HTS-based discovery and optimization of novel positive allosteric modulators of the α7 nicotinic acetylcholine receptor

Article abstract of DOI:10.1016/j.ejmech.2021.113560

HTS campaign of the corporate compound collection resulted in a novel, oxalic acid diamide scaffold of α7 nACh receptor positive allosteric modulators. During the hit expansion, several derivatives, such as 4, 11, 17 demonstrated not only high in vitro potency, but also in vivo efficacy in the mouse place recognition test. The advanced hit molecule 11 was further optimized by the elimination of the putatively mutagenic aromatic-amine building block that resulted in a novel, aminomethylindole compound family. The most balanced physico-chemical and pharmacological profile was found in case of compound 55. Docking study revealed an intersubunit binding site to be the most probable for our compounds. 55 demonstrated favorable cognitive enhancing profile not only in scopolamine-induced amnesia (place recognition test in mice) but also in natural forgetting (novel object recognition test in rats). Compound 55 was, furthermore, active in a cognitive paradigm of high translational value, namely in the rat touch screen visual discrimination test. Therefore, 55 was selected as a lead compound for further optimization. Based on the obtained favorable results, the invented aminomethylindole cluster may provide a viable approach for cognitive enhancement through positive allosteric modulation of α7 nAChRs.

Full text of DOI:10.1016/j.ejmech.2021.113560

European Journal of Medicinal Chemistry 222 (2021) 113560
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
HTS-based discovery and optimization of novel positive allosteric
modulators of the a7 nicotinic acetylcholine receptor
a
,
*
a
a
a
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Istvan Ledneczki , Anita Horvath , Pal Tapolcsanyi , Janos Eles ,
a
a
b
b
c
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ꢀ ꢀ
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Katalin Dudas Molnar , Istvan Vago , Andras Visegrady , Laszlo Kiss , Aron Szigetvari ,
c
c
a
d
b
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b
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b
Janos Koti , Balazs Kramos , Sandor Maho , Patrik Holm , Sandor Kolok ,
b
b
b
b
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b
Laszlo Fodor , Marta Than , Diana Kostyalik , Ottilia Balazs , Monika Vastag ,
e
b
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Istvan Greiner , Gyorgy Levay , Balazs Lendvai , Zsolt Nemethy
a Department of Chemistry, Gedeon Richter Plc., Budapest, Hungary
b Pharmacological and Drug Safety Research, Gedeon Richter Plc., Budapest, Hungary
c Spectroscopic Research Department, Gedeon Richter Plc., Budapest, Hungary
d Orion Corporation, Turku, Finland
e Research Management, Gedeon Richter Plc., Budapest, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
HTS campaign of the corporate compound collection resulted in a novel, oxalic acid diamide scaffold of
7 nACh receptor positive allosteric modulators. During the hit expansion, several derivatives, such as 4,
Received 26 February 2021
Received in revised form
26 April 2021
Accepted 18 May 2021
Available online 29 May 2021
a
11, 17 demonstrated not only high in vitro potency, but also in vivo efficacy in the mouse place recognition
test. The advanced hit molecule 11 was further optimized by the elimination of the putatively mutagenic
aromatic-amine building block that resulted in a novel, aminomethylindole compound family. The most
balanced physico-chemical and pharmacological profile was found in case of compound 55. Docking
study revealed an intersubunit binding site to be the most probable for our compounds. 55 demonstrated
favorable cognitive enhancing profile not only in scopolamine-induced amnesia (place recognition test in
mice) but also in natural forgetting (novel object recognition test in rats). Compound 55 was, further-
more, active in a cognitive paradigm of high translational value, namely in the rat touch screen visual
discrimination test. Therefore, 55 was selected as a lead compound for further optimization. Based on the
obtained favorable results, the invented aminomethylindole cluster may provide a viable approach for
Keywords:
Alpha 7 nicotinic acetylcholine receptor
Positive allosteric modulation
High-throughput screening
Structure-activity relationship
Cognitive enhancement
cognitive enhancement through positive allosteric modulation of a7 nAChRs.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
research groups in the past two decades. Despite the increasing
efforts, symptomatic treatment options for cognitive disturbances
are still limited to acetylcholinesterase inhibitors (AChEi; e.g.
donepezil, rivastigmine, galantamine) and the NMDA receptor
antagonist memantine. ACh exerts its beneficial effects through the
activation of cholinergic receptors. Two types of ACh receptors
(muscarinic and nicotinic receptors) exist, as shown by the different
agonist activities of muscarine and nicotine. Nicotinic ACh re-
ceptors (nAChRs) are pentameric ligand-gated ion channels formed
Cognition is a highly complex function of the central nervous
system (CNS). Acetylcholine (ACh) is essential for the proper
functioning of various domains of cognition (e.g. memory, attention
or executive processes such as cognitive control or flexibility).
Cognitive impairment is prevalent in many neurological (e.g. Alz-
heimer's disease, Parkinson's disease) and psychiatric (e.g. schizo-
phrenia, depression) diseases, as well as in general aging. Due to its
central role in the cognitive processes, cholinergic neurotransmis-
sion was intensively investigated by academic and industrial
by
result in distinct nAChR subtypes, the most predominant nAChR
subtypes in the CNS being the heteromeric ( 4)2( 2)3 and the
homomeric ( 7)5 nAChRs [2]. The 7 nAChR is considered as the
a- and b-subunits [1]. Various a- and b-subunit compositions
a
b
a
a
most common nicotinic receptor subtype in the rat [3] and human
[4] brain, expressed in high densities in “areas of cognition”, e.g. in
* Corresponding author.
E-mail address: ledneczki@richter.hu (I. Ledneczki).
https://doi.org/10.1016/j.ejmech.2021.113560
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
the prefrontal cortex [5], hippocampus and various subcortical
newly invented synthetic methods (Scheme 1). Indolamine build-
ing blocks 22e (6-fluoro-1-methyl-1H-indol-5-amine) and 27e (3-
fluoro-1-methyl-1H-pyrrolo[2,3-b]pyridin-5-amine) were synthe-
sized according to newly invented synthetic methods depicted in
Scheme 1. 6-Fluoro-1H-indole-2,3-dione (22a) was nitrated with
the “mixed acid” system and subsequently in 22b the dioxo func-
tionality was reduced with borane-THF complex to obtain 22c.
Afterwards it was N-methylated and the nitro group of the resulting
22d was reduced to amine moiety (22e). 5-Bromo-7-azaindole
(27a) was fluorinated at position 3 with Selectfluor. The resulted
3-fluoroindole (27b) was N-methylated to obtain 27c, which was
then substituted with benzylamine to 27d in a Buchwald-Hartwig
reaction and finally the benzyl group was cleaved by catalytic hy-
drogenation to obtain 27e.
limbic structures [6,7]. Lower expression levels of
detected in the thalamic regions and basal ganglia [8,9].
occur pre-, post and extrasynaptically in various neuronal cells.
Extrasynaptic localization of 7 nAChRs [10] may constitute the
a7 nAChRs were
a7 nAChRs
a
main target of nicotinic ligands arriving from the extracellular
space [11]. However, ACh may also enter the synapse and activates
synaptic nAChRs even at very low concentrations [12].
Activation of nAChRs yields local excitation of the host cells
mainly by increasing the permeability for Ca2þ ions, which, in turn,
leads to the modulation of key neurotransmitter levels [13].
Orthosteric agonists of the
a7 nAChR bind to the conventional
agonist binding site located at the interface between two adjacent
subunits distinct from the transmembrane ion-channel pore [14].
Unique features of the a7 nAChR activation include the low prob-
ability of channel opening, fast activation kinetics, high perme-
ability to Naþ, Kþ and Ca2þ compared to other nAChR subtypes
[15,16], as well as the extremely fast desensitization [17,18], which
2.1.2. The synthesis for secondary amine intermediates 5d, 9c and
10c
In Scheme 2 the synthesis of the non-purchasable amine in-
termediates was exemplified in case of the azetidinemethyl inter-
mediate 5d (for compounds 5e8, 44, 48e55). tert-Butyl 3-
(hydroxymethyl)azetidine-1-carboxylate (5a) was mesylated (5b)
and then reacted with p-cresol (5c) and finally the BOC protecting
group was removed under acidic conditions to obtain the azetidi-
nemethyl intermediate 5d. The amine intermediates for the pyr-
rolidine (6), the azepane (7) and the azetidine (8) compounds were
synthesized in an analogous way.
Piperidine and piperazine secondary amine intermediates (for
compounds 1e4 and 11) were purchased from commercially
available sources. The syntheses of the non-purchasable 9c and 10c
intermediates are depicted in Scheme 2. N-BOC-nortropine (9a)
was arylated in a Mitsunobu reaction while 10a was alkylated with
4-methylbenzyl bromide and in both cases the BOC protecting
group was removed under acidic media resulting in derivatives 9c
or 10c, respectively.
limits the efficacy of
responsiveness (under extreme conditions,
a
7 nAChR agonists, and leads to reduced
7 nAChR agonists may
a
even behave as antagonists). Suboptimal in vivo efficacy of ligands
binding to the orthosteric site may well be attributed to desensi-
tization. The problem of fast receptor inactivation and the limited
agonist efficacy may be circumvented by using positive allosteric
modulators (PAMs). In the absence of endogenous ligands, proto-
typical PAMs normally lack any intrinsic activity, however, they can
enhance the effect of the orthosteric agonists. One extreme PAM
phenotype (generally described as “Type I”) predominantly affects
the peak current upon a7 nAChR activation with little or no effect
on desensitization kinetics [19,20] while the so called “Type II”
PAMs modulate not only the peak current but also the speed of
current decay due to the inhibition of desensitization [21]. This
terminology proved to be as an oversimplification as several PAMs
were described with intermediate characteristics. Positive modu-
lation of a7 nAChRs has been shown to have cognitive benefits in
various preclinical models [22,23]using both rodents [24,25] and
non-human primates [26]. Agonists were investigated extensively
2.1.3. The synthesis of oxoacetic acid intermediates 11c, 12d, 13g,
14b, 15h, 16b, 17b and 18e
in clinical trials [27,28]. Distribution of
a
7 nAChRs across brain
The synthesis of the oxoacetic acid intermediates was exem-
plified in the piperazine derivative 11c depicted in Scheme 3. The
amine intermediate 11a was reacted with ethyl chlorooxoacetate to
gain the oxoacetic ester derivative 11b. It was then hydrolyzed
under basic conditions to yield the oxoacid compound 11c. This
synthetic methodology was applied for the oxoacetic intermediates
of compounds 1e11 and 19e30.
areas associated with cognition and the association of CHRNA7
gene with inhibitory sensory gating deficit in schizophrenic pa-
tients suggest that nAChR activation may have benefits in the
treatment of schizophrenia. Some clinical observations in the past
two decades also point to this direction [29]. For representative
structures, see Fig. 1 (see Refs. [20,21,23,30e40]).
In our previous paper [41] a scaffold hopping approach to
identify a7 nAChR positive modulator compounds with procogni-
Different syntheses of the oxoacetic acid intermediates for
compounds 12e18 are also depicted in Scheme 3. 4-Piperidone
(12a) was N-alkylated with 1-bromomethyl-4-methylbenzene
and the resulted 12b was added to a solution of NaH and ethyl
isocyanoacetate yielding 12c. It was subjected first to acidic and
subsequently basic hydrolysis to obtain 12d. 6-Methylpyridin-3-ol
(13a) was reacted in a Mitsunobu reaction with cyclohexanol in-
termediate 13b. The ester functionality in 13c was hydrolyzed
(13d), followed by the reaction with cyano-phosphorane reagent
13e. The resulted 13f cyanoketo phosphorane was heated with
oxone to gain the methyl ester derivative 13g. For the synthesis of
14b, pyrazole was N-alkylated with p-chlorobenzyl bromide, then
the formed intermediate 14a was C-alkylated with ethyl 2-chloro-
2-oxoacetate. The synthesis of 15h started with the bromination of
ethyl 2-aminothiazole-4-carboxylate (15a) in a Sandmeyer reac-
tion. 15b was reacted with 4-chlorophenol and the ester function of
15c was reduced to aldehyde 15d with DIBAL-H. Cyanohydrin 15e
was formed in the reaction with methylmorpholine-N-oxide and
trimethylsilyl cyanide (TMSCN). The nitrile moiety in 15e was
subsequently hydrolyzed with cc. HCl to 2-hydroxycarboxylic acid
derivative 15f, which was then transformed to a methyl ester 15g.
tive potential was reported. Hereinafter we present the results of a
separate approach based on new chemical starting points emerging
from a high throughput screening (HTS) campaign. Modification of
an initial HTS hit series resulted in an advanced chemotype, that,
after further optimization, provided a novel, benzylic amine type of
a
7 nAChR positive modulator lead compound with improved
physico-chemical characteristics, favorable in vitro properties and
procognitive potential [42]. General formulas of the investigated
chemotypes and the main phases of the optimization process can
be seen in Fig. 2.
2. Results and discussion
2.1. Chemistry
2.1.1. The synthesis of new indole-amine intermediates 27e and 22e
Indolamine and aminomethyl-indole building blocks were
purchased from commercially available sources or their synthesis
was based either on the chemical literature (in case of 21, 24) or on
2

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European Journal of Medicinal Chemistry 222 (2021) 113560
Fig. 1. Selected positive allosteric modulators of
a
7 nAChR with highly variable degree of inhibition of the agonist-evoked channel desensitization. If available, the clinical status and
indication of the compounds are also indicated.
Fig. 2. The optimization process from the HTS hits to the Lead molecule with general and representative formulas of the investigated chemotypes.
Using Dess-Martin periodinane the desired 2-oxocarboxylic acid
methyl ester 15h was isolated. To have intermediate 16b, 16a was
Friedel-Crafts acylated with ethylchlorooxoacetate, while in case of
17b, the pyridine derivative 17a was lithiated and then reacted with
diethyloxalate to isolate 17b. The necessary hydrolysis of 13g, 14b,
15h, 16b and 17b was accomplished in an analogous way as
described in case of 11c. For the synthesis of 18e, dibromopyrazine
18a was reacted with p-cresol and then 18b was further reacted
with 1-ethoxy-1-(tributhylstannylethylene) (18c) using palladium
catalysis to obtain the acetyl derivative 18d. It was further oxidized
with SeO2 to yield the desired derivative.
2.1.4. Amide couplings for the synthesis of oxalic acid diamide
products (1e30)
The applied four different methods for the amide coupling to
obtain the desired, oxalic acid diamide final products (1e30) are
shown in Scheme 4. The synthesis of compounds 1e4, 9e12, 17e20
was achieved in an analogous way as it was exemplified via the
3

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European Journal of Medicinal Chemistry 222 (2021) 113560
Scheme 1. The synthesis of non-purchasable, novel indole-amine intermediates 22e and 27e. Reagents and conditions: (a) NaNO3, cc. H2SO4, 0 ꢀC; (b) Borane-THF, THF, 0 ꢀC to rt; (c)
MeI, NaH, dry THF, 0 ꢀC to rt.; (d) H2, Pd/C, MeOH, rt (e) Selectfluor, MeCN, cc. CH3COOH, 80 ꢀC; (f) benzylamine, NaOtBu, RuPhos, Pd2(dba)3, dry toluene, rfx.; (g) H2, Pd/C, MeOH,
EtOAc, rt.
Scheme 2. The synthesis for secondary amine intermediates 5d, 9c and 10c. Reagents and conditions: (a) methanesulfonyl chloride, Et3N, CH2Cl2, rt.; (b) p-cresol, Cs2CO3, DMF,
110 ꢀC; (c) TFA, CH2Cl2, 0 ꢀC; (d) p-cresol, PPh3, DIAD, THF, rt.; (e) HCl in 1,4-dioxane, 1,4-dioxane, rt; (f) 4-methylbenzyl bromide, MeCN, Et3N, 0 ꢀC to rt.
synthesis of 11. Its oxoacetic acid intermediate 11c was amidated
with the corresponding indole-amine 11d in the presence of 1-
hydroxybenzotriazole hydrate (HOBt$H2O) and of N-(3-
dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC.
HCl) with Et3N base.
The synthesis of compounds 6e7, 13e16, 21e26, 28e30 was
achieved in an analogous way as it was exemplified by the synthesis
of compound 23. The corresponding aryl-amine (23a) and the
oxoacetic acid derivative (11c) were reacted with the help of HATU
(Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium)
coupling agent in the presence of DIPEA base.
Compounds 5 and 8 were synthesized starting from the indole-
amine 11d, which was amidated with ethyl oxalyl chloride and the
isolated intermediate 8a was hydrolyzed to 8b. In this compound
the newly formed carboxylic acid functionality was amidated with
the corresponding azetidine or azetidinemethyl (5d) intermediates
in the presence of HBTU (N,N,N0,N0-tetramethyl-O-(1H-benzo-
triazol-1-yl)uronium hexafluorophosphate) and Et3N. For the syn-
thesis of compound 27 intermediate 11c was transformed into an
acid chloride with 1-chloro-N,N,2-trimethyl-1-propenylamine
(Ghosez's reagent) and the obtained activated derivative was
coupled with indole-amine (27e).
resulted 31c was hydrolyzed under basic conditions to obtain 31d.
For the synthesis of compound 31 intermediates 31d and 11a were
coupled with HATU/DIPEA system. Compound 32 was synthesized
from indole-carbonitrile 32a, which was first N-methylated to 32b
followed by conversion to carboxamide 32c with H2O2 and then
reacting with oxalyl chloride the crude benzoyl isocyanate deriva-
tive (32d) was yielded. Compound 32 was obtained in the reaction
of 32d with 4-(4-methylbenzyl)piperidine hydrochloride. Com-
pound 33 was synthesized from indole-carboxylic acid 33a, which
was transformed to the ester 33b which was then N-methylated
with dimethyl sulphate. Intermediate 33c was then converted to
hydrazide 33d and was further reacted with ethyl piperazine-
oxoacetate 11b to oxoacetohydrazide 33e in the presence of HBTU
as the coupling agent. The final compound 33 was yielded in a
cyclization reaction of 33e with thionyl chloride. The synthesis of
compound 34 started with the condensation reaction of 5-
acetylindole 34a with diethyl oxalate and then 34b was cyclized
with methylhydrazine to the pyrazole derivative 34c. The ester
functionality was hydrolyzed and the carboxylic acid 34d was
coupled with 1-(4-chlorobenzyl)piperazine in the presence of 1-
propanephosphonic acid cyclic anhydride (PPAA) to 34. For the
synthesis of compound 35, ethyl piperidine-4-carboxylate (35a)
was N-alkylated with the corresponding benzyl bromide and 35b
was transformed to hydrazide 35c. This derivative was then reacted
with ethyl oxalyl chloride to form the oxadiazole core in 35d and
after ester hydrolysis (using the same methodology as in case of
11b) carboxylic acid 35e was obtained. The amide coupling with
11d was accomplished with N,N,N0,N0-tetramethyl-O-(1H-
2.1.5. The syntheses of final compounds with modified linker region
(31e37)
The syntheses of compounds with modified linker region
(31e37) are depicted in Scheme 5. In a Suzuki reaction, 3-bromo-2-
propenoate 31a was reacted with indoleboronic acid 31b and the
4

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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
Scheme 3. The synthesis of oxoacetic acid intermediates 11c, 12d, 13g, 14b, 15h, 16b, 17b and 18e. Reagents and conditions: (a) ethyl chlorooxoacetate, Et3N, CH2Cl2, rt.; (b) NaOH,
EtOH, 0 ꢀC to rt; (c) DMF, K2CO3, 1-bromomethyl-4-methlybenzene, rt.; (d) NaH, dry THF, ethyl-isocyanoacetate, 0e5 ꢀC to rt.; (e) 1. EtOH, 10% HCl, 0e5 ꢀC to rt. 2. KOH, EtOH, water,
rt; (f) PPh3, DIAD, THF, 0 ꢀC; (g) MeOH, K2CO3, 70 ꢀC; (h) EDC, DMAP, CH2Cl2, 0 ꢀC; (i) oxone, CH2Cl2, MeOH, 40 ꢀC; (j) K2CO3, DMF, 0e80 ꢀC; (k) 2-chloro-2-oxoacetate, MeCN, 0 ꢀC to
rfx.; (l) MeCN, CuBr2, tBuONO, 0 ꢀC to rt.; (m) 1. 4-chlorophenol, THF, tBuOK, 0 ꢀC, 2. DMSO, 85 ꢀC; (n) CH2Cl2, DIBAL-H, citric acid, ꢁ70 ꢀC to rt.; (o) CH2Cl2, methylmorpholine-N-
oxide, TMSCN, 0 ꢀC to rt; (p) cc. HCl, rfx.; (q) MeOH, cc. H2SO4, 0 ꢀC to rt; (r) CH2Cl2, Dess-Martin periodinane, 0 ꢀC to rt; (s) ethylchlorooxoacetate, AlCl3, CHCl3, 0 ꢀC to rt; (t) toluene,
BuLi in hexane, diethyloxalate, ꢁ78 ꢀC; (u) p-cresol, K2CO3, DMF, rfx.; (v) toluene, Pd(PPh3)4, rfx; (w) SeO2, pyridine, 70 ꢀC.
benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) and Et3N
to isolate 35. Compound 36 was synthesized from methyl 2-bromo-
3-methoxypropanoate (36a) with 4-(4-methylphenoxy)piperidine.
The resulted methyl ester 36b was hydrolyzed to the acid com-
pound 36c, which was subsequently amidated with 11d in the
presence of HATU/DIPEA system to yield compound 36. Compound
37 was synthesized in the reaction of 1-methyl-1H-indole-5-
carbonyl azide (37a) and 1-[(4-methylphenyl)methyl]piperidin-4-
amine (37b). The syntheses of the aminomethylindole derivatives
38, 45e47 were exemplified in case of 38. The aminomethylindole
derivative 38a was coupled with the carboxylic acid 38b using the
corresponding coupling agent. The synthesis of derivatives 39e44
and 48e55 were exemplified in case of compound 44. The ami-
nomethylindole derivative 38a was activated with 4-nitrophenyl
chloroformate and the resulted active carbamate was one-pot
further reacted with 3-{[4-(trifluoromethyl)phenoxy]methyl}
5

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European Journal of Medicinal Chemistry 222 (2021) 113560
Scheme 4. Amide couplings for the synthesis of oxalic acid diamide derivatives (1e30). Reagents and conditions: (a) 1-hydroxybenzotriazole hydrate (HOBt$H2O), N-(3-
dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC. HCl), DMF, Et3N; (b) 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexa-
fluorophosphate (HATU), DMF, DIPEA, rt., (c) DIPEA, CH2Cl2, ethyl oxalyl chloride, 0 ꢀC to rt; (d) LiOH$H2O, water, MeOH, rt.; (e) amine, CH2Cl2, N,N,N0,N0-tetramethyl-O-(1H-
benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), Et3N, rt.; (f) DMF, 1-chloro-N,N, 2-trimethyl-1-propenylamine (Ghosez's reagent), rt.
azetidine (5d).
nitrogen in the connection point. In certain cases e such as the
saturated reversed piperidine (12) and cyclohexyl (13) derivatives
e moderate, while in the case of the 5-membered heteroaromatic
analogues (14, 15), suboptimal or even complete lack of activity was
found. On the other hand, the aromatic phenyl, pyridyl and pyr-
azine derivatives (16, 17, 18) showed significant activity. Further-
more, the pyridyl analogue 17 was active not just in vitro but also
showed in vivo efficacy in the mouse place recognition test (Fig. 3).
Unfortunately, drug-likeness of these planar structures with four
aromatic rings seemed to be suboptimal as they had low solubility,
high logP with no improvement in metabolic stability. Therefore,
the less planar piperazine-based compound 11 seemed the most
favorable choice. It showed a considerably balanced profile and
fulfilled the criteria of an advanced hit molecule and it was sub-
jected to further characterization.
2.2. Identification of the oxalic acid diamide scaffold
In order to identify structurally novel positive modulators of the
human
collection was undertaken utilizing a highly sensitive in vitro
functional [Ca2þ
assay. Multiple hit series were identified, in
a7 nAChR, a HTS campaign of our corporate compound
]
i
particular the presence of HTS hits 1, 2 lead to robust [Ca2þ]i re-
sponses upon agonist addition in the primary assay (Table 1.).
During hit expansion two structurally close, methylated analogues
were also identified as active. Though the 2-methyl derivative 3
showed complete inactivity, the nitrogen methylation in compound
4 exerted beneficial impact on the activity. Furthermore, the in vivo
evaluation of this compound revealed procognitive effects in the
mouse place recognition test at 3 mg/kg with appropriate brain
penetration, indicating optimization potential (Fig. 3).
2.3. Biological characterization of the advanced hit molecule 11
Based on these promising initial findings comprehensive SAR
evaluation of the central core was initiated and versatile saturated
and aromatic rings were applied. Basic physico-chemical and
in vitro characteristics of the synthesized derivatives are presented
in Table 1. In case of the azetidinemethyl (5) analogue, moderate
activity was detected. Unfortunately, introduction of the pyrroli-
dine (6), azepine (7) and azetidine (8) structural elements resulted
in suboptimal activity while the usage of nortropine (9) or a
dimethylated piperazine building blocks (10) led to complete
inactivity. Interestingly, the non-substituted piperazine derivative
(11) proved to be very potent. The in vitro metabolic stabilities
characterized with intrinsic clearance (CLint) values in human and
mouse microsomes remained in the acceptable range, while the
metabolic stability in rat microsomes was rather low. While
searching for “metabolic soft points”, we found that certain inactive
derivatives lacking the nitrogen on right-hand side amide part
showed higher metabolic stability (data not shown). Therefore, the
piperazine central ring was attempted to be replaced with several
5- and 6-membered aromatic and saturated rings not having a
The piperazine-centered chemotype's chemical accessibility
was excellent, and 11 showed proper physicochemical parameters
with appropriate kinetic solubility and experimental logP values
(41.6 mM and 3.7, respectively). Compound 11 showed robust ac-
tivity in the functional assay with an EC50 value of 90 nM. Using
patch clamp a considerable inhibition of the choline-evoked cur-
rent desensitization and enhancement of peak current amplitude
was observed with an EC50 value of 710 nM (Fig. 4., upper panel).
The compound proved to be highly selective in a set of
displacement binding assays for 68 molecular targets, showing
affinity to 5-HT2B and sigma-1 receptors (98 and 55% displacement
at 10
displacement at 10
m
M, respectively). Importantly, 11 did not show >15%
M in binding assays relevant for ligand affinity
m
to 5-HT3 receptors (human [3H] GR-65630 binding in recombinant
HEK-293 cells), NMDA receptors ([3H]kainic acid, [3H]MDL105,519,
[3H]CGP-39653 or [3H]TCP binding in Wistar rat cerebral cortex
preparations) or GABAA receptors ([3H]flunitrazepam or [3H]mus-
cimol binding in Wistar Rat brain preparations minus cerebellum).
6

