1,2,4-Thiadiazoles as promising multifunctional agents for treatment of neurodegenerative diseases

Article abstract of DOI:10.1007/s11172-016-1486-9

Detailed studies of properties of new 3-substituted 5-anilino-1,2,4-thiadiazoles containing different substituents at position 3 of the thiadiazole ring were carried out, in particular, their esterase profile and antioxidant properties. It was found that the presence in the molecule of 2-aminopropyl fragment determines an efficient and selective inhibition of butyrylcholinesterase as compared to acetylcholinesterase and carboxylesterase, with radical-scavenging activity being weak. The compounds containing a 2-aminopropenyl fragment possess a high radicalscavenging activity, weakly inhibit cholinesterases, and exhibit anticarboxylesterase activity. A wide spectrum of activity of 3-substituted 5-anilino-1,2,4-thiadiazoles as inhibitors of cholinesterases and highly efficient scavengers of free radicals gives a basis for the optimization of structure and development in this series of original agents for therapy of neurodegenerative diseases.

Full text of DOI:10.1007/s11172-016-1486-9

Russian Chemical Bulletin, International Edition, Vol. 65, No. 6, pp. 1586—1591, June, 2016
1586
1,2,4ꢀThiadiazoles as promising multifunctional agents for treatment
of neurodegenerative diseases
G. F. Makhaeva, A. N. Proshin, N. P. Boltneva, E. V. Rudakova, N. V. Kovaleva, O. G. Serebryakova,
I. V. Serkov, and S. O. Bachurin
Institute of Physiologically Active Compounds, Russian Academy of Sciences,
1 Severny prꢀd, 142432 Chernogolovka, Moscow Region, Russian Federation.
Eꢀmail: gmakh@ipac.ac.ru; bachurin@ipac.ac.ru
Detailed studies of properties of new 3ꢀsubstituted 5ꢀanilinoꢀ1,2,4ꢀthiadiazoles containing
different substituents at position 3 of the thiadiazole ring were carried out, in particular, their
esterase profile and antioxidant properties. It was found that the presence in the molecule of
2ꢀaminopropyl fragment determines an efficient and selective inhibition of butyrylcholinesterase
as compared to acetylcholinesterase and carboxylesterase, with radicalꢀscavenging activity
being weak. The compounds containing a 2ꢀaminopropenyl fragment possess a high radicalꢀ
scavenging activity, weakly inhibit cholinesterases, and exhibit anticarboxylesterase activity.
A wide spectrum of activity of 3ꢀsubstituted 5ꢀanilinoꢀ1,2,4ꢀthiadiazoles as inhibitors of
cholinesterases and highly efficient scavengers of free radicals gives a basis for the optimization
of structure and development in this series of original agents for therapy of neurodegenerative
diseases.
Key words: 1,2,4ꢀthiadiazole, 3ꢀsubstituted 5ꢀanilinoꢀ1,2,4ꢀthiadiazoles, acetylcholinestꢀ
erase, butyrylcholinesterase, carboxylesterase, antioxidants, neurodegenerative diseases,
Alzheimer´s disease.
Among a wide range of different neurodegenerative
protection system is insufficient.13,14 In this connection,
the compounds possessing an ability to quench free radiꢀ
cals can be used as neuroprotectors.15—17
diseases, a special place in its negative value to society
takes Alzheimer´s disease (AD) characterizing by a gradual
steady development of disorder of memory and higher corꢀ
tical functions of the brain. A promising approach to the
treatment of such complex, multiꢀfactor diseases1 is the
development of multifunctional agents possessing cogniꢀ
tiveꢀstimulating (inhibitors of cholinesterases) and neuroꢀ
protective properties.2—5
Inhibitors of acetylcholinesterase (AChE, EC 3.1.1.7)
improve cognitive functions at dementias of different oriꢀ
gins,6,7 increasing concentration of neurotransmitter aceꢀ
tylcholine in the brain synapses. Inhibitors of butyrylꢀ
cholinesterase (BChE, EC 3.1.1.8) also improve cognitive
functions,8,9 which is especially important at AD progresꢀ
sion, when activity of AChE is decreased and its function
in hydrolysis of acetylcholine is taken over by BChE.9—11
Apart from that, BChE inhibitors do not have such a danꢀ
gerous side effects as acute cholinergic toxicity, as well as
other unwanted effects characteristic of AChE inhibitors.12
Disorders in neurotransmitting and metabolic neuron
processes are one of the key pathological changes in neuroꢀ
degenerative diseases. The processes of neuron metabolism
disorder first of all include oxidation stress, arising as
a result of a sharp enhancement of oxidation processes in
the brain cells when the functions of the antioxidant
1,2,4ꢀThiadiazole derivatives are of great interest also
because of their very wide spectrum of biological activiꢀ
ty.18,19 In particular, the compounds containing a 1,2,4ꢀ
thiadiazole fragment are described as potential agents of
therapy of AD and other neurodegenerative disorders.
Thus, it is known20 that 1,2,4ꢀthiadiazole Nꢀbenzylpiperiꢀ
dine derivatives are efficient inhibitors of AChE. 3ꢀ(1,2,4ꢀ
Thiadiazolyl)pyridine 1ꢀoxides are found to have an abiliꢀ
ty to efficiently bind to muscarinic cholinergic receptors
and exhibit antioxidant activity in the DPPHꢀassay.21 The
neuroprotective properties and the ability to quench
free radicals were demonstrated for 1,2,4ꢀthiadiazolylꢀ
nitrones.22 Among 1,2,4ꢀthiadiazole derivatives, inhibiꢀ
tors of glycogen synthase kinase 3β,23 inhibitors of betaꢀ
secretase,24 and modulators of gammaꢀsecretase25 were
found. To sum up, the analysis of the literature data shows
that the 1,2,4ꢀthiadiazole scaffold is promising for the
development of multifunctional agents for treatment of
neurodegenerative disorders.
The present work is aimed on the directed synthesis of
3ꢀsubstituted 5ꢀanilinoꢀ1,2,4ꢀthiadiazoles containing difꢀ
ferent substituents at position 3 of the thiadiazole ring,
studies of their esterase profile and antioxidant activity.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 6, pp. 1586—1591, June, 2016.
1066ꢀ5285/16/6506ꢀ1586 © 2016 Springer Science+Business Media, Inc.

