Development of 1,3-thiazole analogues of imidazopyridines as potent positive allosteric modulators of GABAA receptors

Article abstract of DOI:10.1016/j.bioorg.2019.103334

Structure–activity relationship studies were conducted in the search for 1,3-thiazole isosteric analogs of imidazopyridine drugs (Zolpidem, Alpidem). Three series of novel γ-aminobutyric acid receptor (GABA_AR) ligands belonging to imidazo[2,1-b]thiazoles, imidazo[2,1-b][1,3,4]thiadiazoles, and benzo[d]imidazo[2,1-b]thiazoles were synthesized and characterized as active agents against GABA_AR benzodiazepine-binding site. In each of these series, potent compounds were discovered using a radioligand competition binding assay. The functional properties of highest-affinity compounds 28 and 37 as GABA_AR positive allosteric modulators (PAMs) were determined by electrophysiological measurements. In vivo studies on zebrafish demonstrated their potential for the further development of anxiolytics. Using the OECD “Fish, Acute Toxicity Test” active compounds were found safe and non-toxic. Structural bases for activity of benzo[d]imidazo[2,1-b]thiazoles were proposed using molecular docking studies. The isosteric replacement of the pyridine nuclei by 1,3-thiazole, 1,3,4-thiadiazole, or 1,3-benzothiazole in the ring-fused imidazole class of GABA_AR PAMs was shown to be promising for the development of novel hypnotics, anxiolytics, anticonvulsants, and sedatives drug-candidates.

Full text of DOI:10.1016/j.bioorg.2019.103334

Journal Pre-proofs
Development of 1,3-Thiazole Analogues of Imidazopyridines as Potent Posi-
tive Allosteric Modulators of GABAA Receptors
Tatyana A. Tikhonova, Irina V. Rassokhina, Eugeny A. Kondrakhin, Mikhail
A. Fedosov, Julia V. Bukanova, Alexey V. Rossokhin, Irina N. Sharonova,
Georgy I. Kovalev, Igor V. Zavarzin, Yulia A. Volkova
PII:
S0045-2068(19)30317-7
DOI:
https://doi.org/10.1016/j.bioorg.2019.103334
YBIOO 103334
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To appear in:
Bioorganic Chemistry
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28 February 2019
17 September 2019
30 September 2019
Please cite this article as: T.A. Tikhonova, I.V. Rassokhina, E.A. Kondrakhin, M.A. Fedosov, J.V. Bukanova,
A.V. Rossokhin, I.N. Sharonova, G.I. Kovalev, I.V. Zavarzin, Y.A. Volkova, Development of 1,3-Thiazole
Analogues of Imidazopyridines as Potent Positive Allosteric Modulators of GABAA Receptors, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103334
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Development of 1,3-Thiazole Analogues of Imidazopyridines as Potent
Positive Allosteric Modulators of GABAA Receptors
Tatyana A. Tikhonova,† Irina V. Rassokhina,† Eugeny А. Kondrakhin,‡ Mikhail A. Fedosov,†
Julia V. Bukanova,§ Alexey V. Rossokhin,§ Irina N. Sharonova,§ Georgy I. Kovalev,‡ Igor V.
Zavarzin,† Yulia A. Volkova*†
†
N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 47 Leninsky
prosp., 119991 Moscow, Russia
‡
V. V. Zakusov Institute of Pharmacology, Russian Academy of Sciences, 8 Baltiyskaya str.,
125315 Moscow, Russia
§ Research Center of Neurology, 5 By-str. Obukha, 105064 Moscow, Russia
1

ABSTRACT
Structure–activity relationship studies were conducted in the search for 1,3-thiazole isosteric
analogs of imidazopyridine drugs (Zolpidem, Alpidem). Three series of novel γ-aminobutyric
acid receptor (GABAAR) ligands belonging to imidazo[2,1-b]thiazoles, imidazo[2,1-
b][1,3,4]thiadiazoles, and benzo[d]imidazo[2,1-b]thiazoles were synthesized and characterized
as active agents against GABAAR benzodiazepine-binding site. In each of these series, potent
compounds were discovered using a radioligand competition binding assay. The functional
properties of highest-affinity compounds 28 and 37 as GABAAR positive allosteric modulators
(PAMs) were determined by electrophysiological measurements. In vivo studies on zebrafish
demonstrated their potential for the further development of anxiolytics. Using the OECD “Fish,
Acute Toxicity Test” active compounds were found safe and non-toxic. Structural bases for
activity of benzo[d]imidazo[2,1-b]thiazoles were proposed using molecular docking studies. The
isosteric replacement of the pyridine nuclei by 1,3-thiazole, 1,3,4-thiadiazole, or 1,3-
benzothiazole in the ring-fused imidazole class of GABAAR PAMs was shown to be promising
for the development of novel hypnotics, anxiolytics, anticonvulsants, and sedatives drug-
candidates.
2

INTRODUCTION
γ-Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the
mammalian central nervous system (CNS) and is essential for the overall balance between
neuronal excitation and inhibition [1, 2]. γ-Aminobutyric acid released from GABAergic axon
terminals influences neurons via GABAA receptors (GABAARs), which are ligand-gated chloride
ion channels that mediate fast synaptic inhibition when activated by GABA. GABAARs are
transmembrane protein complexes composed of five subunits. To date, 19 different human
GABAAR subunits were identified and classified as α (α1−α6), β (β1−β3), γ (γ1−γ3), δ, ε, π, and
θ [3]. The heteropentameric protein α1β2γ2 membrane composed of 2α1, 2β2, and γ2 subunits
represents the most widespread adult isoform of GABAAR in the central nervous system [4-6].
Individual subunit combinations generate various GABAARs and are responsible for distinct
electrophysiological and pharmacological properties leading to specific functions [7, 8].
GABAAR bears several allosteric sites,[9-12] including those for barbiturates,[13]
benzodiazepines,[14] neurosteroids,[15, 16] and avermectins [17]. GABAARs are widely
distributed in the CNS [1] and the clinical use of drugs that alter GABAAR function as
anxiolytics, hypnotics, and anticonvulsants provides ample proof of the concept that the
GABAAR is a verified target for medicinal chemistry. Meanwhile, multiple GABAAR subtypes
that mediate these clinical effects raised the need for the development of novel highly efficient
ligands for this receptor.
Benzodiazepines (BZDs) are the most important drugs for GABAARs. These compounds mainly
have anxiolytic, anticonvulsant, muscle relaxant, and sedative-hypnotic effects[18]. Hence,
BZDs and other chemical compounds capable of high-affinity binding to GABAAR are
considered as very important therapeutic agents to treat specific diseases in the CNS. While the
first ‘benzodiazepine drugs’ were 1,4-benzodiazepines (such as Diazepam), many ligands of the
high-affinity BZD binding site designed more recently have a non-benzodiazepine structure. The
most prominent chemotypes include a heterogeneous group of 'Z-drugs' (such as Zolpidem,
Zaleplon, and Zopiclone) and pyrazoloquinolinones (such as CGS 8216). Most chemotypes
comprise positive allosteric modulators (PAMs), null (silent) modulators, and negative allosteric
modulators. Non-selective PAMs exhibit anticonvulsant, sedative-hypnotic, and anxiolytic
activity, whereas nonselective negative allosteric modulators have proconvulsant and anxiogenic
actions. Null or silent modulators, such as the BZD antidote Flumazenil, antagonize these effects
in vivo [19]. BZDs and 'Z-drugs' act via binding at the extracellular interface between the γ2
subunit and adjacent α subunits [20-23].
The search for new ligands of the central BZD receptor with more selective effects compared to
classical 1,4-benzodiazepines has been stimulated by evidence that specific GABAAR subtypes
discriminate between BZD site ligands [24, 25]. Among various structurally diverse compounds
that bind with high affinity to the BZD binding site, imidazopyridines have been extensively
studied for their biological activity. BZDs and imidazopyridines exhibit essentially different
receptor affinity for different GABAAR subtypes. BZDs show similar affinity for the α1, α2, α3,
and α5 receptor subunits. In contrast, one imidazopyridine, Zolpidem, displays higher affinity for
alpha subunits, mainly the α1 receptor subunit [26-28]. Although many structural modifications
of the imidazopyridine core were proposed,[26, 28-32] the most prominent PAM effect was
observed for imidazo[1,2-a]pyridines with aromatic and amide substituents (Figure 1). Among
other, the FDA-approved drug Zolpidem remains a gold-standard medication (‘sleeping pill’) for
3

treating primary insomnia,[33, 34] while Alpidem, Necopidem, and Saripidem were marketed as
anxiolytic drugs. The compounds DS-1 (4-chloro-N-[6,8-dibromo-2-(2-thienyl)imidazo[1,2-
a]pyridine-3-yl benzamide) and DS-2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl
benzamide) are PAMs selective for δ-GABAARs. The compound DS-1 potently (low nM)
enhances and directly activates GABA-evoked currents mediated by α4β3δ receptors and is the
most potent known agonist of these receptors [35]. The compound DS-2 is the first PAM
selective for α4/6βxδ receptors. The effects of DS2 are primarily determined by the δ subunit but
are additionally influenced by the α, but not the β, subunit (α4/6βxδ > α1βxδ >> γ2-GABAARs>
α4β3) [36].
Sedative-hypnotic
selective componds

Br
N
N
S
N
S
Me
N
N
N
Me
Br
NH
NH
O
O
O
N
Me
Me
DS-1
DS-2
Zolpidem
Cl
Cl
Anxiolytics
N
N
N
Et
Cl
Cl
N
N
N
Me
Cl
O
Me
N
Me
N
N
O
O
Pr
Pr
i-Bu
Pr
Necopidem
Alpidem
Saripidem
Figure 1. Overview of imidazopyridines developed as GABAARs PAMs.
In the present paper, we report the results of our study on a new series of GABAAR modulators
derived by rational design from imidazopyridines. In order to obtain compounds with higher
activity and taking into account that a relatively small change in the ligand structure brings about
an alteration of receptor functionality, we focused our efforts on the aromatic portion of
imidazopyridines and synthesized new compounds in which the pyridine ring of known
imidazo[1,2-a]pyridine drugs (Zolpidem, Alpidem) is replaced by 1,3-thiazole providing
imidazo[2,1-b]thiazole isosteres. Interestingly, the pharmacomodulation by replacing the
aromatic moiety in drugs by sulfur-containing heterocycles was successfully used for the
development of novel GABAAR PAMs, such as the benzodiazepine class (leading to clotiazepam
[37]) or the pyridine class (leading to SB 205384, [38] DS-1, [35] DS-2 [36]).
The pyridine/1,3-thiazole switch is expected to give rise to the following three different
regioisomeric thiazole systems: imidazo[2,1-b]thiazoles (I), imidazo[2,1-b][1,3,4]thiadiazoles
(II), and benzo[d]imidazo[2,1-b]thiazoles (III) (Figure 2). It is important to note that a few
benzo[d]imidazo[2,1-b]thiazoles were described as analgesics and anxiolytics [39-41]; however,
in some cases no precise data on their activity were reported in original documents, and in other
cases their mechanism of action was not established and target validation was not performed.
Here, we report comprehensive in vitro and in vivo studies of compounds I-III as GABAAR
PAMs. Keeping in mind that the most potent in vivo results for GABAAR PAMs were mostly
4

obtained for imidazopyridines with halogen substituents,[29, 42] we focused our efforts on
compounds bearing chlorine, bromine, and fluorine atoms.
S
S
N N
O
S
N
N
N
X
X
N
N
X
Ar
Ar
Ar
O
O
R
R
R
I
II
III
Figure 2. Three 1,3-regioisomeric thiazole systems investigated in this work.
RESULTS AND DISCUSSION
Chemistry. The structures of the compounds investigated in this study are shown in Table 1. The
choice of these compounds was based on the results of the preliminary in silico screening of a
virtual library of imidazo[2,1-b]thiazoles (see Supporting Information). We used a general
synthetic approach to 1,3-thiazole-annulated heterocycles that was elaborated in our recent
studies [43, 44]. Alcohol ester 1-4, 6-8, and 11-20 and alkyl amides 5, 9, 10, and 36-40 were
prepared by the copper-catalyzed three-component coupling reaction of heterocyclic
thioamidines with aryl aldehydes and propiolic acid derivatives (Scheme 1). This protocol was
efficient for the construction of various aryl-substituted imidazo-[2,1-b]thiazoles, -[2,1-
b][1,3,4]thiadiazoles, and -[2,1-b]benzothiazoles. The products yields ranged 33–78 % and were
the lowest among imidazo-[2,1-b]thiazoles. High efficiency and functional group tolerance
allowed the selective incorporation of aryls containing fluorine, chlorine, bromine, methyl,
methoxy, trifluoromethyl, or nitro substituents into the imidazo[2,1-b]thiazole ring.
Scheme 1a
S
N
O
S
N
R2
i
N
R2 CHO
R3
NH2
X
+
+
R3
X
R1
X = C,N
O
R1 = OMe, OEt, NMe2, NEt2, NPr2
R1
R2 = C6H5, p-MeC6H4, p-MeOC6H4, o-MeOC6H4, o,p-MeOC6H3,
p-FC6H4, o-ClC6H4, m-ClC6H4, p-ClC6H4, p-BrC6H4, o,p-Cl2C6H,
m,p-Cl2C6H4, p-CF3C6H4, p-NO2C6H4
1-20,
36-40
R3 = H, Et, iso-Pr, CH2OMe
a Reaction conditions: (i) CuOTf•C6H6 (10 mol %), Cu(OTf)2 (10 mol %), 4Å molecular sieves,
toluene, 120 oC, 2 h.
Propiolamide (R1 = NH2) proved to be inefficient in the three-component coupling reaction
giving unsubstituted imidazo[2,1-b]thiazole amides; multiple side reactions occurring under
standard conditions. Therefore, the synthesis of compounds 21–35 was achieved from an
appropriate ethyl ester by stepwise saponification and amidation of in situ generated acyl
chloride with ammonia gas (Scheme 2).
Scheme 2a
5

