Discovery of novel positive allosteric modulators of the α7 nicotinic acetylcholine receptor: Scaffold hopping approach

European Journal of Medicinal Chemistry 214 (2021) 1 13189

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

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

Discovery of novel positive allosteric modulators of the

acetylcholine receptor: Scaffold hopping approach

a7 nicotinic

,

,

ꢀ

Istvan Ledneczki , Pal Tapolcsanyi , Eszter Gabor , Andras Visegrady , Marton Vass ,

a

e

a

ꢀ

Janos Eles , Patrik Holm , Anita Horvath , Aniko Pocsai , Sandor Maho ,

Istvan Greiner , Balazs Kramos , Zoltan Beni , Janos Koti , Anna E. Kancz ,

ꢀ

ꢀ

Marta Than , Sandor Kolok , Judit Laszy , Ottilia Balazs , Gyula Bugovits ,

b

a

b

ꢀ

€

ꢀ

Jozsef Nagy , Monika Vastag , Agota Szajli , Eva Bozo , Gyorgy Levay , Balazs Lendvai ,

b

ꢀ

Zsolt Nemethy

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

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

^cResearch Management, Gedeon Richter Plc., Budapest, Hungary

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

^eOrion Pharma, Turku, Finland

a r t i c l e i n f o

a b s t r a c t

Article history:

The paper focuses on the scaffold hopping-based discovery and characterization of novel nicotinic alpha

Received 8 October 2020

Received in revised form

8 January 2021

Accepted 8 January 2021

Available online 14 January 2021

7 receptor positive modulator (a7 nAChR PAM) ligands around the reference molecule (A-867744). First,

substantial efforts were carried out to assess the importance of the various pharmacophoric elements on

the in vitro potency (SAR evaluation) by chemical modiﬁcations. Subsequently, several new derivatives

with versatile, heteroaromatic central cores were synthesized and characterized. A promising, pyrazole-

containing new chemotype with good physicochemical and in vitro parameters was identiﬁed. Retro-

spective analysis based on homology modeling was also carried out. Besides its favorable in vitro char-

acteristics, the most advanced derivative 69 also showed in vivo efﬁcacy in a rodent model of cognition

(scopolamine-induced amnesia in the mouse place recognition test) and acceptable pharmacokinetic

properties. Based on the in vivo data, the resulting molecule with advanced drug-like characteristics has

Keywords:

Nicotinic alpha 7 receptor

Positive allosteric modulator

Scaffold hopping

the possibility to improve cognitive performance in

a biologically relevant dose range, further

Structure activity relationship

Cognitive improvement

strengthening the view of the supportive role of

a7 nACh receptors in the cognitive processes.

1. Introduction

ligand-gated ion channel superfamily are made up of ﬁve subunits

[1,2] and divided into two groups, based on their amino acid se-

Cognition, a highly complex function of the central nervous

system (CNS) consists of various domains (e.g. learning, memory,

attention, executive functions, etc.). Marketed drugs targeting

cognitive functions (e.g. cholinesterase inhibitors) traditionally

focus on enhancing the cholinergic neurotransmission. The

neurotransmitter acetylcholine (ACh) exerts its biological functions

through binding to muscarinic (mAChR) and/or nicotinic (nAChR)

acetylcholine receptors. nAChRs, members of the pentameric

quences: one containing a, and another containing b subunits.

Pentameric assemblies of various subunit compositions result in

different nicotinic receptor subtypes with distinct pharmacological

characteristics. The most predominant nAChR subtypes in the CNS

are the (

a

4)₂(b2)₃and the homopentameric (a7)₅receptors [2].

Given that

a

7 nAChRs, a major nicotinic receptor subtype in the rat

[3,4] and human [5] brain are abundantly expressed in the so-called

“areas of cognition” (the prefrontal cortex [6,7], the hippocampus

and other subcortical limbic structures [8,9]), activity changes of

the

ditions such as learning and memory disfunctions and other

cognitive deﬁcits as well. Procognitive role of 7 nAChR ligands are

supported by in vivo studies in rodents [10,1 1] and non-human

a7 nAChRs have been implicated in a number of related con-

* Corresponding author. Gedeon Richter Plc., P.O. Box 27, H-1475, Budapest,

Hungary.

a

E-mail address: ledneczki@richter.hu (I. Ledneczki).

https://doi.org/10.1016/j.ejmech.2021.113189

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I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

primates [12] as well as in humans [13,14]. As the speciﬁc distri-

bution of a7 nAChRs in brain areas associated with cognition and

iodoaniline, respectively, the amino-pyrrole ring formations were

achieved (29, 35e2). tert-Butyl nitrite was used for the removal of

the amino moiety (26, 35). After the ester hydrolysis of 26 and the

acid chloride formation from 5e2, the amide of N,O-dimethylhy-

droxylamine (5e3) was synthesized. Further reacted it according to

the Weinreb-Nahm ketone synthesis with EtMgBr compound 5 was

yielded. The N-methylated derivatives (6, 7) were synthesized

starting from compound 1 using the MeI/NaH reagent system in

THF. Starting from diketone 8e1 using 1-benzylpiperidin-4-amine

the pyrrole derivative 8e2 was isolated. The application of the

Vilsmeier-Haack conditions resulted in acylated derivative 8e3 and

after the benzyl deprotection (8e4) and piperidine N-amidation

compound 8 was obtained. Intermediate 9e1 and compound 3

were acylated (Friedel-Crafts conditions) to afford 9 and 34e1,

36e1, respectively. After amide hydrolysis in 34e1, compound 34

while after catalytic reduction of 36e1 and subsequent amide hy-

drolysis in 36e2, compound 36 was obtained.

the association of CHRNA7 gene with inhibitory sensory gating

deﬁcit in patients with schizophrenia suggests that activation of the

nAChRs may play a role in the treatment of schizophrenia.

Activation of the nAChR ion channel is primarily controlled by

ligand binding to the conventional agonist binding site. It is, how-

ever, also regulated by either negative or positive allosteric mod-

ulators (NAMs and PAMs). Allosteric model of nAChR activation

involves at least a resting and an activated state as well as a

desensitized state. Although the a7 nAChR is characterized by fast

activation kinetics and high permeability to Ca^2þions compared to

other nAChR subtypes [15], it also exhibits rapid desensitization

following exposure to orthosteric agonists [16e18]. Desensitiza-

tion, therefore, limits the extent of agonist action, resulting in the

insensitivity of the receptor to further stimulation. Suboptimal ef-

ﬁcacy of the various a7 nAChR-selective agonists and partial ago-

nists in clinical trials may well be attributed to this desensitization-

induced receptor loss-of-function.

The phenomenon of rapid receptor desensitization and the

subsequent insensitivity to stimulation can be overwhelmed by the

application of PAMs by enhancing the endogenous agonist-induced

receptor activation. Furthermore, selectivity issues often experi-

2.1.2. Synthesis of derivatives 38e39 and 52e86 with pyrazole

central cores

The pyrazole-centered compound 37 was synthesized according

to the method cited in reference [26]. General synthesis of 1,5-

diaryl-pyrazole family was exempliﬁed in cases of 39 and 69

depicted in Scheme 2. The chemical structures are shown in Table 4.

The synthesis started from benzene-1-sulfonamide derivatives

(38e1, 39e1). After protection of the sulfonamide moiety in 38e1

with bis(4-methoxybenzyl) group or in 39e1 with (dimethyla-

mino)methylidene by the reaction with DMF-DMA, the acyl groups

of 38e1 and 39e2 were elongated by Claisen condensation with

diethyl oxalate upon deprotonation with LiHMDS. Cyclization of

dioxoester 38e3 and 39e3 with 4-chlorophenylhydrazine resulted

in the regioisomeric mixture of corresponding pyrazols. In case of

38e3 only one regioisomer was isolated, which after deprotection

led to 38. While in case of the reaction of 39e3 the two isomers

were separated (39e4, 39e5). 39e4 was hydrolyzed to carboxylic

acid 39e6, which was then transformed to acyl chloride 39e7 as a

key intermediate suitable for carboxamide formation including

Weinreb-Nahm amid 39e8. Grignard reaction of 39e8 with eth-

ylmagnesium bromide afforded ketone 39e9. Basic deprotection of

(dimethylamino)methylidene derivatives (39e9) yielded 39. In

case of the amide product 69 intermediate 39e7 was amidated

using 3-azabicyclo[3.1.0]hexane and the resulted 69e1 was

deprotected to gain compound 69. For the 3,5-diaryl-pyrazol family

(80e86) the applied synthesis was the same, however, it was

started from intermediate 39e5. The chemical structures of 80e86

are shown in Table 5.

enced with

a7 nAChR agonists (especially towards 5-HT₃receptors)

are not usually reported for PAMs. Positive modulation of

a7

nAChRs has been shown to have cognitive beneﬁts in various pre-

clinical models [19,20] and investigated extensively in clinical tri-

als. The speciﬁc localization of

the positive modulation aiming at the inhibition of

channel desensitization and the advanced features of

a

7 nAChRs in brain “cognitive areas”,

7 nAChR

7-selective

a

nAChR ligands may lead to the development of clinically success-

ful cognitive enhancers [14] (for representative structures,

see Fig. 1, [21e23]).

The present paper aims at describing the synthetic activities at

Gedeon Richter Plc. to discover novel positive modulators of the a7

nAChR. In the light of the limited knowledge on SAR from the

available patents and scientiﬁc literature, generation of such com-

pounds with sufﬁcient potency and selectivity is a highly chal-

lenging task. Therefore, various complementary approaches (such

as scaffold hopping, HTS and virtual screening) were utilized in a

parallel manner to identify novel PAM compounds with signiﬁcant

afﬁnity to the a7 nAChR. The focus of the present paper was put on

the scaffold hopping approach applied on the selected A-867744

(1) reference structure. As a result, 8 chemotypes with various

central cores were identiﬁed and characterized. Their general for-

mulas or representative chemical structures can be seen in Fig. 2.

2. Results and discussions

2.1.3. Synthesis of derivatives with differently substituted pyrrole

(40e42) and pyrazole (43e46) central cores

2.1. Chemistry

The synthesis of the 4,5-diarylpyrrole derivatives 40e42 is

depicted in Scheme 3. The chemical structures are shown in Table 2.

According to the Weinreb-Nahm ketone synthesis the bis(4-

methoxybenzyl) protected sulfonamide derivative of benzoic acid

40e1 was amidated, followed by a Grignard reaction resulting in

ketone 40e3. After alkylation (40e4) and ketal removal the alde-

hyde derivative 40e5 was obtained. In a microwave assisted

cyclization reaction pyrrole derivative 40e6 was yielded, which

was then either directly (40e7) or after N-methylation (42e1) was

acylated (42e2). After PMB deprotection with TFA compounds 40

and 42 were obtained. (In case of 41 the same methodology was

applied.) Synthesis of compounds 43e46 was exempliﬁed in case of

43. Having hydrolyzed the ester derivative 43e1 to carboxylic acid

43e2 the Weinreb-Nahm ketone synthesis was applied resulting in

the amide (43e3) and subsequently the ketone (43e4) in-

termediates. After the removal of (dimethylamino)methylidene

2.1.1. Synthesis of compounds 1e36 with 1,5-diarylpyrrole central

cores

The pyrrole-centered derivatives 1, 3, 10e33 were synthesized

according to the synthesis depicted in Refs. [24,25]. In cases, where

the synthesis was different (2, 4, 5, 6, 7, 8, 9, 34, 35, 36) the applied

methods can be seen in Scheme 1. The chemical structures are

shown in Table 1. Compound 2 was synthesized in a Vilsmeier-

Haack acylation. The synthesis of 4 was started from a hydrox-

yketone 4e1, which was oxidized with pyridinium chlorochromate

to dioxo compound 4e2 and then it was cyclized to a pyrrole de-

rivative 4e3 using sulfanylamide in acetic acid. After a Friedel-

Crafts acylation (4e4) and amide hydrolysis 4 was obtained.

Starting from the corresponding 2-cyano-4-oxobutanoate de-

rivatives (5e1, 35e1) and reacting them with sulfanilamide and 4-

2

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I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

Fig. 1. PAM compounds 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. General formulas and representative structures of the tested chemotypes.

3

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I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

Scheme 1. Synthesis of the derivatives 2, 4, 5, 6, 7, 8, 9, 34, 35, 36. Reagents and conditions: (a) EtCON(Me)₂, POCl₃, ClCH₂CH₂Cl, 0e80 ^ꢀC; (b) PCC, CH₂Cl₂, rt; (c) sulfanylamide,

AcOH, MeCN, rfx; (d) (EtCO)₂O, AlCl₃, ClCH₂CH₂Cl, 0 ^ꢀC to rt; (e) NaOH, H₂O, rfx.; (f) sulfanylamide, cc HCl, rfx; (g) 4-iodoaniline, cc. HCl, rfx.; (h) ^tBuONO, DMF, 50 ^ꢀC; (i) NaOH, H₂O,

EtOH, rfx.; (j) 1. (COCl)₂, CH₂Cl₂, DMF, 2. MeHNOMe.HCl, CH₂Cl₂, Et₃N; (k) EtMgBr, THF, rfx.; (l) MeI, NaH, THF, rt.; (m) 1-benzylpiperidin-4-amine, toluene, AcOH, rfx; (n)

ClCOOCH₂CH₂Cl, CH₂Cl₂, 0 ^ꢀC to rt; (o) sulfamide, dioxane, rfx.; (p) H₂(atm.), Pd/C, MeOH, THF, rt.

protecting group 43 was obtained. The chemical structures are

shown in Table 3.

potency [27] (SAR evaluation) by chemically modifying compound

1. Compounds were characterized for 7 nAChR PAM characteristics

in

7 nAChR-expressing HEK293 cells by a functional [Ca^2þ] inﬂux

a

assay that is capable of detecting subtle [Ca^2þ]_ichanges (it must

also be noted that the assay is highly sensitive to the inhibition of

desensitization). In addition to that kinetic solubility data

measured by HPLC (in pH 7.4 phosphate-buffer with 1% DMSO) are

summarized in Table 1.

2.1.4. Synthesis of derivatives with furan (47, 48) or oxazole

(49e51) central cores

Syntheses of derivatives with furan (47, 48) or oxazole (49e51)

central cores are depicted in Scheme 4. For the furyl compounds,

the starting material ester 47e1 was condensed with a 4-

ﬂuorophenylacetonitril into 47e2, which was then O-alkylated

with 1-bromobutan-2-one. The resulting 47e3 was cyclized into

the aminofuryl derivative 47e4. It was either directly deprotected

forming compound 48 or the amino moiety was ﬁrst removed by

tert-butyl nitrite and after then the PMB deprotection resulted in

compound 47. The oxazole compound 49 was synthesized from

49e1 oxime reacted with ethyl oxalyl chloride to obtain 49e2 ester.

It was reacted with chlorosulfuric acid and then with aqueous

ammonia solution to derivative 49. Similarly, compound 51 was

synthesized from the oxime derivative 50e1 (which was formed

from the ketone 40e3) reacted with ethyl oxalyl chloride. Ester 51

was transformed to 50 according to the following reaction

sequence: hydrolysis (50e2), Weinreb-Nahm ketone formation

(50e3, 50e4) and deprotection.

Upon systematic removal of the substituents around the pyrrole

central core, structures differing only in a single substituent from

compound 1 were obtained. Somewhat surprisingly, removal of the

R¹, R³, R⁵substituents resulted in only inactive derivatives in the

primary in vitro [Ca^2þ]_iassay (2e4), with the sole exception of the

removal of the small R²methyl group (5). Beside this truncation,

compound 1 was subjected to further systematic chemical modi-

ﬁcations. Derivatives either with a single (6¡20) or with multiple

modiﬁcations (21e36) were synthesized and characterized. Un-

fortunately, the original 4-sulfamoylphenyl moiety (R¹) was not

replaceable, since almost all modiﬁcations of this kind resulted in

inactivity in vitro (6¡11). Only certain modiﬁcations, such as in the

R³(acetyl- and ester-derivatives, 12e13) and R⁵groups (substitu-

tion pattern of phenyl group, 14e20) yielded in vitro active

compounds.

The analysis of these results further supported the steepness of

2.1.5. SAR evaluation of the derivatives of reference compound 1

First, substantial efforts were carried out to assess the impor-

tance of the various pharmacophoric elements on the in vitro

SAR for

a7 nAChR PAMs, already described in the scientiﬁc litera-

ture [28,29]. Therefore, establishing a continuous SAR seemed to be

4

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I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

Table 1

Systematically modiﬁed derivatives of A-867744 reference compound (1).

Comp.

R¹

R²

R³

R⁴

R⁵

Kin. sol. (

mM)

[Ca^2þ

]

i

[Ca^2þ]_iEC₅₀

(mM)

% response at 10 mM

1

2

4-sulfamoylphenyl

H

Me

EtCO

H

4-Cl-Ph

5.2

0.9

84

inact.

0.6

e

3

4

4-sulfamoylphenyl

Me

H

4-Cl-Ph

H

6.1

inact.

e

EtCO

100

e

5

6

4-sulfamoylphenyl

4-(N-methylsulfamoyl)phenyl

H

Me

EtCO

H

4-Cl-Ph

19.5

8.7

100

inact.

0.4

e

7

4-(N,N-dimethylsulfamoyl)phenyl

Me

EtCO

H

4-Cl-Ph

1.2

inact.

e

-

8

9

N-sulfamoylpiperidine-1-yl

4-methylsulphonylphenyl

Me

EtCO

H

4-Cl-Ph

27.8

4.8

12

inact.

e

10

11

3-sulfamoylphenyl

indol-6-yl

Me

EtCO

H

4-Cl-Ph

0.2

5.1

inact.

e

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

4-sulfamoylphenyl

Me

H

MeCO

EtOCO

EtCO

EtOCO

EtCO

EtOCO

COOH

H

4-Cl-Ph

Ph

2.7

4.2

57

29

74

89

86

93

94

97

89

49

46

51

1.9

1.4

1.7

1.2

1.3

1.2

1.3

3.1

2.3

0.7

1.0

2.1

14.1

29.3

12.8

1.0

12.6

38.2

7.7

12.6

16.1

9.2

13.7

1.3

10.8

63.0

36.4

4.4

7.5

50

4-F-Ph

4-MeO-Ph

4-Me-Ph

3-Cl-Ph

2-Cl-Ph

2,4-Di-Cl-Ph

4-F-Ph

4-MeO-Ph

3-Cl-Ph

2-Cl-Ph

4-Me-Ph

4-Cl-Ph

4-F-Ph

55

3.5

6%^a

99

-

0.3

1.1

0.4

3.0

1.1

H

NH₂

Me

100

31

56

inact.

4-F-Ph

4-Cl-Ph

4-F-Ph

e

32

33

34

35

36

4-sulfamoylphenyl

4-iodophenyl

Me

H

COOH

H

4-Me-Ph

3-Cl-Ph

4-Cl-Ph

4-F-Ph

Ph

10

inact.

H

100

6.9

0.3

3.8

EtCO

H

EtOCO

nPr

4-sulfamoylphenyl

Me

H

a

% measured @1 mM.

a highly difﬁcult task, as subtle structural differences led to inac-

tivity or signiﬁcant activity loss.

tested using that pyrazole ring.

Merging compounds 13 and 37 resulted in compound 38, which

showed almost similar potency as the corresponding pyrrole

compound 13 (Fig. 3.).

Considering the improved in vitro potency upon the elimination

of the methyl group in the original pyrrole series (5), this modiﬁ-

cation was applied also in this novel sub-cluster. As a result,

increased in vitro potency was detected with compound 39

As the next logical step, combination of the most useful struc-

tural elements led to several in vitro active compounds (21e28),

alongside with two newly identiﬁed amine derivatives (in the po-

sition of R², 29e30). However, use of carboxylic and propyl groups

as R³(31e33, 36), propionyl as R⁴(34) or iodophenyl as R²(35)

resulted again in loss of in vitro activity.

compared to 38 (the PAM EC₅₀was improved from 2 to 0.5 mM in

the primary [Ca^2þ]_iassay).

2.1.6. Identiﬁcation of the new, pyrazole-based scaffold

Based on this promising result, derivatives with other hetero-

aromatic rings were synthesized and characterized. As a result,

several new chemotypes containing pyrrole, pyrazole, furan and

oxazole rings were synthesized. Main characteristics of these de-

rivatives are summarized in Table 2., Table 3.

After thorough analysis of the obtained SAR of reference com-

pound 1, it was hypothesized that the correct substitution pattern

around the central core is more important than the core structure

itself. This hypothesis was tested by synthesizing new derivatives

with versatile heteroaromatic rings as central cores. Since the only

somewhat potent compound emerging from the virtual screen of

the corporate compound collection was 37 possessing a pyrazole

central core (a summary of the virtual screening can be seen in the

Supplementary Data); the scaffold hopping approach was ﬁrst

Having analyzed the above in vitro data for the corresponding

active derivatives with novel pyrrole and pyrazole central cores (39

and 41 showed EC₅₀values of 0.5 and 1.0

mM, respectively, while 44

resulted in 11% enhancement at 10

mM), also, taking into

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European Journal of Medicinal Chemistry 214 (2021) 113189

Scheme 2. Synthesis of compound 38, 39 and 69. Reagents and conditions: (a) DMF-DMA, DMF, EtOAc, rt; (a’) 4-MeOBnCl, EtCOMe, NaI, K₂CO₃, rfx; (b) 1. LiHMDS, THF, ꢁ70 ^ꢀC, 2.

(COOEt)₂, ꢁ70 ^ꢀC to rt; (c) 4-ClPhNHNH₂.HCl, EtOH, HCl, H₂O; (c’) TFA, anisole, CH₂Cl₂, rt.; (d) KOH, H₂O, EtOH, rfx; (e) (COCl)₂, CH₂Cl₂, DMF, (f) MeHNOMe$HCl CH₂Cl₂, Et₃N; (g)

EtMgBr, THF, rfx.; (h) KOH, MeOH, THF, rt; (i) 3-azabicyclo[3.1.0]hexane, CH₂Cl₂, Et₃N.

consideration the difﬁculties with the synthesis of the pyrrole ring

system in the family of 41 as well as the narrow SAR in the che-

motype of compound 44, the conclusion was drawn that the pyr-

azole ring in compound 39 was the most favorable choice.

Furthermore, application of furan (47e48) and oxazole (49e51)

rings as central cores were also attempted. The chemical structures

and synthesis are shown in Scheme 4. Their synthesis, however,

proved to be rather difﬁcult and e somewhat surprisingly e none of

the completed derivatives showed eligible in vitro activity. These

results proved that despite our hypothesis, the central core may

also exert some effect on the in vitro functional activity (one

possible explanation is described later in section concerning in

silico docking).

modiﬁed only in R¹and R²positions were attempted to be syn-

thesized (Table 4.). Results of the initial in vitro characterization of

ketone and amide derivatives (R¹: alkyls or amines) are summa-

rized in Table 4 below. Almost all synthesized ketones (39 and

52e68) proved to be potent in vitro in this series (however, with no

improvement in potency). In contrast, almost half of the amides

(69e79) showed inactivity in the primary in vitro assay. Regarding

R¹group the ethyl and 3-azabicyclo[3.1.0]hexane seemed to be the

most favorable. As R²moiety the 4-chlorophenyl structural element

seemed to be the most useful.

Based on the [Ca^2þ]_idata, the most potent ketone (39) and

amide (69) compounds were chosen for detailed pharmacological

characterization.

Results with the third, new pyrazole chemotype are summa-

rized in Table 5. These derivatives derived from the 3,5-diaryl-

pyrazol byproduct intermediate which was described in Scheme 4

(in that case 39e4). Having further reacted these intermediates to

the corresponding ketone and amide derivatives a new pyrazole

compound family was identiﬁed. Unfortunately, apart from com-

pound 80, all synthesized analogues proved to be inactive.

2.1.7. Optimization of the new chemotype

Based on the result above, all further optimization activities

were carried out in the most favorable 1,5-diaryl-pyrazole com-

pound family.