1,2,4ꢀThiadiazoles
Russ.Chem.Bull., Int.Ed., Vol. 65, No. 6, June, 2016
1587
Table 1. Esterase profile of 3ꢀsubstituted 5ꢀanilinoꢀ1,2,4ꢀthiaꢀ
diazoles
The studies of esterase profile includes evaluation of the
inhibitory activity of compounds against AChE, BChE,
and structurally close enzyme carboxylesterase (CaE, EC
3.1.1.1).26—29 The enzyme CaE hydrolyzes multitude of
therapeutic agents containing ester groups30 and the inhiꢀ
bition of this enzyme with anticholinesterase compounds
used for a long time by a patient for treatment of AD can
be the reason for unwanted drugꢀdrug intereactions.31
Antioxidant activity of compounds was determined based
on their ability to quench free radicals in DPPHꢀ and
ABTSꢀassays.32,33
The target 1,2,4ꢀthiadiazole derivatives were syntheꢀ
sized according to the procedure developed by us earlier,
namely, by the reaction of 5ꢀaminoꢀ3ꢀ(2ꢀoxopropyl)ꢀ
1,2,4ꢀthiadiazoles (1) with primary amines (2). The enamꢀ
ines (3a—e) formed in the first step were reduced with
sodium borohydride at 40—50 °C to obtain the target 1,2,4ꢀ
thiadiazole amino derivatives (4a—e).
Comꢀ
pound
Inhibitory activity of compounds against serine
esterases, IC50/μmol L–1 (% of inhibition of enzyme
activity by compound in a concentration
of 20 μmol L–1, n = 3)
AChE
BChE
CaE
3a
4a
3b
4b
3c
4c
3d
4d
3e
4e
3f
>20 (3.1)
>20 (18.9)
—*
>20 (11.2)
—*
81
5.6 0.4
—*
7.4 0.5
>20 (23.9)
1.7 0.1
20
1.4 0.1
311 34
7
25
110
36
130 13
8.6 0.8
>20 (5.8)
4.2 0.4
>20 (11.6)
11.8 1.3
>20 (24.5)
3
9
3
>20 (21.6)
—*
2
>20 (6.1)
>20 (7.3)
>20 (18.5)
>20 (8.0)
>20 (11.8)
>20 (6.0)
>20 (9.3)
>20 (13.5)
265 27
>20 (29.8)
>20 (13.3)
>20 (10.4)
>20 (12.8)
>20 (14.7)
10 1
3g
3h
3i
41.0 3.9
11.2 0.9
3.12 0.02
26.7 1.9
Scheme 1
3j
* No activity.
inhibition degree of BChE and CaE, as it is seen from
Table 1, depends on the structure of substituents at posiꢀ
tion 3 of the thiadiazole ring, in particular, it is considerꢀ
ably determined by the presence of 2ꢀaminopropyl or
2ꢀaminopropenyl fragments. Compounds 4a and 4b conꢀ
taining benzyl substituents in the 2ꢀaminopropyl fragment
selectively and efficiently inhibit BChE with the IC50 values
in the micromolar region (5.6 0.4 and 7.4 0.5 μmol L–1
,
respectively). Conversely, the compounds with the same
benzyl substituents in the 2ꢀaminopropenyl fragment inꢀ
hibit BChE weakly (3a, IC50 = 81 7 μmol L–1) or do not
inhibit at all (3b). The replacement in compounds bearing
2ꢀaminopropyl fragment of benzyl groups with the cycloꢀ
hexyl and cycloheptyl substituents leads to the enhanceꢀ
ment of the inhibitory activity against BChE. Thus, comꢀ
pounds 4c and 4d efficiently and selectively inhibit BChE
with the values IC50 = 1.7 0.1 and 1.4 0.1 μmol L–1
,
respectively, while similar compounds 3c,d containing
a 2ꢀaminopropenyl fragment inhibit weakly or virtually do
not inhibit BChE.
Concerning inhibition of CaE, the presence in the molꢀ
ecule of 2ꢀaminopropyl or 2ꢀaminopropenyl fragment has
the opposite influence. The compounds with 2ꢀaminoꢀ
propyl fragment and benzyl substituents at position 3 of
i. MeOH, ∼20 °C, 2—5 h; ii. MeOH, NaBH4, 40—50 °C, 2 h.
Comꢀ
pound
3a, 4a 3ꢀClꢀ4ꢀF
R
R´
Comꢀ
pound
3f
R
R´
Bn
3ꢀFꢀ4ꢀCl
4ꢀCF3
4ꢀCl
C2H4OH
C2H4ONO2
C2H4ONO2
C2H4ONO2
3b, 4b 3ꢀClꢀ4ꢀF 4ꢀMeC6H4CH2 3g
3c, 4c
3d, 4d
3e, 4e
4ꢀCl
3,4ꢀCl2
4ꢀCl
cycloꢀC6H11
cycloꢀC7H13
C2H4OH
3h
3i
4ꢀAc
3j
3ꢀFꢀ4ꢀCl C2H4ONO2
the thiadiazole ring 4a and 4b weakly inhibit CaE (IC50
=
The studies of inhibitory activity of compounds against
three structurally close serine esterases and analysis of
the esterase profile showed that all the studied 3ꢀsubstitutꢀ
ed 5ꢀanilinoꢀ1,2,4ꢀthiadiazoles in a concentration of
20 μmol L–1 virtually do not inhibit AChE (Table 1). The
= 110 9 and 130 13 μmol L–1, respectively), whereas
similar compounds with 2ꢀaminopropenyl fragment 3a and
3b inhibit CaE stronger (IC50 = 25 3 and 36 3 μmol L–1
,
respectively). The antiꢀCaE activity of compounds with
2ꢀaminopropenyl fragment increases upon introduction

1588
Russ.Chem.Bull., Int.Ed., Vol. 65, No. 6, June, 2016
Makhaeva et al.
Table 2. Antiradical activity of 3ꢀsubstituted 5ꢀanilinoꢀ1,2,4ꢀ
thiadiazoles in DPPHꢀ and ABTSꢀassays
of cyclic aliphatic substituents at position 3 of the thiadiꢀ
azole ring (3c and 3d, IC50 = 8.6 0.8 and 4.2 0.4 μmol L–1,
respectively). Such a selectivity of action against BChE in
compounds with 2ꢀaminopropyl fragment and preferred
inhibition of CaE by compounds with 2ꢀaminopropenyl
fragment should be taken into account when developing
medicinal agents based on 3ꢀsubstituted 5ꢀanilinoꢀ1,2,4ꢀ
thiadiazoles, since inhibition of CaE by these compounds
taken by a patient for a long time for treatment AD can be
a reason for unwanted drugꢀdrug intereactions.
