Structure-Function Evaluation of Imidazopyridine Derivatives Selective for δ-Subunit-Containing γ-Aminobutyric Acid Type A (GABAA) Receptors

Article abstract of DOI:10.1021/acs.jmedchem.7b01484

δ-Selective compounds 1 and 2 (DS1, compound 22; DS2, compound 16) were introduced as functionally selective modulators of δ-containing GABA type A receptors (GABA_AR). In our hands, [³H]EBOB-binding experiments with recombinant GABA_AR and compound 22 showed no proof of δ-selectivity, although there was a minimally higher preference for the α4β3δ and α6β2/3δ receptors with respect to potency. In order to delineate the structural determinants of δ preferences, we synthesized 25 derivatives of DS1 and DS2, and investigated their structure-activity relationships (SAR). Four of our derivatives showed selectivity for α6β3δ receptors (29, 38, 39, and 41). For all of them, the major factors that distinguished them from compound 22 were variations at the para-positions of their benzamide groups. However, two compounds (29 and 39), when tested in the presence of GABA, revealed effects at several additional GABA_AR. The newly synthesized compounds will still serve as useful tools to investigate α6β3δ receptors.

Full text of DOI:10.1021/acs.jmedchem.7b01484

Subscriber access provided by UNIVERSITY OF THE SUNSHINE COAST
Article
Structure-function evaluation of imidazopyridine derivatives selective
A
for # subunit containing #-aminobutyric acid type A (GABA) receptors
Kirsten Yakoub, Sascha Jung, Christian Sattler, Helen Damerow, Judith
Weber, Annika Kretzschmann, Aylin S. Cankaya, Markus Piel, Frank Rösch,
Anne S. Haugaard, Bente Frølund, Tanja Schirmeister, and Hartmut Lüddens
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01484 • Publication Date (Web): 16 Feb 2018
Downloaded from http://pubs.acs.org on February 17, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Structure-function evaluation of imidazopyridine derivatives
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
selective for δ subunit containing γ-aminobutyric acid type A
(
GABA ) receptors
A
a#
b#
a
b
c
Kirsten Yakoub , Sascha Jung , Christian Sattler , Helen Damerow , Judith Weber , Annika
c
c
c
c
d
Kretzschmann , Aylin S. Cankaya , Markus Piel , Frank Rösch , Anne S. Haugaard , Bente
d
b*
a*
Frølund , Tanja Schirmeister , Hartmut Lüddens
a
Institutes Dept. of Psychiatry and Psychotherapy, University Medical Center Mainz, Faculty of Health and Medical
b
c
Sciences, Dꢀ55131 Mainz, Germany; Dept. of Pharmacy & Biochemistry and Dept. of Nuclear Chemistry,
d
Johannes Gutenberg University Mainz, Dꢀ55128 Mainz, Germany; Dept. of Drug Design and Pharmacology,
University of Copenhagen, DKꢀ2100 Copenhagen, Denmark
GABA receptors; δꢀselectivity; DS1; DS2; structureꢀactivity relationship
A
ABSTRACT: Delta selective compounds 1 and 2 (DS1, compound 22; DS2, compound 16) were introduced as functionally
3
selective modulators of δꢀcontaining GABA receptors type A (GABA R). In our hands [ H]EBOB binding experiments with
A
recombinant GABA R using compound 22, showed no proof of δꢀselectivity, though a minimally higher preference with respect
A
to potency for α4β3δ and α6β2/3δ receptors. In order to delineate the structural determinant for δꢀpreference, we synthesized 25
derivatives of DS1 and DS2, and investigated their structureꢀactivity relationship (SAR). Four of our derivatives showed
selectivity for α6β3δ receptors (29, 38, 39, 41). All of them possess a variation at the paraꢀposition of the benzamide group as the
major difference to compound 22. However, two compounds (29, 39), when tested in the presence of GABA, revealed an effect
at several additional GABA R. The newly synthesized compounds will still serve as useful tools to investigate α6β3δ receptors.
A
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
INTRODUCTION
The main inhibitory neurotransmitter system in the brain, the γꢀaminobutyric acid (GABA) system, with its predominant
GABA receptors type A (GABA R) system is a target for several molecules, including benzodiazepines, neurosteroids,
A
1ꢀ6
barbiturates, propofol, volatile anesthetics and, putatively, ethanol. Their action leads to anxiolytic, sedative, memoryꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
modifying, anticonvulsant, as well as hypnotic effects.
2
GABA R assemble as heteropentameric complexes from a variety of subunits (α1ꢀ6, β1ꢀ3, γ1ꢀ3, δ, ε, θ, π and ρ1ꢀ3). There are
A
two different forms of inhibition mediated by GABA R. The subunit composition differs with different synaptic localization as
A
well as type of inhibition mediated. Synaptic receptors, typically composed of two α, two β and one γ subunit, lead to phasic
7
inhibition, whereas tonic inhibition is mediated mostly by periꢀ and extrasynaptic δꢀcontaining receptors. These receptors are
characterized by their enhanced GABA sensitivity and reduced desensitization properties and can be regarded as prototypic
7
extrasynaptic receptors at ambient GABA concentrations. Extrasynaptic GABA R containing the δ subunit are found in the
A
8ꢀ9
cerebellum, thalamus, olfactory bulb, cortex and hippocampus, where the δ subunit is frequently coꢀdistributed with α4 and α6
9
10ꢀ11
subunits. The GABA R subtypes α6β2/3δ are expressed at high levels exclusively by mature cerebellar granule neurons.
In
A
forebrain areas, e.g., in thalamic relay cells, neostriatum, dentate gyrus and some layers of the cortex, δ subunits are combined
1
2ꢀ14
15
with α4 and β2/3 subunits.
The existence of α4β1δ receptors is so far only postulated and still under investigation. In
16
hippocampal interneurons tonic inhibition seems to be also conveyed via α1βδ receptors. The major fraction of nonꢀ δꢀ
7
, 17
containing receptors found in areas other than synapses may be constituted by α5βγ2ꢀtype receptors in the hippocampus.
18ꢀ19
In contrast to γ2ꢀcontaining GABA R, which follow a strict arrangement order,
δꢀcontaining receptors may assemble less
A
20ꢀ22
stringently.
Although the occurrence of δꢀcontaining GABA R in different brain areas is well documented, the coꢀassembly
A
and composition of these receptors, native or recombinant, as well as their detailed function is not completely understood. These
receptors may be linked to several disorders like alcoholism, stroke, depression, epilepsy, schizophrenia and traumatic brain
23ꢀ24
injury.
In addition to the occurrence of δꢀcontaining GABA R in the brain, δ subunit protein and mRNA was detected in the
A
25ꢀ27
lamina II tissue of the spinal cord, where they could play a role in analgesia.
Taken together, the uncertainties of δꢀcontaining GABA R concerning physiological processes, pharmacological interactions,
A
assembly order and partnering subunits demand novel pharmacological tools to differentiate these receptors from other subtypes
in vitro and in vivo. Although many ligands, such as general anesthetics, neurosteroids and gaboxadol show enhanced potency at
14
δꢀcontaining GABA R, especially when partnered with α4/6 and β3 subunits, their selectivity is insufficient for detailed
A
investigations. Later, the imidazopyridines DS1 and DS2 (Delta Selective compounds 1 and 2) were introduced by Wafford et al.
2
ACS Paragon Plus Environment

Page 3 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
in 2009. In electrophysiological experiments, using human recombinant GABA R, the authors showed δꢀselectivity for both
A
28
compounds in combination with α4 and β3 subunits. Later experiments revealed a GABAꢀenhancing effect of DS2 at additional
29
δꢀcontaining receptors (α1β2δ, α4β1/2/3δ, α6β2δ), while numerous other GABA R remained unaffected. However, the site of
A
29
action for DS1 and DS2 is still unknown.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
In the present study we used [ H]ethynylbicycloorthobenzoate ([ H]EBOB) binding to rat recombinant GABA R as an assay
A
3
for the modulatory effect on GABA R function of the studied compounds. [ H]EBOB as well as tꢀ
A
35
35
[
S]butylbicyclophosphorothionate ([ S]TBPS), both so called “cage convulsants”, are potent nonꢀcompetitive GABA R
A
30ꢀ31
3
antagonists binding to the picrotoxinin binding site within the GABA R ion channel.
The binding of [ H]EBOB and
A
35
[ S]TBPS is sensitive to conformational changes in the chloride channel, e.g. being reduced by increasing concentrations of the
3
2ꢀ35
agonist GABA.
This effect is potentiated by modulators such as benzodiazepines and neurosteroids, or in turn, reversed by
33, 35ꢀ39
3
35
inverse agonists.
Allosteric modulation of [ H]EBOB or [ S]TBPS binding is also shown in the absence of GABA,
3
2, 35, 38ꢀ40
indicating a direct effect on receptor conformation.
The correlation between allosteric modulation of GABA R binding of the cage convulsants and ionophore function has been
A
3
5
32, 41ꢀ43
3
31, 33, 40
shown numerous times, mostly for [ S]TBPS,
but also for [ H]EBOB.
The pharmacological profile of allosteric
35
3
31, 34ꢀ35
agents for [ S]TBPS and [ H]EBOB binding modulation is very similar and therefore comparable.
3
In initial [ H]EBOB binding experiments we experienced that DS1 (compound 22, Figure 1) recognizes receptor subtypes
additional to α6βδ. In electrophysiological experiments this was also shown for DS2 (compound 16, Figure 1), although, in both
cases, with higher effects on δꢀcontaining receptors. Thus, we set out to identify the pharmacophore of these δꢀpreferring
candidates. A systematic structureꢀactivity relationship (SAR study) was performed by synthesis of 25 derivatives of DS1 and
DS2 (Figure 1), followed by their characterization in radioligand binding assays employing the ability of these ligands to
3
modulate the [ H]EBOB binding.
3
We identified important positions in the chemical scaffold of DS1/DS2 for modulation of [ H]EBOB binding and provide
evidence that the α6 subunit is more critical than the δ subunit. Our SAR study shows that replacing the chloride of the original
3
DS1 molecule by a fluorinated or nonꢀfluorinated butoxy group led to novel, now truly δꢀselective modulators of [ H]EBOB
binding using rat recombinant GABA R.
A
RESULTS AND DISCUSSION
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Chemistry. The synthesis of Nꢀ[2ꢀarylimidazo[1,2ꢀa]pyridinꢀ3ꢀyl]amides (16–36, 41–44) was typically achieved in a twoꢀstep
procedure. In the first step, the respective 2ꢀarylꢀimidazo[1,2ꢀa]pyridinꢀ3ꢀamines (1–11) were synthesized by multicomponent
reactions starting from commercially available 2ꢀaminoꢀpyridines and aromatic aldehydes, cf. Scheme 1.
Amines 1–7 were synthesized using a modified version of the multicomponent reaction reported by van Niel et al. for the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
44
preparation of 8ꢀmethylꢀ2ꢀthiophenꢀ2ꢀylꢀimidazo[1,2ꢀa]pyridineꢀ3ꢀylamine (Method A, Scheme 1). The aldehyde is converted
into the bisulfite adduct with Na S O and then refluxed with the respective 2ꢀaminoꢀpyridine to form the corresponding Schiff
2
2
5
base. Addition of KCN to the reflux followed by slowly cooling to room temperature precipitated the desired amines, which were
collected and further purified (yield range 41ꢀ68%). Another version of this reaction was used for the synthesis of amines 10 and
11 (Method B, Scheme 1). Here, the reaction was performed in ethanol/water with added BiCl to enhance the electrophilicity of
3
the aldehyde. However, amine 8 could not be derived with the above procedures. The double bromination of the 2ꢀaminopyridine
starting material likely results in reduced nucleophilicity of the amine group and poor solubility in aqueous solution. In a first
attempt, amine 8 was synthesized in a mixture of water/ethanol (2.25/1) and refluxing for several hours after KCN addition
(Method C, Scheme 1), but yields ranged between 9% and 17%. Therefore, the approach by Bienaymé et al. was envisaged for
4
5
this compound (Method D, Scheme 1). Bienaymé et al. use isocyanides instead of KCN to form secondary 2ꢀarylꢀimidazo[1,2ꢀ
a]pyridinꢀ3ꢀamines in methanol at room temperature. The use of tertꢀbutyl isocyanide should allow acidic removal of the tertꢀ
butyl group afterwards. The tertꢀbutyl protected intermediate was isolated in an acceptable yield (52%) and could be deprotected
quantitatively to the desired amine 8 using 5 M hydrobromic acid. Amine 9 was obtained from 6 and 1,3ꢀdibromobutane in a
nucleophilic substitution step using K CO as base (Method E, Scheme 1).
2
3
The second step in the reaction sequence was the amide bond formation (Scheme 2). The amines 1ꢀ11 were reacted with the
respective acid chlorides in a mixture of toluene and pyridine at room temperature giving the desired Nꢀ[2ꢀarylimidazo[1,2ꢀ
a]pyridinꢀ3ꢀyl]amides (16–36, 41–44) in good yields (60ꢀ80%). Attempts to form the amide bond via standard coupling protocols
(HBTU/TBTU, PPA, IBCF) failed due to the low nucleophilicity of the aromatic amine. Compounds with a free hydroxyl group
(37, 40) were synthesized either from the acetoxy protected precursor 30 or from the TBDPS protected compound 36 (Scheme 3).
Removal of the acetoxy group was accomplished with 5 M NaOH in THF at room temperature. Cleavage of the TBDPS group
was achieved with a 1 M solution of tetrabutyl ammonium fluoride. Both reactions provided the desired products in excellent
yields (88ꢀ92%). Compound 37 was used for further derivatization. It was reacted in typical Williamson ether syntheses to form
products 38 and 39 with a terminal fluorine as analogues to compound 29 (Scheme 4).
4
ACS Paragon Plus Environment

