Development of tricyclic N-benzyl-4-hydroxybutanamide derivatives as inhibitors of GABA transporters mGAT1-4 with anticonvulsant, antinociceptive, and antidepressant activity

European Journal of Medicinal Chemistry 221 (2021) 1 13512

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European Journal of Medicinal Chemistry

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

Development of tricyclic N-benzyl-4-hydroxybutanamide derivatives

as inhibitors of GABA transporters mGAT1-4 with anticonvulsant,

antinociceptive, and antidepressant activity

,

Paula Zare˛ba ^a, Kinga Sałat , Georg C. Hofner , Kamil Ła ˛tka , Marek Bajda ,

€

Gniewomir Latacz ^a, Krzysztof Kotniewicz ^a, Anna Rapacz ^a, Adrian Podkowa ^a,

c

a

b

ꢀꢀ

Maciej Maj , Krzysztof Jozwiak , Barbara Filipek , Klaus T. Wanner ,

Barbara Malawska ^a, Katarzyna Kulig ^a

,

1

a

ꢀ

Faculty of Pharmacy, Jagiellonian University Medical College, 9 Medyczna St, 30e688, Krakow, Poland

b

€

Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universitat München Butenandtstr, 5-13, 81377, Munich, Germany

^cDepartment of Biopharmacy, Medical University of Lublin, ul. W. Chodzki 4a, 20-093, Lublin, Poland

a r t i c l e i n f o

a b s t r a c t

Article history:

g

-Aminobutyric acid (GABA) neurotransmission has a signiﬁcant impact on the proper functioning of the

Received 21 November 2020

Received in revised form

20 April 2021

Accepted 22 April 2021

Available online 4 May 2021

central nervous system. Numerous studies have indicated that inhibitors of the GABA transporters

mGAT1-4 offer a promising strategy for the treatment of several neurological disorders, including epi-

lepsy, neuropathic pain, and depression. Following our previous results, herein, we report the synthesis,

biological evaluation, and structure-activity relationship studies supported by molecular docking and

molecular dynamics of a new series of N-benzyl-4-hydroxybutanamide derivatives regarding their

inhibitory potency toward mGAT1-4. This study allowed us to identify compound 23a (N-benzyl-4-

hydroxybutanamide bearing a dibenzocycloheptatriene moiety), a nonselective GAT inhibitor with a

Keywords:

GABA transporter

mGAT1

mGAT2

mGAT3

slight preference toward mGAT4 (pIC₅₀

¼

5.02

0.11), and compound 24e (4-hydroxy-N-[(4-

methylphenyl)-methyl]butanamide bearing a dibenzocycloheptadiene moiety) with relatively high

inhibitory activity toward mGAT2 (pIC₅₀¼ 5.34 0.09). In a set of in vivo experiments, compound 24e

successively showed predominant anticonvulsant activity and antinociception in the formalin model of

tonic pain. In contrast, compound 23a showed signiﬁcant antidepressant-like properties in mice. These

results were consistent with the available literature data, which indicates that, apart from seizure con-

trol, GABAergic neurotransmission is also involved in the pathophysiology of several psychiatric diseases,

however alternative mechanisms underlying this action cannot be excluded. Finally, it is worth noting

that the selected compounds showed unimpaired locomotor skills that have been indicated to give

reliable results in behavioral assays.

mGAT4

GAT inhibitors

N-benzylamides

[³H]GABA uptake

Molecular docking

Molecular dynamics

Anticonvulsant activity

Antinociceptive activity

Antidepressant-like properties

1. Introduction

disorders, including epilepsy [2e5], neuropathic pain (NP) [6e8],

depression [1], anxiety [9], and Parkinson's [10], Huntington's [1 1],

g

-Aminobutyric acid (GABA, 1, Fig. 1) is the main inhibitory

and Alzheimer's diseases [12]. For years, a number of mechanisms

inﬂuencing the imbalance between inhibitory and excitatory

neurotransmission have been studied. Among these mechanisms,

regulation of the synthesis, release, and removal of synaptically

released GABA is of fundamental importance for the maintenance

of brain function at all levels. As a result, numerous studies have

conﬁrmed that inhibition of membrane-bound GABA transporters

(GATs) is a successful approach for the enhancement of decreased

GABA function. GABA transporters of the solute carrier family 6

neurotransmitter found in the central nervous system (CNS) of

diverse species [1]. A growing body of research has shown that

pathologically decreased GABA concentrations in the synaptic cleft

are involved in the etiology of several neurological and psychiatric

ꢀ

* Corresponding author. 9 Medyczna Street, 30e688, Krakow, Poland.

E-mail address: paula.zareba@uj.edu.pl (P. Zare˛ba).

Author is deceased.

1

https://doi.org/10.1016/j.ejmech.2021.113512

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P. Zare˛ba, K. Sałat, G.C. Hofner et al.

European Journal of Medicinal Chemistry 221 (2021) 113512

Fig. 1. Structure of a GABA transport substrate e GABA (1), which itself is a potent inhibitor of [³H]-GABA uptake [44], and known GAT inhibitors with their inhibitory activities

toward GABA transporters and their pIC₅₀values: tiagabine (2) [29], DDPM-2571 (3) [29], (S)-SNAP-5114 (4) [35], 5 [36], SBV2-114 (6) [22], 7 [45] n.d. e not described.

(SLC6) are divided into four types, and have been cloned from

mouse, rat, and human tissues [13]. It has been established that

mouse GAT1 (mGAT1), GAT2 (mGAT2), GAT3 (mGAT3), and GAT4

(mGAT4) correspond to human and rat GAT1, BGT1 (betaine/GABA

transporter 1), GAT2, and GAT3, respectively [2]. However, the

species-dependent nomenclature can seem rather confusing; thus,

the International Union of Basic and Clinical Pharmacology

(IUPHAR) has proposed standardization of the terminology as fol-

lows: GAT1 (SLC6A1), GAT2 (SLC6A13), GAT3 (SLC6A11), and BGT1

(SLC6A12) [14]. In our current study, we utilized the mGAT1em-

GAT4 nomenclature since we used in vitro assays based on GABA

transporters cloned from mouse cells and in vivo mouse models of

seizures, pain, depression, and anxiety. The four GAT subtypes differ

in their expression and pharmacological roles. mGAT1 and mGAT4

are the most prevalent GAT and are almost exclusively located in

the CNS. mGAT1 is distributed throughout the brain, with a high

density in the neocortex, hippocampus, cerebellum, and basal

ganglia. mGAT4 is predominantly observed at high concentrations

in astrocytes and at lower densities in the spinal cord, brain stem,

thalamus, and hypothalamus [13,14]. The high levels of brain

mGAT1 and mGAT4 cause these transporters to play an important

role in the inactivation of GABA and have been identiﬁed as a main

pharmacological target. In contrast, the betaine-GABA transporter

(mGAT2) and mGAT3 represent potentially minor prospective sig-

niﬁcance for the inactivation of GABAergic neurotransmission in

the CNS since it was demonstrated that both are primarily

expressed in peripheral organs and only at a low level in the brain

[15e19]. However, BGT1 is still under investigation [20e24]. The

effective development and registration of tiagabine (2, Fig. 1), a

selective mGAT1 inhibitor, has made mGAT1 a well-known anti-

convulsant target [25,26]. Furthermore, evidence from both clinical

and preclinical studies have noted the importance of tiagabine (2)

in anxiety, pain, and depression [27,28]. Other selective or nonse-

lective mGAT1 inhibitor representatives, in particular the guvacine

analog DDPM-2571 (3, Fig. 1) [29] and N-substituted nipecotic acid

analog (S)-SNAP-5114 (4, Fig. 1) [30], have also been used as phar-

macological tools to evaluate their CNS effects and the biological

functions of diverse GABA transporters [31]. DDPM-2571 (3), a

prominent mGAT1 inhibitor with the highest selectivity and afﬁnity

of mGAT1 inhibitors to date, presents antidepressant-like, anxio-

lytic-like, and antinociceptive properties [32]. (S)-SNAP-5114 (4),

the ﬁrst potent and slightly selective mGAT3/4 inhibitor, was

described as an effective anticonvulsive and antinociceptive agent

against different types of pain, including acute, persistent, and

chronic pain [2,30,31,33,34]. However, with respect to the poor

chemical stability of (S)-SNAP-5114 (4), which led to limitations in

in vivo studies, modiﬁcation of its lipophilic scaffold was found to

be a promising strategy to improve selectivity toward mGAT1-4. As

a consequence, performed studies allowed us to obtain, among

others, compound 5 (Fig. 1) with higher inhibitory potency toward

mGAT4 than lead molecule 4 with comparable subtype selectivity

[35,36]. Compound 5 became a relevant molecule for the future

development of more potent and selective mGAT4 inhibitors.

Furthermore, several reports have indicated that SBV2-114 (6) has

demonstrated moderate potency with improved selectivity toward

BGT1 [20,37e40]. Even so, in a recent study, SBV2-114, the cis-

cyclohexyl-b-amino acid 6, has attracted attention as a potential

target for seizure management [41]. As pointed out above, despite

the low density of BGT1 in the brain, its inhibitors appear to be

attractive for the elucidation of the pharmacological activity of

BGT1 inhibitors. However, it should be noted that obtaining highly

selective GAT inhibitors is difﬁcult due to the high sequence iden-

tities of mGAT2-4 [1 7,42,43].

Based on the above considerations, GABA uptake inhibitors were

found to be beneﬁcial for the treatment of different disorders

involving GABAergic neurotransmission. In this context, our pre-

vious work identiﬁed compound 7 (Fig. 1) which shows higher

inhibitory potency toward mGAT3 (pIC₅₀¼ 5.61) than the well-

known mGAT3/4 inhibitor

e

(S)-SNAP-5114 (4, pIC₅₀

(mGAT3) ¼ 5.29). In parallel, two selected structural analogs of 7

displayed an analgesic effect in two mouse models of chronic pain

induced by streptozotocin (STZ) and oxaliplatin administration

[45]. These results encouraged us to extend the development of

novel GABA uptake inhibitors. Thus, in the present study, a set of

new 4-hydroxybutanamide, 4-aminobutanamide, and 4-

acetamidobutanamide derivatives was designed and synthesized.

The subject of this study was to transform the diaromatic moiety of

the parent compounds 8 [46] and 9 [47] into tricyclic systems

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European Journal of Medicinal Chemistry 221 (2021) 113512

Fig. 2. The general structures of the target compounds based on modiﬁcation of parent compounds 8 [46] and 9 [47] and known tricyclic derivatives of nipecotic acid 10 [49], and 11

[49].

(Fig. 2). We investigated the diverse lipophilic and electronegative

side chains present in the structures of reference compounds, i.e.,

dibenzocycloheptadiene and ﬂuorene moieties (10 [48] and 11

[49]) or dibenzocycloheptatriene moiety. Other modiﬁcations

included the introduction of substituents into the benzyl fragment

of the molecules, such as a chlorine atom at the ortho or para po-

sition or a ﬂuorine atom or methyl group at the para position

(Fig. 2). The whole novel series of butanamide derivatives were

tested for their inhibitory potency at mGAT1-4. In addition, to

explore the molecular interactions of target compounds with GABA

transporters, we performed computational docking and molecular

dynamics studies. Finally, to conﬁrm the therapeutic potential of

the obtained compounds, we tested lead compounds in animal

seizure models. Additionally, bearing in mind the wide spectrum of

indications of tiagabine (2) we also studied the antinociceptive,

antidepressant-like, and anxiolytic-like activity for selected

compounds.

desired 4-aminobutanamide derivatives 27aee. 4-Amino de-

rivatives 27aee were then converted into suitable 2-substituted 4-

acetamidobutanamide derivatives 28aee via an N-acylation reac-

tion [45]. All compounds were synthesized as racemic mixtures.

2.2. In vitro evaluation and structureeactivity relationships

The inhibitory potencies of all the obtained ﬁnal compounds

22aee, 23aee, 24aee, 27aee, and 28aee were determined for the

four mouse GABA transporter subtypes (mGAT1e4). The assay was

based on [³H]GABA uptake using human embryonic kidney cells

(HEK-293) stably expressing mouse GABA transporters according to

the literature [26]. The speciﬁc afﬁnity to mGAT1 was determined

by a competitive MS Binding Assay with NO711 as an unlabeled

marker quantiﬁed by LCeESIeMS/MS [53]. The compounds that

could reduce GABA uptake or NO711 binding by at least 50% at an

inhibitor concentration of 100

mM were considered active. The

pIC₅₀or pK_ivalues from the [³H]GABA uptake or MS Binding Assays

2. Results

were determined in triplicate in three independent experiments. If

the test compounds at a screening concentration of 100 mM could

not reduce [³H]GABA uptake or NO711 binding below 50%

2.1. Chemistry

(pIC₅₀¼ 4.00), the percent of remaining [³H]GABA uptake or NO711

2.1.1. Synthesis of the 4-hydroxybutanamide derivatives 22aee,

23aee and 24aee

binding was given (in the presence of 100 mM inhibitor).

The inﬂuence on the inhibitory activity of the more rigid side

chain of the N-benzyl-4-hydroxybutanamide derivatives toward

mGAT1-4 was determined with reference to parent compounds 8

and 9 (Fig. 2). As seen from the in vitro data in Table 1 and Fig. 3,

exchange of the diaryl moiety in parent compounds 8 and 9 into a

tricyclic moiety generally led to increased GABA uptake inhibition,

except for compounds with ﬂuorene moiety (22aee) that displayed

reduced activity especially toward mGAT1-2 and mGAT4. It was

found that the type of bridge connecting both aryl groups together

and the substituents at position 4 of butanamide are substantial for

GABA uptake inhibition. Among all the investigated 4-

The synthetic procedure for the 4-hydroxybutanamide de-

rivatives 22aee, 23aee, and 24aee is outlined in Scheme 1. The

primary strategy used for the synthesis of target compounds

involved N-alkylation of the amine building blocks 15, 16, and 17

[50] with the appropriate alkyl halide, such as 3-

bromodihydrofuran-2(3H)-one (18), to form 19e21. As the initial

step for the synthesis of building blocks with a tricyclic system, 15,

16, and 17 [50] and the corresponding bromides 12e14 [51] were

obtained as described previously and further converted to suitable

amines 15 [50],16, and 17 by a previously used general method [47].

Using N-benzylamine and its derivatives, aminolysis reactions

[47,52] of intermediates 19e21 afforded the designed 4-

hydroxybutanamide derivatives 22aee, 23aee, and 24aee.

hydroxybutanamide derivatives with

a tricyclic system, an

optimal bridge seemed to be obtained with the dibenzocyclo-

heptadiene derivatives 24aee. For compounds 24d and 24e with an

ethylene bridge linking the two aryl groups together, we observed a

noticeable increased activity in favor of mGAT2. The most potent

mGAT2 inhibitor was compound 24e, with a methyl group in the

para position of the benzyl moiety (pIC₅₀5.34). However, con-

necting both aromatic rings with a direct covalent bond caused a

decrease in the activity of the obtained compounds 22aee to pIC₅₀

4.35e4.60 for mGAT4 compared to the nonrigid analogs 8 and 9 to

pIC₅₀5.00e5.06 for mGAT4, and maintenance of the preference for

mGAT3 was observed but on a slightly lower level (pIC₅₀4.70e4.99

and 5.00e5.14 for mGAT3, respectively; Table 1, Fig. 3). In addition,

introduction of a double bond between C10 and C11 in the linker of

2.1.2. Synthesis of the 4-aminobutanamide and 4-

acetamidobutanamide derivatives 27aee and 28aee

The designed 4-aminobutanamide and 4-acetamidobutanamide

derivatives 27aee and 28aee were prepared in accordance with

previously described synthetic procedures [45]. As outlined in

Scheme 2, in the ﬁrst step, the introduction of a suitable tricyclic

system with a butenyl linker into position 2 of the N-benzyl-2-

bromo-4-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)butanamide de-

rivatives (25aee) was performed to obtain compounds 26aee.

Subsequent hydrolysis of the phthalimide group yielded the

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European Journal of Medicinal Chemistry 221 (2021) 113512

Scheme 1. Synthesis route for the 2-substituted derivatives of 4-hydroxybutanamide 22aee, 23aee, and 24aee. Reagents and conditions: (i) MeNH₂solution (33 wt % in absolute

ethanol), rt, 24 h; (a) tetra-n-butylammonium bromide (TBAB), K₂CO₃, CH₃CN, 15 min at 0 ^ꢀC and 16 h at rt; (b) argon, dry THF, reﬂux, 48 h.

Scheme 2. Synthesis route for the 4-aminobutanamide and 4-acetamidobutanamide derivatives 27aee and 28aee. Reagents and conditions: (a) amine (17), KI, K₂CO₃, MeCN,

reﬂux, 24 h; (b) NH₂NH₂(50%e60%), EtOH, 2 h at 60 ^ꢀC and 5 h at rt; (c) DMAP, DCC, CH₃COOH, DCM, 10 min at 0 ^ꢀC and 20 h at rt.

the tricyclic moiety (23aee) showed lower activity to pIC₅₀

4.16e4.68 for mGAT1 compared with their structural analogs

24aee; their pIC₅₀values ranged from 4.70 to 5.00 for mGAT1.

Relatively good inhibitory activity against mGAT4 were obtained as

a result of replacing the hydroxyl group in the dibenzocyclo-

heptadiene derivatives (24aee) with an amino group at the 4-

position of butanamide (27aee); their pIC₅₀values ranged from

5.19 to 5.36 for mGAT4. Among 4-aminobutanamide derivatives,

compound 27d, with a ﬂuorine atom in the para position of the

benzyl moiety, showed the highest pIC₅₀value toward mGAT4 (27d,

pIC₅₀5.36). In turn, in the group of 4-acetamidobutanamide de-

rivatives (28aee), notably a decrease in inhibitory activity toward

mGAT2 was observed although the preference for mGAT1 and

mGAT3-4 was maintained compared to 4-hydroxybutanamide de-

rivatives (24aee). Nevertheless, an acetamide derivative with a

methyl group at the 4-position of the benzyl moiety (28e, pIC₅₀

5.25) retained activity toward mGAT3 at the same level as its

structural analog with a free amino group at the 4-position (27e,

pIC₅₀5.32, Table 1, Fig. 3).

2.3. Molecular modeling

In order to elucidate how the obtained compounds interact with

GABA transporters, we conducted molecular docking and molecu-

lar dynamics studies using homology models of the targets. For this

purpose, we used previously developed models of human GAT-1,

GAT-2, GAT-3 and BGT-1 which correspond to mouse GAT-1, GAT-

3, GAT-4 and GAT-2, respectively [43]. Mouse and human GABA

transporters have very high homology of the amino acid sequence

[55e59]. The differences mainly concern the amino acid residues at

the N- and C-terminus, away from the ligand binding sites. In

addition, previous studies indicate that the activity of compounds is

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European Journal of Medicinal Chemistry 221 (2021) 113512

Table 1

Inhibitory potencies (pIC₅₀SEM) toward mGAT1e4 obtained from the [³H]GABA uptake experiments and binding afﬁnities (pK_iSEM) toward mGAT1 from the MS Binding

Assays of the obtained compounds. [54]

Fig. 3. Bar graph showing the pIC₅₀values of compounds 22aee, 23aee, 24aee, 27aee, and 28aee compared to the values of the parent compounds 8 and 9.