As it is seen from Table 1, the compounds with
2ꢀaminopropenyl fragment 3g—j containing a nitroxyl radꢀ
ical and their precursors, the corresponding alcohols 3e,
3f, and 4e, weakly inhibit BChE and moderately CaE. The
maximal antiꢀCaE activity among them is possessed by
compound 3i with IC50 = 3.12 0.02 μmol L–1, that, as it
was mentioned above, can be the reason of unwanted drugꢀ
drug interactions.
The studies of the mechanism of BChE inhibition with
3ꢀsubstituted 5ꢀanilinoꢀ1,2,4ꢀthiadiazoles showed that
these compounds are mixedꢀtype reversible inhibitors.
Figure 1 exemplifies the data on the dependence of residuꢀ
al activity of BChE on the concentration of inhibitor (comꢀ
pound 4b) at different concentrations of the substrate givꢀ
en in LineweaverꢀBurk reciprocal plots. The intersection
of lines of dependencies 1/V = f(1/S) in the left upper
quadrant indicates a mixed type of the BChE inhibition by
the test compound. The inhibition constants of BChE by
compound 4b are as follows: Ki = 1.84 0.17 μmol L–1
(competitive component) and αKi = 6.80 0.74 μmol L–1
(noncompetitive component).
Comꢀ
pound
Antiradical activity of compoundsa
DPPH, ABTS
% of binding
% of binding
IC50
3a
4a
3b
4b
3c
4c
3d
4d
3e
4e
3f
3g
3h
3i
3j
Trolox
51.0 4.2
0
58.0 1.6
0
96.0 3.8
19.2 4.8
96.0 2.3
34.0 5.2
96.2 4.3
16.8 3.8
95.3 3.3
20.4 3.8
92.8 4.5
18.3 3.6
89.6 4.3
78.0 4.2
91.3 1.76
96.0 4.2
95.0 3.8
100 2.2
100 2.7
35 2.4
—
b
29 3.2
b
—
52.6 3.2
3.1 4.2
48.6 2.8
2.1 1.8
50.6 3.5
2.2 3.2
46.8 3.2
37.1 2.1
52.2 3.4
59.8 3.2
50.3 2.6
100 1.2
100 1.8
31.8 2.3
b
—
26.4 2.8
b
—
36.5 2.4
b
—
42.6 3.4
67.2 6.8
26.9 2.45
20.3 2.54
33.5 2.85
12.3 1.6
21.4 2.25
Ascorbic acid
a Percentage of quenching of DPPHꢀ and ABTSꢀradicals by
compound in a concentration of 100 μmol L–1 or IC50/μmol L–1
,
n = 3.
b Not determined.
the DPPHꢀassay and weak activity in the ABTSꢀassay is
observed. At the same time, virtually all the studied comꢀ
pounds containing 2ꢀaminopropenyl fragment (3a—j) exꢀ
hibit pronounced antiradical activity in the DPPHꢀassay
(∼50% binding at a concentration of 100 μmol L–1) and
high radicalꢀscavenging activity in the ABTSꢀassay, which is
comparable with the activity of standard antioxidants,
ascorbic acid and trolox (see Table 2). The radicalꢀscavenꢀ
ging ability of the most active (3i, IC50 = 20.3 2.54 μmol L–1)
and least active (3g, IC50 = 67.2 6.8 μmol L–1) comꢀ
pounds differ only by a factor of 3, i.e., the structure of
substituent at position 3 of 5ꢀanilinoꢀ1,2,4ꢀthiadiazoles
weakly affects their antiradical activity. The presence in
the molecule of these compounds of 2ꢀaminopropenyl
fragment is a determining factor for exhibiting the ability
to quench free radicals.
In conclusion, the present studies of the esterase proꢀ
file and antioxidant properties of 3ꢀsubstituted 5ꢀanilinoꢀ
1,2,4ꢀthiadiazole derivatives showed that depending on the
presence in the molecule of 2ꢀaminopropyl or 2ꢀaminoꢀ
propenyl fragment, these compounds exhibit specific bioꢀ
logical activity. The presence of 2ꢀaminopropyl fragment
determines an efficient and selective inhibition of BChE
as compared to AChE and CaE, with their radicalꢀscavꢀ
enging activity being weak. At the same time, compounds
containing 2ꢀaminopropenyl fragment possess high radiꢀ
calꢀscavenging activity, but weakly inhibit cholinesterases
The studies of antioxidant properties of 3ꢀsubstituted
5ꢀanilinoꢀ1,2,4ꢀthiadiazole derivatives in the assays with
model radicals DPPH and ABTS (Table 2) showed that in
compounds with 2ꢀaminopropyl fragment 4a—e, the radiꢀ
calꢀscavenging activity is virtually completely absent in
V
–1/min A–1
1
2
3
4
0.024
0.021
0.018
0.015
0.012
0.009
0.006
–3 –2 –1
0
1
2
3
[S]–1/L mmol–1
Fig. 1. Kinetics of inhibition of BChE in the presence of comꢀ
pound 4b in a concentration of 9 (1), 6 (2), and 3 μmol L–1 (3),
as well as in the absence of the inhibitor (4).

1,2,4ꢀThiadiazoles
Russ.Chem.Bull., Int.Ed., Vol. 65, No. 6, June, 2016
1589
CHH); 2.04 (s, 3 H, Me); 3.37 (m, 1 H, NCH); 5.05 (s, 1 H,
HC=C); 7.16 (dd, 1 H, Harom, J1 = 8.8 Hz, J2 = 2.3 Hz); 7.45 (d,
1 H, Harom, J = 8.8 Hz); 7.48 (d, 1 H, Harom, J = 2.3 Hz); 8.42
(d, 1 H, CHNH, J = 9.3 Hz); 10.19 (br.s, 1 H, ArNH).
and exhibit antiꢀCaE activity. Thus, a number of 3ꢀsubstiꢀ
tuted 5ꢀanilinoꢀ1,2,4ꢀthiadiazole derivatives were found
to possess properties of selective inhibitors of BChE or
highly efficient free radical scavengers, that makes promꢀ
ising the further search in this series for the agents for the
neurodegenerative diseases therapy.
