Novel Functionalized Amino Acids as Inhibitors of GABA Transporters with Analgesic Activity

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Research Article

Novel Functionalized Amino Acids as Inhibitors of GABA

Transporters with Analgesic Activity

Beata Gryz ł o, Paula Zar ę ba,* Katarzyna Malawska, Gabriela Mazur, Anna Rapacz, Kamil Ł a t ̧ ka,

Georg C. H ö fner, Gniewomir Latacz, Marek Bajda, Kinga Sa ł at, Klaus T. Wanner, Barbara Malawska,

and Katarzyna Kulig

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ABSTRACT: Neuropathic pain resistance to pharmacotherapy has encouraged researchers to develop eﬀective therapies for its

treatment. γ-Aminobutyric acid (GABA) transporters 1 and 4 (mGAT1 and mGAT4) have been increasingly recognized as

promising drug targets for neuropathic pain (NP) associated with imbalances in inhibitory neurotransmission. In this context, we

designed and synthesized new functionalized amino acids as inhibitors of GABA uptake and assessed their activities toward all four

mouse GAT subtypes (mGAT1−4). According to the obtained results, compounds 2RS,4RS-39c (pIC₅₀(mGAT4) = 5.36), 50a

(pIC₅₀(mGAT2) = 5.43), and 56a (with moderate subtype selectivity that favored mGAT4, pIC₅₀(mGAT4) = 5.04) were of

particular interest and were therefore evaluated for their cytotoxic and hepatotoxic eﬀects. In a set of in vivo experiments, both

compounds 50a and 56a showed antinociceptive properties in three rodent models of NP, namely, chemotherapy-induced

neuropathic pain models (the oxaliplatin model and the paclitaxel model) and the diabetic neuropathic pain model induced by

streptozotocin; however compound 56a demonstrated predominant activity. Since impaired motor coordination is also observed in

neuropathic pain conditions, we have pointed out that none of the test compounds induced motor deﬁcits in the rotarod test.

KEYWORDS: GABA transporters, mGAT1−4 inhibitors, [³H]GABA uptake, neuropathic pain models, antiallodynic activity,

antihyperalgesic activity

GABA speciﬁc transport systems represent a mechanism that

regulates the eﬃciency of GABA transmission; thus, since

1990, the family of GAT sodium symporters has become an

interesting biological target. Cloning of several GATs has led

to a better understanding of the molecular properties of this

solute carrier family, Figure 1. GATs have a confusing

numbering system; hence, we present a summary of the

current nomenclature based on that from the International

1. INTRODUCTION

The γ-aminobutyric acid (GABA) is a neurotransmitter known

for its inhibitory modulation of neuronal networks.^1,2

Endogenous GABA is synthesized from glutamate^3,4and

controls the generation of membrane potential oscillations by

acting on two types of receptors, ionotropic (GABA_A) and

metabotropic (GABA_B) as summarized in Figure 1.⁵Plasma

membrane transporters of GABA (GATs) are components of

one of the pathways responsible for terminating inhibitory

signaling. GATs expression in diﬀerent cell types is highly

dynamic and can be modiﬁed depending on the activity.

Reuptake achieved through GATs occurs in nerve terminals

(allowing GABA to be recycled as a neurotransmitter) and/or

the surrounding glial cells, whereby glial GATs are responsible

for 20% reuptake of GABA, Figure 1.⁶

Received: May 29, 2021

Accepted: July 16, 2021

American Chemical Society

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A

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Figure 1. Diagram of GABA transport systems in neurons and glia (A) and stoichiometry between GABA and co-ions in GAT1 and GAT3 (B).

Figure 2. Structures and inhibitory activities (pIC₅₀values) of GAT substrates (1,¹⁸2,¹⁸3,¹⁹4¹⁹) and GAT inhibitors (5−12) separated according

to their transporter subtype selectivity: mGAT1 (tiagabine (5),²⁰DDPM-2571 (6),²⁰7,²¹NO711 (8)¹⁹) and mGAT4 ((S)-SNAP-5114 (9),²²

DDPM-859 (10),²²11,²³12²⁴).

Union of Basic and Clinical Pharmacology (IUPHAR). Human

(and rat) GAT1 (SLC6A1), GAT2 (SLC6A13), GAT3

(SLC6A11), and BGT1 (SLC6A12)⁷correspond to mouse

mGAT1, mGAT2, mGAT3, and mGAT4, respectively.⁸In this

paper, we present the results of an in vitro test on murine

GATs, and the mouse nomenclature (mGAT1−GAT4) will be

used in a later section.

levels observed in the kidneys and at the brain surface in the

leptomeninges.^9,10mGAT3 is a second GABA transporter

distributed mainly in the peripheral tissues, and since there is a

lack of selective and potent mGAT3 inhibitors, the function of

mGAT3 remains unclear.⁹⁻¹¹Due to the diversity in GAT

subtype localization and function, researchers have focused on

the synthesis of subtype-selective inhibitors.

mGAT1 (representing neuronal uptake) and mGAT4

(mediating transport into glial cells) are mainly localized in

the central nervous system (CNS). The peripherally located

mGAT2 (BGT1) is largely expressed in the liver, with lower

Small amino acids are known to be GAT substrates;

moreover, some substrate preferences toward GAT subtypes

are well established. All four GATs can transport GABA (1).

Additionally, β-alanine (2) is a substrate for both mGAT3 and

B

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Figure 3. Schematic summary of the structural modiﬁcation approach from parent compound 13.²⁵

Scheme 1. Synthesis of the 4-Hydroxypentanamide (37a−c, 39a−c, 41, 43a,b, 45) and 4-Hydroxybutanamide (47a−c)

a

Derivatives

a

Reagents and conditions: (a) suitable amine (17 (A), 21 (C), 25 (D), 26 (E), or 32 (F)), TBAB, K₂CO₃, CH₃CN, 15 min at 0 °C and 16 h at rt;

(b) argon, dry THF, reﬂux, 48 h.

mGAT4 with a low aﬃnity for mGAT1 and mGAT2. A

functional approach based on small molecules, such as (R)-

nipecotic acid (3) or guvacine (4), has resulted in the synthesis

of many subtype-selective inhibitors (Figure 2). Eﬀective

blockade of the uptake toward GAT is believed to have

therapeutic value for not only epileptic seizures but also

neuropathic pain (NP) and several abnormalities, including

tremors, ataxia, and nervousness.¹²mGAT1 inhibitors are the

most potent compounds, and a wide range of these subtype-

selective inhibitors are known (5−8, Figure 2). One example is

the mGAT1 selective compound tiagabine (5), which has been

approved by the FDA for adjunctive treatment of seizures.