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European Journal of Medicinal Chemistry 221 (2021) 113512

very similar toward the transporters from both species [60]. All

models were built with the SWISS-MODEL server based on the

crystal structure of the Drosophila melanogaster dopamine trans-

porter as a template. As the activity of compounds was investigated

for the racemates, we considered both the R- and S- isomer in the

molecular modeling studies. The general orientation of both ste-

reoisomers was similar due to the large conformational freedom of

the fragment containing the chiral carbon atom. For each type of

the transporter, we present with details this isomer which obtained

higher docking score and a more coherent binding mode (for in-

formation about absolute conﬁguration see Fig. 4). Even though the

stereoisomers of compounds differ from each other in value of

scoring function, there is no clear trend and the differences are too

small to indicate which isomer is more active.

The results indicated that all studied compounds bind mainly

within the vestibule of the transporters (Fig. 4). It is a different

binding mode from that observed for reference compounds (e.g.

tiagabine, (S)-SNAP-5114) described in our previous work [43]. In

their case, the nipecotic acid fragment was located within the main

binding site (S1), where the carboxylic group interacted with the

sodium ion (Na1). In turn, the aromatic groups reached the S2 site

in the vestibule which is situated above the extracellular gate

(Suppl. Fig. S1). The compounds investigated here represent a

different chemotype and contain the N-benzylamide group instead

of an acid group. Such extension of the molecule limits the in-

teractions within the S1 site.

PHE294, TYR452 and MET458 in GAT-1; PHE293 and TYR453 in

BGT-1; TYR448, TYR449, TYR515 and MET454 in GAT-2; and

PHE531, TYR469, TYR535 and MET474 in GAT-3. The planar ﬂuo-

rene moiety creates weaker hydrophobic interactions and does not

ﬁt the described cavities as well as the larger coplanar dibenzocy-

cloheptatriene and dibenzocycloheptadiene moieties. In addition,

the poses obtained for compounds with ﬂuorene fragments were

less coherent, which is reﬂected in the lower activity of these de-

rivatives. The main core containing the tertiary amine and amide

moieties is arranged along the vestibule, and the benzyl group is

directed toward the S1 site, which is located inside this site or near

the extracellular gate. The exact arrangements of the main core and

benzyl group vary depending on the type of GABA transporter.

The prediction of protonation states at physiological pH (7.4)

indicates that in the case of 4-aminobutanamide derivatives, pro-

tonated primary amines and non-protonated tertiary amine are the

dominant forms. For GAT-1, GAT-2 and GAT-3, the location of the

positive charge makes it possible to create a beneﬁcial salt bridge

between the protonated amine of the ligand and the carboxyl group

of an aspartic acid residue (ASP451 in GAT-1; ASP447 in GAT-2;

ASP467 in GAT-3) while at the same time reach the main binding

site (S1) through the benzyl group. The abovementioned salt bridge

with the aspartic acid residue that is part of the extracellular gate

seems to be one of the key interactions for ligand binding. This salt

bridge was very stable during the molecular dynamics simulations

carried out for the GAT-1, GAT-2, and GAT-3 complexes with rep-

resentatives of the most active compounds, i.e., 27c, 27e and 27d,

respectively (Fig. 5). In turn, the benzyl moieties can form hydro-

phobic interactions with TYR60, TYR140, LEU300 and LEU136 in

GAT-1; PHE288, TYR129, LEU294 and LEU125 in GAT-2; and

In most poses for all derivatives and each type of GAT, the

dibenzocycloheptatriene, dibenzocycloheptadiene and ﬂuorene

fragments are located closest to the entrance to the transporter.

These moieties form hydrophobic interactions mainly with:

Fig. 4. Binding modes of compounds (R)-27d (green) and (R)-27c (pink) in GAT-1 (panel A); compounds (R)-24d (orange) and (R)-24e (blue) in BGT-1 (panel B); compounds (S)-27d

(green) and (S)-27e (yellow) in GAT-2 (panel C); and compounds (S)-23a (purple) and (S)-27d (green) in GAT-3 (panel D). Ionic interactions and hydrogen bonds are represented by

yellow dashes.

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European Journal of Medicinal Chemistry 221 (2021) 113512

Fig. 5. RMSD changes for the compounds over the course of the molecular dynamics simulations (panel A). Distance changes between the carboxyl moiety of the aspartic acid

residue from the extracellular gate and protonated amino group of each ligand, during molecular dynamics simulations (panel B). Analyzed ligand-protein complexes: GAT-1 e (R)-

27c; BGT-1 e (R)-24e; GAT-2 e (S)-27e; and GAT-3 e (S)-27d.

LEU314, LEU143, PHE308 and TYR147 in GAT-3. Such a favorable

arrangement may explain the increased activity of the 4-amino

derivatives toward GAT-1, GAT-2 and GAT-3. For the 4-

hydroxybutanamide and 4-acetamidobutanamide derivatives, in

the absence of a primary amine, the tertiary amine is protonated.

Different positions of the positive charge make it difﬁcult to form a

salt bridge with aspartic acid while maintaining the beneﬁcial in-

teractions from the benzyl fragment. However, the 4-hydroxy and

4-acetamido compounds can create ionic bonds between the amine

group and aspartic acid residue and hydrogen bonds with the

carboxyl group of the aforementioned residue through the hy-

droxyl group (Fig. 4D, compound 23a) or, less frequently, the

acetamide group.

During the molecular dynamics calculations for the complexes

of 27e with GAT-2 and 27d with GAT-3, the formation of a hydrogen

bond between the amide group of the compounds and the hydroxyl

group of residue SER452 (GAT-2) or SER472 (GAT-3) was observed.

In the case of GAT-1, the amide group of 27c initially formed a

hydrogen bond with the TYR140 hydroxyl group and then moved

toward the nonhelical fragment of the TM1 domain, forming water-

mediated hydrogen bonds with atoms on the GLY65 and ASN66

main chains.

coherent, which may explain their lower activity. The reported

binding mode within BGT-1 for the most active compound (24e)

was generally preserved during the course of the molecular dy-

namics simulations. A signiﬁcant shift from the original position

was observed only for the benzyl group as described above.

2.4. Hepatotoxicity and cytotoxicity

From all ﬁnal compounds, we selected two representative de-

rivatives (23a and 24e) for in vitro studies in order to verify their

safety in the HepG2 and HEK-293 cell lines for the purpose of

testing the selected compounds in vivo. Compound 24e was

selected as one of two compounds with the highest activity toward

mGAT2 (pIC₅₀¼ 5.34 0.09, Table 1, Fig. 3). Compound 23a dis-

played balanced activity as a nonselective GAT inhibitor with a

slight preference toward mGAT4 and was obtained using a simpler

and more effective synthesis procedure than the 4-

aminobutanamide analogs. Thus, initially, the HepG2 hepatoma

cell-based hepatotoxicity assay was used to investigate the safety of

compounds 23a and 24e. The results revealed that neither of the

selected compounds induced signiﬁcant hepatotoxicity after 72 h of

In the case of BGT-1 transporters, it was observed that the tested

compounds bind higher in the vestibule, as shown in Fig. 4B with

two of the most active compounds toward BGT-1, 24d and 24e. Such

binding implies a different placement of the benzyl group. Initially,

this group interacts mainly with TYR133, LEU56 and ILE136, and

then, over the course of the simulation (carried out for compound

incubation at concentrations up to 25

(p < 0.0001) decrease in HepG2 cell viability was observed for 23a

and 24e only at the highest concentrations of 50 and 100

(Fig. 6A).

Compound 24e was slightly more toxic compared with 23a, as it

completely eradicated cell viability at 50 M, whereas 23a was

showed 72% viability compared to the control (1% DMSO in culture

media). In general, taking into account the results for the reference

mM. A statistically signiﬁcant

mM

m

24e), the benzyl group moves toward TYR132, creating

p-p in-

teractions with this residue. Therefore, the benzyl group does not

reach the S1 site, unlike it does in other types of GABA transporters.

Another relatively active compound, 24d, is almost identically

placed within BGT-1 compared to 24e. This arrangement seems to

be beneﬁcial because the S1 site in BGT-1 is highly polar due to the

presence of the residues GLU52, GLU126 and GLN299. The upper

position of the compound in the vestibule also enables the creation

of a salt bridge between ASP452 and the protonated tertiary amine

of the 4-hydroxybutanamide derivatives. During MD simulations,

this salt bridge was initially broken, resulting in ionic interactions.

However, at the end of the simulation, the salt bridge was restored.

4-Hydroxybutanamide derivatives additionally form two hydrogen

bonds with ASP452 via hydroxyl and amide groups (Fig. 4B). The 4-

aminobutanamide derivatives generally have a similar arrange-

ment within BGT-1 but form one hydrogen bond less with ASP452

because at physiological pH, the tertiary amine moiety of the 4-

aminobutanamide derivatives is deprotonated, so it cannot be a

hydrogen bond donor. This result is similar to the case of the 4-

acetamidobutanamide derivatives. The acetamide group of these

compounds does not form a hydrogen bond with ASP452 due to

steric hindrance. In addition, the poses of these compounds are less

cytostatic drug doxorubicin at 1 mM, the tested compounds showed

weak (23a) or moderate (24e) hepatotoxic effects. It is crucial to

note that we did not observe a statistically signiﬁcant decrease in

HepG2 cell viability for concentrations corresponding to the IC₅₀

values obtained for individual GAT subtypes (IC₅₀values in the

range of 4e14 mM for compounds 23a and 24e). Analogical studies

were conducted with the use of the HEK-293 cell line. In general,

both compounds showed stronger toxic effects compared to the

results from the HepG2 assay, as a signiﬁcant (p < 0.0001) decrease

in HEK-293 cell viability was observed not only at the concentra-

tions of 50 and 100

(p < 0.05) slight induction in cell proliferation was observed after

treatment with 1 M 23a and 24e. Nevertheless, the observed

antiproliferative effects at 25 M were still weaker than those for

the cytostatic drug doxorubicin at 1 M. Therefore, we assumed

mM but also at 25 mM. Interestingly, a signiﬁcant

m

that selected compounds showed an acceptable level of safety and

an appropriate in vitro proﬁle for further veriﬁcation of their anti-

convulsant, antinociceptive, antidepressant-like, and anxiolytic-

like properties in mouse models of seizures, pain, depression, and

anxiety.

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European Journal of Medicinal Chemistry 221 (2021) 113512

Fig. 6. The inﬂuence of 23a, 24e, and the reference cytostatic drug doxorubicin (DX) on the viability of hepatoma HepG2 cells (panel A) and HEK-293 cells (panel B) after 72 h of

incubation. Statistical signiﬁcance (*p < 0.05, ****p < 0.0001) was analyzed by GraphPad Prism 8.0.1 software using one-way ANOVA and Bonferroni's multiple comparison post hoc

test.

2.5. In vivo pharmacological evaluation

2.5.1. Anticonvulsant activity

effective in this assay (Fig. 8A and B).

In our previous studies, tiagabine revealed protective anticon-

vulsant properties against PTZ-induced seizures. In comparison to

the control group, it prolonged the latency to ﬁrst clonus at a wide

range of doses (6.25e50 mg/kg, i.p.) and reduced the number of

seizure episodes in PTZ-treated mice [28]. DDPM-2571, compared

to vehicle, signiﬁcantly prolonged the latency to seizure onset at

doses of 1 mg/kg - 10 mg/kg. It also reduced the number of seizure

episodes at doses of 0.5 mg/kg - 10 mg/kg [32]. (S)-SNAP-5114 at

doses of 30 and 100 mg/kg was not effective in this assay (un-

published data), which is in line with previously reported data [61].

2.5.1.1. Electroconvulsive threshold (ECT) test. In the ECT test

(Fig. 7A), both compounds 23a and 24e used at the dose of 100 mg/

kg elevated the threshold for electroconvulsions and 24e was

particularly efﬁcacious in this assay (elevation of seizure threshold:

23a: 43% and 24e: 90% vs. vehicle-treated mice). In our previous

studies [28,32], we tested selective GAT1 inhibitors, i.e., tiagabine at

a dose of 100 mg/kg (i.p.), which was only weakly effective in

elevating the threshold for electroconvulsions (31.6% vs. control),

and DDPM-2571 at the maximal tolerated dose (10 mg/kg, i.p.). The

latter did not elevate the electroconvulsive threshold. The mixed

GAT3/4 inhibitor (S)-SNAP-5114 (100 mg/kg, i.p.), was also not

effective in this assay [46].

2.5.1.4. Pilocarpine-induced seizures. In the pilocarpine-induced

model of seizures, one-way ANOVA showed a statistically signiﬁ-

cant effect of treatment on the latency to status epilepticus (F

[2,28] ¼ 5.249, p < 0.05). In this test, compared to the control group,

none of the compounds was effective at the dose 100 mg/kg (i.p.);

24e signiﬁcantly (p < 0.01) reduced the latency to the occurrence of

status epilepticus, while 23a did not affect the latency to status

epilepticus in pilocarpine-treated mice (Fig. 9).

In our previous studies, intraperitoneal administration of tia-

gabine at a dose of 50 mg/kg prolonged the latency to prodromal

seizure symptoms, latency to the occurrence of status epilepticus

and latency to death [28]. In the pilocarpine test, pretreatment with

DDPM-2571 signiﬁcantly prolonged the latency to status epilepticus

at doses of 5 and 10 mg/kg [32]. (S)-SNAP5114 at doses of 100 and

200 mg/kg (i.p.) prolonged the latency to status epilepticus and la-

tency to death [46].

2.5.1.2. Maximal electroshock seizure (MES) test. In the MES test,

neither 23a nor 24e, both examined at 100 mg/kg, were effective

(Fig. 7B). As in the control group, seizures were observed in 83% of

the mice tested. Additionally, the reference compounds tested in

our previous studies, tiagabine, DDPM-2571 and (S)-SNAP-5114,

were not effective in the MES test, either [28,32,46].

2.5.1.3. Pentylenetetrazole-induced seizures (PTZ). In the pentyle-

netetrazole test, the dose 100 mg/kg of both compounds 23a and

24e was tested. One-way ANOVA revealed an overall treatment

effect on the latency to the ﬁrst clonus (F [2,27] ¼ 3.411, p < 0.05)

and on the number of seizure episodes (F [2,29] ¼ 5.126, p < 0.05).

Post hoc analyses showed that compared to the control group, 24e

signiﬁcantly prolonged the mean latency to the occurrence of the

ﬁrst clonus (p < 0.05, Fig. 8A). This compound also reduced the

number of seizure episodes, but this activity did not reach statis-

tical signiﬁcance (Fig. 8B). Compound 23a at 100 mg/kg was not

2.5.2. Antinociceptive activity

2.5.2.1. Hot plate test. In the hot plate test, both compounds 23a

and 24e were tested at a dose of 30 mg/kg. One-way ANOVA did not

demonstrate an overall effect of treatment (F [2,21] ¼ 0.8202,

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European Journal of Medicinal Chemistry 221 (2021) 113512

Fig. 9. Anticonvulsant activity of the test compounds 23a and 24e (100 mg/kg, i.p.)

measured in a mouse pilocarpine model of seizures. Results are shown as mean latency

[s]

( SEM) to status epilepticus. Statistical analysis: one-way analysis of variance

(ANOVA), followed by Dunnett's post hoc comparison. Signiﬁcance vs. vehicle-treated

group: p < 0.01; n ¼ 10e12.

both examined compounds 23a and 24e were tested at a dose of

30 mg/kg. In the neurogenic phase, one-way ANOVA did not reveal

an overall treatment effect (F [2,18] ¼ 3.161, p > 0.05), but an overall

treatment effect was observed in the second phase of the formalin

test (F [2,1 7] ¼ 8.915, p < 0.01). As demonstrated in Fig. 1 1A, in the

neurogenic phase, only 24e reduced the duration of the nocifensive

response (p < 0.05 vs. control). Both 23a and 24e showed anti-

nociceptive properties in the second phase of the formalin test

(p < 0.05 and p < 0.01 vs. control, respectively, Fig. 1 1B).

Previously, we demonstrated that tiagabine at a dose of 8 mg/kg

attenuated the nociceptive response in both phases of the formalin

test. Compared to the control, it reduced the duration of the noci-

fensive response by 81.7% and 95.9% in the acute and late phases of

the formalin test, respectively [28]. In the formalin test, in the ﬁrst

(neurogenic) phase of the test, DDPM-2571 did not demonstrate

antinociceptive properties. In contrast, DDPM-2571 was highly

effective in the second phase of this test, attenuating nocifensive

behavior in a dose-dependent manner [32]. (S)-SNAP-5114 was not

effective in this assay (unpublished data).

Fig. 7. Effect of compounds 23a and 24e (100 mg/kg, i.p.) on the threshold for elec-

troconvulsions measured in the mouse ECT test (panel A), and anticonvulsant activity

of compounds 23a and 24e (100 mg/kg, i.p.) measured in the maximal electroshock

seizure (MES) test (panel B). Results are shown as median current strength (CS₅₀

:

current intensity required to induce tonic hind limb extension in 50% of the mice

challenged; panel A) or % of mice with tonic hind limb extension in each group (panel

B); n ¼ 6.

p > 0.05). Although both 23a and 24e showed a tendency to prolong

the latency pain reaction, these results did not reach statistical

signiﬁcance (Fig. 10).

2.5.3. Antidepressant-like activity

Our previous studies showed that in the hot plate test, tiagabine

at a dose of 8 mg/kg signiﬁcantly prolonged the latency to noci-

ceptive response [28]. DDPM-2571 prolonged the latency to pain

reaction at doses of 1 mg/kg, 2.5 mg/kg and 5 mg/kg [32]. (S)-SNAP-

5114 did not demonstrate antinociceptive activity in the hot plate

test [46].

2.5.3.1. Forced swim test. In the forced swim test, one-way ANOVA

revealed an overall effect of treatment (F [6,49] ¼ 5.144, p < 0.001).

Compared to the control group, 23a showed dose-dependent, sta-

tistically signiﬁcant antidepressant-like activity in this test

(Fig. 12A). Compound 24e was also effective in the forced swim test,

but its signiﬁcant antidepressant-like activity was shown only at

the dose of 30 mg/kg (p < 0.05 vs. control, Fig. 12A).

2.5.2.2. Formalin test. In the formalin test, as in the hot plate test,

Previously, tiagabine (doses: 8 and 30 mg/kg, i.p.) signiﬁcantly

Fig. 8. Effects of the tested compounds 23a and 24e (100 mg/kg, i.p.) on latency to ﬁrst clonus (panel A) and the number of seizure episodes (panel B) in a PTZ-induced seizure

model. Results are shown as mean latency [s] to the occurrence of seizures ( SEM, panel A) and mean number of seizure episodes ( SEM, panel B). Statistical analysis: one-way

ANOVA followed by Dunnett's post hoc test. Signiﬁcance vs. control group: *p < 0.05; n ¼ 10e12.

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in the forced swim test. Previously, we established that tiagabine,

DDPM-2571 and (S)-SNAP-5114 were not effective in this assay

(unpublished data).

2.5.4. Anxiolytic-like activity

2.5.4.1. Four-plate test. In the four-plate test, one-way ANOVA did

not show an overall effect of treatment (F [2,25] ¼ 2.247, p > 0.05).

Neither 23a nor 24e used at the dose 30 mg/kg (i.p.) were effective

in this assay (Fig. 13A).