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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
Scheme 5. The syntheses of compounds with modified linker region (31e37 and 44). Reagents and conditions: (a) K2CO3, Pd(PPh3)4, 1,4-dioxane, 90 ꢀC; (b) EtOH, 1,4-dioxane, sat.
NaHCO3, rfx.; (c) 11a, HATU, DIPEA, DMF, rt.; (d) MeI, NaH, dry THF, 0 ꢀC to rt.; (e) 30% aq. H2O2 sol., K2CO3, DMSO, rt.; (f) oxalyl chloride, CH2Cl2, 0 ꢀC to rfx., (g) 4-(4-methylbenzyl)
piperidine hydrochloride, Et3N, 1,2-dichloroethane, rt.; (h) EtOH, cc. H2SO4, rfx., (i) MeCN, K2CO3, Me2SO4, 95 ꢀC; (j) EtOH, hydrazine hydrate, rfx., (k) 11b, CH2Cl2, Et3N, HBTU, rt.; (l)
1,4-dioxane, Et3N, SOCl2, 100 ꢀC; (m) Na, EtOH, 0e5 ꢀC, diethyl oxalate, rfx., (n) EtOH, methylhydrazine, rfx., (o) EtOH, water, KOH, rt., (p) 1-(4-chlorobenzyl)piperazine, CH2Cl2, Et3N,
1-propanephosphonic acid cyclic anhydride 50% in EtOAc, rt., (q) DMF, K2CO3, 1-(bromomethyl)-4-methylbenzene, 80 ꢀC, (r) hydrazine hydrate, 120 ꢀC, (s) 1. CH2Cl2, Et3N, ethyl
oxalyl chloride, rt., (t) NaOH, EtOH, 0 ꢀC to rt.; (u) 11d (in case of 35) or 38a (in case of 38), CH2Cl2, Et3N, HBTU, rt.; (v) 4-(4-methylphenoxy)piperidine hydrochloride, K2CO3, DMF,
90 ꢀC; (w) MeOH, water, LiOH$H2O, rt., (x) 11d, HATU, DIPEA, DMF, rt.; (y) toluene, rfx; (z) 1. CH2Cl2, DIPEA, 0 ꢀC, 2. 5d, CH2Cl2, rt.
To further characterize the effects of 11 with respect to functional
selectivity, electrophysiological measurements were done in cells
(Dr. Hugh Chapman, personal communication. For further details,
see Table S4. in the Supplementary Materials. Metabolic stability
remained in the medium-to-low range both in human and rodent
expressing recombinant human
revealed no effect.11 exhibited neither inhibition nor enhancement
of the 1 M GABA-evoked currents (effects were <7% in 1 or 10 M)
in HEK-293 cells expressing 2 or
2 GABAARs (n ¼ 5e9).
In addition, 11 showed no agonism (up to 30 M) or PAM activity
(up to 10 M) in 4 nACh-expressing cells in QPatch experiments
a1 and a5 GABAA receptors that
microsomes (with CLint of 15, 158 and 55
mL/min/mg protein in
m
m
human, rat and mouse microsome preparations, respectively, while
its penetration through vinblastine-selected Caco-2 (VB-Caco-2)
monolayers expressing high level of P-gp [43] was excellent with a
a
1
b3
g
a5b3g
m
m
a3
b
PDR value of 0.7 and PappA-B of 30.7 ꢂ 10ꢁ6 cm sꢁ1 (tested @10
mM),
7

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European Journal of Medicinal Chemistry 222 (2021) 113560
Table 1
The piperidine-based HTS hits and derivatives with modified central core (aromatic and aliphatic central rings).
Kin. sol.a
(
m
M)
cx_logP
[Ca2þ]i EC50 (nM) or % response at 1* or 10
m
M
CLint
m
L/min/mg protein)
a
Cmpd
R
L
1
H
14.8
4.2
910
23/148/242
2
3b
4
H
43.5
2.8
8.7
1.8
6.7
7.7
3.1
4.6
3.4
3.4
3.3
3.8
260
inact.
120
18/138/184
H
19/160/112
Me
Me
Me
Me
19/74/78
5
3000
25%
e
e
e
6
7
13%*
8
9
Me
H
0.1
8.1
3.3
3.6
47%
e
inact.
12/61/40
10
H
14.9
3.9
inact.
2/ꢁ/ꢁ
11 Advanced hit
Me
Me
Me
Me
Me
Me
41.6
24.8
e
3.3
4.8
3.9
4.4
5.5
5.5
90
600
670
52%
inact.
60
15/158/55
28/42/169
133/65/64
e
12
13c
14d
15d
16
3.0
0.1
2.0
e
34/48/46
17
18
Me
Me
3.0
4.9
3.9
130
650
51/59/33
10.0
90/127/156
a
Kin. sol.: kinetic solubility; cx_logP: calculated logP; CLint.: intrinsic clearance measured in isolated microsomes; h: human; r: rat; m: mouse.
On the indole ring position 2 was methylated.
On the right-hand side of the molecule, 6-methyl-pyridin-4-yl was used, instead of the 4-methylphenyl moiety.
b
c
d
On the right-hand side of the molecule, 4-chlorophenyl was used, instead of the 4-methylphenyl moiety. (Since the chlorine substitution provided equipotency with the
methyl one (44 vs 49 in Table 5.), in case of the 4-methyl substituted derivatives the activities most probably would still not be high enough.).
respectively. Inhibitory activity of 11 on the hERG channel activity
remained also in the acceptable range with an IC50 value of 7.5 M.
The compound, furthermore, resulted in significant alleviation of
test in the dose range of 1e10 mg/kg, but remained ineffective in
m
the rat novel object recognition paradigm (Fig. 4., middle panel).
Brain-to-plasma ratios were favorable (above 1.2 and 3.8 in mice
and rats, respectively), as revealed by pharmacokinetic analysis of
the scopolamine-induced amnesia in the mouse place recognition
8

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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
Fig. 3. Upper panel: Effect of 4 and 17 on [Ca2þ]i in the presence of an agonists. Middle panel: Impact on recognition index of the ip. administration of 4 and 17 on the 1 mg/kg (ip.)
scopolamine-induced amnesia in the mouse place recognition test. þ p < 0.05 (ANOVA, followed by post-hoc Dunnett-test). Lower panel: results of the pilot pharmacokinetic
measurement from brain and plasma samples taken from the experimental animals.
the samples taken from the experimental animals in both cognitive
tests (Fig. 4., lower panel).
with no genotoxic liability. According to the initial hypothesis, the
different substituents may differently affect the electron density
both on the indole ring and the amine moiety, consequently they
may differently influence the resulting compounds’ behavior in
these regards. Several, non-purchasable, substituted amino-indole/
azaindole derivatives were synthesized, however only few active
ones (19e27) were found due to the steep SAR. Table 2 summarizes
2.4. Optimization in the selected structural regions
However, besides the mixed cognitive profile, another disad-
vantageous feature also emerged. Despite the fact that 11 did not
show any genotoxic liability in the Ames test and the presence of
the corresponding indole-amine building block was never experi-
enced in vitro during the stability measurements, putative in vivo
formation of this aromatic amine metabolite could not be excluded
(according to the Benigni/Bossa rulebase there was some risk for
mutagenicity and carcinogenicity [44]). However, numerous
ethical, biological, and technical difficulties dissuaded us from the
demonstration of the formation of this metabolite in vivo. Another
problematic feature identified was the chemically reactive nature
of the indole motif. Therefore, the focus of the hit-to-lead optimi-
zation campaign was put on either the modification/replacement of
the non-druglike, indole structural element (Region I.) or the total
omission of the problematic aromatic-amine (indole-amine) moi-
ety through modification of the oxalic acid diamide structural
element (Region II.). (Fig. 5.). These approaches were mapped with
focused libraries using conventional and parallel syntheses.
only the active derivatives of this synthetic campaign, where [Ca2þ
]
i
responses were ꢃ10% at 10
mM or higher.
Contrary to the results of monomethylation in position R1 (11),
the dimethylated analogues (R1, R3, as well as R1, R2: 19 and 20,
respectively), along with the 6-aminoindole (21) and 6-
fluoroindole derivatives (22) showed suboptimal functional activ-
ity. However, substitution of the indole at position 3 (R3) or 7 (R7)
with halogens e especially with fluorine e resulted in compounds
of robust activity (23, 24, 25): their activity proved to be compa-
rable with the lead (11) itself. In contrast, however, no improve-
ment in metabolic stability was achieved with these modifications.
In addition to that the in silico evaluation of the corresponding
indolamine building blocks indicated some risk of mutagenic lia-
bility for all tested intermediates (using the Toxtree software [45],
working with the Benigni/Bossa rules [44]. See results in Supple-
mentary Material, in Table S1.)
Next, a diverse set of more druglike (less lipophilic, less reactive)
azaindole-amine building blocks were synthesized and applied in
coupling reactions (Fig. 6). Among them, only the 7-aza derivatives
showed activity in the [Ca2þ]i assay that was accompanied with
somewhat improved rodent metabolic stability (26, 27).
2.4.1. Optimization in region I
2.4.1.1. The usage of modified indole building blocks. First, versatile
substitution patterns were applied on the indole ring with the aim
of identifying indole-amines with improved metabolic stability and
9

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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
Fig. 4. Upper panel: In vitro characterization of the effect of 11. On the left side, concentration-response curves measured in the [Ca2þ]i assay (upper figure) or in electrophysiological
experiments (lower figure) are depicted. On the right side, electrophysiological characterization of the effect of 11 on the choline-induced current is demonstrated. Note the robust
potentiation of the small, choline-induced current by 11. Middle panel: Impact on recognition index of the ip. administration of 11 on the 1 mg/kg (ip.) scopolamine-induced amnesia
in the mouse place recognition test (left) and on the delay-induced natural forgetting in the rat novel object recognition paradigm (right). þ p < 0.05; þþ p < 0.01; þþþ p < 0.001
(ANOVA, followed by post-hoc Dunnett-test). Lower panel: results of the pilot pharmacokinetic measurement from brain and plasma samples taken from the experimental animals
in place recognition (mouse) and novel object recognition (rat) models.
literature data) non-genotoxic aromatic amine building block li-
braries were applied in a parallel synthetic setup. Although the very
steep SAR e and consequently, the low hit rate e is a widely known
phenomenon in the field of allosteric modulators, it was still a
surprise that only two of the approximately 200 synthesized de-
rivatives (see a summary about the structures in the Supplemen-
tary Material, in Fig. S1.) exhibited significant in vitro activity.
Compounds 28 (an indolizine analogue) and 29 (an indoline
analogue) were identified as the most active ones, however, the
activity remained in the non-acceptable (moderate) range ((See 28
and 29 in Table 2 and other 5 structures with subtle, however valid
Fig. 5. The selected structural regions for optimization of the lead molecule 11.
activity can be seen in Supplementary Material in Table S2.).
Summarizing the results of the optimization in Region I, it could
be stated that the bioisoteric replacement of the indole building
block seemed impossible, since only slight modifications of the
substitution pattern were tolerated (and even in these cases the
putative in vivo formation of mutagenic intermediates could not be
excluded).
Unfortunately, the 4- and 5-azaindole derivatives showed no ac-
tivity. Furthermore, significant amount of inactive substituted
indole derivatives was also identified: their lack of activity high-
lighted the steepness of the observed SAR and lead us to abandon
this approach.
2.4.1.2. The usage of non-indole aromatic and non-aromatic amine
building blocks. Second, replacement of the entire indole moiety
was attempted. Versatile non-aromatic and putatively (based on
10

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European Journal of Medicinal Chemistry 222 (2021) 113560
Table 2
Modification of Region I of the advanced hit 11. Active indole/azaindole and non-indole derivatives.
R1
R2
R3
R6
R7
X
Connec. point
Kin.a sol. (
m
M)
cx_ logPa
[Ca2þ]i EC50 (nM) or % response at 10
m
M
CLint
m
L/min/mg protein)
a
Cmpd
19
20
21
22
23
24
25
26
27
28
Me
H
H
Me
H
H
H
H
H
H
H
Me
Me
F
H
F
Cl
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
F
C
C
C
C
C
C
C
N
N
5
5
6
5
5
5
5
5
5
5.0
23.0
12.5
47.1
16.5
6.8
42.7
47.4
e
3.8
3.9
3.5
3.5
3.5
3.9
3.2
2.4
2.6
2.9
920
10%
1400
900
85
420
110
400
210
3300
17/140/198
13/56/151
e
Me
Me
Me
Me
H
e
21/192/214
e
17/130/151
0.3/56/46
12/41/39
e
Me
Me
H
H
F
5.6
29
95.0
3.2
820
71/23/18
a
Kin. sol.: kinetic solubility; cx_logP: calculated logP; CLint.: intrinsic clearance measured in isolated microsomes; h: human; r: rat; m: mouse.
2.4.2. Optimization in region II. Trials to modify the oxalic acid
diamide linker and the identification a benzylic-amine type
chemotype
extended the available chemical space, optimization of the struc-
tures led to inactivity in almost all cases (See in the Supplementary
material in Table S3.). Replacement of the right-hand side amide
moiety by an oxadiazole ring (35) resulted also inactivity. Finally,
several oxo-group replacement methodologies were attempted,
however, due to synthetic difficulties only two inactive analogues, a
methoxymethyl (36) and a urea derivative (37) were successfully
synthesized.
The experienced SAR in Region II proved to be so steep that the
identification of the highly active benzyl-amine derivative 38
happened to be a serendipitous finding instead of the originally
planned oxo-carboxylic acid derivative. 38 proved to be our first,
in vitro active derivative lacking the problematic aromatic amine
structural element. Evaluating compound 38 and the applied 1-(1-
methylindol-5-yl)methanamine (38a) building block no structural
Omission of the problematic aromatic-amine (indole-amine)
moiety through modification of the oxalic acid diamide linker was
attempted via the synthesis of several interesting molecules (see
the derivatives in Table 3.). Synthesis of the lengthier amino-
methylindole derivative 30 was also accomplished, but it proved to
be inactive. The replacement of the amine with a methylene (31)
and the usage of the reversed-type amide (32) were attempted. As a
result, significant activity loss was experienced in both cases.
Furthermore, different amide bioisosteric heterocyclic rings were
applied. Two of the resulting compounds e 33 (an oxadiazole de-
rivative) and 34 (a methyl-pyrazole derivative) e showed moderate
activity. Despite that these new chemotypes should have further
Fig. 6. Selected inactive substituted indole and azaindole derivatives (para-methyl/chlorine substituted piperazine analogues).
11

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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
Table 3
Modifications of Region II. Trials for the elimination of aromatic amine building block.
Cmpd
Structure
[Ca2þ]i EC50 (nM) or % response at 10
mM
30
inact.
31
32
7%
37%
33
34
660
2500
35
36
inact.
inact.
37
38
inact.
40
alerts were estimated by the Toxtree software (see results in Sup-
plementary Material, in Table S1). According to this promising
result, all further efforts were concentrated on the optimization of
this newly identified chemotype.
pyrrolidine (42), pyrrolidinemethyl (43) and azetidinemethyl (44)
derivatives e were synthesized and found to be active (only a few
analogues e namely the reversed piperidine (45), trans-cyclohexyl
(46) and the nipecotic acid (47) derivatives e showed moderate or
no activity). The results are summarized in Table 4.
Further optimization was performed using the most potent
azetidinemethyl series, aiming at mainly the improvement of the
metabolic stability. As the methyl substituent on the right-hand
side phenyl ring seemed to be one of the “metabolic soft points”
e, the evaluation of the phenyl substitution pattern was achieved.
The p-fluorine (48), p-chlorine (49) p-methoxy (50), p-tri-
fluoromethoxy (51) and even p-hydrogen (52) derivatives showed
potent in vitro efficacy, however, their metabolic stabilities proved
2.5. Optimization of the newly identified, aminomethylindole
cluster
Based on the high level of structural similarity between the new
and the original chemotypes, it was hypothesized that the previ-
ously identified SAR (in Regions I and II) could at least partly be
utilized in this new chemotype. Indeed, several new analogues e
such as the piperidine (39), piperazine (40), azetidine (41),
12

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European Journal of Medicinal Chemistry 222 (2021) 113560
Table 4
Derivatives of the new benzylic amine chemotype with versatile central cores.
Kin. sol.a
(m
M)
cx_logPa
[Ca2þ]i EC50 (nM) or % response at 1* or 10
m
M
CLint
m
L/min/mg protein)
a
Cmpd
Z
39
12.6
3.7
210
130/ꢁ/ꢁ
40
18.9
3.6
490
24/ꢁ/ꢁ
41
42
43
26.8
28.8
15.9
3.5
3.6
3.7
620
170
280
128/ꢁ/ꢁ
89/ꢁ/ꢁ
265/ꢁ/ꢁ
44
45
34.7
93.8
3.6
6.5
40
161/93/80
12%
e
46
47
5.5
4.9
4.2
16%*
e
e
84.1
inact.
a
Kin. sol.: kinetic solubility; cx_logP: calculated logP; CLint.: intrinsic clearance measured in isolated microsomes; h: human; r: rat; m: mouse.
Table 5
Evaluation of the phenyl substitution pattern in derivatives with azetidinemethyl central core.
Kin. Sol.a
(
mM)
cx_logPa
[Ca2þ]i EC50 (nM)
CLint m
L/min/mg protein)
a
ID
R
44
48
49
50
51
52
53
54
55b
CH3
F
Cl
OCH3
OCF3
H
CN
CF3
CF3
34.7
1.6
0.7
42.3
0.4
18.0
5.0
3.6
3.2
3.6
2.8
4.8
3.1
2.9
4.0
3.7
40
220
55
60
25
180
350
80
161/93/80
43/ꢁ/ꢁ
72/ꢁ/ꢁ
348/ꢁ/ꢁ
23/100/109
19/134/91
18/39/45
19/42/45
22/56/58
0.1
18.4
160
4.3 (meas.)
a
Kin. sol.: kinetic solubility; cx_logP: calculated logP; CLint.: intrinsic clearance measured in isolated microsomes; h: human; r: rat; m: mouse.
On the left-hand side of the structure the indole nitrogen was not methylated.
b
to be suboptimal. The highest metabolic stability was experienced
improved kinetic solubility with retained activity and moderate
metabolic stability. (In cases of 55 and its non-methylated 1-(1H-
indol-5-yl)methanamine (55a) building block there were also no in
silico toxicity alerts estimated (see results in Supplementary Ma-
terial, in Table S1).
with the p-nitrile derivative 53, however with decreased activity.
The usage of p-trifluororomethyl group (54) resulted in somewhat
improved rodent metabolic stability with highly retained potency,
but the compound had very poor solubility (Table 5.). Fortunately,
the non-methylated (NeH) indole derivative provided enough
balance between these two features: compound 55 showed
13

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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
2.6. Docking study for the determination of the most probable
binding site
allosteric modulator PNU-120596) could not unequivocally clarify
the location of the binding site [50]. To order to determine the most
probable binding sites of our derivatives, the SiteMap module of the
€
€
The possible binding site of
a
7 nAChR PAMs is suggested to be in
Schrodinger software package was applied (Schrodinger Release
€
the transmembrane region of the homopentameric channel.
However, it is not entirely clear whether the PAMs bind within a
subunit or between two subunits of the ion-channel. Earlier studies
based on site-directed mutagenesis complemented by homology
modeling proposed an intrasubunit binding site located between
2020e2: Schrodinger, LLC, New York, NY, 2020. Using the recently
published open-state alpha-7 nicotinic acetylcholine receptor
structure (PDB ID: 7KOX [50]), we were able to identify all above-
mentioned allosteric binding sites (II-IV) and an additional
external lipophilic site (V) was also found (see them in Fig. 7.). (It
should be noted that other sites (mainly in the extracellular
domain) were also found, however they had no relevance, either
because of their size or their highly hydrophilic character.)
Induced-fit docking [51] of compound 55 was carried out using the
allosteric binding sites (II-IV). According to the obtained docking
scores (III: ꢁ12.4 vs. II: ꢁ7.2, IV: ꢁ9.1, V: ꢁ10.8, see in Supple-
mentary Material, in Figure S2.), the intersubunit binding site (III)
seemed to be the most favorable. Furthermore, this binding site
provided specific ligand-protein interactions such as a hydrogen
bond between the carbonyl oxygen of 55 and the S248 residue and
the four transmembrane a-helices for both type I- and type II-like
positive allosteric modulators [19,46]. On the other hand, another
study [47] showed that a specific mutation of M260 to leucine only
type II-like PAMs were converted into non-desensitizing agonists.
These unexpected results pointed out that either the binding site
and/or the binding modes of nAChR allosteric modulators needed
to be revisited. It was also reported that different mutations could
have different effect on the functional character of the ligands even
in case of chemically similar type II-like PAMs [48]. The obtained
results suggested that in the case of several of the investigated type
II-like PAMs, the intersubunit site was more favored. In addition to
that, in case of the ago-PAM GAT107, a second binding site (DAA
site) located at the extracellular domain vestibule of the channel
was proposed [35,49] with the assumption to be responsible for
direct allosteric activation. Unfortunately, even the recently pub-
a
p-p interaction between the ligand and the F252 residue. In
parallel a lipophilic site formed by some apolar residues (e.g. L220,
A223, I279 and L283) was found which was suitable to accommo-
date the substituted phenyl ring of the ligand. With these in-
teractions, the molecule was complexed in a low energy elongated
conformation. (For other binding sites, the best obtained poses can
be seen in Supplementary Material, in Figure S2.) To gain further
lished results using the experimental cryo-EM structures of
a7
nAChR (stabilized by the agonist epibatidine and the positive
Fig. 7. Binding sites. A) The most relevant binding sites determined with SiteMap: I: Neurotransmitter site, II: DAA site [44], III: Intersubunit site, IV: Intrasubunit site, V: External
lipophilic site; B) Refined intersubunit site (III): Compound 55 is placed with the induced-fit protocol.
14

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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
confirmation, compounds 1e55 were docked into these refined
binding sites (IIeV) using the standard precision protocol in Glide
[52,53]. The compounds were scored and ranked, and significantly
more favorable docking scores were obtained for the active com-
pounds (pEC50>5) using the intersubunit binding site model III,
than with the other ones. Furthermore, the model was able to
enrich the active compounds based on the receiver operating
characteristic (ROC) curves (see in Supplementary Material, in
Figure S3.).
putative formation of mutagenic metabolites could not be
excluded.
Second, replacement of the entire indole moiety was attempted.
Diverse non-aromatic and putatively non-genotoxic aromatic
amine building block libraries were applied in a parallel synthetic
setup. Due to the very steep SAR only two derivatives (28e29)
exhibited significant activity.
Third, the omission of the problematic aromatic-amine (indole-
amine) moiety through the modification of the oxalic acid diamide
linker was attempted via the synthesis of new compounds (30e38)
and as a result a novel, aminomethylindole compound family
(38e55) emerged, in which the applied benzylic amine building
blocks possessed no risk for mutagenicity. In addition to that,
detailed docking studies using the recently published open-state
alpha-7 nicotinic acetylcholine receptor structure were carried
out, revealing four possible allosteric binding sites, among them the
intersubunit site seems to be the most probable for our compounds.
The most balanced physico-chemical and pharmacological profiles
were experienced in case of the azetidinemethyl derivative 55,
which demonstrated favorable cognitive enhancing profile not only
in cognitive models involving scopolamine-induced amnesia (place
recognition test in mice) but also natural forgetting (novel object
recognition test in rats). Compound 55 was, furthermore, active in a
cognitive paradigm of higher translational value, namely in the rat
touch screen visual discrimination test. Therefore, 55 was selected
as a lead compound for further optimization.
2.7. Biological evaluation of the best azetidinemethyl derivative 55
Compound 55 was selected for further detailed characterization
on the basis of its excellent functional activity ([Ca2þ]i EC50 of
160 nM) with a robust inhibition of response desensitization
(Fig. 8., upper panel) and enhancement of peak current amplitude
(EC50 ¼ 590 nM) and acceptable metabolic stability (with CLint
values between 22 and 58). The efflux ratio (PDR) of 55 was 1.3
(tested at 1
mM) indicating no efflux liability of the compound
(PappA-B value of 55 ꢂ 10ꢁ6 cm sꢁ1). The compound e investigated
in a panel of commercially available binding assays of 68 molecular
targetse did not show serious off-target liabilities, as >50% ligand
displacement was seen only for NET, DAT and 5-HT2B at 10 mM (For
further details, see Table S4. in the Supplementary Materials).
Furthermore, 55 did not show any mutagenicity in the Ames test
and its hERG IC50 value remained in the acceptable range (4.6 mM).
The compound showed not only in vitro activity, but also significant
efficacy in rodent spatial memory tests in vivo. Following ip.
administration, it alleviated the scopolamine-induced amnesia or
the natural forgetting in the mouse place recognition vs. the rat
novel object recognition tests, respectively (Fig. 8., middle panel)
with a minimal effective dose of 1 mg/kg in both species. Analysis of
brain and blood samples taken from the experimental animals
were also carried out (for results, see Fig. 8., middle panel), where
relatively high expositions with favorable B/P ratios were measured
in both species. The compound, furthermore, showed significant
reversal of the 0.075 mg/kg (sc.) scopolamine-induced amnesia in
the rat touch screen visual discrimination test (Fig. 8., lower panel),
a cognitive model with high translational value. Based on all the
above findings, 55 e fulfilling all lead criteria e was selected to be
our lead molecule for future optimization.
Despite the experienced steep SAR, the identified structural el-
ements provided a chance for further optimizing the lead com-
pound. Based on all these considerations, cognitive enhancement
through positive modulation of a7 nAChRs using compounds from
the above described chemotype may seem a viable approach.
4. Experimental section
4.1. Chemistry and chemical methods
Commercially available reagents and reactants were used
without further purification. Solvents and gases were dried ac-
cording to standard procedures. Organic solvents were evaporated
with reduced pressure using a rotary evaporator.
Purity of the final compounds was verified using an Agilent
1200 HPLC system equipped with a diode array detector and with
an Agilent 6410 triple quadrupole mass spectrometer (QQQ-MS).
Eluent A was 0.1% TFA in water and eluent B was the mixture of
acetonitrile and water at 95:5 with 0.1% TFA. The mobile phase flow
rate was 1.2 mL/min and the applied linear gradient profile was:
0e4 min 0ꢁ100% B, 6 min 100%B, 6.01 min 0% B. Chromatograms
were recorded at 220 4 nm and the applied injection volume was
3. Conclusions
Summarizing the above results, a promising new chemotype
with good physicochemical and in vitro parameters was identified
by an HTS-based approach. A novel, piperidine centered, oxalic acid
diamide a7 nACh receptor positive modulator chemotype (1, 2) was
found. A close derivative 4 showed not just improved in vitro but
also in vivo activity (e.g. in the mouse place recognition test). During
hit expansion, the modification of the central core was investigated
(5e18) and as a result, the saturated piperazine ring-centered 11,
the aromatic, phenyl ring-centered (16) and the pyridyl ring-
centered (17) in vitro potent derivatives were highlighted and 11
and 17 demonstrated in vivo efficacy in the mouse place recognition
test as well. Especially, the less planar compound 11 proved to be a
promising derivative having the most balanced physicochemical
and pharmacological profiles fulfilling the criteria of an advanced
hit molecule.
The focus of the hit-to-lead optimization campaign was put on
three synthetic approaches. First, versatile substitution patterns
were applied on the indole ring with the aim of identifying de-
rivatives with improved metabolic stability and building blocks
possessing no genotoxic liability. Unfortunately, only few active
derivatives (19e27) were found and even in these cases the
1.0 mL. MassHunter Workstation software (B.03.01) was used for
data acquisition and processing. Chromatographic analyses were
carried out at 40 ꢀC on an Ascentis Express C18 50 ꢂ 3.0 mm, 2.7
mm
(Sigma-Aldrich) column. The QQQ-MS operating parameters were
the following: scanning in positive ionization (ESIþ) mode, drying
gas temperature of 350 ꢀC, nitrogen flow rate of 12 L/min, nebulizer
pressure of 45 psi, capillary voltage of 4 kV, fragmentor voltage
135 V.
1H NMR and 13C NMR measurements were performed on a
Bruker Avance III HDX 400 MHz spectrometer equipped with
15Ne31P{1He19F} 5 mm CryoProbe Prodigy and a Bruker Avance III
HDX 500 MHz NMR spectrometer equipped with 1H {13C/15N}
5 mm TCI CryoProbe (Bruker Corporation, Billerica, MA, USA).
Chemical shifts are given on the delta scale as parts per million
(ppm) relative to tetramethylsilane (TMS) (1H: 0.00 ppm, 13C:
0.0 ppm), coupling constants are given in Hz. All experiments were
performed at 298 K. NMR spectra were processed using Bruker
15