S
S
S
N
N
N
R2
R2
R2
N
N
N
i
ii, iii
R3
R3
R3
O
O
O
NH2
OEt
OH
R2 = C6H5, p-MeC6H4, p-MeOC6H4, o-MeOC6H4, o,p-MeOC6H3, p-FC6H4,
o-ClC6H4, m-ClC6H4, p-ClC6H4, o,p-Cl2C6H4, m,p-Cl2C6H4, p-BrC6H4,
p-CF3C6H4; R3 = H, OMe, Cl
5, 21-35
a Reaction conditions: (i) KOH, EtOH, rt, 10 min; (ii) PCl5, CH2Cl2, rt, 24 h; (iii) NH3, CH2Cl2,
rt, 1 h
The overall yields over two steps were 47 to 90%. Notably, this procedure is efficient with
secondary amines; alkyl amides 5, 9, 10, and 36–42 can also be synthesized in quantitative yields
from esters by treatment with appropriate amines.
Affinity for the benzodiazepine-binding site of GABAARs. All synthesized compounds 1–42
were evaluated on GABAARs at the BZD binding site. The affinity of compounds was measured
using a radioligand competition binding assay by displacement of [3H]Flunitrazepam binding
from rat cortical membrane. Zolpidem (ZLP), a sedative-hypnotic medicine, was used as the
reference compound. The corresponding inhibitory concentrations IC50 (IC50 is the half-maximal
concentration of the ligand required to inhibit [3H]Flunitrazepam binding by 50%) are listed in
Table 1.
6

Table 1. The IC50 values of imidazothiazoles tested in vitro for [3H]Flunitrazepam binding to rat
cortical membranes
S
N N
O
S
R3
S
N
N
N
R3
N
R2
R2
N
R2
O
O
R1
R1
R1
1-5
6-10
11-42
IC50
IC50
Comp.
R1
R2
R3
Comp.
R1
R2
R3
value
(nM)*
value
(nM)*
1
2
OMe
OMe
OEt
p-ClC6H4
p-NO2C6H4
p-ClC6H4
o,p-Cl2C6H
p-ClC6H4
p-ClC6H4
p-ClC6H4
p-ClC6H4
p-ClC6H4
p-ClC6H4
C6H5
>2000
>2000
>2000
>2000
839±240
>2000
>2000
>2000
74±8
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
p-MeC6H4
p-MeOC6H4
o-MeOC6H4
o,p-MeOC6H3
o-ClC6H4
73.0±0.2
187±6
>2000
>2000
141±5
373±6
10.0±0.7
>2000
>2000
178±6
>2000
69±6
3
4
OEt
5
NMe2
OEt
6
Et
m-ClC6H4
p-ClC6H4
7
OEt
iso-Pr
8
OEt
CH2OMe
p-ClC6H4
Cl
9
NMe2
NPr2
OEt
Et
Et
p-ClC6H4
MeO
10
11
12
13
14
15
160±10
956±45
1025±41
ne
o,p-Cl2C6H4
m,p-Cl2C6H4
p-FC6H4
OEt
p-MeC6H4
p-MeOC6H4
o-MeOC6H4
OEt
p-BrC6H4
198±8
471±5
320±10
OEt
>2000
>2000
p-CF3C6H4
p-MeC6H4
OEt
o,p-MeOC6H3
NMe2
O
O
16
OEt
712±10
37
NMe2
o-ClC6H4
6.0±0.5
17
18
19
20
21
OEt
OEt
OEt
OEt
NH2
p-FC6H4
p-ClC6H4
p-BrC6H4
p-ClC6H4
C6H5
>2000
>2000
ne
38
39
40
41
42
NMe2
NMe2
NMe2
NEt2
m-ClC6H4
p-ClC6H4
o,p-Cl2C6H4
p-ClC6H4
p-ClC6H4
48±1
70±2
12.0±0.5
75±8
Cl
>2000
191±5
NPr2
>2000
43±1
Zolpidem
*Each IC50 value is the mean of three independent measurements ± SD; ne, not established because of poor solubility of
compound under experimental conditions.
We found that esters 1–4 and 6–8 of imidazo[2,1-b]thiazole and imidazo[2,1-b][1,3,4]thiadiazole
series are inactive at concentrations lower than 2000 nM (Table 1), whereas the corresponding
alkyl amides 5, 9, and 10 exhibited potency for binding to BZD site at a nanomolar level (their
IC50 values were 839, 74, and 160 nM, respectively). The affinity of imidazo[2,1-
7

b][1,3,4]thiadiazole 9 was just 1.7 times lower compared with the reference drug ZLP (IC50 = 43
nM).
In contrast, the evaluation of the benzo[d]imidazo[2,1-b]thiazole series (esters 11–20) affinity
revealed that compounds 11, 12, 13, and 16 bearing p-methyl, p-methoxy, and m,p-
methylenedioxy substituents at the aryl moiety have a low activity with IC50 ranging from 700 to
956 nM. Compounds 14 and 15 containing a methoxy substituent in the ortho position, as well as
compounds 17–20 bearing halogens (F, Cl, Br) in the para position, proved to be inactive.
Benzo[d]imidazo[2,1-b]thiazole amides 21–42, except for 24, 25, 29, 30, 32 and 42, were found
to be more potent; almost all of them displayed a nanomolar affinity below 471 nM. Compound
23 bearing a methoxy substituent in the para position of the aryl moiety displayed a weak
activity with IC50 value of 187 nM, which is similar to that of unsubstituted amide 21 (IC50 = 191
nM). Tolyl-substituted derivative 22 exhibited an above-average potency for binding with IC50 =
73 nM.
Compound 28 with the p-ClC6H4 pendant showed the affinity higher to that of the drug ZLP
(IC50 = 43 nM) with IC50 of 10 nM. A change in the position of chlorine atom at the aryl moiety,
as well as an increase in the number of substituents in 28, led to a decrease in its activity. Thus,
the IC50 values of ortho- and meta-substituted analogs 26 and 27 were 141 nM and 373 nM,
respectively. Compound 31 containing an o,p-Cl2C6H3 substituent exhibited IC50 of 178 nM,
while m,p-Cl2C6H3-substituted 32 was inactive. The insertion of chlorine and methoxy
substituents (compounds 29 and 30) into the benzothiazole moiety of compound 28 resulted in
the loss of affinity. The replacement of chlorine atom in structure 28 by other halogen
substituents, such as bromine, fluorine, or trifluoromethyl (compounds 33, 34, and 35), led to a
decrease in its activity and an increase in IC50 to 69 nM, 198 nM, and 471 nM, respectively.
Thus, o-methoxy- and o,p-dimethoxy substituted analogs 24 and 25 were found to be inactive.
The activity of derivatives of compound 28 alkylated at the amide moiety correlates with a
substituent chain length. Compound 39 containing a dimethylamide group exhibited an above-
average potency for binding with IC50 = 70 nM, which is slightly lower than that of compound
41 with a diethylamide group (IC50 = 75 nM). However, an analog bearing a dipropylamide
substituent was active only at concentrations above 2000 nM.
The SAR evaluation of a series of dimethyl amides 36–40 demonstrated that general trends in
effects of their substituents at the aryl moiety on the potency for receptor binding differ from
those observed for unsubstituted amides. Thus, tolyl-substituted 36 showed a weak affinity (IC50
= 320 nM) when compared to analog 22 (IC50 = 73 nM). Compound 38 meta-Cl substituted at
the aryl moiety (IC50 = 48 nM) had a higher potency for binding compared to p-Cl-substituted
compound 39 (IC50 = 70 nM). Amide 40 containing an o,p-Cl2C6H4 substituent was among the
most active compounds (IC50 = 12 nM). Benzo[d]imidazo[2,1-b]thiazole 37 bearing an o-Cl
substituent at the aryl moiety was the most active compound (IC50 = 6 nM) of all the tested
compounds, with IC50 being seven times lower than that of the reference drug ZLP (IC50 = 43
nM).
Behavioral effects on adult zebrafish and toxicity. Benzo[d]imidazo[2,1-b]thiazoles were
selected for further in vivo evaluations based on their pronounced activity revealed by in vitro
screening. Zebrafish displays a high homology to humans with an intact and highly conserved
neurotransmitter system[45] and exhibits similar neurobehavioural phenotypes that can easily be
quantified [46]. Zebrafish (Danio rerio) were used as an efficacious type of experimental
8

animals, which proved to be an especially effective model organism for GABAAR ligands in in
vivo studies [46]. The “novel tank” test was used to assess behavioral indices [47]. It was
previously shown that the adult zebrafish is sensitive to the effects of known anxiolytics and
sedative agents in the novel tank tests. This test, conceptually similar to the open field test in
rodents, is the most extensively studied model of anxiety in the zebrafish. The novel tank test
exploits the fact that adult zebrafish spend about 50% of a 5 min session in the bottom of a novel
tank, an effect which is reduced by Diazepam, Buspirone, or acute ethanol treatment [48]. In
accordance with the evaluation protocol fish treated with benzo[d]imidazo[2,1-b]thiazoles were
exposed to the water-filled tank divided into two equal virtual horizontal portions by a line
marking the outside walls. Zebrafish behavior was then processed to score behavioral endpoints,
such as the time spent in top, top entries, the latency to top entry (s), and the number of freezing
bouts (see Methods). We characterized drug-induced behavioral responses in adult zebrafish
following their acute exposure to compounds 11–42. The drug ZLP was used as the reference
compound [49]. It was of particular interest in terms of the predictive value to compare the
results of activity studies in the zebrafish model with the radioligand competition displacement
assay data [50]. Latency to enter the upper half, transitions into the upper half of the tank, and
the time spent in the upper half of the tank were found the most informative parameters for
compounds 11-42 to be divided into three groups A-C, while freezing bouts were statistically
undisruptive.
Compounds 11, 12, 14–17, 18, 20, 24, 25, 27, 29, 30, 32, and 35 (group A’) with IC50 values
higher than 370 nM (Table 1) had no pronounced effect on Zebrafish behavior (see Supporting
information). There was no significant difference in latency to enter the upper half, transitions
into the upper half of the tank, and the time spent in the upper half of the tank between the
control group and groups of the tested compounds (some representative case studies are depicted
in Figure 3A).
Amides 21, 23, and 31 (group A”) with IC50 ranging from 178 to 191 nM had a weak effect on
adult zebrafish behavior (Figure 3A). Behavioral effects were most clearly observed upon the
administration of compound 31 (IC50 = 178 nM), resulting in a slightly shorter time spent in the
upper half of the tank, a reduced number of transitions into the upper half of the tank, and higher
latency to enter the upper half compared to the control group.
Similar, but more pronounced, trends in behavioral effects were observed for compounds 13, 19,
28, 33, 34, and 39–41 (group B, Figure 3B) with IC50 of 10-75 nM [51]. Compounds 13, 19, and
28 at 1 mg/L concentrations led to a 20-fold decrease in the time spent in the upper half of the
tank and a 13-fold decrease in transitions into the upper half of the tank compared to the control
group. Compounds 33, 34, 39, 40, and 41 caused the reduction of the time spent in the upper half
of the tank to zero along with a decrease to zero in the number of transitions into the upper half
of the tank. Latency to enter the upper half in group B was unprecedentedly long compared with
the control group.
Upon exposure to acute compounds 22 and 36–38 (IC50 = 6–320 nM, group С, Figure 3C), adult
zebrafish showed the opposite behavior. These compounds at a 1 mg/L concentration produced
anxiolytic-like behavioral effects similar to those of Zolpidem. Along with the loss of normal
motor function in zebrafish, we registered long time spent in the upper half of the tank, a high
9

number of transitions into the upper half of the tank, and low latency to enter the upper half
compared to the control group. Compounds 37 and 38 were found to be most allied to Zolpidem.
It is remarkable that the observed behavioral effects in groups B and C were fully eliminated
upon treatment of fish with Flumazenil,[52] which is considered to be a strong antagonist acting
at the GABAARs BZD binding site (for experimental details see Supporting Information, Figure
S1). This clearly shows that the altered behavior compared to the control group upon
administration of benzo[d]imidazo[2,1-b]thiazoles 13, 19, 22, 28, 33, 34, and 36–41 was caused
by their in vivo binding to GABAAR BZD binding sites.
Figure 3. Effects of the studied compounds (1 mg/L, acute 20-min exposure) on adult zebrafish
behavior assessed in the 6-min “novel tank” test. A group of 10 fish (approximately 50 : 50 male
: female ratio) was used for each compound. The data are presented as mean ± SEM. (A) The
results for representative compounds 11, 12, 16, 27, 35 from group A’ (have no effects) and 21,
23, and 31 from group A” (poorly affected). (B) The results for compounds 13, 19, 28, 33, 34,
and 39–41 from group B. (C) The results for compounds 22 and 36–38 from group С.
The comparison of the results obtained using the radioligand competition displacement assay and
behavioral responses in zebrafish shows that the IC50 values determined by the radioligand assay
and the “novel tank” test corroborate each other and, hence, may be considered a predictive
value, allowing a preliminary screening.
Having evaluated the in vivo efficacy, we assessed the safety of active compounds 13, 19, 21–23,
28, 31, 33, 34, and 36–41 using the OECD “Fish, Acute Toxicity Test”,[53] which is widely
10