Page 5 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Screening for δ direct acting DS derivatives. In order to identify the structural characteristics of a directly acting, δꢀselective
3
[
H]EBOB modulator on GABA R, the binding assay was initially executed in the absence of GABA. Promising candidates were
A
then tested in the presence of GABA, thus mimicking a more physiological condition.
At first, the compounds were tested on α6β3δ GABA R, one of the two δꢀcontaining receptors in cerebellar granule cells of the
A
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
10ꢀ11
mammalian brain, the other being the α6β2δ receptor.
If no [³H]EBOB binding modulation in concentrations up to 10 µM
was observed the compounds were not further analyzed. Promising candidates displaying effects on α6β3δ GABA R (Table 1,
A
data highlighted in bold) were tested at α1ꢀ, α4ꢀ and α6ꢀcontaining receptors, as in vivo δ subunits only occur together with these
11, 13, 15ꢀ16
three α subunits in addition to any of the three β variants.
StructureꢀActivity Relationship. All studied compounds possess an imidazopyridine moiety as the core structure. The lead
compounds 16 and 22 were modified at three sites, i.e., the thiophene group, the chlorinated benzamide group and the bromo
substituents at the pyridine moiety to investigate the structureꢀactivity relationship. A broad range of derivatives (16ꢀ21, 23ꢀ27,
30, 32ꢀ33, 40, 42ꢀ44), including the published compound 16 could be dismissed after the first testing on α6β3δ GABA R as they
A
showed no effect in the absence of GABA.
Compounds lacking the thiophene group (44) or having it replaced by a phenyl (17, 19), a pꢀmethoxyphenyl (24), a
3
bromopropoxyphenyl (25), a furanyl (42) or a methylthiophene group (43) showed no [ H]EBOB binding modulation. This
3
indicates an essential role for the thiophene group for [ H]EBOB binding modulation.
Compounds with less than two bromide atoms (16, 18, 29, 33) showed no or little activity, while their doubleꢀbrominated
counterparts (22, 31) were potent modulators with IC50 values of 0.31 and 0.86 µM, respectively (Table 1). The bromination state
3
seems to play a major part in the potency of [ H]EBOB binding modulation (see section below). Replacement of a bromide atom
by a chloride atom (27, 32) led to loss of activity, indicating the crucial role of this site.
The third site in the core scaffold of DS1 investigated in more detail was the benzamide group, in particular the paraꢀposition
of the benzamide group. Compound 26, possessing an acetamide group instead of the benzamide group showed no effect,
indicating that the benzamide group is indeed crucial for action. Interestingly, even with only one bromide substituent, compound
3
2
8 with a cyclohexylcarboxamide group instead of the benzamide group was able to modulate [ H]EBOB binding, but only with
a negligible potency (Table 1, IC50 = 10.1 ± 2.8 µM). For the group of analogs where only the paraꢀposition of the benzamide
group is varied, a fluoro atom (35) but neither a methoxy (23) nor a fluoroethoxy (34) group is tolerated. However, elongating the
carbon chain to a propoxy (38) or butoxy (29, 39) group, fluorinated in two cases (38, 39), seem to be beneficial for activity and
3
selectivity for δꢀcontaining GABA R in [ H]EBOB binding assays (Table 1). Compound 41 bears a benzoxy group at this
A
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
position and showed the lowest modulatory effect of the δꢀselective compounds with no measurable IC50 value and an observable
effect only at 10 µM (max. inhibition = 54 ± 11%), which may simply due to absence of bromide atoms. For the rest of the active
compounds, 31, 35 and 22 no δꢀselectivity but a preference for most δꢀcontaining GABA R as revealed by their differential
A
potency (Table 1) was observed. However, in case of compound 31 these effects were not significant.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The detailed SAR analysis revealed that a structural scaffold consisting of an imidazopyridine with at least one bromo
substituent in the pyridine part and a thiophene and benzamidine group connected to the imidazo part were crucial for direct
3
modulation of [ H]EBOB binding. The chloro substituent in the paraꢀposition of the benzamide group proved to be exchangeable
3
by specific moieties, but was still important for [ H]EBOB binding modulation and partly responsible for δꢀselectivity.
3
Modulation of [ H]EBOB binding by compounds 22 and 16 and comparison with electrophysiological data. As we
primarily searched for direct modulators, compound 16 was dismissed as a candidate for further investigation as no direct
3
[
H]EBOB modulation was observed (max. inhibition = 20 ± 10 % at 10 µM). In contrast, compound 22 showed a robust
3
modulation of recombinant δꢀcontaining GABA R in [ H]EBOB binding experiments (Table 1). The assumption of a direct
A
effect by compound 22 and a GABAꢀmodulatory effect of compound 16 is in agreement with previously published,
2
8
3
electrophysiological data. However, in [ H]EBOB binding assays with additional recombinant GABA R, we experienced a
A
preference, but no selectivity for δꢀcontaining receptors in terms of potency (Table 1, Figure 2). Comparison of α4β3δ with other
α4ꢀcontaining receptors showed increased IC50 values of 7ꢀfold (p < 0.005) for α4β3γ2 and 4ꢀfold for α4β3 receptors (difference
nearly reaching significance with p = 0.051) (Figure 2B). In case of α6ꢀ and β2ꢀcontaining receptors the increase was 4ꢀ and 7ꢀ
fold (p < 0.005 for α6β2 and α6β2γ2) compared to α6β2δ GABA R (Figure 2D). The smallest difference in potency was
A
observed for α6ꢀ and β3ꢀcontaining receptors with only a 2.5ꢀ to 3ꢀfold shift compared to α6β3δ receptors (p < 0.005 for α6β3, p
< 0.05 for α6β3γ2) (Figure 2C). Although the potency of compound 22 for δꢀcontaining GABA R was for the most part
A
significantly higher, no differences in efficacy between δꢀ and nonꢀδꢀcontaining receptors could be measured (Table 1, Figure 2).
3
A slight modulation of [ H]EBOB binding in α1ꢀcontaining receptors by 22 was only observed at the highest concentration (10
3
µM) (Figure 2A). Compound 22 was the most potent modulator of [ H]EBOB binding of all tested compounds, however, a 2.5ꢀ7ꢀ
fold preference for δꢀcontaining receptors might be insufficient for investigational use.
3
The results of [ H]EBOB binding modulation by 22 contradict the reported δꢀselectivity of this compound in previously
28
published electrophysiological tests. Still, recent findings from Ahring et al. support our results. These findings also indicate a
lack of selectivity of 22, as the compound modulated the activity of human recombinant α4β2γ2 receptors in the presence of
4
6
GABA, as shown electrophysiologically. In order to allow a better comparison between published electrophysiological data and
3
our findings from [ H]EBOB binding assays, we also performed patchꢀclamp experiments of the lead compounds on rat
6
ACS Paragon Plus Environment

Page 7 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
recombinant GABA R. There is no major publication that hints to crucial differences in the function of human as compared to rat
A
GABA R subunits. Thus, this difference between our and previously reported data cannot be regarded as a potential pitfall.
A
3
Additionally, we were able to further investigate compound 16, which did not show any effect in [ H]EBOB binding modulation.
3
This approach supported our results from [ H]EBOB binding modulation, as compound 16 was unable to sufficiently activate
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
recombinant GABA R in the absence of GABA (Table 2, Figure 3A). Even at a concentration of 10 µM, 16 only slightly
A
activated α4/6β3 and α4/6β3δ GABA R. In the presence of a GABA EC , compound 16 modulated α1/4/6β3δ receptors, as well
A
20
2
8
as α6β3 ones (Table 3). These results confirm the assumption of compound 16 being solely a positive allosteric modulator, but
2
9
3
also challenge the reported δꢀselectivity. Consistent with the [ H]EBOB binding data compound 22 induced robust currents in
the patchꢀclamp experiment in the absence of GABA (Table 2, Figure 3B). However, no δꢀselectivity was observed as the
3
compound was highly efficient at α4/6βδ receptors, as well as at α6β3 and α6β3γ2. The only discrepancy to [ H]EBOB binding
modulation data is the lack of activation of α4β3 and α4β3γ2 receptors in electrophysiological experiments. In the presence of a
GABA EC , the effect of compound 22 on not δꢀcontaining receptors was even more pronounced (Table 3). Especially the
20
binary receptors α1/4/6β3 exhibited similar EC50 values as the corresponding δꢀcontaining ones.
δꢀselectivity of compound 29 and effect of bromo substituents on potency and efficacy. Compound 29 with its butoxy
moiety attached to the benzamide group showed selectivity for α6β2/3δ receptors with IC50 values of 2.18 ± 0.40 µM and 2.43 ±
0.78 µM, respectively with no measurable potency on α6β2/3 or α6β2/3γ2 receptors (Figure 4A,B). However, a marginal
3
modulation of α6β3γ2 receptors could be observed at a concentration of 10 µM (10 ± 4%). [ H]EBOB binding in GABA R
A
containing α1 and α4 subunits was unaffected (Table 1).
Compound 31 represents the double brominated derivative of 29. Compared to compound 29 it showed an increased potency
and efficacy on α6β3δ receptors (IC50 = 0.86 ± 0.30 µM; max. inhibition = 71 ± 7%). However, in contrast to 29 it modulated the
3
[
H]EBOB binding of other α6ꢀ and β3ꢀcontaining receptors (Table 1). Compound 33 as the nonꢀbrominated derivative of 29 was
devoid of any activity. The dissimilar results for these three compounds, only differing in the bromination state, underline its
crucial role in the action on GABA R.
A
With the butoxy moiety not being exclusively responsible for δꢀselectivity and bromination probably being a key factor for
potency, the selectivity of compound 29 could be explained by concentration, obscuring the effect on other receptors in the low
3
micromolar range. Nevertheless, in [ H]EBOB binding assays compound 29 remains selective up to 20 µM which approaches the
solubility limit. This notion is further supported by the loss of selectivity of this compound in the presence of GABA (see section
below).
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
Effect of GABA on δꢀselectivity of DS derivatives 22, 29 and 39. [ H]EBOB binding experiments were performed in the
3
presence of GABA for three compounds (22, 29, 39). Surprisingly, the potent effect of compound 22 on [ H]EBOB binding on
nearly all α6ꢀ and β3ꢀcontaining receptors tested was independent of GABA in the test solution (Table 4). Similar findings have
28
previously been described in electrophysiological experiments of 22 at α4β3δ receptors.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compounds 29 and 39 were both selective for α6β3δ receptors in comparison to the α6β3 and α6β3γ2 ones in the absence of
GABA. However, both lost their selectivity in the presence of GABA (Table 4). While the modulatory effect on α6β3 and
α6β3γ2 receptors was enhanced in the presence of GABA, no significant effect on α6β3δ receptor modulation was observed,
neither in potency nor in efficacy. In the presence of GABA, compound 29 modulated α6β3 receptors to a maximal effect of 26 ±
6% and α6β3γ2 receptors to 40 ± 5%, compound 39 modulated α6β3 receptors to a maximal effect of 57 ± 11% and α6β3γ2
receptors to 44 ± 7%. The effect of GABA on the efficacy of both compounds was highly significant (Table 4).
The fact that both compounds modulate additional receptors in presence of GABA may be explained in several ways. One
possibility is that GABA activates receptors and leads to an IC50 shift and thus visualizes an effect that is obscured in its absence.
That is a reasonable possibility for low affinity ligands like 29 where a further shift in potency between α6β3δ receptors and other
α6ꢀ and β3ꢀcontaining receptors would result in IC50 values approaching the solubility limit. Alternatively or additionally, GABA
changes the receptor conformation in such a way that it enables the compounds to enter the now available binding site. As a third
option, selective compounds may bind to α6β3 and α6β3γ2 receptors in the absence of GABA but lack the ability to directly
activate these receptors, thus they are positive allosteric modulators solely increasing the affinity for GABA. Differentiating
between these possibilities was beyond the scope of this series of experiments.
Question of binding site and binding properties. The modulatory effect of the DS derivatives is clearly dependent on the α
variant of GABA R. Receptors containing α6 subunits are preferred by compound 22 over α4ꢀcontaining ones with regard to
A
potency (Table 1). However, this effect was only significant for α4/6β3γ2 receptors (p = 0.008). The δꢀselective compounds 29
3
and 39 exclusively modulated α6ꢀcontaining receptors (Table 1). None of the tested compounds was able to modulate [ H]EBOB
binding to α1ꢀcontaining receptors. The β subunit seems to play a minor role, though for compound 39 selectivity for δꢀ
containing receptors was only observed in combination with the β3 variant (Table 1, Figure 4C,D). For this compound the
potency for α6β2δ receptors (0.27 ± 0.02 µM) was increased 3ꢀfold (p < 0.005) compared to α6β3δ receptors (0.76 ± 0.08 µM).
For compound 22 the effect was similar (0.15 ± 0.03 µM vs. 0.31 ± 0.04 µM) with a 2ꢀfold higher (p < 0.05) potency on α6β2δ
receptors. No significant difference could be measured for the α6β2/3 (p = 0.185) and α6β2/3γ2 (p = 0.486) receptor pairs.
Further, compound 22 did not differentiate between β2/3ꢀcontaining receptors regarding its efficacy (Table 1). Compound 29 did
not distinguish between α6β2/3δ receptors, neither in potency (p = 0.812) nor in efficacy (p = 0.615). The significantly (p < 0.05)
8
ACS Paragon Plus Environment