Furthermore, tiagabine (5) turned out to be highly eﬀective in

various rodent neuropathic pain models.^13,14The guvacine

derivative DDPM-257 (6) is another selective mGAT1

inhibitor that is eﬀective in mouse models of seizures, anxiety,

depression, and acute and tonic pain.¹⁵Moreover, mGAT4

remains also a challenging target, especially since mGAT4

inhibitors seem to be suitable for antinociceptive activity.^16,17

In this context, the GABA transporters were found to be

interesting biological targets in the search for new treatment of

NP. Nevertheless, due to the low to moderate subtype

selectivity of 9−12, the development of new selective

inhibitors remains an important approach for distinguishing

non-mGAT1 pharmacology (Figure 2).

We previously obtained a series of GABA analogs with

mGAT3/4 subtype preference with the most interesting

compound 13 that could reduce tactile allodynia in neuro-

pathic mice.²⁵In this paper, we present a continuation of our

previous work with new derivatives that can be classiﬁed as

C

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a

Scheme 2. Synthesis of the 4-Aminobutanamide (50a,b) and 4-Acetamidobutanamide (51a,b) Derivatives

a

Reagents and conditions: (a) KI, K₂CO₃, CH₃CN, reﬂux, 24 h; (b) NH₂NH₂(50−60%), 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.

a

Scheme 3. Synthesis of 3-Hydroxypropanamide Derivatives (54a−c, 55a−e, 56a−e, 57, 58, and 59a−c)

a

Reagents and conditions: (a) T3P, TEA, N-benzylamine derivative, dry DCM, −17 °C for 30 min and rt for 1 h, argon; (b) suitable amine (17

(A), 20 (B), 21 (C), 25 (D), 26 (E), or 32 (F)), DIPEA, TBAB, dry DMF, reﬂux, 12 h.

analogs of parent compound 13. A summary of these

modiﬁcations is presented in Figure 3. The ﬁrst purpose of

this study was to investigate how stiﬀening the lipophilic

fragment in the second position of the N-benzylamide

derivatives aﬀects subtype preference and/or mGAT1−4

transporter inhibition compared to the more ﬂexible analogs.

Therefore, we introduced a piperidine ring to replace the

ﬂexible carbon chain (the lipophilic fragment; blue rectangle,

Figure 3). To maintain an analogous structure, bisthiophene,

ﬂuorenyl, or suberenone was introduced in the 4-position of

the piperidine ring (fragment 3, Figure 3). Moreover,

motivated by the inhibitory activity of NO711 (8), we decided

to introduce an oxime subunit into the 4-position of the 4-

hydroxy- and 4-aminobutanamide derivatives. This moiety is

interesting for the structure−activity relationship (SAR)

discussion due to its potential ability to impact the binding

mode of mGAT1 inhibitors.²⁶Second, on the basis of the fact

that a large number of mGAT1 ligands possess a carboxylic

acid fragment, we previously synthesized propanoic acid ethyl

and benzyl ester derivatives for hydrolysis into the correspond-

ing carboxylic analogs of the parent N-benzamides (fragment

1, Figure 3). The last structural change was to introduce

variation into the length of the main carbon chain. Therefore,

the synthesized compounds are 3−5 carbon atoms in length.

We exchanged the hydroxyl/amino groups for methyl or

isopropyl groups to determine whether the presence of

hydrogen bond donors aﬀects GAT inhibitory potency in the

present group of compounds. To explore the molecular

interactions of novel obtained GABA uptake inhibitors with

GABA transporters, computational docking and molecular

dynamics studies have been performed. Finally, to conﬁrm the

therapeutic potential of the obtained compounds, we tested

selected the most potent compounds in in vitro assays, for their

antiallodynic and antihyperalgesic activities in three rodent

models of NP.

2. RESULTS AND DISCUSSION

2.1. Chemistry. To synthesize the target compounds, we

used secondary amines 17 (A), 20 (B), 21 (C), 25 (D), 26

(E), 32 (F), and 33 (G). Amines were prepared according to

D

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a

Scheme 4. Synthesis of 2-Substituted Derivatives of Propanoic Acid and Butanoic Acid (64a,b, 65, 66a,b, 67, 68, and 71−75)

a

Reagents and conditions: (a) T3P, TEA, N-benzylamine derivative, dry DCM, −17 °C for 30 min and 1 h at rt, argon; (b) suitable amine (17 (A),

32 (F), or 33 (G)), DIPEA, TBAB, dry DCM, reﬂux, 12 h; (c) suitable amine (20 (B) or 33 (G)), K₂CO₃, DCM reﬂux, 12 h; (d) MeOH, 10 wt %

NaOH, 35 °C, 4.5 h.

the synthetic route presented in Scheme S1 (see Supporting

Information).^{27−30,32−39}

2.1.3. Synthesis of Propanoic Acid and Butanoic Acid

Derivatives (64a,b, 65, 66a,b, 67, 68, and 71−75). The 2-

substituted derivatives of propanoic acid and butanoic acid

(64a,b, 65, 66a,b, 67, 68, and 71−75) were formed from N-

benzylamides (62 or 63a,b, Scheme 4 panel A) or

commercially available esters (69 or 70, Scheme 4 panel B)

following the synthetic route shown in Scheme 4. Alkylation of

amines 17 (A), 32 (F), or 33 (G) with 2-bromo-3-propanoic

acid (60) or 2-bromobutanoic acid (61) gave N-benzylamides

64a,b, 65, 66a,b, 67, and 68. A similar reaction was carried out

for amines 20 (B) and 33 (G) with 2-bromo-3-propanoic ethyl

ester (69) or 2-bromo-3-propanoic benzyl ester (70) to obtain

desired compounds 71−73. Carboxylic acid analogs 74 and 75

were the alkaline hydrolysis products of parent compounds 71

and 73.

2.2. In Vitro Evaluation and SAR. The inhibitory

potencies of all obtained ﬁnal compounds were determined

for the four mouse GABA transporter subtypes (mGAT1−4).

The assay utilized was based on [³H]GABA uptake using

human embryonic kidney cells (HEK-293) stably expressing

mouse GATs according to the literature.¹⁹Speciﬁc binding

aﬃnity toward mGAT1 was determined via a competitive mass

spectrometry (MS) binding assay quantiﬁed by liquid

chromatography−electrospray ionization tandem mass spec-

trometry (LC−ESI-MS/MS) with NO711 as an unlabeled

marker.⁴⁸The compounds that could reduce GABA uptake or

NO711 binding by at least 50% at an inhibitor concentration

of 100 μM were considered active. The pIC₅₀or pK_ivalues

from the [³H]GABA uptake or MS binding assays were

determined in triplicate samples for competition and in three

2.1.1. Synthesis of the 4-Hydroxypentanamide (37a−c,

39a−c, 41, 43a,b, 45), 4-Hydroxybutanamide (47a−c), 4-

Aminobutanamide (50a,b), and 4-Acetamidobutanamide

(51a,b) Derivatives. The designed target 4-hydroxypentana-

mide (37a−c, 39a−c, 41, 43a,b, 45) and 4-hydroxybutana-

mide (47a−c) derivatives were obtained in the two main steps.