employed in the pharmaceutical industry to estimate the potential acute neurotoxicity of new
compounds and select most promising candidates for in-depth biological testing. Compounds 13,
19, 21–23, 31, 33, 34, 36, 37, 40, and 41 were found to be non-toxic; one hundred percent
survival of fish was observed at the maximum attainable concentrations in water (up to 47 mg/L)
after a four-day exposure. Only at the maximum attainable concentrations in water (4.8 mg/L),
compounds 28, 38, and 39 caused the loss of normal motor function in 100% of the tested
zebrafish, leading to death in 2/7-3/7 animals after two days. Therefore, the lead compounds 28
and 37 were found to be safe and non-toxic.
Electrophysiological studies. Thus, we found that all three types of isosteric analogs can exert an
effect on the BZD binding site of GABAARs. Among the tested substances, the most active were
imidazopyridines 28 and 37. They showed the lowest IC50 values for [3H]Flunitrazepam
displacement activity (10 and 6 nM, respectively). Therefore, we chose these compounds and
evaluated their ability to potentiate GABAAR currents.
The functional properties of compounds 28 and 37 as GABAAR PAMs were studied in
electrophysiological experiments performed in freshly isolated rat Purkinje neurons. In situ
hybridization studies showed a high expression of the α1, β2, β3, and γ2 mRNA in rat cerebellar
Purkinje cells [54, 55]. All other GABAARs receptor subunit genes are either absent, or
expressed at much lower levels. The functional properties of GABAARs from Purkinje cells also
suggest the presence of α1, β2, β3, and γ2 subunits. These include high sensitivity to ZLP,[56,
57] indicating the presence of the α1 subunit. The type of the receptor expressed in Purkinje cells
is the most common type of GABAARs in the brain with the subunit composition α1β2γ2 [5].
We performed this set of experiments using the whole-cell patch-clamp recording and fast
perfusion techniques. In all the investigated neurons, GABA application at concentrations from 1
to 300 µM at a holding potential of -70 mV elicited an inward current, whose amplitude
increased with the increasing GABA concentration. The half-maximal GABA concentration for
activation of GABAARs was 5.2 ± 0.4 µM (n = 6). The modulatory effects of BZ compounds on
GABA-induced currents were investigated using the co-application of 2 µM GABA (near EC20)
and the tested compounds in the concentration range of 10 nM–10 µM. The data were collected
from 24 Purkinje neurons, all of which demonstrated the potentiation of GABA current by the
tested compounds during the 2-s co-application. The sample recordings demonstrating whole-
cell responses to the application of 2 µM GABA in the absence and presence of compounds 28
and 37 are shown in Figure 4.
The chloride currents were concentration-dependently increased by the application of these
compounds with a maximal potentiation at about 1 µM for 28 and 3 µM for 37. At higher
concentrations, the potentiation was less pronounced, giving rise to a bell-shaped concentration
effect curve for compound 28. The threshold concentration for the potentiating effect of both
compounds was 10 nM. The concentration-response relationships for the compounds under study
were constructed using peak amplitudes normalized to a control response (see Methods).
Compound 28 enhanced GABAAR currents to a maximum of 297 ± 25% of the control current in
the absence of the drug with EC50 of 39 ± 11 nM and a Hill slope of 1.2 ± 0.4 (n = 5-6). For
compound 37, the maximum enhancement was 198 ± 10% of the control, EC50 was 81 ± 24 nM,
the Hill slope was 0.8 ± 0.2 (n = 8–15). The differences in EC50 and the maximal effects were
statistically significant (p = 5 × 10-4 for EC50 and p = 10-4 for the maximal effect). In several
neurons, we performed a direct comparison of the potentiating effects of these compounds
11

(Figure 5). This example demonstrates that the maximal potentiation of the GABA current
achieved at a saturating concentration of compound 37 (3 µM) is about 200%, while an increase
in the GABA response induced by the application of 1 µM compound 28 is near 300%.
To investigate the pharmacological specificity of the potentiation of the GABA-evoked currents
described above, we also examined the effects of Flumazenil (Ro15-1788) that is generally
considered as an antagonist acting on the BZD binding site [52]. We found that 5 µM Flumazenil
completely suppressed the potentiating effect of both drugs without affecting the control
response (not shown), thus indicating the involvement of the BZD binding site in the effects of
compounds 28 and 37.
Figure 4. Potentiation of GABA-gated chloride current by various concentrations of compounds
28 and 37. (A) Representative currents in acutely isolated cerebellar Purkinje cells induced by 2
µM GABA. The control current (black trace) is superimposed onto the current induced by
GABA co-application at different concentrations of 28 (0.03, 0.1, and 1 µM); VH is –70mV.
Horizontal bars above the current responses show the application of 2 µM GABA and different
concentrations of 28. (B) Concentration-response analysis for potentiation of GABA-activated
currents by 28. Data points represent average values (mean  S.E.M.) from 5–6 cells normalized
to the current obtained under control conditions (2 µM GABA, –70 mV) and the fit by Equation
2 with the following parameters: EC50=39 ± 11 nM and nH = 1.2 ± 0.4. The current amplitude
was measured at the point of the maximal effect following agonist applications. (C)
Representative currents induced by 2 µM GABA (black trace) and superimposed currents
induced by GABA co-application at different concentrations of 37 (0.1, 1 and 3 µM). (D)
Concentration-response analysis for potentiation of GABA-activated currents by 37. Data points
represent average values (mean  S.E.M.) from 8 – 15 cells. EC50 = 81 ± 24 nM, nH = 0.8 ± 0.2 (
n= 8 – 15).
12

Figure 5. Comparison of the effects produced by compounds 28 and 37 on GABA currents
recorded in the same neuron. Upper panel: the time course of the experiment with 28 and 37.
Each point represents the amplitude of the current induced by isolated GABA (2 µM) application
(black dots) or GABA + 28 (squares)/37 (triangles) at different concentrations (colored points,
the higher the concentration, the larger the size of the symbol). Lower panel: current traces
recorded at the points indicated by arrows in the upper panel. The dotted line indicates the
current amplitude by GABA control (2 µM).
Therefore, our electrophysiological experiments with native GABAARs expressed in rat
cerebellar Purkinje neurons demonstrated that as with traditional BZDs, the tested compounds
act by enhancing the GABAergic inhibitory drive. We found that 28 and 37 potentiate GABA
currents via the BZD binding site. Compound 28 potentiates this GABA-induced current with
the affinity similar to the estimates for the reference drug ZLP in these neurons (33 nM) [56].
However, the EC50 value (39 nM) for 28 was about half that of 37 (81 nM), and the maximal
potentiation achieved with 28 was significantly higher than that obtained with 37 (297% and
198%, respectively). A difference in modulatory potency between these two compounds can be
related to different molecular determinants responsible for the interaction of 28 and 37 with the
BZD binding site.
Proposed Structural Basis for Activity of Benzo[d]imidazo[2,1-b]thiazoles and Molecular
Docking. Molecular docking simulations were performed to gain insights into the structural basis
for the activity of imidazo[2,1-b]thiazoles. Benzo[d]imidazo[2,1-b]thiazoles 28 and 37, lead
compounds selected on the basis of in vitro and in vivo experiments, were used as representative
compounds (Figure S2). A comparison between the binding modes of these compounds and the
reference drug Zolpidem was performed.
Binding of the tested molecules (28 and 37) at the α(+)/γ(-) intersubunit interface.
13

We first docked the reference drug ZLP at the α(+)/γ(-) intersubunit interface relying rather on
the shape of the binding pocket than on the specific interaction with surrounding residues. We
placed the molecule in the BZD site in four different starting positions. The ZLP starting modes
were defined in accordance with the direction of two different moieties (imidazopyridine ring
and dimethyl amide group). Hereinafter, we refer to these modes as i) down-right (the
imidazopyridine ring is directed downward toward the membrane and the dimethyl amide group
points right toward the γ2 subunit), ii) down-left (the imidazopyridine ring is directed as in the
down-right mode and the carbonyl-dimethyl amide group points to the left of the γ2 subunit), iii)
up-right (the imidazopyridine ring is directed upward from the membrane and the dimethyl
amide group is directed as in the down-right mode), and iv) up-left (the imidazopyridine ring is
directed as in the up-right mode and the dimethyl amide group is directed as in the down-left
mode) ones.
In each case, an extensive two-stage MCM protocol (see Methods) was used to find the
minimum-energy conformations (MECs) of the ligand receptor complexes. After the
convergence of the MCM trajectories, one binding mode never changes to another one,
indicating that the BZD binding pocket is rather tight. The structural models of ZLP binding are
shown in Figure S3 and the corresponding ligand–receptor interaction energies are given in
Table 2. As it can be seen in Table 2, the down-left binding mode is energetically more favorable
(–37.1 kcal/mol). Three other binding modes have similar ligand–receptor interaction energies.
Most of the residues of the α1 and γ2 subunits, which were identified in the extensive mutational
studies [58-62] as affecting ZLP efficacy, strongly contribute to ligand–receptor interactions in
all found MECs (Figure S3 A-D).
Table 2. Energies (kcal/mol) of the ligand–receptor interaction corresponding to different
binding modes of the tested ligands*
Molecule
Zolpidem
28
Down-right
- 31.8
Down-left
- 37.1
Up-right
- 33.1
- 33.5
- 37.8
Up-left
- 32.5
- 37.1
- 34.2
- 35.0
- 37.5
37
- 26.6
- 27.6
*Down-right (the imidazopyridine ring is directed downward toward the
membrane and the dimethyl amide group points right toward the γ2
subunit), Down-left (the imidazopyridine ring is directed as in the down-
right mode and the carbonyl-dimethyl amide group points to the left of the
γ2 subunit), Up-right (the imidazopyridine ring is directed upward from
the membrane and the dimethyl amide group is directed as in the down-
right mode), and Up-left (the imidazopyridine ring is directed as in the up-
right mode and the dimethyl amide group is directed as in the down-left
mode)
To dock the tested molecules (28 and 37) into the BZD pocket, we initially superimposed their
C4 and C15 atoms (Figure S32A) onto the same atoms of ZLP in the four found MECs of ZLP-
receptor complexes (Figure S3A–D). Then, the two-stage MCM protocol was again employed to
find the MECs of the ligand-receptor complexes. Hereinafter, we refer to the binding modes of
the tested molecules as accepted for ZLP. As in the case of the ZLP binding, the starting modes
14

never change to other ones. The energies of ligand–receptor interactions evaluated for different
binding modes of the tested molecules are given in Table 2. It is evident that the down-left and
up-left MECs are more favorable for the binding of molecule 28 in the BZD pocket. For
molecule 37, the up-right binding mode is the most favorable one. The MECs of the ligand
receptor complexes in these binding modes are shown in Figure 6.
The interaction energy of molecule 37 in the up-right mode is higher (in absolute values) than
the energy of ZLP in the same mode (–37.8 vs –33.1 kcal/mol). The main difference is associated
with an increase in the contribution of van-der-Waals interactions to the energy (–34.9 vs –31.6
kcal/mol) due to the stronger interactions between aromatic residues and the
benzo[d]imidazo[2,1-b]thiazole core of molecule 37. The set of residues involved in ligand–
receptor interactions is essentially the same in both cases (Figures 6A and S3C).
In the BZD pocket, tested molecule 28 forms energetically more favorable contacts in the up-left
mode (–37.1 vs –32.5 kcal/mol) compared to ZLP (Table 2). In this case, the energy gain is
associated with an increase in the contribution of both van-der-Waals (–34.9 vs –31.9 kcal/mol)
and electrostatic (–2.1 vs –0.6 kcal/mol) interactions. The interaction energies of molecules 28
and ZLP bound in the down-left modes have similar values (Table 2). The sets of residues,
which essentially stabilize molecule 28 and ZLP in the binding site, are basically the same in
both the binding modes (Figures 6B,C and S3B,D). In the up-left mode the benzo[d]imidazo[2,1-
b]thiazole core of molecule 28 provides stronger interactions with surrounding aromatic
residues. For example, the energy of interaction of Phe77 (γ2, loop D) and Phe100 (α1, loop A)
with molecule 28 is –6.3 and –1.1 kcal/mol, respectively, vs –4.0 and –0.5 kcal/mol in the ZLP
MEC.
15

Figure 6. Favorable binding modes of the tested molecules at the α(+)/γ(-) intersubunit interface.
A, D molecule 37 bound in the up-right mode. B, E and C, F molecule 28 bound in the down-left
and up-left modes, respectively. In A, B and C the binding modes are shown for the α1-GlyR
based model (3JAE), whereas in D, E and F the binding modes are shown for the α1β3γ2
GABAAR (6HUP). Tested molecules and side chains of residues strongly contributing to the
ligand–receptor energy (> 1 kcal/mol) are represented by sticks. H-bonds are presented by red
dash lines. The α1 subunit is in cyan, and γ2 is in magenta. Hydrogens are not shown.
16