As we highlighted above, the experienced, steep SAR did not

allow much space for additional optimization, therefore derivatives

6

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I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

Scheme 3. Synthesis of compounds 40e42 and 43. Reagents and conditions: (a) 1. (COCl)₂, CH₂Cl₂, DMF, 2. MeHNOMe.HCl, CH₂Cl₂, Et₃N; (b) 1. 4-F-BnCl or 4-Cl-BnBr, Mg, Et₂O, rt, 2.

Weinreb amide, THF, rt; (c) 2-bromomethyl-1,3-dioxolane, NaH, THF, DMF, 0 ^ꢀC to rfx; (d) HCl, H₂O, acetone, rt; (e) NH₄OAc, AcOH, 4 Å m. s., W, 170 ^ꢀC; (f) EtCON(Me)₂, POCl₃,

m

ClCH₂CH₂Cl, 0e80 ^ꢀC; (g) TFA, anisole, CH₂Cl₂, rt; (h) MeI, NaH, DMF, 0 ^ꢀC to rt; (i) KOH, H₂O, EtOH, rfx; (a) 1. (COCl)₂, CH₂Cl₂, DMF, 2. MeHNOMe.HCl, CH₂Cl₂, Et₃N; (j) EtMgBr, THF,

rfx; (k) KOH, MeOH, THF, rt.

Table 2

Table 3

New, potent pyrrole derivatives.

New, potent pyrazole derivatives.

Comp. R¹

R²

Kin. sol. (

m

M) [Ca^2þ

]

i

[Ca^2þ]_iEC₅₀

(mM)

% response at 10 mM

43

44

4-F-Ph Et

4-Cl-Ph Et

88.9

37.8

5

e

11

e

Comp. R¹

R²Kin. sol.(

m

M) Ca^2þ

]

[Ca^2þ]_iEC₅₀

(mM)

i

45

46

4-F-Ph EtO 56.4

4-Cl-Ph EtO 53.3

90

95

7.4

2.0

% response at 10 mM

40

41

42

4-F-Ph

4-Cl-Ph

4-F-Ph Me 1.4

H

15.6

1.8

100

77

0.9

1.0

2.2

indirect relationship between the binding position and the ob-

tained functional readouts. So far, there has been no experimental

3D structures of the human

a7 nAChR available in literature,

2.2. In silico evaluation

2.2.1. Retrospective analysis of the observed SAR

In order to attempt the retrospective analysis of the observed

SAR, docking calculations were carried out based on a hypothesized

binding mode. However, it should be noted that the process start-

ing with the ligand binding event and leading to the functional

behavior of the target protein is highly complex, so there is only

however reﬁned open- and closed-state homology models were

reported with our reference compound 1 [30]. In our docking

studies, the open-state homology model was used which contained

compound 1 bound in the transmembrane region between the two

a

7 subunits (Fig. 4A and B.). This inter-subunit binding site, which

was identiﬁed by Newcombe et al. in the course of a comprehensive

docking study, differs from the earlier assumptions where an intra-

subunit binding site was hypothesized [31]. According to our

7

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I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

Table 4

1,5-diaryl-pyrazole derivatives.

Compound

R¹

R²

Kin. sol. (

m

M)

[Ca^2þ

]

[Ca^2þ

]

I

% response at 10

mM

EC₅₀(mM)

39

52

53

54

4-Cl-Ph

Et

Pr

Me

tBu

41.9

25.8

86.8

6.6

84

100

63

0.5

1.1

9.2

29

e

55

56

4-Cl-Ph

iPr

10

28

e

cPr

25.6

e

57

58

4-Cl-Ph

cBu

cPe

17.2

4.6

51

18

2.5

e

59

60

4-Cl-Ph

cBuMe

cPeMe

4.3

2.6

89

23

2.5

e

61

62

63

64

4-CF₃-Ph

4-F-Ph

3-Cl-Ph

2-Cl-Ph

Et

7.5

96.8

6.9

100

90

73

0.9

2.6

5.8

4.7

inact.

e

65

Ph

Et

47.2

33

e

66

67

4-MeO-Ph

Et

64.3

99.4

71

75

7.4

5.9

68

Et

33.7

inact.

e

69

70

71

72

4-Cl-Ph

36.6

57.9

35.2

7.6

100

94

1.2

2.1

2.3

2.2

100

95

73

74

75

4-Cl-Ph

65.9

9.9

5

e

inact.

30.9

76

77

78

79

4-Cl-Ph

99.6

72.6

53.7

8.8

inact.

22

e

inact.

results, during the protein-ligand-complex reﬁnement, a slightly

modiﬁed binding pose was obtained (Fig. 4C.). In this binding mode

the oxygen of the carbonyl seemed to be capable to form a

hydrogen bond with the Ser-271/248 (sequence number in UniProt

sequence P36544 and in the model of Newcombe et al. respectively

[29]) and this interaction might explain the observed in vitro ac-

tivity change in case of compounds 14 and 36. After a visual in-

spection, it can be seen that the majority of the binding site had a

lipophilic character, with the exception of that part, where the

hydroxyl groups of Thr-300/277 and Tyr-296/273 were located. The

8

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I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

Table 5

3,5-diaryl-pyrazole derivatives..

Compound

R¹

R²

Kin. sol. (

m

M)

[Ca^2þ

]

[Ca^2þ

]

i

I

% response at 10

mM

EC₅₀(mM)

80

81

4-Cl-Ph

3-Cl-Ph

Et

49.6

0.7

100

inact.

1.0

e

82

83

4-Cl-Ph

nPr

0.4

inact.

e

11.4

e

84

85

4-Cl-Ph

14.8

5.2

inact.

e

86

4-Cl-Ph

8.0

inact.

e

Scheme 4. Synthesis of compounds 47e51. Reagents and conditions: (a) 4-F-PhCH₂CN, NaH, THF, rt to rfx; (b) 1-bromobutan-2-one, Et₃N, DMF, 50 ^ꢀC; (c) NaOEt, EtOH, 100 ^ꢀC; (d)

TFA, anisole, CH₂Cl₂, rt; (e) tBuONO, DMF, 50 ^ꢀC; (f) EtOOCCOCl, 120 ^ꢀC; (g) 1. ClSO₃H, 0 ^ꢀC to rt, 2. NH₄OH, H₂O, rt; (h) NH₂OH$HCl, NaOAc, EtOH, rfx; (i) KOH, H₂O, EtOH; (j) 1.

(COCl)₂, CH₂Cl₂, DMF, 2. MeHNOMe.HCl, CH₂Cl₂, Et₃N; (k) EtMgBr, THF, rfx, (l) KOH, MeOH, THF, rt.

hydrophilicity of this region might be favorable for the polar sul-

fonamide moiety (R¹, in Table 1.). On the other hand, the environ-

ment around the methyl group (R², in Table 1.) was lipophilic, thus,

hydrogen bond donor and acceptor groups were most probably not

favored in this region and it might contribute to the observed

inactivity of compounds 47e51. However, on the opposite site of

the central core acceptors might have an advantageous effect in the

stabilization of the binding due to the proximity of the polar Ser-

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European Journal of Medicinal Chemistry 214 (2021) 113189

Fig. 3. Hybridization of compounds 37 and 13 resulted in the ﬁrst potent pyrazole derivative 38.

Fig. 4. Homology model of homopentameric

a

7 nAChR published by Newcombe et al. (A., B.) which contains A-867744, 1 in the original (C., magenta) and reﬁned (C., green)

binding mode as well as compounds 39 (D., blue) and 69 (D., brown). The site map (C.) shows the areas favorable for lipophilic parts (orange grid) hydrogen bond acceptors (red

grid) and donors (blue grid). The amino acid sequence numbers shown in the ﬁgure derive from the P36544 UniProt sequence and from the model of Newcombe et al. respectively.

271/248 side chain. In order to demonstrate it, the observed

binding position of the two most potent compounds (39, 69) was

presented (Fig. 4D.). This position was more or less in accordance

with the observed experimental trends, however it should be noted

that the resolution of contradictions in the literature requires

further calculations [29,30].

and 26 were judged suboptimal due to the metabolic liability of the

ester group, as well as their inferior patentability potential. On the

other hand, pyrazolo derivatives possessed the highest kinetic

solubility values. Compounds 39 and 69 showed almost one order

of magnitude increments in the kinetic solubility compared to the

original Abbott reference compound 1 (5.2, 41.9 and 37.0 mM for 1,

39 and 69, respectively). This analysis supported the selection the

best in vitro active 1,5-diaryl pyrazole ketone and amide derivatives

2.2.2. Drug-likeness evaluation of the new chemotype

(39 and 69, with [Ca^2þ]_iPAM EC₅₀values of 0.53 and 1.2

mM,

In order to evaluate drug-likeness of the compound series

containing different central heteroaromatic cores, lipophilic ligand

efﬁciency (LLE) diagram was analyzed (Fig. 5.) based on available

potency data.

respectively) for detailed in vitro/in vivo biological characterization.

2.3. Biological characterization of compounds 39 and 69

According to this, compounds having the highest lipophilic

ligand efﬁciency belonged to either the original series (1e36,

highlighted by red) or the most promising new series (39, 52e79,

highlighted by green). In particular, compounds 69, 39, 28 and 26

possessed the highest LLE values. However, pyrrolo compounds 28

Effects of both 39 and 69 were characterized on the 10 mM

choline-induced inward currents in the automated patch clamp

system. Both compounds showed one order of magnitude larger

enhancement of the choline-induced current amplitude than that

10

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I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

Fig. 5. Lipophilic ligand efﬁciency (LLE ¼ pEC₅₀- logD) diagram of active compounds. The size of the markers is in line with the kinetic solubility values, while the colors represent

different chemotypes. Numbers on the ﬁgure indicate the respective compounds.

for off-target liabilities in a commercial panel of 68 radioligand

binding assays (Lead Proﬁling Screen, Euroﬁns). Both compounds

proved to be speciﬁc at 10 mM with >50% displacement at only two

off-targets (54 and 53% at the CB₁receptor for 39 and 69, respec-

tively. 39, furthermore resulted in 83% displacement of the

phosphodiesterase-4 inhibitor rolipram, while 69 proved to be

active at the androgen receptor with 73% displacement). Impor-

tantly, neither 39 nor 69 showed >20% displacement at 10 mM in

binding assays relevant for ligand afﬁnity to 5-HT₃receptors ([³H]

GR-65630 binding in recombinant HEK cells), NMDA receptors

([³H]CGP-39653, [³H]MDL105,519 or [³H]TCP binding in Wistar rat

cerebral cortex preparations) or GABA_Areceptors ([³H]ﬂuni-

trazepam or [³H]muscimol binding in Wistar Rat brain preparations

minus cerebellum), with the sole exception of 39 showing 28%

displacement of [³H]ﬂunitrazepam binding that suggests a mod-

erate afﬁnity to GABA_Areceptors (data provided by Euroﬁns). To

further characterize the effects of 69 in functional assays, electro-

Fig. 6. 10 mM choline-induced current responses in the presence of 1 (A-867744), 39

and 69 on the representative current traces.

physiological measurements were done in

a4/b2 nACh- and

of 1, demonstrated by the 16-, 168- and 162-fold enhancements by

1, 39 and 69, respectively (representative current traces for 39 and

69 are depicted in Fig. 6.). The molecules had moderate (human)/

low (mouse, rat) (39) or low (human, mouse, rat) (69) intrinsic

clearances in the liver microsomes of different species. In

vinblastine-selected Caco-2 (VB-Caco-2) cultures of high level of P-

gp expression [32], the efﬂux ratio (PDR) of 39 was 3.6 (tested at

GABA_AR-expressing recombinant cells. 69 elicited a concentration-

dependent inhibition of the acethylcholine-evoked currents in

a

4

b

2 nAChR-expressing HEK cells, with an IC₅₀value of 1.8

minimum efﬁcient concentration was 1 M), while evoked no

agonist current, when applied per se at 1 or 10 M. In contrast, 69

showed no agonism or PAM activity in the same concentration

range in 4 nACh-expressing cells in QPatch experiments (Dr.

mM (the

m

a3b

10

mM), indicating a role of efﬂux in drug transport; no efﬂux was

Hugh Chapman, personal communication). Furthermore, 69

detected for 69 with

a

PDR value of 0.7 and Papp_A-B

showed concentration-dependent enhancement of GABA-evoked

currents with a minimum efﬁcient concentration of 1 mM on both

20 ꢂ 10^ꢁ6cm s^ꢁ1. Compounds 39 and 69 were further investigated

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European Journal of Medicinal Chemistry 214 (2021) 113189

Table 6

Comparison of the in vitro and in vivo properties of 39 and 69 with 1 (A-867744). FI: fold increase of the 10 mM choline-induced inward current by 10

place recognition test. RI: recognition index. NA: not available. n.e.: no effect.

m

M test substance. PR:

69

1

39

Kinetic solubility

logP

m

M

5.2

4.7

0.6

16/NA

3, 26, 10

9/2

41.9

3.8

0.5

168/6.1

18, 16, 20

20/0.7

36.6

3.1

1.2

162/4.5

4, 5, 10

18/3.6

@10 mM

2 off-targets

3 mg/kg

[Ca^2þ

]

EC₅₀

FI/EC₅₀

CL_int

(

m

M)

i

Patch clamp

Microsomal stability (Intrinsic clearance)

Penetration

VB-Caco-2

Off-targets

In vivo (PR)

(m

(

m

L/min/mg/protein) (h,m,r)

Papp_A-B£ 10^¡6cm s^¡1/PDR

@1

NA

mM

@10

m

M

Displacement > 50%

RI (sign. effect)

2 off-targets

3 mg/kg or n.e.^a

n.e.^b

a

Depending on the route of administration. 1 resulted in a signiﬁcant effect on the recognition index with a minimal effective dose of 3 mg/kg following ip. administration,

but had no effect after oral administration.

b

No signiﬁcant effect following ip. administration on the recognition index was found. However, there was a signiﬁcant difference between times spent in the novel vs.

familiar arms of the maze in one single dose (3 mg/kg).

Fig. 7. Upper panel: Effect of ip. administration of 39 and 69 on the 1 mg/kg (ip.) scopolamine-induced amnesia in the mouse place recognition test (A., B.: times spent in the novel

and familiar arms of the maze; C., D.: recognition index). N: novel; F: familiar. ***p < 0.001, *p < 0.05 (ANOVA, followed by post-hoc Dunnett-test). Lower panel: results of the pilot

pharmacokinetic measurements from brain and blood samples taken from the experimental animals.

a

1

b

3

g

2 and

tion of

respectively, while the enhancement values for

were 32%, 36% and 123% at 1, 3 and 10 M, respectively (mean

a

g

5

b

3

g

2 GABA_ARs (the compound increased the func-

2 GABA_AR by 24%, 56% and 163% at 1, 3 and 10 M,

2 GABA_AR

by pilot pharmacokinetic measurements from brain and blood

samples taken from the experimental animals. In this model, mice

are placed in a Y-maze where one arm is known by the animals

from a former session and one is completely novel for them; time

spent in familiar arm (F) and unfamiliar arm (N: novel arm) is

measured as well as recognition index [RI¼(N/N þ F) ꢂ 100)] is

calculated for the animals. Despite its better brain penetration, 39

did not change the recognition index in the place recognition test

(Fig. 7C.), while signiﬁcant time differences were measured across

the novel and familiar arms of the maze at 3 mg/kg dose (Fig. 7A.).

a

1

b3

m

a5b3g

m

values, n ¼ 5e10)). Results of the in vitro and in vivo characteriza-

tion are summarized in Table 6.

Based on their promising in vitro properties, both 39 and 69

were further characterized in vivo in rodents. In mice, 39 and 69

were tested in the scopolamine-induced (ip., 1 mg/kg) amnesia

model using the place recognition (Y-maze) paradigm, supported

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European Journal of Medicinal Chemistry 214 (2021) 113189

nACh receptors in cognitive functions.

4. Experimental section

4.1. Chemistry and chemical methods

Commercially available reagents were used without further

puriﬁcation. Solvents and gases were dried according to standard

procedures. Organic solvents were evaporated with reduced pres-

sure 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 1.0

mL. MassHunter Workstation software

(B.03.01) was used for data acquisition and processing. Chromato-

graphic 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. NMR measurements were per-

formed in DMSO‑d₆at 25 ^ꢀC on a 400 or 500 MHz Bruker Avance III

HD spectrometer equipped with a ¹He¹⁹F/¹⁵Ne³¹P Prodigy or a

¹H/¹³C/¹⁵N TCI Extended Temperature CryoProbe, respectively.

Standard ¹H and ¹³C NMR spectra were collected in all cases. In

some cases, HSQC, HMBC and ROESY data were collected as well

using the pulse programs available in the Topspin 3.5 p7 sequence

library. Chemical shifts are referenced to internal TMS

(0.00 ppm/¹H) or to residual solvent signal (39.5 ppm/¹³C). For data

interpretation and reporting the ACD/Spectrus Processor 2017.1.3

software (ACD/Labs, Canada) was used.

Electron impact high-resolution MS (HESI-HRMS) measure-

ments were performed on a Thermo Velos Pro Orbitrap Elite

(Thermo Fisher Scientiﬁc, Bremen, Germany) system. The ioniza-

tion 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 4.0 (Thermo Fisher

Scientiﬁc).

Fig. 8. Rat pharmacokinetic parameters of 69.

In contrast, the amide 69 showed signiﬁcant time changes at all

doses (Fig. 7B.) as well as signiﬁcant increase of the recognition

index at 3 mg/kg with a bell-shaped dose-response relationship

(Fig. 7D.) The more expressed in vivo efﬁcacy of 69 (compared to

that of 39) may bolster our initial theory that the more pronounced

inhibition of channel desensitization leads to better cognitive per-

formance (see the different current traces for 39 and 69 in Fig. 6.).

Based on in vitro and in vivo performance, 69 was selected as

lead molecule. To gain more information in rats, a detailed phar-

macokinetic study was carried out with 69 in 3 mg/kg following ip.

and p.o. administration to non-fasted, male Wistar rats. Compared

to mice, the compound showed excellent brain penetration with

relatively low bioavailability (3.83%). Like in mice, p.o. adminis-

tration of 69 resulted in low, but relevant brain and plasma levels

(Fig. 8.; Table 7.). Half-life of 69 following iv. administration was

~1 h.

3. Conclusions

4.2. General methods for syntheses

Summarizing the above data, a promising new chemotype with

good physicochemical and in vitro parameters was identiﬁed. The

most advanced derivative (69) showed not only in vitro, but also

in vivo efﬁcacy in a rodent model of learning and memory with

acceptable pharmacokinetic properties. Despite the steep SAR

(which is not unusual in the ﬁeld of allosteric modulators), the

identiﬁed structural elements may allow further optimization of

the presented lead compound. Based on the in vivo data with 69,

other highly advanced molecules from this chemotype also have

the possibility of being cognitive enhancers in a biologically rele-

General method 1A (Vilsmeier acylation reaction)

Under an inert atmosphere, N,N-dimethylpropionamide

(0.071 mL, 1.3 eq) was treated with POCl₃(0.061 mL, 1.3 eq) at

0e5 ^ꢀC. The resulting reaction mixture was then allowed to reach

room temperature and stirred for another hour, diluted with 1,2-

dichloroethane (2 mL) cooled to 0 ^ꢀC and the corresponding pyr-

role derivative (0.5 mmol) dissolved in 1,2-dichloroethane or neat

was added. After 1e5 h of heating under reﬂux, the mixture was

cooled to 40 ^ꢀC, an aqueous solution of NaOAc (0.21 g in 0.5 mL, 5

eq) was added and the heating was continued for another 30 min.

vant dose range, further strengthening the supportive role of a7

Table 7

Pharmacokinetic parameters of 69 after 3 mg/kg iv. and p.o. treatment in rats (mean, CV%, n ¼ 4 males/dose group).

Route of admin.

Dose (mg/kg)

AUC_0e5h(ng/mL*h plasma; ng/g brain)

Clearance (mL/min/kg)

14.1 (14.2)

V_d(L/kg)

1.17 (5.8)

T_half(h)

C_max(ng/mL)

T_max(h)

plasma

brain

iv.

3

3492 (13.3)

134

0.97 (14.9)

e

p.o.

NC

31.6

2.0

e

158

36.8

2.0

13

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European Journal of Medicinal Chemistry 214 (2021) 113189

Water (15 mL) was added, the mixture was extracted with CH₂Cl₂,

the combined organic phase was washed with sat. NaHCO₃solution

and water, dried, concentrated, and the residue was puriﬁed by

column chromatography on silica.

In an inert atmosphere, to a stirred mixture of Mg turnings

(0.72 g, 30 mmol, 5 eq) and iodine (1 crystal) in diethyl ether (5 mL)

was added dropwise a solution of the corresponding alkyl halide

(32 mmol) in diethyl ether (30 mL). After consumption of the

magnesium turnings, a solution of the Weinreb amide (6 mmol) in

THF (100 mL) was added dropwise while slightly cooling with tap

water (T < 30 ^ꢀC) and the stirring was continued at room temper-

ature or heated under reﬂux.

The reaction mixture was quenched by dropwise addition of sat.

NH₄Cl solution and water while cooling with ice-water and

extracted with EtOAc. The combined organic phase was washed

with sat. NH₄Cl solution and water, dried and concentrated. The

residue was puriﬁed by column chromatography.

General method 1B (Friedel-Crafts acylation)

Under an inert atmosphere (Ar) dry AlCl₃(3 g, 22 mmol, 3 eq)

was suspended in 1,2-dichloroethane or CH₂Cl₂(45 mL). Into this

suspension, propionic anhydride (1.46 mL, 11 mmol, 1.5 eq) was

added dropwise under ice/water cooling and it was stirred for

additional 15 min. Into the resulting solution, the corresponding

pyrrole derivative (7.5 mmol) in 1,2-dichloroethane or dichloro-

methane (20 mL) was added and it was stirred for 3e6 h at room

temperature. The reaction mixture was poured slowly into ice and

the phases were separated. The aqueous phase was extracted with

dichloromethane (5 ꢂ 100 mL) and the combined organic phase

was dried over anhydrous sodium sulphate and ﬁltered and evap-

orated. The crude product was puriﬁed by column chromatography

on silica.

General method 6 (bis-PMB-sulfonamide deprotection)

A mixture of the corresponding sulfonamide (0.41 mmol), tri-

ﬂuoroacetic acid (4.5 mL) and anisole (0.27 mL, 6 eq) in CH₂Cl₂

(6 mL) was stirred at RT overnight. The pH of the reaction mixture

was adjusted to pH ¼ 8 by the addition of saturated Na₂CO₃solu-

tion, the mixture was extracted with CH₂Cl₂, the combined organic

phase was dried, concentrated and the residue was puriﬁed by

column chromatography on silica.

General method 2 (reductive deamination)

To N,N-dimethylformamide (1 mL) preheated to 50 ^ꢀC was

added tert-butyl nitrite (0.09 mL, 1.5 eq) followed by the addition of

a solution of the corresponding heteroaromatic amino compound

(0.5 mmol) in N,N-dimethylformamide (2 mL) and the mixture was

heated at this temperature for further 20 min. After cooling the

reaction mixture to 0 ^ꢀC, the separated gummy material was

ﬁltered off, dissolved in CH₂Cl₂, (or the mixture was extracted with

EtOAc), dried, concentrated. The residue was puriﬁed by column

chromatography on silica.

General method 7 (N-methylation)

To a solution of the corresponding pyrrole derivative (0.4 mmol)

in DMF (3 mL), NaH (18 mg, 0.45 mmol, 60% in mineral oil) was

added at 0 ^ꢀC under an inert atmosphere and the mixture was

stirred for 10 min at this temperature. Methyl iodide (0.03 mL,

0.48 mmol) was added and the mixture was allowed to reach RT

and reacted for further 3 h. After cooling in an ice-water bath, water

was added, the obtained precipitate was ﬁltered off (or extracted

with EtOAc) washed with water, dried and puriﬁed by column

chromatography on silica.