First, nucleophilic substitution between building blocks 17

(A), 21 (C), 25 (D), 26 (E), or 32 (F) and 3-bromo-5-

methyldihydrofuran-2(3H)-one (34) or 3-bromodihydrofuran-

2(3H)-one (35) was performed to obtain compounds 36, 38,

40, 42, 44, and 46 (Scheme 1).⁴⁰Then, aminolysis was

conducted following previously described synthetic proce-

dures.^25,40−43Compounds 37a−c, 39a−c, 41, 43a,b, and 45

were isolated in pure form as a mixture of racemic

diastereomers, e.g., 2RS,4RS-45 and 2RS,4SR-45, with a

diastereoselectivity (ds) of approximately 7:3 (see the

experimental section).

The 4-aminobutanamide derivatives (50a,b) and 4-acet-

amidobutanamide derivatives (51a,b) were prepared according

to the synthetic route shown in Scheme 2. Compounds 50a

and 50b were obtained from previously described synthetic

procedures^44,45via the N-alkylation of 32 (F) with 48a,b and

the hydrazinolysis of 49a,b. Acetylation of 50a and 50b gave

51a and 51b, respectively (Scheme 2).^25,46

2.1.2. Synthesis of 3-Hydroxypropanamide Derivatives

(54a−c, 55a−e, 56a−e, 57, 58, and 59a−c). 3-Hydrox-

ypropanamide derivatives were obtained according to a

previously reported synthetic route shown in Scheme 3.^25,47

2-Bromo-3-hydroxypropanoic acid (52) has been obtained

from unprotected serine. Designed amides (53a−e) were

formed after activation of acid 52 by n-propanephosphoric acid

anhydride (T3P).

Alkylation of amine 17 (A), 20 (B), 21 (C), 25 (D), 26 (E),

or 32 (F) by 2-bromo-3-hydroxypropanoic acid N-benzyla-

mide (53a−e) was carried out overnight at reﬂux in dry

dimethylformamide (DMF) with N,N-diisopropylethylamine

(DIPEA) and tetra-n-butylammonium bromide (TBAB).

independent experiments only for compounds with pIC₅₀

≥

5.00. If at a screening concentration of 100 μM the test

compounds could not reduce [³H]GABA uptake or NO711

binding below 50% (pIC₅₀= 4.00), the percent of remaining

[³H]GABA uptake or NO711 binding is given in the presence

of 100 μM inhibitor.

In the current approach, we focused on diversifying

lipophilic side chain substituents at the α position of N-

benzylamides. Newly synthesized 4-hydroxypentanoic acid

derivatives possessing a suberone-N-methylpropan-1-amine

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Table 1. Inhibitory Potencies (pIC₅₀Standard Error of the Mean (SEM)) toward mGAT1−4 Determined by the [³H]GABA

Uptake Experiments and mGAT1 Binding Aﬃnities (pK_iSEM) from the MS Binding Assays of the Obtained Compounds

a

pIC₅₀

mGAT2

4.61

SEM

mGAT3

pK_i

SEM

compd

R¹

R²

R³

R⁴

mGAT1

4.58

4.53

100 μM: 52%

100 μM: 50%

4.75

mGAT4

4.14

mGAT1

2RS,4RS-37a

2RS,4SR-37a

2RS,4RS-37b

2RS,4SR-37b

2RS,4RS-37c

2RS,4SR-37c

2RS,4RS-39a

2RS,4RS-39b

2RS,4SR-39b

2RS,4RS-39c

2RS,4SR-39c

2RS,4RS-41a

2RS,4SR-41a

2RS,4RS-43a

2RS,4SR-43a

2RS,4RS-43b

2RS,4SR-43b

2RS,4RS-45

2RS,4SR-45

47a

47b

47c

50a

50b

51a

51b

tiagabine (5)¹⁹

(S)-SNAP-5114 (3)¹⁹

DDPM-859 (4)²²

DDPM-2571 (5)²⁰

13²⁵

Me

H

A

C

D

E

F

H

OH

NH₂

4.38

100 μM: 74%

100 μM: 78%

100 μM: 94%

100 μM: 108%

100 μM: 76%

100 μM: 73%

100 μM: 110%

100 μM: 84%

100 μM: 87%

100 μM: 69%

100 μM: 73%

100 μM: 92%

100 μM: 89%

100 μM: 77%

100 μM: 73%

100 μM: 86%

100 μM: 102%

100 μM: 100%

100 μM: 86%

100 μM: 87%

100 μM: 86%

100 μM: 82%

4.48 0.13

100 μM: 60%

100 μM: 58%

100 μM: 69%

4.80

4.23

4.90

100 μM: 73%

4.69

5.09

4.13

4.21

4.03

4.14

4.94 0.07

4.55

4.83

4.62

4.99

5.18 0.07

5.10 0.07

4.19

4.26

4.67

4.55

4.42

100 μM: 87%

4.70 0.05

4.74 0.09

4.74

5.04 0.04

4.96 0.05

5.07 0.10

5.32 0.10

5.03 0.05

5.17 0.07

100 μM: 64%

5.29 0.04

4.85 0.04

4.35

4.56

4.21

100 μM: 56%

4.71

4.8

2-Cl

4-F

4-Cl

4-Me

H

4.71

100 μM: 51%

100 μM: 56%

100 μM: 51%

100 μM: 52%

100 μM: 51%

100 μM: 49%

4.07

4.77

4.71

4.15

100 μM: 54%

4.36 0.08

4.24 0.11

4.78

4.50 0.12

4.66

4.70 0.06

4.91

4.74

4.60

5.04 0.10

5.36 0.10

5.16 0.09

52%

4.14

4.58

4.80

4.32

100 μM: 62%

4.55 0.12

4.40 0.06

4.83

4.73 0.12

5.15 0.04

4.69 0.09

5.31 0.09

4.90

H

4.05

4.25

4.23

4-F

100 μM: 59%

100 μM: 80%

4.34 0.07

4.50 0.18

4.44

4.86 0.10

4.99

5.43 0.11

5.31 0.05

4.88

4.92 0.06

100 μM: 52%

100 μM: 56%

4.12 0.08

4.31

H

2-Cl

2,4-di-Cl

2-Cl

3,4-di-Cl

2-Cl

3,4-di-Cl

4.48

NH(CO)CH₃

100 μM: 74%

100 μM: 72%

7.43 0.11 ⁴⁹

4.56 0.02

4.93

4.95

6.88 0.12

4.07 0.09

4.19 0.07

8.27 0.03

4.86 0.10

100 μM: 73%

5.81 0.10

5.78 0.03

4.07

nd

8.29 0.02

4.98 0.05

5.31 0.04

5.24 0.05

100 μM: 77%

a

Data are given as the mean SEM of three independent experiments that were performed in triplicate. The results presented as a percent

represent [³H]GABA uptake or NO711 binding in the presence of 100 μM inhibitor. Data without the SEM imply that only one experiment was

performed in triplicate. nd: not determined.