We built a homology model of GABAAR based on the structure of α1 GlyR (PDB entry 3JAE).
Most recently in 2019, several new cryo-EM structures of the full-length human α1β3γ2
GABAAR in complex with GABA and the classical benzodiazepines alprazolam and diazepam
become available [63]. The last ones were reconstituted in lipid nanodiscs and have undisrupted
five-fold quasi-symmetrical architecture. A comparison of the cryo-EM structure of α1β3γ2
GABAAR and our homology model showed that our sequence alignment of the loop F does not
correctly reflect all structural facts.
A low sequence identity in the loop F and varying sequence lengths result in many gaps in the
alignment and make the last one most ambiguous among other regions of ECD of GABAAR
(Fig. S2 B). The existence of the conserved V-X-V motif in the loop F, which forms a short β-
strand of three residues, was not reflected in our alignment. Obviously, this misalignment cannot
affect the geometry of the BZD pocket, but some interactions of the tested molecules with
surrounding residues may be affected. Thus, in order to validate our models we performed the
docking of the tested molecules (28 in the up-left/down-left modes and 37 in the up-right mode)
to the α(+)/γ(-) intersubunit interface of the α1β3γ2 GABAAR (PDB entry 6HUP). The docking
and MC minimization protocols were similar to the ones used before for the α1-GlyR based
models.
The structural models of binding of the tested molecules at the α(+)/γ(-) intersubunit interface of
the 6HUP structure are shown in Figures 6. The values of the ligand-receptor interaction energy
of the molecules 28 and 37, bound in the up-left and up-right modes in the 6HUP structure (Fig.
6 D, F), are close to the ones obtained with the α1-GlyR based model (-35.5 vs -37.1 kcal/mol
and -36.3 vs -37.8 kcal/mol, respectively). The main difference is that in the 6HUP structure
residues Met130, Thr142 (loop E, γ2) and Val203, Ser206 (α1, loop C) less strongly involved in
stabilization of tested molecules. The molecule 28 bound in the down-left mode more strongly
interacts with the loop C (α1) residues in the 6HUP structure and forms two H-bonds with
Ser205 and Ser206 (Fig. 6 E). However, in this mode compound 28 practically does not interact
with residues Glu189, Val190 and Asp192 (loop F, γ2) and loses in the ligand-receptor energy
compare to the α1-GlyR based model (-33.2 vs -37.5 kcal/mol).
We superimposed our homology model of the α1β2γ2 GABAAR and cryo-EM structure of the
α1β3γ2 GABAAR in complex with GABA and Diazepam (DZP) [63] (Figure S4). It is easy to
see that all main loops that form the BZD binding site in the interface between α1 and γ2
subunits coincide well (Figure S4 A, B). The main difference can be found in the conformations
of the loops C (α1) and F (γ2). The loop F (γ2) in the 6HUP structure is more distant from the
BZD pocket than in the α1-GlyR based model (Fig. S4 B). Therefore, the misalignment of the
loop F residues did not affect our docking results.
Superpositions of DZP in the 6HUP structure and tested molecules 37 and 28 bound in the up-
right and up-left modes, respectively, in our homology model are shown in Figure S4 C, D. Both
ligands (DZP and 37/28) lean on the BZD pocket floor, which is delineated by the side chain of
Asn60 (γ2). The chlorine atoms in DZP and 37/28 molecules interact with the His102 (α1) and
Asn60 (γ2) side chains. Extensive aromatic-aromatic interactions between these molecules and
surrounding residues (Phe100, H102, Tyr210 in α1 and Tyr58, Phe77 in γ2) can be detected. The
molecules 37 and 28 are bulkier compared to DZP. However, the positions of the ligands in
general are rather well matched to each other that clearly indicates the preferred binding modes
of the tested molecules at the α(+)/γ(-) intersubunit interface.
17

Below are summarized key points of this work. GABAARs are the site of action of a variety of
pharmacologically and clinically important drugs, including BZDs barbiturates, anesthetics,
neuroactive steroids, and convulsants [19, 64]. BZDs have been used clinically for more than 50
years to treat disorders such as insomnia, anxiety, and epilepsy, as well as to aid muscle
relaxation and anesthesia [19]. While the first ‘benzodiazepine drugs’ were 1,4-benzodiazepines
(such as Diazepam, Flunitrazepam), a variety of structurally diverse ligands, which bind to the
BZD site, were designed more recently. Each such chemical class includes compounds that
enhance (positive allosteric modulators or BZD site agonists) or reduce (negative allosteric
modulators or BZD site inverse agonists) GABA-induced chloride ion flux via the same BZD
binding site. There are approximately 20 chemical classes of ligands that bind to the BZD site of
GABAAR, including the structures of approximately 40 compounds. Among compounds of
several chemical classes, which can interact with the BZD binding site, there are heterocycles
containing an imidazole ring. Probably, imidazo[1,2-a]pyridine is the most widely used
heterocyclic system of this group. It is present in marketed drugs, including anxiolytic and
sedative drugs, such as zolpidem, necopidem, saripidem, alpidem, and olprinone [65].
The aim of the present study is to investigate a series of new imidazole derivatives, in which the
pyridine ring of known imidazo[1,2-a]pyridine drugs (Zolpidem, Alpidem) is replaced by 1,3-
thiazole providing imidazo[2,1-b]thiazole isosteres, and to establish whether these compounds
can act as active ligands of GABAAR BZD binding site. First, the potential GABAergic activity
of these compounds was tested by investigating their ability to displace [3H]Flunitrazepam from
the BZD binding site of the GABAAR using a radioligand competition binding assay. These
radioligand binding assays were performed using a GABAAR preparation from the rat cerebral
cortex. [3H]Flunitrazepam was found to be a photoaffinity label for the BZD sites in the brain
and proved to have sufficient affinity (KD ~10 nM) to detect binding in crude homogenates.[66]
We found that some of the tested imidazo[2,1-b]thiazoles and imidazo[2,1-b][1,3,4]thiadiazoles
(5, 9, and 10) have IC50 in the range of 74–936 nM. More importantly, experiments revealed that
benzo[d]imidazo[2,1-b]thiazoles interact with the BZD-binding site as high-affinity ligands with
IC50 of 6 nM and 10 nM for the most active compounds 28 and 37, respectively.
These experiments showed that 1,3-thiazole isosteres of imidazo[1,2-a]pyridines are ligands of
the BZD site of GABAAR. It is well established that classical BZDs, such as Diazepam or
Flunitrazepam, predominantly interact with receptors composed of α1βγ2, α2βγ2, α3βγ2, or
α5βγ2. They exhibit no activity against α4βγ2 or α6βγ2 receptors and weak activity against
receptors containing γ1 or γ3 subunits [64]. A GABAAR preparation from the rat cerebral cortex,
which was used in our study, contains different GABAAR isoforms. Therefore, our radioligand
binding assays using classical BZD does not allow us to make a conclusion about the GABAAR
subtypes with which the drugs under study interact. At the same time, the discovery of a
different subpopulation of GABAAR subtypes in the mammalian brain suggested that the
sedative-hypnotic, anxiolytic, myorelaxant, and anticonvulsant effects of BZDs may be mediated
by different GABAAR subtypes localized in distinct brain regions [5, 67]. The function of at least
some GABAAR subtypes in the brain was identified using a combined molecular genetic and
pharmacological approach [68, 69]. Based on an in vivo point mutation strategy, α1-GABAARs
were found to mediate sedation, anterograde amnesia, and part of the seizure protection, whereas
α2-GABAARs, but not α3-receptors, mediate anxiolysis [70].
18

Next, we evaluated the neurobehavioral effects of the newly synthesized benzo[d]imidazo[2,1-
b]thiazoles 11-42 using zebrafish models. Zebrafish have become a powerful tool in
neuroscience research due to due to their molecular/physiological conservation, small body size,
ease of experimental manipulations in vivo, and rich behavioral repertoire. Zebrafish models and
tests are particularly useful in CNS drug screening, as well as in modeling complex neurological
and psychiatric disorders [71]. The zebrafish genome also shares 70% homology with the human
genome, which is valuable for translational medicine, drug discovery, and large-scale chemical
screens [72]. Benzo[d]imidazo[2,1-b]thiazoles 11–42 were tested for behavioral activity in vivo
in order to perform structure–activity relationship analysis. The mostly used “novel tank” test was
applied to assess behavioral stress and anxiety indices [47]. Anxiolytic drugs, including
Diazepam,[73] can modulate zebrafish “novel tank” behaviors tending to increase time spent in
top [74]. We observed anxiety-like behavior for compounds 22, 36, 37, and 38 comprising the
group C, and, what is surprising, for the GABAAR α1-selective sedative-hypnotic drug Zolpidem
(Figure 4). Moreover, the administration of compounds of the group B, including 28, led to a
decrease in time spent in top, alike sedative agents [75]. This results can be attributed to
difficulties in distinguishing between anxiety-like and stress-like behavior of zebrafish.
Moreover, little is known about GABAAR diversity and expression in the CNS of zebrafish.
Recently, it was shown that zebrafish contain orthologs for most of the GABAAR subunits found
in mammals, but the diversity of subfamilies is bigger [76]. The ability to interact with
differently composed GABAARs, including those specific only for zebrafish, may be responsible
for the differences in the observed the drug-induced behavioral effects.
Compounds 28 and 37 were found to be the most active in the series under study and were
chosen for detailed biological studies. The observed reduction of the behavioral effects of lead
compounds 28 and 37 by after-treatment with Flumazenil suggests the involvement of the α1-
containing GABAAR in drug-induced behavioral responses in zebrafish. The ability of lead
compounds 28 and 37 to modulate activity of α1-containing GABAAR subtypes was tested using
electrophysiological experiments in rat cerebellar Purkinje cells, which express the α1β2/3γ2
type of GABAAR [54]. We found that, as with traditional BZDs, the compounds under study
work by enhancing GABAergic inhibitory drive. The molecular modeling confirmed that
molecules 28 and 37 interact with the GABAAR BZD binding site. The different character of
interactions of the tested molecules with the BZD binding pocket in the up-right (37) and up-left
(28) modes may also finally result in diverse intrinsic activity evaluated by GABA current
measurements and specific features of behavioural effects caused by these drugs. This suggestion
is indirectly supported by the finding that even minor transformations (methyl substitution in a
single aromatic ring) of 4MP-TQS, which is an nAChR allosteric agonist, resulted in dramatic
changes in the ligand action. Derivatives of this ligand exhibit different pharmacological effects
on α7 nAChR, from positive to negative, and a silent modulation [77].
Thus, the interactions of lead compound 28 and 37 with α1-containing GABAAR subtypes may
endow these compounds with sedative-hypnotic properties. However, it cannot be ruled out that
the effects of these compounds, like most BZD site ligands, are mediated by their complex
activity at multiple α subunits and the resulting different behavioral effects, including anxiolytic-
like, depend on their affinity/efficacy for modulating activity of different GABAAR isoforms.
The full potential of lead compounds and the exploration of a greater variety of compounds
along with elucidation of their GABAAR selectivity must await additional studies.
19

CONCLUSION
In the present work, we explored the potential of pyridine/1,3-thiazole bioisosteric replacement
in imidazopyridine drugs. Three new classes, imidazo[2,1-b]thiazoles, imidazo[2,1-
b][1,3,4]thiadiazoles, and benzo[d]imidazo[2,1-b]thiazoles, bearing aromatic and amide
substituents were shown to have potency for binding to the GABAAR BZD binding site at a
nanomolar level. A number of benzo[d]imidazo[2,1-b]thiazoles exhibited the affinity for
GABAARs (IC50 = 6 – 12 nM) that is 3 – 7 times higher compared to the reference drug
Zolpidem (IC50 = 43 nM). Active compounds were safe and non-toxic at the maximum attainable
concentrations in water (up to 47 mg/L). Lead compounds potentiate this GABA-induced current
in Purkinje cells with the affinity similar to the estimates for the reference drug Zolpidem in
these neurons. We demonstrated an in vivo biological efficacy of benzo[d]imidazo[2,1-b]thiazole
ligands in behavioral assays using zebrafish as experimental animals. To our delight, the data
obtained by the radioligand competition displacement assay and the results of the “novel tank”
test providing anxiety behavioral responses in zebrafish corroborate each other and, hence, may
be considered to be of a predictive value, allowing a preliminary screening of GABAAR PAMs.
Two groups of active compounds inducing “Zolpidem-alike” and “Zolpidem-like” behavioral
responses were formed. The hit compounds from each group, namely 28 and 37 proved to act
upon the same mechanism as GABAAR PAMs potentiating neurons. The structural bases for
activity of benzo[d]imidazo[2,1-b]thiazoles were proposed using molecular docking studies.
To sum up, 1,3-thiazole bioisosteric replacement in imidazopyridine drugs has a high potential
for the development of new effective and selective GABAAR PAMs. This approach can be
efficient for the elaboration of novel hypnotics, anxiolytics, anticonvulsants, and sedatives.
Imidazo[2,1-b]thiazoles, imidazo[2,1-b][1,3,4]thiadiazoles, and benzo[d]imidazo[2,1-b]thiazoles
constitute new classes of potent GABAAR PAMs. The further exploration of these series will be
aimed at evaluating their activity in mammals and discovering new candidates for the treatment
of central nervous system disorders.
EXPERIMENTAL SECTION
Instruments and Materials
NMR spectra were acquired on Bruker Avance 600 and 300 spectrometers at room temperature;
the chemical shifts δ were measured in ppm relative to the solvent (1Н: CDCl3, δ = 7.27 ppm,
13
DMSO-d6, δ = 2.50 ppm; C: CDCl3, δ = 77.00 ppm, DMSO-d6, δ = 39.50 ppm). Splitting
patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd,
double doublet. The coupling constants (J) are in Hertz. The structures of all compounds were
established using 1D NMR (1H, 13C, JMOD) spectroscopy. Infrared spectra were measured on a
FT-IR spectrometer in KBr pellets. High-resolution and accurate mass spectra were obtained on
Bruker micrOTOF-QTM ESI-TOF (Electrospray Ionization/Time of Flight) and Thermo
Scientific* LTQ Orbitrap mass spectrometers. Melting points (mp) were measured on a Boetius
capillary melting point apparatus and are uncorrected. Analytical thin-layer chromatography
(TLC) was carried out on silica gel plates (silica gel 60 F254 aluminum supported plates); the
20

