑d6) δ: 165.1, 163.2, 159.0, 154.3, 133.6,
130.7, 130.0, 129.5, 128.5, 127.6, 127.5, 126.8, 126.6, 125.5, 125.4,
122.7, 107.8, 102.1, 22.1, 14.1 ppm; Anal. calcd for C20H16N2O2S: C,
68.94; H, 4.63; N, 8.04. Found: C, 68.72; H, 4.65; N, 8.01%.
Yield: 76%; m.p.: 258–259 ◦C; EI MS (m/z, %): 390.1 (42), 389.1
(20), 388.1 (M+, 100), 219.0 (17), 205.0 (17), 201.0 (22), 187.0 (17),
69.3 (15); 1H NMR (500 MHz, DMSO‑d6) δ: 11.43 (s, 1H, HO-C(1)),
–
10.89 (s, 1H, HO-C(2), 8.17 (s, 1H, H- C(6)), 8.16 (d, J = 8.7 Hz, 2H,
′
′
′
′
–
–
H- C(2 ,6 )), 7.54 (d, J = 8.3 Hz, 2H, H- C(3 ,5 )), 6.78 (s, 1H, C(2)-H)
ppm; 13C NMR (150 MHz, DMSO‑d6) δ: 165.7, 162.2, 156.9, 155.3,
4.2.12. 4-Chloro-6-(5-(naphthalen-2-yl)-1,3,4-thiadiazol-2-yl)benzene-
1,3-diol (17)
150.2, 129.8 (2C), 129.7, 128.2, 122.2 (2C), 119.9 (q, J (19F,13C) =
1
258 Hz, CF3), 112.4, 109.7, 103.9 ppm. Anal. calcd for C15H8ClF3N2O3S:
C, 46.34; H, 2.07; N, 7.21. Found: C, 46.54; H, 2.08; N, 7.18%.
Yield: 79%; m.p.: 261–262 ◦C; EI MS (m/z, %): 356.1 (15), 355.1
(11), 354.1 (M+, 38), 187 (8), 186 (16), 185 (100), 171 (10), 141.1 (6),
127.1 (14), 126.1 (8); 1H NMR (500 MHz, DMSO‑d6) δ: 11.60 (s, 1 H,
–
HO-C(3)), 11.07 (s, 1 H, HO-C(1), 8.71 (d, 1H, J = 8.3 Hz, H- C(Ar)),
4.2.8. 4-(5-(2,5-Bis(2,2,2-trifluoroethoxy)phenyl)-1,3,4-thiadiazol-2-yl)
benzene-1,3-diol (13)
–
8.24 (s, 1H, C(5)-H), 8.14 (d, 1H, J = 8.2 Hz, H- C(Ar)), 8.07 (d, 1H, J
Yield: 71%; m.p.: 280–281 ◦C; EI MS (m/z, %): 467.1 (21), 466.1
(M+, 100), 397.1 (30), 300.2 (10), 167.1 (14), 153.1 (13), 135.1 (11); 1H
NMR (500 MHz, DMSO‑d6) δ: 11.30 (s, 1 H, HO-C(3)), 8.10 (d, J = 9.0
= 7.4 Hz, H- C(Ar)), 7.97 (d, 1H, J = 6.5 Hz, H- C(Ar)), 7.67 (m, 3H,
–
–
H- C(Ar)), 6.90 (s, 1H, C(2)-H) ppm; 13C NMR (150 MHz, DMSO‑d6) δ:
–
166.5, 162.2, 156.9, 155.5, 134.0, 131.4, 130.4, 130.1, 129.1, 128.2,
128.1, 127.2, 127.1, 126.0, 125.8, 112.3, 109.8, 104.1 ppm. Anal. calcd
for C18H11ClN2O2S: C, 60.93; H, 3.12; N, 7.90. Found: C, 60.71; H, 3.13;
N, 7.93%.