In our previous studies with this same test, we demonstrated

potential anxiolytic properties of tiagabine (8 mg/kg, i.p.).

Compared to the control group, tiagabine increased the mean

number of punished crossings by 79% [28]. In the four-plate test,

DDPM-2571 at the dose of 5 mg/kg signiﬁcantly increased the mean

number of punished crossings compared to the control group [32].

(S)-SNAP-5114 was effective at a dose of 30 mg/kg (unpublished

data).

Fig. 10. Antinociceptive activity of the compounds 23a and 24e (30 mg/kg, i.p.) in the

hot plate test. Results are shown as latency to pain reaction [s] ( SEM) in response to

thermal stimulus (55 ^ꢀC). Statistical analysis: one-way analysis of variance (ANOVA),

followed by Dunnett's post hoc comparison: p > 0.05; n ¼ 8.

reduced immobility by 59% and 97.4%, respectively vs. the control

[28]. In the forced swim test, DDPM-2571 signiﬁcantly reduced the

duration of immobility at doses of 2.5 and 5 mg/kg [32]. (S)-

SNAP5114 (30 mg/kg, i.p.) also slightly but statistically signiﬁcantly

reduced immobility by 20% vs. control mice [46].

2.5.4.2. Elevated plus maze. In the elevated plus maze test, one-way

ANOVA did not show an overall effect of treatment on the time

spent in open arms (F [2,26] ¼ 0.7630, p > 0.05) or % entry into the

open arms (F [2,23] ¼ 3.384, p > 0.05). The results obtained in this

test conﬁrmed that neither 23a nor 24e at the dose of 30 mg/kg

possessed anxiolytic-like properties in mice and they did not in-

ﬂuence either time spent in open arms (Fig. 13B) or % of open arm

entries (Fig. 13C).

In our previous studies, tiagabine (8 mg/kg, i.p.) prolonged the

time spent in the open arms of the elevated plus maze and raised

the ratio of the number of entries into the open arms to the number

of entries to all arms of the maze in a statistically signiﬁcant

manner [28]. In the elevated plus maze test, the dose of 5 mg/kg

DDPM-2571, compared to the control, increased the % time spent in

2.5.3.2. Tail suspension test. Since 30 mg/kg dose of 23a and 24e

showed signiﬁcant antidepressant-like activity in the forced swim

test, this dose was also assessed for its ability to reduce immobility

in the tail suspension test. One-way ANOVA showed an overall

effect of treatment (F [2,19] ¼ 5.211, p < 0.05). Dunnett's post hoc

test showed a signiﬁcant effect from both compounds on the

duration of immobility in this test (p < 0.05, Fig. 12B). These results

conﬁrmed the antidepressant-like activity of 23a and 24e observed

Fig. 11. Antinociceptive activities of compounds 23a and 24e used at 30 mg/kg (i.p.) in the mouse formalin test. The results are shown as the duration of the licking/biting response

[s] ( SEM) in the formalin-injected paw during the neurogenic phase (0e5 min, panel A) and inﬂammatory phase (15e30 min, panel B) of the test. Statistical analysis: one-way

ANOVA followed by Dunnett's post hoc comparison. Signiﬁcance vs. vehicle-treated group in the respective phase: *p < 0.05, **p < 0.01; n ¼ 6e8.

Fig. 12. Antidepressant-like activity of compounds 23a and 24e in the forced swim test (panel A) and in the tail suspension test (panel B). The results are shown as the duration of

immobility [s] ( SEM) in drug-treated and vehicle-treated mice. Statistical analysis: one-way ANOVA followed by Dunnett's post hoc comparison. Signiﬁcance vs. vehicle-treated

group: *p < 0.05, **p < 0.01; n ¼ 7e9.

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Fig. 13. Anxiolytic-like activity of compounds 23a and 24e used at the dose of 30 mg/kg (i.p.) in the mouse four-plate test (panel A) and in the elevated plus maze test (panels B and

C). Results are shown as mean number of punished crossings ( SEM) in drug-treated and vehicle-treated groups (panel A), time [s] spent in open arms ( SEM, panel B) or % entry

into open arms (panel C). Statistical analysis: one-way ANOVA, followed by Dunnett's post hoc comparison: p > 0.05; n ¼ 7e10.

the open arms of the elevated plus maze and reduced the % time

spent in the closed arms. Compared to vehicle-treated animals,

DDPM-2571 also increased the number of entries into the open

arms [32]. (S)-SNAP-5114 (30 mg/kg, i.p.) was not effective in this

test (data unpublished).

derivatives with dibenzocycloheptadiene moieties (24aee) were

found to be the most potent mGAT2 inhibitors. Based on molecular

modeling studies, we assume that this could be a result of the

formation of two additional hydrogen bonds with mGAT2 (BGT-1)

via hydroxyl and amide groups in comparison with the 4-

aminobutanamide derivatives (27aee), which form only one

hydrogen bond. On the other hand, among all the compounds,

relatively high inhibitory activity toward mGAT3/4 with moderate

mGAT1 binding afﬁnities displayed compounds 27aee. This may be

the result of the appropriate position of the positive charges on

27aee, which makes the creation of a beneﬁcial salt bridge with an

aspartic acid residue in mGAT1/3/4 possible to reach the main

binding site (S1) through the benzyl group. As one of the most

potent mGAT2 inhibitors, we identiﬁed compound 24e with pIC₅₀

5.34 0.09. An additional compound that revealed attractive and

well-balanced activity toward mGAT4 was 23a. Both compounds

23a and 24e showed rather weak hepatotoxic and cytotoxic effects

against hepatoma HepG2 and HEK-293 cells, respectively, and thus

were selected for in vivo studies. In a set of in vivo experiments, the

pharmacological properties of novel GAT inhibitors were demon-

strated. The selected compounds (23a and 24e) had varied anti-

convulsant, antinociceptive and antidepressant-like properties in

mice. However, neither test compound demonstrated anxiolytic-

like activity in mice. Numerous GAT1 inhibitors are regarded as

potential drug candidates for the treatment of epilepsy, and tia-

gabine, a selective GAT1 inhibitor, has been used for the treatment

of partial seizures in humans [62,63]. Of note, this drug shows

effectiveness only in selected mouse models of seizures [28].

Hence, in our research, to increase the complementarity of the

methods, we used a variety of behavioral tests that assessed the

potential anticonvulsant properties of 23a and 24e. The ECT test is a

model that mimics partial tonic-clonic seizures in men [64]. Both

23a and 24e elevated the seizure threshold for electroconvulsions,

but 24e was more potent than 23a. In the MES test, which reﬂects

human grand mal epilepsy [65], neither 23a nor 24e revealed

anticonvulsant activity. Additionally, the pilocarpine model did not

show anticonvulsant properties from either 23a or 24e. Since

previously tested GAT1 inhibitors (tiagabine, DDPM-2571) were

more effective in this seizure model, it can be concluded that GAT1

inhibition rather than the inhibition of other GAT isoforms plays a

role in the abolition of seizures induced by pilocarpine. In contrast,

24e showed anticonvulsant properties in the PTZ test. Considering

the pharmacological effects of 23a and 24e and their anticonvul-

sant activity, we were interested in whether the observed effects

could be a result of synergistic effects between GABA reuptake and

the calcium inﬂux inhibition. Although, it is assumed that drugs

acting by blockade of voltage-gated sodium and to a lesser degree

calcium channels (with the exception of ethosuximide) are effec-

tive in MES test, but not in PTZ model [66,67], our studies showed

that in the MES test, which reﬂects human grand mal epilepsy,

2.5.5. Locomotor activity test

In the locomotor activity test, repeated measures ANOVA

showed an overall treatment effect on the locomotor activity of

animals (F[2,105]

¼

6.505, p < 0.01). The time effect and

drug ꢁ time interaction were not signiﬁcant (F[4,105] ¼ 0.8336,

p > 0.05 and F[8,105] ¼ 0.5726, p > 0.05, respectively). As shown in

Fig. 14, 23a reduced the locomotor activity of mice only between

the 24th and 30th min of the test (p < 0.05 vs. control). Compound

24e at a dose of 30 mg/kg did not affect the locomotor activity of

mice.

In our previous studies, intraperitoneal tiagabine signiﬁcantly

reduced locomotor activity at doses of 8 and 30 mg/kg [28]. DDPM-

2571 at doses of 5 mg/kg and 10 mg/kg did not affect the locomotor

activity of mice [32]. Similarly, (S)-SNAP-5114 (30 mg/kg, i.p.) did

not affect the locomotor activity of the animals (unpublished data).

3. Discussion

We designed a novel series of mGAT1-4 ligands from a group of

GABA derivatives. SAR studies and molecular modeling conﬁrmed

that the introduction of a dibenzocycloheptadiene moiety con-

nected with a propylene chain on the nitrogen atom in the second

position of N-benzyl-4-hydroxybutanamide derivatives increases

the GABA uptake inhibitory activity. 4-Hydroxybutanamide

Fig. 14. Inﬂuence of the test compounds 23a and 24e used at the dose of 30 mg/kg on

the locomotor activity of mice. The results are shown as the mean number of light-

beam crossings ( SEM) measured at selected time intervals: 0e6 min, 6e12 min,

12e18 min, 18e24 min and 24e30 min. Statistical analysis of the results was con-

ducted using repeated measures ANOVA followed by Bonferroni's multiple comparison

test; n ¼ 8.

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European Journal of Medicinal Chemistry 221 (2021) 113512

neither 23a nor 24e revealed anticonvulsant activity. In contrast,

24e showed anticonvulsant properties in the PTZ test, additionally,

23a and 24e showed rather low ability to inhibit the calcium inﬂux

from both 23a and 24e on the motor skills of the animals seems to

be a beneﬁcial property of these compounds, as it precludes false

positive interpretation of data obtained in the behavioral models

used in the present study. Of note, selective GAT1 inhibitors or

GAT1 knockout mice were previously reported to possess

anxiolytic-like and antidepressant-like properties in animal

models. Our present research could indicate the potential role of

non-GAT1 inhibitors in the treatment of depression or the obtained

physiological effects could result from activity at other neuro-

transmitter systems or being synergistic effects between different

systems. To verify the antidepressant-like properties obtained from

these in vivo tests we performed a pan-screening of 23a and 24e on

other targets in preliminary studies. Thus, we decided to evaluate

23a and 24e for their speciﬁc afﬁnity to three monoamine trans-

porters: serotonin (hSERT), noradrenaline (hNET) and dopamine

(hDAT) that were determined by a competitive MS Binding Assay

with the triple reuptake inhibitor (1R,3S)-indatraline as native

marker quantiﬁed by LCeESIeMS/MS [75,76]. As a result, 23a and

24e did not show afﬁnities to monoamine reuptake transporters at

the concentration of 100 nM. For detailed data, see Supporting

Information Table S1). Based on the obtained results we could

expect that obtained signiﬁcant antidepressant-like properties for

23a and to a lesser degree for 24e could not result in synergistic

effects between GABA neurotransmitter systems and NET and SERT

inhibition. Finally, assays performed by Euroﬁns Pharma Discovery

Services toward serotonin 5-HT_2Areceptor shed light on the po-

tential mechanism responsible for antidepressant-like effects

observed for the obtained compounds. It turned out that the novel

at the concentration of 10 mM (40 9 and 48 18, respectively vs.

nimodipine as a reference 59 6, Table S1 in Supplementary ma-

terials). Bearing that in mind, we can speculate that the inﬂuence of

the test compounds 23a or 24e on calcium channels might be

excluded as a potential mechanism responsible for their anticon-

vulsant action. Taken together, in this part of the present research,

various mouse models of seizures were utilized to study the anti-

convulsant properties of 23a and 24e. Notably, these screening

assays not only identiﬁed a potential lead compound with anti-

convulsant properties (24e) but also allowed us to predict its efﬁ-

cacy against different types of seizures in humans [68]. The PTZ

model of clonic seizures generally describes nonconvulsive

(absence or myoclonic) seizures in humans [69]. The efﬁcacy of 24e

in the PTZ model suggests that this compound might be potentially

effective in absence seizures in humans.

In the present study, we also demonstrated the antinociceptive

properties of 23a and 24e in the formalin test but not in the hot

plate test. Compound 24e attenuated pain responses in both phases

of the formalin test, while 23a was effective only in the late phase.

The formalin model of tonic pain reﬂects persistent pain, which is

regarded to be dependent on sensory C-ﬁber activation (acute/early

phase of this test) and inﬂammation and sensitization within the

spinal cord dorsal horn and brain (the second phase of the test).

This ﬁnding predicts that 24e might have a wider spectrum of

antinociceptive activity than 23a. Obtained results attracted our

attention since they are found to be similar to those obtained for

ReN 1869, a tricyclic derivative of nipecotic acid, presented by Novo

Nordisk [70]. Interestingly, ReN 1869 is a selective histamine H₁

receptor antagonist possessing antinociceptive activity in tests of

chemical nociception in rodents, e.g., the formalin test but not in

thermal tests, e.g., the hot plate test. Therefore, we decided to

describe in detail and justiﬁed our concerns regarding the obtained

in vivo effects and the activity toward GATs. For that account, we

decided for screening our selected compounds (23a and 24e) to

assess their afﬁnity toward histamine H₁receptors in the Euroﬁns

CEREP, France. The obtained results conﬁrmed their potential anti-

histaminic activity. Compounds binding, calculated as a % inhibition

compounds showed inhibition of control speciﬁc binding at 10 mM

toward 5-HT_2Areceptor (see Supporting Information Table S1).

4. Conclusion

Our current SAR studies identiﬁed a new series of N-benzyl-4-

hydroxybutanamide derivatives with inhibitory potency toward

mGAT1-4. Interesting results obtained in the primary in vitro

screening highlight the need for further investigation to produce a

clear explanation of the pharmacological activity of presented

herein GAT inhibitors. Finally, the broad studies containing exper-

imental data presented in this article will shed light on a different

structural model of known GAT inhibitors and their pharmacolog-

ical proﬁle.

of control speciﬁc binding at 10 mM, was impressively high: H₁(h)

antagonist radioligand 98% (23a) and 99% (24e, see Supporting

Information Table S1). In this particular case, we have provided

evidence that the antinociceptive properties of 23a and 24e could

result from their dual activity toward GAT transporters and hista-

mine H₁receptors. These interesting results constitute only a very

primary in vitro screening. Further investigation to determine pK_i

values as well as the functional assays for the H₁receptors is rec-

ommended in the next stage of our research in the future.

Furthermore, available literature data indicate that, apart from

seizure control, GABAergic neurotransmission is also involved in

the pathophysiology of several psychiatric diseases, including

anxiety and depression, and GAT inhibitors might be potentially

useful for the treatment of these disorders [71e74]. Our present

study did not conﬁrm the anxiolytic-like activity of both com-

pounds tested, but these compounds, in particular 23a and to a

lesser degree 24e, showed signiﬁcant antidepressant-like proper-

ties in the two mouse models used for antidepressant drug

screening, i.e., the forced swim test and the tail suspension test.

Additionally, the effects of 23a and 24e on locomotor activity were

assessed. Unimpaired locomotion is pivotal to obtain reliable re-

sults in behavioral assays. In particular, it is sometimes difﬁcult to

differentiate between antinociceptive effects and anxiety- or

depression-related behavioral activities of drugs from indices

related to impaired locomotion. The lack of signiﬁcant inﬂuence

5. Experimental section

5.1. Chemistry

Solvents for the reactions, such as THF and methylene chloride,

were dried, distilled, and collected under an argon atmosphere

before use. THF was distilled from a mixture of sodium and

benzophenone, while methylene chloride was distilled from cal-

cium hydride. Thionyl chloride and TEA were distilled under vac-

uum before use. Puriﬁcation of the chemical compounds by column

chromatography was carried out using silica gel (mesh:

0.063e0.200 mm, Sigma-Aldrich) as a stationary phase. The re-

actions were monitored by thin-layer chromatography (aluminum

sheets precoated with silica gel 60 F254 (Merck) and visualized via

UV light (254 nm). In addition, the TLC plate was stained with a 0.5%

solution of ninhydrin in n-propanol or in a solution of 5%

(NH₄)₆Mo₇O₂₄and 0.2% Ce(SO₄)₂in 5% H₂SO₄. The retention factor

(R_f) was deﬁned using the following solvent systems: S₁(chloro-

form/acetone 1:1 v/v), S₂(hexane/ethanol/TEA 7:2:1 v/v/v), S₃

(DCM/acetone 7:3 v/v), S₅(PE/EtOAc 1:1 v/v), S₇(DCM/acetone 7:3

v/v), S₉(DCM/acetone 9:1 v/v), and S₁₀(NH₃/methanol/DCM/pe-

troleum ether 9:45:120:18 v/v/v/v). ¹H NMR and ¹³C NMR spectra

12

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European Journal of Medicinal Chemistry 221 (2021) 113512

were recorded using Varian Mercury-VX 300, with ¹H at

chromatography over silica gel (S₉: DCM/acetone ¼ 9:1) to yield 19

(1.82 g, 73%, R_f¼ 0.71 (S₁: chloroform/acetone ¼ 1:1)) as a yellow

oil. Formula C₂₁H₂₁NO₂, MW 319.16. ¹H NMR (300 MHz, CDCl₃)

300.08 MHz and ¹³C at 75.46 MHz. The chemical shifts (

d) are re-

ported in ppm and were calculated in relation to the frequency of

the deuteron ﬁeld stabilization signal. Coupling constants, J, are

reported in Hz. The purities of the ﬁnal compounds were recorded

using an Acquity™ UPLC (Waters, Milford, MA, USA) coupled to a

tandem quadrupole (TQD, Waters) mass spectrometer (electro-

spray ionization (ESI) mode-tandem quadrupole). Chromato-

graphic separations were carried out using an Acquity UPLC

d

ppm 2.30e2.38 (m, 2 H (NCH₂CH₂)), 2.50 (s, 3 H (Me)), 2.91e3.00

(m, 2 H (OCH₂CH₂)), 3.02e3.11 (m, 2 H (CH₂N)), 4.07e4.25 (m, 2 H

(NCH; OCH₂)), 4.33e4.43 (m, 1 H (OCH₂)), 6.77 (t, J ¼ 6.92 Hz,

1 H (C ¼ CH)), 7.24e7.40 (m, 4 H (Ar)), 7.65e7.72 (m, 2 H (Ar)), 7.75

(dt, J ¼ 7.37, 1.06 Hz, 1 H (Ar)), 7.83e7.88 (m, 1 H (Ar)).

bridged ethyl hybrid (BEH) C₁₈column; 2.1 ꢁ 100 mm, 1.7

particle size, equipped with Acquity UPLC BEH C18 VanGuard

m particle size. All of the ﬁnal com-

pounds showed purities of >95%. Elemental analyses (C, H, N, and S)

were carried out on a Vario Elementar EL III (Elementar Analy-

sensysteme, Hanau, Germany) and determined to be within 0.4% of

the theoretical values.

m

5 .1. 3 . 2 . 3 - [ M e t h yl ( 3 - { t r i c y c l o [ 9 . 4 . 0 . 0 ³, ⁸ ] p e n t a d e c a -

1(11),3,5,7,9,12,14-heptaen-2-ylidene}propyl)amino]oxolan-2-one

(20). Synthesis followed GP1 with 16 (15 mmol, 3.89 g), 3-

bromooxolan-2-one (18) (15 mmol, 2.47 g), TBAB (1.5 mmol,

50 mg), and anhydrous K₂CO₃(15 mmol, 2.08 g) in acetonitrile

(25 mL). The crude product was puriﬁed by column chromatog-

raphy over silica gel (S₉: DCM/acetone ¼ 9:1) to yield 20 (4.23 g,

98%, R_f¼ 0.72 (S₁: chloroform/acetone ¼ 1:1)) as a yellow oil.

precolumn; 2.1 ꢁ 5 mm, 1.7

m

5.1.1. [3-(9H-ﬂuoren-9-ylidene)propyl](methyl)amine (15)

Formula C₂₃H₂₃NO₂, MW 345.44. ¹H NMR (300 MHz, CDCl₃)

d

ppm

A

mixture of 9-(3-bromopropylidene)-9H-ﬂuorene (12,

2.10e2.23 (m,

2

H

(-CH₂-CH₂-N)), 2.24e2.45 (m,

5

H

(Me,

25.6 mmol, 7.3 g) and a 33% solution of methylamine in ethanol

(35 mL) was stirred at room temperature for 48 h. After the reaction

was complete, water (20 mL) was added to the residue, and the

product was extracted with DCM (2 ꢁ 15 mL). The combined

organic fractions were dried over Na₂SO₄, and the obtained product

was puriﬁed by column chromatography over silica gel (started

with S₃: DCM/acetone ¼ 7:3 and changed eluent to S₁₀: 25% NH₃/

methanol/DCM/petroleum ether ¼ 9:45:120:18) to yield 15 (3.73 g,

62%, R_f¼ 0.56 (S₁₀)) as a yellow oil. Formula C₁₇H₁₇N, MW 235.33.