visualization was accomplished with an UV lamp (365 nm). Column chromatography was
performed on silica gel 60 (60-120 mesh, Merck) or neutral aluminum oxide 0.063 – 0.200 mm
(70 - 230 mesh ASTM). Parent N,N-dipropyl- and N,N-dimethylpropiolamides were prepared
according to the published procedure [78]. All reactions were carried out using freshly distilled
and dry solvents. Purity was determined by HPLC and confirmed by inspection of NMR spectra
(1 H and 13C NMR). HPLC analysis was performed using Agilent 1260 equipped with
autosampler (Agilent Poroshell 120 C18 column (50 mm × 3.0 mm I.D., 2.7 μm); 0.05% TFA in
water/acetonitrile gradient; UV detection at 215 and 254 nm) and electrospray ionization. All test
compounds were of ≥ 95% purity. None of the test compounds contain substructures associated
with pan-assay interfering activities (PAINS) [79] by inspection or by screening for PAINS or
aggregators at http://zinc15.docking.org/patterns/home.
Typical experimental procedure for ethers 1-7, 11-20. A screw-capped V-shaped vial (2.0 mL)
was charged with 2-aminothiazole (0.36 mmol, 1.0 equiv), aldehyde (0.40 mmol, 1.1 equiv),
freshly activated 4Å molecular sieves (300 mg) and dry toluene (1.5 mL). The vial was capped
o
with a pressure cap and stored at 120 C for 14-24 h until complete consumption of 2-
1
aminothiazole (monitored by H NMR). The resulting mixture containing imine was cooled to
room temperature and the solvent was removed under reduced pressure. Under inert atmosphere,
the residue was redissolved in dry toluene (1 mL) and then CuOTf•C6H6 (18 mg, 0.036 mmol, 10
mol %), Cu(OTf)2 (13 mg, 0.036 mmol, 10 mol %), alkyne (0.72 mmol, 2 equiv) and 4Å
molecular sieves (100 mg) were added. The reaction mixture was additionally stirred in the vial
o
capped with a pressure cap at 120 C for 2 h. After the completion of the reaction, the mixture
was filtered through a plug of neutral aluminum oxide (eluent – EtOAc). The filtrate was
concentrated under reduced pressure to give a crude material, which was purified by column
chromatography on silica gel (eluent Et3N:EtOAc/petroleum ether) or neutral aluminum oxide
(eluent EtOAc/petroleum ether) to give product.
Methyl 2-(6-(4-chlorophenyl)imidazo[2,1-b]thiazol-5-yl)acetate (1) [44]. Yield 66% (73 mg),
1
yellow solid, mp 123−124 °C; Rf = 0.68 (petroleum ether/EtOAc, 1:3). H NMR (600 MHz,
CDCl3): δ 7.68 (d, J = 8.78 Hz, 2H), 7.53 (d, J = 4.53 Hz, 1H), 7.43 (d, J = 8.78 Hz, 2H), 6.89
13
(d, J = 4.53 Hz, 1H), 3.94 (s, 2H), 3.79 (s, 3H). C NMR (150 MHz, CDCl3) δ 169.7 (CO),
149.3 (C), 144.4 (C), 133.5 (C), 132.6 (C), 129.0 (2×CH), 128.7 (2×CH), 117.9 (CH), 114.4 (C),
112.8 (CH), 52.7 (CH2), 31.4 (CH3).
Methyl 2-(6-(4-nitrophenyl)imidazo[2,1-b]thiazol-5-yl)acetate (2) [44]. Yield 56% (64 mg),
1
yellow solid, mp 194−195 °C; Rf = 0.54 (hexane/EtOAc, 1:2). H NMR (600 MHz, CDCl3) δ
8.35 (d, J = 8.73 Hz, 2H), 7.97 (d, J = 8.73 Hz, 2H), 7.64 (d, J = 4.51 Hz, 1H), 7.08 (d, J = 4.51
Hz, 1H), 4.03 (s, 2H), 3.83 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 168.9 (CO), 149.6 (C), 147.2
(C), 141.5 (C), 139.0 (C), 128.3 (2×CH), 124.2 (2×CH), 118.1 (CH), 116.3 (CH), 114.8 (C), 53.0
(CH2), 31.4 (CH3).
Ethyl 2-(6-(4-chlorophenyl)imidazo[2,1-b]thiazol-5-yl)acetate (3) [44]. Yield 46% (53 mg),
1
yellow solid, mp 111−112 °C; Rf = 0.22 (petroleum ether - EtOAc, 4:1). H NMR (300 MHz,
CDCl3): δ 7.70 (d, J = 8.81 Hz, 2H), 7.53 (d, J = 4.41 Hz, 1H), 7.44 (d, J = 8.81 Hz, 2H), 6.89
(d, J = 4.41 Hz, 1H), 4.23 (q, J = 7.33 Hz, 2H), 3.91 (s, 2H), 1.30 (t, J = 7.33 Hz, 3H). 13C NMR
21

(75 MHz, CDCl3): δ 169.3 (CO), 149.2 (C), 144.4 (C), 133.3 (C), 132.9 (C), 129.0 (2×CH),
128.9 (2×CH), 117.9 (CH), 114.5 (C), 112.6 (CH), 61.7 (CH2), 31.6 (CH2), 14.3 (CH3).
Ethyl 2-(6-(2,4-dichlorophenyl)imidazo[2,1-b]thiazol-5-yl)acetate (4) [44]. Yield 60% (76 mg),
1
yellow solid, mp 80−82 °C; Rf = 0.22 (petroleum ether - EtOAc, 4:1). H NMR (300 MHz,
CDCl3): δ 7.49−7.50 (m, 3H), 7.32 (dd, J = 2.20, 8.76 Hz, 1H), 6.88 (d, J = 4.40 Hz, 1H), 4.17
(q, J = 7.37 Hz, 2H), 3.76 (s, 2H), 1.25 (t, J = 7.37 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 169.1
(CO), 149.2 (C), 141.7 (C), 134.6 (C), 134.3 (C), 133.4 (CH), 131.7 (C), 129.7 (CH), 127.2
(CH), 118.2 (CH), 116.7 (C), 112.7 (CH), 61.5 (CH2), 31.3 (CH2), 14.1 (CH3).
2-(6-(4-Chlorophenyl)imidazo[2,1-b]thiazol-5-yl)-N,N-dimethylacetamide (5). Yield 46% (53
1
mg), yellow solid, mp 136-138 °C; Rf = 0.33 (petroleum ether - EtOAc, 1:1). H NMR (300
MHz, CDCl3): δ 7.63 (d, J = 4.70 Hz, 1Н), 7.54 (d, J = 8.61 Hz, 2Н), 7.39 (d, J = 8.61 Hz, 2Н),
6.79 (d, J = 4.70 Hz, 1Н), 4.00 (s, 2Н), 2.92 (s, 3Н), 2.83 (s, 3Н). 13C NMR (75 MHz, CDCl3): δ
168.1 (CO), 149.4 (C), 143.2 (C), 133.6 (C), 131.9 (C), 129.2 (2×CH), 128.9 (2×CH), 119.1
(CH), 114.1 (C), 112.5 (CH), 61.2 (CH2), 36.6 (CH3), 36.1 (CH3). IR (KBr) 3433 (w), 2980 (s),
1721 (s), 1582 (s), 1498 (s), 1198 (s), 1014 (s), 739 (w) сm-1. HRMS (ESI/Q-TOF) 320.0606,
calcd. for C15H15ClN3OS+ 320.0619.
Ethyl 2-(6-(4-chlorophenyl)-2-ethylimidazo[2,1-b][1,3,4]thiadiazol-5-yl)acetate (6) [44]. Yield
55% (69 mg), yellow solid, mp 125−127 °C; Rf = 0.32 (petroleum ether - EtOAc, 4:1). 1H NMR
(300 MHz, CDCl3): δ 7.62 (d, J = 8.24 Hz, 2H), 7.38 (d, J = 8.24 Hz, 2H), 4.20 (q, J = 7.32 Hz,
2H), 4.03 (s, 2H), 3.02 (q, J = 7.33 Hz, 2H), 1.41 (t, J = 7.32 Hz, 3H), 1.24 (t, J = 7.33 Hz, 3H).
13C NMR (75 MHz, CDCl3): δ 169.3 (CO), 153.9 (C), 142.5 (C), 133.2 (C), 132.9 (C), 128.7
(2×CH), 128.4 (2×CH), 116.3 (C), 106.7 (C), 61.4 (CH2), 30.7 (CH2), 25.7 (CH2), 14.1 (CH3),
13.1 (CH3).
Ethyl 2-(6-(4-chlorophenyl)-2-isopropylimidazo[2,1-b][1,3,4]thiadiazol-5-yl)acetate (7) [44].
Yield 41% (54 mg), yellow solid, mp 137−138 °C; Rf = 0.32 (petroleum ether - EtOAc, 5:1). 1H
NMR (300 MHz, CDCl3): δ 7.65 (d, J = 8.20 Hz, 2H), 7.40 (d, J = 8.20 Hz, 2H), 4.22 (q, J =
7.24 Hz, 2H), 4.06 (s, 2H), 3.29− 3.38 (m, 1H), 1.46 (s, 3H), 1.44 (s, 3H), 1.25 (t, J = 7.24 Hz,
13
3H). C NMR (75 MHz, CDCl3): δ 170.9 (CO), 169.4 (C), 144.0 (C), 142.4 (C), 133.3 (C),
133.0 (C), 128.8 (2×CH), 128.5 (2×CH), 116.3 (CH), 61.5 (CH2), 32.5 (CH), 30.8 (CH2), 22.3
(2×CH3), 14.2 (CH3).
Ethyl 2-(6-(4-chlorophenyl)-2-(methoxymethyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)acetate (8)
[44]. Yield 35% (46 mg), yellow solid, mp 109−112 °C; Rf = 0.20 (petroleum ether - EtOAc,
5:1). 1H NMR (300 MHz, CDCl3): δ 7.65 (d, J = 8.75 Hz, 2H), 7.41 (d, J = 8.75 Hz, 2H), 4.75 (s,
2H), 4.22 (q, J = 7.28 Hz, 2H), 4.06 (s, 2H), 3.51 (s, 3H), 1.27 (t, J = 7.28 Hz, 3H). 13C NMR (75
MHz, CDCl3): δ 169.1 (CO), 162.8 (C), 144.4 (C), 142.8 (C), 133.5 (C), 132.8 (C), 128.8
(2×CH), 128.6 (2×CH), 116.4 (C), 69.4 (CH2), 61.6 (CH2), 59.2 (CH3), 30.6 (CH2), 14.1 (CH3).
2-(6-(4-сhlorophenyl)-2-ethylimidazo[2,1-b][1,3,4]thiadiazol-5-yl)-N,N-dimethylacetamide (9).
Yield 50% (63 mg), yellow solid, mp 140-142 °C; Rf = 0.32 (petroleum ether - EtOAc, 1:1). 1H
NMR (300 MHz, CDCl3): δ 7.65 (d, J = 8.47 Hz, 2Н), 7.37 (d, J = 8.47 Hz, 2Н), 4.03 (s, 2Н),
22