General method 3 (ester hydrolysis)

To a solution or a suspension of the corresponding ester

(1 mmol) in ethanol (10 mL), aqueous 5 M NaOH or KOH solution (5

eq, 1 mL) was added and the mixture was heated under reﬂux for

1e4 h. The mixture was concentrated under reduced pressure,

water was added, acidiﬁed by the addition of 10% HCl solution, and

the precipitated solid was ﬁltered off, washed with water and dried,

or the acidic mixture was extracted with ethyl acetate.

General method 4 (amidation)

To a suspension of the corresponding carboxylic acid (15 mmol)

in CH₂Cl₂(100 mL) was added DMF (4 drops if only catalyst, 2

equivalent if both catalyst and reagent) followed by the dropwise

addition of oxalyl chloride (4.0 mL, 3 eq) at 0e5 ^ꢀC and the mixture

General method 8 (N-(dimethylamino)methylidene sulfonamide

deprotection)

A mixture of a solution of the corresponding N-(dimethylamino)

methylidene derivative (0.15 mmol) in THF (2 mL) and a solution of

KOH (40 mg, 0.6 mmol, 85%, 4 eq) in MeOH (1 mL) was stirred at RT

overnight. The reaction mixture was acidiﬁed by adding 10% HCl

solution while cooling. The volatile organic solvents were removed

under reduced pressure and the residue was extracted with CH₂Cl₂.

The combined organic phase was dried over Na₂SO₄, concentrated

and the residue was puriﬁed by column chromatography on silica.

4-[5-(4-chlorophenyl)-2-methyl-3-propanoyl-1H-pyrrol-1-

yl]benzenesulfonamide (1) [24,25]

was stirred at room temperature for 1.5

atmosphere.

h under an inert

The solvent and excess oxalyl chloride were evaporated under

reduced pressure, the residue was dissolved in CH₂Cl₂(100 mL),

N,O-dimethylhydroxylamine hydrochloride or the corresponding

amine (1.2 eq) was added, followed by dropwise addition of triethyl

amine (5.4 mL, 2.5 eq) at 0e5 ^ꢀC and the mixture was stirred at

room temperature overnight.

Mp 253e256 ^ꢀC; HESI-HRMS: calcd for C₂₀H₂₀O₃N₂ClS [MþH]^þ:

403.08777; found: 403.08800; delta

¼

0.58 ppm. ¹H NMR

(400 MHz, DMSO‑d₆)

d

7.89 (d, J ¼ 7.8 Hz, 2H), 7.51 (s, 2H), 7.44e7.50

(m, 2H), 7.30 (s, 1H), 7.28 (s, 1H), 7.05e7.10 (m, 2H), 6.97 (s, 1H), 2.85

(d, J ¼ 7.3 Hz, 2H), 2.33 (s, 3H), 1.08 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR

(101 MHz, DMSO‑d₆)

d 196.4, 143.4, 139.1, 136.2, 131.2, 131.0, 130.0,

The reaction mixture was diluted with CH₂Cl₂(150 mL),

extracted with 10% HCl-solution, sat. NaHCO₃solution and water,

dried over Na₂SO₄, concentrated and the residue was puriﬁed by

column chromatography on silica.

129.0, 128.5, 127.7, 126.3, 120.4, 110.3, 32.6, 11.9, 7.9.

1-[5-(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl]propan-1-

one (2)

Prepared according to General method 1A starting from 2-(4-

chlorophenyl)-5-methyl-1H-pyrrole (2e1) [33] (0.25 g, 1.3 mmol).

Yield: 49 mg (15%). See in the Supplementary material in Schemes

Se1. Mp 216e217 ^ꢀC; HESI-HRMS: calcd for C₁₄H₁₅ONCl [MþH]^þ:

General method 5A (Grignard reaction)

In an inert atmosphere, to a stirred solution of the Weinreb

amide (0.7 mmol) in THF (25 mL) was dropwise added ethyl

magnesium bromide solution (1 M in THF, 3.5 mL 5 eq) while

slightly cooling with tap water (T < 30 ^ꢀC) and the mixture was

heated under reﬂux for 3 h.

The reaction mixture was quenched by dropwise addition of sat.

NH₄Cl solution and water while cooling with ice-water and

extracted with EtOAc. The combined organic phase was washed

with sat. NH₄Cl solution and water, dried and concentrated. The

residue was puriﬁed by column chromatography.

248.08367; found: 248.08397; delta

(400 MHz, DMSO‑d₆)

¼

1.20 ppm. ¹H NMR

11.68 (br s, 1H), 7.64e7.70 (m, 2H), 7.41e7.45

d

(m, 2H), 7.00 (d, J ¼ 2.7 Hz,1H), 2.75 (q, J ¼ 7.3 Hz, 2H), 2.48e2.49 (s,

3H), 1.04 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 195.8,

135.4, 130.3, 129.6, 128.1, 127.4, 124.4, 120.5, 106.9, 32.1, 12.8, 7.9.

4-[2-(4-chlorophenyl)-5-methyl-1H-pyrrol-1-yl]benzene-

sulfonamide (3) [24]

Mp 215e218 ^ꢀC; HESI-HRMS: calcd for C₁₇H₁₆O₂N₂ClS [MþH]^þ:

General method 5B (Grignard reaction)

347.06155; found: 347.06159; delta ¼ 0.11 ppm. ¹H NMR (400 MHz,

14

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European Journal of Medicinal Chemistry 214 (2021) 113189

DMSO‑d₆)

d

7.83e7.88 (m, 2H), 7.47 (s, 2H), 7.37e7.42 (m, 2H),

sulfanylamide crystalized out was ﬁltered off, the ﬁltrate was

concentrated and puriﬁed by column chromatography on silica,

eluent: EtOAc-cyclohexane,1:2. Yield: 2.12 g (47%) of white crystals.

7.22e7.27 (m, 2H), 6.98e7.03 (m, 2H), 6.38 (d, J ¼ 3.6 Hz, 1H), 6.11

(dq, J ¼ 0.9, 3.6 Hz, 1H), 2.09 (br, s, 3H). ¹³C NMR (101 MHz,

DMSO‑d₆)

d 142.4, 140.7, 131.5, 131.3, 131.1, 129.9, 128.3, 128.1, 127.7,

126.2, 109.5, 107.9, 12.4.

Step 2. ethyl 5-(4-chlorophenyl)-1-(4-sulfamoylphenyl)-1H-pyr-

4-(2-Methyl-3-propanoyl-1H-pyrrol-1-yl)benzenesulfona-

role-3-carboxylate (26)

mide (4)

See in Scheme 1.

Prepared according to General method 2 (reductive deamination)

starting from 29 (2.0 g, 4.8 mmol). Yield: 0.60 g (31%) of light pink

crystals.

Step 1. 4-oxopentanal (4e2)

Pyridinium chlorochromate (12.8 g, 60 mmol) was dissolved in

dry dichloromethane (50 mL), 5-hydroxy-2-pentanone (4e1)

(4 mL, 40 mmol) was added dropwise and the mixture was stirred

at room temperature for 2 h. The reaction mixture was ﬁltered

through a pad of silica (approx. 3 cm) and washed with diethyl

ether twice. The combined organic phase was evaporated and the

residue was used without any further puriﬁcation.

Step 3. 5-(4-chlorophenyl)-1-(4-sulfamoylphenyl)-1H-pyrrole-3-

carboxylic acid (5e2)

Prepared according to General method 3 (ester hydrolysis) start-

ing from 26 (0.64 g, 1.6 mmol). Yield: 0.59 g (99%) of beige crystals.

Step 4. 5-(4-chlorophenyl)-1-(4-{[(E)-[(dimethylamino)methyl-

idene]amino]sulfonyl}phenyl)-N-methoxy-N-methyl-1H-pyrrole-

3-carboxamide (5e3)

Step 2. 4-(2-methyl-1H-pyrrol-1-yl)benzenesulfonamide (4e3)

To intermediate 4e2 acetic acid (40 mL), acetonitrile (60 mL)

and sulfanylamide (3.5 g, 20 mmol) were added and the solution

was reﬂuxed for 2 h. After completion of the reaction, the solvent

was evaporated, the obtained crude product was dissolved in EtOAc

(250 mL), washed with sat. NaHCO₃solution (100 mL) and brine

(100 mL). The organic phase was dried over anhydrous Na₂SO₄,

ﬁltered and evaporated. The crude product was puriﬁed by column

chromatography on silica, eluent: dichloromethane - methanol

(95:5). Yield: 4.48 g (95%).

The product was synthesized according to General method 4

(amidation) starting from 5e2 (0.58 g, 1.55 mmol). Yield: 0.34 g

(46%) colourless amorphous solid.

Step 5. 4-[2-(4-chlorophenyl)-4-propanoyl-1H-pyrrol-1-yl]benze-

nesulfonamide (5)

Prepared according to General method 5A (Grignard reaction)

starting from 5e3 (0.34 g, 0.71 mmol). Yield: 0.10 g (36%) white

crystals. Mp 189e190 ^ꢀC; HESI-HRMS: calcd for C₁₉H₁₈O₃N₂ClS

[MþH]^þ: 389.07212; found: 389.07282; delta ¼ 1.81 ppm. ¹H NMR

Step 3. N-[4-(2-methyl-3-propanoyl-1H-pyrrol-1-yl)benzene-

sulfonyl]propanamide (4-4)

(400 MHz, DMSO‑d₆)

d

8.02 (d, J ¼ 1.8 Hz, 1H), 7.83e7.87 (m, 2H),

7.48 (br s, 2H), 7.41e7.46 (m, 2H), 7.35e7.40 (m, 2H), 7.13e7.18 (m,

2H), 6.85 (d, J ¼ 1.8 Hz,1H), 2.84 (q, J ¼ 7.3 Hz, 2H),1.08 (t, J ¼ 7.3 Hz,

Prepared according to General method 1B (Friedel-Crafts reaction)

starting from 4e3 (1.7 g, 7.5 mmol). Yield: 0.15 g (6%) of light pink

crystals.

3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 196.0, 143.6, 141.6, 133.9, 132.7,

130.6, 130.4, 130.3, 129.0, 127.4, 126.5, 126.3, 111.1, 32.4, 9.1.

4-[5-(4-chlorophenyl)-2-methyl-3-propanoyl-1H-pyrrol-1-

Step 4. 4-(2-methyl-3-propanoyl-1H-pyrrol-1-yl)benzenesulfona-

mide (4)

yl]-N-methylbenzenesulfonamide

chlorophenyl)-2-methyl-3-propanoyl-1H-pyrrol-1-yl]-N,N-

dimethylbenzenesulfonamide (7)

(6)

and

4-[5-(4-

A mixture of 4e4 (76 mg, 0.22 mmol) and a solution of NaOH

(150 mg, 3.3 mmol) in water (3 mL) was reﬂuxed for 4 h. It was

quenched by the addition of. cc. HCl (0.3 mL), extracted with CH₂Cl₂

(4 ꢂ 8 mL) and dried over anhydrous Na₂SO₄, ﬁltered and evapo-

rated. The residue was puriﬁed by column chromatography on

silica, eluent: CH₂Cl₂-methanol (99:1). Yield: 52 mg (81%). Mp

199 ^ꢀC; HESI-HRMS: calcd for C₁₄H₁₇O₃N₂S [MþH]^þ: 293.09544;

found: 29309591; delta ¼ 1.6 ppm. ¹H NMR (400 MHz, DMSO‑d₆)

See in Scheme 1.

Under an argon atmosphere, compound 1 (315 mg, 0.78 mmol)

was dissolved in dry THF (40 mL) and the solution was cooled to

0e5 ^ꢀC using ice-water cooling bath. Into this solution, NaH (60 mg,

2eq, 60% dispersion in mineral oil) and methyl iodide (0.29 mL, 6eq)

were added. The solution was stirred at room temperature for 3 h.

The reaction mixture was diluted with water and ethyl acetate

(50 mL). After separation of the phases the aqueous phase was

extracted with ethyl acetate (4 ꢂ 50 mL). The combined organic

phase was washed with water and brine, dried over Na₂SO₄, ﬁltered

and evaporated. The crude product was puriﬁed and separated by

column chromatography on silica using dichloromethane-ethyl

acetate as elution system (solvent ratio was changed from 95:5 to

50:50). Yield for 6: 39 mg (12%). Mp 220e222 ^ꢀC; HESI-HRMS: calcd

d

7.89e7.99 (m, 2H), 7.58e7.67 (m, 2H), 7.51 (s, 2H), 6.99 (d,

J ¼ 3.2 Hz, 1H), 6.75 (d, J ¼ 3.2 Hz, 1H), 2.79 (q, J ¼ 7.3 Hz, 2H), 2.43

(s, 3H), 1.06 (s, 3H) ¹³C NMR (101 MHz, DMSO‑d₆)

197.0, 143.3,

d

140.9, 133.9, 130.4, 126.9, 126.6, 126.4, 121.6, 110.4, 33.1, 12.4, 8.5.

4-[2-(4-chlorophenyl)-4-propanoyl-1H-pyrrol-1-yl]benzene-

sulfonamide (5)

See in Scheme 1.

for

C

₂₁H₂₂O₃N₂ClS [MþH]^þ: 417.10342; found: 417.10392;

delta ¼ 1.20 ppm. ¹H NMR (400 MHz, DMSO‑d₆)

d 7.82e7.87 (m,

Step 1. ethyl 2-amino-5-(4-chlorophenyl)-1-(4-sulfamoylphenyl)-

1H-pyrrole-3-carboxylate (29)

2H), 7.58 (q, J ¼ 4.97 Hz, 1H), 7.49e7.53 (m, 2H), 7.26e7.31 (m, 2H),

7.02e7.07 (m, 2H), 6.99 (s, 1H), 2.86 (q, J ¼ 7.3 Hz, 2H), 2.43 (d,

J ¼ 5.0 Hz, 3H), 2.35 (s, 3H), 1.08 (t, J ¼ 7.3 Hz, 4H). ¹³C NMR

A mixture of ethyl 4-(4-chlorophenyl)-2-cyano-4-oxobutanoate

(5e1) [34] (2.87 g, 10.8 mmol), sulfanylamide (13.0 g, 75.5 mmol)

and cc. hydrochloric acid (37% aqueous, 5 drops) was heated under

reﬂux for 2 days. (After 1 day of reﬂux, another 6 drops of cc. HCl

was added.) After cooling to room temperature, the excess

(101 MHz, DMSO‑d₆) d 196.4, 139.7, 138.5, 136.2, 131.2, 130.9, 129.9,

128.9, 128.8, 127.7, 127.4, 120.4, 110.3, 32.6, 28.1, 11.9, 7.9.

Yield for 7: 132 mg (40%). Mp 191e193 ^ꢀC; HESI-HRMS: calcd for

C

₂₂H₂₄O₃N₂ClS

[MþH]^þ:

431.11907;

found:

431.11892;

delta ¼ ꢁ0.34 ppm. ¹H NMR (400 MHz, DMSO‑d₆)

d

7.80e7.85 (m,

15

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European Journal of Medicinal Chemistry 214 (2021) 113189

2H), 7.52e7.57 (m, 2H), 7.25e7.32 (m, 2H), 7.00e7.04 (m, 2H), 7.01

(s, 1H), 2.86 (q, J ¼ 7.3 Hz, 2H), 2.61 (s, 6H), 2.36 (s, 3H), 1.08 (t,

dichloromethane to 14% methanol in dichloromethane as an eluent.

Yield: 53 mg (68%).

J ¼ 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 196.4, 140.3, 136.2,

133.8, 131.2, 130.9, 129.8, 128.9, 128.8, 128.1, 127.7, 120.5, 110.3, 37.0,

32.6, 11.9, 7.9.

Step 4. 4-[5-(4-chlorophenyl)-2-methyl-3-propanoyl-1H-pyrrol-

1-yl]piperidine-1-sulfonamide (8)

4-[5-(4-chlorophenyl)-2-methyl-3-propanoyl-1H-pyrrol-1-

yl]piperidine-1-sulfonamide (8)

Under an argon atmosphere

a mixture of 8e4 (98 mg,

See in Scheme 1.

0.30 mmol) and sulfamide (144 mg, 1.50 mmol) were heated in 1,4-

dioxane (12 mL) under reﬂux for 4 h and 20 min. Upon completion

of the reaction (as determined by TLC) the mixture was cooled and

was diluted with ethyl acetate (45 mL) and water (15 mL). The

mixture was extracted with saturated NaHCO₃solution (25 mL), the

layers were separated, the aqueous layer was extracted with EtOAc

(20 mL), the combined organic layer was washed with water

(5 ꢂ 40 mL) and dried over anhydrous Na₂SO₄. The solvent was

evaporated under reduced pressure and the residue was puriﬁed by

column chromatography using 0.6% methanol in CH₂Cl₂to yield

solid product, which was then triturated with diethyl ether, ﬁltered

and dried. Yield: 85 mg (69%).

Step 1. 1-benzyl-4-[2-(4-chlorophenyl)-5-methyl-1H-pyrrol-1-yl]

piperidine (8e2)

Under a nitrogen atmosphere_,to a solution of 8e1 [35] (0.83 g,

3.96 mmol) in toluene (5 mL) was added 1-benzylpiperidin-4-

amine (0.80 mL, 3.92 mmol) and glacial acetic acid (0.060 mL),

and the mixture was heated under reﬂux conditions in a ﬂask

equipped with a Dean-Stark apparatus. The progress of the reaction

was monitored by TLC. After 145 min of heating, 1-benzylpiperidin-

4-amine (0.20 mL, 0.98 mmol) was added, after further 80 min two

drops of acetic acid, 1-benzylpiperidin-4-amine (2 mL, 9.8 mmol)

and toluene (2 mL) were added. Finally, after 7 h of reﬂux the re-

action solution was cooled to room temperature and stirred over-

night. Toluene (100 mL), saturated aqueous NaHCO₃solution

(30 mL), and water (10 mL) were added. The two layers were

separated, the aqueous layer was extracted with toluene (40 mL).

The combined organic layer was washed with water (6 ꢂ 70 mL)

and dried over anhydrous Na₂SO₄. The solvent was evaporated

under reduced pressure to obtain a crude product, which was then

puriﬁed by column chromatography on silica using 17% ethyl ace-

tate in cyclohexane as an eluent to obtain the title compound.

Yield: 1.11 g (77%). Mp 222e226 ^ꢀC; HESI-HRMS: calcd for

1-[5-(4-chlorophenyl)-1-(4-methanesulfonylphenyl)-2-

methyl-1H-pyrrol-3-yl]propan-1-one (9)

See in Scheme 1. Prepared according to General method 1B

(Friedel-Crafts reaction) starting from 9e1 2-(4-chlorophenyl)-1-[4-

(methanesulfonyl)phenyl]-5-methyl-1H-pyrrole(9e1)

[24]

(345 mg, 1 mmol). Yield: 0.21 g (52%). Mp 216e218 ^ꢀC; HESI-HRMS:

calcd for C₂₁H₂₁O₃NClS [MþH]^þ: 402.09252; found: 402.09278;

delta ¼ 0.66 ppm. ¹H NMR (400 MHz, DMSO‑d₆)

d 8.00e8.04 (m,

2H), 7.54e7.58 (m, 2H), 7.27e7.32 (m, 2H), 7.03e7.08 (m, 2H), 6.99

(s, 1H), 3.29e3.30 (m, 3H), 2.86 (q, J ¼ 7.3 Hz, 2H), 2.35 (s, 3H), 1.24

(br s, 2H), 1.08 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d

196.4, 140.7, 140.1, 136.2, 131.2, 131.0, 129.8, 128.9, 128.9, 127.8,

C

₁₉H₂₅O₃N₃ClS

[MþH]^þ:

410.12997;

found:

410.12994;

7.49e7.53 (m,

127.7, 120.5, 110.5, 42.7, 32.6, 12.0, 7.9.

3-[5-(4-chlorophenyl)-2-methyl-3-propanoyl-1H-pyrrol-1-

yl]benzenesulfonamide (10)

Synthesized in a manner analogous to the method described for

compound 1 using 3-aminobenzenesulfonamide instead of 4-

aminobenzenesulfonamide. Mp 236e240 ^ꢀC; HESI-HRMS: calcd

delta ¼ ꢁ0.08 ppm. ¹H NMR (400 MHz, DMSO‑d₆)

d

2H), 7.37e7.41 (m, 2H), 6.78 (s, 2H), 6.54 (s, 1H), 4.00e4.09 (m, 1H),

3.46e3.61 (m, 2H), 2.72 (q, J ¼ 7.3 Hz, 2H), 2.67 (s, 3H), 2.40e2.49

(m, 2H), 2.06e2.21 (m, 2H), 1.84e1.91 (m, 2H), 1.01 (t, J ¼ 7.3 Hz,

3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 196.3, 134.6, 132.1, 131.5, 131.3,

131.0, 127.9, 110.2, 54.2, 45.5, 32.3, 29.3, 12.5, 8.0.

for

C

₂₀H₂₀O₃N₂ClS [MþH]^þ: 403.08777; found: 403.08785;

delta ¼ 0.20 ppm. ¹H NMR (400 MHz, DMSO‑d₆)

7.90 (ddd, J ¼ 1.1,

d

Step 2. 1-[1-(1-benzylpiperidin-4-yl)-5-(4-chlorophenyl)-2-

methyl-1H-pyrrol-3-yl]propan-1-one (8e3)

1.7, 7.8 Hz, 1H), 7.68 (t, J ¼ 7.8 Hz, 1H), 7.65e7.66 (m, 1H), 7.51 (ddd,

J ¼ 1.2, 2.1, 8.1 Hz, 2H), 7.49 (s, 1H), 7.25e7.30 (m, 2H), 7.05e7.10 (m,

2H), 6.99 (s, 1H), 2.86 (q, J ¼ 7.3 Hz, 2H), 2.32 (s, 3H), 1.08 (t,

Prepared according to General method 1 (Friedel-Crafts reaction)

starting from 8e2 (1.0 g, 2.74 mmol). Yield: 0.61 g (52%).

J ¼ 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆)

¼ 196.4,144.7,136.8,

d

136.3, 131.3, 130.9, 129.8, 129.8, 128.8, 127.8, 125.2, 124.8, 120.3,

110.2, 32.5, 11.9, 7.9.

Step 3. 1-[5-(4-chlorophenyl)-2-methyl-1-(piperidin-4-yl)-1H-

pyrrol-3-yl]propan-1-one (8e4)

Ethyl

5-(4-chlorophenyl)-1-(1H-indol-6-yl)-2-methyl-1H-

pyrrole-3-carboxylate (11)

Synthesized in a manner analogous to the method described for

compound 13 using 6-aminoindol instead of sulfanylamide. Mp

186e187 ^ꢀC; HESI-HRMS: calcd for C₂₂H₂₀O₂N₂Cl [MþH]^þ:

379.12078; found: 379.12156; delta ¼ 2.05 ppm. ¹H NMR (400 MHz,

To a solution of 8e3 (99 mg, 0.235 mmol) in dichloromethane

(6 mL) was added 1-chloroethyl chloroformate (0.033 mL,

0.31 mmol) in dichloromethane (2.5 mL) at 0e5 ^ꢀC under argon

atmosphere. The reaction mixture was stirred at the same tem-

perature for 40 min, then allowed to warm to room temperature.

After being stirred at the same temperature for 2.5 h the reaction

mixture was evaporated. The residue was dissolved in methanol

(9 mL) and heated to reﬂux for 1 h. The solution was allowed to cool

to room temperature and concentrated. To the residue was added

diethyl ether, after trituration and ﬁltration the hydrogen chloride

salt of the crude product was obtained. The salt was taken up in a

mixture CH₂Cl₂(90 mL) and 3% NaOH solution (30 mL), the layers

were separated, aqueous layer was extracted with CH₂Cl₂(10 mL),

the combined organic layer was washed with water (5 ꢂ 20 mL)

and dried over anhydrous Na₂SO₄. The solvent was evaporated

under reduced pressure to obtain a crude amine which was then

puriﬁed by column chromatography using 10% methanol in

DMSO‑d₆)

d 11.37 (br s, 1H), 7.42e7.48 (m, 2H), 7.15e7.20 (m, 2H),

7.04e7.09 (m, 2H), 6.91 (dd, J ¼ 2.0, 8.5 Hz, 1H), 6.73 (s, 1H),

6.45e6.48 (m, 1H), 4.23 (q, J ¼ 7.1 Hz, 2H), 2.28 (s, 2H), 1.29 (t,

J ¼ 7.1 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 163.8, 138.0, 134.6,

131.8, 130.6, 130.4, 128.4, 128.2, 127.5, 127.0, 126.6, 120.5, 119.1, 111.5,

111.1, 108.9, 101.1, 58.4, 13.9, 11.7.