Page 9 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
increased efficacy of compound 39 for α6β2 receptors (33 ± 4%) in comparison to α6β3 receptors (11 ± 6%), and the shift in
potency for δꢀcontaining receptors by compounds 22 and 39, indicates a slight influence of the β subunit on modulatory
properties of the mentioned compounds.
3
Compounds 22, 28, 35, 31 modulated [ H]EBOB binding also in the absence of a δ subunit, but with a preference for most δꢀ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
containing receptors (Table 1), however, these effects were not significant for compound 31. Compounds 29, 38, 39, 41 showed
selectivity in the absence of GABA for α6β3δ receptors and in the case of 29 for α6β2δ receptors as well (Table 1, Figure 4).
These results lead to the question whether nonꢀselective compounds address the same, an additional or a completely different
binding site as δꢀselective compounds. An additional binding site would explain the loss of selectivity for certain substances, with
one binding site located on the δ subunit and an additional one located on another part of the receptor complex. The two binding
site model could as well extend the three possibilities to explain the differences of selectivity in the absence and presence of
GABA, i.e., one binding site being GABAꢀmimetic and thus enabling access to the modulatory site.
Regarding the high efficacy (84 ± 8%) and potency (0.31 ± 0.04 µM) of compound 22 in comparison to compound 29 (46 ±
9
%; 2.18 ± 0.40 µM) at α6β3δ receptors, the question of a correlation between potency and efficacy arises. After comparing
measurements of several compounds at α6β3δ receptors this assumption can be dismissed. Compound 35 has a high efficacy (87
9%), combined with a modest potency (1.20 ± 0.24 µM). An even stronger phenotype is observed for compound 28, where
±
potency is especially low (10.1 ± 2.8 µM) but efficacy is quite high (85 ± 14%). On the contrary, compound 39 showed high
potency (0.76 ± 0.08 µM), but only modest efficacy (53 ± 12%). Thus, potency and efficacy of the compounds for modulating
GABA R seem to be independently influenced by the structural composition of the molecules.
A
CONCLUSION
28
Compound 22 was originally published as a δꢀselective GABA R modulator. In our hands this compound did not show
A
selectivity but a significant preference for δꢀcontaining receptors, which caused us to investigate the SAR and further optimize
the structure to gain a selective derivative.
3
Four of the synthesized compounds stood out because they selectively and directly modulated the [ H]EBOB binding of δꢀ
containing GABA R. These compounds have comparable alkyl groups in paraꢀposition of the benzamide group, which is the
A
major difference to compound 22 possessing a chloro substituent at this position. Two of these compounds (29, 39) were further
3
investigated because of their promising modulatory effect on [ H]EBOB binding. Both compounds showed selectivity in the
absence of GABA for α6β3δ receptors, compound 29 additionally for α6β2δ receptors. Besides the δ subunit selectivity, both
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compounds also showed α6ꢀselectivity. In the presence of GABA, compounds 29 and 39 lost their δꢀselectivity and modulated
other α6ꢀ and β3ꢀcontaining receptors.
Since no correlation between potency and efficacy of the compounds for α6β3δ receptors could be observed the structural
determinants are apparently different and have to be investigated by more detailed SAR studies.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Altogether we identified two promising candidates, compounds 29 and 39. Especially with regard to the in vitro investigation
of recombinant δꢀcontaining GABA R, these compounds may help to further investigate the assembly of α6β2/3δ receptors.
A
EXPERIMENTAL SECTION
Syntheses.
General.
All reagents and solvents were of analyticalꢀgrade quality and purchased from SigmaꢀAldrich, Alfa Aesar, Acros, or TCI.
Commercial chemicals were used without further purification, unless otherwise noted. Solvents were purified by distillation and
1
13
desiccated by standard methods if necessary. H and C spectra were recorded on a Bruker Fourier ACꢀ300, on a Bruker Avance
1
19
4
00 MHz spectrometer equipped with a 5 mm PABBO BB) H, F) ZꢀGRD probe or a Bruker Avance 600 MHz spectrometer
13
1
equipped with a cryogenically cooled 5 mm CPDCH C( H) ZꢀGRD probe at 300 K using TMS as internal standard. DMSOꢀd ,
6
CDCl or CD OD were used as solvent. Chemical shifts δ are given in parts per million (ppm) using residual proton peaks of the
3
3
1
13
solvent as reference ( H / C: DMSO: 2.50 / 39.52 ppm, CHCl : 7.26 / 77.16 ppm, MeOH: 3.31 / 49.00 ppm). Mass spectra were
3
recorded on an ESIꢀMicromass LCT from Micromass. High resolution mass spectra were obtained on a Waters QꢀTOFꢀUltima 3
instrument or on a Thermo QExactive Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with an APꢀ
SMALDI 10 ion source (TransmitMIT, Gießen, Germany) and operated at mass resolving power 140,000@m/z200 in positive ion
mode with MALDI ionization. FDꢀMS was measured on a Finnigan MAT 95 instrument. Purity of the compounds was
determined using analytical reverseꢀphase HPLC and was > 95% for all compounds. Analytical HPLC was executed with a
2
50*4.6 mm Kromasil 100 C18 7µm column from MZꢀAnalysentechnik. The pump was a PKꢀ2080 from Jasco. The detector was
a UVꢀ2075 Plus from Jasco. Detection wavelength was λ = 280 nM. The flow rate was 1 mL/min. Analytical HPLC was also
performed on a MerckꢀHitachi HPLC system consisting of an Lꢀ7100 pump, an Lꢀ7200 autosampler, and an Lꢀ7400 UV detector
(254 nm), using an Chromolith SpeedROD RPꢀ18 column (4.6 * 50 mm) eluted at a flow rate of 4.0 mL/min. A linear gradient
elution was performed with eluent A (H O/TFA 100:0.1) containing 0% of solvent B (MeCN/H O/TFA, 90:10:0.01) rising to
2
2
1
00% of B during 5 min. Column chromatography was performed with silica gel (0.06 – 0.02 mm or 0.040 – 0.063 mm)
10
ACS Paragon Plus Environment

Page 11 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
purchased from MachereyꢀNagel or Merck. All reactions were monitored by thinꢀlayer chromatography using MachereyꢀNagel
ALUGRAM Xtra SIL G/UV254 silica gel 60 plates for detection at 254 nm.
Syntheses of 2ꢀarylꢀimidazo[1,2ꢀa]pyridinꢀ3ꢀamines.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General Procedure.
0.015 mol (0.5 eq.) Na S O were dissolved in 80 mL of distilled water. Then 0.03 mol (1.0 eq.) of the respective aldehyde
2
2
5
was added and the mixture stirred for 30 min at rt. After addition of 0.03 mol (1.0 eq.) of the respective 2ꢀaminoꢀpyridine, the
resulting solution was refluxed for 2 h. The solution was allowed to cool to 80 °C before 0.03 mol (1.0 eq.) KCN were added in
one portion. After stirring for 1 h at 80 °C, the mixture was cooled to rt and stirred for additional 3 h. In the following, the
mixture was cooled with an ice bath and the precipitate was collected by vacuum filtration. The residue was washed several times
with cold water and once with 10 mL cooled (– 18 °C) ethyl acetate. Recrystallization of the crude product from H O/EtOH 4:1
2
yielded the respective 2ꢀarylꢀimidazo[1,2ꢀa]pyridinꢀ3ꢀamines (1–7) as yellow solids.
2ꢀ(Thiophenꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀamine (1)
Synthesized following the general procedure using 2.85 g Na S O5, 3.36 g thiopheneꢀ2ꢀcarbaldehyde, 2.82 g 2ꢀaminoꢀpyridine
2
2
1
and 1.95 g KCN. Yield: 4.4 g (68%). R (ethyl acetate/petroleum ether 1:1) = 0.26. H NMR (300 MHz, DMSOꢀd )
δ
[ppm] =
f
6
8
9
.19 (dt, J = 6.9, 1.1 Hz, 1H), 7.55 (dd, J = 3.6, 1.0 Hz, 1H), 7.46 – 7.29 (m, 2H), 7.11 (dd, J = 5.1, 3.6 Hz, 1H), 7.03 (ddd, J =
13
.1, 6.7, 1.2 Hz, 1H), 6.83 (td, J = 6.7, 1.1 Hz, 1H), 5.28 (s, 2H). C NMR (75 MHz, DMSOꢀd6)
δ [ppm] = 138.8, 138.5, 127.8,
125.5, 123.6, 123.2, 122.4, 121.9, 121.8, 116.2, 111.0.
4
7
2
ꢀPhenylimidazo[1,2ꢀa]pyridinꢀ3ꢀamine (2)
Synthesized following the general procedure using 2.85 g Na S O , 3.18 g benzaldehyde, 2.82 g 2ꢀaminoꢀpyridine and 1.95 g
2
2
5
1
KCN. Yield: 4.1 g (65%). R (ethyl acetate/petroleum ether 1:3) = 0.47. H NMR (300 MHz, DMSOꢀd )
δ
[ppm] = 8.24 (d, J =
f
6
6.9 Hz, 1H), 8.05 (d, J = 8.0 Hz, 2H), 7.46–7.36 (m, 3H), 7.23 (t, J = 7.3 Hz, 1H), 7.04 (t, J = 6.7 Hz, 1H), 6.83 (t, J = 6.7 Hz,
1
3
1
1
H), 5.17 (s, 2H). C NMR (75 MHz, DMSOꢀd6) δ [ppm] = 138.8, 135.2, 128.3, 127.3, 126.5, 126.1, 125.9, 122.5, 121.8, 116.6,
10.8.
6
ꢀBromoꢀ2ꢀ(thiophenꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀamine (3)
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Synthesized following the general procedure using 2.85 g Na S O , 3.36 g thiopheneꢀ2ꢀcarbaldehyde, 5.19 g 2ꢀaminoꢀ5ꢀ
2
2
5
1
bromoꢀpyridine and 1.95 g KCN. Yield: 4.9 g (56%). R (ethyl acetate/petroleum ether 1:1) = 0.65. H NMR (300 MHz, DMSOꢀ
f
d )
δ [ppm] = 8.50 (s, 1H), 7.55 (d, J = 3.4 Hz, 1H), 7.42 (d, J = 5.0 Hz, 1H), 7.37 (d, J = 9.5 Hz, 1H), 7.18–7.03 (m, 2H), 5.49
6
13
(
s, 2H). C NMR (75 MHz, DMSOꢀd6)
δ
[ppm] = 138.2, 136.7, 127.9, 126.2, 124.2, 124.0, 123.5, 122.2, 122.2, 117.3, 105.2.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
48
6
ꢀBromoꢀ2ꢀphenylimidazo[1,2ꢀa]pyridinꢀ3ꢀamine (4)
Synthesized following the general procedure using 2.85 g Na S O , 3.18 g benzaldehyde, 5.19 g 2ꢀaminoꢀ5ꢀbromoꢀpyridine
2
2
5
1
and 1.95 g KCN. Yield: 4.5 g (52%). R (ethyl acetate/petroleum ether 1:1) = 0.62. H NMR (300 MHz, DMSOꢀd )
δ
[ppm] =
f
6
8
.54 (s, 1H), 8.02 (d, J = 7.7 Hz, 2H), 7.53–7.35 (m, 3H), 7.24 (t, J = 7.3 Hz, 1H), 7.12 (dd, J = 9.5, 1.5 Hz, 1H), 5.38 (s, 2H).
13
C NMR (75 MHz, DMSOꢀd6) δ [ppm] = 136.9, 134.7, 128.4, 127.6, 127.3, 126.1, 124.2, 122.3, 117.7, 105.1.
6ꢀBromoꢀ2ꢀ(4ꢀmethoxyphenyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀamine (5)
Synthesized following the general procedure using 2.85 g Na S O , 4.08 g 4ꢀmethoxyꢀbenzaldehyde, 5.19 g 2ꢀaminoꢀ5ꢀbromoꢀ
2
2
5
1
pyridine and 1.95 g KCN. Yield: 3.9 g (41%). R (ethyl acetate/petroleum ether 1:1) = 0.51. H NMR (300 MHz, DMSOꢀd )
δ
f
6
[
ppm] = 8.50 (s, 1H), 7.97 (d, J = 8.7 Hz, 2H), 7.38 (d, J = 9.4 Hz, 2H), 7.10 (d, J = 5.5 Hz, 1H), 6.99 (d, J = 8.7 Hz, 2H), 5.20
1
3
(
s, 2H), 3.79 (s, 3H). C NMR (75 MHz, DMSOꢀd6)
δ [ppm] = 157.9, 136.9, 128.6, 127.5, 127.2, 126.0, 123.9, 122.2, 117.4,
113.8, 104.9, 55.1.
4
ꢀ(3ꢀAminoꢀ6ꢀbromoimidazo[1,2ꢀa]pyridinꢀ2ꢀyl)phenol (6)
Synthesized following the general procedure using 2.85 g Na S O , 3.66 g 4ꢀhydroxybenzaldehyde, 5.19 g 2ꢀaminoꢀ5ꢀbromoꢀ
2
2
5
1
pyridine and 1.95 g KCN. Yield: 3.7 g (43%). R (ethyl acetate/petroleum ether 1:1) = 0.16. H NMR (300 MHz, DMSOꢀd )
δ
f
6
[ppm] = 9.45 (s, 1H), 8.48 (s, 1H), 7.85 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 9.4 Hz, 1H), 7.09 (d, J = 9.4 Hz, 1H), 6.82 (d, J = 8.5
1
3
Hz, 2H), 5.13 (s, 2H). C NMR (75 MHz, DMSOꢀd6)
04.8.
δ [ppm] = 156.1, 136.9, 129.2, 127.6, 125.6, 123.7, 122.1, 117.3, 115.2,
1
6
ꢀChloroꢀ2ꢀ(thiophenꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀamine (7)
Synthesized following the general procedure using 2.85 g Na S O , 3.36 g thiopheneꢀ2ꢀcarbaldehyde, 3.86 g 2ꢀaminoꢀ5ꢀ
2
2
5
1
chloroꢀpyridine and 1.95 g KCN. Yield: 4.3 g (58%). R (ethyl acetate/petroleum ether 1:1) = 0.64. H NMR (300 MHz, DMSOꢀ
f
d )
δ
[ppm] = 8.44 (s, 1H), 7.56 (d, J = 3.6 Hz, 1H), 7.48–7.39 (m, 2H), 7.12 (t, J = 8.7 Hz, 1H), 7.04 (d, J = 11 Hz, 1H), 5.50 (s,
6
13
2
H). C NMR (75 MHz, DMSOꢀd6)
δ
[ppm] = 138.2, 136.7, 127.9, 126.4, 124.0, 123.8, 122.19, 122.18, 120.1, 118.3, 117.1.
12
ACS Paragon Plus Environment