NeCHeCH₂-)), 2.53e2.66 (m, 1 H (NeCH₂-)), 2.66e2.80 (m, 1 H

(-CH₂-N)), 3.50e3.66 (m, 1 H (NeCH-)), 4.08e4.17 (m, 1 H (OeCH₂-

)), 4.25e4.37 (m, 1 H (OeCH₂-)), 5.56 (dd, J ¼ 8.21, 6.54, 4.49 Hz,

1 H (C]CH-)), 6.85 (s, 2 H (-CH-CH-)), 7.20e7.39 (m, 8 H (Ar)).

5.1.3.3. 3-[Methyl(3-{tricyclo[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,12,14-

hexaen-2-ylidene}propyl)amino]oxolan-2-one (21). Synthesis fol-

lowed GP1 with 17 (15 mmol, 3.95 g), 3-bromooxolan-2-one (18)

(15 mmol, 2.47 g), TBAB (1.5 mmol, 50 mg), and anhydrous K₂CO₃

(15 mmol, 2.08 g) in acetonitrile (25 mL). The crude product was

puriﬁed by column chromatography over silica gel (S₉: DCM/

acetone ¼ 9:1) to yield 21 (4.63 g, 89%, R_f¼ 0.70 (S₁: chloroform/

acetone ¼ 1:1)) as a yellow oil. Formula C₂₃H₂₅NO₂, MW 347.46. ¹H

¹H NMR (CDCl₃)

d ppm 2.51 (s, 3 H (Me)), 2.92e2.97 (m, 2 H

(NHCH₂CH₂)), 3.00e3.06 (m, 2 H (NHCH₂)), 6.74 (t, J ¼ 7.18 Hz,

1 H (C]CH)), 7.27e7.40 (m, 4 H (Ar)), 7.64e7.89 (m, 4 H (Ar)).

5.1.2. Methyl(3-{tricyclo[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,9,12,14-

heptaen-2-ylidene}propyl)amine (16)

NMR (300 MHz, CDCl₃) d ppm 2.11e2.24 (m, 2 H (NeCH₂eCH₂-)),

2.30 (s, 3 H (-N-CH₃)), 2.31e2.38 (m, 2 H (OeCH₂eCH₂-)), 2.65e2.81

(m, 3 H (Ar-CH₂-CH₂-)), 3.27 (br. s., 1 H (Ar-CH₂-CH₂-)), 3.59 (t,

J ¼ 9.62 Hz, 1 H (NeCH-)), 4.08e4.18 (m, 2 H (NeCH₂-)), 4.28e4.36

(m, 2 H (OeCH₂-)), 5.86 (t, J ¼ 7.31 Hz, 1 H (¼CH-), 7.03e7.27 (s, 8 H

(Ar)).

A mixture of 2-(3-bromopropylidene)tricyclo[9.4.0.0³,⁸]penta-

deca-1(11),3,5,7,9,12,14-heptaene (13, 30 mmol, 9.3 g) and a 33%

solution of methylamine in ethanol (41 mL) was stirred at room

temperature for 48 h. After the reaction was complete, water

(25 mL) was added to the residue, and the product was extracted

with DCM (2 ꢁ 15 mL). The combined organic fractions were dried

over Na₂SO₄, and the obtained product was puriﬁed by column

chromatography over silica gel (started with S₃: DCM/acetone ¼ 7:3

and changed eluent to S₁₀: 25% NH₃/methanol/DCM/petroleum

ether ¼ 9:45:120:18) to yield 16 (6.5 g, 83%, R_f¼ 0.81 (S₁₀)) as a

5.1.4. General procedure for the synthesis of N-benzyl-2-{[3-(9H-

ﬂuoren-9-ylidene)propyl](methyl)amino}-4-hydroxybutanamide

derivatives 22aee

Under an argon atmosphere, 3-{[3-(9H-ﬂuoren-9-ylidene)pro-

pyl](methyl)amino}oxolan-2-one (19) (1 equiv) was heated with

the relevant N-benzylamine derivative (2 equiv) in dry THF under

reﬂux for 48 h. After the reaction was complete, the mixture was

ice-cooled, and a 1 M solution of HCl (1.5 mL) was added to the

mixture. The mixture was then extracted with dichloromethane

(3 ꢁ 10 mL). The combined organic fractions were dried over

Na₂SO₄and evaporated under vacuum. The crude product was

puriﬁed by column chromatography.

yellow oil. Formula C₁₉H₁₉N, MW 261.37. ¹H NMR (CDCl₃)

d ppm

2.30 (m, 5 H (H₃CeNHeCH₂eCH₂-)), 2.61 (td, J ¼ 6.86, 4.49 Hz, 2 H

(-CH₂-NH-)), 5.53 (dd, J ¼ 8.03, 6.91 Hz, 1 H (C]CH)), 6.86 (d,

J ¼ 1.03 Hz, 2 H (ArCHCHAr)), 7.21e7.37 (m, 8 H (Ar)).

5.1.3. General procedure (GP1) for the synthesis of 3-substituted

oxolan-2-one derivatives 19e21

A mixture of anhydrous K₂CO₃(1 equiv), the suitable amine (1

equiv) and tetrabutylammonium bromide (TBAB, 0.1 equiv) in

acetonitrile was stirred at 0 ^ꢀC for 15 min. Then, a solution of 3-

bromooxolan-2-one (18) (1 equiv) was added dropwise followed

by stirring for an additional 20 h at room temperature. After the

reaction was complete, the precipitate was ﬁltered off, and the

ﬁltrate was concentrated under vacuum. The crude product was

puriﬁed by column chromatography over silica gel.

5.1.4.1. N-Benzyl-2-{[3-(9H-ﬂuoren-9-ylidene)propyl](methyl)

amino}-4-hydroxybutanamide (22a). Compound 22a was prepared

using 19 (2.05 mmol, 0.65 g) and N-benzylamine (4.10 mmol,

0.44 g) in 10 mL of dry THF. The obtained crude product was pu-

riﬁed by column chromatography over silica gel (S₉: CH₂Cl₂/

acetone ¼ 9:1) to yield 22a (330 mg, 38%, R_f¼ 0.60 (S₁)) as a yellow

oil. Formula C₂₈H₃₀N₂O₂, MW 426.55. ¹H NMR (CDCl₃)

d ppm

1.90e2.01 (m, 2 H (CH₂CH₂OH)), 2.32 (s, 3 H (NMe)), 2.78e2.85 (m,

2 H (C]CHCH₂)), 2.93e3.10 (m, 2 H (CH₂N)), 3.37 (dd, J ¼ 7.82,

4.74 Hz, 1 H (NCH)), 3.61e3.71 (m, 1 H (CH₂OH)), 3.86e3.94 (m, 1 H

(CH₂OH)), 4.29 (d, J ¼ 6.16 Hz, 2 H (CONHCH₂)), 6.63 (t, J ¼ 7.18 Hz,

1 H (C]CH)), 7.17e7.42 (m, 8 H (ﬂuorene)), 7.46e7.82 (m, 6 H (Ar;

CONH)). The proton signal from the hydroxyl group was not

5.1.3.1. 3-{[3-(9H-Fluoren-9-ylidene)propyl](methyl)amino}oxolan-

2-one (19). Synthesis followed GP1 with 15 (8.17 mmol, 1.84 g), 3-

bromooxolan-2-one (18) (8.18 mmol, 0.80 g), TBAB (0.82 mmol,

0.26 g), and anhydrous K₂CO₃(8.10 mmol, 1.12 g) in acetonitrile

(10 mL). The crude product was puriﬁed by column

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European Journal of Medicinal Chemistry 221 (2021) 113512

identiﬁed. ¹³C NMR (CDCl₃)

d

ppm 24.81 (-CH₂-CH₂-N), 34.21 (-CH₂-

J ¼ 7.31 Hz, 1 H (C]CH)), 6.72e6.86 (m, 2 H (ﬂuorene)), 6.94e7.02

(m, 2 H (ﬂuorene)), 7.21e7.43 (m, 6 H (ﬂuorene; Ar)), 7.48e7.54 (m,

1 H (Ar)), 7.67e7.75 (m, 2 H (Ar; CONH)). The proton signal from the

CH₂OH), 41.60 (NeCH₃), 43.81 (eCONHeCH₂-), 57.64 (-CH₂-N),

58.44 (-CH₂-OH), 72.40 (NeCH<), 115.41 (C]CH), 121.21 (2 C, ﬂu-

orene), 124.11 (2 C, ﬂuorene), 126.72 (4 C, Ar), 127.54 (2 C, ﬂuorene),

128.64 (2 C, ﬂuorene), 128.74 (Ar), 137.91 (Ar), 138.40 (2 C, ﬂuorene),

141.21 (C]CH), 141.30 (2 C, ﬂuorene), 173.21 (1 C, C]O). MS: m/z

427 [MþH^þ].

hydroxyl group was not identiﬁed. ¹³C NMR (CDCl₃)

d ppm 24.12

(-CH₂-CH₂-N), 34.31 (-CH₂-CH₂OH), 41.51 (NeCH₃), 43.12

(eCONHeCH₂-), 57.91 (-CH₂-N), 58.30 (-CH₂-OH), 72.62 (NeCH<),

115.58 (2 C, -CH-C-F), 115.92 (C]CH), 121.05 (2 C, ﬂuorene), 123.91

(2 C, ﬂuorene), 127.51 (2 C, ﬂuorene), 128.35 (2 C, ﬂuorene), 128.45

(Ar), 133.54 (2 C, Ar), 138.41 (2 C, ﬂuorene), 140.10 (C]CH), 140.21

(2 C, ﬂuorene), 162.13 (d, ¹J_C-F¼ 245.24 Hz, AreF), 173.19 (C]O). MS:

m/z 445 [MþH^þ].

5.1.4.2. N-[(2-Chlorophenyl)methyl]-2-{[3-(9H-ﬂuoren-9-ylidene)

propyl](methyl)amino}-4-hydroxybutanamide (22b).

Compound 22b was prepared using 19 (1.75 mmol, 0.56 g) and (2-

chlorophenyl)methanamine (3.5 mmol, 0.50 g) in 5 mL of dry THF.

The obtained crude product was puriﬁed by column chromatog-

raphy over silica gel (S₉: CH₂Cl₂/acetone ¼ 9:1) to yield 22b

(323 mg, 40%, R_f¼ 0.64 (S₁)) as a yellow oil. Formula C₂₈H₂₉ClN₂O₂,

5.1.4.5. 2-{[3-(9H-Fluoren-9-ylidene)propyl](methyl)amino}-4-

hydroxy-N-[(4-methylphenyl)methyl]butanamide

(22e).

Compound 22e was prepared using 19 (1.8 mmol, 0.57 g) and p-

tolylmethanamine (3.6 mmol, 0.44 g) in 7 mL of dry THF. The ob-

tained crude product was puriﬁed by column chromatography over

silica gel (CH₂Cl₂/acetone ¼ 9:1) to yield 22e (284 mg, 36%, R_f¼ 0.65

(S₁)) as a yellow oil. Formula C₂₉H₃₂N₂O₂, MW 440.58. ¹H NMR

MW 460.19. ¹H NMR (CDCl₃)

d ppm 1.88e1.97 (m, 2 H (CH₂CH₂OH)),

2.31 (s, 3 H (NMe)), 2.76e2.85 (m, 2 H (C]CHCH₂)), 2.94e3.07 (m,

2 H (CH₂N)), 3.33e3.40 (m, 1 H (NCH)), 3.60e3.69 (m, 1 H (CH₂OH)),

3.84e3.92 (m, 1 H (CH₂OH)), 4.40 (dd, J ¼ 8.85, 6.28 Hz, 2 H

(CONHCH₂)), 6.63 (t, J ¼ 7.31 Hz, 1 H (C]CH)), 7.06e7.17 (m, 2 H

(ﬂuorene)), 7.19e7.41 (m, 7 H (ﬂuorene; Ar)), 7.54 (d, J ¼ 8.72 Hz, 1 H

(Ar)), 7.66e7.77 (m, 2 H (Ar)), 7.88 (t, J ¼ 6.03 Hz, 1 H (CONH)). The

proton signal from the hydroxyl group was not identiﬁed. ¹³C NMR

(CDCl₃)

d d ppm 1.91e1.99 (m, 2 H

ppm ¹H NMR (CDCl₃)

(CH₂CH₂OH)), 2.25 (s, 3 H (ArMe)), 2.32 (s, 3 H (NMe)), 2.76e2.85

(m, 2 H (C]CHCH₂)), 2.98 (quin, J ¼ 6.92 Hz, 2 H (CH₂N)), 3.36 (dd,

J ¼ 7.57, 5.00 Hz, 1 H (NCH)), 3.63e3.70 (m,1 H (CH₂OH)), 3.86e3.93

(m, 1 H (CH₂OH)), 4.26 (d, J ¼ 6.16 Hz, 2 H (CONHCH₂)), 6.62 (t,

J ¼ 7.31 Hz, 1 H (C]CH)), 6.95e7.03 (m, 4 H (ﬂuorene)), 7.20e7.41

(m, 6 H (ﬂuorene; Ar)), 7.47e7.52 (m,1 H (Ar)), 7.68e7.80 (m, 2 H (Ar;

CONH)). The proton signal from the hydroxyl group was not iden-

(CDCl₃)

d ppm 24.05 (-CH₂-CH₂-N), 34.31 (-CH₂-CH₂OH), 41.82

(NeCH₃), 42.93 (eCONHeCH₂-), 57.13 (-CH₂-N), 58.04 (-CH₂-OH),

72.61 (NeCH<), 115.44 (C]CH), 121.74 (2 C, ﬂuorene), 124.05 (2 C,

ﬂuorene), 126.64 (Ar), 127.40 (2 C, ﬂuorene), 128.10 (Ar), 128.21 (Ar),

128.35 (Ar),128.45 (2 C, ﬂuorene),128.85 (Ar),132.90 (AreCl),138.41

(2 C, ﬂuorene), 140.12 (ﬂuorene), 140.51 (ﬂuorene), 142.41 (Ar), 173.10

(C]O). MS: m/z 461 [MþH^þ].

tiﬁed. ¹³C NMR (CDCl₃)

d ppm 21.15 (Ar-CH₃), 24.81 (-CH₂-CH₂-N),

34.14 (-CH₂-CH₂OH), 41.50 (NeCH₃), 43.41 (eCONHeCH₂-), 57.61

(-CH₂-N), 58.62 (-CH₂-OH), 72.42 (NeCH<), 115.42 (C]CH), 121.02

(2 C, ﬂuorene), 124.71 (2 C, ﬂuorene), 127.40 (2 C, ﬂuorene), 128.12

(2 C, Ar), 128.40 (2 C, ﬂuorene), 128.61 (2 C, Ar), 134.71 (Ar), 136.42

(Ar), 138.54 (2 C, ﬂuorene), 140.13 (C¼ CH), 140.24 (2 C, ﬂuorene),

173.80 (C]O). MS: m/z 441 (MþH^þ).

5.1.4.3. N-[(4-Chlorophenyl)methyl]-2-{[3-(9H-ﬂuoren-9-ylidene)

propyl](methyl)amino}-4-hydroxybutanamide

(22c). Compound

22c was prepared using 19 (1.70 mmol, 0.54 g) and (4-

chlorophenyl)methanamine (3.4 mmol, 0.48 g) in 5 mL of dry

THF. The obtained crude product was puriﬁed by column chroma-

tography over silica gel (S₉: CH₂Cl₂/acetone ¼ 9:1) to yield 22c

(335 mg, 43%, R_f¼ 0.61 (S₁)) as a yellow oil. Formula C₂₈H₂₉ClN₂O₂,

5.1.5. General procedure for the synthesis of N-benzyl-4-hydroxy-2-

[methyl(3-{tricyclo[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,9,12,14-

heptaen-2-ylidene}propyl)amino]butanamide derivatives 23aee

Under an argon atmosphere, 3-[methyl(3-{tricyclo[9.4.0.0³,⁸]

pentadeca-1(11),3,5,7,9,12,14-heptaen-2-ylidene}propyl)amino]

oxolan-2-one (20) (1 equiv) was heated with the relevant N-ben-

zylamine derivative (2 equiv) in dry THF under reﬂux for 48 h. After

the reaction was complete, the mixture was ice-cooled, and a 1 M

solution of HCl (1.5 mL) was added to the mixture. The mixture was

then extracted with dichloromethane (3 ꢁ 10 mL). The combined

organic fractions were dried over Na₂SO₄and evaporated under

vacuum. The crude product was puriﬁed by column chromatog-

raphy over silica gel (S₇: DCM/acetone ¼ 7:3).

MW 460.19. ¹H NMR (CDCl₃)

d ppm 1.88e1.98 (m, 2 H (CH₂CH₂OH)),

2.32 (s, 3 H (NMe)), 2.79e2.87 (m, 2 H (C]CHCH₂)), 2.93e3.07 (m,

2 H (CH₂N)), 3.35 (dd, J ¼ 7.95, 4.36 Hz, 1 H (NCH)), 3.66 (td, J ¼ 7.57,

3.85 Hz, 1 H (CH₂OH)), 3.85e3.93 (m, 1 H (CH₂OH)), 4.08e4.21 (m,

2 H (CONHCH₂)), 6.64 (t, J ¼ 7.44 Hz, 1 H (C]CH)), 6.91 (m,

J ¼ 8.46 Hz, 2 H (Ar)), 7.09 (m, J ¼ 8.46 Hz, 2 H (Ar)), 7.21e7.43 (m,

5 H (Ar)), 7.51 (d, J ¼ 7.69 Hz, 1 H (Ar)), 7.65e7.83 (m, 3 H (Ar;

CONH)). The proton signal from the hydroxyl group was not iden-

tiﬁed. ¹³C NMR (CDCl₃)

d ppm 23.85 (-CH₂-CH₂-N), 34.34 (-CH₂-

CH₂OH), 41.84 (NeCH₃), 43.92 (eCONHeCH₂-), 57.72 (-CH₂-N),

58.34 (-CH₂-OH), 72.61 (NeCH<), 115.90 (C]CH), 121.41 (2 C, ﬂu-

orene), 124.51 (2 C, ﬂuorene), 127.32 (2 C, ﬂuorene), 128.34 (2 C,

ﬂuorene), 128.54 (2 C, Ar), 132.84 (AreCl), 134.84 (2 C, Ar), 136.00

(Ar), 138.74 (2 C, ﬂuorene), 140.50 (C]CH), 140.41 (2 C, ﬂuorene),

173.22 (C]O). MS: m/z 461 (MþH^þ).