3.16 (s, 3Н), 2.99-3.03 (m, 5Н), 1.40 (t, J = 7.70 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 168.3
(CО), 165.9 (C), 143.7 (C), 142.5 (C), 133.2 (C), 133.1 (С), 128.7 (2×CH), 128.6 (2×CН), 117.6
(С), 37.6 (CH3), 36.0 (СН3), 29.6 (CH2), 25.8 (СН2), 13.1 (СН3). IR (KBr) 3436 (s), 2918 (s),
1643 (s), 1492 (s), 1396 (s), 1155 (s), 1139 (s), 840 (s) сm-1. HRMS (ESI/Q-TOF) 371.0705,
calcd. for C16H17ClN4OSNa+ 371.0704.
2-(6-(4-сhlorophenyl)-2-ethylimidazo[2,1-b][1,3,4]thiadiazol-5-yl)-N,N-dipropylacetamide (10).
1
Yield 55% (80 mg), yellow solid, mp 89-90 °C; Rf = 0.32 (petroleum ether - EtOAc, 1:1). H
NMR (300 MHz, CDCl3): δ 7.67 (d, J = 8.46 Hz, 2Н), 7.39 (d, J = 8.46 Hz, 2Н), 4.05 (s, 2Н),
3.37 (t, J = 7.41 Hz, 4Н), 3.02 (q, J = 7.23 Hz, 2Н), 1.58-1.72 (m, 4H), 1.42 (t, J = 7.23 Hz, 3Н),
13
0.89-0.99 (m, 6Н). C NMR (75 MHz, CDCl3): δ 168.1 (CО), 165.8 (C), 143.7 (C), 142.5 (C),
133.3 (C), 133.1 (С), 128.8 (2×CH), 128.7 (2×CН), 118.1 (С), 50.1 (СН2), 48.3 (СН2), 29.7
(CH2), 25.8 (СН2), 22.4 (СН2), 21.1 (СН2), 13.1 (CH3), 11.4 (СН3) 11.3 (СН3). IR (KBr) 3428
(s), 2936 (s), 1631 (s), 1494 (s), 1235 (s), 1146 (s), 1088 (s), 813 (s) сm-1. HRMS (ESI/Q-TOF)
405.1511, calcd. for C20H26ClN4OS+ 405.1510.
Ethyl (2-phenylimidazo[2,1-b][1,3]benzothiazol-3-yl)acetate (11) [44]. Yield 46% (56 mg),
1
yellow solid, mp 80-82 °C; Rf = 0.20 (petroleum ether - EtOAc, 5:1). H NMR (300 MHz,
CDCl3): δ 7.70-7.81 (m, 4Н), 7.47 (dd, J = 7.64 Hz, 3Н), 7.31-7.41 (m, 2Н), 4.27 (q, J = 7.12
13
Hz, 2Н), 4.21 (s, 2Н), 1.28 (t, J = 7.12 Hz, 3H). C NMR (75 MHz, CDCl3): δ 169.9 (CО),
147.6 (C), 146.0 (C), 134.0 (C), 133.2 (С), 130.5 (C), 129.1 (2×CH), 128.7 (2×CH), 127.8 (CH),
126.1 (CH) 124.7 (CH), 124.5 (CH), 117.0 (C), 113.1 (CН), 61.8 (CН2), 31.8 (CH2), 14.2 (CH3).
Ethyl [2-(4-methylphenyl)imidazo[2,1-b][1,3]benzothiazol-3-yl]acetate (12) [44]. Yield 52% (66
mg), yellow solid, mp 74-77 °C; Rf = 0.23 (petroleum ether - EtOAc, 4:1). 1H NMR (300 MHz,
CDCl3): δ 7.80 (d, J = 8.05 Hz, 1Н), 7.79 (d, J = 7.53 Hz, 1Н), 7.86 (d, J = 8.05 Hz, 2Н), 7.43
(dd, J = 7.53 Hz, 1Н), 7.36 (dd, J = 8.05 Hz, 1Н), 7.29 (d, J = 8.05 Hz, 2Н), 4.28 (q, J = 7.29 Hz,
13
2Н), 4.21 (s, 2Н), 2.42 (s, 3Н), 1.29 (s, J = 7.29 Hz, 3H). C NMR (75 MHz, CDCl3): δ 170.0
(CО), 147.5 (C), 146.2 (C), 137.5 (C), 133.2 (C), 131.1 (C), 130.5 (С), 129.4 (2×CH), 128.0
(2×CH), 126.0 (CH), 124.5 (CH), 124.4 (CН), 116.7 (C), 113.0 (CH), 62.0 (CН2), 31.9 (CH2),
21.3 (CH3), 14.2 (CH3).
Ethyl [2-(4-methoxyphenyl)imidazo[2,1-b][1,3]benzothiazol-3-yl]acetate (13) [44]. Yield 45%
1
(59 mg), yellow solid, mp 90-92 °C; Rf = 0.32 (petroleum ether - EtOAc, 4:1). H NMR (300
MHz, CDCl3): δ 7.83 (d, J = 8.25 Hz, 1Н), 7.75 (d, J = 8.25 Hz, 1Н), 7.69 (d, J = 8.24 Hz, 2H),
7.46 (dd, J = 7.33, 8.24 Hz, 1Н), 7.27 (dd, J = 7.33, 8.24 Hz, 1Н), 7.02 (d, J = 8.24 Hz, 2Н), 4.28
13
(q, J = 7.32 Hz , 2Н), 4.19 (s, 2Н), 3.87 (s, 3Н), 1.28 (t, J = 7.32 Hz, 3Н). C NMR (75 MHz,
CDCl3): δ 169.9 (CО), 159.6 (C), 134.9 (C), 133.1 (C), 132.4 (C), 130.5 (C), 129.5 (2×СH),
126.3 (CH), 125.6 (C), 124.9 (CH), 124.6 (CH), 116.4 (C), 114.3 (2×CH), 113.1 (CH), 61.9
(CН2), 55.4 (ОCH3), 31.9 (CH2), 14.3 (CH3).
Ethyl [2-(2-methoxyphenyl)imidazo[2,1-b][1,3]benzothiazol-3-yl]acetate (14) [44]. Yield 42%
(55 mg), yellow solid, mp 175-177 °C; Rf = 0.34 (petroleum ether - EtOAc, 4:1). 1H NMR (300
MHz, CDCl3): δ 7.70 (d, J = 8.49 Hz, 1Н), 7.62 (d, J = 8.49 Hz, 2Н), 7.28-7.42 (m, 3Н), 7.07
(dd, J = 7.36 Hz, 1Н), 7.00 (d, J = 8.49 Hz, 1H), 4.20 (q, J = 7.32 Hz, 2Н), 4.04 (s, 2Н), 3.79 (s,
23

3Н), 1.22 (t, J = 7.32 Hz, 3Н). 13C NMR (75 MHz, CDCl3): δ 170.1 (CО), 156.5 (C), 147.3 (C),
142.2 (C), 133.2 (C), 132.0 (CH), 130.5 (C), 129.5 (СH), 126.0 (CH), 124.4 (CH), 124.3 (CH),
123.0 (C), 121.0 (CH), 118.8 (C), 113.0 (CH), 111.3 (CH), 61.3 (CН2), 55.6 (ОCH3), 31.9 (CH2),
14.2 (CH3).
Ethyl [2-(2,4-dimethoxyphenyl)imidazo[2,1-b][1,3]benzothiazol-3-yl]acetate (15) [44]. Yield
1
33% (47 mg), yellow solid, mp 88-90 °C; Rf = 0.31 (petroleum ether - EtOAc, 4:1). H NMR
(300 MHz, CDCl3): δ 7.87 (d, J = 8.07 Hz, 1Н), 7.75 (d, J = 7.34 Hz, 1Н), 7.47 (dd, J = 7.34 Hz,
1Н), 7.30-7.40 (m, 3Н), 6.98 (d, J = 8.07 Hz, 1H), 4.29 (q, J = 7.34 Hz, 2Н), 4.22 (s, 2Н), 3.99
(s, 3Н), 3.95 (s, 3Н), 1.29 (t, J = 7.34 Hz, 3Н). 13C NMR (75 MHz, CDCl3): δ 169.8 (CО), 149.3
(C), 149.2 (C), 147.3 (C), 144.9 (C), 133.0 (C), 130.4 (C), 126.3 (СH), 125.8 (C), 125.0 (CH),
124.9 (CH), 120.4 (CH), 116.5 (C), 113.3 (CH), 111.5 (CH), 111.3 (CH), 61.9 (CН2), 56.1
(ОCH3), 56.0 (ОCH3), 31.9 (CH2), 14.2 (CH3).
Ethyl [2-(1,3-benzodioxol-5-yl)imidazo[2,1-b][1,3]benzothiazol-3-yl]acetate (16) [44]. Yield
1
50% (68 mg), yellow solid, mp 79-81 °C; Rf = 0.32 (petroleum ether - EtOAc, 4:1). H NMR
(300 MHz, CDCl3): δ 7.80 (d, J = 8.07 Hz, 1Н), 7.70 (d, J = 7.34 Hz, 1Н), 7.42 (dd, J = 7.34,
8.07 Hz, 1Н), 7.33 (dd, J = 7.34, 8.07 Hz, 1Н), 7.21-7.28 (m, 2Н), 6.90 (d, J = 8.07 Hz, 1Н),
13
6.01 (s, 2Н), 4.28 (q, J = 7.33 Hz, 2Н), 4.18 (s, 2Н), 1.29 (t, J = 7.33 Hz, 3Н). C NMR (75
MHz, CDCl3): δ 169.9 (CО), 147.9 (C), 147.3 (C), 146.0 (C), 133.1 (C), 130.4 (С), 128.1 (C),
126.0 (CH), 124.5 (CH), 124.4 (CH), 121.8 (CН), 116.4 (C), 112.9 (CH), 110.1 (C), 108.7 (СН),
108.5 (СН), 101.2 (CН2), 61.8 (СН2), 31.8 (CH2), 14.2 (CH3).
Ethyl [2-(4-fluorophenyl)imidazo[2,1-b][1,3]benzothiazol-3-yl]acetate (17) [44]. Yield 48% (61
mg), yellow solid, mp 86-87 °C; Rf = 0.22 (petroleum ether - EtOAc, 5:1). 1H NMR (300 MHz,
CDCl3): δ 7.68-7.81 (m, 4Н), 7.41 (dd, J = 7.76 Hz, 1H), 7.32 (dd, J = 7.76 Hz, 1H), 7.15 (dd, J
= 8.52 Hz, 2Н), 4.28 (q, J = 7.33 Hz, 2Н), 4.16 (s, 2Н), 1.28 (t, J = 7.33 Hz, 3H). 13C NMR (75
MHz, CDCl3): δ 169.8 (CО), 162.5 (d, JC-F = 247.1 Hz, C), 147.5 (C), 145.2 (C), 133.0 (C),
130.3 (C), 130.1 (d, JC-F = 3.2 Hz, C), 129.7 (d, JC-F = 8.0 Hz, 2×CH), 126.1 (CH), 124.6 (CH),
124.4 (CH), 116.7 (C), 115.5 (d, JC-F = 21.4 Hz, 2×CH), 113.0 (СН), 61.8 (CН2), 31.7 (CH2),
14.2 (CH3).
Ethyl 2-(2-(4-chlorophenyl)benzo[d]imidazo[2,1-b]tiazole-3-yl)acetate (18) [44]. Yield 75% (99
1
mg), yellow solid, mp 100-102 °C; Rf = 0.20 (petroleum ether - EtOAc, 5:1). H NMR (300
MHz, CDCl3): δ 7.84 (d, J = 8.07 Hz, 1H), 7.71-7.73 (m, 3H), 7.43-7.48 (m, 3H), 7.37 (dd, J =
7.33, 8.07 Hz, 1H), 4.29 (q, J = 7.33 Hz, 2H), 4.19 (s, 2H), 1.30 (t, J = 7.33 Hz, 3H). 13C NMR
(75 MHz, CDCl3): δ 169.6 (CO), 147.7 (C), 144.9 (C), 133.7 (C), 133.0 (C), 132.2 (С), 130.4
(C), 129.2 (2×CH), 128.8 (2×CH), 126.1 (CH), 124.8 (CH), 124.4 (CH), 117.1 (C), 113.1 (CH),
61.9 (CH2), 31.7 (CH2), 14.2 (CH3).
2-(2-(4-Bromophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (19). Yield 78% (117 mg),
1
white solid, mp 105-109 °C; Rf = 0.20 (petroleum ether - EtOAc, 5:1). H NMR (300 MHz,
CDCl3): δ 7.84 (d, J = 8.15 Hz, 1H), 7.73 (d, J = 8.15 Hz, 1H), 7.67 (d, J = 8.11 Hz, 2H), 7.60
(d, J = 8.11 Hz, 2H), 7.46 (dd, J = 6.67, 8.15 Hz, 1H), 7.37 (dd, J = 6.67, 8.15 Hz, 1H), 4.29 (q, J
13
= 7.26 Hz, 2H), 4.19 (s, 2H), 1.30 (t, J = 7.26 Hz, 3H). C NMR (75 MHz, CDCl3): δ 169.7
24

(CO), 147.8 (C), 145.0 (C), 133.1 (C), 132.9 (C), 131.9 (2×СH), 130.6 (C), 129.6 (2×CH), 126.2
(CH), 124.8 (CH), 124.5 (CH), 121.9 (C), 117.2 (C), 113.1 (CH), 62.0 (CH2), 31.8 (CH2), 14.2
(CH3). IR (KBr) 3347 (w), 2361 (s), 2343 (s), 1736 (s), 1498 (s), 1370 (s), 1193 (s), 847 (s), 738
(s) сm-1. HRMS (ESI/Q-TOF) 415.0098, calcd. for C19H16BrN2O2S+ 415.0110.
Ethyl [7-chloro-2-(4-chlorophenyl)imidazo[2,1-b][1,3]benzothiazol-3-yl]acetate (20) [44]. Yield
40% (58 mg), yellow solid, mp 188-190 °C; Rf = 0.32 (petroleum ether - EtOAc, 5:1). 1H NMR
(300 MHz, CDCl3): δ 7.77 (d, J = 8.24 Hz, 1Н), 7.68-7.70 (m, 3Н), 7.40-7.45 (m, 3Н), 4.27 (q, J
13
= 7.32 Hz, 2Н), 4.14 (s, 2Н), 1.29 (t, J = 7.32 Hz, 3Н). C NMR (75 MHz, CDCl3): δ 169.5
(CО), 147.5 (C), 145.4 (C), 133.9 (C), 132.2 (С), 131.9 (C), 131.6 (С), 130.4 (СН), 129.2
(2×CH), 128.8 (2×CН), 126.4 (СН), 124.2 (СН), 117.3 (С), 113.9 (СН), 62.0 (CН2), 31.6 (CH2),
14.2 (CH3).
Typical experimental procedure for amides 21-42. Potassium hydroxide (67 mg, 1.20 mmol, 2.0
equiv) in ethanol (1 mL) was added to a solution of ethyl 2-benzo[d]imidazo[2,1-b]tiazole-3-yl
acetate (0.60 mmol, 1.0 equiv) in ethanol (10 mL). The reaction mixture was stored at rt for 10
min until the complete conversion of starting material (TLC monitoring). Next, the solvent was
removed under reduced pressure, residue was dissolved in water (5 mL) and then glacial acetic
acid (0.8 mL) was added. Precipitate formed was filtered, dried under reduced pressure at rt, and
suspended in CH2Cl2 (5 mL). Phosphorus pentachloride (125 mg, 0.60 mmol, 1.0 equiv) was
o
added in portion. Resulting mixture was heated at 40 C for 40 min and stored at rt overnight.
The flow of ammonia gas was passed through reaction mixture over 30 min (for compounds 21-
35) or alkyl amine (0.60 mmol, 1.0 equiv) was added in one portion with following stirring at rt
for 60 min (for compounds 36-42). The resulting mixture was washed with ice-water (20 mL),
and was extracted with CH2Cl2 (3×10 mL). Organic layer was dried over Na2SO4, and
concentrated in vacuo. The crude product was purified by column chromatography using
petroleum ether/ethyl acetate or petroleum ether/EtOAc, 1:2 eluent.
2-(2-Phenylbenzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (21). Yield 74% (136 mg), white
solid, mp 243-245 °C (mplit[40] 246-248 °C); Rf = 0.21 (petroleum ether - EtOAc, 1:1). 1H NMR
(300 MHz, DMSO-d6): δ 8.06 (d, J = 8.07 Hz, 1H), 7.84-7.87 (m, 2H), 7.68 (d, J = 8.07 Hz, 2H),
7.35-7.57 (m, 6H), 4.12 (s, 2H). 13C NMR (75 MHz, DMSO-d6): δ 170.4 (CO), 146.3 (C), 144.3
(C), 134.1 (C), 132.7 (C), 129.2 (С), 128.5 (2×CH), 127.3 (2×CH), 127.2 (CH), 126.3 (CH),
124.9 (CH), 124.7 (CH), 119.5 (C), 113.5 (CH), 31.7 (CH2). IR (KBr) 3438 (w), 2365 (s), 2345
(s), 1676 (s), 1497 (s), 1388 (s), 1073 (s), 745 (s), 703 (s) сm-1. HRMS (ESI/Q-TOF) 308.0850,
calcd. for C17H14N3OS+ 308.0852.
2-(2-(p-Tolyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (22). Yield 65% (125 mg), white
solid, mp 252-253 °C (mplit[40] 253-254 °C); Rf = 0.20 (petroleum ether - EtOAc, 1:1). 1H NMR
(300 MHz, DMSO-d6): δ 8.04 (d, J = 8.28 Hz, 1H), 7.82-7.85 (m, 2H), 7.50-7.57 (m, 3H), 7.42
(dd, J = 7.21, 8.28 Hz, 1H), 7.34 (br.s, 1H), 7.28 (d, J = 8.19 Hz, 2H), 4.09 (s, 2H), 2.36 (s, 3H).
13C NMR (75 MHz, DMSO-d6): δ 170.6 (CO), 144.5 (C), 136.5 (C), 132.7 (C), 131.4 (С), 129.2
(2×CH), 129.1 (C), 129.0 (C), 127.3 (2×CH), 126.3 (CH), 124.9 (CH), 124.6 (CH), 119.1 (C),
113.4 (CH), 31.8 (CH2), 20.8 (CH3). IR (KBr) 3437 (w), 2361 (s), 2342 (s), 1679 (s), 1508 (s),
25