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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
Fig. 8. Upper panel: In vitro characterization of the effect of 55. On the left side, concentration-response curves measured in the [Ca2þ]i assay (upper figure) or in electrophysiological
experiments (lower figure) are depicted. On the right side, electrophysiological characterization of the effect of 55 on the choline-induced current is demonstrated. Note the robust
potentiation of the small, choline-induced current by 55. Middle left panel: Effect of ip. administration of 55 on the 1 mg/kg (ip.) scopolamine-induced amnesia in the mouse place
recognition test. þ p < 0.05, þþ p < 0.01, þþþ p < 0.001 (ANOVA, followed by post-hoc Dunnett-test). Middle right panel: Effect of ip. administration of 55 on the natural forgetting
in the rat novel object recognition test. þ p < 0.05 (ANOVA, followed by post-hoc Dunnett-test). Results of the pilot pharmacokinetic measurements from brain and plasma samples
taken from the experimental animals are also included. Lower panel: Effect of 55 on the 0.075 mg/kg (sc.) scopolamine-induced amnesia in the rat touch screen visual discrimination
test (
D
correct%). *p < 0.05 and **p < 0.01 represent Dunn's post hoc test results following significant Kruskal-Wallis test (H ¼ 13.49, p < 0.05).
16

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European Journal of Medicinal Chemistry 222 (2021) 113560
TopSpin 3.5 pl 7 (Bruker Corporation, Billerica, MA, USA) and ACD/
Spectrus Processor version 2017.1.3 (Advanced Chemistry Devel-
opment, Inc., Toronto, ON, Canada).
(DMSO‑d6, 500 MHz) d 10.55 (s, 1H), 7.9e7.9 (m, 1H), 7.4e7.4 (m,
1H), 7.3e7.3 (m, 2H), 7.1e7.1 (m, 2H), 6.9e6.9 (m, 2H), 6.39 (dd, 1H,
J ¼ 0.8, 3.1 Hz), 4.62 (tt, 1H, J ¼ 3.6, 7.6 Hz), 3.8e3.9 (m, 1H), 3.77 (s,
3H), 3.70 (ddd, 1H, J ¼ 4.0, 6.4, 13.5 Hz), 3.4e3.5 (m, 2H), 2.23 (s,
3H), 1.9e2.0 (m, 2H), 1.6e1.7 (m, 2H); 13C NMR (DMSO‑d6, 126 MHz)
Electrospray high-resolution MS measurements (HESI-HRMS)
were performed on a Thermo Velos Pro Orbitrap Elite Hybrid Mass
spectrometer (Thermo Fisher Scientific, Bremen, Germany). The
ionization method was HESI and operated in positive ion mode. The
capillary temperature was set at 390 ꢀC. Resolving power of 60,000
(FWHM) at m/z 400. Data acquisition and analysis were accom-
plished with Xcalibur software version 3.0 (Thermo Fisher Scien-
tific Inc.).
d
163.0, 161.7, 154.5, 133.6, 130.4, 129.8 (overlapping peaks), 129.5,
127.7, 115.9, 114.8, 111.4, 109.6, 100.3, 71.4, 42.8, 37.8, 32.4, 30.7, 29.8,
20.0.
4.2.5. N-(1-methyl-1H-indol-5-yl)-2-{3-[(4-methylphenoxy)
methyl]azetidin-1-yl}-2-oxoacetamide (5)
Step 1. tert-Butyl 3-{[(methanesulfonyl)oxy]methyl}azetidine-1-
4.2. Methods for syntheses and analytical characterization of the
carboxylate
(5b).
tert-Butyl
3-(hydroxymethyl)azetidine-1-
final compounds
carboxylate (5a, 2.88 g, 15.4 mmol) and Et3N (4.3 mL, 30.8 mmol,
2 eq) were dissolved in CH2Cl2 (130 mL) at room temperature. To
the solution was dropwise added methanesulfonyl chloride (2.0
mL, 30 mmol,1.95 eq), followed by stirring at the same temperature
for 2.5 h, then the solvent was removed under reduced pressure.
The residue was dissolved in EtOAc and the solution was washed
with a saturated aqueous solution of NaHCO3 and with brine, dried
over anhydrous Na2SO4, filtered and concentrated under reduced
pressure to afford 5b (4.0 g, quant.) as a yellow liquid which was
used in the next step without further purification.
Step 2. tert-Butyl 3-[(4-methylphenoxy)methyl]azetidine-1-
carboxylate (5c). A mixture of 5b (3.8 g, 14 mmol), p-cresol (1.95
g, 18 mmol), Cs2CO3 (9.85 g, 30.2 mmol) and DMF (100 mL) was
stirred at 110 ꢀC overnight. The inorganic solid material was filtered
off, the filtrate was concentrated under vacuum. The residue was
dissolved in CH2Cl2, washed in turn with water, 2 M aqueous NaOH
solution and brine. The organic phase was dried over anhydrous
Na2SO4, filtered and concentrated. The residue was purified by
column chromatography on silica gel (eluent: CH2Cl2/MeOH ¼
98:2) to afford 5c (2.3 g, 58%) as light yellow oil.
Step 3. 3-[(4-Methylphenoxy)methyl]azetidine (5d). Trifluoro-
acetic acid (24 mL, 313 mmol) was added to a solution of 5c (2.3 g,
8.3 mmol) in CH2Cl2 (50 mL) cooled to 0 ꢀC in an ice water bath and
the solution was stirred for 1 h at this temperature. The solvent was
removed under reduced pressure at 40 ꢀC. Ice water was added to
the residue and the pH of the mixture was adjusted to 9 by addition
of saturated NaHCO3 solution. The mixture was extracted with
CH2Cl2, the combined organic layer was washed with brine, dried
over anhydrous Na2SO4 and filtered. The solvent was removed
under reduced pressure to afford 5d (1.37 g, 7.7 mmol, 93%).
4.2.1. N-(1H-indol-5-yl)-2-{4-[(4-methylphenyl)methyl]piperidin-
1-yl}-2-oxoacetamide (1)
The corresponding oxoacetic acid intermediate and the final
compound were synthesized in an analogous way as it is described
in case of 11. Yield 43.1%. Mp 86e88 ꢀC. HESI-HRMS: calcd for
C
23H26O2N3
[MþH]þ:
376.20195;
found:
376.20144;
11.05 (br s, 1H),
delta ¼ ꢁ1.37 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d
10.47 (s, 1H), 7.90 (d, 1H, J ¼ 2.0 Hz), 7.3e7.4 (m, 2H), 7.25 (dd, 1H,
J ¼ 2.0, 8.7 Hz), 7.0e7.1 (m, 4H), 6.39 (ddd, 1H, J ¼ 0.8, 2.0, 2.9 Hz),
4.2e4.4 (m, 1H), 3.7e3.8 (m, 1H), 3.0e3.1 (m, 1H), 2.67 (dt, 1H,
J ¼ 3.0, 12.7 Hz), 2.5e2.5 (m, 2H), 2.26 (s, 3H), 1.7e1.9 (m, 1H),
1.6e1.7 (m, 2H), 1.1e1.2 (m, 2H); 13C NMR (DMSO‑d6, 126 MHz)
d
162.9, 161.9, 136.6, 134.7, 133.0, 129.7, 128.8, 128.7, 127.3, 126.1,
114.8, 111.23, 111.21, 101.1, 45.7, 41.5, 40.6, 37.3, 31.8, 30.9, 20.5.
4.2.2. N-(1H-indol-5-yl)-2-[4-(4-methylphenoxy)piperidin-1-yl]-
2-oxoacetamide (2)
The oxoacetic acid intermediate and the final compound were
synthesized in an analogous way as it is described in case of 11.
Yield 31.9%. Mp 116e118 ꢀC. HESI-HRMS: calcd for C22H24O3N3
[MþH]þ: 378.18122; found: 378.18053; delta ¼ ꢁ1.82 ppm. 1H NMR
(DMSO‑d6, 500 MHz)
d 11.07 (br s, 1H), 10.52 (s, 1H), 7.92 (d, 1H,
J ¼ 2.0 Hz), 7.3e7.4 (m, 2H), 7.27 (dd, 1H, J ¼ 2.0, 8.7 Hz), 7.09 (d, 2H,
J ¼ 8.2 Hz), 6.9e6.9 (m, 2H), 6.41 (ddd, 1H, J ¼ 0.7, 2.0, 2.9 Hz), 4.62
(tt, 1H, J ¼ 3.6, 7.5 Hz), 3.8e3.9 (m, 1H), 3.70 (ddd, 1H, J ¼ 3.8, 6.4,
13.4 Hz), 3.4e3.5 (m, 2H), 2.23 (s, 3H), 1.9e2.0 (m, 2H), 1.6e1.7 (m,
2H); 13C NMR (DMSO‑d6, 126 MHz)
d 163.1, 161.7, 154.5, 133.0,129.8,
129.7, 129.5, 127.3, 126.1, 115.9, 114.8, 111.2, 111.2, 101.1, 71.4, 42.8,
37.8, 30.7, 29.8, 20.0.
Step
4.
N-(1-methyl-1H-indol-5-yl)-2-{3-[(4-methylphenoxy)
solution of [(1-
methyl]azetidin-1-yl}-2-oxoacetamide (5). To
a
4.2.3. N-(2-methyl-1H-indol-5-yl)-2-{4-[(4-methylphenyl)methyl]
piperidin-1-yl}-2-oxoacetamide (3)
methyl-1H-indol-5-yl)carbamoyl]formic acid (8b) (135 mg, 0.8
mmol) in dry CH2Cl2 (10 mL) was added N,N,N0,N0-tetramethyl-O-
(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (304
The oxoacetic acid intermediate and the final compound were
synthesized in an analogous way as it is described in case of 11. Mp
132e134 ꢀC. HESI-HRMS: calcd for C24H28O2N3 [MþH]þ:
mg, 0.8 mmol) and Et3N (335 mL, 2.4 mmol, 3.0 eq) at room tem-
perature under argon. The mixture was stirred for 20 min then 3-[(4-
methylphenoxy)methyl]azetidine (5d) (213 mg, 0.8 mmol) was
added. The mixture was stirred at room temperature overnight. After
completion of the reaction (monitored by TLC), the reaction mixture
was quenched by addition of saturated NaHCO3 solution (10 mL), the
organic layer was washed with water (10 mL), then the organic
phase was dried over anhydrous Na2SO4, filtered and concentrated in
vacuo. The residue was purified by column chromatography on silica
gel eluting with ethyl acetate: cyclohexane (67:33) to yield 91.5 mg
(30%) of the title compound 5. LC-MS (ESI) m/z [MþH]þ¼ 378.1. Mp
185e186 ꢀC. HESI-HRMS: calcd for C22H24O3N3 [MþH]þ: 378.18122;
found: 378.18052; delta ¼ ꢁ1.85 ppm. 1H NMR (DMSO‑d6, 500 MHz)
390.21760; found: 390.21690; delta
¼
ꢁ1.80 ppm. 1H NMR
(DMSO‑d6, 500 MHz)
d 10.86 (s, 1H), 10.40 (s, 1H), 7.74 (d, 1H,
J ¼ 1.9 Hz), 7.2e7.2 (m, 1H), 7.1e7.2 (m, 1H), 7.0e7.1 (m, 4H), 6.1e6.1
(m, 1H), 4.3e4.3 (m, 1H), 3.7e3.8 (m, 1H), 3.0e3.1 (m, 1H), 2.6e2.7
(m, 1H), 2.48 (s, 1H), 2.36 (d, 3H, J ¼ 0.7 Hz), 2.26 (s, 3H), 1.7e1.8 (m,
1H), 1.6e1.7 (m, 2H), 1.0e1.2 (m, 2H); 13C NMR (DMSO‑d6, 126 MHz)
d
163.0, 161.9, 136.6, 136.4, 134.7, 133.2, 129.5, 128.8, 128.7, 128.3,
113.6, 110.3, 110.2, 99.2, 45.7, 41.5, 40.6, 37.3, 31.8, 30.9, 20.5, 13.3.
4.2.4. N-(1-methyl-1H-indol-5-yl)-2-[4-(4-methylphenoxy)
piperidin-1-yl]-2-oxoacetamide (4)
The oxoacetic acid intermediate and the final compound were
synthesized in an analogous way as it was described in case of 11.
Yield 37.9%. Mp 143e145 ꢀC. HESI-HRMS: calcd for C23H26O3N3
[MþH]þ: 392.19687; found: 392.19613; delta ¼ ꢁ1.88 ppm. 1H NMR
d
10.35 (s, 1H), 8.01 (d, 1H, J ¼ 2.0 Hz), 7.49 (dd, 1H, J ¼ 2.0, 8.8 Hz),
7.37 (d, 1H, J ¼ 8.8 Hz), 7.30 (d,1H, J ¼ 3.1 Hz), 7.1e7.1 (m, 2H), 6.8e6.9
(m, 2H), 6.38 (dd, 1H, J ¼ 0.8, 3.1 Hz), 4.67 (ddd, 1H, J ¼ 0.8, 8.5, 10.6
Hz), 4.37 (ddd, 1H, J ¼ 1.0, 5.7, 10.6 Hz), 4.17 (ddd, 1H, J ¼ 1.0, 8.5, 10.7
17

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European Journal of Medicinal Chemistry 222 (2021) 113560
Hz), 4.14 (d, 2H, J ¼ 6.6 Hz), 3.86 (dd, 1H, J ¼ 1.0, 5.4, 10.7 Hz), 3.76 (s,
3H), 3.0e3.1 (m, 1H), 2.23 (s, 3H); 13C NMR (DMSO‑d6, 126 MHz)
8.0%. Mp 218e219 ꢀC. HESI-HRMS: calcd for C21H22O3N3 [MþH]þ:
364.16557; found: 364.16501; delta
¼
ꢁ1.53 ppm. 1H NMR
d
159.5, 157.9, 156.3, 133.6, 130.3, 129.7, 129.6, 129.3, 127.5, 115.2,
(DMSO‑d6, 500 MHz)
d
10.41 (s, 1H), 8.01 (d, 1H, J ¼ 2.0 Hz), 7.49 (dd,
114.4, 111.8, 109.4, 100.3, 68.9, 55.9, 51.0, 32.4, 28.9, 20.0.
1H, J ¼ 2.0, 8.8 Hz), 7.37 (d, 1H, J ¼ 8.8 Hz), 7.31 (d, 1H, J ¼ 3.0 Hz),
7.1e7.2 (m, 2H), 6.7e6.8 (m, 2H), 6.38 (dd,1H, J ¼ 0.7, 3.0 Hz), 5.0e5.1
(m, 2H), 4.4e4.5 (m, 2H), 3.9e4.0 (m, 1H), 3.76 (s, 3H), 2.24 (s, 3H);
4.2.6. N-(1-methyl-1H-indol-5-yl)-2-[3-(4-methylphenoxy)
pyrrolidin-1-yl]-2-oxoacetamide (6)
13C NMR (DMSO‑d6, 126 MHz)
d 159.6, 157.7, 154.0, 133.7, 130.3, 130.1,
The oxoacetic acid intermediate and the final compound were
synthesized in an analogous way as it was depicted in case of 11.
The corresponding pyrrolidine intermediate was synthesized in an
analogous way as it is described in case of the azetidinemethyl
intermediate 5d. The amide coupling was achieved in an analogous
way as in case of 23. HESI-HRMS: calcd for C22H24O3N3 [MþH]þ:
130.0, 129.6, 127.5, 115.3, 114.4, 111.8, 109.4, 100.3, 66.3, 60.4, 55.6,
32.4, 20.0.
4.2.9. N-(1H-indol-5-yl)-2-[3-(4-methylphenoxy)-8-azabicyclo
[3.2.1]octan-8-yl]-2-oxoacetamide (9)
Step
1.
tert-butyl
3-phenoxy-8-azabicyclo[3.2.1]octane-8-
378.18122; found: 378.18054; delta
(DMSO‑d6, 500 MHz)
¼
ꢁ1.79 ppm. 1H NMR
carboxylate (9b). Under an Ar atmosphere, N-BOC-nortropine (9a)
(4.55 g, 20 mmol) was dissolved in abs. THF, p-cresole (2.38 g, 22
mmol) and triphenylphosphine (6.82 g, 26 mmol) were added
followed by the dropwise addition of DIAD (diisopropyl azodi-
carboxylate, 5.26 g, 26 mmol) at room temperature. The mixture
was stirred at room temperature for 2 days. After completion, 1 M
NaOH solution (12 mL) and 2 ꢂ EtOAc (2 ꢂ 25 mL) were charged.
After separation of the phases, the organic phase was dried over
Na2SO4, and evaporated to dryness. The crude product was purified
with flash chromatography using cyclohexane/EtOAc as the eluent.
Yield: 2.65 g (42%).
Step 2. 3-phenoxy-8-azabicyclo[3.2.1]octane (9c). 9b (2.55 g, 8.4
mmol) was dissolved in 1,4-dioxane (30 mL), HCl solution in 1,4
dioxane (80 mL) was added and the mixture was stirred at room
temperature overnight. The resulting solution was evaporated and
the solid crude product 9c was crystalized from diethyl ether. Yield:
1.01 g (58%).
d
10.46 (s, 0.5H), 10.46 (s, 0.5H), 7.99 (d, 0.5H,
J ¼ 2.0 Hz), 7.96 (d, 0.5H, J ¼ 2.0 Hz), 7.4e7.5 (m, 1H), 7.3e7.4 (m,
1H), 7.3e7.3 (m, 1H), 7.1e7.1 (m, 2H), 6.8e6.9 (m, 2H), 6.39 (dd,
0.5H, J ¼ 0.8, 3.1 Hz), 6.38 (dd, 0e5H, J ¼ 0.8, 3.1 Hz), 5.0e5.1 (m,
1H), 3.9e4.0 (m, 1.5H), 3.8e3.8 (m, 0.5H), 3.77 (s, 1.5H), 3.76 (s,
1.5H), 3.6e3.7 (m, 1.5H), 3.54 (ddd, 0.5H, J ¼ 7.7, 10.1, 12.0 Hz), 2.24
(s, 2H), 2.22 (s, 2H), 2.1e2.2 (m, 2H); 13C NMR (DMSO‑d6, 126 MHz)
d
161.2, 160.9,160.0,159.8,154.45, 154.41, 133.6, 130.3,129.9,129.82,
129.75, 129.69, 129.63, 127.59, 127.57, 115.43, 115.37, 115.1, 111.7,
109.44, 109.42, 100.3, 76.3, 73.9, 52.8, 51.8, 45.5, 44.6, 32.4, 31.2,
28.6, 19.99, 19.97.
4.2.7. N-(1-methyl-1H-indol-5-yl)-2-[4-(4-methylphenoxy)
azepan-1-yl]-2-oxoacetamide (7)
The corresponding azepane intermediate was synthesized in an
analogous way as it is described in case of the azetidinemethyl
intermediate 5d. The amide coupling was achieved in an analogous
way as in case of 23. Yield 50.0%. Mp 134e138 ꢀC. HESI-HRMS: calcd
Step 3e5. The oxoacetic acid intermediate and the final com-
pound 9 were synthesized in an analogous way as it is described in
case of 11. Yield 8.2%. Mp 96e97 ꢀC. HESI-HRMS: calcd for
for
C
24H28O3N3 [MþH]þ: 406.21252; found: 406.21161;
delta ¼ ꢁ2.24 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d
10.55 (s, 0.5H),
C
24H26O3N3 [MþH]þ: 404.19687; found: 404.19594; delta ¼ ꢁ2.30
10.54 (s, 0.5H), 7.9e7.9 (m, 1H), 7.4e7.4 (m, 1H), 7.3e7.4 (m, 2H),
7.0e7.1 (m, 2H), 6.8e6.9 (m, 2H), 6.4e6.4 (m, 1H), 4.5e4.6 (m, 1H),
3.77 (2 ꢂ s, 3H), 3.4e3.7 (m, 4H), 2.23 (s, 1.5H), 2.21 (s, 1.5H),
2.0e2.1 (m, 1H), 1.8e2.0 (m, 4H), 1.7e1.8 (m, 1H); 13C NMR
ppm. 1H NMR (DMSO‑d6, 500 MHz)
d 11.06 (br s, 1H), 10.48 (s, 1H),
7.96 (s, 1H), 7.3e7.4 (m, 3H), 7.0e7.1 (m, 2H), 6.9e6.9 (m, 2H),
6.4e6.4 (m, 1H), 4.8e4.9 (m, 1H), 4.7e4.7 (m, 1H), 4.6e4.7 (m, 1H),
2.22 (s, 3H), 2.2e2.2 (m, 2H), 1.8e2.1 (m, 4H), 1.7e1.8 (m, 1H),
(DMSO‑d6, 126 MHz)
d
164.6, 164.5, 162.1, 162.0, 154.71, 154.66,
1.6e1.6 (m, 1H); 13C NMR (DMSO‑d6, 126 MHz)
d 160.6, 159.0, 154.8,
133.59, 133.57, 130.3, 129.88, 129.84, 129.80, 129.23, 129.19, 127.7,
115.8, 115.7, 114.9, 114.8, 111.55, 111.46, 109.6, 100.3, 75.0, 74.3, 47.1,
44.4, 42.4, 34.2, 32.4, 32.0, 31.3, 30.1, 22.1, 20.7, 20.00, 19.98.
133.0, 129.8, 129.7, 129.4, 127.3, 126.0, 115.8, 115.1, 111.4, 111.1, 101.1,
68.9, 54.0, 50.9, 38.6, 36.8, 28.0, 26.3, 20.0.
4.2.10. 2-[(2R,5S)-2,5-dimethyl-4-[(4-methylphenyl)methyl]
piperazin-1-yl]-N-(1H-indol-5-yl)-2-oxoacetamide (10)
Step 1. tert-Butyl (2S,5R)-2,5-dimethyl-4-[(4-methylphenyl)
4.2.8. N-(1-methyl-1H-indol-5-yl)-2-[3-(4-methylphenoxy)
azetidin-1-yl]-2-oxoacetamide (8)
Step 1. ethyl [(1-methyl-1H-indol-5-yl)amino](oxo)acetate (8a). 1-
Methyl-1H-indol-5-amine (11d, 1.18 g, 8.07 mmol) and DIPEA (2.1
mL, 12.06 mmol) were dissolved in CH2Cl2 (50 mL) and ethyl oxalyl
chloride (0.98 mL, 8.77 mmol) was added dropwise to the resulting
reaction mixture at 0 ꢀC. The mixture was stirred for 3 h at room
temperature. The reaction mixture was treated with saturated
aqueous NaHCO3 solution and the organic phase was washed with
water, dried over Na2SO4 and evaporated to dryness to result in 8a.
Yield: 1.2 g (61%).
Step 2. [(1-methyl-1H-indol-5-yl)carbamoyl]formic acid (8b). 8a
(1.2 g, 4.87 mmol) was hydrolyzed with 2.1 equivalent of aqueous
LiOH$H2O solution (426 mg, 10.15 mmol in 6 mL of water) in
methanol (30 mL). The reaction mixture was stirred for 4 h. The pH of
the mixture was adjusted to 3 by addition of 1 M HCl solution (ca. 10
mL) and extracted with EtOAc (40 mLx2). The organic phase was
dried over Na2SO4 and evaporated to dryness. Yield: 1.5 g of crude
8b. The synthesis of the corresponding azetidine intermediate was
achieved in an analogous way as it was described in case of the
azetidinemethyl intermediate 5d. The amide coupling of 5d and 8b
was achieved in an analogous way as it is described in case of 5. Yield
methyl]piperazine-1-carboxylate
(10b).
tert-Butyl-(2S,5R)-2,5-
dimethylpiperazine-1-carboxylate (10a) (829 mg, 3.87 mmol) was
dissolved in acetonitrile (20 mL) and of 4-methylbenzylbromide
(716 mg, 3.87 mmol) was added. The solution was cooled down
to 0 ꢀC, triethylamine (539
mL, 7.34 mmol) was dropwise added and
stirred overnight at room temperature. The reaction mixture was
evaporated to dryness, water and CH2Cl2 were added. After sepa-
ration the aqueous phase was extracted 3 times with CH2Cl2. The
organic phase was dried and evaporated. Yield: 1.1 g, (89%).
Step 2. The BOC protecting group was removed according to the
method used in case of 9c to obtain (2R,5S)-2,5-dimethyl-1-[(4-
methylphenyl)methyl]piperazine 10c.
Step 3e5. The oxoacetic acid intermediate and the final com-
pound 10 were synthesized in an analogous way as it is described in
case 11. Yield 30.3%. Mp 90e92 ꢀC. HESI-HRMS: calcd for
C
24H29O2N4 [MþH]þ: 405.22850; found: 405.22790; delta ¼ ꢁ1.49
ppm. 1H NMR (DMSO‑d6, 500 MHz)
d 11.05 (br s,1H), 10.52 (s, 0.5H),
10.49 (s, 0.5H), 7.91 (d, 0.5H, J ¼ 1.8 Hz), 7.89 (d, 0.5H, J ¼ 1.9 Hz),
7.3e7.4 (m, 2H), 7.2e7.3 (m, 3H), 7.1e7.2 (m, 2H), 6.4e6.4 (m, 1H),
4.4e4.5 (m, 0.5H), 4.00 (d, 0.5H, J ¼ 12.9 Hz), 3.9e4.0 (m, 0.5H),
18

