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

European Journal of Medicinal Chemistry 222 (2021) 1 13560

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

HTS-based discovery and optimization of novel positive allosteric

modulators of the a7 nicotinic acetylcholine receptor

a

,

*

a

ꢀ

Istvan Ledneczki , Anita Horvath , Pal Tapolcsanyi , Janos Eles ,

a

b

c

ꢀ

ꢀ ꢀ

ꢀ

Katalin Dudas Molnar , Istvan Vago , Andras Visegrady , Laszlo Kiss , Aron Szigetvari ,

ꢀ

ꢀ

Janos Koti , Balazs Kramos , Sandor Maho , Patrik Holm , Sandor Kolok ,

b

ꢀ

b

Laszlo Fodor , Marta Than , Diana Kostyalik , Ottilia Balazs , Monika Vastag ,

e

b

ꢀ

€

ꢀ

Istvan Greiner , Gyorgy Levay , Balazs Lendvai , Zsolt Nemethy

^aDepartment of Chemistry, Gedeon Richter Plc., Budapest, Hungary

^bPharmacological and Drug Safety Research, Gedeon Richter Plc., Budapest, Hungary

^cSpectroscopic Research Department, Gedeon Richter Plc., Budapest, Hungary

^dOrion Corporation, Turku, Finland

^eResearch 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.

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)₃and the

homomeric ( 7)₅nAChRs [2]. The 7 nAChR is considered as the

a- and b-subunits [1]. Various a- and b-subunit compositions

a

b

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

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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 [1 1]. 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 Ca^2þions, 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 Na^þ, K^þand Ca^2þcompared to other nAChR subtypes

[15,16], as well as the extremely fast desensitization [1 7,18], 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

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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 SeO₂to 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

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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 H₂O₂and 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

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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}

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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 [Ca^2þ

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 [Ca^2þ]_ire-

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 (CL_int) 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 EC₅₀value 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 EC₅₀value 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-HT_2Band 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-HT₃receptors (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 GABA_Areceptors ([³H]ﬂunitrazepam or [³H]mus-

cimol binding in Wistar Rat brain preparations minus cerebellum).

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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. H₂O₂sol., 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 GABA_ARs (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 GABA_Areceptors that

microsomes (with CL_intof 15, 158 and 55

mL/min/mg protein in

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

a3

b

PDR value of 0.7 and Papp_A-Bof 30.7 ꢂ 10^ꢁ6cm s^ꢁ1(tested @10

mM),

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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

[Ca^2þ]_iEC₅₀(nM) or % response at 1* or 10

m

M

CL_int

m

L/min/mg protein)

a

Cmpd

R

L

1

H

14.8

4.2

910

23/148/242

2

3^b

4

H

43.5

2.8

8.7

1.8

6.7

7.7

3.1

4.6

3.4

3.3

3.8

260

inact.

120

18/138/184

H

19/160/112

Me

19/74/78

5

3000

25%

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

41.6

24.8

e

3.3

4.8

3.9

4.4

5.5

90

600

670

52%

inact.

60

15/158/55

28/42/169

133/65/64

e

12

13^c

14^d

15^d

16

3.0

0.1

2.0

e

34/48/46

17

18

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; CL_int.: 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 IC₅₀value 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

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European Journal of Medicinal Chemistry 222 (2021) 113560

Fig. 3. Upper panel: Effect of 4 and 17 on [Ca^2þ]_iin 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 [Ca^2þ

]

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 [Ca^2þ]_iassay 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 [Ca^2þ]_iassay (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.^asol. (

m

M)

cx_ logP^a

[Ca^2þ]_iEC₅₀(nM) or % response at 10

m

M

CL_int

m

L/min/mg protein)

a

Cmpd

19

20

21

22

23

24

25

26

27

28

Me

H

Me

H

Me

F

H

F

Cl

H

F

H

F

C

N

5

6

5

5.0

23.0

12.5

47.1

16.5

6.8

42.7

47.4

e

3.8

3.9

3.5

3.9

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

H

e

21/192/214

e

17/130/151

0.3/56/46

12/41/39

e

Me

H

F

5.6

29

95.0

3.2

820

71/23/18

a

Kin. sol.: kinetic solubility; cx_logP: calculated logP; CL_int.: 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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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

[Ca^2þ]_iEC₅₀(nM) or % response at 10

mM

30

inact.