Page 13 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Synthesis of 6,8ꢀdibromoꢀ2ꢀ(thiophenꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀamine (8)
Procedure 1. 0.01 mol (0.5 eq.) Na S O were dissolved in 90 mL distilled water and 0.02 mol (1.0 eq.) thiopheneꢀ2ꢀ
2
2
5
carbaldehyde were added. The resulting solution was stirred at rt for 30 min. Then 36 mL EtOH and 0.02 mol (1.0 eq.) 2ꢀaminoꢀ
,5ꢀdibromopyridine were added and the mixture was refluxed for 2 h. 0.02 mol (1.0 eq.) KCN were added in small portions to
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the refluxing mixture. After heating at reflux for 2 h, the mixture was allowed to cool to rt and stirred for additional 3 h. EtOH
was removed under reduced pressure and the remaining aqueous solution was cooled in an ice bath. The precipitate was collected
by vacuum filtration and washed several times with cold water and once with cooled (– 18 °C) ethyl acetate. Pure 8 was obtained
after recrystallization from H O/MeOH 4:1 as brownꢀyellow solid (1.6 g, 17%).
2
Procedure 2. 2ꢀAminoꢀ3,5ꢀdibromopyridine (1.00 g, 3.97 mmol) was dissolved in methanol (10 mL). Thiopheneꢀ2ꢀ
carbaldehyde (557 µL, 5.96 mmol), tertꢀbutylisonitrile (517 µL, 4.57 mmol) and a 1 M solution of perchloric acid in methanol
(
500 µL) were added before the solution was stirred for 24 h at room temperature. After additional isonitrile was added (135 µL,
1.20 mmol), the reaction mixture was again stirred for 24 h before it was diluted with dichloromethane (50 mL) and washed with
aqueous NaHCO (2 × 50 mL) and brine (50 mL). The organic layer was dried with Na SO and the solvent was evaporated in
3
2
4
vacuo. The obtained residue was purified by column chromatography (cyclohexane:ethyl acetate, 30:1 + 3% triethylamine) to
yield 6,8ꢀdibromoꢀNꢀ(tertꢀbutyl)ꢀ2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀamine as yellow crystals (888 mg, 2.07 mmol), yield
1
52%. H NMR (300 MHz, DMSOꢀd )
δ [ppm] = 8.57 (d, J = 1.7 Hz, 1H), 7.82 (dd, J = 3.6, 1.2 Hz, 1H), 7.74 (d, J = 1.7 Hz, 1H),
6
13
7.53 (dd, J = 5.1, 1.2 Hz, 1H), 7.12 (dd, J = 5.1, 3.6 Hz, 1H), 4.83 (s, 1H), 1.10 (s, 9H). C NMR (75 MHz, DMSOꢀd6)
δ [ppm]
=
137.4, 136.9, 135.7, 128.7, 127.5, 126.0, 125.7, 125.2, 123.8, 110.9, 104.4, 56.6, 30.1. This compound (716 mg, 1.67 mmol)
was suspended in 5 M hydrobromic acid (20 mL) and stirred at 110 °C for 2 h, then at 80 °C for 1.5 h. After alkalization with 5
M solution of NaOH and extraction with ethyl acetate (2 × 50 mL), the organic layer was dried with Na SO . Removal of the
2
4
1
solvent in vacuo yielded 8 as yellow solid (610 mg, 1.64 mmol), yield 98%. R (ethyl acetate/petroleum ether 1:3) = 0.47. H
f
NMR (300 MHz, DMSOꢀd6)
δ [ppm] = 8.57 (s, 1H), 7.57 (d, J = 3.6 Hz, 1H), 7.52 (s, 1H), 7.47 (d, J = 5.1 Hz, 1H), 7.14 (t, J =
1
3
8
1
.6 Hz, 1H), 5.68 (s, 2H). C NMR (75 MHz, DMSOꢀd6) δ [ppm] = 137.6, 134.3, 128.0, 125.6, 124.4, 123.7, 122.7, 121.9,
10.4, 104.1.
Synthesis of 6ꢀbromoꢀ2ꢀ[4ꢀ(3ꢀbromopropoxy)phenyl]ꢀimidazo[1,2ꢀa]pyridinꢀ3ꢀamine (9)
0
.3 g (1 mmol, 1.0 eq.) 6, 0.4 g (0.2 mL, 2 mmol, 2.0 eq.) 1,3ꢀdibromopropane and 0.17 g (1.2 mmol, 1.2 eq.) K CO were
2 3
refluxed in 10 mL acetone for 8 h. Acetone was removed in vacuo and the residue was purified by column chromatography
SiO , petroleum ether/ethyl acetate 1:1) yielding 9 as yellow solid. Yield: 0.31 g (74%). R (ethyl acetate/petroleum ether 1:1) =
(
2
f
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
0
.60. H NMR (300 MHz, DMSOꢀd )
δ
[ppm] = 8.51 (s, 1H), 7.96 (d, J = 8.6 Hz, 2H), 7.38 (d, J = 9.4 Hz, 1H), 7.11 (d, J = 9.5
6
1
3
Hz, 1H), 7.01 (d, J = 8.6 Hz, 2H), 5.22 (s, 2H), 4.12 (t, J = 5.9 Hz, 2H), 3.69 (t, J = 6.5 Hz, 2H), 2.27 (quint, J = 6.1 Hz, 2H).
NMR (75 MHz, DMSOꢀd6) [ppm] = 157.0, 136.9, 128.3, 127.6, 127.4, 126.1, 124.1, 122.2, 117.4, 114.4, 105.0, 65.3, 31.9,
1.3.
C
δ
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthesis of 2ꢀ(furanꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀamine (10)
To a solution of furanꢀ2ꢀcarbaldehyde (0.2 mL, 2.41 mmol) in a mixture of H O (0.5 mL) and EtOH (0.5 mL) was added 2ꢀ
2
aminopyridine (229 mg, 2.43 mmol), KCN (164 mg, 2.52 mmol) and BiCl (37 mg, 0.117 mmol). H O (1 mL) was additionally
3
2
added and the mixture was stirred at 110 °C for 1.5 h. The resulting dark brown mixture was evaporated directly on celite and
1
purified by flash column chromatography (7:3 EtOAc/heptane) yielding the product as an orange semiꢀsolid (147 mg, 31%). H
NMR (400 MHz; DMSOꢀd6):
δ [ppm] = 8.16—8.13 (m, 1H), 7.69—7.68 (m, 1H), 7.35 (dt, J = 9.1, 1.1 Hz, 1H), 7.04—7.00 (m,
13
1
H), 6.83—6.79 (m, 1H), 6.69 (dd, J = 3.3, 0.7 Hz, 1H), 6.58 (dd, J = 3.3, 1.8 Hz, 1H), 5.42—5.35 (m, 2H). C NMR (101
MHz, DMSOꢀd6): δ [ppm] = 151.0, 141.6, 139.1, 127.5, 122.8, 122.1, 118.9, 116.9, 111.9, 111.4, 104.8.
Synthesis of 2ꢀ(5ꢀmethylthiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀamine (11)
To a solution of 5ꢀmethylthiopheneꢀ2ꢀcarbaldehyde (0.87 mL, 7.93 mmol) in EtOH (10 mL) was added BiCl (250 mg, 0.792
3
mmol) to form a suspension, which was stirred at ambient temperature for 20 min followed by addition of 2ꢀaminopyridine (761
mg, 8.08 mmol). The reaction was refluxed at 95 °C for 1.5 h after which KCN (1.03 g, 15.9 mmol) was added along with H O (5
2
mL) and heating was continued at 107 °C for 6 h. The dark mixture was quenched with NaOH (1 M, 6 mL) and extracted with
EtOAc (3 x 200 mL). The combined organic phase was dried over MgSO and purified by flash column chromatography (8:2
4
1
EtOAc/heptane). The product was precipitated from EtOAc/heptane to afford an orange solid (315 mg, 17%). H NMR (400
MHz; CD OD):
δ
[ppm] = 8.15 (dt, J = 6.9, 1.2 Hz, 1H), 7.38 (dt, J = 9.1, 1.1 Hz, 1H), 7.32 (d, J = 3.6 Hz, 1H), 7.17 (ddd, J =
3
13
9
.1, 6.7, 1.3 Hz, 1H), 6.90—6.86 (m, 1H), 6.78 (dt, J = 3.5, 1.1 Hz, 1H), 2.52—2.50 (m, 3H). C NMR (100 MHz, CD OD):
δ
3
[ppm] = 140.2, 138.6, 134.4, 125.5, 123.4, 123.3, 122.1, 115.2, 111.6, 13.7.
Syntheses of acid chlorides.
4
9ꢀ50
4
ꢀButoxybenzoyl chloride (12)
5
.0 g (0.033 mol, 1.0 eq.) methylꢀ4ꢀhydroxybenzoate, 6.75 g (0.05 mol, 1.5 eq.) 4ꢀbromobutane and 5.5 g (0.04 mol, 1.2 eq.)
K CO were refluxed in 65 mL acetone for 10 h. The solution was cooled to rt and the precipitated inorganic salts were filtered
2
3
14
ACS Paragon Plus Environment

Page 15 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
off before acetone was removed under reduced pressure. The residue was purified by column chromatography (SiO2,
petroleum/ethyl acetate 9:1) yielding methylꢀ4ꢀbutoxybenzoate as colorless oil (4.40 g, 64%). R (ethyl acetate/petroleum ether
f
1
1
3
:1) = 0.71. HꢀNMR (300 MHz, CDCl )
δ
[ppm] = 7.97 (d, J = 8.8 Hz, 2H), 6.90 (d, J = 8.8 Hz, 2H), 4.01 (t, J = 6.5 Hz, 2H),
3
13
.88 (s, 3H), 1.78 (quint, J = 6.6 Hz, 2H), 1.49 (sext, J = 7.4 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H). C NMR (75 MHz, CDCl3)
δ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
[ppm] = 167.0, 163.1, 131.7, 122.4, 114.2, 68.0, 51.9, 31.3, 19.3, 13.9. 1.0 g (5 mmol, 1.0 eq.) methylꢀ4ꢀbutoxybenzoate and 1.0
g (25 mmol, 5 eq.) NaOH in 10 mL distilled water were refluxed for 2 h. After cooling to rt, the solution was acidified to pH 1 by
addition of 1 M HCl. The mixture was extracted three times with ethyl acetate, the combined organic layers were dried with
Na SO4 and the solvent was evaporated to yield 4ꢀbutoxybenzoic acid as colorless solid (0.93 g, 96%). Rf (ethyl
2
1
acetate/petroleum ether 3:1) = 0.23. HꢀNMR (300 MHz, CDCl )
δ [ppm] = 8.06 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.7 Hz, 2H),
3
13
4.03 (t, J = 6.5 Hz, 2H), 1.80 (quint, J = 6.6 Hz, 2H), 1.51 (sext, J = 7.3 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H). C NMR (75 MHz,
CDCl3) [ppm] = 172.1, 163.8, 132.5, 121.5, 114.3, 68.1, 31.3, 19.3, 14.0. 0.7 g 4ꢀbutoxybenzoic acid was dissolved in 3 mL
SOCl and refluxed for 2 h. Then SOCl was removed in vacuo yielding 12 as lightꢀyellow oil, which was used directly in the
δ
2
2
next step without further purification or characterization.
5
1
4ꢀ(2ꢀfluoroethoxy)benzoyl chloride (13)
4
ꢀHydroxybenzoic acid (200 mg, 1.45 mmol) and 40% aqueous nꢀBu POH solution (2.02 mL, 2.896 mmol) was dissolved in
4
dry THF (3 mL). After the mixture was cooled in an ice bath, 1ꢀbromoꢀ2ꢀfluoroethanol (184 mg, 1.45 mmol) was added dropwise
and the mixture was stirred for 16 h at room temperature. The solvent was removed under vacuum and the crude 4ꢀ(2ꢀ
fluoroethoxy)benzoic acid was purified by column chromatography (methanol:dichloromethane, 1:40 + 3% formic acid) to give a
1
colorless solid (95 mg, 0.52 mmol), yield 36%. H NMR (300 MHz, CD OD)
δ [ppm] = 7.95 (d, J = 8.7 Hz, 2H), 6.98 (d, J = 9.0
3
51
Hz, 2H), 4.79 (m, 1H), 4.63 (m, 1H), 4.31 (m, 1H), 4.21 (m, 1H). Spectral data match that reported in literature. Thionyl
chloride (0.50 mL, 6.9 mmol) was added dropwise to 4ꢀ(2ꢀfluoroethoxy)benzoic acid (85 mg, 0.46 mmol) under inert
atmosphere. After heating at 85 °C for 4 h the excess of thionyl chloride was removed under high vacuum to give the title
compound as light yellow oil. It was used without further purification or characterization.
52
4
ꢀFluorobenzoyl chloride (14)
Thionyl chloride (77.7 µL, 1.07 mmol) was added dropwise to 4ꢀfluorobenzoic acid (50.0 mg, 0.357 mmol) under inert
atmosphere. After heating at 85 °C for 1.5 h the excess of thionyl chloride was removed under high vacuum to give the desired
product 14 as colorless oil at room temperature, which sets under cooling. It was used without further purification or
characterization in the next step.
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
3
4
ꢀ((tertꢀButyldiphenylsilyl)oxy)benzoyl chloride (15)
To a solution of 4ꢀhydroxybenzaldehyde (1.00 g, 8.20 mmol) and imidazole (1.45 g, 31.3 mmol) in DMF (15 mL) tertꢀ
butylchlorodiphenylsilane (2.77 mL, 10.7 mmol) was added and the mixture was stirred for 42 h at room temperature. After
repeated addition of TBDPSꢀCl (640 µL, 2.46 mmol), the solution was stirred for another 24 h before saturated aqueous NH Cl
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(20 mL) was added and extracted with diethyl ether (3 × 50 mL). The combined organic extracts were washed with brine (50 mL)
and dried with Na SO before the solvent was removed in vacuo. Purification of the crude product by column chromatography
2
4
(
cyclohexan:ethyl acetate, 30:1) gave 4ꢀ((tertꢀbutyldiphenylsilyl)oxy)benzaldehyde as colorless crystals (2.13 g, 5.92 mmol),
1
yield 72%. H NMR: (300 MHz, CDCl )
δ
[ppm] = 9.81 (s, 1H), 7.74 – 7.67 (m, 4H), 7.67 – 7.61 (m, 2H), 7.49 – 7.34 (m, 6H),
[ppm] = 191.1, 161.3, 135.5, 132.0, 131.9, 130.4, 128.1, 127.9,
20.4, 26.5, 19.6. 4ꢀ((tertꢀButyldiphenylsilyl)oxy)benzaldehyde (102 mg, 0.283 mmol) was dissolved in a mixture of
3
1
3
6.90 – 6.82 (m, 2H), 1.11 (s, 9H). C NMR: (75 MHz, CDCl3)
δ
5
3
1
dichloromethane/water (4 mL, ratio 1:1) before tetrabutylammonium hydrogensulfate (8.00 mg, 0.0248 mmol) was added.
KMnO (44.7 mg, 0.283 mmol) was subsequently added portion wise under ice cooling. The reaction mixture was stirred for 3 h
4
at room temperature before dichloromethane (15 mL) and 2 M hydrochloric acid (15 mL) were added and separated. After
extraction of the aqueous phase with dichloromethane (2 × 20 mL) the organic layers were dried with Na SO and the solvent
2
4
removed in vacuo. Purification by column chromatography (cyclohexane:ethyl acetate, 9:1 + 3% formic acid) gave 4ꢀ((tertꢀ
1
butyldiphenylsilyl)oxy)benzoic acid as colorless solid (74 mg, 0.197 mmol), yield 70%. H NMR: (300 MHz, CDCl )
δ [ppm] =
3
13
7
.92 – 7.81 (m, 2H), 7.78 – 7.66 (m, 4H), 7.50 – 7.33 (m, 6H), 6.86 – 6.75 (m, 2H), 1.12 (s, 9H). C NMR: (75 MHz, CDCl3)
δ
54
[ppm] = 171.8, 160.7, 135.5, 132.2, 130.3, 128.1, 122.2, 119.8, 26.5, 19.6.
4ꢀ((tertꢀButyldiphenylsilyl)oxy)benzoic acid
(
374 mg, 0.99 mmol) was combined with oxalyl chloride (1 mL) under inert atmosphere and stirred for 3 h at room temperature.
Excess oxalyl chloride was removed under high vacuum to yield 15 as slightly yellow liquid, which was used directly in the next
step without further purification or characterization.
Syntheses of Nꢀ[2ꢀarylimidazo[1,2ꢀa]pyridinꢀ3ꢀyl]amides.
General Procedure.
1
.7 mmol (1.0 eq.) of the respective 2ꢀarylꢀimidazo[1,2ꢀa]pyridinꢀ3ꢀamine (1–9) were dissolved in 8 mL dry toluene and 4 mL
dry pyridine. Then 1.9 mmol (1.1 eq.) of the specified acid chloride were added in one portion and the resulting mixture stirred
for 1h at rt. 6 mL of distilled water were added and stirred for additional 15 min. The solution was cooled with an ice bath and the
precipitate was collected by vacuum filtration, then washed several times with water and once with cooled (– 18 °C) acetone. The
1
6
ACS Paragon Plus Environment