(C) moiety ((2RS,4RS/2RS,4SR)-39a−c) and diphenylmetha-

none O-(2-(methylamino)ethyl) oxime (F) moiety

((2RS,4RS/2RS,4SR)-45) in general showed inhibitory

potency comparable with parent compound 13 except for

compounds with a more rigid moiety such as 4-(bis(3-

methylthiophen-2-yl)methylene)piperidine (A) moiety

((2RS,4RS/2RS,4SR)-37a−c), 4-(suberone)piperidine (E)

moiety ((2RS,4RS/2RS,4SR)-43a,b), or 4-(ﬂuorene)piperidine

(D) moiety ((2RS,4RS/2RS,4SR)-41), which displayed

reduced activity toward all GABA transporter subtypes

(Figure 4, Table 1). On the other hand, when we compared

4-hydroxybutanamide derivatives with diphenylmethanone O-

(2-(methylamino)ethyl) oxime (F) moiety 47a−c with 4-

hydroxypentanoic acid analogs 2RS,4RS-45 and 2RS,4SR-45,

slightly higher inhibitory activity was observed for the 4-

hydroxybutanamide analogs (Table 1, Figure 4). Moreover, in

the group of oximes containing benzamides ((2RS,4RS/

2RS,4SR)-45, 47a−c, 50a,b, and 51a,b), the 4-amino-

butanamide derivatives (50a,b) also displayed slightly

improved inhibitory activity. Hence, among the oxime

derivatives, compound 50a with a chlorine atom in the 2-

position of the benzyl moiety exhibited the highest inhibitory

potency toward mGAT2 among all compounds obtained (50a,

pIC₅₀(mGAT2) = 5.43). Acylation of the amino functionality

at the 4-position of butanoic acid (51a,b) did not change the

inhibitory potency proﬁle against mGAT1−4 (Table 1, Figure

4). An interesting eﬀect was observed for derivatives 39a−c

with a suberone-N-methylpropan-1-amine (C) moiety. In this

group of compounds, we identiﬁed two dual mGAT3/4

subtype selective inhibitors (2RS,4RS-39b and 2RS,4SR-39b)

with a similar GAT preference to that of (S)-SNAP-5114 (9)

but with reduced potency. On the other hand, compound

2RS,4RS-39c, with a methyl group in the para position of the

benzyl moiety, was found to be the most potent mGAT4

inhibitor (pIC₅₀(mGAT4) = 5.36) with favorable GAT

subtype selectivity.

The exchange of a hydroxyl group for a methyl group is

represented in many of the 3-hydroxypropanamide derivatives

(54a−c, 55a−e, 56a−e, 57, 58, and 59a−c) and propanoic

acid and butanoic acid derivatives (57, 64a,b, 65, 66a,b, 67,

F

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Figure 4. Bar graphs of the pIC₅₀values for the library of 4-hydroxypentanamide (37a−c, 39a−c, 41a, 43a,b, 45), 4-hydroxybutanamide (47a−c),

4-aminobutanamide (50a,b), and 4-acetamidobutanamide (51a,b) derivatives compared to the values for the reference compounds (S)-SNAP-

5114 (9) and 13. Error bars are not present when only one experiment was performed in triplicate. Additionally, bars are not presented when the

pIC₅₀value is lower than 4.00.

68, and 71−75; Table 2, Figure 5). The most potent

synthesized serine derivatives have a tricyclic building block

as the lipophilic substituent (dibenzocycloheptadiene moiety;

C) in the α position of the N-benzylamides, 56a−e (Table 2,

Figure 5). 56a−e showed the highest inhibitory activity toward

mGAT3/4 (pIC₅₀in a range of 5.46−5.04). Notably, one

mGAT1-selective butanoic acid derivative (64a,

pIC₅₀(mGAT1) = 5.15) has a rigid bisthiophene moiety (A)

in the α position. Surprisingly, the small structural change in

64a, with a hydrogen substituted for the chlorine atom in the

N-benzylamide moiety (64b), results in a loss of activity.

Unfortunately, we failed to observe an increase in inhibitory

activity when the N-benzylamide moiety was exchanged for a

carboxylic acid group (74 and 75), ethyl ester (71 and 73), or

benzyl ester (72) (Table 3).

2.3. Molecular Modeling. To determine the binding

mode of the tested compounds with GATs, molecular

modeling calculations were performed. For this purpose,

models of human GAT-1, BGT-1, GAT-2, and GAT-3 were

used, as they are the targets for the new inhibitors. Although

the activity of the compounds was tested on mouse

transporters, we assumed that the compounds could bind to

human and mouse in the same place and with comparable

aﬃnity due to only slight diﬀerences in the amino acid

sequences, mainly concerning the N- and C-terminus.^31,50

Homology modeling of the GABA transporters and the

diﬀerences between the structures of the particular types of

these proteins were described in more detail in our previous

work.⁵¹

on representatives of the most active compounds toward

particular types of transporters to conﬁrm the stability of the

presented binding modes and created interactions (Figure 7,

Figure S1). For compounds with an undeﬁned absolute

conﬁguration, all possible stereoisomers were investigated. For

the tested compounds, more consistent arrangements and

beneﬁcial interactions among all types of transporters were

generally observed for the α carbon S isomers.

In the case of GAT-1, the bisthiophene fragment in the most

active compound 64a creates hydrophobic interactions and

CH−π stacking with TYR452 and PHE294 near the entrance

to the transporter (Figure 6, panel A). The amino group of 64a

is located between the aromatic ring of PHE294 and the

carboxyl moiety of ASP451, i.e., residues that are part of the

extracellular gate. This arrangement allows for the formation of

a stable ionic bond with ASP451 and cation−π interaction with

PHE294. During molecular dynamics simulation, as a result of

the bending of the nonhelical fragment of domain 10, the side

chain of SER454 approached the protonated amino group of

the compound. This enabled the creation of a stable hydrogen

bond between these groups (Figure 7, panel A). The ethyl

substituent in the α position reaches TRP68, creating

hydrophobic interactions. The amide carbonyl group forms a

hydrogen bond with the side chain of SER456. During the

dynamic calculations, a hydrogen bond with the hydroxyl

group of SER454 was also observed. The benzyl fragment

reaches into the S1 site, creating hydrophobic interactions

mainly with LEU300, LEU460, LEU136, and PHE294. This

arrangement appears to be beneﬁcial considering that the S1

site in GAT-1 is the most hydrophobic among all types of

transporters.