–
–
Hz, 1H, H C(5)), 8.01 (d, J = 3.5 Hz, 1H, H C(Ar)), 7.37 (d, J = 9.6
′
–
–
Hz, 1H, H C(Ar)), 7.30 (dd, J = 3.3 and 9.5 Hz, 1H, H- C(4 )), 6.54 (d,
–
J = 2.3 Hz, 1H, C(2)-H), 6.46 (dd, 1H, J = 2.2 and 9.4 Hz, H- C(6)),
5.02 (q, J = 9.0 Hz, 2H, CH2), 5.02 (m, J = 9.0 Hz, 2H, CH2); 13C NMR
(125 MHz, DMSO‑d6) δ: 164.6, 161.2, 159.6, 156.4, 152.1, 148.3, 128.8,
124.1 (q, 1J (19F,13C) = 265 Hz, CF3), 123.7 (q, 1J (19F,13C) = 258 Hz,
CF3), 120.2, 118.6, 115.2, 113.5, 108.5, 108.1, 102.4, 65 (q, 2J (19F,13C)
= 30 Hz, 2C, CH2) ppm; 19F NMR (470 MHz, DMSO‑d6) δ: –71.95 (td, J
= 281 and 9.1 Hz, CF3), –72.55 (t, 3F, J = 9.1 Hz, CF3) ppm. Anal. calcd
for C18H12F6N2O4S: C, 46.36; H, 2.59; N, 6.01. Found: C, 46.54; H, 2.60;
4.2.13. 4-Ethyl-6-(5-(furan-2-yl)-1,3,4-thiadiazol-2-yl)benzene-1,3-diol
(18)
Yield: 81%; m.p.: 265–267 ◦C; EI MS (m/z, %): 288.0 (M+, 65), 274.0
(18), 273.0 (100), 111.1 (9); 1H NMR (500 MHz, DMSO‑d6) δ: 10.98 (s, 1
–
H, HO-C(3)), 10.06 (s, 1 H, HO-C(1), 7.97 (s, 1H, H- C(2)), 7.96 (s, 1H,
′
′
′
–
–
–
H- C(5 )), 7.75 (d, J = 3.3 Hz, 1H, H- C(3 )), 7.25 (m, 1H, H- C(4 )),
8

A. Skrzypek et al.
Bioorganic Chemistry 107 (2021) 104617
6.62 (s, 1H, -C(2)-H), 2.52 (m, 2H, Et), 1.15 (t, J = 7.4 Hz, 3H, Et); 13
C
4.5. Inhibition of self-mediated Aβ42 aggregation
NMR (150 MHz, DMSO‑d6) δ: 162.2, 159.5, 156.9, 154.8, 145.9, 145.8,
127.9, 123.2, 113.1, 111.5, 108.1, 103.5, 22.6, 17.8 ppm. Anal. calcd for
The presence of fibrils is a crucial pathomechanism of AD. The assay
was monitored by thioflavin T (ThT)-based fluorometric one. The inhi-
bition studies were carried out upon incubating Aβ42 with and without
the test compounds. Amyloid β-peptide (1–42) (Aβ42), was purchased
from Aldrich as a lyophilized powder and stored at ꢀ 20 ◦C. This Aβ
C
14H12N2O3S: C, 58.32; H, 4.20; N, 9.72. Found: C, 58.07; H, 4.22; N,
9.69%.