Nꢀ(3,4ꢀDichlorophenyl)ꢀ3ꢀ[2ꢀ(cyclohexylamino)propyl]ꢀ1,2,4ꢀ
thiadiazolꢀ5ꢀamine (4c). The yield was 67%. A yellow powder,
m.p. 177—179 °C. Found (%): C, 53.10; H, 5.82; N, 14.54.
C17H22Cl2N4S. Calculated (%): C, 52.99; H, 5.75; N, 14.54. MS,
1
m/z: 386.3 [M + H]+. H NMR (CDCl3), δ: 1.03 (d, 3 H, Me,
Experimental
J = 5.6 Hz); 0.85—1.35 (m, 6 H, (CH2)3); 1.61 (m, 2 H, CHHꢀ
1,2,4ꢀThiadiazole derivatives 3a—j and 4a—e were syntheꢀ
sized according to the procedures described earlier.34 1H NMR
spectra were recorded on a Bruker CХРꢀ200 spectrometer
(200 MHz, Germany), chemical shifts are given in δꢀscale relaꢀ
tive to Me4Si. Melting points were determined on a Boetius
heating stage without correction. Elemental analysis was carried
out using a CarloꢀErba C,H,Nꢀanalyzer. Mass spectra were reꢀ
corded on an Exactive mass spectrometer (ThermoFisher Scienꢀ
tific, Germany).
Nꢀ(3ꢀChloroꢀ4ꢀfluorophenyl)ꢀ3ꢀ[2ꢀ(benzylamino)propꢀ1ꢀ
enyl]ꢀ1,2,4ꢀthiadiazolꢀ5ꢀamine (3a). The yield was 92%. A yellowꢀ
ish powder, m.p. 155—157 °C. Found (%): C, 57.95; H, 4.42;
N, 14.35. C18H16ClFN4S. Calculated (%): C, 57.57; H, 4.30;
N, 14.95. MS, m/z: 375.9 [M + H]+. 1H NMR (CDCl3), δ: 2.02
(s, 3 H, CCH3); 4.53 (d, 2 H, ArCH2, J = 6.3 Hz); 5.19 (s, 1 H,
HC=C); 7.17 (m, 2 H, Harom); 7.36 (m, 6 H, Harom); 8.77 (t, 1 H,
NHCH2, J = 6.3 Hz).
CHCHH); 1.80 (m, 2 H, CHHCHCHH); 2.48 (m, 1 H, NCH);
2.80 (d, 2 H, CH2); 3.30 (d, 1 H, NCH); 7.11 (dd, 1 H, Harom
,
J1 = 8.8 Hz, J2 = 2.2 Hz); 7.39 (d, 1 H, Harom, J = 8.8 Hz); 7.43
(d, 1 H, Harom, J = 2.2 Hz).
Nꢀ(4ꢀChlorophenyl)ꢀ3ꢀ[2ꢀ(cycloheptylamino)propꢀ1ꢀenyl]ꢀ
1,2,4ꢀthiadiazolꢀ5ꢀamine (3d). The yield was 88%. A white powꢀ
der, m.p. 154—156 °C. Found (%): C, 59.66; H, 6.50; N, 15.49.
C
18H23ClN4S. Calculated (%): C, 59.57; H, 6.39; N, 15.44. MS,
m/z: 363.9 [M + H]+. 1H NMR (DMSOꢀd6), δ: 1.60 (m, 10 H,
(CH2)4, CHHCHCHH); 1.91 (m, 2 H, CHHCHCHH); 1.98
(s, 3 H, Me); 3.55 (m, 1 H, NCH); 4.99 (s, 1 H, HC=C); 7.22
(d, 2 H, Harom, J = 8.7 Hz); 7.54 (d, 2 H, Harom, J = 8.7 Hz);
8.27 (d, 1 H, CHNH, J = 9.75 Hz); 10.46 (br.s, 1 H, ArNH).
Nꢀ(4ꢀChlorophenyl)ꢀ3ꢀ[2ꢀ(cycloheptylamino)propyl]ꢀ1,2,4ꢀ
thiadiazolꢀ5ꢀamine (4d). The yield was 71%. A white powder,
m.p. 187—189 °C. Found (%): C, 59.20; H, 6.96; N, 15.29.
C
18H25ClN4S. Calculated (%): C, 59.24; H, 6.90; N, 15.35. MS,
1
Nꢀ(3ꢀChloroꢀ4ꢀfluorophenyl)ꢀ3ꢀ[2ꢀ(benzylamino)propyl]ꢀ
1,2,4ꢀthiadiazolꢀ5ꢀamine (4a). The yield was 65%. A white powꢀ
der, m.p. 82—84 °C. Found (%): C, 58.93; H, 4.37; N, 14.72.
C18H18ClFN4S. Calculated (%): C, 58.68; H, 4.67; N, 14.41.
m/z: 365.9 [M + H]+. H NMR (CDCl3), δ: 1.09 (d, 3 H, Me,
J = 6.7 Hz); 1.21—1.70 (m, 10 H, (CH2)4, CHHCHCHH); 1.81
(m, 2 H, CHHCHCHH); 2.75 (m, 1 H, NCH); 2.85 (m, 2 H,
CH2); 3.28 (m, 1 H, NCH); 7.24 (d, 2 H, Harom, J = 8.7 Hz);
7.36 (d, 2 H, Harom, J = 8.7 Hz).
1
MS, m/z: 377.9 [M + H]+. H NMR (CDCl3), δ: 1.21 (d, 3 H,
CH2CHCH3, J = 6.5 Hz); 2.87 (dd, 1 H, CHHCHMe, J1 = 6.0 Hz,
J2 = 14.4 Hz); 3.01 (dd, 1 H, CHHCHMe, J1 = 6.7 Hz,
J2 = 14.2 Hz); 3.27 (sextet, 1 H, CH2CHMe, J = 6.3 Hz); 3.79,
3.91 (both d, 2 H, ArCHH, J = 13.0 Hz); 7.22 (m, 7 H, Harom);
7.42 (m, 1 H, Harom).