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European Journal of Medicinal Chemistry 222 (2021) 113560
3.5e3.6 (m, 1.5H), 3.4e3.5 (m, 1.5H), 3.18 (dd, 0.5H, J ¼ 3.7, 12.9 Hz),
3.0e3.1 (m, 0.5H), 2.9e3.0 (m, 0.5H), 2.69 (dd, 0.5H, J ¼ 4.0,11.8 Hz),
2.61 (dd, 0.5H, J ¼ 4.3, 12.0 Hz), 2.28 (s, 1.5H), 2.27 (s, 1.5H), 2.2e2.3
(m, 1H), 1.35 (d, 1.5H, J ¼ 6.6 Hz), 1.24 (d, 1.5H, J ¼ 6.8 Hz), 0.98 (d,
1.5H, J ¼ 6.6 Hz), 0.95 (d, 1.5H, J ¼ 6.6 Hz); 13C NMR (DMSO‑d6, 126
under this condition. After the completion of the reaction (moni-
tored by TLC- visualized by iodine - o-toluidine) the mixture was
poured onto water (80 mL) and extracted with EtOAc (3 ꢂ 30 mL).
The combined organic layer was washed with brine (3 ꢂ 30 mL)
dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
The residue was chromatographed on silica gel eluting with a
mixture of cyclohexane - EtOAc 1:1 to yield 2.54 g (64%) of 12b.
Step 2. Ethyl 2-formamido-2-(1-[4-methylphenyl]methylpiperidin-
4-ylidene)acetate (12c). To a stirred suspension of NaH (60% in
mineral oil) (751 mg, 18.7 mmol, 1.5 eq) in THF (100 mL) was added
a solution of ethyl isocyanoacetate (1.78 mL, 16.25 mmol) in THF (5
mL) over a period of 15 min at 0e5 ꢀC under argon. The mixture was
stirred for 40 min under this condition then a solution of 12b (2.54
g, 12.5 mmol) in THF (45 mL) was added dropwise at 0e5 ꢀC. The
temperature allowed to warm to room temperature and the
mixture was stirred overnight. After the completion of the reaction
(monitored by TLC) the mixture was poured onto water (100 mL)
and extracted with EtOAc (2 ꢂ 80 mL). The combined organic layer
was washed with brine (2 ꢂ 30 mL) dried over anhydrous Na2SO4,
filtered and concentrated in vacuo to yield 1.09 g of 12c. It was used
in the next step without further purification.
MHz)
d 163.9, 163.7, 161.9, 161.7, 135.79, 135.75, 135.73, 133.00,
132.98, 129.72, 129.67, 128.7, 128.14, 128.12, 127.3, 126.1, 114.93,
114.90, 111.32, 111.30, 111.2, 101.1, 57.6, 57.5, 51.3, 51.1, 49.5, 48.6,
47.9, 46.5, 44.0, 41.2, 20.6, 16.8, 15.5, 7.1, 6.8.
4.2.11. N-(1-methyl-1H-indol-5-yl)-2-{4-[(4-methylphenyl)methyl]
piperazin-1-yl}-2-oxoacetamide (11)
Step 1. Ethyl 2-{4-[(4-methylphenyl)methyl]piperazin-1-yl}-2-
oxoacetate (11b). To a solution of 1-(4-methylbenzyl)piperazine
(11a, 854 mg, 4.49 mmol) and Et3N (0.94 mL, 6.7 mmol) in CH2Cl2
(25 mL) was added ethyl chlorooxoacetate (0.6 mL, 5.37 mmol) at
0 ꢀC under argon, and the mixture was stirred at room temperature
overnight. Dichloromethane (100 mL) was added, and the mixture
was washed with water, dried over Na2SO4, filtered and concen-
trated under reduced pressure to give the product 11b (1.243 g,
95%), as light yellow oil.
Step 2. 2-{4-[(4-Methylphenyl)methyl]piperazin-1-yl}-2-oxoacetic
acid (11c). To a solution of 11b (3.098 g, 10.67 mmol) in ethanol was
added dropwise a solution of NaOH (515 mg, 12.88 mmol, 1.2 eq) in
water (5 mL) at 0 ꢀC. The mixture was stirred at room temperature
for 2.5 h, and concentrated. The residue was dissolved in water (50
mL) extracted with diethyl ether, acidified with 12.9 mL of 1 M
aqueous HCl solution and extracted with CH2Cl2. The aqueous layer
was freeze dried to afford the crude, title compound (3.546 g) as a
white solid containing 1.2 eq of NaCl. 11c was used in the next step
without further purification.
Step 3. 2-Oxo-2-(1-[4-methylphenyl]methylpiperidin-4-yl)acetic
acid (12d). To a stirred solution of 12c (1.09 g, 3.43 mmol) in EtOH
(21 mL) was added 10% aqueous solution of HCl (21 mL) at 0e5 ꢀ
C
under argon then the mixture was stirred at room temperature
overnight. After the completion of the reaction (monitored by TLC)
to this mixture was added a solution of KOH (3.79 g, 67.7 mmol)
dissolved in EtOH (28 mL) and water (10 mL); (pH ¼ 12). After the
completion of the hydrolysis (monitored by TLC) pH of the mixture
was adjusted to 6 with 1 N HCl solution. The precipitated solid was
removed by filtration, the mother liquor was concentrated in vacuo
to yield 3.2 g (theor. 0.9 g) of the mixture of 12d together with KCl.
It was used in the next step without further purification.
Step
3.
N-(1-Methyl-1H-indol-5-yl)-2-{4-[(4-methylphenyl)
methyl]piperazin-1-yl}-2-oxoacetamide (11). 500 mg of 2-{4-[(4-
methylphenyl)methyl]piperazin-1-yl}-2-oxoacetic acid (11c, 1.52
mmol) was dissolved in dry DMF under an argon atmosphere. To
this solution 1-hydroxybenzotriazole hydrate (HOBt$H2O) (350 mg,
2.287 mmol, 1.5 eq) and N-(3-dimethylaminopropyl)-N0-ethyl-
carbodiimide hydrochloride (EDC. HCl) (438 mg, 2.287 mmol, 1.5
eq) were added and the mixture was stirred for 15 min under argon
atmosphere. Afterwards, 1-methyl-1H-indol-5-amine (11d) (278
mg, 1.9 mmol, 1.25 eq) and Et3N (386 mg, 3.81 mmol, 2.5 eq) were
added. The resulted reaction mixture was stirred for 24 h at room
temperature under argon atmosphere. After the completion of the
reaction (monitored by TLC) the mixture was diluted with water
(20 mL) and extracted with EtOAc (3 ꢂ 20 mL). The combined
organic layer was washed with water, dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. The crude product 11
was purified by column chromatography to afford 71 mg (12%) of
the title compound. LC-MS (ESI) m/z [MþH]þ ¼ 391.2. Mp 125e127
ꢀC. HESI-HRMS: calcd for C23H27O2N4 [MþH]þ: 391.21285; found:
391.21181; delta ¼ ꢁ2.67 ppm. 1H NMR (DMSO‑d6, 500 MHz)
Step 4. The synthesis of 12 was achieved in an analogous way as
it is described in case of 11. Yield 9.1%. Mp 147e148 ꢀC. HESI-HRMS:
calcd for
C
24H28O2N3 [MþH]þ: 390.21760; found: 390.21716;
delta ¼ ꢁ1.14 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d 10.34 (s, 1H),
8.03 (d, 1H, J ¼ 2.0 Hz), 7.49 (dd, 1H, J ¼ 2.0, 8.8 Hz), 7.39 (d, 1H, J ¼
8.8 Hz), 7.31 (d, 1H, J ¼ 3.1 Hz), 7.2e7.2 (m, 2H), 7.1e7.1 (m, 2H), 6.39
(dd, 1H, J ¼ 0.8, 3.1 Hz), 3.77 (s, 3H), 3.42 (s, 2H), 3.28 (tt, 1H, J ¼ 3.8,
11.4 Hz), 2.8e2.9 (m, 2H), 2.28 (s, 3H), 2.02 (dt, 2H, J ¼ 2.1, 11.4 Hz),
1.8e1.9 (m, 2H), 1.4e1.5 (m, 2H); 13C NMR (DMSO‑d6, 126 MHz)
d
201.1, 159.3, 135.8, 135.2, 133.7, 130.3, 129.6, 128.6, 127.6, 115.2,
111.9, 109.5, 100.3, 61.9, 52.2, 41.7, 32.4, 27.0, 20.6.
4.2.13. N-(1-methyl-1H-indol-5-yl)-2-oxo-2-[(1r,4r)-4-[(6-
methylpyridin-3-yl)methyl] cyclohexyl]acetamide (13)
Step 1. Ethyl (1s,4s)-4-[(6-methylpyridin-3-yl)oxy]cyclohexane-1-
carboxylate (13c). To a solution of 6-methylpyridin-3-ol (13a, 400
mg, 3.69 mmol, 1.5 eq) in THF (11 mL) was added ethyl trans-4-
hydroxycyclohexane-1-carboxylate (13b, 0.40 mL, 2.46 mmol)
and triphenylphosphine (974 mg, 3.71 mmol, 1.51 eq) under inert
conditions at 0 ꢀC and stirred for 8 min. Diisopropyl azodicarbox-
ylate (0.73 mL, 3.69 mmol, 1.5 eq) in THF (1 mL) was then added
dropwise (in 20 min). After stirring for 1 h at this temperature, the
reaction mixture was allowed to warm to room temperature and
stirred for further 4 days. The solvent was evaporated under
reduced pressure to obtain a crude oil which was then purified by
column chromatography using cyclohexane and ethyl acetate (3:1)
to cyclohexane and ethyl acetate (1:2) as an eluent to yield (464 mg,
71.63%) of 13c.
d
10.54 (s, 1H), 7.9e7.9 (m, 1H), 7.4e7.4 (m, 1H), 7.3e7.3 (m, 2H),
7.2e7.2 (m, 2H), 7.1e7.2 (m, 2H), 6.38 (dd,1H, J ¼ 0.8, 3.0 Hz), 3.76 (s,
3H), 3.5e3.6 (m, 4H), 3.48 (s, 2H), 2.4e2.5 (m, 4H), 2.28 (s, 3H); 13
NMR (DMSO‑d6, 126 MHz) 162.8, 161.4, 136.0, 134.4, 133.6, 130.3,
C
d
129.8, 128.9, 128.7, 127.6, 114.8, 111.5, 109.5, 100.3, 61.4, 52.5, 51.7,
45.6, 40.7, 32.4, 20.6.
4.2.12. N-(1-methyl-1H-indol-5-yl)-2-{1-[(4-methylphenyl)methyl]
piperidin-4-yl}-2-oxoacetamide (12)
Step 1. 1-[(4-Methylphenyl)methyl]piperidin-4-one (12b). To a
solution of 4-piperidone monohydrate hydrochloride (12a, 3.0 g,
19.5 mmol) in DMF (60 mL) was added K2CO3 (5.4 g, 39 mmol) and
1-bromomethyl-4-methylbenzene (5.42 g, 29.3 mmol, 1.5 eq) at
room temperature under argon. The mixture was stirred overnight
Step
2.
(1s,4s)-4-[(6-methylpyridin-3-yl)oxy]cyclohexane-1-
carboxylic acid (13d). To a solution of 13c (558 mg, 2.12 mmol) in
methanol (15 mL) was added potassium carbonate (586 mg, 4.24
mmol, 2 eq) and water (3.75 mL) then the mixture was heated at 70
19

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European Journal of Medicinal Chemistry 222 (2021) 113560
ꢀC for 2 h. After the reaction mixture had been allowed to cool to
room temperature, 10% HCl solution was added, followed by the
addition of CH2Cl2 (25 mL) and water (25 mL), and the two phases
were separated. The aqueous layer was extracted with CH2Cl2 (5 ꢂ
15 mL). The combined organic layer was washed with water (20
mL) and brine (20 mL). The aqueous layer was extracted again with
CH2Cl2 (3 ꢂ 15 mL). The combined organic layer was dried over
Na2SO4 and concentrated and dried at 40 ꢀC for 24 h. Yield: 204 mg
(41%).
reflux temperature overnight. The reaction mass was diluted with
water and extracted with EtOAc (2 ꢂ 20 mL). The combined organic
layer was washed with brine and evaporated. The crude product
was purified with column chromatography (eluent: 15% EtOAc/
petrolether). Yield: 1.2 g (39%).
Step 3e4. The ester hydrolysis was accomplished in an analo-
gous way as it is described in case of 11c. The amide coupling was
achieved in an analogous way as it is described in case of 23. Mp
154e156 ꢀC. HESI-HRMS: calcd for C21H18O2N4Cl [MþH]þ:
Step 3. 3-{4-[(6-Methylpyridin-3-yl)oxy]cyclohexyl}-3-oxo-2-(tri-
393.11128; found: 393.11107; delta
(DMSO‑d6, 500 MHz)
¼
ꢁ0.53 ppm. 1H NMR
phenyl-
l⁵-phosphanylidene) propanenitrile (13f). To a mixture of 13d
d
10.51 (s, 1H), 8.94 (d, 1H, J ¼ 0.5 Hz), 8.21 (d,
(197 mg, 0.837 mmol) in CH2Cl2 (14 mL) at 0 ꢀC was added EDC (169
mg, 0.879 mmol, 1.05 eq) and DMAP (12 mg, 0.098 mmol, 0.117 eq)
followed by the dropwise addition of a solution of cyano phos-
phorane 13e (506 mg, 1.68 mmol, 2 eq) in CH2Cl2 (15 mL). After
stirring the mixture at 0 ꢀC for 1 h, it was warmed to room tem-
perature and stirred overnight. The reaction mixture was diluted
with CH2Cl2 and washed with water (30 mL). The aqueous layer
was extracted with CH2Cl2 (2 ꢂ 15 mL). The combined organic layer
was washed with water (15 mL) and brine (15 mL), dried over
Na2SO4 and concentrated. The residue was purified by column
chromatography over silica gel, eluting with a mixture of cyclo-
hexane and EtOAc (1:5) to yield 312 mg (72%) of cyanoketo phos-
phorane 13f.
1H, J ¼ 0.5 Hz), 8.10 (d, 1H, J ¼ 2.0 Hz), 7.54 (dd, 1H, J ¼ 2.0, 8.8 Hz),
7.4e7.5 (m, 2H), 7.41 (d, 1H, J ¼ 8.8 Hz), 7.3e7.4 (m, 2H), 7.33 (d, 1H,
J ¼ 3.1 Hz), 6.41 (dd, 1H, J ¼ 0.8, 3.1 Hz), 5.46 (s, 2H), 3.78 (s, 3H); 13
C
NMR (DMSO‑d6, 126 MHz)
d 181.5, 160.4, 142.2, 136.6, 135.3, 133.7,
132.6, 130.4, 129.8, 129.7, 128.6, 127.6, 118.7, 115.3, 112.0, 109.5,
100.4, 54.1, 32.5.
4.2.15. 2-[2-(4-Chlorophenoxy)-1,3-thiazol-5-yl]-N-(1-methyl-1H-
indol-5-yl)-2-oxoacetamide (15)
Step 1. Ethyl 2-bromo-1,3-thiazole-4-carboxylate (15b). 2-
Aminothiazole-4-carboxylate (15a) (2 g, 11.6 mmol) was dissolved
in acetonitrile (20 mL) and to this stirred solution was added cop-
per(II) bromide (2.6 g, 11.6 mmol) at 0 ꢀC. The resulting mixture was
stirred for 20 min at room temperature. Then it was cooled to 0 ꢀC
and tert-butyl nitrite (1.54 g) was added and stirred for further 2 h
at room temperature. After the completion of the reaction, the re-
action mixture was concentrated under reduced pressure and the
residue was purified by column chromatography using a mixture of
20%EtOAc and 80%petrolether as the eluent. Yield: 1.5 g (57%).
Step 2. Ethyl 2-(4-chlorophenoxy)-1,3-thiazole-4-carboxylate
(15c) A stirred solution of 4-chlorophenol (3.78 mL, 38.5 mmol)
in THF (50 mL) was cooled to 0 ꢀC and potassium tert-butoxide (5.15
g, 45.9 mmol) was portionwise added. The reaction mixture was
refluxed for 2 h and the solvent was evaporated. The obtained
potassium salt was suspended in DMSO (60 mL), 15b (9 g, 38.1
mmol) was added and the mixture was heated at 85 ꢀC for 16 h.
After completion of the reaction, the reaction mixture was poured
into cooled water and extracted with EtOAc (3 ꢂ 350 mL). The
combined organic phase was washed with brine, dried (Na2SO4)
and evaporated under reduced pressure to obtain the crude 15c
(6.05 g, 55.4%). It was used without further purification.
Step 3. 2-(4-Chlorophenoxy)-1,3-thiazole-4-carbaldehyde (15d) To
the stirred solution of 15c (1.5 g, 5.3 mmol) in CH2Cl2 (15 mL) cooled
to ꢁ70 ꢀC was dropwise added DIBAL-H (1 M solution, 8.3 mL, 8.3
mmol) and it was stirred for 3 h at ꢁ70 ꢀC. After completion of the
reaction, the reaction mixture was quenched with aqueous citric
acid solution at ꢁ70 ꢀC. The mixture was stirred for 20 min at room
temperature, extracted with CH2Cl2 (2 ꢂ 100 mL) and the combined
organic layer was dried over Na2SO4 and evaporated to dryness.
Yield: 0.95 g (75%).
Step 4. Methyl 2-{4-[(6-methylpyridin-3-yl)oxy]cyclohexyl}-2-
oxoacetate (13g). Under argon, oxone (880 mg, 1.43 mmol, 2.41
eq) and of cyanoketo phosphorane 13f (308 mg, 0.594 mmol) was
dissolved in a mixture of CH2Cl2 (28 mL) and methanol (12 mL) and
heated at 40 ꢀC for 7 days. The reaction mixture was filtered and
evaporated. The residue was purified by column chromatography
over silica gel, eluted with a mixture of cyclohexane and EtOAc
(1:5) to cyclohexane and EtOAc (1:1) then CH2Cl2 and methanol
(10:1) to yield 51 mg (31%) of 13g.
Step 5e6. The hydrolysis was accomplished in the same way as it
is described in case of 11c. The amide coupling was achieved in an
analogous way as it is described in case of 23. Yield 5%. Mp 176e178
ꢀC. HESI-HRMS: calcd for C23H26O3N3 [MþH]þ: 392.19687; found:
392.19625; delta ¼ ꢁ1.58 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d
10.36 (s, 1H), 8.15 (d, 1H, J ¼ 2.9 Hz), 8.04 (d, 1H, J ¼ 2.0 Hz), 7.51
(dd, 1H, J ¼ 2.0, 8.8 Hz), 7.40 (d, 1H, J ¼ 8.8 Hz), 7.33 (dd, 1H, J ¼ 2.9,
8.5 Hz), 7.32 (d, 1H, J ¼ 3.0 Hz), 7.15 (d, 1H, J ¼ 8.5 Hz), 6.40 (dd, 1H,
J ¼ 0.8, 3.0 Hz), 4.3e4.4 (m, 1H), 3.77 (s, 3H), 3.3e3.4 (m, 1H), 2.38
(s, 3H), 2.1e2.2 (m, 2H), 2.0e2.0 (m, 2H), 1.4e1.5 (m, 4H); 13C NMR
(DMSO‑d6, 126 MHz)
d 201.3, 159.3, 151.3, 149.5, 137.9, 133.7, 130.4,
129.6, 127.6, 123.2, 122.9, 115.2, 111.9, 109.5, 100.3, 74.9, 42.4, 32.4,
30.3, 25.4, 22.9.
4.2.14. 2-{1-[(4-Chlorophenyl)methyl]-1H-pyrazol-4-yl}-N-(1-
methyl-1H-indol-5-yl)-2-oxoacetamide (14)
Step 1. 1-[(4-Chlorophenyl)methyl]-1H-pyrazole (14a). Pyrazole (3
g, 44 mmol) was dissolved in DMF (25 mL) and the solution was
cooled down to 0 ꢀC. Potassium carbonate (7.3 g. 53 mmol) and the
p-chlorobenzyl bromide (10.84 g, 53 mmol) were added, the
mixture was warmed up to 80 ꢀC and stirred for 16 h. The progress
of the reaction was monitored by TLC (40% EtOAc/60% petrolether).
After completion of the reaction, the reaction mixture was poured
into cooled water and extracted with EtOAc (3 ꢂ 200 mL). The
combined organic layer was washed with brine, dried and evapo-
rated. The 14a crude product was purified with column chroma-
tography using a mixture of 25%EtOAc and 75% light petroleum as
the eluent. Yield: 2.0 g (23%).
Step 4. [2-(4-Chlorophenoxy)-1,3-thiazol-4-yl](hydroxy)acetoni-
trile (15e). Intermediate 15d (0.9 g, 3.76 mmol) was solved in CH2Cl2
(10 mL) and the mixture was cooled to 0 ꢀC. Methylmorpholine-N-
oxide (0.13 g, 1.1 mmol) and trimethylsilyl cyanide (TMSCN) (0.7
mL, 5.6 mmol) were added to the solution and it was allowed to
warm up to room temperature and stirred for 16 h. After comple-
tion of the reaction, the mixture was poured into water, the
aqueous phase was extracted with CH2Cl2 (2 ꢂ 100 mL). The
organic phase was dried and evaporated to dryness. Yield: 0.85 g
(84%).
Step 2. Ethyl {1-[(4-chlorophenyl)methyl]-1H-pyrazol-4-yl}(oxo)
acetate (14b). To the solution of 14a (2.0 g, 10.4 mmol) in acetoni-
trile was added ethyl 2-chloro-2-oxoacetate (2.02 g, 14.8 mmol) at
Step 5. [2-(4-Chlorophenoxy)-1,3-thiazol-4-yl](hydroxy)acetic
acid (15f). To 15e (0.85 g, 3.19 mmol) was added cc HCl (5 mL) and
the mixture was refluxed for 3 h. It was poured to cooled water and
extracted with EtOAc. The organic layer was dried over Na2SO4 and
0
ꢀC. The solution was stirred at 0 ꢀC for 15 min then heated at
20