4-[3-Acetyl-5-(4-chlorophenyl)-2-methyl-1H-pyrrol-1-yl]

benzenesulfonamide (12) [25]

Mp 259e260 ^ꢀC HESI-HRMS: calcd for C₁₉H₁₈O₃N₂ClS [MþH]^þ:

389.07212; found: 389.07282; delta ¼ 1.8 ppm. ¹H NMR (500 MHz,

DMSO‑d₆)

d 7.82e7.95 (m, 2H), 7.51 (s, 2H), 7.45e7.50 (m, 2H),

7.23e7.33 (m, 2H), 7.03e7.12 (m, 2H), 6.97 (s, 1H), 2.43 (s, 3H), 2.32

(s, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

194.2, 144.1, 139.7, 136.8,

131.8, 131.6, 130.5, 129.6, 129.1, 128.4, 126.9, 121.5, 111.5, 28.8, 12.5

d

16

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European Journal of Medicinal Chemistry 214 (2021) 113189

Ethyl 5-(4-chlorophenyl)-2-methyl-1-(4-sulfamoylphenyl)-

1H-pyrrole-3-carboxylate (13) [24,25]

(m, 2H), 7.18e7.26 (m, 3H), 7.06 (s,1H), 2.86 (q, J ¼ 7.31 Hz, 2H), 2.33

(s, 3H), 1.08 (t, J ¼ 7.32 Hz, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

Mp 218e219 ^ꢀC; HESI-HRMS: calcd for C₂₀H₂₀O₄N₂ClS [MþH]^þ:

d 197.1, 144.2, 139.7, 137.0, 133.7, 133.0, 131.5, 130.0, 129.1, 126.6,

419.08268; found: 419.08343; delta ¼ 1.78 ppm. ¹H NMR (400 MHz,

126.9, 121.0, 111.4, 33.2, 12.5, 8.5.

4-[2-Methyl-5-(2-chlorophenyl)-3-propanoyl-1H-pyrrol-1-

yl]benzenesulfonamide (19)

DMSO‑d₆)

d

7.89 (d, J ¼ 7.9 Hz, 2H), 7.46e7.53 (m, 4H), 7.28 (d,

J ¼ 7.7 Hz, 2H), 7.06 (d, J ¼ 7.7 Hz, 2H), 6.75 (s, 1H), 5.76 (s, 1H), 4.24

(d, J ¼ 7.1 Hz, 2H), 2.33 (s, 3H), 1.29 (t, J ¼ 7.1 Hz, 4H). ¹³C NMR

Synthesized in a manner analogous to the method described for

(101 MHz, DMSO‑d₆)

d

163.6, 143.4, 139.3, 137.3, 131.6, 131.0, 129.8,

compound

instead

1

using 2-bromo-1-(2-chlorophenyl)ethan-1-one

129.0, 128.6, 127.8, 126.3, 112.1, 109.9, 58.6, 13.8, 11.6.

4-(2-Methyl-5-phenyl-3-propanoyl-1H-pyrrol-1-yl)benzene-

sulfonamide (14)

of

2-bromo-1-(4-chlorophenyl)ethan-1-one.

Mp

211e213 ^ꢀC; HESI-HRMS: calcd for C₂₀H₂₀O₃N₂ClS [MþH]^þ:

403.08777; found: 403.08809; delta

(500 MHz, DMSO‑d₆) d 7.76e7.80 (m, 2H), 7.45 (s, 2H), 7.39e7.42

¼

0.80 ppm. ¹H NMR

Synthesized in a manner analogous to the method described for

compound 1 using 2-bromo-1-phenylethan-1-one instead of 2-

bromo-1-(4-chlorophenyl)ethan-1-one. Mp 246e248 ^ꢀC; HESI-

(m, 2H), 7.34e7.39 (m, 2H), 7.25e7.33 (m, 2H), 6.82 (s, 1H), 2.83 (q,

J ¼ 7.3 Hz, 2H), 2.36 (s, 3H), 1.07 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR

HRMS: calcd for

C

₂₀H₂₁O₃N₂S [MþH]^þ: 36912674; found:

(126 MHz, DMSO‑d₆) d 196.9, 143.7, 139.3, 135.4, 133.6, 133.4, 130.9,

369.12705; delta ¼ 0.84 ppm. ¹H NMR (400 MHz, DMSO‑d₆)

130.2, 129.7, 129.2, 128.7, 126.9, 126.3, 120.7, 111.5, 33.1, 12.6, 8.5.

4-[2-Methyl-5-(2,4-dichlorophenyl)-3-propanoyl-1H-pyrrol-

1-yl]benzenesulfonamide (20)

Synthesized in a manner analogous to the method described for

compound 1 using 2-bromo-1-(2,4-dichlorophenyl)ethan-1-one

d

7.85e7.90 (m, 2H), 7.51 (s, 2H), 7.43e7.50 (m, 2H), 7.15e7.26 (m,

2H), 7.05e7.11 (m, 2H), 6.92 (s, 1H), 2.85 (q, J ¼ 7.3 Hz, 2H), 2.33 (s,

3H), 1.08 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d

196.4,

143.3, 139.4, 135.8, 132.6, 131.1, 128.5, 127.7, 127.4, 126.3, 126.2,

120.3, 109.8, 32.5, 11.9, 7.9.

instead

of

2-bromo-1-(4-chlorophenyl)ethan-1-one.

Mp

4-[5-(4-ﬂuorophenyl)-2-methyl-3-propanoyl-1H-pyrrol-1-

yl]benzenesulfonamide (15)

209e210 ^ꢀC; HESI-HRMS: calcd for C₂₀H₁₉O₃N₂Cl₂S [MþH]^þ:

437.04880; found: 437.04912; delta ¼ 0.73 ppm. ¹H NMR (500 MHz,

Synthesized in a manner analogous to the method described for

DMSO‑d₆)

d

7.79e7.83 (m, 2H), 7.58 (t, J ¼ 1.2 Hz, 1H), 7.46 (s, 2H),

compound

instead

1

using 2-bromo-1-(4-ﬂuorophenyl)ethan-1-one

7.39e7.43 (m, 2H), 7.38 (d, J ¼ 1.2 Hz, 2H), 6.85 (s, 1H), 2.83 (q,

of

2-bromo-1-(4-chlorophenyl)ethan-1-one.

Mp

J ¼ 7.3 Hz, 2H), 2.36 (s, 3H), 1.07 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR

239e240 ^ꢀC; HESI-HRMS: calcd for C₂₀H₂₀O₄N₂FS [MþH]^þ:

(126 MHz, DMSO‑d₆) d 196.9, 143.8, 139.1, 135.8, 134.6, 134.5, 133.8,

387.11732; found: 387.11766; delta ¼ 0.88 ppm. ¹H NMR (400 MHz,

130.0, 128.8, 128.7, 128.5, 127.2, 126.4, 120.8, 111.9, 33.1, 12.6, 8.5.

Ethyl 5-(4-ﬂuorophenyl)-2-methyl-1-(4-sulfamoylphenyl)-

1H-pyrrole-3-carboxylate (21)

DMSO‑d₆)

d 7.84e7.91 (m, 2H), 7.50 (s, 2H), 7.43e7.49 (m, 2H),

7.04e7.15 (m, 4H), 6.90 (s, 1H), 2.85 (q, J ¼ 7.3 Hz, 2H), 2.33 (s, 3H),

1.08 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d

196.4, 160.5,

An intermediate for the synthesis of compound 15. Mp

143.4, 139.2, 135.7, 131.5, 129.6, 128.6, 127.6, 126.2, 120.2, 114.7,

109.8, 32.5, 11.9, 7.9.

224e225 ^ꢀC; HESI-HRMS: calcd for C₂₀H₂₀O₄N₂FS [MþH]^þ:

403.11223; found: 403.11220; delta

¼

ꢁ0.08 ppm. ¹H NMR

4-[5-(4-methoxyphenyl)-2-methyl-3-propanoyl-1H-pyrrol-

1-yl]benzenesulfonamide (16)

(500 MHz, DMSO‑d₆)

d

7.88 (d, J ¼ 7.71 Hz, 2H), 7.50 (s, 2H),

7.45e7.49 (m, 2H), 7.03e7.12 (m, 4H), 6.69 (s, 1H), 4.24 (q, J ¼ 7.1 Hz,

Synthesized in a manner analogous to the method described for

2H), 2.33 (s, 3H), 1.29 (t, J ¼ 7.1 Hz, 3H). ¹³C NMR (126 MHz,

compound

instead

1

of

using 2-bromo-1-(4-methoxyphenyl)ethan-1-one

DMSO‑d₆) d 164.3, 161.1, 144.0, 139.9,137.4, 132.4,130.2, 129.2, 128.1,

2-bromo-1-(4-chlorophenyl)ethan-1-one.

Mp

128.1, 126.8, 115.3, 112.5, 110.0, 59.1, 14.4, 12.2.

218e220 ^ꢀC; HESI-HRMS: calcd for C₂₁H₂₃O₄N₂S [MþH]^þ:

Ethyl

5-(4-methoxyphenyl)-2-methyl-1-(4-

399.13730; found: 399.13729; delta

(400 MHz, DMSO‑d₆)

¼

ꢁ0.04 ppm. ¹H NMR

sulfamoylphenyl)-1H-pyrrole-3-carboxylate (22)

An intermediate for the synthesis of compound 16. Mp

181e183 ^ꢀC; HESI-HRMS: calcd for C₂₁H₂₃O₅N₂S [MþH]^þ:

d 7.84e7.89 (m, 2H), 7.50 (s, 2H), 7.42e7.48

(m, 2H), 6.97e7.02 (m, 2H), 6.77e6.82 (m, 3H), 3.69 (s, 3H), 2.84 (d,

J ¼ 7.3 Hz, 2H), 2.31 (s, 3H), 1.07 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR

415.13222; found: 415.13216; delta

(400 MHz, DMSO‑d₆) d 7.82e7.90 (m, 2H), 7.50 (s, 2H), 7.43e7.48

¼

ꢁ0.15 ppm. ¹H NMR

(101 MHz, DMSO‑d₆)

d

196.4, 157.6, 143.2, 139.5, 135.1, 132.5, 128.9,

128.6, 126.1, 123.5, 120.1, 113.2, 108.9, 54.4, 32.5, 12.0, 7.9.

4-[2-Methyl-5-(4-methylphenyl)-3-propanoyl-1H-pyrrol-1-

yl]benzenesulfonamide (17)

(m, 2H), 6.96e7.02 (m, 2H), 6.75e6.80 (m, 2H), 6.59 (s, 1H), 4.23 (q,

J ¼ 7.2 Hz, 1H), 3.69 (s, 3H), 2.31 (s, 3H), 1.28 (t, J ¼ 7.2 Hz, 3H). ¹³

C

NMR (126 MHz, DMSO‑d₆)

d 164.4, 158.2, 143.8, 140.2, 136.9, 133.4,

Synthesized in a manner analogous to the method described for

129.5, 129.2, 126.8, 124.0, 113.8, 112.3, 109.1, 59.1, 55.0, 14.4, 12.2.

compound

instead

1

of

using 2-bromo-1-(4-methylphenyl)ethan-1-one

Ethyl 5-(3-chlorophenyl)-2-methyl-1-(4-sulfamoylphenyl)-

1H-pyrrole-3-carboxylate [23]

2-bromo-1-(4-chlorophenyl)ethan-1-one.

Mp

251e253 ^ꢀC; HESI-HRMS: calcd for C₂₁H₂₃O₃N₂S [MþH]^þ:

An intermediate for the synthesis of compound 18. Mp

197e199 ^ꢀC; HESI-HRMS: calcd for C₂₀H₂₀O₄N₂ClS [MþH]^þ:

419.08268; found: 419.08316; delta ¼ 1.14 ppm. ¹H NMR (500 MHz,

383.14239; found: 383.14312; delta ¼ 1.91 ppm. ¹H NMR (400 MHz,

DMSO‑d₆)

d 7.85e7.89 (m, 2H), 7.51 (s, 2H), 7.43e7.48 (m, 2H),

7.00e7.05 (m, 2H), 6.94e6.98 (m, 2H), 6.85 (s, 1H), 2.84 (q,

DMSO‑d₆) d 7.87e7.92 (m, 2H), 7.53 (s, 2H), 7.48e7.52 (m, 2H),

J ¼ 7.3 Hz, 2H), 2.32 (s, 3H), 2.22 (s, 3H), 1.07 (t, J ¼ 7.3 Hz, 3H). ¹³

C

7.22e7.26 (m, 1H), 7.19e7.22 (m, 1H), 7.17e7.18 (m, 1H), 6.93 (td,

J ¼ 1.6, 7.3 Hz, 1H), 6.81 (s, 1H), 4.24 (q, J ¼ 7.1 Hz, 2H), 2.33 (s, 3H),

NMR (101 MHz, DMSO‑d₆)

d 197.5, 144.3, 140.5, 136.7, 136.6, 133.7,

129.6, 129.4, 129.3, 128.4, 127.2, 121.3, 110.4, 33.6, 21.1, 13.0, 9.0.

4-[2-Methyl-5-(3-chlorophenyl)-3-propanoyl-1H-pyrrol-1-

yl]benzenesulfonamide (18)

1.29 (t, J ¼ 7.1 Hz, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

d 164.2, 144.2,

139.8, 138.1, 133.5, 133.0, 131.8, 130.0, 129.2, 127.5, 126.9, 126.8,

126.4, 112.8, 110.9, 59.2, 14.4, 12.2.

Synthesized in a manner analogous to the method described for

Ethyl 5-(2-chlorophenyl)-2-methyl-1-(4-sulfamoylphenyl)-

1H-pyrrole-3-carboxylate (24)

compound

instead

1

using 2-bromo-1-(3-chlorophenyl)ethan-1-one

of

2-bromo-1-(4-chlorophenyl)ethan-1-one.

Mp

An intermediate for the synthesis of compound 19. Mp

220e221 ^ꢀC; HESI-HRMS: calcd for C₂₀H₂₀O₃N₂ClS [MþH]^þ:

235e237 ^ꢀC; HESI-HRMS: calcd for C₂₀H₂₀O₄N₂ClS [MþH]^þ:

403.08777; found: 403.08786; delta

¼

0.22 ppm. ¹H NMR

419.08268; found: 419.08305; delta

¼

0.89 ppm. ¹H NMR

(500 MHz, DMSO‑d₆)

d

7.88e7.92 (m, 2H), 7.52 (s, 2H), 7.48e7.52

(500 MHz, DMSO‑d₆)

d

7.78 (d, J ¼ 7.7 Hz, 2H), 7.46 (s, 2H),

17

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I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

7.39e7.43 (m, 2H), 7.37 (dd, J ¼ 1.3, 7.9 Hz, 1H), 7.21e7.34 (m, 3H),

6.60 (s, 1H), 4.24 (q, J ¼ 7.1 Hz, 2H), 2.36 (s, 3H), 1.29 (t, J ¼ 7.1 Hz,

4.20 (d, J ¼ 7.1 Hz, 2H), 1.26 (t, J ¼ 7.1 Hz, 3H). ¹³C NMR (126 MHz,

DMSO‑d₆)

d 165.0, 160.6, 148.4, 143.6, 138.7, 129.0, 128.7, 128.4,

3H). ¹³C NMR (126 MHz, DMSO‑d₆)

d

164.3,143.7,139.4,136.5,133.5,

127.0, 125.5, 115.2, 107.7, 92.6, 58.5, 14.6.

133.3, 130.7, 130.2, 130.0, 129.3, 128.8, 126.9, 126.3, 112.3, 111.2, 59.2,

14.4, 12.2.

5-(4-ﬂuorophenyl)-2-methyl-1-(4-sulfamoylphenyl)-1H-pyr-

role-3-carboxylic acid (31)

Ethyl 2-methyl-5-(4-methylphenyl)-1-(4-sulfamoylphenyl)-

1H-pyrrole-3-carboxylate (25)

An intermediate for the synthesis of compound 15.

¹H NMR (500 MHz, DMSO‑d₆)

d 7.68e7.76 (m, 2H), 6.99e7.02

An intermediate for the synthesis of compound 17. Mp

(m, 4H), 6.50 (s, 1H), 2.33 (s, 3H). Mp > 350 ^ꢀC; HESI-HRMS: calcd

217e218 ^ꢀC; HESI-HRMS: calcd for C₂₁H₂₃O₄N₂S [MþH]^þ:

for

C

₁₈H₁₆O₄N₂FS [MþH]^þ: 375.08093; found: 375.08128;

399.13730; found: 399.13719; delta

¼

ꢁ0.30 ppm. ¹H NMR

delta ¼ 0.9 ppm. ¹H NMR (500 MHz, DMSO‑d₆)

d 7.68e7.76 (m, 2H),

(500 MHz, DMSO‑d₆)

d

7.85e7.89 (m, 2H), 7.51 (s, 2H), 7.44e7.48 (m,

6.99e7.02 (m, 4H), 6.50 (s, 1H), 2.33 (s, 3H). ¹³C NMR (126 MHz,

2H), 7.00e7.03 (m, 2H), 6.93e6.96 (m, 2H), 6.64 (s, 1H), 4.23 (q,

DMSO‑d₆) d 169.5, 160.4, 145.2, 140.7, 132.8, 129.8, 129.6, 129.1,

128.6, 126.4, 123.2, 115.0, 112.4, 12.0.

J ¼ 7.1 Hz, 2H), 2.32 (s, 3H), 2.22 (s, 3H), 1.29 (t, J ¼ 7.1 Hz, 3H). ¹³

C

NMR (126 MHz, DMSO‑d₆)

d

164.3, 143.9, 140.2, 137.2, 136.3, 133.5,

2-Methyl-5-(4-methylphenyl)-1-(4-sulfamoylphenyl)-1H-

pyrrole-3-carboxylic acid (32)

129.1, 128.9, 128.7, 127.9, 126.7, 112.5, 109.5, 59.1, 20.6, 14.4, 12.2.

Ethyl 5-(4-chlorophenyl)-1-(4-sulfamoylphenyl)-1H-pyrrole-

3-carboxylate (26)

An intermediate for the synthesis of compound 17. Mp > 350 ^ꢀC;

HESI-HRMS: calcd for C₁₉H₁₉O₄N₂S [MþH]^þ: 371.10600; found:

371.10616; delta ¼ 0.42 ppm. ¹H NMR (500 MHz, DMSO‑d₆)

An intermediate for the synthesis of compound 5. Mp

200e201 ^ꢀC; HESI-HRMS: calcd for C₁₉H₁₈O₄N₂ClS [MþH]^þ:

d

7.75e7.80 (m, 2H), 7.22e7.29 (m, 2H), 6.95e6.98 (m, 2H),

405.06703; found: 405.06729; delta

¼

0.65 ppm. ¹H NMR

6.85e6.89 (m, 2H), 6.58 (br s, 2H), 6.48 (s, 1H), 2.34 (s, 3H), 2.20 (s,

(500 MHz, DMSO‑d₆)

7.48 (br s, 2H), 7.42e7.46 (m, 2H), 7.35e7.38 (m, 2H), 7.13e7.17 (m,

2H), 6.83 (d, J ¼ 1.8 Hz, 1H), 4.25 (q, J ¼ 7.1 Hz, 2H), 1.28 (t, J ¼ 7.1 Hz,

d

7.82e7.86 (m, 2H), 7.81 (d, J ¼ 1.8 Hz, 1H),

3H). ¹³C NMR (126 MHz, DMSO‑d₆)

d

169.7, 134.7, 132.5,130.6,130.4,

128.7, 128.5, 127.1, 126.3, 123.1, 112.0, 20.5, 12.0.

5-(3-chlororophenyl)-2-methyl-1-(4-sulfamoylphenyl)-1H-

pyrrole-3-carboxylic acid (33)

3H). ¹³C NMR (126 MHz, DMSO‑d₆)

d 163.4, 143.2, 141.0, 133.1, 132.2,

130.0, 130.0, 129.4, 128.6, 126.9, 126.1, 116.9, 111.7, 59.6, 14.4.

4-[2-(4-ﬂuorophenyl)-4-propanoyl-1H-pyrrol-1-yl]benzene-

sulfonamide (27)

Synthesized in a manner analogous to the method described for

the synthesis of compound 5 using ethyl 4-(4-ﬂuorophenyl)-2-

cyano-4-oxobutanoate [34] instead of ethyl 4-(4-chlorophenyl)-2-

cyano-4-oxobutanoate. Mp 185e187 ^ꢀC; HESI-HRMS: calcd for

An intermediate for the synthesis of compound 18. Mp

237e240 ^ꢀC; HESI-HRMS: calcd for C₁₈H₁₆O₄N₂ClS [MþH]^þ:

391.05138; found: 391.05171; delta ¼ 0.83 ppm. ¹H NMR (500 MHz,

DMSO‑d₆) d 12.12 (br s, 1H), 7.87e7.92 (m, 2H), 7.51e7.54 (m, 2H),

7.47e7.52 (m, 2H), 7.18e7.25 (m, 2H), 7.15 (t, J ¼ 1.8 Hz,1H), 6.93 (td,

J ¼ 1.8, 6.8 Hz, 1H), 6.78 (s, 1H), 2.32 (s, 3H). ¹³C NMR (126 MHz,

DMSO‑d₆) d 165.8,144.1, 140.0, 137.9, 133.7, 133.0, 131.5, 130.0,129.2,

C

₁₉H₁₈O₃N₂FS

[MþH]^þ:

373.10167;

found:

373.10190;

127.5, 126.8, 126.6, 126.3, 113.6, 111.4, 12.1.

4-[2-(4-chlorophenyl)-5-methyl-3-propanoyl-1H-pyrrol-1-

yl]benzenesulfonamide (34)

delta ¼ 0.6 ppm. ¹H NMR (500 MHz, DMSO‑d₆)

d

8.00 (d, J ¼ 1.8 Hz,

1H), 7.80e7.88 (m, 2H), 7.48 (s, 2H), 7.38e7.45 (m, 2H), 7.09e7.23

(m, 4H), 6.80 (d, J ¼ 1.8 Hz, 1H), 2.84 (q, J ¼ 7.3 Hz, 2H), 1.08 (t,

See in Scheme 1.

J ¼ 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

d 195.5, 161.5, 143.0,

141.2, 133.7, 130.5, 129.4, 127.7, 126.8, 126.0, 125.7, 115.5, 110.2, 31.9,

8.7.

Step 1. N-{4-[2-(4-chlorophenyl)-5-methyl-3-propanoyl-1H-pyr-

rol-1-yl]benzenesulfonyl}-propanamide (34e1)

Ethyl 5-(4-ﬂuorophenyl)-1-(4-sulfamoylphenyl)-1H-pyrrole-

3-carboxylate (28)

Prepared according to General method 1B (Friedel-Crafts reaction)

starting from compound 3 (3.21 g). Compound 34e1 was isolated

by column chromatography from the isomeric mixture of mono and

diacylated compounds. (See regioisomer at compound 36.) Yield:

0.42 g (10%).

An intermediate for the synthesis of compound 27. Mp

200e201 ^ꢀC; HESI-HRMS: calcd for C₁₉H₁₈O₄N₂FS [MþH]^þ:

389.09658; found: 389.09675; delta

¼

0.44 ppm. ¹H NMR

(500 MHz, DMSO‑d₆)

d

7.80e7.84 (m, 2H), 7.79 (d, J ¼ 1.9 Hz, 1H),

7.48 (s, 2H), 7.38e7.45 (m, 2H), 7.09e7.22 (m, 4H), 6.77 (q, J ¼ 1.9 Hz,

1H), 4.24 (q, J ¼ 7.1 Hz, 2H),1.28e1.28 (m,1H),1.28 (t, J ¼ 7.1 Hz, 2H).