Nꢀ(4ꢀChlorophenyl)ꢀ3ꢀ[2ꢀ(hydroxyethylamino)propꢀ1ꢀenyl]ꢀ
1,2,4ꢀthiadiazolꢀ5ꢀamine (3e). The yield was 86%. A white powꢀ
der, m.p. 184—186 °C. Found (%): C, 50.24; H, 4.95; N, 18.11.
C13H15ClN4OS. Calculated (%): C, 50.24; H, 4.86; N, 18.03.
1
MS, m/z: 311.8 [M + H]+. H NMR (CDCl3), δ: 1.96 (s, 3 H,
Nꢀ(3ꢀChloroꢀ4ꢀfluorophenyl)ꢀ3ꢀ[2ꢀ(4ꢀmethylbenzylamino)ꢀ
propꢀ1ꢀenyl]ꢀ1,2,4ꢀthiadiazolꢀ5ꢀamine (3b). The yield was 91%.
Colorless crystals, m.p. 164—166 °C. Found (%): C, 58.36;
H, 4.32; N, 14.81. C19H18ClFN4S. Calculated (%): C, 58.68;
H, 4.67; N, 14.41. MS, m/z: 389.9 [M + H]+. 1H NMR (CDCl3),
δ: 2.03 (s, 3 H, CH3); 2.38 (s, 3 H, ArCH3); 4.49 (d, 2 H, ArCH2,
J = 6.3 Hz); 5.21 (s, 1 H, HC=C); 7.20 (m, 6 H, Harom); 7.42
(m, 1 H, Harom); 8.71 (t, 1 H, NHCH2, J = 6.3 Hz).
Nꢀ(3ꢀChloroꢀ4ꢀfluorophenyl)ꢀ3ꢀ[2ꢀ(4ꢀmethylbenzylamino)ꢀ
propyl]ꢀ1,2,4ꢀthiadiazolꢀ5ꢀamine (4b). The yield was 67%.
A white powder, m.p. 90—92 °C. Found (%): C, 58.22; H, 5.26;
N, 14.37. C19H20ClFN4S. Calculated (%): C, 58.38; H, 5.16;
N, 14.33. MS, m/z: 391.9 [M + H]+. 1H NMR (CDCl3), δ: 1.21
(d, 3 H, CH2CHCH3, J = 6.3 Hz); 2.33 (s, 3 H, ArCH3); 2.90
(dd, 1 H, CHHCHMe, J1 = 6.0 Hz, J2 = 14.4 Hz); 3.01 (dd, 1 H,
CHHCHMe, J1 = 6.7 Hz, J2 = 14.2 Hz); 3.28 (sextet, 1 H,
CH2CHMe, J = 6.3 Hz); 3.77, 3.89 (both d, 2 H, ArCHH,
J = 13.0 Hz); 7.17 (m, 5 H, Harom); 7.29 (m, 1 H, Harom); 7.41
(m, 1 H, Harom).
Me); 3.75 (m, 4 H, (CH2)2); 5.02 (s, 1 H, HC=C); 7.17 (d, 2 H,
Harom, J = 8.7 Hz); 7.36 (d, 2 H, Harom, J = 8.7 Hz); 8.21 (br.s,
1 H, ArNH); 8.35 (t, 1 H, NH, J = 6.1 Hz).
Nꢀ(4ꢀChlorophenyl)ꢀ3ꢀ[2ꢀ(hydroxyethylamino)propyl]ꢀ1,2,4ꢀ
thiadiazolꢀ5ꢀamine (4e). The yield was 66%. A white powder,
m.p. 168—170 °C. Found (%): C, 49.88; H, 5.55; N, 17.99.
C
13H17ClN4OS. Calculated (%): C, 49.91; H, 5.48; N, 17.91.
MS, m/z: 313.8 [M + H]+. H NMR (CDCl3), δ: 1.09 (d, 3 H,
Me, J = 6.7 Hz); 2.91 (m, 4 H, (CH2)2); 3.28 (m, 1 H, NCH);
3.76 (m, 2 H, CH2); 7.17 (d, 2 H, Harom, J = 8.8 Hz); 7.37 (d, 2 H,
Harom, J = 8.8 Hz).
1
Nꢀ(4ꢀChloroꢀ3ꢀfluorophenyl)ꢀ3ꢀ[2ꢀ(hydroxyethylamino)propꢀ
1ꢀenyl]ꢀ1,2,4ꢀthiadiazolꢀ5ꢀamine (3f). The yield was 90%. A white
powder, m.p. 172—174 °C. Found (%): C, 47.56; H, 4.35;
N, 17.10. C13H14ClFN4OS. Calculated (%): C, 47.49; H, 4.29;
N, 17.04. MS, m/z: 329.8 [M + H]+. 1H NMR (CDCl3), δ: 2.04
(s, 3 H, Me); 3.41 (q, 2 H, NCH2, J = 5.7 Hz); 3.71 (t, 2 H,
OCH2, J = 5.7 Hz); 5.17 (s, 1, HC=C); 7.11 (t, 1 H, Harom
,
J = 8.8 Hz); 7.40 (m, 1 H, Harom); 7.71 (dd, 1 H, Harom, J1 = 8.8 Hz,
J2 = 2.4 Hz); 8.35 (t, 1 H, NH, J = 6.1 Hz).
Nꢀ(3,4ꢀDichlorophenyl)ꢀ3ꢀ[2ꢀ(cyclohexylamino)propꢀ1ꢀenyl]ꢀ
1,2,4ꢀthiadiazolꢀ5ꢀamine (3c). The yield was 90%. A white powꢀ
der, m.p. 186—188 °C. Found (%): C, 53.35; H, 5.32; N, 14.61.
Nꢀ(4ꢀTrifluoromethylphenyl)ꢀ3ꢀ[2ꢀ(2ꢀnitroxyethylamino)ꢀ
propꢀ1ꢀenyl]ꢀ1,2,4ꢀthiadiazolꢀ5ꢀamine (3g). The yield was 90%.
Dark brown crystals, m.p. 147—149 °C. Found (%): C, 43.28;
H, 3.66; N, 17.91. C14H14F3N5O3S. Calculated (%): C, 43.19;
H, 3.62; N, 17.99. MS, m/z: 390.4 [M + H]+. 1H NMR (CDCl3),
C
17H20Cl2N4S. Calculated (%): C, 53.27; H, 5.26; N, 14.62.