Page 17 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
residue was purified by column chromatography (SiO ) to yield the respective Nꢀ[2ꢀarylimidazo[1,2ꢀa]pyridinꢀ3ꢀyl]amides (16–
2
3
3) as colorless solids.
44
4
ꢀChloroꢀNꢀ[2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl]benzamide (16)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthesized following the general procedure using 0.37 g 1 and 0.33 g 4ꢀchlorobenzoyl chloride. Column chromatography:
1
SiO , ethyl acetate/petroleum 50:50 to 100:0. Yield: 0.43 g (71%). R (ethyl acetate/petroleum ether 1:1) = 0.14. H NMR (300
2
f
MHz, DMSOꢀd6),
δ
[ppm] = 10.75 (s, 1H); 8.18ꢀ8.14 (m, 3H); 7.71 (d, J = 8.5 Hz, 2H); 7.62 (d, J = 9.0 Hz, 1H); 7.55 (d, J = 5.0
13
Hz, 1H); 7.49 (d, J = 3.6 Hz, 1H); 7.34 (t, J = 8.6 Hz, 1H); 7.13 (t, J = 8.6 Hz, 1H); 6.96 (t, J = 6.8 Hz, 1H). C NMR (75 MHz,
DMSOꢀd6),
δ
[ppm] = 165.7, 142.2, 137.3, 136.2, 134.1, 131.8, 130.0, 128.8, 127.9, 126.2, 125.6, 124.4, 123.9, 116.6, 114.1,
+
1
12.4. ESIꢀMS (m/z): [M+H] calcd for C H ClN OS: 354.05, found: 354.06. RPꢀHPLC: R (MeCN/H O 1:1) = 9.48 min,
1
8
12
3
t
2
44
purity > 99%. Spectral data match that reported in literature.
4
ꢀChloroꢀNꢀ(2ꢀphenylimidazo[1,2ꢀa]pyridinꢀ3ꢀyl)benzamide (17)
Synthesized following the general procedure using 0.36 g 2 and 0.33 g 4ꢀchlorobenzoyl chloride. Column chromatography:
1
SiO , ethyl acetate/petroleum 25:75 to 50:50. Yield: 0.40 g (67%). R (ethyl acetate/petroleum ether 3:1) = 0.46. H NMR (300
2
f
MHz, DMSOꢀd6)
δ
[ppm] = 10.79 (s, 1H), 8.23–8.09 (m, 3H), 7.98 (d, J = 8.0 Hz, 2H), 7.78–7.60 (m, 3H), 7.44 (t, J = 7.6 Hz,
1
3
2
1
3
H), 7.39–7.29 (m, 2H), 6.96 (t, J = 6.8 Hz, 1H). C NMR (75 MHz, DMSOꢀd6)
δ
[ppm] = 165.7, 142.1, 137.8, 137.3, 133.5,
+
31.8, 130.0, 128.8, 128.6, 127.7, 126.6, 125.3, 123.9, 116.9, 115.2, 112.3. ESIꢀMS (m/z): [M+H] calcd for C H ClN O:
20
14
3
48.08, found: 348.09. RPꢀHPLC: R (MeCN/H O 1:1) = 10.68 min, purity: 98%.
t
2
Nꢀ[6ꢀBromoꢀ2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl]ꢀ4ꢀchlorobenzamide (18)
Synthesized following the general procedure using 0.50 g 3 and 0.33 g 4ꢀchlorobenzoyl chloride. Column chromatography:
1
SiO , ethyl acetate/petroleum 50:50 to 100:0. Yield: 0.57 g (79%). R (ethyl acetate/petroleum ether 1:1) = 0.49. H NMR (300
2
f
MHz, DMSOꢀd6),
δ
[ppm] = 10.74 (s, 1H), 8.63 (s, 1H), 8.15 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.63ꢀ7.56 (m, 2H),
1
3
7.50ꢀ7.54 (m, 2H), 7.13 (t, J = 8.7 Hz, 1H). C NMR (75 MHz, DMSOꢀd6)
δ
[ppm] = 165.8, 140.6, 137.2, 135.7, 134.8, 131.9,
+
1
30.2, 128.7, 128.5, 128.0, 126.6, 124.8, 124.1, 117.7, 114.8, 106.5. ESIꢀMS (m/z): [M+H] calcd for C H BrClN OS: 431.95,
18
11
3
found: 431.92. RPꢀHPLC: R (MeCN/H O 1:1) = 18.07 min, purity: 98%.
t
2
Nꢀ(6ꢀBromoꢀ2ꢀphenylimidazo[1,2ꢀa]pyridinꢀ3ꢀyl)ꢀ4ꢀchlorobenzamide (19)
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Synthesized following the general procedure using 0.49 g 4 and 0.33 g 4ꢀchlorobenzoyl chloride. Column chromatography:
1
SiO , ethyl acetate/petroleum 40:60 to 90:10. Yield: 0.44 g (61%). R (ethyl acetate/petroleum ether 1:1) = 0.53. H NMR (300
2
f
MHz, DMSOꢀd6)
δ
[ppm] = 10.78 (s, 1H), 8.60 (s, 1H), 8.14 (d, J = 8.4 Hz, 2H), 7.95 (d, J = 7.6 Hz, 2H), 7.77–7.59 (m, 3H),
1
3
7
.51–7.38 (m, 3H), 7.33 (t, J = 7.3 Hz, 1H). C NMR (75 MHz, DMSOꢀd6)
δ
[ppm] = 165.8, 140.6, 138.6, 137.2, 133.1, 131.8,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
30.2, 128.6, 128.3, 128.0, 126.7, 124.1, 118.1, 115.9, 106.5. ESIꢀMS (m/z): [M+H] calcd for C H BrClN O: 425.99 found:
20
13
3
426.02. RPꢀHPLC: R (MeCN/H O 1:1) = 20.49 min, purity > 99%.
t
2
44
4
ꢀMethoxyꢀNꢀ[2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl]benzamide (20)
Synthesized following the general procedure using 0.37 g 1 and 0.32 g 4ꢀmethoxybenzoyl chloride. Column chromatography:
1
SiO , ethyl acetate/petroleum 50:50 to 100:0. Yield: 0.39 g (66%). R (ethyl acetate/petroleum ether 1:1) = 0.18. H NMR (300
2
f
MHz, DMSOꢀd6)
δ
[ppm] = 10.50 (s, 1H), 8.22–8.00 (m, 3H), 7.61 (d, J = 9.0 Hz, 1H), 7.54 (d, J = 4.2 Hz, 1H), 7.49 (d, J = 3.6
13
Hz, 1H), 7.33 (t, J = 7.9 Hz, 1H), 7.19–7.08 (m, 3H), 6.95 (t, J = 6.8 Hz, 1H), 3.88 (s, 3H). C NMR (75 MHz, DMSOꢀd6)
δ
[
ppm] = 166.0, 162.5, 142.1, 136.4, 134.1, 130.1, 127.8, 126.1, 125.4, 125.1, 124.3, 123.8, 116.6, 114.6, 113.9, 112.3, 55.6. ESIꢀ
+
MS (m/z): [M+H] calcd for C H N O S: 350.09, found: 350.09. RPꢀHPLC: R (MeCN/H O 1:1) = 6.75 min, purity > 99%.
1
9
15
3
2
t
2
4
4
Spectral data match that reported in literature.
Nꢀ[6ꢀBromoꢀ2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl]ꢀ4ꢀmethoxybenzamide (21)
Synthesized following the general procedure using 0.50 g 3 and 0.32 g 4ꢀmethoxybenzoyl chloride. Column chromatography:
1
SiO , ethyl acetate/petroleum 60:40 to 90:10. Yield: 0.54 g (75%). R (ethyl acetate/petroleum ether 1:1) = 0.36. H NMR (300
2
f
MHz, DMSOꢀd6)
δ
[ppm] = 10.51 (s, 1H), 8.50 (s, 1H), 8.12 (d, J = 8.7 Hz, 2H), 7.65–7.54 (m, 2H), 7.51–7.40 (m, 2H), 7.19–
1
3
7
1
4
.09 (m, 3H), 3.88 (s, 3H). C NMR (75 MHz, DMSOꢀd6)
δ
[ppm] = 166.1, 162.5, 140.5, 135.8, 134.8, 130.2, 128.3, 127.9,
+
26.5, 125.2, 124.7, 123.9, 117.7, 115.3, 113.8, 106.4, 55.6. ESIꢀMS (m/z): [M+H] calcd for C H BrN O S: 428.00, found:
1
9
14
3
2
27.99. RPꢀHPLC: R (MeCN/H O 1:1) = 10.27 min, purity > 99%.
t
2
44
4ꢀChloroꢀNꢀ[6,8ꢀdibromoꢀ2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl]benzamide (22)
Synthesized following the general procedure using 0.63 g 8 and 0.33 g 4ꢀchlorobenzoyl chloride. Column chromatography:
1
SiO , ethyl acetate/petroleum 75:25 to 100:0. Yield: 0.51 g (59%). R (ethyl acetate/petroleum ether 3:1) = 0.43. H NMR (300
2
f
MHz, DMSOꢀd6)
δ
[ppm] = 10.84 (s, 1H), 8.75 (s, 1H), 8.15 (d, J = 8.5 Hz, 2H), 7.92 (s, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.61 (d, J
13
= 4.9 Hz, 1H), 7.53 (d, J = 3.4 Hz, 1H), 7.14 (t, J = 8.7 Hz, 3H). C NMR (75 MHz, DMSOꢀd6)
δ
[ppm] = 165.8, 138.5, 137.3,
+
1
35.4, 135.0, 131.8, 130.2, 130.1, 128.6, 128.0, 127.0, 125.3, 124.0, 116.5, 110.7, 105.6. ESIꢀMS (m/z): [M+H] calcd for
18
ACS Paragon Plus Environment