Molecular docking studies indicated that compounds

generally bind in a similar manner in all types of transporters

(Figure 6). The compounds are located along the vestibule of

the transporters, which is consistent with our previous results

obtained for similar 4-amino- and 4-hydroxybutanamide

derivatives.⁵¹Molecular dynamics simulations were performed

The diphenylmethylidene fragment of compound 50a, the

most active toward BGT-1, locates itself in this transporter

close to the EL6 loop compared to the poses observed in

GAT-1. Compound 50a participates in hydrophobic inter-

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Table 2. Inhibitory Potencies (pIC₅₀SEM) toward mGAT1−4 Determined from the [³H]GABA Uptake Experiments and

mGAT1 Binding Aﬃnities (pK_iSEM) from the MS Binding Assays of the Obtained Compounds

a

Data are given as the mean SEM of three independent experiments that were performed in triplicate. The results presented as a percent

represent [³H]GABA uptake or NO711 binding in the presence of 100 μM inhibitor. Data without the SEM imply that only one experiment was

performed in triplicate. n.d.: not determined.

actions mainly with TYR520, TRP540, TYR454, ILE459, and

TYR453 as well as π−π stacking with TYR453 (Figure 6, panel

B). The protonated primary amine is engaged in a salt bridge

with ASP452. The amide group is located close to SER457,

which enables the creation of a hydrogen bond during

molecular dynamics simulation (Figure 7, panel B). During

the simulation, a change in the conformation of the ARG61

side chain was also observed. This provided an additional

hydrogen bond with the aforementioned amide group. The 2-

chlorobenzyl fragment is located, contrary to that observed in

the GAT-1 transporter, above the extracellular gate in the S2

site, creating hydrophobic interactions mainly with TYR133,

TYR132, and TRP60. This arrangement is beneﬁcial because

the S1 site in BGT-1 is more polar than in other types of

transporters. Additionally, a halogen bond between the

chlorine atom of 50a and the carboxyl group of ASP452 was

observed. In the case of another relatively highly active

compound 50b, the diphenylmethylidene and 4-amino-

butanamide fragment retain the interactions described above,

whereas the 3,4-dichlorobenzyl fragment is located at the level

of the extracellular gate, creating hydrophobic and CH−π

interactions with TYR133.

In GAT-2, the most active compound 50b is placed similarly

as in the BGT-1 transporter. The diphenylmethylidene

fragment forms hydrophobic interactions mainly with

TYR448, TYR515, and MET454. The protonated amino

group of this compound creates a stable salt bridge with

ASP447 (Figure 6, panel C). The amide moiety is located near

the nonhelical fragment of TM10. During the molecular

dynamics simulation, the fragment containing this moiety

rotates which enables creation of a hydrogen bond between the

carbonyl oxygen of the amide group and the hydroxyl group of

SER452. In contrast to the position in the BGT-1 transporter,

the 3,4-dichlorobenzyl fragment reaches the inside of the S1

site in GAT-2. It forms hydrophobic interactions mainly with

LEU294, LEU456, and PHE288.

In the case of GAT-3, the diaromatic fragments of the most

active compounds 50b and 2RS,4RS-39c are in a similar

position compared to that observed in the GAT-2 transporter.

However, due to the presence of SER468 and PHE531, which

are replaced by tyrosine and serine residues, respectively, in the

other transporters, the diphenylmethylidene and dibenzocy-

cloheptadiene fragments are bound slightly higher within the

vestibule. These moieties engage in hydrophobic interactions

mainly with the above-mentioned PHE531, as well as with

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Figure 5. Bar graphs of the pIC₅₀values for the library of 3-hydroxypropanamide (54a−c, 55a−e, 56a−e, 57, 58, and 59a−c), propanoic acid, and

butanoic acid derivatives (64a,b, 65, 66a,b, 67, and 68) compared to the values for the reference compounds (S)-SNAP-5114 (9) and 13. Error

bars are not present when only one experiment was performed in triplicate. Additionally, bars are not presented when the pIC₅₀value is lower than

4.00.

Table 3. Inhibitory Potencies (pIC₅₀SEM) toward mGAT1−4 Determined from the [³H]GABA Uptake Experiments and

mGAT1 Binding Aﬃnities (pK_iSEM) from the MS Binding Assays of the Obtained Compounds

a

pIC₅₀

mGAT2

SEM

mGAT3

4.66

100 μM: 86%

4.6

100 μM: 62%

100 μM: 52%

100 μM: 64%

5.29 0.04

4.85 0.04

4.35

pK_i

SEM

compd

R¹

R²

mGAT1

mGAT4

4.20

100 μM: 97%

4.28

100 μM: 77%

100 μM: 60%

100 μM: 73%

5.81 0.10

5.78 0.03

4.07

mGAT1

71

72

73

74

75

−CH₂CH₃

−CH₂Ph

−CH₂CH₃

H

B

G

B

100 μM: 52%

100 μM: 98%

100 μM: 55%

100 μM: 71%

6.88 0.12

4.07 0.09

4.19 0.07

8.27 0.03

4.86 0.10

100 μM: 54%

100 μM: 63%

100 μM: 52%

100 μM: 55%

100 μM: 59%

100 μM: 52%

100 μM: 56%

4.12 0.08

4.31

100 μM: 103%

100 μM: 109%

100 μM: 94%

4.54

100 μM: 81%

7.43 0.11 ⁴⁹

nd

H

tiagabine (5)¹⁹

(S)-SNAP-5114 (3)¹⁹

DDPM-859 (4)²²

DDPM-2571 (5)²⁰

13²⁵

nd

8.29 0.02

100 μM: 77%

4.98 0.05

5.31 0.04

5.24 0.05

a

Data are given as the mean SEM of three independent experiments that were performed in triplicate. The results presented as a percent

represent [³H]GABA uptake or NO711 binding in the presence of 100 μM inhibitor. Data without the SEM imply that only one experiment was

performed in triplicate. nd: not determined.

TYR535 and TYR469. The protonated amino group forms an

ionic bond (compound 2RS,4RS-39c) or a salt bridge

(compound 50b) with ASP467, similar to the previously

described compounds (Figure 6, panel D). During the

molecular dynamics simulation performed for compound

2RS,4RS-39c, this protonated amino group slightly moves

away from ASP467 while simultaneously approaching the

aromatic ring of PHE308, which enables cation−π interaction

while maintaining an ionic bond with ASP467 (Figure 7, panel

D). At the same time the hydroxyl moiety creates a stable

hydrogen bond with ASP467. The amide groups of both

described compounds are also located close to ASP467 being

involved in the hydrogen bond with this residue. However,

over the course of the dynamics simulation for 2RS,4RS-39c it

was observed that the amide moiety can move closer to the

side chain of TYR146, creating a hydrogen bond. The 4-

methylbenzyl and 3,4-dichlorobenzyl fragments are located at

the level of the lower part of the extracellular gate, creating

hydrophobic and CH−π interactions with TYR147. Com-

pound 50b additionally forms a halogen bond with the amide

moiety of GLY71.