MS, m/z: 384.3 [M + H]+. 1H NMR (CDCl3), δ: 1.35 (m, 6 H,
(CH2)3); 1.68 (m, 2 H, CHHCHCHH); 1.80 (m, 2 H, CHHCHꢀ

1590
Russ.Chem.Bull., Int.Ed., Vol. 65, No. 6, June, 2016
Makhaeva et al.
δ: 2.00 (s, 3 H, Me); 3.61 (q, 2 H, NHCH2, J = 5.6 Hz); 4.59
(t, 2 H, CH2ONO2, J = 5.6 Hz); 5.21 (s, 1 H, CH); 7.58 (s, 4 H,
ing concentrations. The kinetic data were analyzed in the Linꢀ
eweaverꢀBurk plot. The kinetic constants of inhibition Ki (comꢀ
petitive component) and αKi (noncompetitive component) were
calculated using the Origin 6.1 program.
H
arom); 8.47 (t, 1 H, NHCH2, J = 6.4 Hz).
Nꢀ(4ꢀChlorophenyl)ꢀ3ꢀ[2ꢀ(2ꢀnitroxyethylamino)propꢀ1ꢀenyl]ꢀ
1,2,4ꢀthiadiazolꢀ5ꢀamine (3h). The yield was 88%. Brown crysꢀ
tals, m.p. 186—188 °C. Found (%): C, 43.80; H, 4.02; N, 19.63.
All the measurements were carried out on a BioRad Benchꢀ
mark Plus microplate spectrophotometer (France).
C
13H14ClN5O3S. Calculated (%): C, 43.88; H, 3.97; N, 19.68.
Studies of antiradical activity of compounds. The antiradical
activity of compounds under study was determined using two the
spectrophotometric methods: DPPHꢀ and ABTSꢀassays. The
compounds were dissolved in DMSO. The content of DMSO in
the reaction mixture was 4%. Ascorbic acid and trolox were used
as a positive control. The radicalꢀscavenging activity of comꢀ
MS, m/z: 356.8 [M + H]+. H NMR (CDCl3), δ: 2.02 (s, 3 H,
Me); 3.61 (q, 2 H, NHCH2, J = 5.7 Hz); 4.60 (t, 2 H, CH2ONO2,
J = 5.5 Hz); 5.12 (s, 1 H, CH); 7.25 (d, 2 H, Harom, J = 9.0 Hz);
7.53 (d, 2 H, Harom, J = 9.0 Hz); 8.36 (t, 1 H, NHCH2, J = 6.5 Hz);
10.57 (s, 1 H, NH).
1
Nꢀ(4ꢀAcetylphenyl)ꢀ3ꢀ[2ꢀ(2ꢀnitroxyethylamino)propꢀ1ꢀenyl]ꢀ
1,2,4ꢀthiadiazolꢀ5ꢀamine (3i). The yield was 87%. Yellow crysꢀ
tals, m.p. 170—172 °C. Found (%): C, 49.64; H, 4.77; N, 19.36.
pounds was evaluated at their concentration of 100 μmol L–1
,
the IC50 values (a concentration of the compound at which the
ABTSꢀradical is bound by 50%) were determined for the most
active compounds in the ABTSꢀassay.
C
15H17N5O4S. Calculated (%): C, 49.58; H, 4.72; N, 19.27.
MS, m/z: 364.4 [M + H]+. 1H NMR (DMSOꢀd6), δ: 1.99 (s, 3 H,
Me); 2.39 (s, 3 H, Me); 3.61 (q, 2 H, NHCH2, J = 5.3 Hz); 4.58
(t, 2 H, CH2ONO2, J = 5.5 Hz); 5.10 (s, 1 H, CH); 7.24 (d, 2 H,
Harom, J = 8.8 Hz); 7.53 (d, 2 H, Harom, J = 8.8 Hz); 8.34 (t, 1 H,
NHCH2, J = 6.4 Hz); 10.57 (br.s, 1 H, NH).
Nꢀ(4ꢀChloroꢀ3ꢀfluorophenyl)ꢀ3ꢀ[2ꢀ(2ꢀnitroxyethylamino)ꢀ
propꢀ1ꢀenyl]ꢀ1,2,4ꢀthiadiazolꢀ5ꢀamine (3j). The yield was 87%.
Yellowish brown crystals, m.p. 148—150 °C. Found (%): C, 41.79;
H, 3.58; N, 18.81. C13H13ClFN5O3S. Calculated (%): C, 41.77;
H, 3.51; N, 18.74. MS, m/z: 374.8 [M + H]+. 1H NMR (DMSOꢀd6),
δ: 2.06 (s, 3 H, Me); 3.65 (q, 2 H, NHCH2, J = 5.7 Hz); 4.63
(t, 2 H, CH2ONO2, J = 5.7 Hz); 5.17 (s, 1 H, CH); 7.17 (m, 1 H,
Harom); 7.44 (m, 1 H, Harom); 7.83 (m, 1 H, Harom); 8.40 (t, 1 H,
NHCH2, J = 6.3 Hz); 10.58 (br.s, 1 H, NH).
The reaction with the stable free radical DPPH (2,2ꢀdiphenꢀ
ylꢀ1ꢀpicrylhydrazyl) (Sigma—Aldrich) was carried out as deꢀ
scribed earlier32 with insignificant modifications. A solution of
DPPH was prepared in 96% ethanol. A concentration of DPPH
in the reaction mixture was 100 μmol L–1. A test compound was
added to the reaction medium containing a DPPHꢀradical and
96% ethanol and thoroughly stirred. The reaction was carried
out at a temperature of 25 °C in dark, the incubation time was
1 h. A discoloration degree of the solution of DPPH upon addiꢀ
tion of the test compounds was determined at a wavelength of
517 nm on a Cary 60 spectrophotometer (Agilent Technologies,
Canada). All the measurements were carried out in triplicate.