Page 19 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
C H Br ClN OS: 509.87, found: 509.89. RPꢀHPLC: R (MeCN/H O 1:1) = 38.73 min, purity > 99%. Spectral data match that
18
10
2
3
t
2
4
4
reported in literature.
44
Nꢀ[6,8ꢀDibromoꢀ2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl]ꢀ4ꢀmethoxybenzamide (23)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthesized following the general procedure using 0.63 g 8 and 0.32 g 4ꢀmethoxybenzoyl chloride. Column chromatography:
1
SiO , ethyl acetate/petroleum 50:50 to 90:10. Yield: 0.53 g (62%). R (ethyl acetate/petroleum ether 1:3) = 0.27. H NMR (300
2
f
MHz, DMSOꢀd6)
δ
[ppm] = 10.59 (s, 1H), 8.62 (s, 1H), 8.12 (d, J = 8.6 Hz, 2H), 7.91 (s, 1H), 7.60 (d, J = 5.0 Hz, 1H), 7.52 (d, J
13
=
3.6 Hz, 1H), 7.27–6.99 (m, 3H), 3.88 (s, 3H). C NMR (75 MHz, DMSOꢀd6)
δ
[ppm] = 166.1, 162.6, 138.4, 135.4, 135.2,
+
1
30.3, 130.0, 128.0, 126.9, 125.2, 125.1, 123.8, 117.0, 113.8, 110.4, 105.5, 55.6. ESIꢀMS (m/z): [M+H] calcd for
C H Br N O S: 505.91 found: 505.95. RPꢀHPLC: R (MeCN/H O 1:1) = 22.03 min, purity: 98%. Spectral data match that
19
13
2
3
2
t
2
4
4
reported in literature.
Nꢀ[6ꢀBromoꢀ2ꢀ(4ꢀmethoxyphenyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl]ꢀ4ꢀchlorobenzamide (24)
Synthesized following the general procedure using 0.54 g 5 and 0.33 g 4ꢀchlorobenzoyl chloride. Column chromatography:
1
SiO , ethyl acetate/petroleum 50:50 to 100:0. Yield: 0.60 g (78%). R (ethyl acetate/petroleum ether 1:1) = 0.38. H NMR (300
2
f
MHz, DMSOꢀd6)
δ
[ppm] = 10.73 (s, 1H), 8.55 (s, 1H), 8.14 (d, J = 8.3 Hz, 2H), 7.89 (d, J = 8.6 Hz, 2H), 7.69 (d, J = 8.3 Hz,
13
2
H), 7.60 (d, J = 9.5 Hz, 1H), 7.43 (d, J = 9.5 Hz, 1H), 7.01 (d, J = 8.6 Hz, 2H), 3.77 (s, 3H). C NMR (75 MHz, DMSOꢀd6) δ
[ppm] = 165.8, 159.2, 140.5, 138.7, 137.2, 131.9, 130.1, 128.6, 128.0, 125.5, 123.9, 117.8, 115.0, 114.1, 106.2, 55.2. ESIꢀMS
+
(
m/z): [M+H] calcd for C H BrClN O : 456.00, found: 456.03. RPꢀHPLC: R (MeCN/H O 1:1) = 20.45 min, purity > 99%.
2
1
15
3
2
t
2
Nꢀ[6ꢀBromoꢀ2ꢀ[4ꢀ(3ꢀbromopropoxy)phenyl]imidazo[1,2ꢀa]pyridinꢀ3ꢀyl]ꢀ4ꢀchloroꢀbenzamide (25)
Synthesized following the general procedure using 0.72 g 9 and 0.33 g 4ꢀchlorobenzoyl chloride. Column chromatography:
1
SiO , ethyl acetate/petroleum 50:50 to 100:0. Yield: 0.68 g (71%). R (ethyl acetate/petroleum ether 1:1) = 0.51. H NMR (300
2
f
MHz, DMSOꢀd6)
δ
[ppm] = 10.73 (s, 1H), 8.56 (s, 1H), 8.14 (d, J = 8.5 Hz, 2H), 7.88 (d, J = 8.7 Hz, 2H), 7.69 (d, J = 8.4 Hz,
2H), 7.61 (d, J = 9.4 Hz, 1H), 7.44 (d, J = 9.5 Hz, 1H), 7.03 (d, J = 8.7 Hz, 2H), 4.10 (t, J = 5.9 Hz, 2H), 3.66 (t, J = 6.5 Hz, 2H),
1
3
2
.24 (quint, J = 6.2 Hz, 2H). C NMR (75 MHz, DMSOꢀd6)
δ
[ppm] = 165.8, 158.3, 140.5, 138.7 137.2, 131.8, 130.1, 128.6,
+
1
28.1, 125.7, 123.9, 117.8, 115.0, 114.7, 106.2, 65.3, 31.8, 31.2. ESIꢀMS (m/z): [M+H] calcd for C H Br ClN O : 561.95,
23
18
2
3
2
found: 561.98. RPꢀHPLC: R (MeCN/H O 1:1) = 35.54 min, purity: 97%.
t
2
Nꢀ[6ꢀBromoꢀ2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl]acetamide (26)
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Synthesized following the general procedure using 0.50 g 3 and 0.15 g acetyl chloride. Column chromatography: SiO , ethyl
2
1
acetate/petroleum 40:60 to 80:20. Yield: 0.44 g (78%). R (ethyl acetate/petroleum ether 1:1) = 0.30. H NMR (300 MHz,
f
DMSOꢀd6)
δ [ppm] = 10.15 (s, 1H), 8.44 (s, 1H), 7.63–7.50 (m, 3H), 7.42 (d, J = 9.5 Hz, 1H), 7.16 (t, J = 8.7 Hz, 1H), 2.24 (s,
13
3
H). C NMR (75 MHz, DMSOꢀd6) δ [ppm] = 170.5, 140.3, 135.9, 134.2, 128.2, 128.0, 126.4, 124.7, 123.9, 117.7, 115.1, 106.3,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
2
2.9. ESIꢀMS (m/z): [M+H] calcd for C H BrN OS: 335.97, found: 335.98. RPꢀHPLC: R (MeCN/H O 1:1) = 4.28 min, purity
1
3
10
3
t
2
> 99%.
44
4
ꢀChloroꢀNꢀ[6ꢀchloroꢀ2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl]benzamide (27)
Synthesized following the general procedure using 0.42 g 7 and 0.33 g 4ꢀchlorobenzoyl chloride. Column chromatography:
1
SiO , ethyl acetate/petroleum 50:50 to 100:0. Yield: 0.48 g (73%). R (ethyl acetate/petroleum ether 1:1) = 0.52. H NMR (300
2
f
MHz, DMSOꢀd6)
δ
[ppm] = 10.76 (s, 1H), 8.58 (s, 1H), 8.16 (d, J = 8.4 Hz, 2H), 7.75–7.63 (m, 3H), 7.57 (d, J = 5.0 Hz, 1H),
13
7
1
.50 (d, J = 3.6 Hz, 1H), 7.39 (d, J = 5.5 Hz, 1H), 7.13 (t, J = 8.7 Hz, 1H). C NMR (75 MHz, DMSOꢀd6)
δ
[ppm] = 165.8,
+
40.6, 137.2, 135.7, 135.1, 131.8, 130.2, 128.7, 128.0, 126.6, 126.4, 124.8, 122.1, 119.6, 117.5, 115.0. ESIꢀMS (m/z): [M+H]
calcd for C H Cl N OS: 380.00 found: 388.01. RPꢀHPLC: R (MeCN/H O 1:1) = 16.19 min, purity > 99%.
18
11
2
3
t
2
Nꢀ[6ꢀBromoꢀ2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl]cyclohexancarboxamide (28)
Synthesized following the general procedure using 0.50 g 3 and 0.28 g cyclohexanecarbonyl chloride. Column
chromatography: SiO , ethyl acetate/petroleum 40:60 to 50:50. Yield: 0.46 g (67%). R (ethyl acetate/petroleum ether 1:3) = 0.45.
2
f
1
H NMR (300 MHz, DMSOꢀd6)
δ
[ppm] = 10.03 (s, 1H), 8.21 (s, 1H), 7.66–7.54 (m, 2H), 7.49 (d, J = 3.2 Hz, 1H), 7.43 (d, J =
9.5 Hz, 1H), 7.16 (t, J = 8.4 Hz, 1H), 2.59 (t, J = 5.5 Hz, 1H), 2.06 (d, J = 6.0 Hz, 2H), 1.81 (d, J = 6.0 Hz, 2H), 1.68 (d, J = 5.5
1
3
Hz, 1H), 1.57–1.12 (m, 5H). C NMR (75 MHz, DMSOꢀd6)
δ
[ppm] = 176.0, 140.4, 135.9, 134.4, 128.2, 127.9, 126.5, 124.7,
+
1
23.5, 117.8, 115.0, 106.3, 43.7, 28.9, 25.4, 25.3. ESIꢀMS (m/z): [M+H] calcd for C H BrN OS: 404.04, found: 404.04. RPꢀ
18
18
3
HPLC: R (MeCN/H O 1:1) = 15.03 min, purity > 99%.
t
2
Nꢀ[6ꢀBromoꢀ2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl]ꢀ4ꢀbutoxybenzamide (29)
Synthesized following the general procedure using 0.50 g 3 and 0.28 g 12. Column chromatography: SiO , ethyl
2
1
acetate/petroleum 50:50 to 90:10. Yield: 0.45 g (57%). R (ethyl acetate/petroleum ether 1:3) = 0.42. H NMR (300 MHz,
f
DMSOꢀd6)
3H), 4.10 (t, J = 6.4 Hz, 2H), 1.75 (quint, J = 6.6 Hz, 2H), 1.47 (m, 2H), 0.96 (t, J = 7.3 Hz, 3H). C NMR (75 MHz, DMSOꢀd6)
[ppm] = 166.1, 162.0, 140.5, 135.8, 134.8, 130.2, 128.3, 127.9, 126.5, 124.9, 124.7, 123.9, 117.7, 115.3, 114.2, 106.4, 67.5,
δ [ppm] = 10.49 (s, 1H), 8.48 (s, 1H), 8.10 (d, J = 8.6 Hz, 2H), 7.64–7.53 (m, 2H), 7.51–7.41 (m, 2H), 7.16–7.09 (m,
13
δ
20
ACS Paragon Plus Environment

Page 21 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
+
30.6, 18.7, 13.7. ESIꢀMS (m/z): [M+H] calcd for C H BrN O S: 470.05, found: 470.04. RPꢀHPLC: R (MeCN/H O 1:1) = 8.45
22
20
3
2
t
2
min, purity > 99%.
4
ꢀ((2ꢀ(Thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl)carbamoyl)phenyl acetate (30)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthesized following the general procedure using 197 mg 1 and 200 mg 4ꢀacetoxybenzoyl chloride. Column chromatography:
1
SiO , ethyl acetate/petroleum 50:50 to 100:0. Yield: 0.23 g (65%). R (ethyl acetate/petroleum ether 1:1) = 0.45. H NMR (300
2
f
MHz, DMSOꢀd6)
δ
[ppm] = 10.69 (s, 1H), 8.25 – 8.11 (m, 3H), 7.66 – 7.47 (m, 3H), 7.43 – 7.29 (m, 3H), 7.13 (t, J = 6.7 Hz,
1
3
1
1
3
H), 6.95 (t, J = 6.7 Hz, 1H), 2.34 (s, 3H). C NMR (75 MHz, DMSOꢀd6)
δ
[ppm] = 169.0, 165.9, 153.6, 142.1, 136.26, 134.1,
+
30.5, 129.7, 127.9, 126.1, 125.5, 124.4, 123.9, 122.2, 116.6, 114.2, 112.4, 20.9. ESIꢀMS (m/z): [M+H] calcd for C H N O S:
20
15
3
3
78.0907, found: 378.0888. RPꢀHPLC: R (MeCN/H O 1:1) = 9.26 min, purity: 98%.
t
2
4ꢀButoxyꢀNꢀ(6,8ꢀdibromoꢀ2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl)benzamide (31)
Synthesized following the general procedure using 200 mg 8 and 150 mg of 12. Column chromatography: SiO , ethyl
2
1
acetate/petroleum 50:50. Yield: 0.21 g (62%). R (ethyl acetate/petroleum ether 1:1) = 0.55. H NMR (300 MHz, DMSOꢀd ) δ
f
6
[ppm] = 10.57 (s, 1H), 8.60 (d, J = 1.6 Hz, 1H), 8.10 (d, J = 8.8 Hz, 2H), 7.90 (d, J = 1.6 Hz, 1H), 7.60 (dd, J = 5.1, 1.2 Hz, 1H),
7
.52 (dd, J = 3.7, 1.2 Hz, 1H), 7.21 – 7.05 (m, 3H), 4.10 (t, J = 6.5 Hz, 2H), 1.74 (dq, J = 8.3, 6.5 Hz, 2H), 1.56 – 1.36 (m, 2H),
1
3
0
.96 (t, J = 7.4 Hz, 3H). C NMR (75 MHz, DMSOꢀd6)
δ
[ppm] = 166.1, 162.0, 138.4, 135.4, 135.2, 130.3, 130.0, 128.0, 126.9,
+
1
25.2, 124.8, 123.7, 116.9, 114.2, 110.7, 105.5, 67.6, 30.6, 18.7, 13.7. ESIꢀMS (m/z): [M+H] calcd for C H Br N O S:
22
19
2
3
2
547.9637, found: 547.9618. RPꢀHPLC: R (MeCN/H O 1:1) = 9.88 min, purity: 97%.
t
2
4
ꢀButoxyꢀNꢀ(6ꢀchloroꢀ2ꢀ(thiopheneꢀ2ꢀyl)ꢀimidazol[1,2ꢀα]pyridinꢀ3ꢀyl)benzamide (32)
Synthesized following the general procedure using 0.50 g 7 and 0.64 g 12. Column chromatography: SiO , ethyl
2
1
acetate/petroleum 50:50. Yield: 0.50 g (66%). R (ethyl acetate/petroleum ether 1:1) = 0.38. H NMR (300 MHz, DMSOꢀd ) δ
f
6
[ppm] = 10.56 (s, 1H), 8.50 (d, J = 1.3 Hz, 1H), 8.11 (d, J = 8.8 Hz, 2H), 7.75 – 7.66 (m, 1H), 7.59 (dd, J = 5.0, 1.1 Hz, 1H), 7.53
(
dd, J = 3.6, 1.0 Hz, 1H), 7.44 (dd, J = 9.5, 1.3 Hz, 1H), 7.18 – 7.08 (m, 3H), 4.10 (t, J = 6.5 Hz, 2H), 1.81 – 1.68 (m, 2H), 1.47
13
(
h, J = 7.6 Hz, 2H), 0.96 (t, J = 7.4 Hz, 3H). C NMR (75 MHz, DMSOꢀd ) δ [ppm] = 166.1, 162.0, 140.1, 135.0, 134.4, 130.3,
6
+
1
27.9, 126.9, 126.8, 125.1, 124.9, 122.1, 119.9, 117.1, 115.6, 114.3, 67.6, 30.6, 18.7, 13.7. ESIꢀMS (m/z): [M+H] calcd for
C H ClN O S: 426.10, found: 426.11. RPꢀHPLC: R (MeCN/H O 1:1) = 6.18 min, purity: 98%.
22 20
3
2
t
2
4
ꢀButoxyꢀNꢀ(ꢀ2ꢀ(thiopheneꢀ2ꢀyl)ꢀimidazol[1,2ꢀα]pyridinꢀ3ꢀyl)benzamide (33)
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Synthesized following the general procedure using 0.30 g 1 and 0.45 g 12. Column chromatography: SiO , ethyl
2
1
acetate/petroleum 60:40. Yield: 0.24 g (60%). R (ethyl acetate/petroleum ether 2:1) = 0.45. H NMR (300 MHz, DMSOꢀd ) δ
f
6
[
ppm] = 10.73 (s, 1H), 8.32 (d, J = 6.8 Hz, 1H), 8.20 – 8.05 (m, 2H), 7.84 – 7.53 (m, 4H), 7.26 – 7.06 (m, 4H), 4.11 (t, J = 6.5
13
Hz, 2H), 1.75 (m, 2H), 1.55 – 1.36 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H). C NMR (75 MHz, DMSOꢀd ) δ [ppm] = 166.3, 162.2,
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
140.4, 132.7, 131.1, 130.3, 128.7, 128.7, 127.9, 127.7, 126.1, 124.6, 115.3, 114.9, 114.4, 114.2, 67.6, 30.6, 18.7, 13.7. ESIꢀMS
+
(
m/z): [M+H] calcd for C H N O S: 392.1427 found: 392.1415. R (MeOH) = 4.82 min, purity: 98%.
2
2
21
3
2
t
Synthesis of Nꢀ(6,8ꢀdibromoꢀ2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl)ꢀ4ꢀ(2ꢀfluoroethoxy)benzamide (34)
To a solution of 8 (132.0 mg, 0.355 mmol) in a mixture of dry toluene (1.5 mL) and dry pyridine (0.9 mL) under inert
atmosphere 13 (93.4 mg, 0.461 mmol) was added and the mixture was stirred for 16 h at room temperature. Water (0.5 mL) was
added and the mixture was stirred for 10 min at room temperature. After cooling in an ice bath the precipitate was filtered off and
washed with cold water. Purification of the crude product by column chromatography (gradient hexane:ethyl acetate, 1:1 to
1
hexane:ethyl acetate, 0:1) yielded 34 as colorless solid (132.6 mg, 0.25 mmol), yield 70%. H NMR: (300 MHz, DMSOꢀd ) δ
6
[
ppm] = 10.62 (s, 1H), 8.64 (s, 1H), 8.12 (d, J = 8.0 Hz, 2H), 7.92 (s, 1H), 7.61 (d, J = 5.0 Hz, 1H), 7.53 (d, J = 1.2, 3.6 Hz, 1H),
13
7
1
.17 (m, 3H), 4.88 (m, 1H), 4.72 (m, 1H), 4.43 (m, 1H), 4.34 (m, 1H). C NMR: (75 MHz, DMSOꢀd ) δ [ppm] = 166.5, 161.9,
6
38.9, 135.9, 135.6, 130.8, 130.5, 128.4, 127.4, 125.8, 125.7, 124.2, 117.3, 114.8, 111.2, 106.0, 83.3, 81.7. FDꢀMS (m/z):
+
[M+H] calcd for C H N O SBr F: 539.9, found: 539.9.
2
0
14
3
2
2
Nꢀ(6,8ꢀdibromoꢀ2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl)ꢀ4ꢀfluorobenzamide (35)
Compound 8 (74.6 mg, 0.200 mmol) was dissolved in a mixture of dry toluene (0.5 mL) and dry pyridine (0.3 mL) under inert
atmosphere before 14 (60.0 µL, 0.508 mmol) was added. The mixture was stirred for 69 h, then dichloromethane (10 mL) was
added. The organic phase was washed with equal volumina of water, 1 M aqueous NaOH and 1 M aqueous hydrochloric acid
before it was dried over Na SO and concentrated in vacuo. The obtained residue was purified by column chromatography
2
4
(
gradient cyclohexane:ethyl acetate, 6:1 + 3% triethylamine to cyclohexane:ethyl acetate, 1:1 + 3% triethylamine) to yield 35 as
1
colorless solid (89.8 mg, 0.181 mmol), yield 91%. H NMR: (300 MHz, DMSOꢀd ) δ [ppm] = 10.78 (s, 1H), 8.73 (d, J = 1.6 Hz,
6
1
2
1
H), 8.26 – 8.16 (m, 2H), 7.92 (d, J = 1.6 Hz, 1H), 7.61 (dd, J = 5.1, 1.2 Hz, 1H), 7.53 (dd, J = 3.7, 1.2 Hz, 1H), 7.50 – 7.41 (m,
1
3
H), 7.15 (dd, J = 5.0, 3.6 Hz, 1H). C NMR: (75 MHz, DMSOꢀd ) δ [ppm] = 165.7, 164.6 (d, J = 250 Hz), 138.6, 135.4, 135.0,
6
31.1 (d, J = 9.3 Hz), 130.2, 129.5 (d, J = 2.8 Hz), 128.0, 127.0, 125.3, 123.9, 116.5, 115.6 (d, J = 22 Hz), 110.7, 105.7. HRꢀMS
+
(ESI): [M+H] calcd for C H N OSBr F: m/z = 493.8974, found 493.8981.
1
8
11
3
2
22
ACS Paragon Plus Environment