2.4. Hepatotoxicity and Cytotoxicity. In this study we

investigated three representative compounds, 2RS,4RS-39c,

50a, and 56a, for in vitro studies to verify their safety in HepG2

and HEK-293 cells. Among all obtained compounds,

compound 2RS,4RS-39c, a 4-hydroxypentanamide derivative,

and compound 50a, a 4-aminobutanamide derivative, were

selected for further studies because they provided the highest

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Figure 6. Binding modes of compound (S)-64a in GAT-1 (A); compound (S)-50a in BGT-1 (B); compound (S)-50b in GAT-2 (C); compound

(2S,4S)-39c in GAT-3 (D). CH−π and π−π interactions are marked with blue dashes, ionic interactions with pink dashes, cation−π interactions

with green dashes, and hydrogen bonds with yellow dashes.

inhibitory activity toward mGAT4 (pIC₅₀= 5.36 0.10) or

mGAT2 (pIC₅₀= 5.43 0.11), respectively. Compound 56a, a

3-hydroxypropanamide derivative, was selected as one of two

compounds with moderate subtype selectivity for mGAT4.

To investigate the safety of compounds 2RS,4RS-39c, 50a,

and 56a, a HepG2 hepatoma cell-based hepatotoxicity assay

was used. Compounds were tested at six concentrations (0.1−

100 μM). The results showed that at lower compound

concentrations (0.1 and 1 μM), none of the tested compounds

caused a statistically signiﬁcant decrease in HepG2 cell viability

and thus were not hepatotoxic in comparison to doxorubicin

(DX) at 1 μM (Figure 8, panel A). However, only compound

56a did not induce signiﬁcant hepatotoxicity after 72 h of

incubation at concentrations up to 25 μM. A statistically

signiﬁcant (p < 0.0001) decrease in HepG2 cell viability was

observed for 56a only at the highest concentrations of 50 and

100 μM (Figure 8, panel A). Compound 2RS,4RS-39c was

slightly more toxic than 56a, as it showed a statistically

signiﬁcant (p < 0.0001) decrease in HepG2 cell viability at 25

μM, whereas 56a showed 100% viability compared to the

control at this concentration (1% DMSO in culture media).

Compound 50a signiﬁcantly eradicated cell viability at

concentrations between 10 and 100 μM (p < 0.0001).

Nevertheless, these results are in accordance with the

hepatotoxicity examination of thioridazine, an antipsychotic

agent that is still in use (Figure 8, panel C).⁵²

Subsequently, a similar study was performed with the HEK-

293 cell line. Overall, compounds 2RS,4RS-39c and 56a

showed stronger toxic eﬀects than in the HepG2 assay, where a

signiﬁcant (****p < 0.0001) decrease in HEK-293 cell

viability was observed at the concentration of 25 μM. On

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Figure 7. Distance changes between the particular transporter residues and ligand moieties during molecular dynamics simulations for compound

(S)-64a in GAT-1 (A), compound (S)-50a in BGT-1 (B), compound (S)-50b in GAT-2 (C), and compound (2S,4S)-39c in GAT-3 (D).

the other hand, compound 50a showed a comparable safety

proﬁle in HEK-293 and HepG2 cells.

hyperalgesia in the von Frey, hot plate, or cold plate tests,

respectively. Since oxaliplatin is responsible for inducing cold

hypersensitivity, in both humans and experimental animals,^53,54

the cold plate test was used to assess the eﬀect of the test

compounds on the thermal pain threshold in oxaliplatin-

treated mice. In the two other NP models the hot plate test

was applied to measure heat pain threshold in paclitaxel- and

STZ-treated mice.^55,56Since impaired motor coordination is

also observed in NP conditions, we additionally tested the

inﬂuence of compounds 2RS,4RS-39c, 50a, and 56a on motor

coordination in the rotarod test.

2.5.1. Oxaliplatin-Induced Peripheral Neuropathy: Inﬂu-

ence on Tactile Allodynia (von Frey Test). In the early phase

of oxaliplatin-induced neuropathy, an overall eﬀect of treat-

ment on the mechanical nociceptive threshold was observed

(2RS,4RS-39c, F[1.904, 23.80] = 180.6, p < 0.0001; 50a,

F[3.037, 24.30] = 136.7, p < 0.0001; 56a, F[2.371, 21.34] =

83.50, p < 0.0001). In this early phase of neuropathy, the

administration of oxaliplatin signiﬁcantly lowered the pain

threshold for mechanical stimulation (p < 0.0001 vs vehicle-

treated nonneuropathic mice) (Figure 9). Compound

2RS,4RS-39c was not eﬀective in this phase of oxaliplatin-

induced neuropathy (Figure 9, panel A). Compound 50a at

both doses signiﬁcantly elevated the pain threshold for

mechanical stimulation (p < 0.01 vs predrug paw withdrawal

Considering the observed antiproliferative eﬀects of

compounds 2RS,4RS-39c and 56a at 25 μM and at 1 μM for

compound 50a, it can be generalized that these eﬀects were

still lower than those for DX (at 1 μM) or comparable with

those of thioridazine (over the concentration range of 10−100

μM). In this respect and based on the very promising

biological results, compounds 2RS,4RS-39c, 50a, and 56a were

selected for further investigation to elevate their antinocicep-

tive activity in mouse models of NP. However, taking into

account the obtained range of toxicity of the tested

compounds, we assumed that further drug-like property

optimization is required to obtain an acceptable safety proﬁle,

which will be the next stage of our research.

2.5. In Vivo Pharmacological Evaluation (Mouse

Models of Neuropathic Pain). In this part of the present

research, we assessed if the compounds 2RS,4RS-39c, 50a, and

56a display analgesic (antiallodynic and antihyperalgesic)

properties in NP conditions.