The antiradical activity of compounds (I) was evaluated usꢀ
ing the formula
Biochemical studies. Studies of the esterase profile of comꢀ
pounds. Kinetic studies were carried out using commercial agents:
human erythrocyte AChE, horse serum BChE, and porcine liver
CaE (Sigma, USA). Activity of AChE and BChE was deterꢀ
mined by Ellman method35 (λ = 412 nm), using acetylthioꢀ
choline (1 mmol L–1) and butyrylthiocholine (1 mmol L–1) as
a substrate, respectively; conditions: 100 mM phosphate buffer with
рH 7.5, 25 °C. Activity of CaE was determined spectrophotoꢀ
metrically based on release of 4ꢀnitrophenol (λ = 405 nm), using
4ꢀnitrophenyl acetate (1 mmol L–1) as a substrate;36 conditions:
100 mM phosphate buffer with рH 8.0, 25 °C. The measureꢀ
ments were carried out using a BioRad Benchmark Plus microꢀ
plate spectrophotometer (France). Compounds were dissolved
in DMSO, the incubation mixture contained 2% of solvent. The
primary evaluation of inhibitory activity of compounds was carꢀ
ried out by determination of inhibition degree of enzymes at
a concentration of the compound of 20 μmol L–1. For this, a samꢀ
ple of the corresponding enzyme was incubated with the test
compound for 10 min, then the residual activity of the enzyme was
measured. Each experiment was carried out in triplicate. The IC50
values, a concentration of the inhibitor required to decrease enꢀ
zyme activity by 50%, were determined for the most active comꢀ
pounds. To determine the IC50 of the inhibition of AChE,
BChE, and CaE, a sample of the corresponding enzyme was
incubated with the test compound in the range of concentrations
1•10–11—1•10–4 mol L–1 for 10 min and then a residual activity
of the enzyme was determined. Each experiment was carried out
in triplicate. The IC50 values were calculated using the Origin 6.1
program.
I = [(A0 – Ai)/A0]•100%,
where A0 and Ai are the optical density of a control solution of
DPPHꢀradical in the absence and after addition of a test comꢀ
pound, respectively.
The reaction with ABTS•+ꢀradical was carried out accordꢀ
ing to the method described earlier33 with insignificant modifiꢀ
cations. The ABTS•+ radical was obtained by the reaction of an
aqueous solution of ABTS (2,2´ꢀazinoꢀbis(3ꢀethylbenzthiazolꢀ
inoꢀ6ꢀsulfonic acid (7 mmol L–1) (Sigma—Aldrich)) with a 4.9 mM
aqueous solution of potassium persulfate (Sigma—Aldrich) takꢀ
en in equal volumes. The reaction proceeded over 12—16 h at
room temperature in dark. Before carrying out the measureꢀ
ments, the resulting solution of ABTS•+ꢀradical was diluted with
96% ethanol until the optical density of the sample reached
0.75—0.95 at a wavelength of 734 nm. The test compounds were
added to the solution of ABTS•+ꢀradical and thoroughly stirred.
The reaction was carried out at 30 °C in dark, the incubation
time was 1 h. The discoloration degree of the solution of ABTS•+ꢀ
radical was determined at a wavelength of 734 nm using a Cary
60 spectrophotometer (Agilent Technologies, Canada). All the
measurements were carried out in triplicate. The antiradical acꢀ
tivity of compounds (I) was evaluated as described above for the
DPPHꢀradical. The IC50 values were determined for the most
active compounds. The range of concentrations of test comꢀ
pounds was 1•10–6—1•10–4 mol L–1. The IC50 values were
calculated using the Origin 6.1 program.
The studies were carried out using facilities of Multiꢀ
access Center of the Institute of Physiologically
Active Compounds of the Russian Academy of Sciences
To clarify the inhibition mechanism of BChE, we studied
a dependence of BChE activity on the substrate concentration in
the absence and in the presence of the inhibitor in three increasꢀ

1,2,4ꢀThiadiazoles
Russ.Chem.Bull., Int.Ed., Vol. 65, No. 6, June, 2016
1591
19. Y. Li, J. Geng, Y. Liu, S. Yu, G. Zhao, Chem. Med. Chem.,
2013, 8, No. 1, 27; DOI: 10.1002/cmdc.201200355.
20. A. Martinez, E. Fernandez, A. Castro, S. Conde, I. Rodriguꢀ
ezꢀFranco, J.ꢀE. Bacos, A. Badia, Eur. J. Med. Chem., 2000,
35, 913; DOI: 10.1016/S0223ꢀ5234(00)01166ꢀ1.
21. A. Martinez, D. Alonso, A. Castro, V. Arána, I. Cardelús,
J.ꢀE. Bacos, A. Badia, Arch. Pharm. Pharm. Med. Chem.,
1999, 332, 191.
(Agreement No. 14.621.21.0008, work identifier
RFMEFI62114X0008).
This work was financially supported by the Russian
Science Foundation (Project No. 14ꢀ23ꢀ00160).
References
22. W. Porcal, P. Hernandez, M. Gonzalez, A. Ferreira, C. Oleꢀ
Azar, H. Cerecetto, A. Castro, J. Med. Chem., 2008, 51,
6150; DOI: 10.1021/jm8006432.
1. M. C. Carreiras, E. Mendes, M. J. Perry, A. P. Francisco,
J. MarcoꢀContelles, Curr. Top. Med. Chem., 2013, 13, 1745;
DOI: 10.2174/15680266113139990135.
23. A. Martinez, M. Alonso, A. Castro, C. Perez, F. J. Moreno,
J. Med. Chem., 2002, 45, 1292; DOI: 10.1021/jm011020u.
24. A. S. Gurjar, V. Andrisano, A. D. Simone, V. S. Velingkar,
Bioorg. Chem., 2014, 57, 90; DOI: 10.1016/j.bioorg.
2014.09.002.
2. S. O. Bachurin, Med. Res. Rev., 2003, 23, No. 1, 48; DOI:
10.1002/med.10026.
3. M. L. Bolognesi, E. Simoni, M. Rosini, A. Minarini,
V. Tumiatti, C. Melchiorre, Curr. Top. Med. Chem., 2011, 11,
2797; DOI: 10.1016/j.bmcl.2010.12.093.
25. T. Lübbers, A. Flohr, S. Jolidon, P. DavidꢀPierson, H. Jacoꢀ
bsen, L. Ozmen, K. Baumann, Bioorg. Med. Chem. Lett.,
2011, 21, 6554; DOI: 10.1016/j.bmcl.2011.08.060.
26. G. F. Makhaeva, O. G. Serebryakova, N. P. Boltneva, T. G.
Galenko, A. Y. Aksinenko, V. B. Sokolov, I. V. Martynov,
Dokl. Biochem. Biophys. (Engl. Transl.), 2008, 423, 352 [Dokl.