4.2.14. 4-Chloro-6-(5-(furan-2-yl)-1,3,4-thiadiazol-2-yl)benzene-1,3-diol
(19)
(1–42) was diluted in the phosphate buffer (pH 8.0) to obtain a 12.58 μM
Yield: 80%; m.p.: 241–242 ◦C; EI MS (m/z, %): 296.0 (37), 295.0
(15), 294.0 (M+, 100), 291.0 (22), 290.0 (20), 289.0 (67), 288.0 (21),
255.7 (36), 254.1 (43), 253.1 (23), 200.9 (18), 191.8 (17), 159.9 (15),
159.0 (29), 128.0 (23), 125.1 (30), 111.1 (17), 69.3 (20), 64.3 (50), 44.5
(final concentration) solution. The new compounds under study and the
positive reference (curcumin) were dissolved and then further diluted
with the 50 mM glycine-NaOH buffer (pH 8.5) to the final concentra-
tions: 100, 50, 25, 13 and 3 L of phosphate buffer (pH
μ
M. Firstly, 60
μ
(62); 1H NMR (500 MHz, DMSO‑d6) δ: 11.47 (s, 1 H, HO-C(1)), 10.89 (s,
8.0), next the Aβ42 (10 μL) samples and the tested compounds (10 μL)
′
1 H, HO-C(3), 8.15 (s, 1H, H- C(5)), 7.98 (d, J = 1.4 Hz, 1H, H- C(5 )),
were added to the plate. Then all plates were incubated for 24 h at 30 ◦C,
without stirring. Also, the peptide sample was incubated under identical
conditions but without the inhibitor for the control. The tests were
carried out in the black, flat bottom 96-well plate, in triplicate in three
–
–
′
–
–
7.28 (d, J = 3.4 Hz, 1H, H- C(3 )), 6.78 (s, 1H, H- C(2)), 6.76 (m, 1H,
C(4′)-H); 13C NMR (150 MHz, DMSO‑d6) δ: 160.2, 157.2, 156.4, 154.7,
145.7, 145.1, 128.3, 111.9, 111.4, 111.3, 109.1, 103.5 ppm. Anal. calcd
for C12H7ClN2O3S: C, 48.90; H, 2.39; N, 9.51. Found: C, C, 49.13; H,
2.40; N, 9.54%.
independent experiments. Then 20 μL of 2.5 μM ThT was added into 50
mM glycine–NaOH (pH 8.5). After the 5 min incubation with the dye,
the ThT fluorescence intensity (λex = 440 nm; λem = 490 nm) was
measured (Synergy H1, BioTek Instruments, Inc. Winooski, VT, USA)
[42,43]. The percent inhibition was calculated based on Eq. (1):
4.2.15. Compounds
Ethyl-6-(5-(4-fluorophenylamino)-1,3,4-thiadiazol-2-yl)benzene-
1,3-diol (2), 4-chloro-6-(5-(4-fluorophenylamino)-1,3,4-thiadiazol-2-yl)
benzene-1,3-diol (3), 4-(5-(3-chlorophenylamino)-1,3,4-thiadiazol-2-
yl)-6-ethylbenzene-1,3-diol (6), 4-(5-(butylamino)-1,3,4-thiadiazol-2-
yl)benzene-1,3-diol (8), 4-(5-(dimethylamino)-1,3,4-thiadiazol-2-yl)
benzene-1,3-diol (10) were described previously [23,25,41].
I% = 100 ꢀ (IFI/IF0*100)
(1)
where IF0 and IFi express the fluorescence intensities, in the absence and
presence of the studied derivatives minus the fluorescence intensities
due to the blanks. The presented values were calculated as the mean ±
SD of triplicate of three independent tests.
4.3. In vitro AChE and BuChE inhibition assay
4.6. In vitro antioxidant activity assay
Acetylcholinesterase (AChE, E.C. 3.1.1.7), butyrylcholinesterase
(BuChE, E.C. 3.1.1.8), acetylthiocholine iodide (ATCh), butylthiocho-
line iodide (BTCh), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), done-
pezil hydrochloride, tacrine hydrochloride and neostigmine bromide
were purchased from Sigma–Aldrich (Steinheim, Germany). For the
evaluation of synthesized 1,3,4-thiadiazoles there was applied the
method elaborated by Ellman et al. [29] and the inhibitory activities
against AChE and BuChE were determined. Neostigmine, donepezil and
tacrinne were applied as the standards. This assay is based on the in-
teractions of thiocholine to give a coloured product with a chromogenic
reagent. The compounds were tested in the range from 10ꢀ 3 to 10ꢀ 9 M at
412 nm using a Varian Cary 50 Spectrophotometer. The details were
described previously [22]. The samples were analysed in triplicate. The
50% compound concentration (IC50) was designated from a dos-
e–response curve received by plotting the percentage of inhibition
versus the log concentration applying GraFit 7.0 software. The results
were presented as the mean ± standard error of measurement (SEM).