1488 (s), 1388 (s), 1370(s), 1110 (s), 753 (s), сm-1. HRMS (ESI/Q-TOF) 322.1013, calcd. for
C18H16N3OS+ 322.1009.
2-(2-(4-Methoxyphenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (23). Yield 71% (144
mg), white solid, mp 285-287 °C (mplit[40] 285-287 °C); Rf = 0.23 (petroleum ether - EtOAc,
1:1). 1H NMR (300 MHz, DMSO-d6): δ 8.04 (d, J = 8.22 Hz, 1H), 7.83-7.85 (m, 2H), 7.60 (d, J
= 8.82 Hz, 2H), 7.53 (dd, J = 7.22, 8.22 Hz, 1H), 7.42 (dd, J = 7.22, 8.22 Hz, 1H), 7.32 (br.s,
13
1H), 7.06 (d, J = 8.82 Hz, 2H), 4.08 (s, 2H), 3.82 (s, 3H). C NMR (75 MHz, DMSO-d6): δ
170.6 (CO), 158.6 (C), 145.6 (C), 144.3 (C), 132.7 (C), 129.2 (С), 128.6 (2×CH), 126.7 (C),
126.3 (CH), 124.9 (CH), 124.5 (CH), 118.6 (C), 114.0 (2×CH), 113.3 (CH), 55.1 (CH3), 31.8
(CH2). IR (KBr) 3306 (w), 2362 (s), 2343 (s), 1677 (s), 1509 (s), 1374 (s), 1254 (s), 1176 (s),
1038 (s), 830 (s), 739 (s) сm-1. HRMS (ESI/Q-TOF) 338.0945, calcd. for C18H16N3O2S+
338.0958.
2-(2-(2-Methoxyphenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (24). Yield 75% (152
mg), white solid, mp 264-265 °C; Rf = 0.23 (petroleum ether - EtOAc, 1:1). 1H NMR (300 MHz,
DMSO-d6): δ 8.01 (d, J = 7.74 Hz, 1H), 7.75 (d, J = 7.74 Hz, 1H), 7.35-7.50 (m, 4H), 7.09 (d, J
= 8.05 Hz, 1H), 7.02 (dd, J = 6.85, 7.74 Hz, 1H), 4.05 (s, 2H), 3.66 (s, 3H) (the signal of the NH2
13
was not observed). C NMR (75 MHz, DMSO-d6): δ 169.9 (CO), 155.4 (C), 132.1 (C), 130.9
(CH), 128.9 (CH), 128.6 (С), 125.9 (CH), 125.8 (C), 124.3 (CH), 124.2 (CH), 122.1 (C), 120.0
(CH), 118.7 (C), 113.3 (C), 113.2 (CH), 111.0 (CH), 55.7 (CH3), 30.4 (CH2). IR (KBr) 3372 (w),
2369 (s), 2340 (s), 1669 (s), 1497 (s), 1108 (s), 834 (s), 796 (s) сm-1. HRMS (ESI/Q-TOF)
338.0957, calcd. for C18H16N3O2S+ 338.0957.
2-(2-(2,4-Dimethoxyphenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (25). Yield 63% (139
mg), white solid, mp 261-263 °C; Rf = 0.25 (petroleum ether - EtOAc, 1:1). 1H NMR (300 MHz,
DMSO-d6): δ 8.03 (d, J = 7.33 Hz, 1H), 7.84-7.87 (m, 2H), 7.53 (dd, J = 6.61, 8.13 Hz, 1H),
7.42 (dd, J = 6.61, 8.13 Hz, 1H), 7.36 (s, 1H), 7.27 (br.s, 1H), 7.18 (d, J = 8.13 Hz, 1H), 7.06 (d,
13
J = 8.13 Hz, 1H), 4.11 (s, 2H), 3.83 (s, 3H), 3.82 (s, 3H). C NMR (75 MHz, DMSO-d6): δ
170.8 (CO), 148.7 (C), 148.3 (C), 144.5 (C), 132.8 (C), 129.3 (С), 127.0 (C), 126.3 (CH), 124.9
(2×CH), 124.6 (CH), 119.6 (CH), 118.8 (C), 113.5 (CH), 111.9 (CH), 111.3 (C), 55.6 (CH3),
55.4 (CH3), 31.9 (CH2). IR (KBr) 3409 (w), 2389 (s), 2340 (s), 1663 (s), 1494 (s), 1094 (s), 835
(s), 799 (s) сm-1. HRMS (ESI/Q-TOF) 368.1061, calcd. for C19H18N3O3S+ 368.1063.
2-(2-(2-Chlorophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (26). Yield 59% (121 mg),
white solid, mp 203-205 °C (mplit[40] 244-246 °C); Rf = 0.28 (petroleum ether - EtOAc, 1:1). 1H
NMR (300 MHz, DMSO-d6): δ 8.06 (d, J = 8.06 Hz, 1H), 7.79 (d, J = 7.33 Hz, 1H), 7.46-7.65
(m, 7H), 3.87 (s, 2H). 13C NMR (75 MHz, DMSO-d6): δ 170.2 (CO), 145.7 (C), 142.2 (C), 133.0
(C), 132.7 (C), 132.6 (СH), 132.5 (CH), 129.9 (C), 129.8 (CH), 129.2 (CH), 127.1 (C), 126.3
(CH), 124.92 (2×CH), 121.0 (C), 113.8 (CH), 31.4 (CH2). IR (KBr) 3442 (w), 1727 (s), 1499 (s),
1376 (s), 1256 (s), 1080 (s), 1035 (s), 828 (s), 743 (s) сm-1. HRMS (ESI/Q-TOF) 342.0462,
calcd. for C17H13ClN3OS+ 342.0452.
2-(2-(3-Chlorophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (27). Yield 78% (160 mg),
white solid, mp 243-245 °C (mplit[40] 242-245 °C); Rf = 0.28 (petroleum ether - EtOAc, 1:1). 1H
26

NMR (600 MHz, DMSO-d6): δ 8.06 (d, J = 7.34 Hz, 1H), 7.87-7.93 (m, 2H), 7.73 (s, 1H), 7.65
13
(d, J = 7.34 Hz, 1H), 7.40-7.58 (m, 5H), 4.15 (s, 2H). C NMR (75 MHz, DMSO-d6): δ 170.3
(CO), 146.3 (C), 142.9 (C), 136.2 (C), 133.4 (C), 132.6 (С), 130.5 (CH), 129.4 (C), 127.1 (CH),
126.9 (CH), 126.4 (CH), 125.7 (CH), 125.0 (2×CH), 120.3 (C), 113.6 (CH), 31.7 (CH2). IR
(KBr) 3440 (s), 1675 (s), 1599 (s), 1493 (s), 1368 (s), 1318 (s), 1078 (s), 1032 (s), 755 (s) сm-1.
HRMS (ESI/Q-TOF) 342.0461, calcd. for C17H13ClN3OS+ 342.0462.
2-(2-(4-Chlorophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (28). Yield 61% (125 mg),
white solid, mp 285-287 °C (mplit[40] 288-290 °C); Rf = 0.27 (petroleum ether - EtOAc, 1:1). 1H
NMR (300 MHz, DMSO-d6): δ 8.03 (d, J = 8.15 Hz, 1H), 7.85 (d, J = 7.68 Hz, 2H), 7.68 (d, J =
7.68 Hz, 2H), 7.51-7.54 (m, 3H), 7.42 (dd, J = 6.99, 8.15 Hz, 1H), 7.32 (br.s, 1H), 4.10 (s, 2H).
13C NMR (75 MHz, DMSO-d6): δ 170.3 (CO), 144.0 (C), 143.7 (C), 133.5 (C), 133.1 (C), 132.3
(C), 129.3 (С), 128.9 (2×CH), 128.6 (2×CH), 126.3 (CH), 124.9 (CH), 124.8 (C), 120.3 (C),
113.5 (CH), 31.7 (CH2). IR (KBr) 3343 (s), 1668 (s), 1497 (s), 1395 (s), 1092 (s), 835 (s), 743 (s)
см-1. HRMS (ESI/Q-TOF) 342.0454, calcd. for C17H13ClN3OS+ 342.0462.
2-(7-Chloro-2-(4-chlorophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (29). Yield 56%
(126 mg), white solid, mp 240-242 °C; Rf = 0.25 (petroleum ether - EtOAc, 1:1). 1H NMR (300
MHz, DMSO-d6): δ 8.26 (br.s, 1H), 7.86 (d, J = 7.33 Hz, 2H), 7.69 (d, J = 7.33 Hz, 2H), 7.59-
7.63 (m, 1H), 7.53-7.56 (m, 2H), 7.36 (br.s, 1H), 4.11 (s, 2H). IR (KBr) 3368 (w), 2361 (s), 2343
(s), 1670 (s), 1499 (s), 1296 (s), 1100 (s), 1012 (s), 833 (s), 804 (s) сm-1. HRMS (ESI/Q-TOF)
376.0059, calcd. for C17H12Cl2N3OS+ 376.0072.
2-(2-(4-Chlorophenyl)-7-methoxybenzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (30). Yield 47%
(105 mg), white solid, mp 275-278 °C; Rf = 0.20 (petroleum ether - EtOAc, 1:1). 1H NMR (300
MHz, DMSO-d6): δ 7.92 (br.s, 1H), 7.79 (d, J = 8.79 Hz, 1H), 7.70 (m, 3H), 7.52 (d, J = 8.79
13
Hz, 2H), 7.29 (br.s, 1H), 7.10 (dd, J = 2.47, 8.95 Hz, 1H), 4.09 (s, 2H), 3.85 (s, 3H). C NMR
(75 MHz, DMSO-d6): δ 170.3 (CO), 156.6 (C), 142.6 (C), 133.2 (C), 131.7 (С), 130.7 (C), 128.8
(2×CH), 128.6 (3×CH), 126.7 (C), 119.7 (C), 114.2 (C), 113.4 (CH), 109.4 (CH), 55.8 (CH3),
31.6 (CH2). IR (KBr) 3449 (w), 1674 (s), 1501 (s), 1401 (s), 1225 (s), 829 (s), 806 (s) сm-1.
HRMS (ESI/Q-TOF) 372.0560, calcd. for C18H15ClN3O2S+ 372.0568.
2-(2-(2,4-Dichlorophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (31). Yield 90% (203
mg), white solid, mp 237-239 °C; Rf = 0.27 (petroleum ether - EtOAc, 1:1). 1H NMR (300 MHz,
DMSO-d6): δ 8.05 (d, J = 8.24 Hz, 1H), 7.81 (d, J = 8.24 Hz, 1H), 7.76 (s, 1H), 7.66 (br.s, 1H),
7.50-7.53 (m, 3H), 7.43 (dd, J = 7.43, 8.24 Hz, 1H), 7.20 (br.s, 1H), 3.88 (s, 2H). 13C NMR (75
MHz, DMSO-d6): δ 170.0 (CO), 145.8 (C), 141.1 (C), 134.0 (C), 133.7 (CH), 133.5 (C), 132.5
(С), 131.8 (C), 129.3 (CH), 129.2 (C), 127.3 (CH), 126.3 (CH), 124.8 (2×CH), 121.4 (C), 113.8
(CH), 31.3 (CH2). IR (KBr) 3350 (w), 2362 (s), 2344 (s), 1671 (s), 1493 (s), 1389 (s), 1314 (s),
1125 (s), 1028 (s), 830 (s), 744 (s) сm-1. HRMS (ESI/Q-TOF) 376.0055, calcd. for
C17H12Cl2N3OS+ 376.0072.
2-(2-(3,4-Dichlorophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (32). Yield 68% (153
mg), white solid, mp 230-232 °C; Rf = 0.27 (petroleum ether - EtOAc, 1:1). 1H NMR (300 MHz,
DMSO-d6): δ 8.07 (d, J = 7.33 Hz, 1H), 7.90-7.92 (m, 3H), 7.75 (d, J = 8.06 Hz, 1H), 7.67 (d, J
27