Step 2. 4-[2-(4-chlorophenyl)-5-methyl-3-propanoyl-1H-pyrrol-

1-yl]benzenesulfonamide (34)

¹³C NMR (126 MHz, DMSO‑d₆)

d

163.4, 161.4, 143.0, 141.1, 133.4,

130.5, 128.9, 127.6, 126.8, 126.1, 116.7, 115.5, 111.2, 59.5, 14.4.

Ethyl 2-amino-5-(4-chlorophenyl)-1-(4-sulfamoylphenyl)-

1H-pyrrole-3-carboxylate (29)

A mixture of a suspension of 34e1 (415 mg, 0.9 mmol) in

ethanol (20 mL) and a solution of KOH (1.3 g) in water (5 mL) was

heated under reﬂux overnight. Water was added and the mixture

was acidiﬁed by the addition of 10% HCl during cooling with ice.

The precipitate obtained was collected by ﬁltration, washed until

neutral, dried and recrystallized from methanol. Yield: 120 mg

(33%) white crystals. Mp 250e251 ^ꢀC; HESI-HRMS: calcd for

An intermediate for the synthesis of compound 5. Mp

240e243 ^ꢀC; HESI-HRMS: calcd for C₁₉H₁₉O₄N₃ClS [MþH]^þ:

420.07793; found: 420.07808; delta

(500 MHz, DMSO‑d₆) d 7.86e7.90 (m, 2H), 7.50 (br s, 2H), 7.42e7.46

¼

0.34 ppm. ¹H NMR

(m, 2H), 7.18e7.23 (m, 2H), 6.96e7.00 (m, 2H), 6.51 (s, 1H), 5.94 (s,

2H), 4.20 (q, J ¼ 7.1 Hz, 2H), 1.27 (t, J ¼ 7.1 Hz, 3H). ¹³C NMR

C

₂₀H₂₀O₃N₂ClS

[MþH]^þ:

403.08777;

found:

403.08763;

7.77e7.79 (m,

(126 MHz, DMSO‑d₆)

d

164.9, 148.7, 143.7, 138.6, 130.7, 130.4, 128.7,

delta ¼ ꢁ0.35 ppm. ¹H NMR (500 MHz, DMSO‑d₆)

d

128.3, 128.2, 127.1, 125.2, 108.5, 92.8, 58.5, 14.6.

2H), 7.46 (s, 2H), 7.37e7.41 (m, 2H), 7.28e7.31 (m, 2H), 7.18e7.21

(m, 2H), 6.60 (q, J ¼ 1.0 Hz, 1H), 2.55 (q, J ¼ 7.3 Hz, 2H), 2.03 (d,

J ¼ 0.9 Hz, 3H), 0.93 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

Ethyl

2-amino-5-(4-ﬂuorophenyl)-1-(4-sulfamoylphenyl)-

1H-pyrrole-3-carboxylate (30)

An intermediate for the synthesis of compound 27. Mp

d 195.7, 143.7, 139.7, 134.9, 132.9, 132.7, 130.8, 130.2, 129.2, 127.7,

216e218 ^ꢀC; HESI-HRMS: calcd for C₁₉H₁₉O₄N₃FS [MþH]^þ:

126.4, 122.1, 108.6, 33.2, 12.6, 8.3.

404.10748; found: 404.10739; delta

¼

ꢁ0.23 ppm. ¹H NMR

Ethyl

5-(4-ﬂuorophenyl)-1-(4-iodophenyl)-1H-pyrrole-3-

(500 MHz, DMSO‑d₆)

d

7.85e7.89 (m, 2H), 7.50 (s, 2H), 7.40e7.44

carboxylate (35)

See in Scheme 1.

(m, 2H), 7.02 (s, 2H), 7.00 (d, J ¼ 1.4 Hz, 2H), 6.43 (s, 1H), 5.89 (s, 2H),

18

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I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

Step 1. ethyl 2-amino-5-(4-ﬂuorophenyl)-1-(4-iodophenyl)-1H-

pyrrole-3-carboxylate (35e2)

(piperidin-1-yl)-1H-pyrazole-3-carboxamide (37)

Synthesized in a manner analogous to the method described in

the literature [26] for 1-(2-chlorophenyl)-5-(4-

Synthesized in a manner analogous to the method described for

the synthesis of compound 29 using ethyl 4-(4-ﬂuorophenyl)-2-

cyano-4-oxobutanoate (35e1) [34] instead of ethyl 4-(4-

chlorophenyl)-2-cyano-4-oxobutanoate and 4-iodoaniline instead

of sulfanylamide. Mp 41e43 ^ꢀC; HESI-HRMS: calcd for C₁₉H₁₆O₂NFI

[MþH]^þ: 436.02043; found: 436.02021; delta ¼ ꢁ0.51 ppm. ¹H

methanesulfonylphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-

carboxamide using 2,4-dichlorophenylhydrazine instead of 2-

chlorophenylhydrazine. Mp 275e276 ^ꢀC; HESI-HRMS: calcd for

C

₂₃H₂₅O₃N₄Cl₂S

[MþH]^þ:

507.10189;

found:

507.10199;

7.92e7.99 (m,

delta ¼ 0.20 ppm. ¹H NMR (500 MHz, DMSO‑d₆)

d

4H), 7.87 (d, J ¼ 8.5 Hz, 1H), 7.81 (d, J ¼ 2.3 Hz, 1H), 7.64 (dd, J ¼ 2.3,

8.5 Hz, 1H), 7.46e7.56 (m, 2H), 3.39 (br s, 4H), 3.26 (s, 3H), 2.32 (s,

3H), 1.78e1.89 (m, 4H), 1.37e1.60 (m, 2H). ¹³C NMR (126 MHz,

NMR (500 MHz, DMSO‑d₆)

d

7.74e7.79 (m, 2H), 7.69 (d, J ¼ 1.9 Hz,

1H), 7.12e7.18 (m, 4H), 7.02e7.06 (m, 2H), 6.74 (d, J ¼ 1.9 Hz, 1H),

4.23 (q, J ¼ 7.1 Hz, 2H), 1.27 (s, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

DMSO‑d₆) d 159.8, 158.4, 142.7, 141.8, 141.2, 135.7, 135.2, 132.7, 132.0,

d

163.5, 161.4, 138.4, 138.1, 133.3, 130.4, 128.8, 127.9, 127.6, 116.4,

130.5, 129.8, 128.6, 127.3, 118.6, 115.2, 82.7, 56.4, 43.1, 23.1, 21.1, 9.0.

Ethyl 1-(4-chlorophenyl)-4-methyl-5-(4-sulfamoylphenyl)-

1H-pyrazole-3-carboxylate (38)

115.4, 110.8, 93.8, 59.4, 14.4.

Step 2. ethyl 5-(4-ﬂuorophenyl)-1-(4-iodophenyl)-1H-pyrrole-3-

carboxylate (35)

See in Scheme 2.

Step

1.

N,N-bis[(4-methoxyphenyl)methyl]-4-

Prepared according to General method 2 (reductive deamination)

starting from 35e2 (138 mg, 0.31 mmol). Yield: 34 mg (25%) of

white solid.

4-(2-Methyl-5-phenyl-3-propyl-1H-pyrrol-1-yl)benzene-

sulfonamide (36)

propanoylbenzenesulfonamide (38e2)

In an inert atmosphere, a mixture of 4-propanoylbenzene-1-

sulfonamide 38e1 (1.16 g, 5.4 mmol), 4-methoxybenzyl chloride

(1.5 mL), K₂CO₃(1.51 g) and NaI (1.63 g) in methyl ethyl ketone

(90 mL) was heated under reﬂux for 2 days while 3 times 4-

methoxybenzyl chloride (1.5 mL), K₂CO₃(1.51 g) and methyl ethyl

ketone were repeatedly added. After cooling, water was added and

the mixture was extracted with CH₂Cl₂, dried over Na₂SO₄,

concentrated and puriﬁed with column chromatography on silica,

eluent: EtOAc- cyclohexane (1:4). Yield: 1.82 g (75%).

See in Scheme 1.

Mp 158e162 ^ꢀC; HESI-HRMS: calcd for C₂₀H₂₃O₂N₂S [MþH]^þ:

355.14748; found: 355.14752; delta ¼ 0.13 ppm. ¹H NMR (500 MHz,

DMSO‑d₆)

d 7.79e7.85 (m, 2H), 7.46 (s, 2H), 7.30e7.39 (m, 2H),

7.14e7.19 (m, 2H), 7.05e7.11 (m, 1H), 6.96e7.01 (m, 2H), 6.27 (s, 1H),

2.37e2.43 (m, 2H), 1.99 (s, 3H), 1.57 (sxt, J ¼ 7.3 Hz, 2H), 0.96 (t,

J ¼ 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

d 142.7, 141.9, 132.8,

132.1, 128.8, 128.2, 127.3, 127.2, 126.6, 125.7, 120.9, 110.6, 27.9, 23.8,

14.0, 10.7.

Step 2. ethyl 4-(4-{bis[(4-methoxyphenyl)methyl]sulfamoyl}

phenyl)-3-methyl-2,4-dioxobutanoate (38e3)

Step 1. N-{4-[5-(4-chlorophenyl)-2-methyl-3-propanoyl-1H-pyr-

rol-1-yl]benzenesulfonyl}-propanamide (36e1)

In an inert atmosphere, to a mixture of diethyl ether (10 mL) and

1 M LiHMDS solution in THF (4.9 mL) was dropwise added a solu-

tion of 38e2 (2.18 g, 4.8 mmol) in a mixture of diethyl ether (3 mL)

and THF (3 mL) at ꢁ70 ^ꢀC and the mixture was stirred at ꢁ70 ^ꢀC for

1 h. Diethyl oxalate (0.74 mL, 1.1 eq) was dropwise added and

stirring was continued for further 2 h at ꢁ70 ^ꢀC, then overnight at

RT. The precipitated solid was ﬁltered off, washed with ether, dried

in vacuo, suspended in ethanol (4 mL), acidiﬁed with 6 M HCl

(1 mL), water was added and the mixture was extracted with EtOAc,

dried over Na₂SO₄, concentrated. Yield: 610 mg (23%).

Prepared according to General method 1B (Friedel-Crafts reaction)

starting from 3 (3.21 g). The desired product was isolated by col-

umn chromatography from the isomeric mixture of mono and

diacylated compounds. (See regioisomer at compound 34.) Yield:

0.95 g (23%) of yellow solid.

Step 2. N-[4-(2-methyl-5-phenyl-3-propyl-1H-pyrrol-1-yl)benze-

nesulfonyl]propanamide (36e2)

Step 3. ethyl 5-(4-{bis[(4-methoxyphenyl)methyl]sulfamoyl}

phenyl)-1-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxylate

(38e4)

A solution of 36e1 (0.94 g, 2 mmol) in a mixture of tetrahy-

drofuran (20 mL) and methanol (40 mL) was vigorously stirred

under hydrogen gas at atmospheric pressure in the presence of Pd/

C (100 mg, 10%Pd) at room temperature overnight. After removal of

the catalyst by ﬁltration, the ﬁltrate was concentrated and puriﬁed

by column chromatography on silica using a 5 to 95 mixture of

EtOAc and CH₂Cl₂as the eluent. Yield: 0.42 g (50%) of white crystals.

A

mixture of

38e3

(609

mg, 1.1

mmol),

4-

chlorophenylhydrazine hydrochloride (207 mg, 1.1 eq), ethanol

(4 mL) and aqueous HCl solution (6 M, 0.2 mL) was heated under

reﬂux for 24 h. The reaction mixture was concentrated, puriﬁed by

column chromatography on silica, eluent: EtOAc-cyclohexane (1:4)

Yield: 101 mg (14%).

Step 3. 4-(2-methyl-5-phenyl-3-propyl-1H-pyrrol-1-yl)benzene-

sulfonamide (36)

Step 4. ethyl 1-(4-chlorophenyl)-4-methyl-5-(4-sulfamoylphenyl)-

A mixture of a suspension of 36e2 (410 mg, 1 mmol) in ethanol

(20 mL) and a solution of KOH (1.3 g) in water (5 mL) was heated

under reﬂux overnight. Water was added and the mixture was

acidiﬁed by the addition of 10% HCl during cooling with ice. The

precipitate obtained was collected by ﬁltration, washed until

neutral, dried and puriﬁed by column chromatography on silica

using a 25 to 75 mixture of EtOAc and cyclohexane as the eluent.

Yield: 130 mg (36%) white crystals.

1H-pyrazole-3-carboxylate (38)

Prepared according to General method 6 (bis-PMB-sulfonamide

deprotection) starting from 38e4 (101 mg, 0.15 mmol). Yield: 28 mg

(44%). Mp 244e249 ^ꢀC; HESI-HRMS: calcd for C₁₉H₁₉O₄N₃ClS

[MþH]^þ: 420.07793; found: 420.07861; delta ¼ 1.61 ppm. ¹H NMR

(500 MHz, DMSO‑d₆)

d 7.83e7.86 (m, 2H), 7.47e7.52 (m, 2H),

7.42e7.48 (m, 4H), 7.27e7.32 (m, 2H), 4.34 (q, J ¼ 7.2 Hz, 2H), 2.23

(s, 3H), 1.33 (t, J ¼ 7.2 Hz, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

d 162.1,

1-(2,4-dichlorophenyl)-5-(4-methanesulfonylphenyl)-N-

19

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European Journal of Medicinal Chemistry 214 (2021) 113189

144.2,141.8,140.7,137.8,132.9,131.9,130.6,129.2,127.2, 126.0,119.6,

60.3, 14.2, 9.4.

4-[1-(4-chlorophenyl)-3-propanoyl-1H-pyrazol-5-yl]ben-

zene-1-sulfonamide (39)

was added followed by a dropwise addition of a solution of oxalyl

chloride (0.26 mL, 3 eq) in CH₂Cl₂(1.5 mL) while cooling with ice.

The mixture was stirred at RT for 4 h, the solvent was evaporated in

vacuo and the residue was dissolved in CH₂Cl₂(10 mL). Next, N,O-

dimethylhydroxylamine hydrochloride (110 mg, 1.1 eq) was added,

followed by a dropwise addition of triethylamine (0.36 mL, 2.5 eq)

during cooling in an ice-water bath and the mixture was stirred

overnight at RT. The reaction mixture was diluted with CH₂Cl₂

(20 mL), extracted with 5% HCl, saturated NaHCO₃solution and

water, dried over Na₂SO₄and concentrated. The residue was puri-

ﬁed with column chromatography on silica, eluent: MeOHe CH₂Cl₂

(1:99). Yield: 270 mg of light yellow amorphous solid. MS:

[M þ H^þ] ¼ 476.1.

See in Scheme 2.

Step 1. N’-(4-acetylbenzene-1-sulfonyl)-N,N-dimethylmethanimi-

damide (39e2)

To a suspension of 4-acetylbenzenesulfonamide (39e1) (2.68 g

13.4 mmol) in a mixture of EtOAc (25 mL) and DMF (5 mL), DMF-

DMA (2 mL, 1.1 eq) was dropwise added. After stirring for 4 h at

RT, the reaction mixture was concentrated in vacuo, triturated with

diethyl ether (20 mL), the solid was collected, washed with ether

and dried. Yield: 3.22 g of beige solid. MS: [M þ H^þ] ¼ 255.1.

Step 6. N’-{4-[1-(4-chlorophenyl)-3-propanoyl-1H-pyrazol-5-yl]

benzene-1-sulfonyl}-N,N-dimethylmethanimidamide (39e9)

Step 2. ethyl 4-(4-{[(dimethylamino)methylidene]sulfamoyl}

phenyl)-2,4-dioxobutanoate (39e3)

Under an inert atmosphere, Mg turnings (68 mg, 2.8 mmol) and

a small I₂crystal was covered with anhydrous THF (1 mL). A solu-

tion of ethyl bromide (0.21 mL, 2.8 mmol) in THF (1 mL) was

dropwise added during vigorous stirring. After all Mg had reacted, a

suspension of the Weinreb amide 39e8 (261 mg) in THF (5 mL) was

added and the mixture was heated under reﬂux for 3 h. After

cooling with ice, a saturated NH₄Cl solution (10 mL) was dropwise

added and the mixture was extracted with CH₂Cl₂, dried over

Na₂SO₄, concentrated and puriﬁed with column chromatography

on silica, eluent: EtOAc-CH₂Cl₂(5:95). Yield: 159 mg of white

crystals. MS: [M þ H^þ] ¼ 445.1.

To a mixture of THF (20 mL) and 1 M LiHMDS solution in THF

(10 mL) was dropwise added a suspension of 39e2 (2.54 g,

10 mmol) in THF (30 mL) under ꢁ70 ^ꢀC in an inert atmosphere and

the mixture was stirred at ꢁ70 ^ꢀC for 1 h. Diethyl oxalate (1.5 mL,1.1

eq) was dropwise added and stirring was continued for further 2 h

at ꢁ70 ^ꢀC, then overnight at RT (TLC eluent: MeOHeCH₂Cl₂, 5:95).

The precipitated solid was ﬁltered off, washed with ether, dried in

vacuo, suspended in water (200 mL), acidiﬁed with cc. HCl (1 mL)

and stirred for 15 min. The solid was collected by ﬁltration, washed

till neutral with water and dried. Yield: 2.40 g of beige solid. MS:

[M þ H^þ] ¼ 355.3.

Step 7. 4-[1-(4-chlorophenyl)-3-propanoyl-1H-pyrazol-5-yl]ben-

zenesulfonamide (39)

Step 3. ethyl 1-(4-chlorophenyl)-5-(4-sulfamoylphenyl)-1H-pyr-

azole-3-carboxylate (39e5)

Compound 39e9 (153 mg, 0.34 mmol) was dissolved in THF

(5 mL), a solution of KOH (94 mg, ~4.5 eq, 90%) in MeOH (2.5 mL)

was added and the mixture was stirred overnight at RT. (TLC eluent:

EtOAc). After cooling with ice, water (10 mL) was added, acidiﬁed

with 10% HCl and the organic solvents were evaporated. The

aqueous residue was extracted with CH₂Cl₂, the organic phase was

washed with water, dried over Na₂SO₄, concentrated and puriﬁed

with column chromatography on silica, eluent: EtOAc-CH₂Cl₂(1:9).

Yield: 82 mg of white crystals. HPLC purity: 99.7%.

A mixture of dioxoester (39e3) (1.20 g, 3.39 mmol), 4-

chlorophenylhydrazine hydrochloride (667 mg, 1.1 eq), ethanol

(15 mL) and aqueous HCl solution (20%, 0.6 mL) was heated under

reﬂux for 12 h (TLC eluent: MeOHeCH₂Cl₂, 5:95). The solid

precipitated upon cooling overnight was ﬁltered off, washed with

cold ethanol and dried affording 126 mg of ethyl 1-(4-

chlorophenyl)-3-(4-sulfamoylphenyl)-1H-pyrazole-5-carboxylate

(39e4). The ﬁltrate was concentrated, puriﬁed with column chro-

matography on silica, eluent: EtOAc- cyclohexane (1:3, then 2:3)

resulting in 31 mg of 39e4 and 550 mg of ethyl 1-(4-chlorophenyl)-

5-(4-sulfamoylphenyl)-1H-pyrazole-3-carboxylate (39e5). Yield:

550 mg. MS: [M þ H^þ] ¼ 406.0.

Mp 202e204 ^ꢀC; HESI-HRMS:

M

þ

H

¼

390.06658

(delta ¼ ꢁ2.0 ppm; C₁₈H₁₇O₃N₃ClS). HR-ESI-MS-MS (CID ¼ 45%; rel.

int. %): 373(100); 334(35); 309(1); 292(1).

¹H NMR (500 MHz, DMSO‑d₆)

d

¼ 7.79e7.82 (2H, m, H-16, 15),

7.55e7.59 (2H, m, H-10, 9), 7.46e7.50 (2H, m, H-14, 13), 7.44 (2H, br

s, H-22), 7.39e7.43 (2H, m, H-8, 7), 7.21 (1H, s, H-4), 3.05 (2H, q,

J ¼ 7.3 Hz, H-24), 1.12 (3H, t, J ¼ 7.3 Hz, H-26); ¹³C NMR (125.7 MHz,

Step 4. 1-(4-chlorophenyl)-5-(4-sulfamoylphenyl)-1H-pyrazole-3-

carboxylic acid (39e6)

DMSO‑d₆)

d

¼ 195.8 (C-23), 150.8 (C-5), 144.2 (C-17), 143.5 (C-2),

137.8 (C-6), 133.3 (C-11), 131.9 (C-12), 129.5 (C-10, 9), 129.2 (C-14,

To a solution of 39e5 (540 mg, 1.3 mmol) in ethanol (20 mL) was

added a solution of KOH (390 mmol, 6 eq) in water (1 mL) and the

mixture was heated under reﬂux 30 min. The mixture was cooled

with ice, water (40 mL) was added and the pH was adjusted to 2 by

adding 1 M HCl solution (6 mL). The mixture was extracted with

CH₂Cl_2,and EtOAc, the collected organic layer was dried over

Na₂SO₄and concentrated under vacuum. Yield: 390 mg of light

yellow solid. MS: [M þ H^þ] ¼ 378.0.

13), 127.4 (C-8, 7), 126.0 (C-16, 15), 108.4 (C-4), 31.5 (C-24), 7.9 (C-

26).

4-[3-(4-ﬂuorophenyl)-5-propanoyl-1H-pyrrol-2-yl]benzene-

sulfonamide (40)

See in Scheme 3.

Step 1. N-methoxy-N-methyl-4-[N,N-bis(4-methoxybenzyl)sulfa-

moyl]benzamide (40e2)

Step 5. 1-(4-chlorophenyl)-5-(4-{[(dimethylamino)methylidene]

sulfamoyl}phenyl)-N-methoxy-N-methyl-1H-pyrazole-3-

carboxamide (39e8)

Prepared according to General method 4 (amidation) starting

from 4-[N,N-bis(4-methoxybenzyl)sulfamoyl]benzoic acid (40e1)

[36,37] (6.75 g, 15.3 mmol). Yield: 7.14 g (95%) of colourless oil.

Under an inert atmosphere, compound 39e6 (385 mg,

1.0 mmol) was suspended in CH₂Cl₂(10 mL), DMF (0.15 mL, 1.9 eq)

Step 2. N,N-bis(4-methoxybenzyl)-4-[(4-ﬂuorophenyl)acetyl]ben-

zenesulfonamide (40e3)

20

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I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

General method 5B (Grignard reaction) starting from 40e2 (2.9 g,

6 mmol). Yield: 2.29 g (72%) of white solid.

131.9, 131.6, 130.2, 128.9, 125.6, 123.2, 117.7, 115.4, 30.7, 9.0.

4-[3-(4-chlorophenyl)-5-propanoyl-1H-pyrrol-2-yl]benzene-

sulfonamide (41)

Step 3. N,N-bis(4-methoxybenzyl)-4-[3-(1,3-dioxolan-2-yl)-2-(4-

ﬂuorophenyl)propanoyl]-benzenesulfonamide (40e4)

Synthesized in a manner analogous to the method described for

the synthesis of compound 40 using 4-chlorobenzyl bromide

instead of 4-ﬂuorobenzyl chloride. Mp 288e289 ^ꢀC; HESI-HRMS:

calcd for C₁₉H₁₈O₃N₂ClS [MþH]^þ: 389.07212; found: 389.07273;

Under an inert atmosphere, a solution of 40e3 (2.285 g,

4.28 mmol) in a mixture of DMF (12 mL) and THF (12 mL) cooled

with ice-water was treated with NaH (206 mg, 1.2 eq, 60% in

mineral oil) in portions. 2-Bromomethyl-1,3-dioxolane (0.5 mL, 1.1

eq) was added and the mixture was heated under reﬂux for 10 h

(TLC eluent: EtOAc-cyclohexane, 3:7).