Page 23 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Synthesis of 4ꢀ((tertꢀButyldiphenylsilyl)oxy)ꢀNꢀ(6,8ꢀdibromoꢀ2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl)benzamide
(
36)
Compound 8 (185 mg, 0.496 mmol) was dissolved in a mixture of dry toluene (3 mL) and dry pyridine (1.5 mL) before it was
added to 15 (353 mg, 0.893 mmol) under inert atmosphere. The solution was stirred for 43 h. Ethyl acetate (30 mL) was added
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and the organic layer was washed with 1 M aqueous hydrochloric acid and 1 M aqueous NaOH, dried over Na SO and
2
4
concentrated in vacuo. Purification of the crude product by column chromatography (cyclohexane:ethyl acetate, 8:1) gave 36 as
1
colorless solid (271 mg, 0.370 mmol), yield 75%. H NMR: (300 MHz, DMSOꢀd )
δ [ppm] = 10.54 (s, 1H), 8.63 (d, J = 1.6 Hz,
6
1
H), 7.99 – 7.91 (m, 2H), 7.89 (d, J = 1.6 Hz, 1H), 7.77 – 7.67 (m, 4H), 7.59 (dd, J = 5.1, 1.2 Hz, 1H), 7.56 – 7.44 (m, 7H), 7.12
13
(dd, J = 5.1, 3.6 Hz, 1H), 6.94 – 6.87 (m, 2H), 1.08 (s, 9H). C NMR: (75 MHz, DMSOꢀd6)
δ [ppm] = 166.0, 158.5, 138.4,
1
35.4, 135.1, 135.0, 131.5, 130.5, 130.2, 130.0, 128.2, 128.00, 126.9, 126.0, 125.3, 123.8, 119.2, 116.7, 110.7, 105.5, 26.2, 19.0.
Synthesis of Nꢀ(6,8ꢀdibromoꢀ2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl)ꢀ4ꢀhydroxybenzamide (37)
Compound 36 (129 mg, 0.176 mmol) was dissolved in freshly distilled tetrahydrofuran (2 mL) before a 1 M solution of
tetrabutylammonium fluoride in THF (264 µL, 0.264 mmol) was added. The reaction mixture was stirred for 24 h. After removal
of the solvent under reduced pressure the crude product was obtained. Purification by column chromatography (gradient
cyclohexane:ethyl acetate, 4:1 + 3% formic acid until 100% ethyl acetate + 3% formic acid) yielded product 37 as colorless solid
1
(
76.6 mg, 0.155 mmol), yield 88%. H NMR: (300 MHz, DMSOꢀd )
δ
[ppm] = 10.49 (s, 1H), 10.28 (s, 1H), 8.57 (d, J = 1.7 Hz,
6
1H), 8.07 – 7.95 (m, 2H), 7.91 (d, J = 1.6 Hz, 1H), 7.60 (dd, J = 5.0, 1.2 Hz, 1H), 7.51 (dd, J = 3.7, 1.2 Hz, 1H), 7.14 (dd, J = 5.1,
1
3
3
.6 Hz, 1H), 6.99 – 6.88 (m, 2H). C NMR: (75 MHz, DMSOꢀd6)
δ
[ppm] = 166.2, 161.3, 138.4, 135.4, 135.2, 130.4, 130.0,
+
1
28.0, 126.9, 125.2, 123.7, 123.4, 117.1, 115.1, 110.8, 105.5. HRꢀMS (ESI): [M+H] calcd for C H N O SBr : m/z = 491.9017,
18
12
3
2
2
found 491.9023.
Synthesis of Nꢀ(6,8ꢀdibromoꢀ2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl)ꢀ4ꢀ(3ꢀfluorpropoxy)benzamide (38)
To a solution of 3ꢀfluoropropanꢀ1ꢀol (324 µL, 4.00 mmol) in dichloromethane (4 mL) was added 4ꢀmethylbenzenesulfonyl
chloride (839 mg, 4.40 mmol) portionꢀwise and pyridine (360 µL, 4.40 mmol, 1.10 eq.) dropwise under ice cooling. The reaction
mixture was then stirred for 7 h at room temperature. After addition of water (20 mL) and saturated aqueous NH Cl solution
4
(10 mL) the mixture was extracted with ethyl acetate (3 × 60 mL). The combined organic extracts were dried over Na SO and
2
4
concentrated in vacuo before purification of the crude product by column chromatography (cyclohexane:ethyl acetate, 7:1) which
1
yielded 3ꢀfluoropropyl 4ꢀmethylbenzenesulfonate as highly viscous, colorless oil (684 mg, 2.95 mmol), yield 74%. H NMR:
(
300 MHz, CDCl3) δ [ppm] = 7.90 – 7.71 (m, 2H), 7.43 – 7.30 (m, 2H), 4.48 (dt, J = 47, 5.7 Hz, 2H), 4.16 (t, J = 6.1 Hz, 2H),
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
2
.45 (s, 3H), 2.16 – 1.91 (m, 2H). C NMR: (75 MHz, CDCl3)
(d, J = 4.9 Hz), 30.2 (d, J = 20 Hz), 21.8. This compound (31.5 mg, 0.134 mmol) was added to a solution of 37 (50.0 mg,
.103 mmol) and K CO (18.5 mg, 0.134 mmol) in DMF (4 mL). The resulting mixture was heated to 40 °C and stirred for 24 h
δ [ppm] = 145.1, 132.9, 130.1, 128.0, 79.7 (d, J = 166 Hz), 66.3
0
2
3
under inert atmosphere. Next ethyl acetate (40 mL) was added and the organic phase washed with 1 M aqueous NaOH
3 × 30 mL) and dried over Na SO4 before the solvent was removed under reduced pressure. Purification by column
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
2
chromatography (cyclohexane:ethyl acetate, 5:1) yielded the title compound as colorless solid (17.5 mg, 0.0316 mmol), yield
1
3
1
1%. H NMR: (300 MHz, DMSOꢀd )
δ
[ppm] = 10.60 (s, 1H), 8.63 (d, J = 1.6 Hz, 1H), 8.20 – 8.04 (m, 2H), 7.91 (d, J = 1.6 Hz,
6
H), 7.60 (dd, J = 5.1, 1.2 Hz), 7.52 (dd, J = 3.7, 1.2 Hz, 1H), 7.25 – 7.09 (m, 3H), 4.64 (dt, J = 47, 5.9 Hz, 2H), 4.21 (t, J = 6.3
13
Hz, 2H), 2.16 (dp, J = 26, 6.1 Hz, 2H). C NMR: (75 MHz, DMSOꢀd6)
δ [ppm] = 166.1, 161.7, 138.5, 135.4, 135.2, 130.3,
130.1, 128.0, 127.0, 125.2, 125.1, 123.8, 116.9, 114.3, 110.8, 105.6, 80.8 (d, J = 162 Hz), 64.0 (d, J = 5.4 Hz), 29.7 (d, J = 20
+
Hz). HRꢀMS (ESI): [M+H] calcd for C H N O FSBr : m/z = 551.9392, found 551.9404.
2
1
17
3
2
2
Synthesis of Nꢀ(6,8ꢀdibromoꢀ2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl)ꢀ4ꢀ(4ꢀfluorbutoxy)benzamide (39)
Compound 37 (38.7 mg, 0.0785 mmol) was dissolved in acetone (2 mL) and K CO (14.1 mg, 0.102 mmol) was added. The
2
3
mixture was stirred shortly before 1ꢀbromoꢀ4ꢀfluorobutane (11.0 µL, 0.102 mmol) and a catalytic amount of KI were added. The
reaction mixture was then stirred for 24 h and heated under reflux, whereupon additional alkyl halogenide (0.0306 mmol) in
acetone (0.5 mL) was added. The reaction mixture was finally stirred and heated under reflux for another 23 h before ethyl
acetate (30 mL) was added and the organic phase was washed with 1 M aqueous NaOH solution (3 × 30 mL), dried over Na SO
2
4
and concentrated under reduced pressure. The crude product was then purified by column chromatography (cyclohexane:ethyl
1
acetate, 6:1 + 3% triethylamine) to yield 39 as colorless solid (15.3 mg, 0.0270 mmol), yield 35%. H NMR: (300 MHz, DMSOꢀ
d )
δ
[ppm] = 10.59 (s, 1H), 8.62 (d, J = 1.7 Hz, 1H), 8.15 – 8.06 (m, 2H), 7.91 (d, J = 1.6 Hz, 1H), 7.60 (dd, J = 5.1, 1.2 Hz,
6
1
H), 7.52 (dd, J = 3.7, 1.2 Hz, 1H), 7.17 – 7.10 (m, 3H), 4.52 (dt, J = 47, 5.9 Hz, 2H), 4.14 (t, J = 6.0 Hz, 2H), 1.93 – 1.72 (m,
13
4H). C NMR: (75 MHz, DMSOꢀd6)
δ
[ppm] = 166.1, 161.9, 138.5, 135.4, 135.2, 130.3, 130.0, 128.0, 127.0, 125.2, 124.9,
+
1
23.8, 116.9, 114.3, 110.8, 105.6, 83.6 (d, J = 162 Hz), 67.4, 26.6 (d, J = 19 Hz), 24.6 (d, J = 5.3 Hz). HRꢀMS (ESI): [M+H]
calcd for C H N O SBr F: m/z = 565.9549, found 565.9551.
22
19
3
2
2
Synthesis of 4ꢀhydroxyꢀNꢀ(2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl)benzamide (40)
0
.15 g of compound 30 were dissolved in 3 mL THF and 3 mL 5 M NaOH were added. The biphasic mixture was vigorously
stirred for 3 h at rt. THF was removed under reduced pressure and the residue was diluted with 10 mL H O. The solution was
2
acidified to pH 1 by addition of 3 M HCl. The precipitate formed was collected by vacuum filtration and dried in vacuo. The
24
ACS Paragon Plus Environment