= 8.06 Hz, 1H), 7.56 (dd, J = 7.33 Hz, 1H), 7.46 (dd, J = 7.33 Hz, 1H), 7.39 (s, 1H), 4.15 (s, 2H).
IR (KBr) 3433 (w), 1727 (s), 1495 (s), 1366 (s), 1254 (s), 1088 (s), 1030 (s), 828 (s), 743 (s) сm-
1. HRMS (ESI/Q-TOF) 376.0006, calcd. for C17H12Cl2N3OS+ 376.0072.
2-(2-(4-Fluorophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (33). Yield 84% (163 mg),
white solid, mp 266-268 °C (mplit[40] 268-270 °C); Rf = 0.21 (petroleum ether - EtOAc, 1:1). 1H
NMR (300 MHz, DMSO-d6): δ 8.05 (d, J = 7.33 Hz, 1H), 7.87 (d, J = 8.79 Hz, 2H), 7.71 (dd, J
= 5.60, 8.79 Hz, 2H), 7.54 (dd, J = 6.58, 7.33 Hz, 1H), 7.43 (dd, J = 6.58, 7.33 Hz, 1H), 7.29-
7.35 (m, 3H), 4.10 (s, 2H). 13C NMR (75 MHz, CDCl3): δ 170.4 (CО), 162.0 (d, JC-F = 244.0 Hz,
C), 146.3 (C), 144.0 (C), 133.1 (C), 131.1 (d, JC-F = 3.0 Hz, C), 129.9 (C), 129.8 (d, JC-F = 8.1
Hz, 2×CH), 126.1 (CH), 125.5 (CH), 125.2 (CH), 119.9 (C), 115.5 (d, JC-F = 21.4 Hz, 2×CH),
113.9 (СН), 31.1 (CH2). IR (KBr) 3347 (w), 3149 (s), 2375 (s), 2345 (s), 1668 (s), 1508 (s),
1489 (s), 1396 (s), 1225 (s), 1158 (s), 839 (s), 744 (s) сm-1. HRMS (ESI/Q-TOF) 326.0749,
calcd. for C17H13FN3OS+ 326.0758.
2-(2-(4-Bromophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (34). Yield 76% (176 mg),
white solid, mp 214-216 °C (mplit[41] 214-215 °C); Rf = 0.23 (petroleum ether - EtOAc, 1:1). 1H
NMR (300 MHz, DMSO-d6): δ 8.05 (d, J = 7.67 Hz, 1H), 7.92 (br.s, 1H), 7.88 (d, J = 7.67 Hz,
1H), 7.62-7.69 (m, 4H), 7.54 (dd, J = 7.67 Hz, 1H), 7.44 (dd, J = 7.67 Hz, 1H), 7.35 (br.s, 1H),
13
4.12 (s, 2H). C NMR (75 MHz, DMSO-d6): δ 170.3 (CO), 152.3 (C), 152.2 (C), 143.2 (C),
133.4 (C), 132.6 (C), 131.5 (2×СH), 129.2 (2×CH), 126.4 (CH), 124.9 (CH), 124.8 (CH), 120.4
(C), 120.0 (C), 113.6 (CH), 31.7 (CH2). IR (KBr) 3360 (w), 3152 (w), 2388 (s), 2347 (s), 1666
(s), 1498 (s), 1397 (s), 1071 (s), 831 (s), 743 (s) сm-1. HRMS (ESI/Q-TOF) 387.9931, calcd. for
C17H13BrN3OS+ 387.9937.
2-(2-(4-(Trifluoromethyl)phenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (35). Yield 81%
(182 mg), white solid, mp 229-230 °C; Rf = 0.20 (petroleum ether - EtOAc, 1:1). 1H NMR (300
MHz, DMSO-d6): δ 8.07 (d, J = 7.33 Hz, 1H), 7.86-7.95 (m, 4H), 7.84 (d, J = 8.79 Hz, 2H), 7.56
13
(dd, J = 7.33 Hz, 1H), 7.46 (dd, J = 7.33 Hz, 1H), 7.39 (br.s, 1H), 4.18 (s, 2H). C NMR (75
MHz, DMSO-d6): δ 170.2 (CO), 146.5 (C), 142.8 (C), 138.2 (C), 132.5 (C), 129.4 (С), 127.7
(2×CH), 127.5 (q, JC-F = 33.1 Hz, С), 127.1 (CH), 125.5 (CH), 125.5 (CH), 125.0 (2×CH), 122.5
(q, JC-F = 395.0 Hz, CF3), 121.0 (C), 113.7 (CH), 31.7 (CH2). IR (KBr) 3350 (w), 2363 (s), 2344
(s), 1670 (s), 1496 (s), 1330 (s), 1167 (s), 1112 (s), 1068 (s), 743 (s) сm-1. HRMS (ESI/Q-TOF)
376.0718, calcd. for C18H13F3N3OS+ 376.0726.
N,N-Dimethyl-2-(2-(p-tolyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)acetamide (36). Yield 32% (42
mg), yellow solid, mp 234-236 °C (mplit[40] 238-239 °C); Rf = 0.20 (petroleum ether - EtOAc,
1
1:1). H NMR (300 MHz, CDCl3): δ 7.66 (d, J = 7.84 Hz, 1H), 7.61 (d, J = 7.84 Hz, 1H), 7.50
(d, J = 8.07 Hz, 2H), 7.38 (dd, J = 7.84 Hz, 1H), 7.24-7.31 (m, 3Н), 4.19 (s, 2H), 3.10 (s, 3H),
13
3.05 (s, 3H), 2.40 (s, 3H). C NMR (75 MHz, CDCl3): δ 168.8 (CO), 147.3 (C), 145.7 (C),
137.2 (С), 133.3 (C), 131.5 (C), 130.3 (C), 129.3 (2×СН), 128.0 (2×СН), 126.0 (CH), 124.3
(CH), 124.1 (CH), 118.0 (C), 113.2 (CH), 37.5 (CH3), 36.1 (CH3), 30.9 (CH2), 21.3 (CH3). IR
(KBr) 3424 (s), 2925 (s), 1645 (s), 1509 (s), 1487 (s), 1394 (s), 1138 (s), 824 (s), 743 (s) cm-1.
HRMS (ESI/Q-TOF) 350.1314, calcd. for C20H20N3OS+ 350.1322.
28

2-(2-(2-Chlorophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)-N,N-dimethylacetamide (37). Yield
30% (40 mg), yellow solid, mp 154-156 °C (mplit[40] 159-162 °C); Rf = 0.32 (petroleum ether -
1
EtOAc, 1:1). H NMR (300 MHz, CDCl3): δ 7.74 (d, J = 8.07 Hz, 1H), 7.68 (d, J = 8.07 Hz,
1H), 7.52-7.54 (m, 1H), 7.49-7.50 (m, 1H), 7.40 (dd, J = 7.36, 8.44 Hz, 1Н), 7.30-7.34 (m, 3Н),
13
4.04 (s, 2H), 2.98 (s, 3H), 2.96 (s, 3H). C NMR (75 MHz, CDCl3): δ 168.5 (CO), 147.5 (C),
142.8 (C), 133.8 (С), 133.4 (C), 133.2 (C), 132.9 (CН), 130.4 (С), 129.9 (CH), 129.5 (CH),
126.9 (CH), 126.0 (CH), 124.5 (CH), 124.1 (CH), 120.0 (C), 113.8 (CH), 37.4 (CH3), 36.0
(CH3), 30.9 (CH2). IR (KBr) 3432 (s), 3054 (s), 2927 (s), 1644 (s), 1491 (s), 1396 (s), 1313 (s),
1140 (s), 1037 (s), 760 (s) cm-1. HRMS (ESI/Q-TOF) 370.0762 calcd. for C19H17ClN3OS+
370.0775.
2-(2-(3-Chlorophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)-N,N-dimethylacetamide (38). Yield
30% (40 mg), yellow solid, mp 151-152 °C (mplit[40] 156 °C); Rf = 0.32 (petroleum ether -
EtOAc, 1:1). 1H NMR (300 MHz, CDCl3): δ 7.67 (d, J = 7.89 Hz, 1H), 7.58-7.60 (m, 2H), 7.48
(dd, J = 7.64, 9.20 Hz, 1H), 7.29-7.39 (m, 4Н), 4.19 (s, 2H), 3.10 (s, 3H), 3.07 (s, 3H). 13C NMR
(75 MHz, CDCl3): δ 168.4 (CO), 147.7 (C), 144.2 (C), 136.2 (С), 134.5 (C), 133.2 (C), 130.5
(С), 129.9 (CH), 128.0 (CH), 127.5 (CH), 126.1 (2×CH), 124.6 (CH), 124.3 (CH), 118.9 (C),
113.3 (CH), 37.5 (CH3), 36.1 (CH3), 30.7 (CH2). IR (KBr) 3431 (w), 2921 (s), 1645 (s), 1495 (s),
1396 (s), 1140 (s, 795 (s), 747 (s), 713 (s) сm-1. HRMS (ESI/Q-TOF) 370.0764, calcd. for
C19H17ClN3OS+ 370.0775.
2-[2-(4-Chlorophenyl)imidazo[2,1-b][1,3]benzothiazol-3-yl]-N,N-dimethylacetamide (39) [44].
Yield 61% (81 mg), yellow solid, mp 234-236 °C; Rf = 0.32 (petroleum ether - EtOAc, 1:1). 1H
NMR (300 MHz, CDCl3): δ 7.68 (d, J = 8.12 Hz, 1H), 7.53-7.58 (m, 3H), 7.37-7.42 (m, 3H),
13
7.31 (dd, J = 7.33, 8.12 Hz, 1Н), 4.17 (s, 2H), 3.15 (s, 3H), 3.07 (s, 3H). C NMR (75 MHz,
CDCl3): δ 168.4 (CO), 147.6 (C), 144.5 (C), 133.4 (C), 133.1 (C), 132.8 (C), 130.5 (C), 129.4
(2×CH), 128.8 (2×CH), 126.1 (CH), 124.6 (CH), 124.3 (CH), 118.5 (C), 113.1 (CH), 37.5 (CH3),
36.1 (CH3), 30.8 (CH2).
2-(2-(2,4-Dichlorophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)-N,N-dimethylacetamide
(40).
Yield 30% (72 mg), yellow solid, mp 147-146 °C; Rf = 0.35 (petroleum ether - EtOAc, 1:1). 1H
NMR (300 MHz, CDCl3): δ 7.64-7.70 (m, 2H), 7.52 (d, J = 2.01 Hz, 1H), 7.47 (d, J = 8.24 Hz,
1H), 7.39 (dd, J = 7.36, 8.24 Hz, 1Н), 7.31-7.34 (m, 2Н), 4.01 (s, 2H), 3.03 (s, 3H), 2.97 (s, 3H).
13C NMR (75 MHz, CDCl3): δ 168.3 (CO), 147.7 (C), 141.8 (C), 134.7 (С), 134.5 (C), 133.77
(CН), 133.3 (C), 131.9 (С), 130.5 (C), 129.7 (CH), 127.3 (CH), 126.1 (CH), 124.7 (CH), 124.2
(CH), 120.2 (C), 113.7 (CH), 37.5 (CH3), 36.0 (CH3), 30.9 (CH2). IR (KBr) 3432 (s), 2923 (w),
1650 (s), 1493 (s), 1393 (s), 1127 (s), 824 (s), 744 (s), 727 (с) сm-1. HRMS (ESI/Q-TOF)
404.0377, calcd. for C19H16Cl2N3OS+ 404.0386.
2-(2-(4-Chlorophenyl)benzo[d]imidazo[2,1-b]thiazol-3-yl)-N,N-diethylacetamide (41). Yield
95% (226 mg), yellow solid, mp 229-231 °C; Rf = 0.28 (petroleum ether - EtOAc, 1:1). 1H NMR
(300 MHz, CDCl3): δ 7.64 (d, J = 7.33 Hz, 1H), 7.54 (d, J = 7.33 Hz, 1H), 7.52 (d, J = 8.07 Hz,
2H), 7.38 (d, J = 8.07 Hz, 2H), 7.24-7.34 (m, 2Н), 4.12 (s, 2Н), 3.37-3.48 (m, 4Н), 1.12-1.23 (m,
13
6Н). C NMR (75 MHz, CDCl3): δ 167.4 (CО), 147.3 (C), 144.2 (C), 133.1 (C), 132.9 (C),
132.8 (C), 130.2 (С), 129.1 (2×CH), 128.6 (2×CH), 125.8 (CH), 124.4 (CH), 124.1 (CH), 118.7
29