Water was added, and the mixture was extracted with EtOAc,

dried over Na₂SO₄and concentrated. The residue was puriﬁed by

column chromatography on silica (eluent: EtOAc-cyclohexane, 1:9

to 1:1). (The slower eluted component is the product.) Yield:

807 mg (30%) of yellowish oil.

delta ¼ 1.58 ppm. ¹H NMR (400 MHz, DMSO‑d₆)

d 12.26 (br s, 1H),

7.66e7.84 (m, 2H), 7.49e7.61 (m, 2H), 7.33e7.46 (m, 4H), 7.09e7.30

(m, 3H), 2.86 (q, J ¼ 7.4 Hz, 2H), 1.12 (t, J ¼ 7.4 Hz, 3H). ¹³C NMR

(101 MHz, DMSO‑d₆)

d 190.7, 143.1, 134.4, 134.1, 133.0, 132.0, 131.1,

130.0, 129.0, 128.6, 125.7, 122.8, 117.6, 30.7, 8.9.

4-[3-(4-ﬂuorophenyl)-1-methyl-5-propanoyl-1H-pyrrol-2-

yl]benzenesulfonamide (42)

See in Scheme 3.

Step

1.

N,N-bis(4-methoxybenzyl)-4-[3-(4-ﬂuorophenyl)-1-

methyl-1H-pyrrol-2-yl]benzene-sulfonamide (42e1)

Step

4.

N,N-bis(4-methoxybenzyl)-4-[2-(4-ﬂuorophenyl)-4-

oxobutanoyl]benzenesulfonamide (40e5)

Prepared according to General method 7 (N-methylation) starting

from 40e6 (225 mg, 0.4 mmol). Yield: 292 mg of colourless

amorphous solid.

A solution of 40e4 (800 mg, 1.29 mmol) in acetone (50 mL) was

treated with 3 M HCl solution (10 mL) and the mixture was stirred

at RT overnight. After repeated addition of HCl solution (2 ꢂ 2 mL)

the reaction mixture was stirred at RT for another 2 days. After

neutralization with 3 M NaOH solution during cooling with ice, the

volatile organic solvent was removed under reduced pressure, the

aqueous residue was extracted with ether, dried and concentrated.

Yield: 0.79 g (quant.) of yellowish solid (used without further

puriﬁcation).

Step

2.

N,N-bis(4-methoxybenzyl)-{4-[3-(4-ﬂuorophenyl)-1-

methyl-5-propanoyl-1H-pyrrol-2-yl]}benzenesulfonamide (42e2)

Prepared according to General method 1A (Friedel-Crafts reac-

tion) starting from 42e1 (200 mg, 0.35 mmol). Yield: 125 mg of off-

white solid.

Step 3. 4-[3-(4-ﬂuorophenyl)- 1-methyl-5-propanoyl-1H-pyrrol-

2-yl]benzenesulfonamide (42)

Step 5. N,N-bis(4-methoxybenzyl)-4-[3-(4-ﬂuorophenyl)-1H-pyr-

rol-2-yl]benzenesulfonamide (40e6)

The title compound was synthesized according to General

method 6 (bis-PMB-sulfonamide deprotection) starting from 42e2

(122 mg, 0.2 mmol). Yield: 58 mg of light yellow solid. Mp

250e251 ^ꢀC HESI-HRMS: calcd for C₂₀H₂₀O₃N₂FS [MþH]^þ:

A solution of 40e5 (785 mg, 1.33 mmol) in THF (10 mL) was

divided into 5 equal portions and placed into 5 pieces of 10 mL

microwave reaction vessels, respectively. Acetic acid (2-2 mL),

NH₄OAc (1-1 g) and 4 Å molecular sieves (20-20 pieces) were added

to each portion of the reaction mixture. The vessels were closed and

consecutively irradiated in CEM Voyager microwave reactor at

170 ^ꢀC for 15 min. The reaction mixture portions were united,

diluted with EtOAc (200 mL), washed with sat. NaHCO₃solution

and brine, dried, concentrated, and the residue was puriﬁed by

column chromatography on silica (eluent: CH₂Cl₂). Yield: 403 mg

(54%) of white solid.

387.11732; found: 387.11718; delta

(400 MHz, DMSO‑d₆)

¼

ꢁ0.35 ppm. ¹H NMR

d

7.88 (d, J ¼ 7.71 Hz, 2H), 7.52 (d, J ¼ 8. 6 Hz,

2H), 7.48 (br s, 2H), 7.44 (s, 1H), 7.07 (s, 4H), 3.68 (s, 3H), 2.92 (q,

J ¼ 7.3 Hz, 2H), 1.11 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d

191.2, 160.1, 143.5, 133.4, 130.8, 130.4, 129.9, 128.9, 125.4, 117.8,

114.6, 33.9, 31.3, 8.5.

4-[5-(4-ﬂuorophenyl)-3-propanoyl-1H-pyrazol-1-yl]benze-

nesulfonamide (43)

See in Scheme 3.

Step

6

N,N-bis(4-methoxybenzyl)-{4-[3-(4-ﬂuorophenyl)-5-

propanoyl-1H-pyrrol-2-yl]}benzenesulfonamide (40e7)

Step 1. 5-(4-ﬂuorophenyl)-1-(4-sulfamoylphenyl)-1H-pyrazole-3-

carboxylic acid (43e2)

Prepared according to General method 1A (Friedel-Crafts reac-

tion) starting from 40e6 (280 mg, 0.5 mmol). Yield: 260 mg (85%)

of pinkish solid.

Prepared according to General method 3 (ester hydrolysis) start-

ing from ethyl 5-(4-ﬂuorophenyl)-1-(4-sulfamoylphenyl)-1H-pyr-

azole-3-carboxylate (43e1) [38] (418 mg, 1.1 mmol). Yield: 368 mg

(95%) yellowish crystals.

Step 7. 4-[3-(4-ﬂuorophenyl)-5-propanoyl-1H-pyrrol-2-yl]benze-

nesulfonamide (40)

Step 2. 1-(4-{[(Z)-[(dimethylamino)methylidene]amino]sulfonyl}

phenyl)-5-(4-ﬂuorophenyl)-N-methoxy-N-methyl-1H-pyrazole-3-

carboxamide (43e3)

Prepared according to General method 6 (bis-PMB-sulfonamide

deprotection) starting from 40e7 (250 mg, 0.41 mmol). Yield: 55 mg

(36%) of pinkish solid. Mp 279 ^ꢀC; HESI-HRMS: calcd for

C

₁₉H₁₈O₃N₂FS

[MþH]^þ:

373.10167;

found:

373.10203;

Prepared according to General method 4 (amidation) starting

from 43e2 (702 mg, 1.9 mmol). Yield: 518 mg (58%) white solid.

delta ¼ 0.97 ppm. ¹H NMR (500 MHz, DMSO‑d₆)

d

12.21 (br s, 1H),

7.71e7.80 (m, 2H), 7.48e7.57 (m, 2H), 7.40 (s, 2H), 7.23e7.28 (m,

2H), 7.21 (s, 1H), 7.13e7.19 (m, 2H), 2.86 (q, J ¼ 7.4 Hz, 2H), 1.12 (t,

J ¼ 7.4 Hz, 3H).

Step 3 (Z)-N’-{4-[5-(4-ﬂuorophenyl)-3-propanoyl-1H-pyrazol-1-

yl]benzenesulfonyl}-N,N-dimethylmethanimidamide (43e4)

¹³C NMR (126 MHz, DMSO‑d₆)

d 190.7, 161.0, 142.9, 134.5, 132.8,

21

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I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

Prepared according to General method 5A (Grignard reaction)

starting from 43e3 (513 mg, 1.1 mmol). Yield: 170 mg (35%).

CH₂Cl₂, 0 to 5:95). Yield: 5.51 g (66%) of yellow oil.

Step 2. 4-[(1E)-2-cyano-2-(4-ﬂuorophenyl)-1-(2-oxobutoxy)eth-

1-en-1-yl]-N,N-bis[(4-methoxyphenyl)methyl]benzenesulfona-

mide (47e3)

Step 4. 4-[5-(4-ﬂuorophenyl)-3-propanoyl-1H-pyrazol-1-yl]ben-

zene-1-sulfonamide (43)

Prepared according to General method 8 (N-(dimethylamino)

methylidene sulfonamide deprotection) starting from 43e4 (165 mg,

0.39 mmol). Yield: 104 mg (73%). Mp 199e200 ^ꢀC; HESI-HRMS:

calcd for C₁₈H₁₇O₃N₃FS [MþH]^þ: 374.09692; found: 374.09671;

To a solution of 47e2 (5.50 g, 9.85 mmol) in DMF (17 mL), was

added 1-bromo-2-butanone (0.96 mL, 9.4 mmol) followed by

triethyl amine (1.4 mL,10 mmol). The mixture was placed into an oil

bath preheated to 50 ^ꢀC and stirred for 10 min at this temperature.

After cooling to RT, water was added, the precipitated gummy

material was decantated, dissolved in CH₂Cl₂, dried, concentrated

and puriﬁed by column chromatography on silica (eluent: CH₂Cl₂).

Yield: 3.86 g (62%) of light yellow oil.

delta ¼ ꢁ0.55 ppm. ¹H NMR (400 MHz, DMSO‑d₆)

d 7.86e7.91 (m,

2H), 7.52e7.57 (m, 2H), 7.50 (s, 2H), 7.32e7.39 (m, 2H), 7.24e7.30

(m, 2H), 7.12 (s, 1H), 3.05 (q, J ¼ 7.3 Hz, 2H), 1.12 (t, J ¼ 7.3 Hz, 3H).

¹³C NMR (101 MHz, DMSO‑d₆)

d 195.3, 161.8, 150.4, 143.4, 143.2,

140.7, 130.6, 126.2, 125.2, 115.3, 107.5, 30.9, 7.4.

4-[5-(4-chlorophenyl)-3-propanoyl-1H-pyrazol-1-yl]benze-

nesulfonamide (44)

Step 3. 4-[4-amino-3-(4-ﬂuorophenyl)-5-propanoylfuran-2-yl]-

N,N-bis[(4-methoxyphenyl)-methyl]benzenesulfonamide (47e4)

Synthesized in a manner analogous to the method described for

the synthesis of compound 43 starting from ethyl 5-(4-

chlorophenyl)-1-(4-sulfamoylphenyl)-1H-pyrazole-3-carboxylate

instead of ethyl 5-(4-ﬂuorophenyl)-1-(4-sulfamoylphenyl)-1H-

pyrazole-3-carboxylate (43e1). Mp 188e189 ^ꢀC; HESI-HRMS: calcd

To a solution of 47e3 (3.86 g) in ethanol (26 mL), was added

freshly prepared 0.67 M NaOEt solution (13 mL) and the mixture

was heated under reﬂux in an oil bath preheated to 100 ^ꢀC and for

10 min. After cooling in an ice bath, the crystals separated were

ﬁltered off, washed with cold ethanol and dried. Yield: 2.49 g (65%)

of yellow crystals.

for

C

₁₈H₁₇O₃N₃ClS [MþH]^þ: 390.06737; found: 390.06717;

delta ¼ ꢁ0.50 ppm. ¹H NMR (400 MHz, DMSO‑d₆)

d 7.87e7.91 (m,

2H), 7.53e7.58 (m, 2H), 7.46e7.52 (m, 4H), 7.30e7.36 (m, 2H), 7.16

(s, 1H), 3.05 (q, J ¼ 7.3 Hz, 2H), 1.12 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR

Step 4. 4-[3-(4-ﬂuorophenyl)-5-propanoylfuran-2-yl]-N,N-bis[(4-

methoxyphenyl)methyl]-benzenesulfonamide (47e5)

(101 MHz, DMSO‑d₆)

d 195.2, 150.4, 143.2, 143.2, 140.6, 133.4, 130.0,

128.3, 127.1, 126.3, 125.3, 107.7, 30.9, 7.4.

Ethyl

5-(4-ﬂuorophenyl)-1-(4-sulfamoylphenyl)-1H-pyr-

Prepared according to General method 2 (reductive deamination)

starting from 47e4 (314 mg, 0.5 mmol). Yield: 38 mg (12%) of

yellow oil.

azole-3-carboxylate (45) [38]

Mp 216e217 ^ꢀC; HESI-HRMS: calcd for C₁₈H₁₇O₄N₃FS [MþH]^þ:

390.09183; found: 390.09218; delta

¼

0.89 ppm. ¹H NMR

7.83e7.91 (m, 2H), 7.47e7.56 (m, 4H),

(400 MHz, DMSO‑d₆)

d

Step 5. 4-[3-(4-ﬂuorophenyl)-5-propanoylfuran-2-yl]benzene-

sulfonamide (47)

7.31e7.41 (m, 2H), 7.22e7.32 (m, 2H), 7.17 (s, 1H), 4.35 (q, J ¼ 7.1 Hz,

2H), 1.32 (t, J ¼ 7.1 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 161.4,

161.1, 162.4, 144.1, 143.8, 143.7, 141.2, 131.2, 126.8, 125.9, 125.2, 115.9,

110.4, 60.7, 14.2.

Prepared according to General method 6 (bis-PMB-sulfonamide

deprotection) starting from 47e5) (190 mg, 0.31 mmol). Yield:

72 mg (63%) of yellow solid. Mp 221e225 ^ꢀC; HESI-HRMS: calcd for

Ethyl

5-(4-chlorophenyl)-1-(4-sulfamoylphenyl)-1H-pyr-

azole-3-carboxylate (46) [39]

C

₁₉H₁₇O₄NFS

[MþH]^þ:

374.08568;

found:

374.08542;

7.82e7.87 (m,

Mp 209e210 ^ꢀC; HESI-HRMS: calcd for C₁₈H₁₇O₄N₃ClS [MþH]^þ:

delta ¼ ꢁ0.72 ppm. ¹H NMR (400 MHz, DMSO‑d₆)

d

406.06228; found: 406.06286; delta

¼

1.42 ppm. ¹H NMR

d 7.81e7.94 (m, 2H), 7.51e7.56 (m, 4H),

2H), 7.74 (s, 1H), 7.66e7.72 (m, 2H), 7.45 (s, 4H), 7.27e7.35 (m, 2H),

(400 MHz, DMSO‑d₆)

2.95 (d, J ¼ 7.3 Hz, 2H), 1.12 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR (101 MHz,

7.46e7.51 (m, 2H), 7.29e7.35 (m, 2H), 7.20 (s, 1H), 4.35 (q, J ¼ 7.1 Hz,

DMSO‑d₆) d 188.7, 161.4, 143.6, 131.5, 130.1, 127.6, 126.5, 125.7, 120.5,

2H), 1.32 (t, J ¼ 7.1 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d

161.3,

115.5, 30.5, 7.5.

144.2, 143.9, 143.5, 141.2, 134.0, 130.6, 128.9, 127.5, 126.8, 125.9,

4-[4-Amino-3-(4-ﬂuorophenyl)-5-propanoylfuran-2-yl]ben-

zenesulfonamide (48)

110.5, 60.7, 14.2.

4-[3-(4-ﬂuorophenyl)-5-propanoylfuran-2-yl]benzenesulfo-

See in Scheme 4.

namide (47)

Prepared according to General method 6 (bis-PMB-sulfonamide

deprotection) starting from 4-[(1E)-2-cyano-2-(4-ﬂuorophenyl)-1-

(2-oxobutoxy)eth-1-en-1-yl]-N,N-bis[(4-methoxyphenyl)methyl]ben-

zenesulfonamide (47e4) (95 mg, 0.15 mmol). Yield: 40 mg (69%)

yellow solid. Mp 251e254 ^ꢀC; HESI-HRMS: calcd for C₁₉H₁₈O₄N₂FS

[MþH]^þ: 389.09658; found: 389.09640; delta ¼ ꢁ0.48 ppm. ¹H

See in Scheme 4.

Step

1.

4-[2-cyano-2-(4-ﬂuorophenyl)acetyl]-N,N-bis[(4-

methoxyphenyl)methyl]benzene-1-sulfonamide (47e2)

A

suspension of (4-ﬂuorophenyl)acetonitrile (1.8 mL,

NMR (400 MHz, DMSO‑d₆)

d

7.76e7.80 (m, J ¼ 8.7 Hz, 2H),

14.95 mmol) in THF (80 mL) was treated with NaH (1.43 g, 2.4 eq,

60% in mineral oil) at RT. After 5 min of stirring, a solution of methyl

4-[N,N-bis(4-methoxybenzyl)sulfamoyl]benzoate [36] (47e1)

(6.81 g, 1 eq) in THF (120 mL) was added and the mixture was

heated under reﬂux for 3 h. The reaction was quenched by drop-

wise addition of water at 0 ^ꢀC followed by acidiﬁcation with 1 M

HCl solution. The volatile organic solvent was removed under

reduced pressure and the aqueous residue was extracted with

EtOAc. The combined organic phase was washed with water and

brine, dried over Na₂SO₄, ﬁltered and concentrated. The residue

was puriﬁed by column chromatography on silica (eluent: EtOAc-

7.52e7.61 (m, J ¼ 8.7 Hz, 2H), 7.32e7.45 (m, 6H), 5.94 (s, 2H), 2.80

(q, J ¼ 7.5 Hz, 2H), 1.13 (t, J ¼ 7.5 Hz, 3H). ¹³C NMR (101 MHz,

DMSO‑d₆) d 188.2, 161.5, 148.1, 143.7, 143.5, 133.3, 131.6, 131.4, 126.1,

125.5, 125.4, 115.8, 115.4, 29.6, 7.7.

4-(4-ﬂuorophenyl)-5-(4-sulfamoylphenyl)-1,3-oxazole-2-

carboxamide (49)

See in Scheme 4.

Step

1.

ethyl

4-(4-ﬂuorophenyl)-5-phenyl-1,3-oxazole-2-

carboxylate (49e2)

22

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I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

Step 4. 4-(4-{[(Z)-[(dimethylamino)methylidene]amino]sulfonyl}

phenyl)-5-(4-ﬂuorophenyl)-N-methoxy-N-methyl-1,3-oxazole-2-

carboxamide (50e3)

A

mixture

of

1-(4-ﬂuorophenyl)-N-hydroxy-2-

phenylethanimine (49e1) [40] (5.53 g, 24.1 mmol) and ethyl

chlorooxoacetate (8.1 mL, 3 eq.) was stirred at RT for 15 min then at

120 ^ꢀC for 2 h. After cooling to 0 ^ꢀC, the residue was dissolved in

CH₂Cl₂, washed with saturated NaHCO₃solution, brine, dried and

concentrated. The residue was puriﬁed by column chromatography

on silica (eluent: EtOAc-cyclohexane, 5:95). Yield: 3.55 g (47%) of

white solid.

Prepared according to General method 4 (amidation) starting

from 50e2 (360 mg, 0.92 mmol). Yield: 270 mg (64%) of colourless

oil.

Step 5. N-[(dimethylamino)methylidene]-4-[5-(4-ﬂuorophenyl)-2-

propanoyl-1,3-oxazol-4-yl]benzenesulfonamide (50e4)

Step 2. 4-(4-ﬂuorophenyl)-5-(4-sulfamoylphenyl)-1,3-oxazole-2-

carboxamide (49)

Prepared according to General method 5A (Grignard reaction)

starting from 50e3 (270 mg, 0.59 mmol). Yield: 70 mg (28%) of

white solid.

To chlorosulfonic acid (60 mL) cooled to 0 ^ꢀC was added solid

compound 49e2 (2.58 g, 8.29 mmol) portionwise with stirring. The

mixture was stirred at the same temperature for another 30 min

then at RT for 3 h. The reaction mixture was dropwise added to

crushed ice (Caution! Violent reaction!) The precipitated product

was ﬁltered off with suction, washed with water, dissolved in

dioxane (200 mL) and the solution obtained was dropwise added to

NH₄OH solution (25%) cooled to 0 ^ꢀC. After 64 h of stirring at RT, the

mixture was concentrated under reduced pressure, the residue was

suspended in water, acidiﬁed to pH ¼ 3 by addition of 10% HCl

solution. The precipitate was ﬁltered off, washed with water, dried

and puriﬁed by column chromatography on silica (eluent: EtOAc-

CH₂Cl₂, 1:9 to 1:1). Yield: 965 mg (32%) of white solid. Mp

225e227 ^ꢀC; HESI-HRMS: calcd for C₁₆H₁₃O₄N₃FS [MþH]^þ:

362.06053; found: 362.06035; delta ¼ ꢁ0.51 ppm. ¹H NMR

Step 6. 4-[5-(4-ﬂuorophenyl)-2-propanoyl-1,3-oxazol-4-yl]benze-

nesulfonamide (50)

Prepared according to General method 8 (N-(dimethylamino)

methylidene sulfonamide deprotection) starting from 50e4 (66 mg,

0.15 mmol). Yield: 26 mg (46%) of white solid. Mp 204e209 ^ꢀC;

HESI-HRMS: calcd for C₁₈H₁₆O₄N₂FS [MþH]^þ: 375.08093; found:

375.08046; delta ¼ ꢁ1.26 ppm. ¹H NMR (500 MHz, DMSO‑d₆)

d

7.86e7.93 (m, 2H), 7.76e7.83 (m, 2H), 7.65e7.73 (m, 2H),

7.43e7.53 (m, 2H), 7.35e7.43 (m, 2H), 3.37e3.41 (m, 4H), 3.14 (q,

J ¼ 7.2 Hz, 2H), 1.14 (t, J ¼ 7.2 Hz, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

d

188.4, 163.1, 147.8, 146.5, 144.1, 134.1, 130.0, 128.0, 126.3, 123.5,

116.6, 32.0, 7.5.

(400 MHz, DMSO‑d₆)

7.88e7.92 (m, 1H), 7.77e7.81 (m, 2H), 7.60e7.70 (m, 2H), 7.48 (br s,

2H), 7.25e7.39 (m, 2H). ¹³C NMR (101 MHz, DMSO‑d₆)

161.6, 155.4,

d 8.40e8.40 (m, 1H), 8.43 (s, 1H), 8.04 (s, 1H),

Ethyl 5-(4-ﬂuorophenyl)-4-(4-sulfamoylphenyl)-1,3-oxazole-

2-carboxylate (51)

d

Intermediate of the synthesis of compound 50. Mp 196e197 ^ꢀC;

HESI-HRMS: calcd for C₁₈H₁₆O₅N₂FS [MþH]^þ: 391.07585; found:

391.07574; delta ¼ ꢁ0.26 ppm. ¹H NMR (400 MHz, DMSO‑d₆)

153.4, 144.9, 144.0, 138.4, 135.6, 129.8, 129.6, 126.5, 125.8, 115.4.

4-[5-(4-ﬂuorophenyl)-2-propanoyl-1,3-oxazol-4-yl]benzene-

sulfonamide (50)

d

7.84e7.94 (m, 2H), 7.75e7.82 (m, 2H), 7.64e7.72 (m, 2H), 7.46 (s,

2H), 7.30e7.44 (m, 2H), 4.43 (q, J ¼ 7.09 Hz, 2H), 1.36 (t, J ¼ 7.09 Hz,

Step 1. 4-[2-(4-ﬂuorophenyl)-1-(hydroxyimino)ethyl]-N,N-bis[(4-

methoxyphenyl)methyl]-benzenesulfonamide (50e1)

3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d

162.4, 154.4, 150.2,147.5, 143.5,

134.5, 133.3, 129.5, 127.3, 125.7, 122.8, 116.0, 61.8, 13.4.

Compounds 52e68 were synthesized in a manner analogous to

the method described for the synthesis of compound 39. See it in

Scheme 2.

See in Scheme 4.

A solution of 40e3 (3.73 g, 7 mmol), hydroxylamine hydro-

chloride (7.3 g, 1.5 eq) and NaOAc (8.6 g, 1.5 eq) in ethanol (50 mL)

was heated under reﬂux for 2 h. After cooling to room temperature,

the mixture was concentrated under reduced pressure. The residue

was triturated with ice water, allowed to stand in a refrigerator, the

precipitated solid was ﬁltered off, washed with water and dried.