5.1.5.1. N-Benzyl-4-hydroxy-2-[methyl(3-{tricyclo[9.4.0.0³,⁸]penta-

deca-1(11),3,5,7,9,12,14-heptaen-2-ylidene}propyl)amino]butana-

mide (23a). Compound 23a was prepared using 20 (3 mmol, 0.96 g)

and N-benzylamine (6 mmol, 0.64 g) in 10 mL of dry THF. Yield 23a

(572 mg, 42%, yellow oil, R_f¼ 0.44 (S₇)). Formula C₃₀H₃₂N₂O₂, MW

5.1.4.4. 2-{[3-(9H-Fluoren-9-ylidene)propyl](methyl)amino}-N-[(4-

ﬂuorophenyl)methyl]-4-hydroxybutanamide (22d). Compound 22d

was prepared using 19 (2.10 mmol, 0.67 g) and (4-ﬂuorophenyl)

methanamine (4.2 mmol, 0.53 g) in 8 mL of dry THF. The obtained

crude product was puriﬁed by column chromatography over silica

gel (S₉: CH₂Cl₂/acetone ¼ 9:1) to yield 22d (299 mg, 32%, R_f¼ 0.63

(S₁)) as yellow oil. Formula C₂₈H₂₉FN₂O₂, MW 444.54. ¹H NMR

452.60. ¹H NMR (CDCl₃)

(-CH₂-CH₂-OH)), 1.92 (s,

d

ppm 1.78 (dt, J ¼ 10.84, 3.56 Hz, 2 H

(NeCH₃)), 2.08e2.25 (m,

3

H

2 H

(NeCH₂eCH₂-)), 2.34e2.49 (m, 2 H (NeCH₂-)), 3.07e3.18 (m, 1 H

(NeCH-)), 3.37e3.62 (m, 2 H (-CH₂-OH)), 4.24e4.37 (m, 2 H

(eCONHeCH₂-)), 5.24e5.34 (m, 1 H (C]CH-)), 6.65e6.81 (m, 2 H

(-CH ¼ CH-)), 7.10e7.35 (m, 13 H (Ar)), 7.73e7.80 (m, 1 H (COeNH)).

The proton signal from the hydroxyl group was not identiﬁed. ¹³

C

(CDCl₃)

d

ppm 1.90e2.00 (m, 2 H (CH₂CH₂OH)), 2.32 (s, 3 H (NMe)),

NMR (CDCl₃) d ppm 22.52 (C]CHeCH₂), 27.01 (-CH₂-CH₂-OH),

2.79e2.87 (m, 2 H (C]CHCH₂)), 2.93e3.03 (m, 2 H (CH₂N)), 3.36

(dd, J ¼ 8.08, 4.49 Hz, 1 H (NCH)), 3.61e3.69 (m, 1 H (CH₂OH)),

3.85e3.93 (m, 1 H (CH₂OH)), 4.11e4.24 (m, 2 H (CONHCH₂)), 6.64 (t,

41.21 (NeMe), 43.82 (CO(NH)CH₂), 55.73 (-CH₂-N), 64.51 (-CH₂-

OH), 66.82 (NeCH-), 123.93 (C]CH), 124.34 (Ar), 125.10 (Ar), 125.51

(Ar), 126.31 (Ar), 127.91 (2 C, Ar), 128.20 (Ar), 128.42 (Ar), 129.41 (Ar),

14

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European Journal of Medicinal Chemistry 221 (2021) 113512

130.31 (2 C, Ar), 132.52 (2 C, Ar), 134.90 (Ar), 135.81 (Ar), 136.22 (Ar),

137.82 (Ar), 138.93 (Ar), 143.61 (Ar), 144.70 (Ar), 144.90 (C]C),

171.05 (C]O). MS: m/z 453 (MþH^þ).

143.81 (C ¼ CH), 161.92 (d, ¹J_C-F¼ 245.24 Hz, CeF), 174.21 (C]O).

MS: m/z 471 [MþH^þ].

5.1.5.5. 4-Hydroxy-2-[methyl(3-{tricyclo[9.4.0.0³,⁸]pentadeca-

1(11),3,5,7,9,12,14-heptaen-2-ylidene}propyl)amino]-N-[(4-

methylphenyl)methyl]butanamide (23e). Compound 23e was pre-

pared using 20 (0.50 mmol, 160 mg) and p-tolylmethanamine

(1 mmol,121 mg) in 2 mL of dry THF. Yield 23e (112 mg, 48%, yellow

oil, R_f¼ 0.42 (S₇)). Formula C₃₁H₃₄N₂O₂, MW 466.62. ¹H NMR

5.1.5.2. N-[(2-Chlorophenyl)methyl]-4-hydroxy-2-[methyl(3-{tricy-

clo[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,9,12,14-heptaen-2-ylidene}pro-

pyl)amino]butanamide (23b). Compound 23b was prepared using

20 (0.75 mmol, 239 mg) and (2-chlorophenyl)methanamine

(1.5 mmol, 212 mg) in 4 mL of dry THF. Yield 23b (252 mg, 69%,

yellow oil, R_f¼ 0.50 (S₇)). Formula C₃₀H₃₁ClN₂O₂, MW 487.04. ¹H

(CDCl₃)

d ppm 1.73e1.83 (m, 2 H (-CH₂-CH₂-OH)), 1.93 (d,

NMR (CDCl₃)

d

ppm 1.70e1.87 (m, 2 H (-CH₂-CH₂-OH)), 1.98 (s, 3 H

J ¼ 11.03 Hz, 3 H (NeCH₃)), 2.10e2.30 (m, 2 H (NeCH₂eCH₂-), 2.32

(d, J ¼ 6.92 Hz, 3 H (Ar-CH₃)), 2.34e2.50 (m, 2 H (NeCH₂-)),

3.07e3.22 (m, 1 H (NeCH-)), 3.37e3.59 (m, 1 H (-CH₂-OH))

3.74e3.82 (m, 1 H (-CH₂-OH)) 4.04 (dd, J ¼ 14.62, 5.90 Hz, 1 H

(eNHeCH₂-)), 4.21e4.34 (m, 1 H (eCONHeCH₂-)), 5.23e5.35 (m,

1 H (C]CH-)), 6.71e6.89 (m, 2 H (-CH ¼ CH-)), 6.98e7.38 (m, 12 H

(Ar)), 7.73 (d, J ¼ 5.39 Hz, 1 H (COeNH)). The proton signal from the

(NeCH₃)), 2.18e2.36 (m, 2 H (NeCH₂eCH₂-)), 2.37e2.54 (m, 2 H

(NeCH₂-)), 3.10e3.23 (m, 1 H (NeCH-)), 3.39e3.59 (m, 1 H (-CH₂-

OH)), 3.71e3.82 (m, 1 H (-CH₂-OH)), 4.23e4.33 (m, 1 H (eNHeCH₂-

)), 4.44 (dd, J ¼ 6.28, 4.23 Hz, 1 H (eNHeCH₂-)), 5.42e5.53 (m, 1 H

(¼CH-)), 6.66e6.85 (m, 2 H (-CH ¼ CH-)), 7.04e7.41 (m, 12 H (Ar)),

7.83 (dd, J ¼ 12.70, 6.28 Hz, 1 H (COeNH)). The proton signal from

the hydroxyl group was not identiﬁed. ¹³C NMR (CDCl₃)

d

ppm

hydroxyl group was not identiﬁed. ¹³C NMR (CDCl₃)

d ppm 21.12

23.71 (C]CHeCH₂), 26.11 (-CH₂-CH₂-OH), 40.05 (NeMe), 42.72

(CO(NH)CH₂), 54.90 (-CH₂-N), 63.41 (-CH₂-OH), 69.72 (NeCH-),

125.90 (C]CH), 126.10 (2 C, Ar), 127.41 (4 C, Ar), 128.02 (2 C, Ar),

128.40 (Ar), 129.30 (2 C, Ar), 131.15 (Ar), 131.40 (2 C, Ar), 133.91 (Ar),

134.82 (Ar), 135.13 (Ar), 136.71 (Ar), 137.71 (Ar), 142.51 (Ar), 143.72

(C]CH), 171.01 (C]O). MS: m/z 487 [MþH^þ].

(Ar-Me), 26.62 (C]CHeCH₂), 27.64 (-CH₂-CH₂-OH), 38.32 (NeMe),

42.94 (CO(NH)CH₂), 54.71 (-CH₂-N), 61.81 (-CH₂-OH), 67.70 (NeCH-

), 126.91 (Ar), 127.10 (2 C, Ar), 127.51 (2 C, Ar), 128.00 (Ar), 128.31

(4 C, Ar), 129.11 (Ar), 131.02 (Ar), 131.10 (2 C, Ar), 133.70 (Ar), 134.91

(Ar), 135.21 (Ar), 136.91 (Ar), 137.02 (2 C, Ar), 142.40 (Ar), 143.61

(C ¼ CH), 171.90 (C]O). MS: m/z 467 [MþH^þ].

5.1.5.3. N-[(4-Chlorophenyl)methyl]-4-hydroxy-2-[methyl(3-{tricy-

clo[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,9,12,14-heptaen-2-ylidene}pro-

pyl)amino]butanamide (23c). Compound 23c was prepared using

20 (0.75 mmol, 239 mg) and (4-chlorophenyl)methanamine

(1.5 mmol, 212 mg) in 4 mL of dry THF. Yield 23c (248 mg, 68%,

yellow oil, R_f¼ 0.48 (S₇)). Formula C₃₀H₃₁ClN₂O₂, MW 487.04. ¹H

5.1.6. General procedure for the synthesis of N-benzyl-4-hydroxy-2-

[methyl(3-{tricyclo[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,12,14-hexaen-2-

ylidene}propyl)amino]butanamide derivatives 24aee

Under an argon atmosphere, 3-[methyl(3-{tricyclo[9.4.0.0³,⁸]

pentadeca-1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)amino]oxo-

lan-2-one (21) (1 equiv) was heated with the relevant N-benzyl-

amine derivative (2 equiv) in dry THF under reﬂux for 48 h. After

the reaction was completed, the mixture was ice-cooled, and a 1 M

solution of HCl (1.5 mL) was added to the mixture. The mixture was

then extracted with dichloromethane (3 ꢁ 10 mL). The combined

organic fractions were dried over Na₂SO₄and evaporated under

vacuum. The crude product was puriﬁed by column chromatog-

raphy over silica gel (S₇: DCM/acetone ¼ 7:3).

NMR (CDCl₃)

d ppm 1.67e1.80 (m, 2 H (-CH₂-CH₂-OH)), 1.84 (s, 2 H

(NeCH₃)), 1.89 (s, 1 H (NeCH₃)), 2.06e2.16 (m, 1 H (NeCH₂eCH₂-)),

2.18e2.30 (m, 1 H (NeCH₂eCH₂-), 2.30e2.36 (m, 1 H (NeCH₂-)),

2.37e2.49 (m, 1 H (NeCH₂-)), 3.07e3.20 (m, 1 H (NeCH-)),

3.41e3.57 (m, 1 H (-CH₂-OH)), 3.72e3.82 (m, 1 H (-CH₂-OH)),

4.07e4.42 (m, 2 H (eNHeCH₂-)), 5.17e5.28 (m, 1 H (¼CH-)),

6.76e6.89 (m, 2 H (-CH ¼ CH-)), 6.99 (d, J ¼ 8.72 Hz, 1 H (Ar)),

7.06e7.45 (m, 11 H (Ar)), 7.73e7.89 (m, 1 H (COeNH)). The proton

signal from the hydroxyl group was not identiﬁed. ¹³C NMR (CDCl₃)

5.1.6.1. N-Benzyl-4-hydroxy-2-[methyl(3-{tricyclo[9.4.0.0³,⁸]penta-

deca-1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)amino]butanamide

(24a). Compound 24a was prepared using 21 (0.85 mmol, 271 mg)

and N-benzylamine (1.7 mmol, 182 mg) in 4 mL of dry THF. Yield

24a (351 mg, 91%, yellow oil, R_f¼ 0.31 (S₇)). Formula C₃₀H₃₄N₂O₂,

d

ppm 25.90 (C]CHeCH₂), 27.11 (-CH₂-CH₂-OH), 39.02 (NeMe),

43.80 (CO(NH)CH₂), 55.10 (-CH₂-N), 62.34 (-CH₂-OH), 68.65 (NeCH-

), 126.80 (C]CH), 127.12 (2 C, Ar), 127.40 (4 C, Ar), 128.02 (2 C, Ar),

128.20 (Ar), 128.61 (2 C, Ar), 129.22 (2 C, Ar), 131.23 (Ar), 134.81 (Ar),

135.22 (Ar), 136.83 (Ar), 137.10 (2 C, Ar), 142.31 (Ar), 142.70 (C]CH),

171.45 (C]O). MS: m/z 487 [MþH^þ].

MW 454.29. ¹H NMR (CDCl₃)

d ppm 1.80e1.91 (m, 2 H (-CH₂-CH₂-

OH)), 2.08 (s, 3 H (NeCH₃)), 2.20e2.34 (m, 2 H (Ar-CH₂-CH₂-Ar)),

2.42e2.57 (m, (AreCH₂eCH₂-Ar)), 2.66e2.81 (m, 1 H

2

H

5.1.5.4. N-[(4-Fluorophenyl)methyl]-4-hydroxy-2-[methyl(3-{tricyclo

[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,9,12,14-heptaen-2-ylidene}propyl)

amino]butanamide (23d). Compound 23d was prepared using 20

(0.50 mmol, 160 mg) and (4-ﬂuorophenyl)methanamine (1 mmol,

125 mg) in 2 mL of dry THF. Yield 23d (116 mg, 49%, yellow oil,

R_f¼ 0.33 (S₇)). Formula C₃₀H₃₁FN₂O₂, MW 470.59. ¹H NMR (CDCl₃)

(NeCH₂eCH₂-)), 2.95 (d, J ¼ 12.31 Hz, 1 H (NeCH₂eCH₂-)),

3.09e3.42 (m, 2 H (NeCH₂-)), 3.51e3.61 (m, 1 H (NeCH-)),

3.77e3.88 (m, 1 H (-CH₂-OH)), 4.13e4.27 (m, 1 H (-CH₂-OH)),

4.28e4.49 (m, 2 H (eNHeCH₂-)), 5.68e5.83 (m, 1 H (¼CH-)),

6.97e7.34 (m, 13 H (Ar)), 7.91 (br. s., 1 H (COeNH)). The proton

signal from the hydroxyl group was not identiﬁed. ¹³C NMR (CDCl₃)

d

ppm 1.73e1.79 (m, 2 H (-CH₂-CH₂-OH)), 1.84 (s, 3 H (NeCH₃)),

d ppm 27.90 (C]CHeCH₂), 31.91 (2 C, Ar-CH₂-CH₂-Ar), 33.71 (-CH₂-

2.28e2.39 (m, 3 H (NeCH₂eCH₂-), 2.42e2.49 (m, 1 H (NeCH₂eCH₂-

), 3.10 (dd, J ¼ 8.85, 3.21 Hz, 1 H (NeCH-)), 3.41e3.61 (m, 2 H

CH₂-OH), 41.31 (NeMe), 43.20 (CO(NH)CH₂), 57.31 (-CH₂-OH), 58.41

(-CH₂-N), 67.51 (NeCH-), 125.80 (C]CH), 126.11 (Ar), 127.61 (2 C,

Ar), 128.42 (4 C, Ar), 128.64 (4 C, Ar), 130.20 (Ar), 133.54 (2 C, Ar),

135.32 (2 C, Ar), 136.94 (2 C, Ar), 139.83 (C ¼ CH), 174.12 (C]O). MS:

m/z 455 [MþH^þ].

(NeCH₂-) (-CH₂-OH)), 4.20e4.32 (m,

2 H (eCONHeCH₂-)),

5.19e5.26 (m, 1 H (C]CH-)), 6.70e6.80 (m, 2 H (-CH ¼ CH-)),

7.15e7.40 (m, 12 H (Ar)), 7.82 (br. s.,1 H (COeNH)). The proton signal

from the hydroxyl group was not identiﬁed. ¹³C NMR (CDCl₃)

d ppm

26.43 (C]CHeCH₂), 27.88 (-CH₂-CH₂-OH), 38.36 (NeMe), 42.34

(CO(NH)CH₂), 54.84 (-CH₂-N), 61.77 (-CH₂-OH), 67.65 (NeCH-),

115.08 (C]CH), 115.54 (2 C, -CH-C-F), 127.02 (Ar), 127.12 (2 C, Ar),

128.81 (2 C, Ar), 129.12 (2 C, Ar), 131.32 (3 C, Ar), 133.71 (Ar), 134.24

(Ar), 134.90 (Ar), 136.91 (Ar), 137.00 (Ar), 142.31 (Ar), 143.31 (Ar),

5.1.6.2. N-[(2-Chlorophenyl)methyl]-4-hydroxy-2-[methyl(3-{tricy-

clo[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)

amino]butanamide (24b). Compound 24b was prepared using 21

(0.75 mmol, 239 mg) and (2-chlorophenyl)methanamine

(1.5 mmol, 212 mg) in 5 mL of dry THF. Yield 24b (304 mg, 83%,

15

€

P. Zare˛ba, K. Sałat, G.C. Hofner et al.

European Journal of Medicinal Chemistry 221 (2021) 113512

yellow oil, R_f¼ 0.44 (S₇)). Formula C₃₀H₃₃ClN₂O₂, MW 489.06. ¹H

(1.5 mmol, 182 mg) in 5 mL of dry THF. Yield 24e (267 mg, 76%,

yellow oil, R_f¼ 0.41 (S₇)). Formula C₃₁H₃₆N₂O₂, MW 468.64. ¹H

NMR (CDCl₃)

d ppm 1.73e1.92 (m, 2 H (-CH₂-CH₂-OH)), 2.11 (s, 3 H

(NeCH₃)), 2.23e2.39 (m, 2 H (Ar-CH₂-CH₂-Ar)), 2.52 (d, J ¼ 6.92 Hz,

NMR (CDCl₃) d d ppm 1.77e1.92 (m, 2 H (-CH₂-

ppm ¹H NMR (CDCl₃)