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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
evaporated. Yield: 0.65 g (72%).
(eluent: cyclohexane/EtOAc ¼ 8/2). Yield: 0.331 g (50%).
Step 6. Methyl [2-(4-chlorophenoxy)-1,3-thiazol-4-yl](hydroxy)
acetate (15g). To a stirred and cooled solution of 15f (0.65 g, 2.27
mmol) in MeOH (10 mL) was added cc. sulfuric acid (1 mL, 18.8
mmol). The reaction mixture was allowed to reach room temper-
ature and was stirred for an hour at this temperature. Methanol was
evaporated and the residue was poured into cooled water and
extracted with EtOAc. The organic layer was dried over Na2SO4 and
evaporated. Yield: 0.55 g (81%).
Step 2e3. The hydrolysis was accomplished in the same way as it
is described in case of 11c. The amide coupling was achieved in an
analogous way as it is described in case of 11. Yield 13.0%. Mp
166e168 ꢀC. HESI-HRMS: calcd for C23H20O3N3 [MþH]þ:
386.14992; found: 386.14929; delta
(DMSO‑d6, 500 MHz)
¼
ꢁ1.63 ppm. 1H NMR
d
10.73 (s, 1H), 8.90 (dd, 1H, J ¼ 0.7, 2.4 Hz),
8.48 (dd,1H, J ¼ 2.4, 8.7 Hz), 8.05 (dd,1H, J ¼ 0.5,1.9 Hz), 7.5e7.5 (m,
1H), 7.4e7.4 (m, 1H), 7.34 (d, 1H, J ¼ 3.1 Hz), 7.2e7.3 (m, 2H), 7.16
(dd, 1H, J ¼ 0.7, 8.7 Hz), 7.1e7.1 (m, 2H), 6.43 (dd, 1H, J ¼ 0.7, 3.1 Hz),
Step 7. Methyl [2-(4-chlorophenoxy)-1,3-thiazol-4-yl](oxo)acetate
(15h) To a stirred and cooled solution of 15g (1 g, 3.33 mmol) in
CH2Cl2 (10 mL) was added Dess-Martin periodinane (1.7 g, 4 mmol).
The reaction mixture was allowed to reach room temperature and
stirred for 3 h at this temperature. To the reaction mixture, cold
water was added and it was extracted with EtOAc. The organic layer
was dried over Na2SO4 and evaporated. Yield: 0.5 g (50%).
Step 8e9. The hydrolysis was accomplished in the same way as it
is described in case of 11c. The amide coupling was achieved in an
analogous way as it is described in case of 23. Yield 13.7%. Mp
170e171 ꢀC. HESI-HRMS: calcd for C20H15O3N3ClS [MþH]þ:
3.78 (s, 3H), 2.34 (s, 3H); 13C NMR (DMSO‑d6, 126 MHz)
d 186.9,
166.3, 161.3, 151.2, 150.5, 141.4, 134.6, 133.8, 130.4, 130.2, 129.5,
127.6, 124.5, 121.3, 115.2, 112.0, 111.1, 109.6, 100.4, 32.5, 20.3.
4.2.18. N-(1-methyl-1H-indol-5-yl)-2-[5-(4-methylphenoxy)
pyrazin-2-yl]-2-oxoacetamide (18)
Step 1. 2-Bromo-5-(4-methylphenoxy)pyrazine (18b). A flask was
charged with 2,5-dibromopyrazine (18a, 2 g, 8.4 mmol), p-cresol
(0.91 g, 8.4 mmol), potassium carbonate (2.32 g, 16.8 mmol) and
DMF (15 mL) and the reaction mixture was refluxed overnight.
After completion it was quenched with water and extracted with
CH2Cl2. The organic layer was washed with brine dried and evap-
orated. The crude product 18b was purified with column chroma-
tography (eluent: cyclohexane/EtOAc ¼ 4/1). Yield: 2.15 g (97%).
Step 2. 1-[5-(4-Methylphenoxy)pyrazin-2-yl]ethan-1-one (18d).
Argon was bubbled through toluene (43 mL) for 10 min, 18b (1.45 g,
5.47 mmol), Pd(PPh3)4 (63.2 mg, 0.05 mmol) and [1-ethoxy-1-
(tributhylstannylethylene)] (18c, 2.2 mL, 6.5 mmol) were added.
The reaction mixture was refluxed overnight. To the mixture was
added 1 M HCl solution (37.5 mL) and the mixture was stirred for 2
h at 70 ꢀC. After completion, it was quenched with 2 M NaOH so-
lution and the phases were separated. The organic layer was dried
and evaporated. The 18d crude product was purified with column
chromatography (eluent: cyclohexane/EtOAc ¼ 4/1). Yield: 970 mg
(77%).
Step 3. [5-(4-Methylphenoxy)pyrazin-2-yl](oxo)acetic acid (18e).
A flask was charged with 18d (355 mg,1.56 mmol), pyridine (10 mL)
and SeO2 (518 mg, 4.67 mmol) and the mixture was stirred for 2
days at 70 ꢀC. After completion, the reaction mixture was filtered
through celite and evaporated. To the residue cold water was added
and it was acidified with 50% phosphoric acid to pH ¼ 2. The
resulting solution was extracted with EtOAc, the organic layers
were combined, dried and evaporated. The 18e crude product was
reacted without any further purification. Yield: 121 mg (30%).
Step 4. The amide coupling was achieved in an analogous way as
it is described in case of 11. Yield 3.0%. Mp 155e157 ꢀC. HESI-HRMS:
412.05172; found: 412.05132; delta
(DMSO‑d6, 500 MHz)
¼
ꢁ0.96 ppm. 1H NMR
d
10.66 (s, 1H), 8.63 (s, 1H), 8.0e8.0 (m, 1H),
7.6e7.6 (m, 2H), 7.5e7.5 (m, 2H), 7.4e7.5 (m, 2H), 7.34 (d, 1H, J ¼ 3.1
Hz), 6.42 (d, 1H, J ¼ 3.1 Hz), 3.78 (s, 3H); 13C NMR (DMSO‑d6, 126
MHz)
d 180.6, 172.0, 161.1, 153.2, 144.5, 133.8, 130.46, 130.45, 130.3,
130.1, 129.5, 127.6, 122.3, 115.2, 112.0, 109.6, 100.4, 32.5.
4.2.16. N-(1-methyl-1H-indol-5-yl)-2-[4-(4-methylphenoxy)
phenyl]-2-oxoacetamide (16)
Step 1. Ethyl [4-(4-methylphenoxy)phenyl](oxo)acetate (16b). 1-
Methyl-4-phenoxybenzene (16a, 5.83 g, 31.6 mmol) was dis-
solved in chloroform (90 mL) and cooled down to 0 ꢀC. To the so-
lution was added ethyl chlorooxoacetate (4 mL, 37.3 mmol),
followed by the portionwise addition of AlCl3 (12.66 g, 94.9 mmol).
The solution was stirred for an hour at 0 ꢀC, allowed to warm up to
room temperature and it was stirred overnight. The reaction
mixture was quenched with water, stirred for an hour and extracted
with CH2Cl2. The organic layer was washed with 5% NaHCO3 solu-
tion twice and brine, dried and evaporated. The crude 16b was
purified with column chromatography (eluent: cyclohexane/
EtOAc ¼ 95/5). Yield: 2.608 g (29%).
Step 2e3. The hydrolysis was accomplished in the same way as it
is described in case of 11c. The amide coupling was achieved in an
analogous way as it is described in case of 23. Yield 41.2%. Mp
127e128 ꢀC. HESI-HRMS: calcd for C24H21O3N2 [MþH]þ:
385.15467; found: 385.15422; delta
(DMSO‑d6, 500 MHz)
¼
ꢁ1.17 ppm. 1H NMR
d
10.72 (s, 1H), 8.1e8.1 (m, 2H), 8.0e8.0 (m,
calcd for
C
22H19O3N4 [MþH]þ: 387.14517; found: 387.14432;
1H), 7.4e7.4 (m, 2H), 7.34 (d, 1H, J ¼ 3.0 Hz), 7.3e7.3 (m, 2H), 7.0e7.1
delta ¼ ꢁ2.19 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d 10.75 (s, 1H),
(m, 4H), 6.42 (d, 1H, J ¼ 3.0 Hz), 3.78 (s, 3H), 2.33 (s, 3H); 13C NMR
8.91 (d,1H, J ¼ 1.3 Hz), 8.70 (d,1H, J ¼ 1.3 Hz), 7.98 (d,1H, J ¼ 2.0 Hz),
7.4e7.4 (m, 1H), 7.4e7.4 (m, 1H), 7.34 (d, 1H, J ¼ 3.0 Hz), 7.3e7.3 (m,
2H), 7.2e7.2 (m, 2H), 6.43 (dd, 1H, J ¼ 0.8, 3.0 Hz), 3.79 (s, 3H), 2.34
(DMSO‑d6, 126 MHz)
d 188.3, 163.0, 162.8, 152.1, 134.3, 133.7, 132.6,
130.7, 130.4, 129.7, 127.7, 127.1, 120.2, 116.8, 115.0, 111.8, 109.6, 100.3,
32.5, 20.3.
(s, 3H); 13C NMR (DMSO‑d6, 126 MHz)
d 188.0, 163.0, 161.5, 149.8,
143.5,140.2,135.5,135.2,133.7,130.5,130.2,129.8,127.7,121.2,114.7,
111.4, 109.7, 100.4, 32.5, 20.3.
4.2.17. N-(1-methyl-1H-indol-5-yl)-2-[6-(4-methylphenoxy)
pyridin-3-yl]-2-oxoacetamide (17)
Step 1. ethyl 2-[6-(4-methylphenoxy)pyridin-3-yl]-2-oxoacetate
(17b). Under argon atmosphere, 5-bromo-(4-methylphenoxy)pyr-
idine (17a, 610 mg, 2.3 mmol) was dissolved in toluene and cooled
4.2.19. N-(1,3-dimethyl-1H-indol-5-yl)-2-{4-[(4-methylphenyl)
methyl]piperazin-1-yl}-2-oxoacetamide (19)
The amide coupling was achieved in an analogous way as it is
described in case of 11. Yield 12.4%. Mp 88e91 ꢀC. HESI-HRMS:
calcd for C24H29O2N4 [MþH]þ: 405.22850; found: 405.22788;
down to ꢁ78 ꢀC. To the solution, BuLi (2.5 M in hexane, 924
mL, 2.3
mmol) was dropwise added and it was stirred for 40 min. Diethyl
oxalate (502
m
L, 3.7 mmol) was dropwise added and the reaction
delta ¼ ꢁ1.54 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d 10.53 (s, 1H),
mixture was stirred at ꢁ78 ꢀC for 8 h. After completion it was
quenched with sat. NH4Cl solution and extracted with EtOAc. The
combined organic layer was washed with brine, dried and evapo-
rated. The crude 17b was purified with column chromatography
7.8e7.9 (m, 1H), 7.3e7.3 (m, 1H), 7.3e7.3 (m, 1H), 7.2e7.2 (m, 2H),
7.1e7.2 (m, 2H), 7.07 (q, 1H, J ¼ 1.0 Hz), 3.69 (s, 3H), 3.5e3.6 (m, 4H),
3.48 (s, 2H), 2.4e2.4 (m, 4H), 2.28 (s, 3H), 2.20 (d, 3H, J ¼ 1.0 Hz); 13
C
NMR (DMSO‑d6, 126 MHz)
d 162.8, 161.4, 136.0, 134.4, 133.9, 129.3,
21

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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
128.9, 128.7, 128.0, 127.7, 114.8, 109.7, 109.4, 108.5, 61.4, 52.5, 51.7,
45.6, 40.7, 32.1, 20.6, 9.3.
filtrate was concentrated to afford 22e. Yield: 105 mg (83%).
Step 5. The amide coupling was achieved in an analogous way as
it is described in case of 23. Yield 29.0%. Mp 122e124 ꢀC. HESI-
4.2.20. N-(2,3-dimethyl-1H-indol-5-yl)-2-{4-[(4-methylphenyl)
methyl]piperazin-1-yl}-2-oxoacetamide (20)
HRMS: calcd for
C
23H26O2N4F [MþH]þ: 409.20343; found:
409.20245; delta ¼ ꢁ2.40 ppm. 1H NMR (DMSO‑d6, 500 MHz)
The amide coupling was achieved in an analogous way as it is
described in case of 11. Yield 23.0%. Mp 113e116 ꢀC. HESI-HRMS:
calcd for C24H29O2N4 [MþH]þ: 405.22850; found: 405.22766;
d
10.35 (s, 1H), 7.73 (d, 1H, J ¼ 7.6 Hz), 7.41 (d, 1H, J ¼ 11.3 Hz), 7.34
(d, 1H, J ¼ 3.1 Hz), 7.2e7.2 (m, 2H), 7.1e7.2 (m, 2H), 6.42 (dd, 1H, J ¼
0.8, 3.1 Hz), 3.75 (s, 3H), 3.52 (br s, 4H), 3.48 (br s, 2H), 2.4e2.5 (m,
delta ¼ ꢁ2.08 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d
10.63 (s, 1H),
4H), 2.29 (s, 3H); 13C NMR (DMSO‑d6, 126 MHz)
d 162.7, 162.6, 152.2
10.44 (s, 1H), 7.7e7.7 (m, 1H), 7.2e7.2 (m, 2H), 7.1e7.2 (m, 4H),
3.4e3.6 (m, 6H), 2.3e2.5 (m, 4H), 2.29 (s, 3H), 2.28 (s, 3H), 2.11 (s,
(d, J ¼ 238.4 Hz), 136.0, 134.4, 133.9 (d, J ¼ 11.8 Hz), 130.7 (d, J ¼ 3.2
Hz), 128.9, 128.7, 123.7, 117.0 (d, J ¼ 1.7 Hz), 116.6 (d, J ¼ 15.4 Hz),
100.5, 96.5 (d, J ¼ 25.0 Hz), 61.4, 52.4, 51.6, 45.5, 40.7, 32.6, 20.6.
3H); 13C NMR (DMSO‑d6,126 MHz)
d 162.9,161.3,136.0,134.4,132.3,
132.3, 129.0, 128.9, 128.7, 128.5, 113.6, 110.0, 108.8, 105.0, 61.4, 52.5,
51.6, 45.6, 40.6, 20.6, 11.2, 8.3.
4.2.23. N-(3-fluoro-1-methyl-1H-indol-5-yl)-2-{4-[(4-
methylphenyl)methyl]piperazin-1-yl}-2-oxoacetamide (23)
To a suspension of 2-{4-[(4-methylphenyl)methyl]piperazin-1-
yl}-2-oxoacetic acid (11c, 39.3 mg, 0.15 mmol) and 3-fluoro-1-
methyl-1H-indol-5-amine (23a, 24.6 mg, 0.15 mmol) in dry DMF
(1 mL) was added HATU (57.03 mg, 0.15 mmol, 1 eq) and DIPEA
4.2.21. N-(3-fluoro-1-methyl-1H-indol-6-yl)-2-{4-[(4-
methylphenyl)methyl]piperazin-1-yl}-2-oxoacetamide (21)
3-Fluoro-1-methyl-1H-indol-6-amine was synthesized in an
analogous way as it is described in the following reference [54]. The
amide coupling was achieved in an analogous way as it is described
in case of 11. Yield 26.0%. Mp 127e128 ꢀC. HESI-HRMS: calcd for
(104 mL, 4 eq) at room temperature. The mixture was shaken at RT
overnight. After the completion of the reaction (monitored by TLC)
the mixture was diluted with brine and extracted with EtOAc
(3 ꢂ 20 mL). The combined organic phase was washed with brine
(3 ꢂ 20 mL), dried over anhydrous Na2SO4, filtered and concen-
trated in vacuo. The residue was purified by flash chromatography
on silica gel eluting with 40% EtOAc in cyclohexane. The desired
product 23 was crystallized from Et2O as well to yield 18 mg (30%)
of the title compound as white solid. LC-MS (ESI) m/z
[MþH]þ ¼ 408.5. Yield 29.7%. Mp 138e139 ꢀC. HESI-HRMS: calcd
C
23H26O2N4F
[MþH]þ:
409.20343;
found:
409.20235;
10.77 (s, 1H),
delta ¼ ꢁ2.64 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d
7.9e8.0 (m, 1H), 7.48 (d, 1H, J ¼ 8.5 Hz), 7.31 (d, 1H, J ¼ 2.7 Hz),
7.2e7.2 (m, 3H), 7.1e7.2 (m, 2H), 3.67 (s, 3H), 3.5e3.6 (m, 4H), 3.48
(s, 2H), 2.4e2.4 (m, 4H), 2.28 (s, 3H); 13C NMR (DMSO‑d6, 126 MHz)
d
162.5, 161.7, 143.0 (d, J ¼ 238.8 Hz), 136.0, 134.4, 133.1, 132.5 (d,
J ¼ 5.0 Hz), 128.9, 128.7, 116.4 (d, J ¼ 2.2 Hz), 112.9, 112.8 (d,
J ¼ 16.5 Hz), 112.5 (d, J ¼ 26.6 Hz), 100.9, 61.4, 52.5, 51.6, 45.6, 40.8,
32.3, 20.6.
for
C
23H26O2N4F [MþH]þ: 409.20343; found: 409.20250;
delta ¼ ꢁ2.27 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d 10.66 (s, 1H),
4.2.22. N-(6-fluoro-1-methyl-1H-indol-5-yl)-2-{4-[(4-
7.95 (d, 1H, J ¼ 1.9 Hz), 7.4e7.4 (m, 1H), 7.3e7.4 (m, 2H), 7.2e7.2 (m,
2H), 7.1e7.2 (m, 2H), 3.70 (s, 3H), 3.5e3.6 (m, 2H), 3.5e3.5 (m, 2H),
3.48 (s, 2H), 2.4e2.4 (m, 4H), 2.28 (s, 3H); 13C NMR (DMSO‑d6,
methylphenyl)methyl]piperazin-1-yl}-2-oxoacetamide (22)
Step 1. 6-Fluoro-5-nitro-1H-indole-2,3-dione (22b). To a solution
of NaNO3 (0.5 g, 6.06 mmol) in conc. H2SO4 (3.8 mL, 71 mmol) was
added dropwise a solution of 6-fluoro-1H-indole-2,3-dione (22a)
(1 g, 6.06 mmol) and conc. H2SO4 (10 mL) over a period of 1 h at
126 MHz)
d
162.6, 161.6, 142.9 (d, J ¼ 238.3 Hz), 136.0, 134.4, 130.1,
130.0 (d, J ¼ 5.1 Hz), 128.9, 128.7, 116.0, 115.4 (d, J ¼ 15.4 Hz), 113.3
(d, J ¼ 26.2 Hz),110.4, 106.7 (d, J ¼ 2.0 Hz), 61.4, 52.5, 51.7, 45.6, 40.8,
32.4, 20.6.
0
ꢀC. The reaction mass was then poured to ice water, stirred for
10e15 min, the precipitate was filtered off and dried to get the
desired product 22b. Yield: 1.15 g (90%).
4.2.24. N-(3-chloro-1-methyl-1H-indol-5-yl)-2-{4-[(4-
Step 2. 6-Fluoro-5-nitro-1H-indole (22c). To a solution of 22b (1.1
g, 5.2 mmol) solved in THF (22 mL) was added borane-THF complex
solution (1 M, 12.2 mL, 12.2 mmol) at 0 ꢀC under nitrogen atmo-
sphere. The reaction mixture was allowed to reach room temper-
ature and stirred for 1.5 h 1 M HCl solution was added to reaction
mixture and the volatile components were evaporated and the
aqueous residue was extracted with EtOAc. The combined organic
layer was dried and evaporated, and the residue was purified with
column chromatography. Yield: 0.33 g (35%).
Step 3. 6-Fluoro-1-methyl-5-nitro-1H-indole (22d). NaH (60 m/m
% mineral oil suspension, 0.126 g, 3.15 mmol) was added slowly to
the solution of 22c (0.33 g, 1.83 mmol) in DMF (6.6 mL) under ni-
trogen atmosphere at 0 ꢀC. It was stirred for 0.5 h at 0 ꢀC and then
MeI (0.39 g, 2.74 mmol) was slowly added to the reaction mass over
20e25 min at 0 ꢀC. After the completion of the addition the reaction
mass allowed to warm up to room temperature and the reaction
mixture was stirred for 1 h at this temperature. Ice cold water was
added and reaction mixture was extracted with EtOAc. After
evaporation of the solvent the desired product 22d was obtained.
Yield: 0.35 g (99%).
methylphenyl)methyl]piperazin-1-yl}-2-oxoacetamide (24)
The synthesis of 3-chloro-1-methyl-1H-indol-5-amine is
detailed in the following chemical literature [55]. The amide
coupling was achieved in an analogous way as it is described in case
of 23. Yield 32.4%. Mp 109e110 ꢀC. HESI-HRMS: calcd for
C
23H26O2N4Cl
[MþH]þ:
425.17388;
found:
425.17322;
10.70 (s, 1H),
delta ¼ ꢁ1.55 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d
7.97 (d, 1H, J ¼ 2.0 Hz), 7.53 (s, 1H), 7.47 (d, 1H, J ¼ 8.9 Hz), 7.37 (dd,
1H, J ¼ 2.0, 8.9 Hz), 7.2e7.2 (m, 2H), 7.1e7.2 (m, 2H), 3.76 (s, 3H),
3.5e3.6 (m, 2H), 3.5e3.5 (m, 2H), 3.48 (s, 2H), 2.4e2.4 (m, 4H), 2.28
(s, 3H); 13C NMR (DMSO‑d6, 126 MHz)
d 162.6, 161.6, 136.0, 134.4,
132.5, 130.9, 128.9, 128.7, 127.1, 124.4, 115.9, 110.6, 108.0, 101.9, 61.4,
52.5, 51.7, 45.7, 40.8, 32.7, 20.6.
4.2.25. 4-{[(7-Fluoro-1H-indol-5-yl)carbamoyl]carbonyl}-1-[(4-
methylphenyl) methyl]piperazin-1-ium acetate (25)
The amide coupling was achieved in an analogous way as it is
described in case of 23. Yield 27.8%. Mp 111e113 ꢀC. HESI-HRMS:
calcd for C22H24O2N4F [MþH]þ: 395.18778; found: 395.18678;
delta ¼ ꢁ2.53 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d 11.96 (br s, 0.5H,
Step 4. 6-Fluoro-1-methyl-1H-indol-5-amine (22e). 6-Fluoro-1-
methyl-5-nitro-1H-indole (22d, 150 mg, 0.77 mmol) was dis-
solved in MeOH (10 mL). Pd/C (10 mg) was added and the reaction
mixture was stirred at room temperature under hydrogen atmo-
sphere overnight. Then the reaction solution was filtered and the
A), 11.57 (br s, 1H), 10.66 (s, 1H), 7.70 (d, 1H, J ¼ 1.6 Hz), 7.40 (dd, 1H,
J ¼ 2.4, 3.0 Hz), 7.25 (dd,1H, J ¼ 1.6,13.0 Hz), 7.2e7.2 (m, 2H), 7.1e7.2
(m, 2H), 6.50 (ddd, 1H, J ¼ 2.0, 3.0, 3.4 Hz), 3.5e3.6 (m, 2H), 3.5e3.5
(m, 2H), 3.48 (s, 2H), 2.40 (q, 4H, J ¼ 5.1 Hz), 2.28 (s, 3H), 1.91 (s, 2H,
A); (A: acetic acid); 13C NMR (DMSO‑d6, 126 MHz)
d 172.0 (A), 162.6,
22

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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
161.5, 148.1 (d, J ¼ 241.9 Hz), 136.0, 134.4, 130.8 (d, J ¼ 6.7 Hz), 129.8
(d, J ¼ 8.9 Hz), 128.9, 128.7, 127.2, 120.7 (d, J ¼ 13.1 Hz), 107.0 (d,
J ¼ 3.2 Hz),102.2 (d, J ¼ 1.9 Hz),100.1 (d, J ¼ 20.8 Hz), 61.4, 52.5, 51.7,
45.6, 40.8, 21.0 (A), 20.6; (A: acetic acid).
0.168 mmol, 1.1 eq) and the mixture was stirred at room temper-
ature for 30 min under argon atmosphere. Afterwards, a solution of
3-fluoro-1-methyl-1H-pyrrolo[2,3-b]pyridin-5-amine (27e) (23
mg, 0.14 mmol, 0.91 eq) and Et3N (22 mL, 15.5 mg, 0.155 mmol) in
DMF (0.5 mL) were added and the reaction mixture was stirred for
4 h at room temperature under argon atmosphere. After the
completion of the reaction (monitored by TLC) the mixture was
poured onto water (8 mL) and extracted with EtOAc (2 ꢂ 15 mL).
The organic layer was dried over anhydrous Na2SO4, filtered and
concentrated in vacuo. The residue was chromatographed on silica
gel eluting with EtOAc to yield 16 mg (28%) of the title compound
27. LC-MS (ESI) m/z [MþH]þ ¼ 410.2. Mp 129e131 ꢀC. HESI-HRMS:
calcd for C22H25O2N5F [MþH]þ: 410.19868; found: 410.19808;
4.2.26. N-{1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl}-2-{4-[(4-
methylphenyl)methyl]piperazin-1-yl}-2-oxoacetamide (26)
The amide coupling was achieved in an analogous way as it is
described in case of 23. Yield 31.3%. Mp 85e87 ꢀC. HESI-HRMS:
calcd for C22H26O2N5 [MþH]þ: 392.20810; found: 392.20764;
delta ¼ ꢁ1.18 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d 10.80 (s, 1H),
8.41 (d, 1H, J ¼ 2.3 Hz), 8.28 (d, 1H, J ¼ 2.3 Hz), 7.52 (d, 1H,
J ¼ 3.4 Hz), 7.2e7.2 (m, 2H), 7.1e7.2 (m, 2H), 6.45 (d, 1H, J ¼ 3.4 Hz),
3.80 (s, 3H), 3.5e3.6 (m, 4H), 3.48 (s, 2H), 2.4e2.5 (m, 4H), 2.28 (s,
delta ¼ ꢁ1.46 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d 10.93 (s, 1H),
3H); 13C NMR (DMSO‑d6, 126 MHz)
d
162.4, 161.8, 144.6, 136.0,
8.45 (d, 1H, J ¼ 2.3 Hz), 8.34 (d, 1H, J ¼ 2.3 Hz), 7.57 (d, 1H, J ¼ 2.1
Hz), 7.2e7.2 (m, 2H), 7.1e7.2 (m, 2H), 3.76 (s, 3H), 3.5e3.6 (m, 4H),
3.48 (s, 2H), 2.4e2.4 (m, 4H), 2.28 (s, 3H); 13C NMR (DMSO‑d6, 126
136.0, 134.4, 131.0, 128.9, 128.7, 127.4, 119.9, 119.2, 98.7, 61.4, 52.5,
51.7, 45.7, 40.8, 30.8, 20.6.
MHz)
d
162.2, 161.9, 139.8 (d, J ¼ 4.2 Hz), 140.7 (d, J ¼ 241.7 Hz),
4.2.27. N-{3-fluoro-1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl}-2-{4-
[(4-methylphenyl)methyl ]piperazin-1-yl}-2-oxoacetamide (27)
Step 1. 5-Bromo-3-fluoro-1H-pyrrolo[2,3-b]pyridine (27b). Under
an argon atmosphere, 5-bromo-7-azaindole (27a) (1.86 g, 9.4
mmol) was dissolved in a mixture of dry acetonitrile (225 mL) and
conc. acetic acid (45 mL). The solution was heated to 40 ꢀC,
Selectfluor (5 g, 14.1 mmol, 1.5 eq) was added, the resulted mixture
was heated to 80 ꢀC and stirred overnight under these conditions. It
was evaporated to dryness, the crude product was dissolved in
EtOAc (200 mL) and washed twice with water. The organic layer
was dried over Na2SO4 and evaporated. After flash column chro-
matography (eluent: CH2Cl2/MeOH ¼ 99/1) 362 mg of product was
isolated (18%).
Step 2. 5-Bromo-3-fluoro-1-methyl-1H-pyrrolo[2,3-b]pyridine
(27c). A solution of 27b (300 mg, 1.4 mmol) in dry THF (12 mL) was
cooled to 0 ꢀC and NaH (140 mg, 60 m/m% mineral oil suspension,
3.5 mmol, 2.5 eq) was added in portions. Afterwards the solution
was allowed to warm up to room temperature (approx. 30 min) and
iodomethane (397 mg, 2 eq) was added dropwise and the mixture
was stirred overnight. The reaction was quenched by the addition
of water. The mixture was extracted three times with EtOAc. The
extract was dried over Na2SO4, filtered and evaporated to dryness
to obtain 320 mg of yellowish brown oil (27c), which was used
without further purification.
Step 3. N-benzyl-3-fluoro-1-methyl-1H-pyrrolo[2,3-b]pyridin-5-
amine (27d). Compound 27c (320 mg, 1.4 mmol) was dissolved in
dry toluene(15 mL). Argon was streamed through the solution for
20 min. Afterwards, benzylamine (0.305 mL, 2.8 mmol), NaOtBu
(405 mg, 4.2 mmol), RuPhos (66 mg, 0.14 mmol) and Pd2(dba)3 (65
mg, 0.07 mmol) were added. The reaction mixture was heated to
105 ꢀC overnight. The mixture was allowed to cool to ambient
temperature, water was added and the mixture was extracted with
EtOAc, dried over Na2SO4 and evaporated to dryness. The obtained
27d was used without further purification.
137.4, 136.0, 134.3, 128.9, 128.7, 127.6, 115.9 (d, J ¼ 2.5 Hz), 113.9 (d,
J ¼ 25.4 Hz), 107.5 (d, J ¼ 15.2 Hz), 61.4, 52.5, 51.6, 45.6, 40.9, 30.5,
20.6.
4.2.28. N-(indolizin-7-yl)-2-{4-[(4-methylphenyl)methyl]
piperazin-1-yl}-2-oxoacetamide (28)
The amide coupling was achieved in an analogous way as it is
described in case of 23. Yield 48.8%. Mp 181e182 ꢀC. HESI-HRMS:
calcd for
C
22H25O2N4 [MþH]þ: 377.19720; found: 377.19657;
delta ¼ ꢁ1.68 ppm. 1H NMR (DMSO‑d6, 400 MHz)
d 10.67 (s, 1H),
8.20 (d, 1H, J ¼ 7.5 Hz), 7.88 (d, 1H, J ¼ 2.1 Hz), 7.44 (ddd, 1H, J ¼ 0.5,
1.5, 2.6 Hz), 7.2e7.2 (m, 2H), 7.1e7.2 (m, 2H), 6.69 (dd, 1H, J ¼ 2.6,
3.8 Hz), 6.68 (dd, 1H, J ¼ 2.1, 7.5 Hz), 6.3e6.3 (m, 1H), 3.5e3.6 (m,
2H), 3.4e3.5 (m, 4H), 2.4e2.4 (m, 4H), 2.28 (s, 3H); 13C NMR
(DMSO‑d6, 101 MHz)
d 162.2, 161.7, 136.0, 134.4, 131.2, 128.8, 128.7,
127.8, 126.2, 113.9, 112.4, 106.2, 105.4, 98.4, 61.4, 52.5, 51.6, 45.6,
40.8, 20.6.
4.2.29. N-(1-methyl-2,3-dihydro-1H-indol-5-yl)-2-{4-[(4-
methylphenyl)methyl]piperazin-1-yl}-2-oxoacetamide (29)
The amide coupling was achieved in an analogous way as it is
described in case of 23. Yield 19.0%. Mp 114e115 ꢀC. HESI-HRMS:
calcd for C23H29O2N4 [MþH]þ: 393.22850; found: 393.22753;
delta ¼ ꢁ2.47 ppm. 1H NMR (DMSO‑d6, 500-MHz)
d 10.36 (s, 1H),
7.3e7.4 (m, 1H), 7.24 (dd, 1H, J ¼ 2.1, 8.4 Hz), 7.2e7.2 (m, 2H), 7.1e7.2
(m, 2H), 6.45 (d, 1H, J ¼ 8.4 Hz), 3.5e3.5 (m, 2H), 3.4e3.5 (m, 4H),
3.21 (t, 2H, J ¼ 8.2 Hz), 2.84 (t, 2H, J ¼ 8.2 Hz), 2.66 (s, 3H), 2.3e2.4
(m, 4H), 2.28 (s, 3H); 13C NMR (DMSO‑d6, 126 MHz)
d 162.8, 161.1,
150.2, 136.0, 134.4, 130.2, 128.8, 128.7, 128.4, 119.1, 117.0, 106.5, 61.4,
55.7, 52.5, 51.6, 45.6, 40.7, 36.1, 28.1, 20.6.
Step 4. 3-Fluoro-1-methyl-1H-pyrrolo[2,3-b]pyridin-5-amine
(27e) Compound 27d (96 mg, 0.376 mmol) was hydrogenated in a
mixture of methanol (6 mL) and EtOAc (6 mL) with 10% Pd/C (35
mg) at atmospheric pressure for 48 h. The catalyst was filtered off,
and the solvent was evaporated. The crude product was purified
with flash chromatography, using EtOAc as the eluent to obtain 23
mg of 27e (37%).
4.2.30. N-[(1-methyl-1H-indol-5-yl)methyl]-2-{4-[(4-
methylphenyl)methyl]piperazin-1-yl}-2-oxoacetamide (30)
The amide coupling was achieved in an analogous way as it is
described in case of 23. Yield 27.0%. Mp 140e141 ꢀC. HESI-HRMS:
calcd for C24H29O2N4 [MþH]þ: 405.22850; found: 405.22747;
delta ¼ ꢁ2.55 ppm. 1H NMR (DMSO‑d6, 400 MHz)
d 9.13 (t, 1H,
J ¼ 6.0 Hz), 7.42 (d,1H, J ¼ 1.6 Hz), 7.37 (d,1H, J ¼ 8.4 Hz), 7.30 (d,1H,
J ¼ 3.1 Hz), 7.2e7.2 (m, 2H), 7.1e7.2 (m, 2H), 7.07 (dd, 1H, J ¼ 1.6,
8.4 Hz), 6.37 (dd, 1H, J ¼ 0.7, 3.1 Hz), 4.37 (d, 2H, J ¼ 6.0 Hz), 3.76 (s,
3H), 3.4e3.5 (m, 6H), 2.3e2.4 (m, 4H), 2.28 (s, 2H); 13C NMR
Step 5. N-{3-Fluoro-1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl}-2-
{4-[(4-methylphenyl)methyl] piperazin-1-yl}-2-oxoacetamide (27).
2-{4-[(4-methylphenyl)methyl]piperazin-1-yl}-2-oxoacetic
acid
(11c, 40 mg, 0.152 mmol) was dissolved in dry DMF (1 mL) under an
argon atmosphere. To this solution was added 1-chloro-N,N,2-
(DMSO‑d6, 101 MHz) d 163.2, 163.1, 136.0, 135.5, 134.4, 129.8, 128.9,
128.8, 128.7, 127.8, 120.9, 119.1, 109.5, 100.0, 61.4, 52.4, 51.6, 45.4,
42.0, 40.6, 32.4, 20.6.
trimethyl-1-propenylamine (Ghosez’ reagent) (22.4 mg, 23
mL,
23