Akad. Nauk, 2008, 423, 826].
27. G. F. Makhaeva, E. V. Radchenko, I. I. Baskin, V. A. Palyꢀ
ulin, R. J. Richardson, N. S. Zefirov, SAR QSAR Env. Res.,
2012, 23, 627; DOI: 10.1080/1062936X.2012.679690.
28. G. F. Makhaeva, E. V. Radchenko, V. A. Palyulin, E. V.
Rudakova, A. Yu. Aksinenko, V. B. Sokolov, N. S. Zefirov,
R. J. Richardson, Chem. Biol. Interact., 2013, 203, 231; DOI:
10.1016/j.cbi.2012.10.012.
4. M. Decker, B. Kraus, J. Heilmann, Bioorg. Med. Chem.,
2008, 16, 4252; DOI: 10.1016/j.bmc.2008.02.083.
5. G. F. Makhaeva, S. V. Lushchekina, N. P. Boltneva, V. B.
Sokolov, V. V. Grigoriev, O. G. Serebryakova, E. A. Vikhareꢀ
va, A. Yu. Aksinenko, G. E. Barreto, G. Aliev, S. O. Bachuꢀ
rin, Sci. Rep., 2015, 5, 13164; DOI: 10.1038/srep13164.
6. A. Martinez, A. Castro, Expert. Opin. Investig. Drugs, 2006,
15, 1; DOI: 10.1517/13543784.15.1.1.
7. P. Anand, B. Singh, Arch. Pharm. Res., 2013, 36, 375; DOI:
10.1007/s12272ꢀ013ꢀ0036ꢀ3.
8. N. H. Greig, T. Utsuki, D. K. Ingram, Y. Wang, G. Pepeu,
C. Scali, Q. S. Yu, J. Mamczarz, H. W. Holloway, T. Giorꢀ
dano, D. Chen, K. Furukawa, K. Sambamurti, A. Brossi,
D. K. Lahiri, Proc. Natl. Acad. Sci. USA, 2005, 102, 17213;
DOI: 10.1073/pnas.0508575102.
9. N. H. Greig, T. Utsuki, Q. Yu, X. Zhu, H. W. Holloway,
T. Perry, B. Lee, D. K. Ingram, D. K. Lahiri, Curr. Med. Res.
Opin., 2001, 17, 159; DOI: 10.1185/0300799039117057.
10. R. M. Lane, S. G. Potkin, A. Enz, Int. J. Neuropsychoꢀ
pharmacol., 2006, 9, 101; DOI: http://dx.doi.org/10.1017/
S1461145705005833.
29. G. F. Makhaeva, N. P. Boltneva, S. V. Lushchekina, O. G.
Serebryakova, T. S. Stupina, A. A. Terentiev, I. V. Serkov,
A. N. Proshin, S. O. Bachurin, R. J. Richardson, Bioorg.
Med. Chem., 2016, 24, 1050; DOI: 10.1016/j.bmc.
2016.01.031.
30. T. Imai, Drug Metab. Pharmacokinet., 2006, 21, 173; DOI:
http://doi.org/10.2133/dmpk.21.173.
31. L. G. Tsurkan, M. J. Hatfield, C. C. Edwards, J. L. Hyatt,
P. M. Potter, Chem. Biol. Interact., 2013, 203, 226; DOI:
10.1016/j.cbi.2012.10.018.
11. A. Nordberg, C. Ballard, R. Bullock, T. DarrehꢀShori,
M. Somogyi, Prim Care Companion CNS Disord., 2013, 15,
No. 2; DOI: 10.4088/PCC.12r01412.
32. W. BrandꢀWilliams, M. E. Cuvelier, C. Berset, LWT — Food
Sci. Technol., 1995, 28, 25; DOI: 10.1016/S0023ꢀ
6438(95)80008ꢀ5.
33. R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang,
C. RiceꢀEvans, Free Rad. Biol. Med., 1999, 26, 1231; DOI:
10.1016/S0891ꢀ5849(98)00315ꢀ3.
34. A. N. Proshin, I. V. Serkov, L. N. Petrova, S. O. Bachurin,
Russ. Chem. Bull. (Int. Ed.), 2014, 63, 1148 [Izv. Akad. Nauk,
Ser. Khim., 2014, 1148].
35. G. L. Ellman, K. D. Courtney, V. Jr. Andres, R. M. Fetherꢀ
Stone, Biochem. Pharmacol., 1961, 7, 88.
12. E. Giacobini, Pharmacol. Res., 2004, 50, 433; DOI: 10.1016/
j.phrs.2003.11.017.
13. P. I. Moreira, S. L. Siedlak, G. Aliev, X. Zhu, A. D. Cash,
M. A. Smith, G. Perry, J Neural. Transm., 2005, 112, 921;
DOI: 10.1007/s00702ꢀ004ꢀ0242ꢀ8.
14. R. A. Floyd, K. Hensley, Neurobiol. Aging, 2002, 23, 795;
DOI: 10.1016/S0197ꢀ4580(02)00019ꢀ2.
15. M. Rosini, E. Simoni, M. Bartolini, A. Tarozzi, R. Matera,
A. Milelli, P. Hrelia, V. Andrisano, M. L. Bolognesi,
C. Melchiorre, Eur. J. Med. Chem., 2011, 46, 5435; DOI:
10.1016/j.ejmech.2011.09.001.
36. S. H. Sterri, B. A. Johnesen, F. Fonnun, Biochem. Pharmaꢀ
col., 1985, 34, 2779; DOI: 10.1016/0006ꢀ2952(85)90579ꢀ9.
16. N. Guzior, A. Wieckowska, D. Panek, B. Malawska, Curr.
Med. Chem., 2015, 22, 373; DOI: 10.2174/
0929867321666141106122628.
17. N. Özta k n, Y. Çetinkaya, P. Taslimi, S. Göksu, . Gülçin,
Bioorg. Chem., 2015, 60, 49; DOI: 10.1016/j.bioorg.
2015.04.006.
18. A. Castro, T. Castaco, A. Encinas, W. Porcal, C. Gil, Bioorg.
Med. Chem., 2006, 14, 1644; DOI: 10.1016/j.bmc.
2005.10.012.
Received April 11, 2016;
in revised form April 21, 2016