2

H

(AreCH₂eCH₂-Ar)), 2.68e2.84 (m, 1 (NeCH₂eCH₂-)),

H

CH₂-OH)), 2.10 (s, 3 H (NeCH₃)), 2.19e2.41 (m, 5 H (Ar-CH₂-CH₂-Ar,

Ar-CH₃)), 2.43e2.61 (m, 2 H (AreCH₂eCH₂-Ar)), 2.68e2.81 (m, 1 H

(NeCH₂eCH₂-)), 2.88e3.02 (m, 1 H (NeCH₂eCH₂-)), 3.13e3.41 (m,

3 H (NeCH-; NeCH₂-)), 3.55 (ddd, J ¼ 11.61, 7.25, 4.23 Hz, 1 H (-CH₂-

OH)), 3.83 (dd, J ¼ 10.39, 4.74 Hz,1 H (-CH₂-OH)), 4.30 (dd, J ¼ 16.03,

6.54 Hz, 2 H (eNHeCH₂-)), 5.71e5.79 (m, 1 H (¼CH-)), 6.85e7.25

(m, 12 H (Ar)), 7.83 (br. s., 1 H (COeNH)). The proton signal from the

2.85e3.05 (m, 1 H (NeCH₂eCH₂-)), 3.13e3.44 (m, 3 H ((NeCH-;

NeCH₂-)), 3.49e3.61 (m, 1 H (-CH₂-OH)), 3.74e3.86 (m, 1 H (-CH₂-

OH)), 4.32e4.54 (m, 2 H (eNHeCH₂-)), 5.71e5.85 (m, 1 H (¼CH-)),

7.05e7.37 (m, 12 H (Ar)), 7.95 (br. s., 1 H (COeNH)). The proton

signal from the hydroxyl group was not identiﬁed. ¹³C NMR (CDCl₃)

d

ppm 28.01 (C]CHeCH₂), 30.92 (2 C, Ar-CH₂-CH₂-Ar), 33.71 (-CH₂-

CH₂-OH), 41.31 (NeMe), 43.62 (CO(NH)CH₂), 57.62 (-CH₂-OH), 61.52

(-CH₂-N), 67.21 (NeCH-), 125.82 (C]CH), 126.11 (Ar), 127.01 (Ar),

127.21 (Ar), 127.61 (Ar), 128.01 (2 C, Ar), 128.42 (Ar), 128.92 (2 C, Ar),

129.51 (Ar), 130.13 (2 C, Ar), 133.53 (2 C, Ar), 135.41 (Ar), 136.91 (Ar),

139.31 (2 C, Ar), 139.80 (C ¼ CH), 174.20 (C]O). MS: m/z 489

[MþH^þ].

hydroxyl group was not identiﬁed. ¹³C NMR (CDCl₃)

d ppm 20.91

(Ar-Me), 21.10 (C]CHeCH₂), 32.01 (2 C, Ar-CH₂-CH₂-Ar), 33.27

(-CH₂-CH₂-OH), 41.45 (NeMe), 43.00 (CO(NH)CH₂), 57.61 (-CH₂-

OH), 58.61 (-CH₂-N), 67.42 (NeCH-), 125.82 (C]CH), 126.13 (Ar),

127.23 (Ar), 127.63 (2 C, Ar), 128.00 (2 C, Ar), 128.41 (2 C, Ar), 129.32

(4 C, Ar), 130.20 (2 C, Ar), 135.05 (2 C, Ar), 136.91 (2 C, Ar), 139.70

(C ¼ CH), 173.90 (C]O). MS: m/z 469 [MþH^þ].

5.1.6.3. N-[(4-Chlorophenyl)methyl]-4-hydroxy-2-[methyl(3-{tricy-

clo[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)

amino]butanamide (24c). Compound 24c was prepared using 21

(0.75 mmol, 239 mg) and (4-chlorophenyl)methanamine

(1.5 mmol, 212 mg) in 5 mL of dry THF. Yield 24c (275 mg, 75%,

yellow oil, R_f¼ 0.40 (S₇)). Formula C₃₀H₃₃ClN₂O₂, MW 489.06. ¹H

5.1.7. General procedure for the synthesis of N-benzyl-4-(1,3-dioxo-

2,3-dihydro-1H-isoindol-2-yl)-2-[methyl(3-{tricyclo[9.4.0.0³,⁸]

pentadeca-1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)amino]

butanamide derivatives 26aee

Anhydrous K₂CO₃(2.5 equiv) and KI (1 equiv) were added to a

solution

of

methyl(3-{tricyclo[9.4.0.0³,⁸]pentadeca-1(11),3,5,

NMR (CDCl₃)

d ppm 1.76e1.92 (m, 2 H (-CH₂-CH₂-OH)), 2.06 (br. s.,

3 H (NeCH₃)), 2.20e2.39 (m, 2 H (Ar-CH₂-CH₂-Ar)), 2.39e2.60 (m,

2 H (AreCH₂eCH₂-Ar)), 2.65e2.82 (m, 1 H (NeCH₂eCH₂-)), 2.95 (d,

J ¼ 13.08 Hz, 1 H (NeCH₂eCH₂-)), 3.07e3.42 (m, 2 H (NeCH₂-)),

3.48e3.62 (m, 1 H (-CH₂-OH)), 3.74e3.88 (m, 1 H (-CH₂-OH)),

4.06e4.20 (m, 1 H (NeCH-)), 4.20e4.38 (m, 2 H (eNHeCH₂-)),

5.65e5.81 (m, 1 H (¼CH-)), 6.90e7.30 (m, 12 H (Ar)), 7.88 (br. s., 1 H

(COeNH)). The proton signal from the hydroxyl group was not

7,12,14-hexaen-2-ylidene}propyl)amine (17) (1 equiv) in acetoni-

trile. Then, the relevant N-benzyl-2-bromo-4-(1,3-dioxoisoindolin-

2-yl)butanamide derivative (25aee) (1.76 mmol, 1 equiv) was

added, and the reaction mixture was stirred and under reﬂux for

24 h. After the reaction was complete, the precipitate was ﬁltered,

the ﬁltrate was concentrated under vacuum, and the product was

puriﬁed by column chromatography over silica gel (S₅: PE/

EtOAc ¼ 1:1).

identiﬁed. ¹³C NMR (CDCl₃)

d ppm 27.81 (C]CHeCH₂), 31.90 (2 C,

Ar-CH₂-CH₂-Ar), 33.70 (-CH₂-CH₂-OH), 42.51 (NeMe), 43.92

(CO(NH)CH₂), 57.42 (-CH₂-OH), 58.32 (-CH₂-N), 67.52 (NeCH-),

125.52 (C]CH), 126.12 (2 C, Ar), 127.20 (2 C, Ar), 128.11 (Ar), 128.41

(2 C, Ar), 128.91 (2 C, Ar), 129.00 (2 C, Ar), 130.21 (4 C, Ar), 136.70

(Ar), 136.93 (2 C, Ar), 139.73 (C ¼ CH), 174.20 (C]O). MS: m/z 489

[MþH^þ].

5.1.7.1. N-Benzyl-4-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)-2-

[methyl(3-{tricyclo[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,12,14-hexaen-2-

ylidene}propyl)amino]butanamide (26a). Compound 26a was pre-

pared using 25a (1.5 mmol, 0.60 g), amine 17 (1.5 mmol, 0.40 g), KI

(1.5 mmol, 0.25 g), anhydrous K₂CO₃(3 mmol, 0.41 g), and aceto-

nitrile (15 mL). Yield 26a (461 mg, 52%, yellow oil, R_f¼ 0.51 (S₅)).

5.1.6.4. N-[(4-Fluorophenyl)methyl]-4-hydroxy-2-[methyl(3-{tricyclo

[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)

amino]butanamide (24d). Compound 24d was prepared using 21

(0.55 mmol, 176 mg) and (4-ﬂuorophenyl)methanamine (1.1 mmol,

138 mg) in 5 mL of dry THF. Yield 24d (214 mg, 82%, yellow oil,

R_f¼ 0.42 (S₇)). Formula C₃₀H₃₃FN₂O₂, MW 472.60. ¹H NMR (CDCl₃)

Formula C₃₈H₃₇N₃O₃, MW 583.72. ¹H NMR (CDCl₃)

d ppm 1.81e1.95

(m, 1 H (NCHCH₂)), 2.08 (s, 3 H (Me)), 2.12e2.33 (m, 3 H

(NCHCH₂; ¼CHCH₂)), 2.41e2.57 (m, 2 H (CH₂N)), 2.60e2.78 (m, 1 H

(Ar-CH₂-)), 2.80e3.02 (m, 1 H (Ar-CH₂-)), 3.08e3.44 (m, 3 H (Ar-

CH₂-; NCH)), 3.78 (dt, J ¼ 13.98, 7.12 Hz, 1 H (CH₂N)), 3.86e4.03 (m,

1

H (CH₂N)), 4.20e4.48 (m, 2 H (CH₂NH)), 5.63e5.77 (m,

d

ppm 1.77e1.90 (m, 2 H (-CH₂-CH₂-OH)), 2.06 (s, 3 H (NeCH₃))

1 H (C ¼ CH)), 6.96e7.21 (m, 9 H (Ar)), 7.21e7.34 (m, 4 H (Ar)),

7.36e7.52 (m, 1 H (CONH)), 7.65e7.75 (m, 2 H (phthalimide)),

7.79e7.88 (m, 2 H (phthalimide)).

2.20e2.35 (m, 2 H (Ar-CH₂-CH₂-Ar)), 2.48 (dd, J ¼ 9.62, 5.51 Hz, 2 H

(AreCH₂eCH₂-Ar)), 2.68e2.81 (m, 1 H (NeCH₂eCH₂-)), 2.87e3.02

(m, 1 H (NeCH₂eCH₂-)), 3.12e3.37 (m, 3 H (NeCH-;NeCH₂-)),

3.50e3.60 (m, 1 H (-CH₂-OH)), 3.76e3.87 (m, 1 H (-CH₂-OH)),

4.21e4.37 (m, 2 H (eNHeCH₂-)), 5.67e5.80 (m, 1 H (¼CH-)

6.86e7.26 (m, 12 H (Ar)), 7.92 (br. s., 1 H (COeNH)). The proton

signal from the hydroxyl group was not identiﬁed. ¹³C NMR (CDCl₃)

5.1.7.2. N-[(2-Chlorophenyl)methyl]-4-(1,3-dioxo-2,3-dihydro-1H-

isoindol-2-yl)-2-[methyl(3-{tricyclo[9.4.0.0³,⁸]pentadeca-

1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)amino]butanamide (26b).

Compound 26b was prepared using 25b (1.5 mmol, 0.65 g), amine

17 (1.5 mmol, 0.40 g), KI (1.5 mmol, 0.25 g), anhydrous K₂CO₃

(3 mmol, 0.41 g), and acetonitrile (15 mL). Yield 26b (441 mg, 47%,

yellow oil, R_f¼ 0.58 (S₅)). Formula C₃₈H₃₆N₃ClO₃, MW 618.16. ¹H

d

ppm 27.85 (C]CHeCH₂), 31.96 (2 C, Ar-CH₂-CH₂-Ar), 33.69 (-CH₂-

CH₂-OH), 42.47 (NeMe), 43.92 (CO(NH)CH₂), 57.81 (-CH₂-OH),

58.72 (-CH₂-N), 67.55 (NeCH-), 115.29 (C]CH), 115.58 (2 C,

CHeCeF),125.77 (Ar), 126.13 (Ar),127.30 (2 C, Ar),127.61 (Ar),128.11

(2 C, Ar), 128.43 (2 C, Ar), 129.24 (2 C, Ar), 130.20 (Ar), 133.97 (Ar),

134.03 (Ar),136.91 (Ar),139.70 (C ¼ CH),162.05 (d, ¹J_C-F¼ 245.25 Hz,

CeF), 174.15 (C]O). MS: m/z 473 [MþH^þ].

NMR (CDCl₃) d ppm 1.80e1.95 (m, 1 H (NCHCH₂)), 2.10 (s, 3 H (Me)),

2.15e2.34 (m, 3 H (NCHCH₂; ¼CHCH₂)), 2.44e2.55 (m, 2 H (CH₂N)),

2.62e2.79 (m, 1 H (ArCH₂)), 2.83e2.99 (m, 1 H (ArCH₂)), 3.13e3.18

(m, 1 H (NCH)), 3.20e3.46 (m, 2 H (ArCH₂)), 3.69e3.82 (m, 1 H

(CH₂N)), 3.83e4.00 (m, 1 H (CH₂N)), 4.42 (br. s., 2 H (CH₂NH)), 5.73

(t, J ¼ 7.31 Hz, 1 H (C ¼ CH)), 6.95e7.23 (m, 8 H (Ar)), 7.23e7.34 (m,

2 H (Ar)), 7.42e7.63 (m, 3 H (Ar; CONH)), 7.64e7.73 (m, 2 H

(phthalimide)), 7.78e7.89 (m, 2 H (phthalimide)).

5.1.6.5. 4-Hydroxy-2-[methyl(3-{tricyclo[9.4.0.0³,⁸]pentadeca-

1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)amino]-N-[(4-

methylphenyl)methyl]butanamide (24e). Compound 24e was pre-

pared using 21 (0.75 mmol, 239 mg) and p-tolylmethanamine

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5.1.7.3. N-[(4-Chlorophenyl)methyl]-4-(1,3-dioxo-2,3-dihydro-1H-

isoindol-2-yl)-2-[methyl(3-{tricyclo[9.4.0.0³,⁸]pentadeca-

1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)amino]butanamide (26c).

Compound 26c was prepared using 25c (1.5 mmol, 0.65 g), amine

17 (1.5 mmol, 0.40 g), KI (1.5 mmol, 0.25 g), anhydrous K₂CO₃

(3 mmol, 0.41 g), and acetonitrile (15 mL). Yield 26c (601 mg, 64%,

yellow oil, R_f¼ 0.54 (S₅)). Formula C₃₈H₃₆N₃ClO₃, MW 618.16. ¹H

5.1.8.1. 4-Amino-N-benzyl-2-[methyl(3-{tricyclo[9.4.0.0³,⁸]penta-

deca-1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)amino]butanamide

(27a). Compound 27a was prepared using 26a (0.75 mmol, 0.44 g)

and hydrazine hydrate (1.5 mmol, 75 mg) in ethanol (10 mL). Yield

27a (280 mg, 82%, yellow oil, R_f¼ 0.95 (S₁₀)). Formula C₃₀H₃₅N₃O,

MW 453.62. ¹H NMR (CDCl₃)

d ppm 1.69e1.89 (m, 2 H (NCHCH₂)),

2.07 (s, 3 H (Me)), 2.19e2.31 (m, 2 H (¼CHCH₂)), 2.41e2.59 (m, 2 H

(CH₂NH₂)), 2.84e2.98 (m, 2 H (ArCH₂)), 3.08e3.37 (m, 3 H (ArCH₂;

NCH)), 3.97 (br. s., 2 H (ArCH₂)), 4.28 (dd, J ¼ 12.31, 4.87 Hz, 2 H

(CH₂NH)), 5.74 (br. s., 1 H (C ¼ CH)), 6.95e7.34 (m, 13 H (Ar)),

NMR (CDCl₃) d ppm 1.79e1.96 (m, 1 H (NCHCH₂)), 2.05 (s, 3 H (Me)),

2.08e2.35 (m, 3 H (NCHCH₂; ¼CHCH₂)), 2.37e2.57 (m, 2 H (CH₂N)),

2.61e2.78 (m, 1 H (ArCH₂)), 2.81e3.00 (m, 1 H (ArCH₂)), 3.04e3.40

(m, 3 H (ArCH₂; NCH)), 3.67e3.83 (m, 1 H (CH₂N)), 3.84e4.00 (m,

7.53e7.74 (m, 1 H (CONH)). ¹³C NMR (CDCl₃)

d ppm 26.81

1

H

(CH₂N)), 4.06e4.41 (m,

2

H

(CH₂NH)), 5.60e5.78 (m,

(¼CHeCH₂), 31.00 (NeCHeCH₂), 32.61 (2 C, Ar-CH₂-CH₂-Ar), 42.91

(CH₂eNH₂), 43.71 (NeMe), 44.71 (CO(NH)CH₂), 57.80 (CH₂N), 73.12

(NeCH), 115.55 (¼CH), 124.70 (Ar), 125.11 (Ar), 126.10 (2 C, Ar),

126.33 (Ar), 126.37 (Ar), 126.52 (Ar), 128.00 (Ar), 128.11 (Ar), 128.21

(Ar), 128.41 (Ar), 129.20 (2 C, Ar), 135.83 (2 C, Ar), 137.24 (2 C, Ar),

138.21 (Ar), 138.70 (C]CH), 172.70 (C]O). MS: m/z 454 [MþH^þ].

1 H (C ¼ CH)), 6.93e7.33 (m, 12 H (Ar)), 7.41e7.57 (m, 1 H (CONH)),

7.63e7.76 (m, 2 H (phthalimide)), 7.78e7.89 (m, 2 H (phthalimide)).

5.1.7.4. N-[(4-Fluorophenyl)methyl]-4-(1,3-dioxo-2,3-dihydro-1H-

isoindol-2-yl)-2-[methyl(3-{tricyclo[9.4.0.0³,⁸]pentadeca-

1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)amino]butanamide (26d).

Compound 26d was prepared using 25d (1.75 mmol, 0.73 g), amine

17 (1.75 mmol, 0.46 g), KI (1.75 mmol, 0.29 g), anhydrous K₂CO₃

(3.5 mmol, 0.48 g), and acetonitrile (15 mL). Yield 26d (429 mg, 47%,

yellow oil, R_f¼ 0.50 (S₅)). Formula C₃₈H₃₆N₃FO₃, MW 601.71. ¹H

5.1.8.2. 4-Amino-N-[(2-chlorophenyl)methyl]-2-[methyl(3-{tricyclo

[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)

amino]butanamide (27b). Compound 27b was prepared using 26b

(0.7 mmol, 0.43 g) and hydrazine hydrate (1.4 mmol, 70 mg) in

ethanol (7 mL). Yield 27b (221 mg, 65%, yellow oil, R_f¼ 0.89 (S₁₀)).

Formula

C d ppm

₃₀H₃₄ClN₃O, MW 488.31. ¹H NMR (CDCl₃)

NMR (CDCl₃) d ppm 1.80e1.96 (m, 1 H (NCHCH₂)), 2.05 (s, 3 H (Me)),

2.10e2.32 (m, 3 H (NCHCH₂; ¼CHCH₂)), 2.36e2.54 (m, 2 H (CH₂N)),

2.61e2.78 (m, 1 H (ArCH₂)), 2.94 (dd, J ¼ 13.72, 8.59 Hz, 1 H

(ArCH₂)), 3.14 (d, J ¼ 3.59 Hz, 1 H (NCH)), 3.15e3.43 (m, 2 H

(ArCH₂)), 3.77 (dt, J ¼ 13.91, 7.02 Hz, 1 H (CH₂N)), 3.93 (dd, J ¼ 16.16,

7.18 Hz, 1 H (CH₂N)), 4.25 (dd, J ¼ 13.72, 5.26 Hz, 2 H (CH₂NH)),

5.60e5.78 (m, 1 H (C ¼ CH)), 6.83e7.23 (m, 12 H (Ar)), 7.38e7.53 (m,

1 H (CONH)), 7.62e7.73 (m, 2 H (phthalimide)), 7.76e7.88 (m, 2 H

(phthalimide)).