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ꢀ
I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
4.2.31. 3-(1-Methyl-1H-indol-5-yl)-1-{4-[(4-methylphenyl)methyl]
piperazin-1-yl}propane-1,2-dione (31)
Step 1. Intermediate 31a was synthesized according to the
following reference [56].
was used in the next step without further purification.
Step 3. N-[(1-methylindol-5-yl)carbonyl]-4-[(4-methylphenyl)
methyl]piperidine-1-carboxamide (32). Oxalyl chloride (343 mg, 228
mL, 2.7 mmol) was added dropwise into a stirred solution of the
Step 2. Ethyl 2-(acetyloxy)-3-(1-methyl-1H-indol-5-yl)prop-2-
enoate (31c). A mixture of ethyl 2-acetoxy-3-bromo-2-propenoate
(31a, 287 mg, 1.2 mmol), 1-methyl-1H-indol-5-boronic acid (31b,
175 mg, 1 mmol), K2CO3 (414 mg, 3 mmol) and Pd(PPh3)4 (35 mg, 3
mol%) in dioxane (4 mL) was heated at 90 ꢀC for 4 h under an inert
atmosphere. After cooling the reaction mixture to room tempera-
ture, the solid material was filtered off, the filtrate was concen-
trated and purified by column chromatography on silica (eluent:
EtOAc-cyclohexane, 1:9) to afford 31c as a yellow oil (180 mg, 63%).
Step 3. 3-(1-Methyl-1H-indol-5-yl)-2-oxopropanoic acid (31d). A
mixture of 31c (110 mg, 0.38 mmol), ethanol (3 mL), dioxane (1 mL)
and saturated aqueous NaHCO3 solution (1 mL) was heated under
reflux for 4 h. After cooling to room temperature, the reaction
mixture was concentrated and purified by column chromatography
on silica (eluent: methanol-CH2Cl2, 1:4) to afford 31d as yellow
solid (66 mg, 81%), which was used immediately in the next reac-
tion step.
carboxamide derivative 32c (314 mg, 1.8 mmol) in dichloroethane
(10 mL) at 0 ꢀC. Then, the mixture was heated at reflux for 20e22 h.
The solvent was evaporated in vacuo to give the respective crude
benzoyl isocyanate (32d), which was used immediately in the next
step without purification. The crude benzoyl isocyanate derivative
in dichloroethane (5 mL) was added to a mixture of 4-(4-
methylbenzyl)piperidine hydrochloride (607 mg, 2.7 mmol) and
triethylamine (365 mg, 502 mL, 3.6 mmol) in dichloroethane (5 mL).
It was stirred at room temperature overnight. The reaction was
quenched with water (20 mL) and extracted with CH2Cl2 (3 ꢂ 10
mL). The organic layers were combined, washed with water (2 ꢂ 5
mL), dried over anhydrous Na2SO4, filtered and concentrated in
vacuo and the residue was purified by flash chromatography on
silica gel (gradient elution: n-hexane to n-hexane - EtOAc ¼ 50:50),
to obtain 32 (17 mg, 25%), as white-off solid. Mp 160e162 ꢀC. HESI-
HRMS: calcd for
C
24H28O2N3 [MþH]þ: 390.21760; found:
390.21697; delta ¼ ꢁ1.62 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d 9.92
Step
4.
3-(1-Methyl-1H-indol-5-yl)-1-{4-[(4-methylphenyl)
(s, 1H), 8.17 (d, 1H, J ¼ 1.7 Hz), 7.70 (dd, 1H, J ¼ 1.7, 8.6 Hz), 7.50 (d,
1H, J ¼ 8.6 Hz), 7.43 (d, 1H, J ¼ 3.1 Hz), 7.0e7.1 (m, 4H), 6.55 (dd, 1H,
J ¼ 0.8, 3.1 Hz), 3.83 (s, 3H), 3.7e4.2 (br m, 2H), 2.7e3.0 (br m, 2H),
2.5e2.5 (m, 3H), 2.26 (s, 3H), 1.7e1.8 (m, 1H), 1.5e1.6 (m, 2H),
methyl]piperazin-1-yl}propane-1,2-dione (31). To a suspension of 1-
(4-methylbenzyl)piperazine (11a, 58 mg, 0.30 mmol) and 3-(1-
methyl-1H-indol-5-yl)-2-oxopropanoic acid (31d, 66 mg, 0.30
mmol) in dry DMF (3 mL) was added HATU (142 mg, 0.375 mmol,
1.1e1.2 (m, 2H); 13C NMR (DMSO‑d6, 126 MHz)
d 166.7, 152.8, 138.2,
1.25 eq) and DIPEA (105
m
L, 2 eq) at room temperature. The mixture
136.8, 134.6, 131.2, 128.8, 128.6, 127.2, 124.0, 121.4, 121.1, 109.3, 101.7,
44.8, 41.6, 37.2, 32.6, 31.5, 20.5.
was shaken at RT overnight. After the completion of the reaction
(monitored by TLC) the mixture was diluted with brine and
extracted with EtOAc (3 ꢂ 20 mL). The combined organic phase was
washed with brine (3 ꢂ 20 mL), dried over anhydrous Na2SO4,
filtered and concentrated in vacuo. The residue was purified by flash
chromatography on silica gel eluting with 10% EtOAc in CH2Cl2. The
desired product 31 was crystallized from Et2O as well to yield 14 mg
(12%) of the title compound as white solid. LC-MS (ESI) m/z
[MþH]þ¼ 389.49. Mp 95e97 ꢀC. HESI-HRMS: calcd for C24H28O2N3
[MþH]þ: 390.21760; found: 390.21680; delta ¼ ꢁ2.06 ppm. 1H
4.2.33. 1-Methyl-4-(5-{4-[(4-methylphenyl)methyl]piperazine-1-
carbonyl}-1,3,4-oxadiazol-2-yl)-1H-indole (33)
Step 1. 1H-indole-4-carboxylic acid (33a) (5 g, 31 mmol) was
dissolved in ethanol (100 mL), conc. sulfuric acid (1 mL) was added
and the resulting solution was refluxed for 6 h. The reaction
mixture was evaporated to ca. 15 mL and cold water (60 mL) was
added and the pH was adjusted to 10 with saturated NaOH solution.
The resulting mixture was extracted with CH2Cl2 (2 ꢂ 75 mL) and
the combined organic layer was dried over Na2SO4 and was evap-
orated to dryness. The 33b crude product was purified with column
chromatography (eluent: CH2Cl2/MeOH ¼ 99/1). Yield: 2.97 g (51%).
Step 2. Ethyl 1-methyl-1H-indole-4-carboxylate (33c). A flask was
charged with compound 33b (2.97 g, 15.7 mmol), acetonitrile (60
mL), potassium carbonate (6.51 g, 3 eq) and dimethyl sulphate(2.7
mL, 1.5 eq). The reaction mixture was stirred at 95 ꢀC overnight,
then filtered and evaporated to dryness. The residue was dissolved
in EtOAc (75 mL) washed with water (2 ꢂ 50 mL) and brine (1 ꢂ 50
mL), dried over Na2SO4 and evaporated. The 33c crude product was
purified by column chromatography with CH2Cl2 as the eluent.
Yield: 1.94 g (61%).
NMR (DMSO‑d6, 500 MHz)
d
7.4e7.4 (m, 2H), 7.37 (d, 1H, J ¼ 3.0 Hz),
7.1e7.1 (m, 2H), 7.0e7.0 (m, 2H), 7.0e7.0 (m, 1H), 6.40 (dd, 1H, J ¼
0.7, 3.0 Hz), 4.04 (s, 2H), 3.80 (s, 3H), 3.3e3.4 (m, 2H), 3.11 (s, 2H),
3.0e3.0 (m, 2H), 2.26 (s, 3H), 2.1e2.2 (m, 2H), 1.5e1.6 (m, 2H); 13
C
NMR (DMSO‑d6, 126 MHz)
128.7, 128.7, 128.5, 122.9, 121.7, 121.4, 109.9, 100.1, 61.3, 51.6, 51.6,
46.5, 44.8, 40.5, 32.5, 20.6.
d 198.8, 165.3, 136.0, 135.7, 134.3, 130.1,
4.2.32. N-(1-methyl-1H-indole-5-carbonyl)-4-[(4-methylphenyl)
methyl]piperidine-1-carboxamide (32)
Step 1. 1-Methyl-1H-indole-5-carbonitrile (32b). To a solution of
lH-indole-5-carbonitrile (32a, 356 mg, 2.5 mmol) in THF (16 mL)
was added NaH (250 mg, 60% in oil, 6.25 mmol) with vigorous
stirring at 0 ꢀC under argon atmosphere. The solution was stirred
Step 3. 1-Methyl-1H-indole-4-carbohydrazide (33d). A solution of
33c (1.94 g, 9.55 mmol), and hydrazine hydrate (60 mL) in ethanol
(96 mL) was refluxed overnight and then it was evaporated to
dryness. The residue was taken up in CH2Cl2 and washed with
water (2 ꢂ 50 mL) and brine (50 mL). The organic phase was dried
and evaporated. The residue was treated with diethyl ether and the
remaining solid 33d was filtered off and dried. Yield: 1.05 g (58%).
for 30 min, then iodomethane (710 mg, 312 mL, 5 mmol) was added.
The reaction mixture was stirred at room temperature for 6 h. The
reaction was quenched with water (50 mL) and extracted with
EtOAc (3 ꢂ 120 mL). The organic layers were combined, washed
with water (2 ꢂ 30 mL), dried over anhydrous Na2SO4, filtered and
concentrated in vacuo and the residue (390 mg of 32b) was used in
the next step without further purification.
Step
4.
N'-(1-methyl-1H-indole-4-carbonyl)-2-{4-[(4-
(33e).
methylphenyl)methyl]piperazin-1-yl}-2-oxoacetohydrazide
Step 2. 1-Methyl-1H-indole-5-carboxamide (32c). To a solution of
32b (390 mg, 2.5 mmol) in DMSO (10 mL) was added K2CO3 (120
mg, 0.87 mmol) with vigorous stirring at 10 ꢀC under argon at-
Compound 11b (0.6 g, 2.2 mmol), CH2Cl2 (100 mL), triethylamine
(0.8 mL, 2.5 eq) and HBTU (1.04 g, 1.2 eq) were charged into a flask
and the solution was stirred for 20 h at room temperature. After
that the solution of 33d (0.54 g, 1.2 eq) in CH2Cl2 (70 mL) was added
to the reaction mixture and it was stirred for additional 48 h. The
resulting reaction mixture was washed with water (2 ꢂ 50 mL) and
brine (50 mL). The organic layer was dried over Na2SO4 and
mosphere then H2O2 (270 mL, 2.5 mmol, 30% solution in water) was
added. The reaction mixture was stirred at room temperature for 12
h. The reaction was quenched with water (20 mL) and the precip-
itated material was filtered off and dried to give 330 mg of 31c that
24

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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
evaporated to dryness. After column chromatography (eluent:
CH2Cl2/MeOH ¼ 95/5) the main product was treated with diethyl
ether, 33e was obtained. Yield: 0.22 g (23%).
chromatography. Yield: 170 mg (81%). Mp 73e75 ꢀC. HESI-HRMS:
calcd for C24H25ON5Cl [MþH]þ: 434.17421; found: 434.17311;
delta ¼ ꢁ2.54 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d 11.12 (br s, 1H),
Step 5. 1-Methyl-4-(5-{4-[(4-methylphenyl)methyl]piperazine-1-
carbonyl}-1,3,4-oxadiazol-2-yl)-1H-indole (33). A flask was charged
with compound 33e (0.22 g, 0.49 mmol), 1,4-dioxane (15 mL) and
triethylamine (0.36 mL, 5 eq) and to the mixture was added thionyl
chloride (0.08 mL 2 eq). The reaction mixture (after gas formation)
was heated to 100 ꢀC for 1.5 h. After completion of the reaction it
was evaporated to dryness. The residue was dissolved in CH2Cl2 and
washed with water (2 ꢂ 30 mL) and brine (40 mL). The organic
phase was dried over Na2SO4 and evaporated. The crude product
was crystalized from diethyl ether, the product 33 was filtered off,
washed with diethyl ether and dried. Yield: 65 mg (32%). Mp
134e135 ꢀC. HESI-HRMS: calcd for C24H26O2N5 [MþH]þ:
8.0e8.0 (m, 1H), 7.58 (dd, 1H, J ¼ 1.6, 8.4 Hz), 7.3e7.4 (m, 6H), 6.84
(s, 1H), 6.44 (ddd, 1H, J ¼ 0.8, 2.0, 3.0 Hz), 3.86 (s, 3H), 3.5e3.7 (m,
4H), 3.53 (s, 2H), 2.4e2.5 (m, 4H); 13C NMR (DMSO‑d6, 126 MHz)
d
160.1, 150.1, 136.9, 136.4, 135.6, 131.5, 130.6, 128.1, 127.7, 125.8,
123.6, 118.9, 116.8, 111.4, 102.8, 101.3, 60.7, 52.7, 52.0, 46.9, 41.5, 37.6.
4.2.35. N-(1-methyl-1H-indol-5-yl)-5-{1-[(4-methylphenyl)
methyl]piperidin-4-yl}-1,3,4-oxadiazole-2-carboxamide (35)
Step 1. Ethyl 1-[(4-methylphenyl)methyl]piperidine-4-carboxylate
(35b). In an argon atmosphere, ethyl piperidine-4-carboxylate
(35a) (10 mL, 64.48 mmol) was dissolved in DMF (80 mL), K2CO3
(26.8 g, 3 eq) and 1-(bromomethyl)-4-methylbenzene (14.4 g 1.2
eq) were added. The reaction mixture was stirred at 80 ꢀC over-
night. After completion of the reaction, the mixture was filtered and
then the filtrate was diluted with water (200 mL) and extracted
with CH2Cl2 (3 ꢂ 50 mL). The combined organic phase was washed
with brine, dried and evaporated to dryness. The residue was pu-
rified by column chromatography (eluent: CH2Cl2/MeOH ¼ 98/2).
Yield: 7.9 g (47%).
416.20810; found: 416.20714; delta
(DMSO‑d6, 400 MHz)
7.3e7.4 (m, 1H), 7.2e7.2 (m, 2H), 7.1e7.2 (m, 2H), 7.06 (dd, 1H, J ¼
0.7, 3.1 Hz), 3.9e4.0 (m, 2H), 3.90 (s, 3H), 3.7e3.8 (m, 2H), 3.50 (s,
2H), 2.4e2.5 (m, 4H), 2.29 (s, 3H); 13C NMR (DMSO‑d6, 101 MHz)
¼
ꢁ2.31 ppm. 1H NMR
d
7.8e7.8 (m, 2H), 7.62 (d, 1H, J ¼ 3.1 Hz),
d
164.8, 157.0, 153.2, 136.9, 136.0, 134.4, 132.4, 128.8, 128.7, 125.1,
120.9, 119.5, 114.4, 113.2, 100.6, 61.3, 52.6, 51.7, 46.5, 42.2, 32.7, 20.6.
Step 2. 1-[(4-Methylphenyl)methyl]piperidine-4-carbohydrazide
(35c). A suspension of 35b (7.9 g, 28.9 mmol) and hydrazine hy-
drate (40 mL) was heated at 120 ꢀC for 3 h. The reaction mixture
was filtered and the resulting solid 35c was washed with n-hexane
(3 ꢂ 50 mL). Yield: 6.67 g (93%).
4.2.34. 5-(5-{4-[(4-Chlorophenyl)methyl]piperazine-1-carbonyl}-
1-methyl-1H-pyrazol-3-yl)-1H-indole (34)
Step 1. Ethyl 4-(1H-indol-5-yl)-2,4-dioxobutanoate (34b). In a
nitrogen atmosphere dry ethanol (5 mL) was added to a flask and
sodium was carefully added (0.202 g, 8.79 mmol). The mixture was
cooled to 0e5 ꢀC with an ice bath. Through a septum a solution of
diethyl oxalate (1.02 mL, 7.54 mmol), 5-acetylindole (34a, 1 g, 6.28
mmol) and ethanol (5 mL) were added slowly, in a drop by drop
manner. The resulting solution was stirred in an ice bath for 1 h and
it was warmed up to reflux and stirred for further 2 h. After
completion of the reaction, water was added (carefully at the
beginning) and the mixture was acidified with 1 M HCl. The formed
precipitate was filtered off and dried at 40 ꢀC to give 34b. Yield:
1.353 g (83%).
Step 2. Ethyl 3-(1H-indol-5-yl) -1-methyl-1H-pyrazole-5-
carboxylate (34c). In a nitrogen atmosphere, 34b (1.353 g, 5.22
mmol) was solved in dry ethanol (15 mL) and methylhydrazine
(0.185 mL, 5.74 mmol) was added dropwise. The mixture was
refluxed for 3 h. After completion, the reaction mixture was cooled
down and evaporated to dryness. The solid residue was dissolved in
EtOAc and washed twice with water. The organic phase was dried
and evaporated to dryness. The crude product was purified with
flash chromatography (eluent: heptane/EtOAc) to result in 34c.
Yield: 520 mg.
Step 3. 3-(1H-indol-5-yl)-1-methyl-1H-pyrazole-5-carboxylic acid
(34d). 34c (130 mg, 0.483 mmol) was dissolved in a mixture of
ethanol (5 mL) and water (0.5 mL). Potassium hydroxide was added
(81 mg, 1.448 mmol) and the solution was stirred at room tem-
perature for 3 h. The solvent was evaporated to dryness. Water was
added and the solution was acidified (pH ¼ 1) with 1 M HCl. The
mixture was extracted 3 times with EtOAc. The combined organic
phase was dried and evaporated to dryness. Yield: 135 mg.
Step 4. 5-(5-{4-[(4-Chlorophenyl)methyl]piperazine-1-carbonyl}-
1-methyl-1H-pyrazol-3-yl)-1H-indole (34). In a N2 atmosphere, a
flask was charged with 34d (116 mg, 0.481 mmol), CH2Cl2 (3e5 mL)
and triethylamine (0.268 mL, 1.923 mmol). (If not dissolved, few
drops of DMF could be added.) 1-(4-chlorobenzyl)piperazine (122
mg, 0.577 mmol) and 1-propanephosphonic acid cyclic anhydride
(50% in EtOAc) (0.567 mL, 0.962 mmol) were slowly added. The
mixture was stirred at room temperature for 2 h. It was diluted with
CH2Cl2 and washed with water and dried. The organic phase was
evaporated to dryness. The final product 34 was purified with flash
Step 3. Ethyl 5-{1-[(4-methylphenyl)methyl]piperidin-4-yl}-1,3,4-
oxadiazole-2-carboxylate (35d). A flask was charged with 35c
(6.55 g, 26.48 mmol), CH2Cl2 (106 mL) and triethylamine (6.67 mL,
1.8 eq). The mixture was cooled down on an ice bath and ethyl
oxalyl chloride (3.2 mL, 1.08 eq) was added dropwise (gas evolu-
tion) and the solution was stirred for 3.5 h at room temperature.
The reaction mixture was washed with saturated NaHCO3, dried
and evaporated to dryness. The residue was dissolved in toluene
(106 mL), pyridine (2.67 mL) and thionyl chloride (6.8 mL, 82.3
mmol, 3 eq) were added (gas evolution) and the reaction mixture
was refluxed for 2 h. After ice-water cooling, the precipitated ma-
terial was filtered off and washed with toluene. The crude product
was dissolved in CH2Cl2 (200 mL) and the solution was washed
with sat. NaHCO3 solution (2 ꢂ 75 mL) and water (100 mL). The
organic layer was dried and evaporated to dryness. Yield 5.24 g
(60%).
Step 4. The hydrolysis of 35d was accomplished the same way as
in case of the preparation of 11c resulting in 35e.
Step 5. N-(1-methyl-1H-indol-5-yl)-5-{1-[(4-methylphenyl)methyl]
piperidin-4-yl}-1,3,4-oxadiazole-2-carboxamide (35). To a solution of
1-methyl-1H-indol-5-amine (11d, 480 mg, 3.3 mmol) of in dry
CH2Cl2 (130 mL) was added N,N,N0,N0-tetramethyl-O-(1H-benzo-
triazol-1-yl)uronium hexafluorophosphate (HBTU) (1040 mg, 2.75
mmol) and Et3N (1920 mL, 5.0 eq) at room temperature under argon.
The mixture was stirred for 20 min then was added 5-{1-[(4-
methylphenyl)methyl]piperidin-4-yl}-1,3,4-oxadiazole-2-carboxylic
acid (35e, 2960 mg, 2.75 mmol). The mixture was stirred at room
temperature overnight. After the completion of the reaction (moni-
tored by TLC) the reaction mixture was quenched by addition of
water (50 mL), the organic layer was washed with saturated NaCl
solution (50 mL) then the organic phase was dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. The residue was purified
by column chromatography on silica gel eluting with CH2Cl2:me-
thanol (95:5) to yield 300 mg (27%) of the title compound. LC-MS
(ESI) m/z [MþH]þ ¼ 429.51. Mp 153e154 ꢀC. HESI-HRMS: calcd for
C
25H28O2N5 [MþH]þ: 430.22375; found: 430.22251; delta ¼ ꢁ2.89
ppm. 1H NMR (DMSO‑d6, 400 MHz)
d
10.99 (s,1H), 8.01 (d,1H, J ¼ 2.0
Hz), 7.5e7.5 (m, 1H), 7.4e7.5 (m, 1H), 7.34 (d, 1H, J ¼ 3.1 Hz), 7.2e7.2
25