AChE and BuChE activity data were compared using Kruskal-Wallis test
a significance level of 5%. Data sets were analysed using Statistica 13
software (TIBCO Software Inc. Palo Alto, CA, USA).
The antioxidant potency of all compounds was evaluated based on
the radical scavenging effect of stable DPPH free radical [44]. Quercetin
was used as the reference substance. The incubation was performed at
37 ◦C for 30 min and the decrease in the absorbance of each solution was
measured at 520 nm. The measurements have been made in triplicate.
The details were presented previously [44]. For the evaluation of radical
scavenging potency there were used following formula:
reduction of absorbance = [(A0 ꢀ A1)/A0] × 100%
(2)
in which: A0 – the absorbance of the blank sample; A1 – the absorbance
of the tested substance (t = 30 min). Finally IC50 values were calculated
as the concentration of compound required for scavenging 50% of DPPH
[32]. Antioxidant activity data were compared using one-way ANOVA
test a significance level of 5%. Data sets were analysed using Statistica
13 software (TIBCO Software Inc. Palo Alto, CA, USA).
4.7. Metal ion chelating
Methanol HPLC grade (ChemSolve) and demineralized water ob-
tained by Milli-Q® Advantage AIO with qualification IQ/OQ were used
to prepare all solutions. Copper acetate, zinc acetate and iron chloride
(Sigma Aldrich) were used as ions sources. Potassium dihydrogen
phosphate and potassium hydroxide (ChemPur) were used to prepare
phosphate buffer solutions. pH values were determined using the Met-
tler Toledo FiveEasy pH meter with the electrode LE483. All spectral
measurements were performed in 96 well clear UV plates and using the
multi-reader BioTek Synergy H1. All obtained data were analyzed by
means of MS Excel. Compounds 4, 5, 6, 8 and copper (II), zinc (II) and
iron (III) salts were dissolved in methanol:water mix (1:1). All solutions
had concentration 0.33 mM. pH values of ion solutions were measured.
Three 10 mM phosphate buffer solutions (pH = 5.5, pH = 8 and pH =
11) were prepared in methanol:water mix (1:1). All compounds were
examined for ability to complex all prepared ions. All tested compounds
were examined as regards the influence of pH on the shape of the UV
4.4. Kinetic characterization of AChE inhibition
The kinetics of AChE inhibition was evaluated according to the Ell-
man’s method [29]. Plots of 1/velocity relative to reciprocal substrate
concentration were used at different final concentrations of the ATC
iodide in the range from 0.05 to 0.01 µM. The analyses were made using
the following final concentrations of 8 and 16 thiadiazoles: 0.06, 0.03,
0.015 µM and without them. The studied sample contained: solution of
DTNB (0.4 mg/ml), AChE solution (2 U/ml), solution of the test thia-
diazole, and different concentrations of the substrate ATC iodide solu-
tions. Absorbance was determined at 412 nm after 7 min of incubation
(25 ◦C). The Lineweaver–Burk plot presents the obtained results. To
define the type of AChE inhibition KM and Vmax values were calculated.
9

A. Skrzypek et al.
Bioorganic Chemistry 107 (2021) 104617
spectrum at the concentration 0.33 mM. For determination of the ability
to complex metal ions and the ratio ligand:ion in the complex, all test
compounds were combined with ions solutions and analyzed according
to the Job’s plot methodology [45–48]. Nine solutions of compound:ion
mix (ratios: 1:0, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:0) were measured in
the spectral scan at 290–600 nm with 1 nm step. The well volume was
equal to 120 µL. The maximal concentration in the well of each com-
pound was equal to 0.33 mM. The obtained numerical spectral data were
analyzed and used to prepare Job’s plots for each compound:ion pair. All
calculations were made according to the third Lambert-Beer law – the
additivity of absorption. The obtained data were used to prepare Job’s
plots and to find the most efficient compound:ion ratio which indicates
the compound:ion ratio in complex molecule.
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11