Yield: 3.605 g (94%) white solid.

4-[3-Butanoyl-1-(4-chlorophenyl)-1H-pyrazol-5-yl]benzene-

sulfonamide (52)

Mp 190e191 ^ꢀC; HESI-HRMS: calcd for C₁₉H₁₉O₃N₃ClS [MþH]^þ:

404.08302; found: 404.08305; delta

¼

0.08 ppm. ¹H NMR

(400 MHz, DMSO‑d₆)

d 7.76e7.84 (m, 2H), 7.53e7.61 (m, 2H),

7.45e7.52 (m, 2H), 7.37e7.45 (m, 4H), 7.21 (s, 1H), 3.01 (t, J ¼ 7.2 Hz,

2H), 1.69 (sxt, J ¼ 7.3 Hz, 2H), 0.94 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR

Step 2. Ethyl 5-(4-ﬂuorophenyl)-4-(4-sulfamoylphenyl)-1,3-

oxazole-2-carboxylate (51)

(101 MHz, DMSO‑d₆)

d 194.7, 150.5, 143.6, 143.0, 137.2, 132.8, 131.3,

128.9, 128.6, 126.9, 125.4, 107.8, 39.5, 16.5, 13.1.

4-[3-Acetyl-1-(4-chlorophenyl)-1H-pyrazol-5-yl]benzene-

sulfonamide (53)

A mixture of 50e1 (3.60 g, 6.6 mmol) and ethyl chlorooxoacetate

(2.2 mL, 19.7 mmol) was stirred at RT for 15 min, then at 120 ^ꢀC for

2 h. After cooling in an ice bath, the waxy residue was dissolved in

CH₂Cl₂, washed with brine, dried and concentrated. The residue

was puriﬁed by column chromatography on silica (eluent: EtOAc-

CH₂Cl₂, 1:9). Yield: 515 mg (20%) of beige solid.

Mp 183e184 ^ꢀC; HESI-HRMS: calcd for C₁₇H₁₅O₃N₃ClS [MþH]^þ:

376.05172; found: 376.05207; delta ¼ 0.92 ppm. ¹H NMR (400 MHz,

DMSO‑d₆)

d 7.68e7.89 (m, 2H), 7.52e7.64 (m, 2H), 7.38e7.51 (m,

6H), 7.21 (s, 1H), 2.57 (s, 3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 192.4,

150.7, 143.6, 143.1, 137.1, 132.8, 131.3, 128.9, 128.6, 126.9, 125.4, 107.7,

25.8.

Step 3. 5-(4-ﬂuorophenyl)-4-(4-sulfamoylphenyl)-1,3-oxazole-2-

carboxylic acid (50e2)

4-[1-(4-chlorophenyl)-3-(2,2-dimethylpropanoyl)-1H-pyr-

azol-5-yl]benzenesulfonamide (54)

Mp 178 ^ꢀC; HESI-HRMS: calcd for C₂₀H₂₁O₃N₃ClS [MþH]^þ:

Prepared according to General method 3 (ester hydrolysis) start-

ing from ethyl 5-(4-ﬂuorophenyl)-4-(4-sulfamoylphenyl)-1,3-

oxazole (51) (460 mg, 1.18 mmol). Yield: 360 mg (84%) of off-

white solid.

418.09867; found: 418.09880; delta ¼ 0.33 ppm. ¹H NMR (400 MHz,

DMSO‑d₆)

d 10.10e10.12 (m, 1H), 7.75e7.87 (m, 2H), 7.53e7.64 (m,

2H), 7.36e7.52 (m, 6H), 7.17 (s, 1H), 1.40 (s, 9H). ¹³C NMR (101 MHz,

DMSO‑d₆) 199.1, 149.1, 143.5, 141.9, 137.2, 132.6, 131.4, 128.9, 128.7,

126.6, 125.3, 110.0, 43.0, 26.5.

d

23

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European Journal of Medicinal Chemistry 214 (2021) 113189

4-[1-(4-chlorophenyl)-3-(2-methylpropanoyl)-1H-pyrazol-5-

yl]benzenesulfonamide (55)

7.30e7.39 (m, 2H), 7.21 (s, 1H), 3.04 (q, J ¼ 7.3 Hz, 2H), 1.12 (t,

J ¼ 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO‑d₆)

d 195.9, 161.7, 150.6,

ESI-LRMS: [MþH]^þ: 404. ¹H NMR (400 MHz, DMSO‑d₆)

144.1, 143.6, 132.0, 129.2, 128.1, 125.9, 116.4, 108.0, 31.5, 8.0.

4-[1-(3-chlorophenyl)-3-propanoyl-1H-pyrazol-5-yl]benze-

nesulfonamide (63)

d

7.75e7.87 (m, 2H), 7.52e7.63 (m, 2H), 7.35e7.52 (m, 6H), 7.22 (s,

1H), 3.62e3.74 (m, 1H), 1.17 (d, 6H).

4-[1-(4-chlorophenyl)-3-(cyclopropanecarbonyl)-1H-pyr-

azol-5-yl]benzenesulfonamide (56)

Mp 182 ^ꢀC; HESI-HRMS: calcd for C₁₈H₁₇O₃N₃ClS [MþH]^þ:

390.06737; found: 390.06788; delta

¼

1.32 ppm. ¹H NMR

Mp 215 ^ꢀC; HESI-HRMS: calcd for C₁₉H₁₇O₃N₃ClS [MþH]^þ:

(400 MHz, DMSO‑d₆)

d

7.80 (d, J ¼ 7.9 Hz, 1H), 7.42e7.61 (m, 3H),

402.06737; found: 402.06754; delta

¼

0.43 ppm. ¹H NMR

7.27 (ddd, J ¼ 1.0, 2.0, 7.9 Hz,1H), 7.22 (s, 1H), 3.06 (q, J ¼ 7.3 Hz,1H),

(400 MHz, DMSO‑d₆)

d

7.77e7.84 (m, 2H), 7.54e7.61 (m, 2H),

1.12 (t, J ¼ 7.3 Hz, 1H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 195.3, 150.3,

7.47e7.51 (m, 2H), 7.43e7.47 (m, 4H), 7.21 (s, 1H), 3.02e3.08 (m,

143.6, 143.0, 139.5, 133.0, 131.3, 130.4, 128.7, 128.3, 125.3, 125.0,

123.9, 107.8, 30.9, 7.3.

1H), 1.07 (d, J ¼ 6.2 Hz, 4H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 194.2,

150.8, 143.6, 143.1, 137.2, 132.8, 131.3, 128.9, 128.7, 126.9, 125.4,

107.5, 16.4, 10.6.

4-[1-(2-chlorophenyl)-3-propanoyl-1H-pyrazol-5-yl]benze-

nesulfonamide (64)

4-[1-(4-chlorophenyl)-3-(cyclobutanecarbonyl)-1H-pyrazol-

5-yl]benzenesulfonamide (57)

Mp 214 ^ꢀC; HESI-HRMS: calcd for C₁₈H₁₇O₃N₃ClS [MþH]^þ:

390.06737; found: 390.06772; delta

(400 MHz, DMSO‑d₆) d 7.72e7.80 (m, 3H), 7.55e7.67 (m, 3H),

¼

0.91 ppm. ¹H NMR

Mp 221e222 ^ꢀC; HESI-HRMS: calcd for C₂₀H₁₉O₃N₃ClS [MþH]^þ:

416.08302; found: 416.08298; delta ¼ ꢁ0.08 ppm. ¹H NMR

7.37e7.47 (m, 4H), 7.28 (s, 1H), 3.02 (q, J ¼ 7.3 Hz, 2H), 1.11 (t,

(400 MHz, DMSO‑d₆)

d

7.80 (d, J ¼ 8.0 Hz, 2H), 7.56 (d, J ¼ 7.9 Hz,

J ¼ 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 195.3, 150.5, 144.4,

143.6,136.0,131.3,131.1,130.0,129.8,129.7,128.0,127.7,125.3,106.3,

30.9, 7.4.

2H), 7.37e7.50 (m, 5H), 7.21 (s, 1H), 4.17 (quin, J ¼ 8.3 Hz, 1H),

2.18e2.34 (m, 4H), 1.98e2.10 (m, 1H), 1.70e1.87 (m, 1H). ¹³C NMR

(101 MHz, DMSO‑d₆)

128.8, 128.7, 126.8, 125.3, 108.2, 40.9, 23.8, 17.1.

d

195.1, 149.2, 143.6, 142.9, 137.1, 132.7, 131.3,

4-(1-Phenyl-3-propanoyl-1H-pyrazol-5-yl)benzenesulfona-

mide (65)

4-[1-(4-chlorophenyl)-3-(cyclopentanecarbonyl)-1H-pyr-

azol-5-yl]benzenesulfonamide (58)

Mp 185e186 ^ꢀC; HESI-HRMS: calcd for C₁₈H₁₈O₃N₃S [MþH]^þ:

356.10634; found: 356.10627; delta ¼ 0.20 ppm. ¹H NMR (400 MHz,

Mp 197e200 ^ꢀC; HESI-HRMS: calcd for C₂₁H₂₁O₃N₃ClS [MþH]^þ:

DMSO‑d₆) d 7.74e7.80 (m, 2H), 7.36e7.53 (m, 9H), 7.21 (s, 1H), 3.05

430.09867; found: 430.09893; delta

¼

0.60 ppm. ¹H NMR

(q, J ¼ 7.3 Hz, 2H), 1.12 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR (101 MHz,

(400 MHz, DMSO‑d₆)

d

7.75e7.84 (m, 2H), 7.53e7.62 (m, 2H),

DMSO‑d₆) d 195.3, 150.0, 143.4, 142.8, 138.4, 131.6, 128.8, 128.5,

7.46e7.51 (m, 2H), 7.38e7.46 (m, 4H), 7.22 (s, 1H), 3.88 (quin,

J ¼ 8.0 Hz, 1H), 1.86e2.01 (m, 2H), 1.73e1.85 (m, 2H), 1.52e1.71 (m,

128.3, 125.3, 125.2, 107.5, 30.9, 7.4.

4-[1-(4-methoxyphenyl)-3-propanoyl-1H-pyrazol-5-yl]ben-

zenesulfonamide (66)

4H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 197.6, 150.8, 144.2, 143.5, 137.8,

133.3, 132.0, 129.5, 129.3, 127.4, 126.0, 108.8, 46.4, 29.4, 25.8.

4-[1-(4-chlorophenyl)-3-(cyclobutylacetyl)-1H-pyrazol-5-yl]

benzenesulfonamide (59)

Mp 198e199 ^ꢀC; HESI-HRMS: calcd for C₁₉H₂₀O₄N₃S [MþH]^þ:

386.11690; found: 386.11636; delta

¼

ꢁ1.41 ppm. ¹H NMR

(400 MHz, DMSO‑d₆)

d 7.74e7.81 (m, 2H), 7.36e7.51 (m, 4H),

Mp 171e173 ^ꢀC; HESI-HRMS: calcd for C₂₁H₂₁O₃N₃ClS [MþH]^þ:

7.28e7.34 (m, 2H), 7.18 (s, 1H), 7.00e7.06 (m, 2H), 3.80 (s, 3H), 3.03

430.09867; found: 430.09929; delta

¼

1.44 ppm. ¹H NMR

(q, J ¼ 7.30 Hz, 2H), 1.12 (t, J ¼ 7.34 Hz, 3H). ¹³C NMR (101 MHz,

(400 MHz, DMSO‑d₆)

d

7.74e7.86 (m, 2H), 7.53e7.61 (m, 2H),

DMSO‑d₆) d 195.3, 158.7, 149.7, 143.3, 142.8, 131.7, 131.4, 128.5,126.6,

7.38e7.51 (m, 6H), 7.19 (s, 1H), 3.14 (d, J ¼ 7.3 Hz, 2H), 2.79 (spt,

125.2, 113.9, 107.1, 54.9, 30.8, 7.4.

J ¼ 7.7 Hz, 1H), 1.98e2.20 (m, 2H), 1.58e1.96 (m, 4H). ¹³C NMR

4-{3-Propanoyl-1-[6-(triﬂuoromethyl)pyridin-3-yl]-1H-pyr-

azol-5-yl}benzenesulfonamide (67)

(101 MHz, DMSO‑d₆)

d 194.6, 151.1, 144.2, 143.6, 137.8, 133.4, 131.9,

129.5, 129.3, 127.5, 126.0, 108.3, 45.2, 31.4, 27.9, 18.4.

4-[1-(4-chlorophenyl)-3-(2-cyclopentylacetyl)-1H-pyrazol-5-

yl]benzenesulfonamide (60)

Mp 163 ^ꢀC; HESI-HRMS: calcd for C₁₈H₁₇O₃N₄F₂S [MþH]^þ:

407.09839; found: 407.09815; delta ¼ ꢁ0.61 ppm. ¹H NMR

(400 MHz, DMSO‑d₆)

d

8.81 (d, J ¼ 2.2 Hz, 1H), 8.11e8.15 (m, 1H),

Mp 113e115 ^ꢀC; HESI-HRMS: calcd for C₂₂H₂₃O₃N₃ClS [MþH]^þ:

8.04e8.10 (m, 1H), 7.81e7.86 (m, 2H), 7.53e7.60 (m, J ¼ 8.6 Hz, 2H),

7.47 (br s, 2H), 7.30 (s, 1H), 3.09 (q, J ¼ 7.3 Hz, 2H),1.77e1.78 (m,1H),

444.11432; found: 444.11447; delta ¼ 0.34 ppm. ¹H NMR (400 MHz,

DMSO‑d₆)

d

7.80 (d, J ¼ 7.90 Hz, 1H), 7.57 (d, J ¼ 7.91 Hz, 1H),

1.14 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 195.1, 151.2,

7.40e7.49 (m, 3H), 7.21 (s, 1H), 3.04 (d, J ¼ 7.21 Hz, 1H), 2.35 (td,

145.8, 143.9, 143.6, 144.9, 137.4, 134.2, 130.8, 129.0, 125.5, 121.0,

120.7, 108.6, 31.0, 7.3.

J ¼ 7.89, 15.28 Hz, 1H), 1.73e1.85 (m, 1H), 1.40e1.66 (m, 2H),

1.09e1.35 (m, 2H). ¹³C NMR (101 MHz, DMSO‑d₆)

d

194.6, 150.6,

4-[1-(5-chloropyridin-2-yl)-3-propanoyl-1H-pyrazol-5-yl]

benzenesulfonamide (68)

143.6, 143.0, 137.2, 132.8, 131.3, 128.9, 128.7, 126.9, 125.4, 107.8, 43.6,

34.9, 31.5, 23.9.

HESI-HRMS: calcd for

C

₁₇H₁₆O₃N₄ClS [MþH]^þ: 391.06262;

4-{3-Propanoyl-1-[4-(triﬂuoromethyl)phenyl]-1H-pyrazol-

5-yl}benzenesulfonamide (61)

found: 391.06325; delta ¼ 1.63 ppm. ¹H NMR (500 MHz, DMSO‑d₆)

d

8.44 (dd, J ¼ 0.5, 2.6 Hz, 1H), 8.25 (dd, J ¼ 2.6, 8.6 Hz, 1H), 7.90 (dd,

Mp 209e211 ^ꢀC; HESI-HRMS: calcd for C₁₉H₁₇O₃N₃F₃S [MþH]^þ:

J ¼ 0.5, 8.6 Hz, 1H), 7.74e7.82 (m, 2H), 7.46e7.54 (m, 2H), 7.44 (s,

424.09372; found: 424.09336; delta ¼ ꢁ0.86 ppm. ¹H NMR

2H), 7.22 (s, 1H), 3.08 (q, J ¼ 7.3 Hz, 2H), 1.13 (t, J ¼ 7.3 Hz, 3H). ¹³

C

(400 MHz, DMSO‑d₆)

d

7.86e7.91 (m, 2H), 7.79e7.84 (m, 2H),

NMR (126 MHz, DMSO‑d₆) d 195.8, 151.1, 149.9, 146.8, 143.9, 143.8,

7.58e7.65 (m, 2H), 7.48e7.53 (m, 2H), 7.45 (s, 2H), 7.25 (s, 1H), 3.07

139.5, 132.6, 131.0, 129.0, 125.7, 120.7, 109.1, 31.5, 7.9.

4-(3-{3-azabicyclo[3.1.0]hexane-3-carbonyl}-1-(4-chlor-

ophenyl)-1H-pyrazol-5-yl)-benzene-1-sulfonamide (69)

See in Scheme 2.

(q, J ¼ 7.3 Hz, 2H), 1.13 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR (101 MHz,

DMSO‑d₆) d 195.2, 150.6, 143.7, 143.1, 141.4, 131.3, 128.7, 128.2, 126.1,

125.6, 125.4, 123.2, 108.3, 30.9, 7.3.

4-[1-(4-ﬂuorophenyl)-3-propanoyl-1H-pyrazol-5-yl]benze-

nesulfonamide (62)

Step

1

N’-[4-(3-{3-azabicyclo[3.1.0]hexane-3-carbonyl}-1-(4-

Mp 191e193 ^ꢀC; HESI-HRMS: calcd for C₁₈H₁₇O₃N₃FS [MþH]^þ:

chlorophenyl)-1H-pyrazol-5-yl)-benzenesulfonyl]-N,N-dime-

thylmethanimidamide (69e1)

374.09692; found: 374.09714; delta ¼ 0.6 ppm. ¹H NMR (500 MHz,

DMSO‑d₆)

d 7.74e7.84 (m, 2H), 7.44e7.48 (m, 4H), 7.43 (br s, 2H),

24

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I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

Under an inert atmosphere, the carboxylic acid 39e6

(0.915 mmol) was suspended in CH₂Cl₂(12 mL), DMF (0.135 mL, 1.9

eq) was added followed by a dropwise addition of a solution of

oxalyl chloride (0.23 mL, 3 eq) in CH₂Cl₂(1 mL) while cooling with

ice. The mixture was stirred at RT for 1.5 h, the solvent was evap-

orated in vacuo and the residue was dissolved in CH₂Cl₂(12 mL).

Next, 3-azabicyclo[3.1.0]hexane hydrochloride (219 mg, 2 eq) was

added, followed by a dropwise addition of triethylamine (0.51 mL, 4

eq) during cooling in an ice-water bath and the mixture was stirred

overnight at RT. The reaction mixture was diluted with CH₂Cl₂

(20 mL), extracted with 5% HCl, saturated NaHCO₃solution and

water, dried over Na₂SO₄and concentrated. The residue was puri-

ﬁed with column chromatography on silica, eluent: EtOAc-

cycloxexane (1:1). Yield: 50 mg of light yellow amorphous solid.

MS: [M þ H^þ] ¼ 498.1.

carbonyl)-1H-pyrazol-5-yl]benzenesulfonamide (72)

Mp 138 ^ꢀC; HESI-HRMS: calcd for C₂₀H₁₈O₃N₄ClF₂S [MþH]^þ:

467.07507; found: 467.07514; delta ¼ 0.14 ppm. ¹H NMR (400 MHz,

DMSO‑d₆)

d

7.81 (d, J ¼ 7.3 Hz, 2H), 7.39e7.58 (m, 8H), 7.18 (d,

J ¼ 2.1 Hz,1H), 4.39 (t, J ¼ 12.9 Hz,1H), 4.20 (t, J ¼ 7.4 Hz,1H), 3.95 (t,

J ¼ 13.3 Hz, 1H), 3.77 (t, J ¼ 7.5 Hz, 1H), 2.40e2.62 (m, 2H). ¹³C NMR

(101 MHz, DMSO‑d₆)

d 161.1/160.8,* 148.0/147.8*, 144.6, 142.9/

142.8*, 138.3, 133.6, 132.5, 129.9, 129.7, 128.6, 127.7/127.7,* 111.3/

111.2, 54.9/53.1*, 46.8/44.4*, 34.3/31.8*.

*due to amide rotamers.

4-[1-(4-chlorophenyl)-3-(piperidine-1-carbonyl)-1H-pyr-

azol-5-yl]benzenesulfonamide (73)

Mp 215e216 ^ꢀC; HESI-HRMS: calcd for C₂₁H₂₂O₃N₄ClS [MþH]^þ:

445.10957; found: 445.10950; delta

¼

ꢁ0.16 ppm. ¹H NMR

(400 MHz, DMSO‑d₆)

d 7.78e7.82 (m, 2H), 7.35e7.56 (m, 8H), 7.01

(s, 1H), 3.75e3.89 (m, 2H), 3.45e3.66 (m, 3H), 1.58e1.68 (m, 2H),

Step 2. 4-(3-{3-azabicyclo[3.1.0]hexane-3-carbonyl}-1-(4-chlor-

1.50e1.58 (m, 4H). ¹³C NMR (101 MHz, DMSO‑d₆)

d

161.8, 148.4,

ophenyl)-1H-pyrazol-5-yl)-benzenesulfonamide (69)

144.5, 142.6, 138.3, 133.4, 132.7, 129.9, 129.6, 127.7, 126.5, 110.7, 47.9,

43.2, 26.9, 25.9, 24.6.

Compound 69e1 (50 mg, 0.1 mmol) was dissolved in THF (2 mL),

a solution of KOH (30 mg, ~5 eq, 90%) in MeOH (2 mL) was added

and the mixture was stirred overnight at RT. After cooling with ice,

water (7 mL) was added, acidiﬁed with 10% HCl (1 mL) and the

organic solvents were evaporated. The aqueous residue was

extracted with CH₂Cl₂, organic phase washed with water, dried on

Na₂SO₄, concentrated and puriﬁed with column chromatography

on silica, eluent: EtOAc-hexane. Yield: 25 mg. HPLC purity: 99.1%.

Mp 210e216 ^ꢀC; HRMS: M þ H ¼ 443.09270 (delta ¼ ꢁ2.7 ppm;

4-[1-(4-chlorophenyl)-3-(morpholine-4-carbonyl)-1H-pyr-

azol-5-yl]benzenesulfonamide (74)

Mp 223 ^ꢀC; HESI-HRMS: calcd for C₂₀H₂₀O₄N₄ClS [MþH]^þ:

447.08883; found: 447.08876; delta

¼

ꢁ0.16 ppm. ¹H NMR

(400 MHz, DMSO‑d₆)

d

7.80 (d, J ¼ 7.9 Hz, 2H), 7.37e7.57 (m, 8H),

7.07 (s, 1H), 3.98 (br s, 2H), 3.57e3.73 (m, 6H). ¹³C NMR (101 MHz,

DMSO‑d₆) 161.8,147.8,144.5,142.7,138.3,133.5,132.5,129.9,129.7,

d

127.8, 126.5, 111.2, 67.0, 66.6, 47.7, 42.8.

1-(4-chlorophenyl)-N-(3,3-diﬂuorocyclobutyl)-N-methyl-5-

(4-sulfamoylphenyl)-1H-pyrazole-3-carboxamide (75)

Mp 114e116 ^ꢀC; HESI-HRMS: calcd for C₂₁H₂₀O₃N₄ClF₂S

[MþH]^þ: 481.09072; found: 481.09059; delta ¼ ꢁ0.27 ppm. ¹H

C

₂₁H₂₀O₃N₄ClS). HR-ESI-MS-MS (CID ¼ 35%; rel. int. %): 426(3);

425(6); 389(3); 378(100); 360(45); 325(1).