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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
(m, 2H), 7.1e7.2 (m, 2H), 6.43 (d, 1H, J ¼ 3.1 Hz), 3.79 (s, 3H), 3.45 (s,
2H), 3.08 (tt,1H, J ¼ 3.9,11.1 Hz), 2.8e2.9 (m, 2H), 2.28 (s, 3H), 2.1e2.2
(m, 2H), 2.0e2.1 (m, 2H), 1.7e1.9 (m, 2H); 13C NMR (DMSO‑d6, 101
1H-indole-5-carbonyl azide (37a, 425 mg, 2.13 mmol) in toluene
(5 mL) was heated under reflux for an hour. A solution of 1-[(4-
methylphenyl)methyl]piperidin-4-amine
(37b,
434
mg,
MHz)
d
170.3, 158.8, 151.2, 135.8, 135.1, 133.9, 130.5, 129.4, 128.7,
2.13 mmol) in toluene (5 mL) was added and the heating was
continued for 5 more hours. Upon cooling the reaction mixture to
room temperature, the crystals separated were filtered off, washed
with diethyl ether and dried to yield the title compound 37
(276 mg, 35%). Mp 187e189 ꢀC. HESI-HRMS: calcd for C23H29ON4
[MþH]þ: 377.23359; found: 377.23291; delta ¼ ꢁ1.80 ppm. 1H NMR
128.6, 127.5, 115.7, 112.7, 109.5, 100.4, 61.9, 51.8, 32.5, 32.5, 28.8, 20.6.
4.2.36. 3-Methoxy-N-(1-methyl-1H-indol-5-yl)-2-[4-(4-
methylphenoxy)piperidin-1-yl]propenamide (36)
Step 1. Methyl 3-methoxy-2-[4-(4-methylphenoxy)piperidin-1-yl]
propanoate (36b). Under argon, a mixture of methyl 2-bromo-3-
methoxypropanoate (36a, 250 mg, 1.3 mmol), 4-(4-
methylphenoxy)piperidine hydrochloride (300 mg, 1.3 mmol) and
potassium carbonate (360 mg, 2.6 mmol, 2 eq) in DMF (4 mL) was
heated at 90 ꢀC for 3 h. The reaction mixture was then cooled to
room temperature, diluted with EtOAc (30 mL) and with water (20
mL) then two phases were separated. The aqueous layer was
extracted with EtOAc (2 ꢂ 15 mL. The combined organic layer was
washed with water (3 ꢂ 30 mL), dried over Na2SO4 and concen-
trated. The residue was purified by flash column chromatography
on silica gel applying gradient elution with a 0e20% EtOAc - CH2Cl2
mixture as eluent to yield 246 mg (62%) of 36b.
Step 2. 3-Methoxy-2-[4-(4-methylphenoxy)piperidin-1-yl]prop-
anoic acid (36c). To a solution of methyl ester 36b (237 mg, 0.77
mmol) in methanol (2.3 mL) was added water (2.75 mL) and
lithium hydroxide monohydrate (163 mg, 3.88 mmol, 5 eq) and the
mixture was stirred at room temperature for 4 h. Then lithium
hydroxide monohydrate (40 mg, 0.953 mmol, 5.18 eq) was added
again and the mixture stirred for further 90 min at room temper-
ature. After the reaction had been completed, it was neutralized
with 10% HCl solution. The mixture was evaporated under reduced
pressure to obtain a crude product. It was dried at 40 ꢀC for 24 h.
The carboxylic acid 36c which contains LiCl (393 mg) was used
without purification in next step.
(DMSO‑d6, 500 MHz)
d
8.08 (s, 1H), 7.59 (d, 1H, J ¼ 2.0 Hz), 7.26 (d,
1H, J ¼ 8.8 Hz), 7.22 (d, 1H, J ¼ 3.0 Hz), 7.2e7.2 (m, 2H), 7.1e7.1 (m,
2H), 7.05 (dd, 1H, J ¼ 2.0, 8.8 Hz), 6.28 (dd, 1H, J ¼ 0.8, 3.0 Hz), 5.96
(d, 1H, J ¼ 7.7 Hz), 3.72 (s, 3H), 3.4e3.5 (m, 1H), 3.40 (s, 2H), 2.6e2.7
(m, 2H), 2.28 (s, 3H), 2.0e2.1 (m, 2H), 1.7e1.8 (m, 2H), 1.3e1.4 (m,
2H); 13C NMR (DMSO‑d6, 126 MHz)
d 154.9, 135.7, 135.4, 132.5,
132.4, 129.6, 128.7, 128.6, 128.0, 114.1, 109.3, 109.1, 99.7, 61.9, 51.6,
46.0, 32.4, 32.3, 20.6.
4.2.38. N-[(1-methyl-1H-indol-5-yl)methyl]-4-(4-methylphenoxy)
benzamide (38)
To a solution of (2-methyl-1H-indol-5-yl)methanamine (38a,
160 mg, 1.0 mmol) in dry CH2Cl2 (100 mL) was added N,N,N0,N0-tet-
ramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate
(HBTU) (460 mg, 1.2 mmol) and Et3N (360 mL, 2.5 eq) at room tem-
perature under argon. The mixture was stirred for 20 min then was
added 4-(4-methylphenoxy)benzoic acid (38b, 230 mg, 1.0 mmol).
The mixture was stirred at room temperature overnight. After the
completion of the reaction (monitored by TLC) the reaction mixture
was quenched by addition of water (50 mL), the organic layer was
washed with saturated NaCl solution (50 mL) then the organic phase
was dried over anhydrous Na2SO4, filtered and concentrated in
vacuo. The residue was purified by column chromatography on silica
gel eluting with CH2Cl2 - methanol (9:1) to yield (168 mg, 45%) of the
title compound. LC-MS (ESI) m/z [MþH]þ ¼ 370.45. Mp 139e140 ꢀC.
HESI-HRMS: calcd for C24H23O2N2 [MþH]þ: 371.17540; found:
Step
3.
3-Methoxy-N-(1-methyl-1H-indol-5-yl)-2-[4-(4-
methylphenoxy)piperidin-1-yl]propenamide) (36). To a solution of
36c (393 mg, 0.77 mmol, contains LiCl) in DMF (12 mL) was added
HATU (384 mg, 1 mmol, 1.31 eq) and 1-methyl-1H-indol-5-amine
(11d, 146 mg, 1 mmol, 1.3 eq). The reaction mixture was cooled to
371.17465; delta ¼ ꢁ2.03 ppm. 1H NMR (DMSO‑d6, 400 MHz)
d 8.92
(t, 1H, J ¼ 6.0 Hz), 7.9e7.9 (m, 2H), 7.46 (d, 1H, J ¼ 0.7 Hz), 7.37 (d, 1H,
J ¼ 8.4 Hz), 7.28 (d, 1H, J ¼ 3.1 Hz), 7.2e7.3 (m, 2H), 7.13 (dd, 1H,
J ¼ 1.6, 8.4 Hz), 6.9e7.0 (m, 4H), 6.37 (dd, 1H, J ¼ 0.7, 3.1 Hz), 4.54 (d,
2H, J ¼ 6.0 Hz), 3.76 (s, 3H), 2.31 (s, 3H); 13C NMR (DMSO‑d6,
0
ꢀC and then DIPEA (540
mL, 3.1 mmol, 4 eq) was added to the
solution. After 1 h the reaction mixture was allowed to reach room
temperature and stirred for 2 days. The reaction mixture was
diluted with ethyl acetate (60 mL) and water (60 mL) then two
phases were separated. The aqueous layer was extracted with
ethyl acetate (2 ꢂ 20 mL). The combined organic layer was washed
with water (5 ꢂ 50 mL), dried over Na2SO4 and concentrated. The
residue was purified by column chromatography over silica gel,
eluting with a mixture of cyclohexane and EtOAc (2:1) to get 0.22
g of crude product which was purified again by column chroma-
tography over silica gel eluting with mixture of CH2Cl2 and EtOAc
(20:1) to yield 70.5 mg (22%) of 36. Mp 75e78 ꢀC. HESI-HRMS:
calcd for C25H32O3N3 [MþH]þ: 422.24382; found: 422.24266;
101 MHz)
d 165.1, 159.8, 153.1, 135.4, 133.4, 130.5, 130.1, 129.7, 129.2,
128.8, 127.8, 120.9, 119.5, 118.8, 116.7, 109.3, 100.0, 42.9, 32.4, 20.2.
4.2.39. N-[(1-methyl-1H-indol-5-yl)methyl]-4-(4-methylphenoxy)
piperidine-1-carboxamide (39)
The amide coupling was achieved in an analogous way as it is
described in case of 44. Yield 19.4%. Mp 132e133 ꢀC. HESI-HRMS:
calcd for C23H28O2N3 [MþH]þ: 378.21760; found: 378.21679;
delta ¼ ꢁ2.15 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d 7.40 (dd, 1H,
J ¼ 0.8,1.6 Hz), 7.34 (d, 1H, J ¼ 8.4 Hz), 7.27 (d,1H, J ¼ 3.1 Hz), 7.1e7.1
(m, 3H), 7.03 (t,1H, J ¼ 5.8 Hz), 6.8e6.9 (m, 2H), 6.36 (dd,1H, J ¼ 0.8,
3.1 Hz), 4.47 (tt, 1H, J ¼ 3.6, 8.5 Hz), 4.31 (d, 2H, J ¼ 5.8 Hz), 3.76 (s,
3H), 3.7e3.7 (m, 2H), 3.1e3.2 (m, 2H), 2.22 (s, 3H), 1.8e1.9 (m, 2H),
delta ¼ ꢁ2.74 ppm. 1H NMR (DMSO‑d6, 400 MHz)
d 9.73 (s, 1H),
7.91 (d, 1H, J ¼ 2.0 Hz), 7.3e7.4 (m, 1H), 7.3e7.3 (m, 2H), 7.0e7.1 (m,
2H), 6.8e6.8 (m, 2H), 6.3e6.4 (m, 1H), 4.28 (tt, 1H, J ¼ 3.7, 8.6 Hz),
3.76 (s, 3H), 3.7e3.8 (m, 1H), 3.61 (dd, 1H, J ¼ 5.7, 9.7 Hz), 3.4e3.4
(m, 1H), 3.27 (s, 3H), 2.9e3.0 (m, 1H), 2.8e2.9 (m, 1H), 2.6e2.7 (m,
1H), 2.4e2.5 (m, 1H), 2.21 (s, 3H), 1.9e2.0 (m, 1H), 1.5e1.7 (m, 2H);
1.4e1.5 (m, 2H); 13C NMR (DMSO‑d6, 126 MHz)
d 157.3, 154.7, 135.4,
131.4, 129.8, 129.6, 129.2, 127.7, 120.9, 118.6, 115.8, 109.1, 100.0, 72.2,
43.9, 40.9, 32.4, 30.5, 20.0.
13C NMR (DMSO‑d6, 101 MHz)
d
168.1, 154.8, 133.2, 130.7, 130.1,
4.2.40. N-[(1-methyl-1H-indol-5-yl)methyl]-4-[(4-methylphenyl)
methyl]piperazine-1-carboxamide (40)
129.7, 129.0, 127.7, 115.7, 114.8, 110.9, 109.3, 100.1, 72.3, 70.4, 67.1,
58.2, 47.4, 47.3, 32.4, 31.2, 31.1, 20.0.
The amide coupling was achieved in an analogous way as it is
described in case of 44. Yield 20.0%. Mp 120e122 ꢀC. HESI-HRMS:
4.2.37. 3-(1-Methyl-1H-indol-5-yl)-1-{1-[(4-methylphenyl)methyl]
piperidin-4-yl}urea (37)
The synthesis of 1-methyl-1H-indole-5-carbonyl azide 37a is
described in the following reference [57]. A solution of 1-methyl-
calcd for
C
23H29ON4 [MþH]þ: 377.23359; found: 377.23292;
delta ¼ ꢁ1.77 ppm. 1H NMR (DMSO‑d6, 400 MHz)
d 7.39 (dd, 1H,
J ¼ 0.7,1.5 Hz), 7.33 (d,1H, J ¼ 8.4 Hz), 7.27 (d,1H, J ¼ 3.1 Hz), 7.2e7.2
(m, 2H), 7.1e7.2 (m, 2H), 7.06 (dd, 1H, J ¼ 1.5, 8.4 Hz), 6.97 (t, 1H,
26

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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
J ¼ 5.6 Hz), 6.35 (dd, 1H, J ¼ 0.7, 3.1 Hz), 4.29 (d, 2H, J ¼ 5.6 Hz), 3.75
(s, 3H), 3.42 (br s, 2H), 3.2e3.3 (br m, 4H), 2.28 (s, 3H), 2.2e2.4 (br
compound 44. LC-MS (ESI) m/z [MþH]þ¼ 418.3. Mp 129e130 ꢀC.
HESI-HRMS: calcd for C22H26O2N3 [MþH]þ: 364.20195; found:
m, 4H); 13C NMR (DMSO‑d6, 101 MHz)
d
157.3, 135.9, 135.4, 134.7,
364.20115; delta ¼ 2.21 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d 7.40
131.3, 129.6, 128.8, 128.7, 127.7, 120.9, 118.6, 109.1, 99.9, 61.6, 52.3,
43.8, 43.3, 32.4, 20.6.
(dd, 1H, J ¼ 0.8, 1.6 Hz), 7.34 (d, 1H, J ¼ 8.4 Hz), 7.27 (d, 1H,
J ¼ 3.0 Hz), 7.0e7.1 (m, 3H), 6.8e6.8 (m, 2H), 6.79 (t, 1H, J ¼ 6.1 Hz),
6.36 (dd, 1H, J ¼ 0.8, 3.0 Hz), 4.26 (d, 2H, J ¼ 6.1 Hz), 4.06 (d, 2H,
J ¼ 6.6 Hz), 3.92 (t, 2H, J ¼ 8.2 Hz), 3.76 (s, 3H), 3.63 (dd, 2H, J ¼ 5.4,
8.2 Hz), 2.9e3.0 (m, 1H), 2.22 (s, 3H); 13C NMR (DMSO‑d6, 126 MHz)
4.2.41. N-[(1-methyl-1H-indol-5-yl)methyl]-3-(4-methylphenoxy)
azetidine-1-carboxamide (41)
The amide coupling was achieved in an analogous way as it is
described in case of 44. Yield 47.7%. Mp 141e142 ꢀC. HESI-HRMS:
calcd for C21H24O2N3 [MþH]þ: 350.18630; found: 350.18580;
d 159.6, 156.3, 135.4, 131.2, 129.7, 129.6, 129.2, 127.8, 120.8, 118.6,
114.3, 109.2, 100.0, 69.4, 51.4, 43.1, 32.4, 27.7, 20.0.
delta ¼ ꢁ1.44 ppm. 1H NMR (DMSO‑d6, 400 MHz)
d 7.40 (dd, 1H,
J ¼ 0.7, 1.6 Hz), 7.35 (d,1H, J ¼ 8.4 Hz), 7.28 (d,1H, J ¼ 3.1 Hz), 7.0e7.1
(m, 3H), 6.94 (t,1H, J ¼ 6.0 Hz), 6.7e6.8 (m, 2H), 6.36 (dd,1H, J ¼ 0.7,
3.1 Hz), 4.93 (tt, 1H, J ¼ 3.9, 6.3 Hz), 4.2e4.3 (m, 4H), 3.76 (s, 3H),
3.73 (dd, 2H, J ¼ 3.9, 9.5 Hz), 2.23 (s, 3H); 13C NMR (DMSO‑d6,
4.2.45. N-[(1-methyl-1H-indol-5-yl)methyl]-1-[(4-methylphenyl)
methyl]piperidine-4-carboxamide (45)
To a solution of (2-methyl-1H-indol-5-yl)methanamine (38a,
160 mg, 1.0 mmol) in dry CH2Cl2 (100 mL) was added N,N,N0,N0-tet-
ramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate
101 MHz) d 159.4,154.1,135.4,130.9,130.0,129.8,129.6,127.8,120.8,
118.6, 114.3, 109.2, 100.0, 65.4, 56.0, 43.2, 32.4, 20.0.
(HBTU) (460 mg, 1.2 mmol) and Et3N (350 mL, 2.5 eq) at room tem-
perature under argon. The mixture was stirred for 20 min then was
added 1-[(4-methylphenyl)methyl]piperidine-4-carboxylic acid
(260 mg, 1.0 mmol). The mixture was stirred at room temperature
overnight. After the completion of the reaction (monitored by TLC)
the reaction mixture was quenched by addition of water (50 mL), the
organic layer was washed with saturated NaCl solution (50 mL) then
the organic phase was dried over anhydrous Na2SO4, filtered and
concentrated in vacuo. The residue was purified by column chro-
matography on silica gel eluting with CH2Cl2 - methanol (9:1) to
yield (68 mg 19%) of the title compound. LC-MS (ESI) m/z
[MþH]þ ¼ 375.51. Mp 170e172 ꢀC. HESI-HRMS: calcd for C24H30ON3
[MþH]þ: 376.23834; found: 376.23761; delta ¼ ꢁ1.94 ppm. 1H NMR
4.2.42. N-[(1-methyl-1H-indol-5-yl)methyl]-3-(4-methylphenoxy)
pyrrolidine-1-carboxamide (42)
The amide coupling was achieved in an analogous way as it is
described in case of 44. Yield 57.0%. Mp 123e126 ꢀC. HESI-HRMS:
calcd for C22H26O2N3 [MþH]þ: 364.20195; found: 364.20116;
delta ¼ ꢁ2.18 ppm. 1H NMR (DMSO‑d6, 400 MHz)
d 7.41 (dd, 1H,
J ¼ 0.7,1.6 Hz), 7.33 (d,1H, J ¼ 8.4 Hz), 7.27 (d,1H, J ¼ 3.1 Hz), 7.0e7.1
(m, 3H), 6.8e6.9 (m, 2H), 6.69 (t,1H, J ¼ 6.0 Hz), 6.35 (dd,1H, J ¼ 0.7,
3.1 Hz), 4.9e5.0 (m,1H), 4.29 (d, 2H, J ¼ 6.0 Hz), 3.75 (s, 3H), 3.5e3.6
(m, 1H), 3.4e3.5 (m, 2H), 3.3e3.4 (m, 1H), 2.22 (s, 3H), 2.0e2.2 (m,
2H); 13C NMR (DMSO‑d6,101 MHz)
d 156.4,154.6,135.4,131.4,129.8,
129.5, 129.4, 127.7, 121.0, 118.6, 115.3, 109.1, 99.9, 75.6, 51.0, 43.6,
43.4, 32.4, 30.5, 20.0.
(DMSO‑d6, 500 MHz)
d
8.19 (t, 1H, J ¼ 5.9 Hz), 7.37 (dd, 1H, J ¼ 0.8,
1.6 Hz), 7.35 (d,1H, J ¼ 8.4 Hz), 7.28 (d,1H, J ¼ 3.1 Hz), 7.1e7.2 (m, 2H),
7.1e7.1 (m, 2H), 7.03 (dd, 1H, J ¼ 1.6, 8.4 Hz), 6.36 (dd, 1H, J ¼ 0.8,
3.1 Hz), 4.30 (d, 2H, J ¼ 5.9 Hz), 3.75 (s, 3H), 3.38 (s, 2H), 2.8e2.8 (m,
2H), 2.27 (s, 3H), 2.13 (tt, 1H, J ¼ 4.3,11.2 Hz), 1.8e1.9 (m, 2H), 1.5e1.7
4.2.43. N-[(1-methyl-1H-indol-5-yl)methyl]-3-[(4-methylphenoxy)
methyl]pyrrolidine-1-carboxamide (43)
The amide coupling was achieved in an analogous way as it is
described in case of 44. Yield 29.2%. Mp 121e122 ꢀC. HESI-HRMS:
calcd for C23H28O2N3 [MþH]þ: 378.21760; found: 378.21681;
(m, 4H); 13C NMR (DMSO‑d6, 126 MHz)
d 174.1, 135.7, 135.4, 135.3,
130.0,129.7,128.59,128.62,127.8,120.8,118.7,109.3,100.0, 62.0, 52.6,
42.2, 42.0, 32.4, 28.6, 20.6.
delta ¼ ꢁ2.10 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d 7.41 (dd, 1H,
J ¼ 0.8,1.5 Hz), 7.32 (d,1H, J ¼ 8.4 Hz), 7.26 (d,1H, J ¼ 3.1 Hz), 7.0e7.1
(m, 3H), 6.8e6.9 (m, 2H), 6.63 (t,1H, J ¼ 6.0 Hz), 6.34 (dd,1H, J ¼ 0.8,
3.1 Hz), 4.29 (d, 2H, J ¼ 6.0 Hz), 3.92 (dd, 1H, J ¼ 6.4, 9.5 Hz), 3.86
(dd, 1H, J ¼ 7.5, 9.5 Hz), 3.75 (s, 3H), 3.49 (dd, 1H, J ¼ 7.4, 10.2 Hz),
3.40 (ddd, 1H, J ¼ 4.7, 8.1, 10.1 Hz), 3.26 (td, 1H, J ¼ 7.6, 10.1 Hz), 3.12
(dd, 1H, J ¼ 7.0, 10.2 Hz), 2.60 (dddddd, 1H, J ¼ 6.4, 6.7, 7.0, 7.4, 7.5,
7.9 Hz), 2.22 (s, 3H), 2.00 (dddd, 1H, J ¼ 4.7, 6.7, 7.6, 12.3 Hz), 1.70
(dddd, 1H, J ¼ 7.6, 7.9, 8.1, 12.3 Hz); 13C NMR (DMSO‑d6, 126 MHz)
4.2.46. (1S,4S)eN-[(1-methyl-1H-indol-5-yl)methyl]-4-(4-
methylphenoxy)cyclohexane-1-carboxamide (46)
To
a
suspension of 4-(4-methylphenoxy)cyclohexane-1-
carboxylic acid (80 mg, 0.34 mmol) and (2-methyl-1H-indol-5-yl)
methanamine (38a, 56.4 mg, 1.0 mmol) in dry DMF (3 mL) was
added HATU (129.6 mg, 0.34 mmol, 1.0 eq) and DIPEA (119 mL, 2 eq)
at room temperature. The mixture was shaken at RT overnight.
After the completion of the reaction (monitored by TLC) the
mixture was diluted with brine and extracted with EtOAc
(3 ꢂ 20 mL). The combined organic phase was washed with brine
(3 ꢂ 20 mL), dried over anhydrous Na2SO4, filtered and concen-
trated in vacuo. The residue was purified by flash chromatography
on silica gel eluting with 10% EtOAc in CH2Cl2. The desired product
46 was crystallized from Et2O as well to yield 93.4 mg (73%) of the
title compound as white solid. LC-MS (ESI) m/z [MþH]þ ¼ 376.49.
Mp 149e151 ꢀC. HESI-HRMS: calcd for C24H29O2N2 [MþH]þ:
d
156.4, 156.3, 135.4, 131.6, 129.7, 129.5, 129.1, 127.7, 121.0, 118.6,
114.2, 109.1, 99.9, 69.1, 48.3, 44.6, 43.5, 37.7, 32.4, 27.6, 20.0.
4.2.44. N-[(1-methyl-1H-indol-5-yl)methyl]-3-[(4-methylphenoxy)
methyl]azetidine-1-carboxamide (44)
To a solution of (2-methyl-1H-indol-5-yl)methanamine (38a,
96 mg, 0.6 mmol) in CH2Cl2 (6 mL) was added DIPEA (155 mg,
208 mL, 1.2 mmol) and a solution of 4-nitrophenyl chloroformate
(121 mg, 0.6 mmol) in CH2Cl2 (3 mL) at 0 ꢀC under argon. The
mixture was stirred for 1.5 h under these conditions. After the
activation period 3-[(4-methylphenoxy)methyl]azetidine (5d,
106 mg, 0.6 mmol) in CH2Cl2 (3 mL) was added dropwise and the
mixture was allowed to warm to room temperature and stirred at
this temperature overnight. After the completion of the reaction
(monitored by TLC), the mixture was concentrated in vacuo. The
crude product was chromatographed on silica gel eluting with a
mixture of cyclohexane and EtOAc and the resulted product was
further purified on preparative TLC to yield 64 mg (26%) of the title
377.22235; found: 377.22170; delta
(DMSO‑d6, 500 MHz)
¼
ꢁ1.74 ppm. 1H NMR
d
8.19 (t, 1H, J ¼ 5.9 Hz), 7.39 (dd, 1H, J ¼ 0.8,
1.6 Hz), 7.36 (d, 1H, J ¼ 8.4 Hz), 7.28 (d, 1H, J ¼ 3.1 Hz), 7.1e7.1 (m,
2H), 7.04 (dd, 1H, J ¼ 1.6, 8.4 Hz), 6.8e6.8 (m, 2H), 6.36 (dd, 1H,
J ¼ 0.8, 3.1 Hz), 4.49 (tt, 1H, J ¼ 2.5, 4.2 Hz), 4.32 (d, 2H, J ¼ 5.9 Hz),
3.76 (s, 3H), 2.27 (tt,1H, J ¼ 3.3, 11.0 Hz), 1.9e1.9 (m, 2H), 1.7e1.8 (m,
2H), 1.5e1.6 (m, 4H); 13C NMR (DMSO‑d6, 126 MHz)
d 174.4, 154.8,
135.4,130.1,129.8,129.7,129.0,127.8,120.8,118.7,115.9,109.3,100.0,
70.6, 42.5, 42.2, 32.4, 28.3, 23.6, 20.0.
27