For this purpose, we used three mouse models of NP,

namely, chemotherapy-induced NP models (i.e., the oxaliplatin

model and the paclitaxel model) and the diabetic NP model

induced by streptozotocin (STZ). We assessed the eﬀect of the

test compounds on tactile allodynia and thermal (heat or cold)

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Figure 8. Inﬂuence of 2RS,4RS-39c, 50a, 56a, and the reference

cytostatic drug DX on the viability of hepatoma HepG2 cells (A) and

HEK-293 cells (B) after 72 h of incubation. The inﬂuence of

thioridazine on the viability of hepatoma HepG2 cells (C) after 72 h

of incubation. Statistical signiﬁcance (****p < 0.0001) was analyzed

by GraphPad Prism 8.0.1 software using one-way ANOVA and

Bonferroni’s multiple comparison post hoc test.

threshold) (Figure 9, panel B). Additionally, both doses of

compound 56a reduced tactile allodynia in the acute phase of

oxaliplatin-induced neuropathy (p < 0.05 vs predrug paw

withdrawal threshold) (Figure 9, panel C).

In the late phase of oxaliplatin-induced neuropathy, an

overall eﬀect of treatment on the mechanical nociceptive

threshold was observed (2RS,4RS-39c, F[1.705, 13.64] =

282.0, p < 0.0001; 50a, F[2.354,18.83] = 83.09, p < 0.0001;

56a, F[2.651, 23.86] = 108.3, p < 0.0001). In this phase, only

compound 56a at a dose of 10 mg/kg showed antiallodynic

properties (signiﬁcant at p < 0.05 vs predrug paw withdrawal

threshold) (Figure 9, panel C).

2.5.2. Oxaliplatin-Induced Peripheral Neuropathy: Inﬂu-

ence on Cold Hyperalgesia (Cold Plate Test). In the early

phase of oxaliplatin-induced neuropathy, an overall eﬀect of

treatment on the thermal (cold) nociceptive threshold was

observed (2RS,4RS-39c, F[1.393, 16.02] = 30.05, p < 0.0001;

50a, F[2.634, 21.07] = 16.57, p < 0.0001; 56a, F[2.701, 24.31]

= 27.96, p < 0.0001). In this phase, the administration of

oxaliplatin signiﬁcantly lowered the pain threshold for cold

stimulation (p < 0.001 vs vehicle-treated nonneuropathic

mice) (Figure 10). Compounds 2RS,4RS-39c and 50a were

not eﬀective in this phase of oxaliplatin-induced neuropathy

(Figure 10, panels A and B). Compound 56a at a dose of 30

mg/kg reduced cold hyperalgesia in the acute phase of

oxaliplatin-induced neuropathy (p < 0.05 vs predrug paw

withdrawal threshold) (Figure 10, panel C).

Figure 9. Eﬀects of intraperitoneally administered 2RS,4RS-39c (A),

50a (B), and 56a (C) on the mechanical nociceptive threshold in a

mouse oxaliplatin-induced NP model measured using the von Frey

test in the early phase (on the day of oxaliplatin injection) and in the

late phase (7 days after oxaliplatin injection) of neuropathy. The

results are shown as the mean ( SEM) force applied to elicit paw

withdrawal. Statistical analysis: one-way analysis of variance followed

by Tukey’s post hoc comparison. Signiﬁcance vs paw withdrawal

threshold of control, nonneuropathic mice: ^####p < 0.0001.

Signiﬁcance vs predrug (after oxaliplatin) paw withdrawal threshold:

*p < 0.05, **p < 0.01. In vehicle-treated mice (VEH), pain sensitivity

threshold measurements were taken in the same manner and at the

same time points as in the oxaliplatin-treated groups, but vehicle-

treated mice were not treated with oxaliplatin; n = 8−10.

In the late phase of oxaliplatin-induced neuropathy, an

overall eﬀect of treatment was observed (2RS,4RS-39c,

F[1.171, 14.64] = 23.22, p < 0.001; 50a, F[3.125, 24.38] =

9.974, p < 0.001; 56a, F[2.508, 22.57] = 16.37, p < 0.0001). In

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Figure 10. continued

Statistical analysis: one-way analysis of variance followed by Tukey’s

post hoc comparison. Signiﬁcance vs latency of control, non-

###

neuropathic mice:

p < 0.001, ^####p < 0.0001. Signiﬁcance vs

predrug (after oxaliplatin) latency to pain reaction: *p < 0.05. In the

vehicle-treated mice (VEH), measurements of the pain sensitivity

threshold were taken in the same manner and at the same time points

as in the oxaliplatin-treated groups, but vehicle-treated mice were not

treated with oxaliplatin; n = 8−10.

this phase, none of the tested compounds showed antihyper-

algesic properties (Figure 10).

On the basis of the results obtained in the oxaliplatin-

induced neuropathic pain model, i.e., due to lack of activity of

2RS,4RS-39c, for further pain tests and NP models, only the

compounds 50a and 56a were selected.

2.5.3. Paclitaxel-Induced Peripheral Neuropathy: Inﬂu-

ence on Tactile Allodynia (von Frey Test). In the paclitaxel-

induced NP model, the eﬀect of 50a and 56a on mechanical

nociceptive threshold was assessed at two time points, i.e., on

the day of paclitaxel administration (4 h after paclitaxel

administration) and 7 days later. On the day of paclitaxel

administration, in the von Frey test an overall eﬀect of

treatment was observed for 50a (F[4, 35] = 4.854, p < 0.01)

and 56a (F[4, 37] = 5.655, p < 0.01). The post hoc analysis

revealed that compared to vehicle-treated nonneuropathic

mice, paclitaxel signiﬁcantly lowered mechanical nociceptive

threshold in mice (p < 0.05). The comparison between

predrug and postdrug paw withdrawal thresholds in each

experimental group revealed that on the day of paclitaxel

administration neither 50a nor 56a at doses 10 and 30 mg/kg

was able to elevate the mechanical nociceptive threshold in

paclitaxel-treated mice (Figure 11, panel A).

Seven days after paclitaxel administration, an overall eﬀect of

treatment was noted for 50a and 56a (F[4, 35] = 13.76, p <

0.0001, and F[4, 37] = 18.69, p < 0.0001, respectively). On this

day of experiment vehicle-treated nonneuropathic mice had

still signiﬁcantly elevated mechanical nociceptive threshold as

compared to paclitaxel treated mice (p < 0.01 vs predrug, i.e.,

before compound 50a or 56a administration, values of paw

withdrawal thresholds). Of note, the comparison of predrug

and postdrug paw withdrawal thresholds in 50a-treated

neuropathic mice and in 56a-treated neuropathic mice showed

that on day 7 after paclitaxel injection both compounds 50a

and 56a at the dose of 30 mg/kg elevated mechanical

nociceptive threshold (p < 0.05). The lower dose of 50a or 56a

was not eﬀective in the von Frey test (Figure 11, panel B).