¹H NMR (500 MHz, DMSO‑d₆)

¼ 7.78e7.82 (2H, m, H-9, 10),

d

7.53e7.57 (2H, m, H-15,16), 7.45e7.49 (2H, m, H-8, 7), 7.44 (2H, br s,

H-24), 7.37e7.41 (2H, m, H-14, 13), 7.08 (1H, s, H-5), 4.25 (1H, d,

J ¼ 11.5 Hz, H-27*), 3.89 (1H, d, J ¼ 12.0 Hz, H-26*), 3.84 (1H, dd,

J ¼ 11.5 Hz, J ¼ 4.3 Hz, H-27*), 3.48 (1H, dd, J ¼ 12.1 Hz, J ¼ 4.4 Hz, H-

26*),1.66 (1H, tt, J ¼ 7.5 Hz, J ¼ 3.9 Hz, H-29^#),1.58 (1H, tt, J ¼ 7.5 Hz,

J ¼ 3.9 Hz, H-28^#), 0.71 (1H, td, J ¼ 7.6 Hz, J ¼ 5.0 Hz, H-30), 0.09

NMR (400 MHz, DMSO‑d₆) d 7.78e7.83 (m, 2H), 7.52e7.60 (m, 2H),

7.46e7.51 (m, 2H), 7.44 (br s, 2H), 7.35e7.41 (m, 2H), 7.06 (s, 1H),

4.90e5.16 (m, 0.5H)/4.62e4.87 (m, 0.5H)*, 3.26 (br s, 1.5H)/3.02 (br

s, 1.5H)*, 2.82e3.09 (m, 4H). ¹³C NMR (partial) (101 MHz, DMSO‑d₆)

d

162.8, 147.0, 143.5, 141.6, 137.2, 132.4, 131.5, 128.8, 128.6, 126.6,

125.4, 109.8. *due to amide rotamers.

(1H, q, J ¼ 4.2 Hz, H-30); ¹³C NMR (125.7 MHz, DMSO‑d₆)

d

¼ 161.2

1-(4-chlorophenyl)-N-cyclopropyl-N-methyl-5-(4-

sulfamoylphenyl)-1H-pyrazole-3-carboxamide (76)

Mp 200e201 ^ꢀC; HESI-HRMS: calcd for C₂₀H₂₀O₃N₄ClS [MþH]^þ:

431.09392; found: 431.09367; delta ¼ ꢁ0.56 ppm. ¹H NMR

(C-18), 148.4 (C-4), 144.0 (C-11), 142.1 (C-3), 137.9 (C-12), 133.0 (C-

17),132.1 (C-6),129.4 (C-15,16),129.2 (C-8, 7),127.2 (C-14,13),126.0

(C-9, 10), 110.6 (C-5), 50.3 (C-27*), 48.6 (C-26*), 16.2 (C-29^#), 13.5

(C-28^#), 9.1 (C-30).

(400 MHz, DMSO‑d₆)

d 7.74e7.85 (m, 2H), 7.51e7.57 (m, 2H),

*

^{, #}: interchangeable assignments.

7.45e7.50 (m, 2H), 7.43 (s, 2H), 7.32e7.40 (m, 2H), 3.11 (br s, 1H),

Compounds 70e79 were synthesized in a manner analogous to

the method described for the synthesis of compound 69. See in

Scheme 2.

3.01 (br s, 3H), 0.65e0.80 (m, 2H), 0.59 (br s, 2H). ¹³C NMR

(101 MHz, DMSO‑d₆)

d 164.0, 147.9, 143.4, 141.3, 137.4, 132.2, 131.7,

128.8, 128.5, 126.6, 125.4, 109.2, 109.1, 33.5, 31.9, 8.5.

1-(4-chlorophenyl)-N-cyclopropyl-5-(4-sulfamoylphenyl)-

1H-pyrazole-3-carboxamide (77)

4-[1-(4-chlorophenyl)-3-(pyrrolidine-1-carbonyl)-1H-pyr-

azol-5-yl]benzenesulfonamide (70)

Mp 247 ^ꢀC; HESI-HRMS: calcd for C₂₀H₂₀O₃N₄ClS [MþH]^þ:

Mp 221e224 ^ꢀC; HESI-HRMS: calcd for C₁₉H₁₈O₃N₄ClS [MþH]^þ:

431.09392; found: 431.09357; delta ¼ ꢁ0.80 ppm. ¹H NMR

417.07827; found: 417.07853; delta ¼ 0.63 ppm. ¹H NMR (400 MHz,

(400 MHz, DMSO‑d₆)

d

7.74e7.86 (m, 2H), 7.51e7.60 (m, 2H),

DMSO‑d₆)

d

8.38 (d, J ¼ 4.5 Hz, 1H), 7.70e7.86 (m, 2H), 7.50e7.60

7.45e7.51 (m, 2H), 7.43 (br s, 2H), 7.35e7.41 (m, 2H), 7.11 (s, 1H),

(m, 2H), 7.44e7.49 (m, 2H), 7.43 (s, 2H), 7.37e7.42 (m, 2H), 7.11 (s,

3.89 (t, J ¼ 6.7 Hz, 2H), 3.52 (t, J ¼ 6.7 Hz, 2H), 1.74e1.96 (m, 4H). ¹³

C

1H), 2.77e2.95 (m, 1H), 0.50e0.83 (m, 4H). ¹³C NMR (101 MHz,

NMR (101 MHz, DMSO‑d₆)

d

159.6, 148.1, 143.4, 141.4, 137.3, 132.3,

DMSO‑d₆) d 161.4, 147.0, 143.4, 142.4, 137.2, 132.5, 131.6, 128.7, 128.5,

131.6, 128.8, 128.6, 126.6, 125.4, 110.0, 47.7, 45.9, 25.4, 22.8.

126.9, 125.4, 108.0, 22.0, 5.1.

4-[1-(4-chlorophenyl)-3-(3,3-diﬂuoroazetidine-1-carbonyl)-

1H-pyrazol-5-yl]benzenesulfonamide (71)

1-(4-chlorophenyl)-N-(piperidin-1-yl)-5-(4-

sulfamoylphenyl)-1H-pyrazole-3-carboxamide (78)

Mp 294e296 ^ꢀC; HESI-HRMS: calcd for C₂₁H₂₃O₃N₅ClS [MþH]^þ:

460.12046; found: 460.11957; delta ¼ ꢁ1.9 ppm. ¹H NMR (400 MHz,

Mp 275e278 ^ꢀC; HESI-HRMS: calcd for C₁₉H₁₆O₃N₄ClF₂S

[MþH]^þ: 453.05942; found: 453.05941; delta ¼ ꢁ0.03 ppm. ¹H

NMR (400 MHz, DMSO‑d₆)

d

7.81 (d, J ¼ 7.9 Hz, 2H), 7.52e7.58 (m,

DMSO‑d₆) d 9.24 (s, 1H), 7.75e7.84 (m, 2H), 7.52e7.60 (m, 2H),

2H), 7.46e7.50 (m, 2H), 7.44 (br s, 2H), 7.39e7.44 (m, 2H), 7.20 (s,

7.44e7.48 (m, 2H), 7.38e7.44 (m, 4H), 7.12 (s, 1H), 2.80 (t, J ¼ 5.3 Hz,

1H), 4.98 (t, J ¼ 12.4 Hz, 2H), 4.52 (t, J ¼ 12.4 Hz, 2H). ¹³C NMR

4H), 1.59 (quin, J ¼ 5.5 Hz, 4H), 1.32e1.41 (m, 2H). ¹³C NMR

(101 MHz, DMSO‑d₆)

d

145.8, 143.6, 142.1, 137.1, 132.6, 131.3, 128.8,

(101 MHz, DMSO‑d₆) d 157.6, 146.5, 143.4, 142.3, 137.2, 132.5, 131.5,

128.7, 126.5, 125.4, 116.2, 109.4, 63.6, 59.3.

128.7, 128.5, 127.0, 125.4, 108.3, 54.7, 24.7, 22.4.

4-[1-(4-chlorophenyl)-3-(3,3-diﬂuoropyrrolidine-1-

4-{3-(pyrrolidine-1-carbonyl)-1-[5-(triﬂuoromethyl)pyridin-

25

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I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

2-yl]-1H-pyrazol-5-yl}benzenesulfonamide (79)

Mp 255e257 ^ꢀC; HESI-HRMS: calcd for C₂₀H₁₉O₃N₅F₃S [MþH]^þ:

466.11552; found: 466.11650; delta ¼ 2.10 ppm.

135.0, 132.2, 129.4, 126.3, 125.8, 124.4, 106.3, 38.1, 34.5.

2-Chloro-N-cyclopropyl-N-methyl-5-[3-(4-

sulfamoylphenyl)-1H-pyrazol-1-yl]benzamide (86)

Mp 209e210 ^ꢀC; HESI-HRMS: calcd for C₂₀H₂₀O₃N₄ClS [MþH]^þ:

431.09392; found: 431.09429; delta ¼ 0.88 ppm. ¹H NMR (500 MHz,

¹H NMR (500 MHz, DMSO‑d₆)

d 8.68e8.72 (m, 1H), 8.49 (dd,

J ¼ 2.1, 8.6 Hz, 1H), 8.12 (d, J ¼ 8.6 Hz, 1H), 7.78e7.82 (m, 2H),

7.53e7.57 (m, 2H), 7.44 (s, 2H), 7.13 (s, 1H), 3.93 (t, J ¼ 6.8 Hz, 2H),

3.54 (t, J ¼ 6.8 Hz, 2H), 1.90e1.99 (m, 2H), 1.81e1.90 (m, 2H). ¹³C

DMSO‑d₆)

d 8.06e8.16 (m, 2H), 7.85e7.95 (m, 2H), 7.57e7.66 (m,

2H), 7.50e7.56 (m, 2H), 7.45 (br s, 1H), 7.41 (s, 2H), 2.95 (br s, 3H),

NMR (126 MHz, DMSO‑d₆)

d 159.8, 154.2, 149.5, 145.1, 143.7, 142.9,

2.54e2.62 (m, 1H), 0.43e0.69 (m, 4H). ¹³C NMR (126 MHz,

137.2, 133.1, 129.0, 125.5, 124.3, 123.1, 118.3, 112.1, 48.2, 46.5, 26.0,

23.3.

DMSO‑d₆) d 162.4, 149.6, 143.7, 138.4, 135.1, 132.2, 129.3, 126.3,

125.8, 124.5, 107.0, 34.0, 31.8, 8.3.

Compounds 80e86 were synthesized in an analogous manner to

the synthesis of 39 using intermediate 39e4 instead of 39e5. See in

Scheme 2.

4.3. Biological assay methods

4-[1-(4-chloro-3-propanoylphenyl)-1H-pyrazol-3-yl]benze-

nesulfonamide (80)

4.3.1. Generation of HEK293 cells expressing human a7 nAChR

Recombinant HEK-293 cell lines stably expressing both the

target hCHRNA7 protein and the hRIC3 chaperone (for proper

functionality) were generated at Gedeon Richter Plc. To achieve

robust expression cDNAs were optimized for human codon usage

and the “Flip-In” technique was employed for proper genomic

integration. The following cDNAs and vector constructs were used:

for hRIC3 expression the synthesized cDNA (Construct ID:

12AAHCLC, LifeTechnologies) in a pcDNA3.1-V5-HAS (resistance

gene: Neomycin) vector; for hCHRNA7 expression the synthesized

cDNA (Construct ID: 12AAHDHC, LifeTechnologies) in a pcDNA5/

FRT (resistance gene: Hygromycin B) vector. First the hRIC-3 then

the hCHRNA7 gene were integrated into the HEK-293 Flip-In cells.

Following the consecutive selection steps 8 clones having robust

Mp 277e282 ^ꢀC; HESI-HRMS: calcd for C₁₈H₁₇O₃N₃ClS [MþH]^þ:

390.06737; found: 390.06757; delta

¼

0.51 ppm. ¹H NMR

(500 MHz, DMSO‑d₆)

d

8.05e8.15 (m, J ¼ 8.3 Hz, 2H), 7.99 (s, 1H),

7.87e7.95 (m, J ¼ 8.3 Hz, 2H), 7.50e7.60 (m, 4H), 7.41 (s, 2H), 3.05

(q, J ¼ 7.2 Hz, 2H), 1.05 (t, J ¼ 7.2 Hz, 3H). ¹³C NMR (126 MHz,

DMSO‑d₆) d 191.2, 149.4, 143.6, 141.0, 139.1, 134.7, 132.8, 128.5, 127.5,

126.2, 125.6, 110.6, 33.3, 7.6.

4-[1-(3-chloro-5-propanoylphenyl)-1H-pyrazol-3-yl]benze-

nesulfonamide (81)

Mp 223e224 ^ꢀC; HESI-HRMS: calcd for C₁₈H₁₇O₃N₃ClS [MþH]^þ:

390.06737; found: 390.06768; delta

¼

0.81 ppm. ¹H NMR

(400 MHz, DMSO‑d₆)

d 8.06e8.17 (m, 2H), 8.00 (s, 1H), 7.88e7.95

(m, 2H), 7.65 (t, J ¼ 1.7 Hz, 1H), 7.45e7.60 (m, 3H), 7.41 (s, 2H), 3.07

(D

F/F > 4, PNU-282987 at 1

m ] re-

M) hCHRNA7-related [Ca^2þ

i

(q, J ¼ 7.2 Hz, 2H), 2.20e2.21 (m,1H),1.07 (t, J ¼ 7.2 Hz, 3H). ¹³C NMR

sponses were obtained.

(101 MHz, DMSO‑d₆)

d 190.6, 149.0, 143.2, 140.9, 140.6, 134.2, 132.1,

129.6, 127.8, 125.7, 125.2, 125.1, 124.1, 110.2, 32.8, 7.1.

4-[1-(3-butanoyl-4-chlorophenyl)-1H-pyrazol-3-yl]benzene-

sulfonamide (82)

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

nAChR

a7

Orthosteric agonists of the a7 nAChR (e.g. PNU-282987) per se

Mp 245 ^ꢀC; HESI-HRMS: calcd for C₁₉H₁₉O₃N₃ClS [MþH]^þ:

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).

404.08302; found: 404.08292; delta ¼ ꢁ0.23 ppm. ¹H NMR

(500 MHz, DMSO‑d₆)

d 8.07e8.15 (m, 2H), 8.02 (s, 1H), 7.88e7.95

(m, 2H), 7.54e7.61 (m, 2H), 7.47e7.55 (m, 2H), 7.42 (s, 2H), 3.01 (t,

J ¼ 7.2 Hz, 2H), 1.62 (sxt, J ¼ 7.3 Hz, 2H), 0.93 (t, J ¼ 7.4 Hz, 3H). ¹³

C

NMR (126 MHz, DMSO‑d₆)

d 190.8, 149.5, 143.7, 141.2, 139.2, 134.8,

132.9, 128.6, 127.6, 126.3, 125.7, 110.9, 41.9, 16.9, 13.5.

4-{1-[4-chloro-3-(morpholine-4-carbonyl)phenyl]-1H-pyr-

azol-3-yl}benzenesulfonamide (83)

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

Mp 178e180 ^ꢀC; HESI-HRMS: calcd for C₂₀H₂₀O₄N₄ClS [MþH]^þ:

m

447.08883; found: 447.08923; delta

¼

0.90 ppm. ¹H NMR

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

sation. 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-

(500 MHz, DMSO‑d₆)

7.60e7.67 (m, 2H), 7.55e7.60 (m, 2H), 7.42 (br s, 2H), 7.36 (s, 1H),

3.59 (br s, 4H), 3.40 (br s, 4H). ¹³C NMR (126 MHz, DMSO‑d₆)

159.7,

d 8.07e8.14 (m, 2H), 7.88e7.95 (m, 2H),

d

149.9, 143.7, 137.9, 137.5, 134.9, 132.4, 129.4, 126.3, 125.9, 124.7,

106.6, 65.9, 65.7, 47.0, 42.0.

sphere of 95% air and 5% CO₂. Before the [Ca^2þ]_imeasurement, 50

ml

of the growth medium was aspirated with a cell washer (BioTek

Elx405UCVWS, Biotek, Winooski, VT, USA), then 50 l/well Calcium

4-{1-[4-chloro-3-(3,3-diﬂuoroazetidine-1-carbonyl)phenyl]-

1H-pyrazol-3-yl}benzenesulfonamide (84)

m

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, 50

Mp 277e286 ^ꢀC; HESI-HRMS: calcd for C₁₉H₁₆O₃N₄ClF₂S

[MþH]^þ: 453.05942; found: 453.05963; delta ¼ 0.46 ppm. ¹H NMR

(500 MHz, DMSO‑d₆)

d 8.03e8.15 (m, 2H), 7.84e7.96 (m, 2H),

7.61e7.70 (m, 3H), 7.52e7.61 (m, 2H), 7.42 (s, 2H), 4.89e5.12 (m,

ml/well assay buffer containing vehicle (Dimethyl sulfoxide [DMSO],

2H), 4.35e4.61 (m, 2H). ¹³C NMR (126 MHz, DMSO‑d₆)

d

159.7,

4% added) or test compounds (4 ꢂ of the ﬁnal concentration) 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).Fluor-

escence measurements were carried out at 37 ^ꢀC. The dye was

excited at 485 nm, emission was sampled at 525 nm at 1.4-s in-

tervals. Baseline was recorded for 20 s followed by agonist stimu-

149.6, 143.8, 138.5, 135.2, 134.8, 132.6, 128.7, 126.6, 126.3, 125.8,

108.2, 63.2, 59.9).

2-Chloro-N,N-dimethyl-5-[3-(4-sulfamoylphenyl)-1H-pyr-

azol-1-yl]benzamide(85)

Mp 246e247 ^ꢀC; HESI-HRMS: calcd for C₁₈H₁₈O₃N₄ClS [MþH]^þ:

405.07827; found: 405.07846; delta

¼

0.48 ppm. ¹H NMR

(500 MHz, DMSO‑d₆)

7.54e7.62 (m, 4H), 7.41 (s, 2H), 7.35 (s, 1H), 2.97 (s, 3H), 2.95 (s, 3H).

¹³C NMR (126 MHz, DMSO‑d₆)

161.0, 149.8, 143.7, 138.2, 138.1,

d

8.09e8.12 (m, 2H), 7.89e7.92 (m, 2H),

lation. 50

m

l 4 ꢂ concentrated agonist (PNU-282987, 1

mM) solution

was added to all wells using the pipettor of FlexStation II and

d

ﬂuorescence was monitored for an additional 20 s. Final DMSO

26

ꢀ

I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

concentration 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

potential of ꢁ80 mV at room temperature. Inward currents were

evoked by 3-s-long application of the agonist GABA (1 mM) at 2-4-

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.

before 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.

D

F ¼ maximum ﬂuorescence intensity values

after stimulation minus average ﬂuorescence intensity values

before stimulation). In all experiments, all treatments were

measured in multiple wells in parallel, and the mean

DF/F values

4.3.4. Metabolic stability assay

were used for analysis. F/F data were converted to corrected %

D

In vitro metabolic stability was assessed using human (Xen-

otech, LLC, USA), Wistar rat and NMRI mouse (In vitro Metabolism

Research, Gedeon Richter Plc, Hungary) liver microsomes. Test

compounds were incubated at 1 or 2.5

at longest up to 40 min with the liver microsomes (0.5 mg/mL).

In vitro intrinsic clearance (CL_intL/min/mg protein) was calculated

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.

mM initial test concentration

,

m

using the basic concept of clearance prediction [41] 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

4.3.3. Patch clamp studies

Channel physiology of

a7 nAChRs was studied by automated

ﬁrst order conditions,

incubations.

S

¼

substrate concentration in the

(QPatch) whole-cell patch clamp. Whole-cell patch clamp re-

cordings were made from Flp-In HEK293 cells stably expressing

hRIC3/hCHRNA7 2 days after plating at a holding potential of ꢁ80

mV. Inward currents were evoked by 3-s-long application of the

agonist (choline, 10 mM) at 2-4-min intervals in the absence and in

the presence of test compounds and recorded at 10 kHz sampling

frequency. After agonist application, wash-out was applied 4 times.

The control solution contained the same concentration of the

vehicle (0.1% DMSO) as the solutions with the test compounds.

Solutions containing the test compounds were applied to the cells

for 6e10 min. 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 based on FI values at several test compound

concentrations.

4.3.5. Permeability assay

Bi-directional permeability (Papp_A-Band 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 described in

Hellinger et al., 2010 [31]. Brieﬂy, the permeability of test com-

pounds (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 180 min, using appropriate duration of time

determined based on preliminary studies for each compound

tested. Samples were analyzed using HPLC or UHPLC-MS/MS.

For electrophysiological measurements using

a4/

b

2 nACh re-

4/ 2 re-

ceptors, HEK293 cells stably expressing human nAChR

a

b

ceptors (Euroﬁns; CYL3106SS) were used for automated patch

clamp recordings using the QPatch-HTX system (Sophion,

Denmark) at room temperature. Whole-cell patch clamp re-

cordings were made from cells at a holding potential of ꢁ80 mV in

single-cell mode. The control solution contained the same con-

centration of the vehicle (DMSO) as the solutions of the test com-

pound. When testing modulation, inward currents were evoked by

4.3.6. 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 Animals. 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).

3-s-long applications of the agonist (acethylcholine, 1 mM) in the

absence (control responses) followed by the presence of the test

compound. Peak amplitudes of current evoked by acetylcholine

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. The concen-

tration dependent relative response data of the test compound was

analyzed using Origin 6.0. The concentration causing half-maximal

inhibition (IC50) was used to characterize the potency of the test

compound. When testing agonism, control response was evoked by

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 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)

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.

Differences between the exploration times spent in the familiar vs.

novel arms of the maze for each group were evaluated by MANOVA,

followed by Duncan post hoc test.

application acetylcholine (1

applied once at 1 or 10 M for 3 s.

For electrophysiological measurements using GABA_Areceptors,

HEK293 cells stably expressing human and

mM) then the test compound was

m

a1

b

3g2

a5b3g2

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

27

ꢀ

I. Ledneczki, P. Tapolcsanyi, E. Gabor et al.

European Journal of Medicinal Chemistry 214 (2021) 113189

ꢁ

Novere, L. Marubio, M. Picciotto, M. Zoli, Brain nicotinic receptors: structure

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Neurobiol. 53 (2006) 199e237, https://doi.org/10.1016/s0301-0082(97)

00034-8.

4.3.7. Pharmacokinetics

Pharmacokinetic study was carried out after 3 mg/kg iv. (n ¼ 4)

or p.o. (n ¼ 3/sampling time) administration to non-fasted, male

Hsd Wistar rats (Toxicoop, Hungary). The dosing volume was 2.5

(iv.) or 5 mL/kg (p.o.). The compound (69) was formulated as 30%

PFG-KIT#1 in deionized water solution for IV or 5% Tween 80 in

deionized water suspension for PO administration. Dosing of the

animals was carried out via injection into the tail vein (iv.) or via

gavage (p.o.). Blood samples were taken from the retroorbital

plexus at 5 min, 20 min, 2 h and 5 h (iv.) or 0.5 h, 1 h, 2 h and 5 h

(p.o.) post-dose into Li-heparin tubes and centrifuged at 2000 g for

20 min at 4 ^ꢀC to gain plasma. In the p.o. dose group whole brains

were also removed after exsanguination from vena femoralis. Brain

homogenate samples were prepared by homogenizing the whole

brain with deionized water (brain: water ¼ 1: 2.5, w/w). Plasma

and brain homogenate samples were analyzed after protein pre-

cipitation with acetonitrile using HPLC MS/MS method. Pharma-

cokinetic parameters were calculated from the plasma and brain

concentration-time proﬁles with Kinetica 5.1. software using the

model independent approach.

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Declaration of competing interest

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

Acknowledgements

[19] M.S. Thomsen, H.H. Hansen, D.B. Timmerman, J.D. Mikkelsen, Cognitive

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D. Misner, G.C. Tombaugh, L. Santarelli, K. Brameld, M.E. Milla, D.C. Button,

The whole study was ﬁnancially supported by Gedeon Richter

Plc., Hungary and Orion Corporation, Finland. The authors would

like to thank Dr. Hugh Chapman for the selectivity information of

compound 69 in a3b4 nACh-expressing cells in QPatch.

Characterization of RO5126946,

a

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B. Huyard, E. Poiraud, D. Paul, Ss M. O’Connor, Discovery of BNC375, a potent,

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.1 13189.

selective, and orally available type

7nAChRs, ACS Med. Chem. Lett. 10 (2019) 754e760, https://doi.org/10.1021/

acsmedchemlett.9b00001.

I positive allosteric modulator of

a

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Discovery of novel positive allosteric modulators of the α7 nicotinic acetylcholine receptor: Scaffold hopping approach

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