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European Journal of Medicinal Chemistry 222 (2021) 113560
4.2.47. N-[(1-methyl-1H-indol-5-yl)methyl]-1-[(4-methylphenyl)
methyl]piperidine-3-carboxamide (47)
To a suspension of N-benzylpiperidine-4-carboxylic acid (20 mg,
0.38) and of (2-methyl-1H-indol-5-yl)methanamine (38a, 61.4 mg,
1.0 mmol) in dry DMF (3 mL) was added HATU (145.6 mg,
calcd for C22H26O3N3 [MþH]þ: 380.19687; found: 380.19596;
delta ¼ ꢁ2.39 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d 7.40 (dd, 1H,
J ¼ 0.8, 1.6 Hz), 7.34 (d, 1H, J ¼ 8.4 Hz), 7.27 (d, 1H, J ¼ 3.1 Hz), 7.07
(dd, 1H, J ¼ 1.6, 8.4 Hz), 6.8e6.9 (m, 4H), 6.79 (t, 1H, J ¼ 6.0 Hz), 6.36
(dd, 1H, J ¼ 0.8, 3.1 Hz), 4.26 (d, 2H, J ¼ 6.0 Hz), 4.03 (d, 2H,
J ¼ 6.6 Hz), 3.92 (t, 2H, J ¼ 8.2 Hz), 3.76 (s, 3H), 3.69 (s, 3H), 3.63 (dd,
2H, J ¼ 5.4, 8.2 Hz), 2.9e2.9 (m, 1H); 13C NMR (DMSO‑d6, 126 MHz)
0.38 mmol, 1.0 eq) and DIPEA (133.4 mL, 2 eq) at room temperature.
The mixture was shaken at room temperature overnight. After the
completion of the reaction (monitored by TLC) the mixture was
diluted with brine and extracted with EtOAc (3 ꢂ 20 mL). The
combined organic phase was washed with brine (3 ꢂ 20 mL), dried
over anhydrous Na2SO4, filtered and concentrated in vacuo. The
residue was purified by flash chromatography on silica gel eluting
with 10% EtOAc in CH2Cl2. The desired product 47 was crystallized
from Et2O as well to yield 65.2 mg (45%) of the title compound as
white solid. LC-MS (ESI) m/z [MþH]þ ¼ 375.51. Mp 226e228 ꢀC.
HESI-HRMS: calcd for C24H30ON3 [MþH]þ: 376.23834; found:
376.23746; delta ¼ ꢁ2.34 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d
159.6, 153.4, 152.5, 135.4, 131.2, 129.6, 127.8, 120.9, 118.6, 115.4,
114.5, 109.2, 100.0, 69.9, 55.3, 51.4, 43.1, 32.4, 27.8.
4.2.51. N-[(1-methyl-1H-indol-5-yl)methyl]-3-{[4-
(trifluoromethoxy)phenoxy]methyl} azetidine-1-carboxamide (51)
Intermediate 3-{[4-(trifluoromethoxy)phenoxy]methyl}azeti-
dine was synthesized in an analogous way as it was described in
case of 5d. The amide coupling was achieved in an analogous way as
it is described in case of 44. Yield 30.9%. Mp 181e182 ꢀC. HESI-
HRMS: calcd for
C
22H23O3N3F3 [MþH]þ: 434.16860; found:
d
8.2e8.5 (br m,1H), 7.3e7.4 (m, 2H), 7.29 (d, 1H, J ¼ 3.0 Hz), 7.0e7.2
434.16766; delta ¼ ꢁ2.17 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d 7.41
(br m, 4H), 7.01 (dd, 1H, J ¼ 1.5, 8.4 Hz), 6.35 (dd, 1H, J ¼ 0.8, 3.0 Hz),
4.29 (br d, 2H, J ¼ 5.8 Hz), 3.76 (s, 3H), 3.38 (br s, 2H), 2.7e2.8 (br m,
1H), 2.6e2.7 (br m, 1H), 2.3e2.5 (br m, 1H), 2.26 (br s, 3H), 2.0e2.2
(br m, 1H), 1.9e2.0 (br m, 1H), 1.7e1.8 (br m, 1H), 1.5e1.7 (br m, 1H),
(dd, 1H, J ¼ 0.8, 1.5 Hz), 7.34 (d, 1H, J ¼ 8.4 Hz), 7.3e7.3 (m, 3H), 7.07
(dd, 1H, J ¼ 1.5, 8.4 Hz), 7.0e7.1 (m, 2H), 6.81 (t, 1H, J ¼ 6.0 Hz), 6.36
(dd, 1H, J ¼ 0.8, 3.1 Hz), 4.27 (d, 2H, J ¼ 6.0 Hz), 4.13 (d, 2H,
J ¼ 6.6 Hz), 3.94 (t, 2H, J ¼ 8.2 Hz), 3.76 (s, 3H), 3.65 (dd, 2H, J ¼ 5.4,
1.4e1.5 (br m, 2H); 13C NMR (DMSO‑d6, 126 MHz)
d
173.0, 135.8,
8.2 Hz), 2.9e3.0 (m, 1H); 13C NMR (DMSO‑d6, 126 MHz)
d 159.6,
135.5, 135.0, 129.8, 129.7, 128.7, 128.6, 127.8, 120.9, 118.8, 109.4,
100.0, 62.1, 55.6, 53.0, 42.4, 42.3, 32.4, 27.1, 24.0, 20.6.
157.3, 141.7 (q, J ¼ 1.8 Hz), 135.4, 131.2, 129.6, 127.7, 122.4, 120.9,
120.1 (q, J ¼ 255.1 Hz), 118.6, 115.6, 109.2, 99.9, 69.8, 51.4, 43.2, 32.4,
27.6.
4.2.48. 3-[(4-Fluorophenoxy)methyl]-N-[(1-methyl-1H-indol-5-yl)
methyl]azetidine-1-carboxamide (48)
Intermediate 3-[(4-fluorophenoxy)methyl]azetidine was syn-
thesized in an analogous way as it was described in case of 5d. The
amide coupling was achieved in an analogous way as it is described
in case of 44. Yield 26.1%. Mp 166e168 ꢀC. HESI-HRMS: calcd for
4.2.52. N-[(1-methyl-1H-indol-5-yl)methyl]-3-(phenoxymethyl)
azetidine-1-carboxamide (52)
Intermediate 3-phenoxymethyl]azetidine was synthesized in an
analogous way as it was described in case of 5d. The amide coupling
was achieved in an analogous way as it is described in case of 44.
Yield 17.0%. Mp 143e144 ꢀC. HESI-HRMS: calcd for C21H24O2N3
[MþH]þ: 350.18630; found: 350.18555; delta ¼ ꢁ2.15 ppm. 1H NMR
C
21H23O2N3F[MþH]þ:
368.17688;
found:
368.17621;
7.40 (dd, 1H,
delta ¼ ꢁ1.82 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d
J ¼ 0.8, 1.5 Hz), 7.34 (d, 1H, J ¼ 8.4 Hz), 7.27 (d, 1H, J ¼ 3.1 Hz), 7.1e7.1
(m, 2H), 7.07 (dd, 1H, J ¼ 1.5, 8.4 Hz), 6.9e7.0 (m, 2H), 6.80 (t, 1H,
J ¼ 6.1 Hz), 6.36 (dd, 1H, J ¼ 0.8, 3.1 Hz), 4.27 (d, 2H, J ¼ 6.1 Hz), 4.08
(d, 2H, J ¼ 6.6 Hz), 3.93 (t, 2H, J ¼ 8.2 Hz), 3.76 (s, 3H), 3.64 (dd, 2H,
J ¼ 5.4, 8.2 Hz), 2.9e3.0 (m, 1H); 13C NMR (DMSO‑d6, 126 MHz)
(DMSO‑d6, 500 MHz)
d
7.40 (dd, 1H, J ¼ 0.8, 1.6 Hz), 7.34 (d, 1H,
J ¼ 8.4 Hz), 7.3e7.3 (m, 3H), 7.07 (dd, 1H, J ¼ 1.6, 8.4 Hz), 6.9e7.0 (m,
3H), 6.80 (t, 1H, J ¼ 6.0 Hz), 6.36 (dd, 1H, J ¼ 0.8, 3.1 Hz), 4.27 (d, 2H,
J ¼ 6.1 Hz), 4.10 (d, 2H, J ¼ 6.6 Hz), 3.94 (t, 2H, J ¼ 8.2 Hz), 3.76 (s,
3H), 3.65 (dd, 2H, J ¼ 5.4, 8.2 Hz), 2.9e3.0 (m, 1H); 13C NMR
d
159.6, 156.5 (d, J ¼ 235.8 Hz), 154.8 (d, J ¼ 1.7 Hz), 135.4, 131.2,
(DMSO‑d6, 126 MHz) d 159.6, 158.4, 135.4, 131.2, 129.6, 129.4, 127.8,
129.6, 127.8, 120.9, 118.6, 115.72 (d, J ¼ 22.9 Hz), 115.71 (d,
120.9, 120.6, 118.6, 114.4, 109.2, 100.0, 69.2, 51.4, 43.1, 32.4, 27.7.
J ¼ 8.1 Hz), 109.2, 100.0, 70.0, 51.4, 43.1, 32.4, 27.7.
4.2.53. 3-[(4-Cyanophenoxy)methyl]-N-[(1-methyl-1H-indol-5-yl)
methyl]azetidine-1-carboxamide (53)
Intermediate 3-[(4-cyanophenoxy)methyl]azetidine was syn-
thesized in an analogous way as it was described in case of 5d. The
amide coupling was achieved in an analogous way as it is described
in case of 44. Yield 11.0%. Mp 184e185 ꢀC. HESI-HRMS: calcd for
4.2.49. 3-[(4-Chlorophenoxy)methyl]-N-[(1-methyl-1H-indol-5-yl)
methyl]azetidine-1-carboxamide (49)
Intermediate 3-[(4-chlorophenoxy)methyl]azetidine was syn-
thesized in an analogous way as it was described in case of 5d. The
amide coupling was achieved in an analogous way as it is described
in case of 44. Mp 171e172 ꢀC. HESI-HRMS: calcd for C21H23O2N3Cl
[MþH]þ: 384.14733; found: 384.14687; delta ¼ ꢁ1.20 ppm. 1H NMR
C
22H23O2N4
[MþH]þ:
375.18155;
found:
375.18071;
delta ¼ ꢁ2.25 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d
7.7e7.8 (m, 2H),
(DMSO‑d6, 500 MHz)
d
7.40 (dd, 1H, J ¼ 0.8, 1.6 Hz), 7.34 (d, 1H,
7.40 (dd, 1H, J ¼ 0.8, 1.5 Hz), 7.34 (d, 1H, J ¼ 8.4 Hz), 7.28 (d, 1H,
J ¼ 3.0 Hz), 7.1e7.1 (m, 2H), 7.07 (dd, 1H, J ¼ 1.5, 8.4 Hz), 6.82 (t, 1H,
J ¼ 6.0 Hz), 6.36 (dd, 1H, J ¼ 0.8, 3.0 Hz), 4.27 (d, 2H, J ¼ 6.0 Hz), 4.22
(d, 2H, J ¼ 6.6 Hz), 3.94 (t, 2H, J ¼ 8.2 Hz), 3.76 (s, 3H), 3.65 (dd, 2H,
J ¼ 5.4, 8.2 Hz), 2.9e3.0 (m, 1H); 13C NMR (DMSO‑d6, 126 MHz)
J ¼ 8.4 Hz), 7.3e7.3 (m, 2H), 7.28 (d, 1H, J ¼ 3.0 Hz), 7.07 (dd, 1H,
J ¼ 1.6, 8.4 Hz), 6.9e7.0 (m, 2H), 6.80 (t, 1H, J ¼ 6.1 Hz), 6.36 (dd, 1H,
J ¼ 0.8, 3.0 Hz), 4.26 (d, 2H, J ¼ 6.1 Hz), 4.10 (d, 2H, J ¼ 6.6 Hz), 3.93
(t, 2H, J ¼ 8.2 Hz), 3.76 (s, 3H), 3.64 (dd, 2H, J ¼ 5.4, 8.2 Hz), 2.9e3.0
(m, 1H); 13C NMR (DMSO‑d6, 126 MHz)
d
159.6, 157.3, 135.4, 131.2,
d 161.9, 159.6, 135.4, 134.1, 131.2, 129.6, 127.8, 120.9, 119.0, 118.6,
129.6, 129.1, 127.8, 124.3, 120.9, 118.6, 116.2, 109.2, 100.0, 69.7, 51.4,
43.1, 32.4, 27.6.
115.5, 109.2, 102.8, 100.0, 69.8, 51.3, 43.2, 32.4, 27.5.
4.2.54. N-[(1-methyl-1H-indol-5-yl)methyl]-3-{[4-
4.2.50. N-[(1-methyl-1H-indol-5-yl)methyl]-3-[(4-
methoxyphenoxy)methyl]azetidine-1-carboxamide (50)
Intermediate 3-[(4-methoxyphenoxy)methyl]azetidine was
synthesized in an analogous way as it was described in case of 5d.
The amide coupling was achieved in an analogous way as it is
described in case of 44. Yield 42.6%. Mp 118e119 ꢀC. HESI-HRMS:
(trifluoromethyl)phenoxy]methyl}azetidine-1-carboxamide (54)
Intermediate 3-{[4-(trifluoromethyl)phenoxy]methyl}azetidine
was synthesized in an analogous way as it was described in case of
5d. The amide coupling was achieved in an analogous way as it is
described in case of 44. Yield 25.8%. Mp 218e219 ꢀC. HESI-HRMS:
calcd for C22H23O2N3F3 [MþH]þ: 418.17369; found: 418.17275;
28

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European Journal of Medicinal Chemistry 222 (2021) 113560
delta ¼ ꢁ2.24 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d
7.6e7.7 (m, 2H),
50
m
L/well assay buffer containing vehicle (Dimethyl sulfoxide
7.41 (dd, 1H, J ¼ 0.8, 1.6 Hz), 7.34 (d, 1H, J ¼ 8.4 Hz), 7.28 (d, 1H,
J ¼ 3.1 Hz), 7.1e7.2 (m, 2H), 7.07 (dd, 1H, J ¼ 1.6, 8.4 Hz), 6.82 (t, 1H,
J ¼ 6.1 Hz), 6.36 (dd, 1H, J ¼ 0.8, 3.1 Hz), 4.27 (d, 2H, J ¼ 6.0 Hz), 4.21
(d, 2H, J ¼ 6.6 Hz), 3.95 (t, 2H, J ¼ 8.2 Hz), 3.76 (s, 3H), 3.66 (dd, 2H,
J ¼ 5.4, 8.2 Hz), 2.9e3.0 (m, 1H); 13C NMR (DMSO‑d6, 126 MHz)
[DMSO], 4% added) or test compounds (4 ꢂ of the final concen-
tration) were added. Cells were then incubated for an additional
10 min at 37 ꢀC.
Baseline and agonist-evoked [Ca2þ]i-changes were measured
with a FlexStation II96 (Molecular Devices, San Jose, CA). Fluores-
cence measurements were carried out at 37 ꢀC. The dye was excited
at 485 nm, emission was sampled at 525 nm at 1.4-s intervals.
Baseline was recorded for 20 s followed by agonist stimulation.
d
161.3, 159.6,135.4, 131.2, 129.6,127.8, 126.8 (q, J ¼ 3.8 Hz), 124.5 (q,
J ¼ 270.8 Hz), 121.1 (q, J ¼ 32.0 Hz), 120.9, 118.6, 114.9, 109.2, 100.0,
69.7, 51.3, 43.2, 32.4, 27.5.
50
m
L 4 ꢂ concentrated agonist (PNU-282987, 1
mM) solution was
4.2.55. N-[(1H-indol-5-yl)methyl]-3-{[4-(trifluoromethyl)phenoxy]
methyl}azetidine-1-carboxamide (55)
added to all wells using the pipettor of FlexStation II and fluores-
cence was monitored for an additional 20 s. Final DMSO concen-
tration was 1% for all treatments. To achieve this, a series of DMSO
stock solutions were prepared from all test compounds. These
stocks were stored under 0 ꢀC and were further diluted in assay
buffer to obtain the desired final concentration immediately before
Intermediate 3-{[4-(trifluoromethyl)phenoxy]methyl}azetidine
was synthesized in an analogous way as it was described in case of
5d. The amide coupling was achieved in an analogous way as it is
described in case of 44. Yield 39%. Mp 128e130 ꢀC. HESI-HRMS:
calcd for C21H21O2N3F3 [MþH]þ: 404.158040; found: 404.15699;
the measurement. Positive control: PNU-120596, 2.5
Results were expressed as F/F values using SoftMax Pro soft-
ware (Molecular Devices), where F was the resting fluorescence
preceding agonist application and F was the increase in fluores-
cence at a given time (
mM.
delta ¼ ꢁ2.59 ppm. 1H NMR (DMSO‑d6, 500 MHz)
d
10.98 (br s, 1H),
D
7.6e7.7 (m, 2H), 7.4e7.4 (m, 1H), 7.3e7.3 (m, 2H), 7.1e7.2 (m, 2H),
7.01 (dd, 1H, J ¼ 1.6, 8.4 Hz), 6.80 (t, 1H, J ¼ 6.0 Hz), 6.36 (ddd, 1H,
J ¼ 0.9, 2.0, 3.0 Hz), 4.26 (d, 2H, J ¼ 6.0 Hz), 4.21 (d, 2H, J ¼ 6.6 Hz),
3.95 (t, 2H, J ¼ 8.2 Hz), 3.66 (dd, 2H, J ¼ 5.4, 8.2 Hz), 2.9e3.0 (m,1H);
D
D
F ¼ maximum fluorescence intensity values
after stimulation minus average fluorescence intensity values
before stimulation). In all experiments, all treatments were
13C NMR (DMSO‑d6, 126 MHz)
d
161.3, 159.6, 134.8, 131.0, 127.4,
126.8 (q, J ¼ 3.8 Hz), 125.3, 124.5 (q, J ¼ 271.2 Hz), 121.1 (q,
measured in multiple wells in parallel, and the mean
DF/F values
J ¼ 31.9 Hz), 120.8, 118.3, 114.9, 110.9, 100.7, 69.7, 51.3, 43.3, 27.5.
were used for analysis. F/F data were converted to corrected %
D
response values by normalizing responses to the control PNU-
120596 response. In each individual experiment, the EC50 and
Emax values were determined from 4-parameter sigmoidal
concentration-response curves fitted to the corrected response
data using SoftMax Pro, with the lower asymptote fixed to zero.
EC50 and Emax values from individual experiments were averaged
and presented as mean SD.
4.3. Biological assay methods
Methods used in the present article were already described in
detail in our previous paper [41]. Here, only a brief summary is
given.
4.3.1. In vitro methods using cells expressing human
receptors
4.3.1.1. Cell culture. Generation of the recombinant HEK293-based
a7 nACh
4.3.1.3. High-throughput screening. The HTS was run on the full
Corporate screening library utilizing a FLIPR [Ca2þ]i assay on Flp-In
cell line stably expressing both the target hCHRNA7 protein and
HEK293 cells stably expressing human
average Z’ for the screen was 0.86 with a statistically derived ac-
tivity threshold of 7.3% at 10 M, resulting in 0.3% active rate. Ac-
tives were tested for specificity on a HEK-293 cell line expressing
TRPM8 receptors and for solution integrity using LC/MS.
a7 nAChR and RIC-3. The
the hRIC3 chaperone (
[41]. Cells were maintained in DMEM (Gibco) supplemented by 10%
FBS (Gibco), 2 mM glutamine (Sigma), 50 g/mL hygromycin B,
400 g/mL G418 and 1% penicillin-streptomycin antimycotic solu-
tion (Sigma/Merck). Cells were split twice a week by trypsinization.
a7-nAChR-HEK cells) was already described
m
m
m
4.3.1.4. Patch clamp measurements. Channel physiology of
nAChRs was studied by automated whole-cell patch clamp QPatch-
HTX, Sophion) in single-cell mode. Whole-cell patch recordings
a7
4.3.1.2. Fluorometric [Ca2þ]i-assay on cells expressing human
nAChR. Orthosteric agonists of the 7 nAChR (e.g. PNU-282987) per
a7
a
se evoke no Ca2þ-influx in most in vitro cellular systems, unless they
are co-applied with PAMs that concurrently hinder the channel
desensitization. However, in the presence of agonists, PAMs with
such characteristics result in robust and concentration-dependent
Ca2þ-influx (it must be noted that the assay is insensitive to
PAMs lacking effect on channel desensitization).
were made from a7-nAChR-HEK cells 2 days after plating at a
holding potential of ꢁ80 mV at room temperature. Inward currents
were evoked by 3-s-long application of 10 mM choline with
2e4 min intervals in the absence, then in the presence of test
compounds and recorded at 10 kHz sampling frequency. Solutions
containing the test compounds were applied to the cells for
6e10 min. The control solution contained the same concentration
of the vehicle (0.1% DMSO) as the solutions of the test compound.
Peak amplitudes of current evoked by choline was measured from
the baseline current. The fold increase (FI) value was calculated
from the ratio of the peak current amplitudes evoked by choline in
the presence or in the absence of the test compounds. EC50 values
were calculated from FI values by testing several concentrations of
the test compound.
Flp-In HEK293 cells stably expressing the human
the RIC-3 chaperon were cultured in DMEM (Gibco) supplemented
by 10% FBS (Gibco), 2 mM glutamine (Sigma), 50 g/mL hygromycin
B, 400 g/mL G418 and 1% penicillin-streptomycin antimycotic
a7 nAChR and
m
m
solution (Sigma). Cells were split 1:3e4 twice a week by trypsini-
zation. For the [Ca2þ]i measurements, cells were seeded onto 96-
well microplates at a density of 60.000 cells/well and maintained
overnight in a tissue culture incubator at 37 ꢀC under an atmo-
sphere of 95% air and 5% CO2. Before the [Ca2þ]i measurement,
For electrophysiological measurements using GABAA receptors,
50
mL of the growth medium was aspirated with a cell washer
HEK293 cells stably expressing human a1b3g2 and a5b3g2
(BioTek Elx405UCVWS, Biotek, Winooski, VT, USA), then 50
m
L/well
GABAARs (Eurofins, CYL3053 and CYL3073) were investigated by
whole-cell patch clamp using the QPatch-HTX automated patch
clamp system in single-cell mode. Whole-cell patch clamp re-
cordings were made from cells 2e4 days after plating at a holding
potential of ꢁ80 mV at room temperature. Inward currents were
Calcium 5 kit (diluted 2-fold in assay buffer containing 140 mM
NaCl, 5 mM KCl, 10 mM HEPES, 2 mM MgCl2, 2 mM CaCl2, 10 mM
glucose and 2 mM probenecid; pH ¼ 7.4) was added manually using
an 8-channel pipette. After an incubation period of 20 min at 37 ꢀC,
29

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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.
European Journal of Medicinal Chemistry 222 (2021) 113560
evoked by 3-s-long application of the agonist 1
m
M GABA with 2-4-
histidine-requiring auxotroph strains of Salmonella typhimurium
(Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and
the tryptophan-requiring auxotroph strain of Escherichia coli
(Escherichia coli WP2 uvrA) in the presence and absence of a
metabolic activation system, which is a cofactor-supplemented
post-mitochondrial S9 fraction prepared from rat liver.
min intervals in the absence, then and in the presence of test
compound. Solutions containing the test compound were applied
to the cells for 7e10 min. The control solution contained the same
concentration of the vehicle (0.1% DMSO) as the solutions of the test
compound. Peak amplitudes of current evoked by GABA was
measured from the baseline current. The modulation values were
calculated from the comparison of peak currents in the absence and
in the presence of the test compound.
4.3.2. In vivo methods
4.3.2.1. Place recognition (Y maze) test. Animal maintenance and
experiments were conducted in accordance with the National
Institute of Health Guide for the Care and Use of Laboratory Ani-
mals. All procedures using animals were approved by the local
ethics committee (Institutional Animal Welfare Committee of
Gedeon Richter Plc. and conformed to the rules and principles of
the European Animal Protection Directives (Directive 2010/63/EU).
The task was carried out in a transparent plexiglass Y-maze
(each arm has a length of 40 cm, an inner width of 11 cm and a
height of 30 cm). Numerous visual cues were placed around the
arms and were kept constant during the experiment. The test
consisted of two trials (T1 and T2) separated by an intertrial interval
of 30 min. Male NMRI mice (Toxicoop, Hungary) were placed in the
starting arm (F ¼ familiar arm) of the maze at the beginning of each
trial. In T1, one of the symmetric arms of the maze was closed (it
will be novel in T2, N ¼ novel arm) and the animals could explore
the maze for 5 min (acquisition phase). In T2, mice had free access
to all three arms for 2 min (retrieval phase). Test compounds were
administered 30 min before T1; scopolamine (1 mg/kg, ip.) was
administered after the acquisition trial at a volume of 0.1 mL/10 g.
The time spent with exploration in the novel and familiar arms
during T2 was measured and recognition index [RI¼(N/
NþF) ꢂ 100)] was calculated for the animals. Differences between
the RI values for each group were evaluated by MANOVA, followed
by Duncan post hoc test.
For hERG measurement, CHO cells stably expressing Kv11.1
potassium channel (hERG; bSys) were investigated by whole-cell
patch clamp using the QPatch-HTX automated patch clamp sys-
tem in voltage-clamp mode at room temperature. Cells were held
at ꢁ80 mV and hERG currents were activated by a þ20 mV prepulse
of 5 s duration followed by a step to ꢁ40 mV for 1.5 s. The current
traces were recorded, and the peak of the tail current evoked by
the ꢁ40 mV step was measured as test parameter. Solutions con-
taining the compounds were applied to the cells for 7e10 min. The
control solution contained the same concentration of the vehicle
(0.1 or 0.3% DMSO) as the solution of the test compounds. The in-
hibition was calculated from the peak tail currents in the presence
and absence of the test compound.
4.3.1.5. Selectivity. Selected compounds were subjected to Eurofins
Panlabs Taiwan Ltd. using the Lead Profiling Screen panel
comprising binding and enzyme assays for 68 common molecular
targets including human recombinant receptors, transporters, en-
zymes and channels (if a human recombinant target was not
available, compounds were tested on the corresponding rodent
receptor). Compounds were tested at the nominal test concentra-
tion of 10
major.
mM; inhibition values higher than 70% were considered as
4.3.1.6. Human, rat and mouse liver microsomal stability assays.
In vitro metabolic stability was assessed using human (Xenotech,
LLC, USA), Wistar rat and NMRI mouse (In vitro Metabolism
Research, Gedeon Richter Plc, Hungary) liver microsomes. Test
4.3.2.2. Novel object recognition (NOR) test. Before testing, male
Hannover Wistar rats (Toxicoop, Hungary) were habituated to the
apparatus (65 ꢂ 45 ꢂ 45 cm grey box with sawdust on its floor) in a
3-min session. The test consisted of a sample trial (T1) and a
recognition trial (T2), separated by an inter-trial interval of 24 h,
during which time rats were replaced into their home cages. In the
sample trial (T1), two identical objects were placed in opposite rear
corners of the box and could be explored for 3 min. In the recog-
nition trial (T2), one of the identical objects was replaced by a new
object. The exploration time for the familiar (F) and the new (N)
object was detected in a 3-min session and RI values were calcu-
lated as follows: RI¼(N/NþF) ꢂ 100). Test compounds were
administered orally (standard NOR test), 30 min before the sample
and recognition trials. Differences between RI values were evalu-
ated by ANOVA, followed by Duncan or LSD post hoc tests. For the
comparison of the experimental groups, nonparametric Kruskal-
Wallis analysis was used followed by multiple comparisons of
mean ranks for all groups as a post hoc test using Statistica 8
software.
compounds were incubated at 1
longest up to 40 min with the liver microsomes (0.5 mg/mL). In vitro
intrinsic clearance (CLint L/min/mg protein) was calculated using
mM initial test concentration at
,
m
the basic concept of clearance prediction [58] according to the
following equations: CLint ¼ Vmax/KM, or if S ≪ KM, CLint ¼ V/S;
Vmax ¼ maximal rate of enzyme reaction; KM ¼ affinity constant of
substrate concentration; V ¼ actual rate of enzyme reaction under
first order conditions,
incubations.
S
¼
substrate concentration in the
4.3.1.7. Permeability assay. Bi-directional permeability (PappA-B
and PappB-A) and efflux ratio (PDR ¼ PappB-A/PappA-B) of test
compounds were measured using vinblastine-treated Caco-2 (VB-
Caco-2) cells [43]. Briefly, the permeability of test compounds (at 1
or 10 mM) were measured in the apical-to-basolateral (A-B) and
basolateral-to-apical (B-A) directions in HBSS-HEPES (Hank's
Buffered Salt Solution containing 25 mM HEPES) using iso-pH
conditions (pH 7.4Ae7.4B) at 37 ꢀC with moderate shaking
(120 rpm). The incubations with test compounds were performed
at longest up to 120 min, using appropriate duration of time
determined based on preliminary studies for each compound
tested. Samples were analyzed using HPLC or UHPLC-MS/MS.
4.3.2.3. Rodent touch screen test. Lister Hooded rats (n ¼ 36) were
kept in reversed light-dark cycle (lights on from 6:00 p.m. to 6:00
a.m.) and were tested during their active period. At the time of
testing they were ca. 8-month-old, weighing 400e500 g. To pro-
vide sufficient motivation, food restriction was applied maintaining
their 85% of free-feeding body weight. The visual discrimination
procedure was carried out in automated Bussey-Saksida touch
screen chambers (Campden Instruments Ltd, [59]). Pharmacolog-
ical testing was preceded by several weeks of learning to percep-
tually discriminate two images and associating which image is
rewarded: Touching the correct stimulus on the screen is rewarded
4.3.1.8. Ames test. Mutagenic potential of test compounds was
tested by measuring their ability to induce reverse mutations at
selected loci of several strains of Salmonella typhimurium and
Escherichia coli by Toxi-Coop Zrt. (Balatonfüred, Hungary). The
experiment (a micro-scale Fluctuation Test) was carried out using
30