1.73e1.91 (m, 2 H (NCHCH₂)), 2.08 (s, 3 H (Me)), 2.19e2.33 (m, 2 H

(¼CHCH₂)), 2.42e2.58 (m, 2 H (CH₂NH₂)), 2.66e2.80 (m, 1 H

(CH₂N)), 2.80e3.03 (m, 3 H (ArCH₂; CH₂N)), 3.09e3.24 (m, 2 H

(ArCH₂; NCH)), 3.24e3.40 (m, 1 H (ArCH₂)), 4.22e4.53 (m, 4 H

(CH₂NH; -NH₂)), 5.77 (t, J ¼ 6.67 Hz, 1 H (C ¼ CH)), 7.02e7.42 (m,

12 H (Ar)), 7.76 (br. s., 1 H (CONH)). ¹³C NMR (CDCl₃)

d ppm 27.02

(¼CHeCH₂), 31.00 (NeCHeCH₂), 32.63 (2 C, Ar-CH₂-CH₂-Ar), 40.14

(CH₂eNH₂), 43.44 (NeMe), 44.33 (CO(NH)CH₂), 57.61 (CH₂N), 73.00

(NeCH), 115.81 (¼CH), 124.71 (Ar), 125.05 (Ar), 126.05 (Ar), 126.10

(Ar), 126.50 (Ar), 127.02 (Ar), 127.10 (Ar), 127.31 (2 C, Ar), 127.41 (Ar),

127.72 (Ar), 128.42 (Ar), 129.12 (2 C, Ar), 132.42 (CeCl), 134.42 (2 C,

Ar), 135.83 (Ar), 138.11 (C]CH), 172.81 (C]O). MS: m/z 488

[MþH^þ].

5.1.7.5. 4-(1,3-Dioxo-2,3-dihydro-1H-isoindol-2-yl)-2-[methyl(3-

{tricyclo[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,12,14-hexaen-2-ylidene}

propyl)amino]-N-[(4-methylphenyl)methyl]butanamide

(26e).

Compound 26e was prepared using 25e (1.65 mmol, 0.69 g), amine

17 (1.65 mmol, 0.43 g), KI (1.65 mmol, 0.27 g), anhydrous K₂CO₃

(3.3 mmol, 0.46 g), and acetonitrile (15 mL). Yield 26e (439 mg, 45%,

yellow oil, R_f¼ 0.59 (S₅)). Formula C₃₉H₃₉N₃O₃, MW 597.75. ¹H

5.1.8.3. 4-Amino-N-[(4-chlorophenyl)methyl]-2-[methyl(3-{tricyclo

[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)

amino]butanamide (27c). Compound 27c was prepared using 26c

(0.86 mmol, 0.53 g) and hydrazine hydrate (1.68 mmol, 84 mg) in

ethanol (10 mL). Yield 27c (306 mg, 73%, yellow oil, R_f¼ 0.94 (S₁₀)).

NMR (CDCl₃) d ppm 1.80e1.94 (m, 1 H (NCHCH₂)), 2.08 (s, 3 H (Me)),

2.10e2.25 (m, 3 H (NCHCH₂; ¼CHCH₂)), 2.31 (s, 3 H (Me)),

2.39e2.54 (m, 2 H (CH₂N)), 2.60e2.75 (m, 1 H (ArCH₂)), 2.82e3.00

(m, 1 H (ArCH₂)), 3.15 (dd, J ¼ 8.46, 4.10 Hz, 1 H (NCH)), 3.17e3.42

(m, 2 H (ArCH₂)), 3.70e3.84 (m, 1 H (CH₂N)), 3.85e4.01 (m, 1 H

(CH₂N)), 4.20e4.33 (m, 2 H (CH₂NH)), 5.64e5.76 (m, 1 H (C ¼ CH)),

6.91e7.23 (m, 12 H (Ar)), 7.37 (br. s., 1 H (CONH)), 7.64e7.76 (m, 2 H

(phthalimide)), 7.76e7.89 (m, 2 H (phthalimide)).

Formula C₃₀H₃₄ClN₃O, MW 488.31. ¹H NMR (CDCl₃)

d ppm 1.90 (s,

2 H (NCHCH₂)), 2.05 (s, 3 H (Me)), 2.16e2.35 (m, 2 H (¼CHCH₂)),

2.48 (br. s., 2 H (CH₂N)), 2.63e3.02 (m, 4 H (ArCH₂; CH₂NH₂)),

3.05e3.24 (m, 2 H (ArCH₂; NCH)), 3.24e3.43 (m, 1 H (ArCH₂)),

4.08e4.34 (m, 2 H (CH₂NH)), 5.73 (br. s., 1 H (C ¼ CH)), 6.89e7.24

(m,12 H (Ar)), 7.65 (br. s.,1 H (CONH)). ¹³C NMR (CDCl₃)

d ppm 26.70

(¼CHeCH₂), 31.90 (NeCHeCH₂), 32.60 (2 C, Ar-CH₂-CH₂-Ar), 41.31

(CH₂eNH₂), 43.71 (NeMe), 44.62 (CO(NH)CH₂), 57.92 (CH₂N), 73.33

(NeCH), 115.60 (¼CH), 124.71 (Ar), 125.02 (2 C, Ar), 126.25 (Ar),

126.50 (Ar), 127.02 (Ar), 127.10 (Ar), 127.33 (Ar), 127.60 (Ar), 127.81

(2 C, Ar), 129.12 (Ar), 132.00 (CeCl), 135.61 (2 C, Ar), 135.80 (2 C, Ar),

138.21 (Ar), 138.62 (C]CH), 172.72 (C]O). MS: m/z 488 [MþH^þ].

5.1.8. General procedure for the synthesis of 4-amino-N-benzyl-2-

[methyl(3-{tricyclo[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,12,14-hexaen-2-

ylidene}propyl)amino]butanamide derivatives 27(aee)

Two millimoles of hydrazine hydrate (2 equiv) was added to a

suspension of

1 mmol of the relevant 2-substituted 4-

phthalimidobutanoic acid derivative 26(aee) (1 equiv) in ethanol

(10 mL). The reaction was carried out at 60 ^ꢀC for 2 h then cooled to

room temperature, with continued stirring for an additional 5 h.

Next, the precipitate was ﬁltered and washed with methylene

chloride (5 mL). The ﬁltrate was evaporated, and the product was

extracted with DCM (2 ꢁ 10 mL). The combined organic fractions

were dried over Na₂SO₄, and the obtained product was puriﬁed by

column chromatography over silica gel (started with S₅: PE/

EtOAc ¼ 1:1 and changed eluent to S₁₀: 25% NH₃/methanol/DCM/

petroleum ether ¼ 9:45:120:18).

5.1.8.4. 4-Amino-N-[(4-ﬂuorophenyl)methyl]-2-[methyl(3-{tricyclo

[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)

amino]butanamide (27d). Compound 27d was prepared using 26d

(0.69 mmol, 0.42 g) and hydrazine hydrate (1.43 mmol, 72 mg) in

ethanol (7 mL). Yield 27d (300 mg, 91%, yellow oil, R_f¼ 0.90 (S₁₀)).

Formula C₃₀H₃₄FN₃O, MW 471.61. ¹H NMR (CDCl₃)

d ppm 1.71e1.94

(m, 2 H (NCHCH₂)), 2.03 (s, 3 H (Me)), 2.14e2.34 (m, 2 H (¼CHCH₂)),

2.46 (br. s., 2 H (CH₂NH₂)), 2.64e2.79 (m, 1 H (CH₂N)), 2.91 (br. s.,

2 H (ArCH₂)), 3.04e3.14 (m, 2 H (CH₂N; NCH)), 3.18 (dd, J ¼ 8.34,

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European Journal of Medicinal Chemistry 221 (2021) 113512

4.23 Hz,1 H (ArCH₂)), 3.21e3.39 (m,1 H (ArCH₂)), 4.21 (dd, J ¼ 13.98,

5.00 Hz, 2 H (CH₂NH)), 4.83 (br. s., 1 H (C ¼ CH)), 5.65e5.79 (m, 2 H

(Ar)), 6.81e6.97 (m, 2 H (Ar)), 6.97e7.24 (m, 8 H (Ar)), 7.78 (br. s.,1 H

5.1.9.2. N-[(2-Chlorophenyl)methyl]-4-acetamido-2-[methyl(3-{tri-

cyclo[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)

amino]butanamide (28b). Compound 28b was prepared using

acetic acid (0.43 mmol, 26 mg), DCC (0.43 mmol, 89 mg), 27b

(0.43 mmol, 0.21 g) and DMAP (0.22 mmol, 27 mg) in DCM (7 mL).

Yield 28b (221 mg, 97%, yellow oil, R_f¼ 0.47 (S₁)). Formula

(CONH)). ¹³C NMR (CDCl₃)

d

ppm 27.90 (¼CHeCH₂), 31.00

(NeCHeCH₂), 31.97 (2 C, Ar-CH₂-CH₂-Ar), 42.32 (CH₂eNH₂), 43.63

(NeMe), 44.05 (CO(NH)CH₂), 57.62 (CH₂N), 73.12 (NeCH), 115.25

(eCHeC-F),115.53 (eCHeC-F),115.89 (¼CH),124.2 (Ar), 125.00 (2 C,

Ar), 126.31 (Ar), 126.61 (Ar), 127.02 (Ar), 127.43 (2 C, Ar), 128.12 (Ar),

128.31 (Ar), 129.22 (Ar), 133.00 (Ar), 133.21 (Ar), 135.81 (Ar), 138.22

(Ar), 138.63 (C]CH), 161.99 (d, ¹J_C-F¼ 245.24 Hz, CeF), 173.36 (C]

O). MS: m/z 472 [MþH^þ].

C

₃₂H₃₆N₃ClO₂, MW 530.10. ¹H NMR (CDCl₃)

d ppm 1.71e1.86 (m, 2 H

(NCHCH₂)), 1.91 (s, 3 H (Me)), 2.08 (s, 3 H (Me)), 2.18e2.32 (m, 2 H

(¼CHCH₂)), 2.41e2.57 (m, 2 H (CH₂N)), 2.66e2.84 (m, 1 H (CH₂NH)),

2.84e2.99 (m, 1 H (CH₂NH)), 3.06 (dd, J ¼ 3.46, 8.34 Hz, 1 H (NCH)),

3.08e3.25 (m, 2 H (ArCH₂)), 3.27e3.47 (m, 2 H (ArCH₂)), 4.30e4.53

(m, 2 H (CH₂NH)), 5.77 (t, J ¼ 7.18 Hz, 1 H (C ¼ CH)), 6.89e7.14 (m,

6 H (Ar)), 7.14e7.26 (m, 6 H (Ar)), 7.31 (d, J ¼ 6.16 Hz, 1 H (CONH)),

5.1.8.5. 4-Amino-2-[methyl(3-{tricyclo[9.4.0.0³,⁸]pentadeca-

1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)amino]-N-[(4-

methylphenyl)methyl]butanamide (27e). Compound 27e was pre-

pared using 26e (0.69 mmol, 0.41 g) and hydrazine hydrate

(1.4 mmol, 70 mg) in ethanol (10 mL). Yield 27e (253 mg, 79%,

yellow oil, R_f¼ 0.92 (S₁₀)). Formula C₃₁H₃₇N₃O, MW 467.64. ¹H NMR

7.74e7.88 (m, 1 H (CONH)). ¹³C NMR (CDCl₃)

d ppm 23.25 (NH(CO)

Me), 28.03 (¼CHeCH₂), 32.00 (NeCHeCH₂), 33.74 (2 C, Ar-CH₂-CH₂-

Ar), 41.22 (CH₂eNH), 43.51 (NeMe), 44.11 (CO(NH)CH₂), 57.00

(CH₂N), 72.91 (NeCH), 115.13 (¼CH), 125.81 (Ar), 126.10 (Ar), 126.97

(Ar), 127.19 (Ar), 127.60 (Ar), 128.03 (Ar), 128.09 (Ar), 128.40 (Ar),

128.55 (2 C, Ar), 128.85 (2 C, Ar), 129.54 (Ar), 130.06 (Ar), 133.51

(CeCl), 135.49 (Ar), 136.88 (2 C, Ar), 139.27 (C]CH), 170.34 (C]O),

173.90 (C]O). MS: m/z 488 [MþH^þ].

(CDCl₃)

d ppm 1.63e1.93 (m, 2 H (NCHCH₂)), 2.08 (s, 3 H (Me)),

2.17e2.37 (m, 2 H (¼CHCH₂)), 2.50 (t, J ¼ 6.28 Hz, 2 H (CH₂NH₂)),

2.61e2.83 (m, 4 H (CH₂Ar; Me)), 2.90e3.05 (m, 3 H (ArCH₂; CH₂N)),

3.11 (dd, J ¼ 8.34, 4.49 Hz, 1 H (NCH)), 3.17e3.37 (m, 2 H (ArCH₂)),

4.25 (br. s., 2 H (CH₂NH)), 5.76 (t, J ¼ 6.92 Hz, 1 H (C ¼ CH)),

6.92e7.27 (m, 12 H (Ar)), 7.47e7.66 (m, 1 H (CONH)). ¹³C NMR

5.1.9.3. N-[(4-Chlorophenyl)methyl]-4-acetamido-2-[methyl(3-{tri-

cyclo[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)

amino]butanamide (28c). Compound 28c was prepared using acetic

acid (0.60 mmol, 36 mg), DCC (0.60 mmol, 0.12 g), 27c (0.60 mmol,

0.29 g) and DMAP (0.30 mmol, 37 mg) in DCM (8 mL). Yield 28c

(280 mg, 89%, yellow oil, R_f¼ 0.41 (S₁)). Formula C₃₂H₃₆N₃ClO₂,

(CDCl₃)

d

ppm 20.10 (Ar-Me), 26.91 (¼CHeCH₂), 31.02 (NeCHeCH₂),

32.61 (2 C, Ar-CH₂-CH₂-Ar), 41.81 (CH₂eNH₂), 43.91 (NeMe), 44.12

(CO(NH)CH₂), 57.63 (CH₂N), 73.02 (NeCH), 115.63 (¼CH), 124.74

(Ar), 125.03 (Ar), 126.02 (Ar), 126.20 (Ar), 126.51 (2 C, Ar), 126.92

(2 C, Ar), 127.05 (Ar), 127.44 (2 C, Ar), 128.22 (2 C, Ar), 129.03 (Ar),

134.11 (2 C, Ar), 135.81 (C_Ar-Me), 138.21 (Ar), 138.72 (C]CH), 172.61

(C]O). MS: m/z 468 [MþH^þ].

MW 530.10. ¹H NMR (CDCl₃)

d ppm 1.73e1.86 (m, 2 H (NCHCH₂)),

1.92 (s, 3 H (Me), 2.04 (s, 3 H (Me)), 2.15e2.34 (m, 2 H (¼CHCH₂)),

2.39e2.53 (m, 2 H (CH₂N)), 2.64e2.79 (m, 1 H (CH₂NH)), 2.85e2.99

(m, 1 H (CH₂NH)), 3.05 (dd, J ¼ 3.98, 7.82 Hz, 1 H (NCH)), 3.09e3.25

(m, 2 H (ArCH₂)), 3.27e3.46 (m, 2 H (ArCH₂)), 4.23 (dd, J ¼ 5.90,

14.36 Hz, 2 H (CH₂NH)), 5.66e5.77 (m, 1 H (C ¼ CH)), 6.89e7.27 (m,

13 H (CONH; Ar)), 7.70e7.92 (m, 1 H (CONH)). ¹³C NMR (CDCl₃)

5.1.9. General procedure for the synthesis of N-benzyl-4-acetamido-

2-[methyl(3-{tricyclo[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,12,14-hexaen-

2-ylidene}propyl)amino]butanamide derivatives 28(aee)

A mixture of acetic acid (2 equiv) and DCC (2 equiv) in 5 mL of

DCM stirred at 0 ^ꢀC for 10 min. Then, the relevant 4-amino-N-

benzyl-2-[methyl(3-{tricyclo[9.4.0.0³,⁸]pentadeca-

1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)amino]butanamide de-

rivative 27(aee) (1 equiv) and DMAP (2 equiv) were added to the

reaction mixture, and stirring continued for 20 h at room temper-

ature. The obtained DCU was ﬁltered, the ﬁltrate was evaporated,

and the product was puriﬁed by column chromatography over silica

gel (S₁: chloroform/acetone ¼ 1:1).

d

ppm 23.24 (NH(CO)Me), 27.88 (¼CHeCH₂), 31.99 (NeCHeCH₂),

33.71 (2 C, Ar-CH₂-CH₂-Ar), 42.43 (CH₂eNH), 43.81 (NeMe), 44.10

(CO(NH)CH₂), 57.01 (CH₂N), 73.10 (NeCH), 115.61 (¼CH), 125.79

(Ar), 126.12 (Ar), 127.31 (Ar), 127.61 (2 C, Ar), 128.00 (2 C, Ar), 128.19

(Ar), 128.43 (Ar), 128.72 (Ar), 128.89 (2 C, Ar), 130.17 (2 C, Ar), 133.09

(CeCl), 136.84 (Ar), 136.89 (Ar), 139.35 (Ar), 139.71 (C]CH), 170.36

(C]O), 173.81 (C]O). MS: m/z 488 [MþH^þ].

5.1.9.4. N-[(4-Fluorophenyl)methyl]-4-acetamido-2-[methyl(3-{tri-

cyclo[9.4.0.0³,⁸]pentadeca-1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)

amino]butanamide (28d). Compound 28d was prepared using

acetic acid (0.6 mmol, 36 mg), DCC (0.6 mmol, 0.12 g), 27d

(0.6 mmol, 0.28 g) and DMAP (0.3 mmol, 37 mg) in DCM (8 mL).

Yield 28d (256 mg, 84%, yellow oil, R_f¼ 0.38 (S₁)). Formula

5.1.9.1. N-Benzyl-4-acetamido-2-[methyl(3-{tricyclo[9.4.0.0³,⁸]penta-

deca-1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)amino]butanamide

(28a). Compound 28a was prepared using acetic acid (0.60 mmol,

36 mg), DCC (0.60 mmol, 0.12 g), 27a (0.60 mmol, 0.27 g) and DMAP

(0.30 mmol, 37 mg) in DCM (8 mL). Yield 28a (280 mg, 95%, yellow

oil, R_f¼ 0.41 (S₁)). Formula C₃₂H₃₇N₃O₂, MW 495.66. ¹H NMR

C

₃₂H₃₆N₃FO₂, MW 513.65. ¹H NMR (CDCl₃)

d ppm 1.70e1.88 (m, 2 H

(CDCl₃)

d

ppm 1.72e1.90 (m, 2 H (NCHCH₂)), 1.93 (s, 3 H (Me)), 2.06

(NCHCH₂)), 1.93 (s, 3 H (Me)), 2.05 (s, 3 H (Me)), 2.13e2.34 (m, 2 H

(¼CHCH₂)), 2.48 (br. s., 2 H (CH₂N)), 2.65e2.80 (m, 1 H (CH₂NH)),

2.86e2.99 (m, 1 H (CH₂NH)), 3.05 (dd, J ¼ 3.21, 8.34 Hz, 1 H (NCH)),

3.10e3.27 (m, 2 H (ArCH₂)), 3.27e3.63 (m, 2 H (ArCH₂)), 4.25 (dd,

J ¼ 5.13, 13.85 Hz, 2 H (CH₂NH)), 5.64e5.79 (m, 1 H (C ¼ CH)),

6.85e7.22 (m, 13 H (CONH; Ar)), 7.79 (br. s., 1 H (CONH)). ¹³C NMR

(s, 3 H (Me)), 2.26 (dd, J ¼ 5.51, 12.18 Hz, 2 H (¼CHCH₂)), 2.48 (br. s.,

2 H (CH₂N)), 2.65e2.79 (m, 1 H (CH₂NH)), 2.87e3.01 (m, 1 H

(CH₂NH)), 3.06 (dd, J ¼ 3.85, 7.95 Hz, 1 H (NCH)), 3.11e3.27 (m, 2 H

(ArCH₂)), 3.27e3.52 (m, 2 H (ArCH₂)), 4.31 (dd, J ¼ 6.03, 13.98 Hz,

2 H (CH₂NH)), 5.64e5.85 (m, 1 H (C ¼ CH)), 6.95e7.35 (m, 13 H (Ar)),

7.79 (br. s., 2 H (CONH)). ¹³C NMR (CDCl₃)

d

ppm 23.25 (NH(CO)Me),

(CDCl₃)

d

ppm 23.27 (NH(CO)Me), 27.81 (¼CHeCH₂), 31.96

27.94 (¼CHeCH₂), 31.99 (NeCHeCH₂), 33.71 (2 C, Ar-CH₂-CH₂-Ar),

43.13 (CH₂eNH), 43.71 (NeMe), 44.51 (CO(NH)CH₂), 57.31 (CH₂N),

73.71 (NeCH), 115.31 (¼CH), 125.81 (Ar), 126.12 (2 C, Ar), 127.21 (Ar),

127.42 (Ar), 127.51 (Ar), 127.61 (Ar), 128.01 (Ar), 128.10 (Ar), 128.51

(Ar), 128.64 (Ar), 130.16 (2 C, Ar), 136.92 (2 C, Ar), 138.25 (2 C, Ar),

139.31 (Ar), 139.78 (C]CH), 170.38 (C]O), 173.77 (C]O). MS: m/z

454 [MþH^þ].