31

32

7%

37%

33

34

660

2500

35

36

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),

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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_logP^a

[Ca^2þ]_iEC₅₀(nM) or % response at 1* or 10

m

M

CL_int

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

84.1

inact.

a

Kin. sol.: kinetic solubility; cx_logP: calculated logP; CL_int.: 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_logP^a

[Ca^2þ]_iEC₅₀(nM)

CL_intm

L/min/mg protein)

a

ID

R

44

48

49

50

51

52

53

54

55^b

CH₃

F

Cl

OCH₃

OCF₃

H

CN

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; CL_int.: 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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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 ([Ca^2þ]_iEC₅₀of

160 nM) with a robust inhibition of response desensitization

(Fig. 8., upper panel) and enhancement of peak current amplitude

(EC₅₀¼ 590 nM) and acceptable metabolic stability (with CL_int

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

(Papp_A-Bvalue of 55 ꢂ 10^ꢁ6cm s^ꢁ1). 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-HT_2Bat 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 IC₅₀value 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 [Ca^2þ]_iassay (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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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.

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 NaHCO₃and 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 NaHCO₃solution. The mixture was extracted with

CH₂Cl₂, the combined organic layer was washed with brine, dried

over anhydrous Na₂SO₄and 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 NaHCO₃solution (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 NaHCO₃solution and the organic phase was washed with

water, dried over Na₂SO₄and 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 Na₂SO₄and 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 CH₂Cl₂were 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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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.

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]^þ¼ 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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I. Ledneczki, A. Horvath, P. Tapolcsanyi et al.

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

Na₂SO₄and 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 CH₂Cl₂and 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

Na₂SO₄and 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 Na₂SO₄and 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 CH₂Cl₂and 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 Na₂SO₄and

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 Na₂SO₄and

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 Na₂SO₄and 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% NaHCO₃solu-

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]^þ¼ 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]^þ¼ 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 Na₂SO₄and 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), NaO^tBu

(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 Na₂SO₄and 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 NaHCO₃solution (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 Na₂SO₄and 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 Na₂SO₄and evaporated. The 33c crude product was

puriﬁed by column chromatography with CH₂Cl₂as 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 CH₂Cl₂and 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 Na₂SO₄and

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 CH₂Cl₂and

washed with water (2 ꢂ 30 mL) and brine (40 mL). The organic

phase was dried over Na₂SO₄and 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 N₂atmosphere, 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

CH₂Cl₂and 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. NaHCO₃solution (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]^þ¼ 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 Na₂SO₄and 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]^þ¼ 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 Na₂SO₄and 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 CH₂Cl₂and 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]^þ¼ 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]^þ¼ 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]^þ¼ 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 [Ca^2þ]_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

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 EC₅₀and

E_maxvalues 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.

EC₅₀and E_maxvalues 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 [Ca^2þ]_iassay 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

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 [Ca^2þ]_i-assay on cells expressing human

nAChR. Orthosteric agonists of the 7 nAChR (e.g. PNU-282987) per

a7

a

se evoke no Ca^2þ-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

Ca^2þ-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. EC₅₀values

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

solution (Sigma). Cells were split 1:3e4 twice a week by trypsini-

zation. For the [Ca^2þ]_imeasurements, 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 [Ca^2þ]_imeasurement,

For electrophysiological measurements using GABA_Areceptors,

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

GABA_ARs (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

ꢀ

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 (CL_intL/min/mg protein) was calculated using

mM initial test concentration at

,

m

the basic concept of clearance prediction [58] according to the

following equations: CL_int¼ V_max/K_M, or if S ≪ K_M, CL_int¼ V/S;

V_max¼ maximal rate of enzyme reaction; K_M¼ 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 (Papp_A-B

and Papp_B-A) and efﬂux ratio (PDR ¼ Papp_B-A/Papp_A-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 7.4_Ae7.4_B) 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

Article Doi

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

DOI: 10.1016/j.ejmech.2021.113560

Source and publish data:

Authors:

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

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

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