Page 25 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
product was obtained as colorless solid (0.13 g, 92%). H NMR (300 MHz, DMSOꢀd )
δ [ppm] = 10.78 (s, 1H), 10.41 (s, 1H),
6
8.53 (d, J = 6.7 Hz, 1H), 8.04 (d, J = 8.7 Hz, 2H), 7.96 – 7.72 (m, 4H), 7.37 (t, J = 6.7 Hz, 1H), 7.25 (dd, J = 4.9, 3.9 Hz, 1H),
1
3
6
1
3
.97 (d, J = 8.7 Hz, 2H). C NMR (75 MHz, DMSOꢀd ) δ [ppm] = 166.6, 161.7, 144.7, 130.5, 129.3, 129.3, 128.0, 125.4, 122.9,
6
+
16.04, 115.98, 115.3, 113.40, 113.37, 108.8, 105.6. Purity by HPLC: 99%. ESIꢀMS (m/z): [M+H] calcd for C H N O S:
18
13
3
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
36.0801, found: 336.0788.
4ꢀ(Benzyloxy)ꢀNꢀ(2ꢀ(thiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl)benzamide (41)
1
Compound 1 was reacted with 4ꢀbenzyloxybenzoylchloride following the general procedure. Yield: 90 mg (63%). H NMR
(
300 MHz, DMSOꢀd ) δ [ppm] = 10.49 (s, 1H), 8.18 – 8.04 (m, 3H), 7.60 (d, J = 9.1 Hz, 1H), 7.57 – 7.28 (m, 8H), 7.22 (d, J =
6
13
8.8 Hz, 2H), 7.12 (dd, J = 4.9, 3.7 Hz, 1H), 7.00 – 6.87 (m, 1H), 5.25 (s, 2H). C NMR (75 MHz, DMSOꢀd ) δ [ppm] = 166.1,
6
1
61.6, 139.8, 138.4, 136.6, 135.4, 135.2, 130.3, 128.5, 128.00, 127.98, 127.8, 127.0, 125.3, 123.8, 116.5, 114.7, 112.5, 110.7,
+
1
05.5, 69.4. ESIꢀMS (m/z): [M+H] calcd for C H N O S: 425.13, found: 425.14. RPꢀHPLC: R (MeCN/H O 1:1) = 4.35 min,
2
5
19
3
2
t
2
purity 98%.
Synthesis of 4ꢀchloroꢀNꢀ(2ꢀ(furanꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl)benzamide (42)
Compound 10 (122 mg, 0.612 mmol) was dissolved in dry THF (20 mL). Pyridine (0.74 mL, 9.17 mmol) was added under
nitrogen atmosphere and the reaction mixture was stirred for 5 min followed by addition of 4ꢀchlorobenzoyl chloride (0.086 mL,
0.673 mmol). The reaction was stirred at ambient temperature for 1 h and subsequently quenched with H O (15 mL) and
2
extracted with EtOAc (2 x 100 mL). The combined organic phase was washed with H O and brine, the organic layer was dried
2
over Na SO and reduced in vacuo to afford a light brown solid which was purified by flash column chromatography (8:2
2
4
EtOAc/heptane + 0.1% AcOH) to afford a brown film (162 mg). The product was precipitated from DCM/EtOAc (1:1, 30 mL) as
1
a light brown solid, which was filtered, washed with EtOAc and dried (82.6 mg, 40%). HꢀNMR (600 MHz; DMSOꢀd ):
δ [ppm]
6
=
10.69 (s, 1H), 8.15—8.12 (m, 3H), 7.75 (dd, J = 1.7, 0.8 Hz, 1H), 7.70—7.68 (m, 2H), 7.60 (dt, J = 9.1, 1.0 Hz, 1H), 7.34 (ddd,
13
J = 9.1, 6.7, 1.3 Hz, 1H), 6.96 (td, J = 6.8, 1.1 Hz, 1H), 6.80 (dd, J = 3.3, 0.8 Hz, 1H), 6.60 (dd, J = 3.4, 1.8 Hz, 1H). CꢀNMR
(151 MHz, DMSOꢀd6):
δ
[ppm] = 166.1, 149.1, 143.5, 142.8, 137.6, 132.4, 131.4, 130.4, 129.2, 125.9, 124.4, 117.2, 115.2,
+
1
12.8, 112.1, 108.3. Purity by HPLC: 99%, HRMS (m/z): [M+H] calcd for C H ClN O : 338.0691; found: 338.0693.
18
12
3
2
Synthesis of 4ꢀchloroꢀNꢀ(2ꢀ(5ꢀmethylthiopheneꢀ2ꢀyl)imidazo[1,2ꢀa]pyridinꢀ3ꢀyl)benzamide (43)
Compound 11 (315 mg, 1.37 mmol) was dissolved in dry THF (20 mL) and put under nitrogen atmosphere. Pyridine was
added and the solution was stirred for 5 min followed by addition of 4ꢀchlorobenzoyl chloride (0.19 mL, 1.51 mmol), the reaction
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was stirred at ambient temperature for 17 h and then at 50 °C for 30 min. After cooling to rt, H O (10 mL) was added and the
2
aqueous phase was extracted with EtOAc (1 x 150 mL, 1 x 75 mL). The combined organic phase was washed with H O and brine
2
and dried over MgSO4 after which it was concentrated in vacuo and purified by flash column chromatography (2:1
1
EtOAc/heptane). The product was precipitated from EtOAc/heptane to afford a light yellow solid (264 mg, 52%). HꢀNMR (600
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MHz; DMSOꢀd6):
δ [ppm] = 10.67 (s, 1H), 8.15—8.12 (m, 3H), 7.71—7.69 (m, 2H), 7.59 (dt, J = 9.1, 1.1 Hz, 1H), 7.32 (ddd, J
1
3
=
9.1, 6.7, 1.3 Hz, 1H), 7.28 (d, J = 3.5 Hz, 1H), 6.94 (td, J = 6.8, 1.1 Hz, 1H), 6.81—6.80 (m, 1H), 2.45 (d, J = 0.9 Hz, 3H). Cꢀ
NMR (151 MHz, DMSOꢀd6):
δ
[ppm] = 166.1, 142.6, 140.0, 137.7, 134.8, 134.3, 132.3, 130.4, 129.3, 126.6, 125.8, 124.9, 124.2,
+
1
17.0, 114.1, 112.7, 15.4. Purity by HPLC: 96%, HRMS (m/z): [M+H] calcd for C H ClN OS: 368.0619; found: 368.0625.
19
14
3
Synthesis of 4ꢀchloroꢀNꢀ(imidazo[1,2ꢀa]pyridinꢀ3ꢀyl)benzamide (44)
To a solution of formaldehyde (37% in H O, 0.94 mL, 12.6 mmol) in EtOH (10 mL) BiCl (394 mg, 1.25 mmol) was added to
2
3
form a white suspension, which was stirred at ambient temperature for 20 min. To the reaction mixture 2ꢀaminopyridine (1.2 g,
2.7 mmol) was added and the reaction was refluxed for 1.5 h followed by addition of KCN (1.6 g, 24.5 mmol) along with H O
1
2
(10 mL), the heating was continued for 4 h after which the reaction was quenched with NaOH (1 M, 6 mL) and filtered. The
aqueous phase was extracted with EtOAc (100 mL) and the organic layer was washed with H O and brine and dried over MgSO
2
4
after which it was reduced on celite and purified by flash column chromatography (9:1 DCM/MeOH) to afford a yellow oil which
crystallized over time (216 mg, 13%). The yellow residue was taken up in dry THF (20 mL) and pyridine (1.9 mL, 23.8 mmol)
was added under nitrogen atmosphere followed by addition of 4ꢀchlorobenzoyl chloride (0.25 mL, 1.90 mmol). The mixture was
stirred at ambient temperature until the reaction was shown to be complete after 4 days. At the end of this time, H O (5 mL) was
2
added, and the mixture was extracted with EtOAc (3 x 100 mL). The combined organic phase was washed with H O and brine
2
and dried over MgSO . The residue was evaporated on celite and purified by flash column chromatography (9.5:0.5
4
DCM/MeOH) to afford a mixture of white solid and yellow oil. The product was precipitated from EtOAc/heptane as a white
1
fibrous solid (200 mg, 47%). HꢀNMR (600 MHz; CD OD):
δ [ppm] = 8.11 (d, J = 6.9 Hz, 1H), 8.06—8.05 (m, 2H), 7.60—7.57
3
13
(
m, 4H), 7.38 (ddd, J = 9.1, 6.7, 1.2 Hz, 1H), 7.01 (td, J = 6.8, 1.0 Hz, 1H). CꢀNMR (151 MHz, DMSOꢀd6): δ [ppm] = 165.6,
1
42.8, 137.5, 132.4, 130.4, 129.1, 128.2, 124.6, 124.5, 120.3, 117.7, 112.3. mp: 141.5—143.7 °C, purity by HPLC: 99%, HRMS
+
(m/z): [M+H] calcd for C H ClN O: 272.0585; found: 272.0591.
1
4
10
3
Ligand binding to recombinant receptors. Human embryonic kidney cells [HEK 293 cells; German collection of
microorganisms and cell cultures (DSMZ), Braunschweig, Germany] were grown to < 50% confluency on 15ꢀcm tissue plates in
2
0 mL DMEM supplemented with 10% heatꢀinactivated fetal calf serum, 1.8 mM glutamine as well as penicillin (1785 units) and
2+
streptomycin (1.8 mg). Transfection was carried out with a Ca ꢀphosphate precipitation method essentially as previously
26
ACS Paragon Plus Environment

Page 27 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
5
5
described. Briefly, plasmids were diluted in 1 mL/plate of 0.3125 M CaCl in H O. One mL/plate of 2x HBS (274 mM NaCl;
2
2
1
.5 mM Na HPO ; 54.6 mM HEPES/ NaOH; pH 7.0) was added to the DNA and incubated for 90 s. Two mL of the mixture
2 4
were pipetted immediately onto 15ꢀcm plates. These were incubated for 18ꢀ24 h before the transfection medium was replaced by
37, 56ꢀ57
fresh medium. Double and triple combinations of rat GABA receptor cDNAs in eukaryotic expression vectors
of the α1,
A
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
α4, α6, β2, β3, γ2S and δ subunits were employed. For optimal receptor expression, final concentrations (µg vector DNA per 15ꢀ
cm tissue culture plate) were: α1, 2.5; α4, 12; α6, 2.5; β2, 12; β3, 0.5; γ2S, 0.375 and δ, 2.5.
58
Cell membranes were prepared as previously described. Briefly, cells were washed in PBS and harvested from tissue plates
4
8 h after transfection. Cells were homogenized in an Ultraturrax homogenizer for 15 s. Crude membranes were obtained after
two centrifugation steps at 23000 g. Pellets were used immediately or frozen at ꢀ20 °C. Membrane pellets were resuspended in 50
mM Tris/citrate buffer, pH 7.3. Resuspended cell membranes (150ꢀ200 µg protein per tube) were incubated in a final volume of
3
0
.5 mL of 50 mM Tris/citrate buffer (pH 7.3), supplemented with 0.2 M NaCl, 3 nM [ H]EBOB (Perkin Elmer, Waltham, MA,
USA) and increasing concentrations of DS compounds in presence (0.1 µM for α6β3γ2/δ and 0.5 µM for α6β3 receptors) or
absence of GABA. DS derivatives were diluted in 50 mM Tris/citrate buffer (pH 7.3) from a stock of 10 mM in DMSO (giving a
total DMSO concentration of 0.1%). GABA was diluted from a 10 mM solution in 50 mM Tris/citrate buffer (pH 7.3). Nonꢀ
3
specific binding of [ H]EBOB was determined by addition of 10 µM of EBOB. The binding assay procedure was essentially
5
8
35
performed as described earlier, though in the cited publication binding assays were executed with [ S]TBPS. As the allosteric
31, 34ꢀ35
interaction of tested ligands on the binding properties of both convulsants has proven to be similar,
we adopted the
3
3
procedure to assays executed with [ H]EBOB. Briefly, after addition of [ H]EBOB, cell membranes were incubated at room
3
temperature for 90 min. We performed [ H]EBOB binding assays under these preꢀequilibrium conditions (90 min incubation at
35
32
21 °C). The preꢀequilibrium conditions were previously investigated and applied to [ S]TBPS binding studies and adopted to
3
40
[
H]EBOB binding experiments. Assay mixtures were then rapidly diluted to 5 mL with iceꢀcold Tris/HCl, pH 7.5, and filtered
through glass mircofibre filters, grade GF/C (GE Healthcare, Buckinghamshire, UK). The procedure was repeated once. Filters
were incubated in 3.5 mL of Aquasafe 300 Plus scintillation fluid (Zinsser Analytic, Frankfurt, Germany). The radioactivity was
determined in a Beckman liquid scintillation counter using external standardization. Protein quantification was performed
5
9
according to the Bradford method using RotiꢀQuant 5x concentrate (Roth, Karlsruhe, Germany).
Electrophysiology. HEK 293 cells were grown on glass cover slips and transiently transfected with final concentrations (µg
vector DNA/94 mm tissue culture plate) of α1: 1, α4: 5, α6: 1, β3: 0.2, γ2: 0.4, δ: 1.5 and a Green Fluorescent protein (eGFP;
Clontech, Heidelberg, Germany): 0.5. 24 h after transfection the medium was replaced by fresh solution and 48 h after
transfection cover slips were transferred to a recording chamber under an upright microscope (Zeiss Axioskop FS, Jena,
Germany). Cells were perfused at 3 mL/min with an extracellular solution (135 mM NaCl, 5.3 mM KCl, 2 mM CaCl , 2 mM
2
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
MgSO , 10 mM HEPES, pH 7.4, adjusted to 320 mOsm with sucrose). Recordings of single, isolated and fluorescent cells were
4
obtained in the whole cell configuration of the patchꢀclamp technique. The pipette solution contained 10 mM NaCl, 80 mM KCl,
5
0 mM KOH, 2 mM MgCl , 2 mM CaCl , 3.1 mM ATP, 0.4 mM GTP, 10 mM EGTA and 10 mM HEPES, pH 7.2. Drugs were
2 2
applied using a fast perfusion stepper system (SFꢀ77B, Warner Instruments, Inc., Midwest, USA) enabling solution exchange
times of <20 ms (controlled by junction potential measurements). DS compounds were applied alone or in test solutions
containing GABA at the receptor specific EC20 of GABA for 4 s with 60 s between successive applications. Data were amplified,
filtered at 1 kHz (4ꢀpole Bessel) and recorded on a standard personal computer at a sampling rate of 3ꢀ5 kHz using EPCꢀ9 patchꢀ
clamp amplifier and Pulse 8.11 (HEKA electronics, Lambrecht, Germany).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
Calculation. [ H]EBOB binding analysis: Nonlinear regression was performed in Prism, version 6.05, on the fourꢀparametric
(
(LogIC50 – x) * η)
logarithmic doseꢀresponse equation formula (y = min + (max – min) / (1+10
) with min and max being the minimum
and maximum value, x the concentration of the derivative in µM and η Hill coefficient. The term “Hill coefficient” should be
read as “pseudoꢀHill coefficient” when dealing with allosteric as contrasting to direct effects. 100% was defined as the value in
the absence of any modulator; 0% was defined by the blank value, i.e., in the presence of an excess of unlabelled EBOB. The
maximum was set constant to 100%, the minimum was fitted. Data are given as the mean ± S.E.M. for the IC50 curves and mean
±
S.D. for effects of single concentration. For statistical comparison Student’s tꢀtest was used. * p < 0.05; ** p < 0.01; *** p <
0.001. Electrophysiology: Data analysis was performed using ClampFit 8.1 (Axon Instruments) and Origin 8.5 (Microcal,
Northampton, MA, USA). Direct activation by compounds 16 and 22 were expressed by the ratio I1µM X/I1mM GABA. To calculate
concentrations eliciting 50% of the maximal modulation of GABA induced currents (EC ), peak currents (Imax) were plotted
50
Hill
against the ligand concentration (LIG) and fitted with the logistic function y = Imin + (ImaxꢀImin) / (1 + ([LIG]/EC50) using a least
square fitting routine, where Imin represents the current induced by the receptor specific EC20 for GABA, Imax the maximal
modulation, and Hill the Hill coefficient. The term “Hill coefficient” should be read as “pseudoꢀHill coefficient” when dealing
with allosteric as contrasting to direct effects. Responses of compounds 16 and 22 were expressed as percentage of control
responses.
It should be explicitly mentioned that in this manuscript all shown Hill coefficients should be read as pseudoꢀHill coefficients.
True Hill coefficients reflect the number of binding sites of a ligand inducing the measured effect, i.e., the effect of GABA on
GABA R has to be seen in this context. In contrast, a modulatory or indirect effect can assume any positive or negative figure,
A
i.e., independent of the number of binding sites. It has to be called pseudoꢀHill coefficient as the mathematics behind its
determination are identical to that for the true Hill coefficient.
28
ACS Paragon Plus Environment

Page 29 of 51
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
AUTHOR INFORMATION
Corresponding Authors
Please address correspondence to Tanja Schirmeister and Hartmut Lüddens
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
*
H.L. Eꢀmail: lueddens@uniꢀmainz.de Phone: (+49)6131175371
T.S. EꢀMail: schirmei@uniꢀmainz.de. Phone: (+49)61313925742
*
Current Author Addresses
A.K.: Max Planck Institute for Polymer Research, Johannes Gutenberg University Mainz, Ackermannweg 10, Dꢀ55128 Mainz,
Germany
J.W.: 1) Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge,
CB2 0RE, U.K. 2) Department of Chemistry, University of Cambridge, Lensfield road, Cambridge, CB2 1EW, U.K.
C.S.: Institute of Physiology II, University Medical Center Jena, Kollegiengasse 9, Dꢀ07743 Jena, Germany
Author Contributions
#
K.Y. and S.J. contributed equally. T.S. and H.L. share senior authorship.
C.S. contributed electrophysiological data.
Funding Sources
MaiFor 2013ꢀ2015
ACKNOWLEDGMENT
The authors would like to thank Mrs. Darcie Mulhearn for critically reading the manuscript.
ABBREVIATIONS USED
3
3
DS1, delta selective compound 1; DS2, delta selective compound 2; [ H]EBOB, [ H]ethynylbicycloorthobenzoate; GABA, γꢀ
35
aminobutyric acid; GABA R, γꢀaminobutyric acid receptors type A; HEK, Human embryonic kidney; [ S]TBPS, tertꢀ
A
35
[
S]butylbicyclophosphorothionate
ASSOCIATED CONTENT
Supporting Information
29
ACS Paragon Plus Environment