(NeCHeCH₂), 33.69 (2 C, Ar-CH₂-CH₂-Ar), 42.42 (CH₂eNH), 43.51

(NeMe), 44.41 (CO(NH)CH₂), 57.71 (CH₂N), 73.10 (NeCH), 115.30

(¼CH), 115.41 (2 C, eCHeC-F), 115.59 (2 C, CHeCeF), 125.79 (Ar),

126.12 (Ar), 127.43 (Ar), 127.61 (Ar), 128.02 (Ar), 128.43 (Ar), 129.15

(Ar), 129.26 (Ar), 130.21 (Ar), 134.01 (Ar), 134.10 (Ar), 136.91 (Ar),

1

139.31 (Ar), 139.70 (C]CH), 162.05 (d, J_C-F¼ 246.00 Hz, CeF),

170.37 (C]O), 173.72 (C]O). MS: m/z 472 [MþH^þ].

18

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P. Zare˛ba, K. Sałat, G.C. Hofner et al.

European Journal of Medicinal Chemistry 221 (2021) 113512

5.1.9.5. 4-Acetamido-2-[methyl(3-{tricyclo[9.4.0.0³,⁸]pentadeca-

1(11),3,5,7,12,14-hexaen-2-ylidene}propyl)amino]-N-[(4-

methylphenyl)methyl]butanamide (28e). Compound 28e was pre-

pared using acetic acid (0.51 mmol, 31 mg), DCC (0.51 mmol, 0.11 g),

27e (0.51 mmol, 0.24 g) and DMAP (0.26 mmol, 31 mg) in DCM

(7 mL). Yield 28e (212 mg, 81%, yellow oil, R_f¼ 0.42 (S₁)). Formula

and solvated with TIP3P water molecules. The system size was

100 Å ꢁ 100 Å. A water pore for each complex was generated. So-

dium and chloride ions (0.15 M NaCl) were added to provide

standard physiological ionic strength. The system was equilibrated

via a six-step protocol recommended by CHARMM-GUI for the

NAMD program. MD simulations were run at 303.15 K with a

timestep of 2 fs and a total duration of 10 ns. The interval for both

the energy and trajectory recordings were 10 ps. The results were

analyzed with the VMD program.

C

₃₃H₃₉N₃O₂, MW 509.68. ¹H NMR (CDCl₃)

d ppm 1.72e1.88 (m, 2 H

(NCHCH₂)), 1.94 (s, 3 H (Me)), 2.06 (s, 3 H (Me)), 2.19e2.29 (m, 2 H

(¼CHCH₂)), 2.32 (s, 3 H), 2.39e2.55 (m, 2 H (CH₂N)), 2.72 (br. s., 1 H

(CH₂NH)), 2.85e3.01 (m, 1 H (CH₂NH)), 3.05 (dd, J ¼ 3.21, 8.34 Hz,

1 H (NCH)), 3.19 (br. s., 2 H (ArCH₂)), 3.30e3.56 (m, 2 H (ArCH₂)),

4.27 (dd, J ¼ 5.13, 12.82 Hz, 2 H (CH₂NH)), 5.74 (br. s., 1 H (C ¼ CH)),

6.89e7.29 (m, 13 H (CONH; Ar)), 7.64e7.81 (m, 1 H (CONH)). ¹³C

5.3. Hepatotoxicity and cytotoxicity

Hepatotoxicity and cytotoxicity were estimated according to

previously described protocols [77] using the hepatoma HepG2

(ATCC HB-8065™) and human embryonic kidney HEKe293 (ATCC®

CRL:1573) cell lines, respectively. In brief, cells were seeded in 96-

well plates at a concentration of 0.7 ꢁ 10⁴and cultured at 37 ^ꢀC in

an atmosphere containing 5% CO₂. Next, the compounds were

added and investigated in quadruplicate at concentrations ranging

NMR (CDCl₃)

d ppm 21.10 (Ar-Me), 23.27 (NH(CO)Me), 27.92

(¼CHeCH₂), 31.99 (NeCHeCH₂), 33.71 (2 C, Ar-CH₂-CH₂-Ar), 42.93

(CH₂eNH), 43.61 (NeMe), 44.11 (CO(NH)CH₂), 57.41 (CH₂N), 72.90

(NeCH), 115.51 (¼CH), 125.79 (Ar), 126.09 (Ar), 127.21 (Ar), 127.54

(Ar), 127.61 (2 C, Ar), 128.05 (Ar), 128.15 (Ar), 128.51 (2 C, Ar), 129.31

(2 C, Ar), 130.12 (Ar), 135.18 (2 C, Ar), 136.94 (C_Ar-Me), 139.78 (2 C,

Ar), 139.81 (C]CH), 170.35 (C]O), 173.69 (C]O). MS: m/z 468

[MþH^þ].

from 0.1 to 100 mM for 72 h. The antiproliferative drug doxorubicin

was used as the reference. The CellTiter 96® AQueous Non-

Radioactive Cell Proliferation Assay (MTS) purchased from Prom-

ega (Madison, WI, USA) was used for the determination of cell

viability. The absorbance at 492 nm was measured using a micro-

plate reader (PerkinElmer).

5.2. Molecular modeling

5.2.1. Docking studies

We used models of human GABA transporters that were

selected in our previous work [43]. They were built with the SWISS-

MODEL server based on the 4XP9 (GAT-1, GAT-2, GAT-3) or 4XP4

(BGT-1) template from the PDB. We used sequence alignment

generated automatically by SWISS-MODEL. N- and C-termini were

omitted because of their low homology. Sodium and chloride ions

were transferred directly from the templates.

5.4. In vitro activity

5.4.1. [³H] GABA uptake assay

The inhibitory activities of the synthesized compounds were

determined in a [³H]GABA uptake assay as described [26], and all

compounds were tested at a screening concentration of 100 mM.

The ligand 3D structures were created in the Maestro program.

Ionization states were predicted under physiological conditions

(pH 7.4) using the Marvin program. Ligands were optimized in the

LigPrep module. All possible stereoisomers for each ligand were

generated. Models were prepared in Protein Preparation Wizard

using default settings.

5.4.2. MS Binding Assays

MS Binding Assays for mGAT1 were performed as described

earlier [53]. Inhibition of mGAT1 binding by the synthesized com-

pounds was determined in MS Binding Assays at a screening con-

centration of 100 mM.

To better ﬁt the models to the examined compounds, the most

active representatives were initially docked into the models of each

type of GABA transporter using the induced ﬁt docking protocol

5.5. In vivo evaluation

€

available in the Schrodinger Suite. The box center was deﬁned by

5.5.1. Animals and housing conditions

residues PHE294, TYR140, TYR452, and ARG69 in GAT-1 and by the

corresponding amino acids in BGT-1, GAT-2, and GAT-3. The box

size was 10 Å ꢁ 10 Å ꢁ 10 Å. The obtained complexes were then

visually inspected in terms of the created interactions, frequency

and score of the poses as well as their coherency between the

particular types of GABA transporters. The compliance with previ-

ous observations of the binding mode for other 4-

aminobutanamide and 4-hydroxybutanamide derivatives was also

taken into account [43]. After selecting the best optimized models,

all studied compounds were docked into the models using the

GLIDE program, and the ﬁnal models were selected based on ligand

pose coherency. The grid center in Glide docking was set as the

centroid of the ligand from the complex, and the inner box size was

10 Å ꢁ 10 Å ꢁ 10 Å. The OPLS2005 force ﬁeld was applied during

grid generation as well as GLIDE and IFD docking. The binding

modes were visualized in the PyMOL program.

Adult male Albino Swiss (CD-1) mice weighing between 18 g

and 22 g were used in all in vivo experiments, except for the tail

suspension test in which C57BL/6J mice of the same age and body

weight were used (Animalab, Poland). The animals were kept in

groups of 10 mice in cages at room temperature (22 2 ^ꢀC) under a

light/dark (12:12) cycle and had free access to food and water

before the experiments. The ambient temperature of the room and

humidity were kept consistent throughout all the tests. For the

experiments, the animals were selected randomly. Each group

consisted of 6e12 animals/dose, and each mouse was used only

once. The experiments were performed between 8 a.m. and 3 p.m.

Immediately after each assay, the animals were euthanized by

cervical dislocation. All procedures were approved by the Local

Ethics Committee of Jagiellonian University in Cracow (ZI/595/

2011). To avoid potential bias in data recording, the investigators

who were involved in behavioral tests were blinded to the exper-

imental groups.

5.2.2. Molecular dynamics

MD simulations were performed in NAMD using the

CHARMM36 m force ﬁeld. Before simulations, all models were

positioned in the membrane using the OPM server, and input ﬁles

for NAMD were prepared with the CHARMM-GUI online server. The

protein-ligand complexes were embedded in a POPC membrane

5.5.2. Test compounds

For the in vivo tests. Compounds 23a and 24e were suspended in

1% Tween 80 solution (Polskie Odczynniki Chemiczne, Poland).

Control mice received 1% Tween 80 solution. Both vehicle and test

compounds were injected intraperitoneally (i.p.) 60 min before the

19

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P. Zare˛ba, K. Sałat, G.C. Hofner et al.

European Journal of Medicinal Chemistry 221 (2021) 113512

experiments. In tests assessing anticonvulsant activity, 100 mg/kg

was the starting dose. In the other behavioral assays, 30 mg/kg was

the highest (and starting) dose. These doses were selected based on

our previous preliminary experiments as well as earlier studies that

assessed pharmacological properties of other GAT inhibitors [46].

5.5.4. Pain tests

5.5.4.1. Hot plate test. This study was conducted based on the

method previously described by Eddy and Leimbach [79]. The de-

vice used in the hot plate test (hot/cold plate, Bioseb, France) is a

heated metal plate surrounded by Plexiglas walls and covered by a

glass lid to prevent the mice from escaping from the plate. The

animals were individually placed on the center of the plate (heated

to a constant temperature of 55 ^ꢀC) for a maximum period of 1 min

(to prevent tissue damage). The measured parameter was the la-

tency to the occurrence of nociceptive reaction (i.e., hind paw

licking or jumping).

5.5.3. Anticonvulsant activity

Two mouse models of electrically induced seizures (i.e., elec-

troconvulsive threshold test and maximal electroshock seizure

test) and two models of chemically induced seizures (i.e., pentyle-

netetrazole seizures and pilocarpine-induced seizures) were used

to assess whether compounds 23a and 24e could protect mice from

seizures.

5.5.4.2. Formalin test. The formalin test was performed based on

the method previously described by Laughlin et al. [80]. In rodents,

the injection of diluted formalin solution evokes a biphasic noci-

fensive behavioral response (licking or biting the injected paw). The

ﬁrst (acute, neurogenic) phase of the test lasts for 5 min, while the

second (late, inﬂammatory) phase occurs between 15 and 30 min

5.5.3.1. Electroconvulsive threshold test. In the electroconvulsive

threshold test (ECT test), the anticonvulsant efﬁcacy of the test

compounds at a dose of 100 mg/kg was evaluated at their previ-

ously established time of peak drug effect (60 min after i.p. Injec-

tion) according to

a

procedure recently described [28].

after formalin injection. In this assay, 20 ml of 5% formalin solution

Electroconvulsions were produced by an alternating current

(duration of the stimulus: 0.2 s; 50 Hz) delivered via standard

auricular electrodes by an electroshock generator (Hugo Sachs ro-

dent shocker, Germany). Tonic hind limb extension (i.e., the hind

limbs outstretched 180^ꢀto the plane of the body axis) was an in-

dicator of seizure episodes. For evaluation of the ECT, at least four

groups of animals per dose were used (each group consisted of 8

animals). These mice were challenged with electroshocks of various

intensities to yield 10e30%, 30e50%, 50e70% and 70e90% of ani-

mals with seizures. Then, a median current strength value (CS₅₀, in

mA), deﬁned as the current intensity required to induce tonic hind

limb extension in 50% of the challenged mice, was estimated by

means of the log-probit method [78].

was injected into the dorsal surface of the right hind paw of each

mouse. Then, the mice were placed separately into glass beakers

and observed for the next 30 min. The total time spent licking or

biting the formalin-injected paw was measured during the ﬁrst

5 min of the test and then between 15 and 30 min of the test in both

drug-treated and vehicle-treated mice.

5.5.5. Antidepressant-like activity

5.5.5.1. Forced swim test. The forced swim test was performed

based on the method previously described by Porsolt et al. [81]. The

mice were dropped individually into glass cylinders (height 25 cm,

diameter 10 cm) ﬁlled with water to a height of 10 cm (maintained

at 23e25 ^ꢀC). In this assay, after an initial 2-min period of vigorous

activity, each mouse assumes an immobile posture. The duration of

immobility in the experimental groups was recorded during the

ﬁnal 4 min of the total 6-min testing period. Mice were judged to be

immobile when they remained ﬂoating passively in the water,

making only small movements to keep their heads above the water

surface.

5.5.3.2. Maximal electroshock seizure test. The maximal electro-

shock test (MES test) was performed as previously described [28].

Brieﬂy, the vehicle-treated mice and drug-treated mice received a

stimulus of sufﬁcient intensity (25 mA) delivered by an electro-

shock generator (Hugo Sachs rodent shocker, Germany) to induce

maximal seizures (tonic extension) of hind limbs. Electro-

convulsions were produced with the use of auricular electrodes,

and the stimulus duration was 0.2 s. The endpoint was the tonic

extension of the hind limbs.

5.5.5.2. Tail suspension test. The tail suspension test was performed

based on the method previously described by Steru et al. [82].

C57BL/6J mice were i.p. administered the test compound or vehicle.

Each mouse was suspended for 6 min by the tail (2 cm from the end

of the tail) using adhesive tape, 75 cm above the surface of the

table. The experiment lasted 6 min, and the total duration of

immobility (in seconds) was measured. Immobility was deﬁned as

the absence of any limb or body movements, except for those

caused by respiration.

5.5.3.3. Pentylenetetrazole-induced seizures test. This study was

performed based on the method previously described by Kowalc-

zyk et al. [46]. Mice were administered a test compound or vehicle,

and 60 min later, they were subcutaneously (s.c.) injected with

pentylenetetrazole at a dose of 100 mg/kg to induce seizures. Then,

the animals were individually placed under glass cylinders for

30 min for the purpose of observation. Measured parameters were

latency to the ﬁrst seizure episode and the number of seizure ep-

isodes during the 30-min observation period.

5.5.6. Anxiolytic-like activity

5.5.6.1. Four-plate test. A four-plate test was performed based on

the method previously described by Aron et al. [83]. The four-plate

test device (Bioseb, France) consists of a rectangular chamber

(18 cm ꢁ 26 cm ꢁ 16 cm) covered by a PVC-constructed lid. The

bottom of the apparatus contains four metal plates (each with di-

mensions of 8 cm ꢁ 11 cm), each separated by a 4 mm-long gap

between them. The bottom of the chamber is connected to the

electric impulse generator. Mice were individually placed in the

device. The ﬁrst 15 s was the habituation period, while during the

next 60 s, the test was carried out. Each time a mouse moved from

one metal plate to another, an electric impulse (0.8 mA, duration of

0.5 s) was applied. The parameter measured in the four-plate test

was the number of “punished” crossings made by the mouse when

moving from one plate to another.

5.5.3.4. Pilocarpine-induced seizures test. The study was performed

based on the method previously described by Kowalczyk et al. [46].

Mice were administered a test compound or vehicle. Sixty minutes

later, pilocarpine (Sigma Aldrich, Poland) was intraperitoneally

(i.p.) injected at a dose of 400 mg/kg in order to induce seizures. To

reduce the effects resulting from peripheral parasympathetic

stimulation, 45 min before pilocarpine administration, the mice

were injected (i.p.) with scopolamine hydrobromide (Sopharma,

Poland) at a dose of 1 mg/kg. After the administration of pilocar-

pine, the mice were individually placed under glass cylinders for a

60-min observation period. Latency to status epilepticus was

measured in each group.

20

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P. Zare˛ba, K. Sałat, G.C. Hofner et al.

European Journal of Medicinal Chemistry 221 (2021) 113512

5.5.6.2. Elevated plus maze. The elevated plus maze test was per-

formed based on the method previously described by Walf and Frye

[84]. The elevated plus maze for mice consists of two opposing

National Science Center in Poland as part of grants DEC-2012/05/B/

NZ7/02705 (anticonvulsant, antidepressant-like and anxiolytic-like

activities) and DEC-2014/15/B/NZ7/00930 (analgesic activity).

open arms (30 cm

ꢁ

5

cm) and two enclosed arms

(30 cm ꢁ 5 cm ꢁ 25 cm) connected by a central platform forming

the shape of a plus sign. The dimensions of the central ﬁeld that

connects the open and closed arms are 5 cm ꢁ 5 cm. In this test,

each mouse was individually placed at the central ﬁeld of the

apparatus with its head turned toward one of the closed arms. The

behavior of each animal over 5 min was observed and recorded by

means of a video camera (Sony Digital, Japan), which was ﬁxed to

the wall above the elevated plus maze. In this test, the following

parameters were measured in drug-treated and control animals:

time spent in the open arms and the percentage ratio between the

number of entries into the open arms and the number of entries

into all of the arms. To exclude the impact of excrements or smell

left by a previous mouse on the behavior of the next mouse, the

device was carefully cleaned after each testing session.

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113512.

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g-Aminobutyric acid transporters as relevant

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Development of tricyclic N-benzyl-4-hydroxybutanamide derivatives as inhibitors of GABA transporters mGAT1-4 with anticonvulsant, antinociceptive, and antidepressant activity

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