2.5.4. Paclitaxel-Induced Peripheral Neuropathy: Inﬂu-

ence on Heat Nociceptive Threshold (Hot Plate Test). Both

compounds 50a and 56a were also assessed for their ability to

aﬀect thermal (heat) nociceptive threshold in paclitaxel-treated

mice. As shown in Figure 12 (panel A), on the day of paclitaxel

administration a signiﬁcantly prolonged latency to pain

reaction and an increased heat nociceptive threshold were

noted in all groups treated with this taxane derivative (p < 0.05

vs vehicle-treated nonneuropathic mice). This eﬀect indicated

that paclitaxel induced hypoalgesia in mice, which was noted

on the day of paclitaxel administration but not 7 days later

(Figure 12, panel A vs panel B).

Figure 10. Eﬀects of intraperitoneally administered 2RS,4RS-39c (A),

50a (B), and 56a (C) on the thermal (cold) pain threshold in a

mouse oxaliplatin-induced NP model measured using the cold plate

test in the early phase (on the day of oxaliplatin injection) and in the

late phase (7 days after oxaliplatin injection) of neuropathy. The

results are shown as the mean ( SEM) latency to pain reaction.

On the day of paclitaxel administration, in the hot plate test,

one-way ANOVA showed an overall eﬀect of treatment for 50a

(F[4, 39] = 6.075, p < 0.001) and 56a (F[4, 39] = 14.27, p <

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Figure 11. Eﬀects of intraperitoneally administered 50a and 56a on the mechanical nociceptive threshold in a mouse paclitaxel-induced NP model

measured using the von Frey test on the day of paclitaxel injection A) and 7 days after single-dose paclitaxel injection (B). The results are shown as

the mean ( SEM) force applied to elicit paw withdrawal. Statistical analysis: one-way analysis of variance followed by Tukey’s post hoc

#

comparison. Signiﬁcance vs paw withdrawal threshold of control, nonneuropathic mice: p < 0.05, ^##p < 0.01. Signiﬁcance vs predrug (after

paclitaxel) paw withdrawal threshold: *p < 0.05. In vehicle-treated mice (VEH), pain sensitivity threshold measurements were taken in the same

manner and at the same time points as in the paclitaxel-treated groups, but vehicle-treated mice were not treated with paclitaxel; n = 8−10.

Figure 12. Eﬀects of intraperitoneally administered 50a and 56a on the thermal (heat) pain threshold in a mouse paclitaxel-induced NP model

measured using the hot plate test on the day of paclitaxel injection (A) and 7 days after paclitaxel injection (B). The results are shown as the mean

( SEM) latency to pain reaction. Statistical analysis: one-way analysis of variance followed by Tukey’s post hoc comparison. Signiﬁcance vs latency

of control, nonneuropathic mice: ^#p < 0.05. Signiﬁcance vs predrug (after paclitaxel) latency to pain reaction: *p < 0.05, **p < 0.01, *p < 0.001. In

the vehicle-treated mice (VEH), measurements of the pain sensitivity threshold were taken in the same manner and at the same time points as in

the paclitaxel-treated groups, but these vehicle-treated mice were not treated with paclitaxel; n = 8−10.

Figure 13. Eﬀects of intraperitoneally administered 50a and 56a on the mechanical nociceptive threshold measured using the von Frey test (A) and

eﬀects of 50a and 56a on the thermal nociceptive threshold measured using the hot plate test (B) in a mouse STZ-induced NP model. The results

are shown as the mean ( SEM) force applied to elicit paw withdrawal or the mean ( SEM) latency to pain reaction. Statistical analysis: one-way

analysis of variance followed by Tukey’s post hoc comparison. Signiﬁcance vs paw withdrawal threshold of control, normoglycemic

(nonneuropathic) mice: ^####p < 0.0001. Signiﬁcance vs predrug (after STZ) paw withdrawal threshold: ***p < 0.001, ****p < 0.0001. In vehicle-

treated mice (VEH), pain sensitivity threshold measurements were taken in the same manner as in the STZ-treated groups, but vehicle-treated mice

were not treated with STZ; n = 8−10.

0.0001). The post hoc analysis demonstrated that in paclitaxel-

treated mice the compound 50a reduced latency to pain

reaction at the dose of 30 mg/kg (p < 0.05 vs predrug latency

to pain reaction; Figure 12, panel A) and 56a reduced latency

to pain reaction at doses 10 mg/kg (p < 0.001 vs predrug

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latency to pain reaction) and 30 mg/kg (p < 0.01 vs predrug

latency to pain reaction; Figure 12, panel A).

not show a neurotoxic eﬀect in mice at the doses displaying

analgesic eﬀect. In a set of in vivo experiments, two

compounds, 50a and 56a at doses of 10 and 30 mg/kg,

showed antiallodynic properties in rodent models of NP

induced by oxaliplatin. Interestingly, compound 56a at a dose

of 10 mg/kg showed antiallodynic properties in the acute and

late phases of oxaliplatin-induced neuropathy, and additionally

at the dose of 30 mg/kg 56a reduced cold hyperalgesia in the

acute phase. In the paclitaxel model of NP both 50a and 56a

were able to reduce tactile allodynia when administered 7 days

after paclitaxel injection. This eﬀect was noted for the dose of

30 mg/kg of both test compounds. Interestingly, in this model

of NP, on the day of paclitaxel administration, both 50a and

56a reduced hypoalgesia induced by paclitaxel and they

restored a physiological heat nociceptive threshold in

paclitaxel-treated mice. The compound 56a was more eﬀective

in reducing heat hypoalgesia than 50a. As for the STZ model

of NP, both compounds were able to reduce tactile allodynia in

diabetic, neuropathic mice. Finally, compound 56a demon-

strated predominant antinociceptive properties in rodent

models of NP and has provided a great contribution to the

current knowledge on the importance of GABA reuptake in the

pathophysiology and pharmacotherapy of NP.

Seven days after paclitaxel administration, one-way ANOVA

did not show an overall eﬀect of treatment for 50a (F[4,35] =

1.407, p > 0.05) in the hot plate test. In contrast to this, in this

assay, an overall eﬀect of treatment was noted for 56a (F[4,

37] = 2.765, p < 0.05). At this time point of testing, Tukey’s

post hoc analysis did not reveal the eﬀect of paclitaxel and

compounds 50a and 56a on the thermal nociceptive threshold

in the hot plate test (Figure 12, panel B).

2.5.5. Diabetic, STZ-Induced Peripheral Neuropathy:

Inﬂuence on Tactile Allodynia (von Frey Test). In the

mouse model of painful diabetic neuropathy induced by STZ,

an overall eﬀect of treatment was demonstrated for both 50a

and 56a in the von Frey test (F[4, 42] = 33.02, p < 0.0001, and

F[4, 40] = 26.59, p < 0.0001, respectively). As shown in Figure

13 (panel A), STZ lowered mechanical nociceptive threshold

in mice (p < 0.0001 vs normoglycemic control). In the von

Frey test, compounds 50a and 56a were eﬀective only at the

dose of 30 mg/kg (50a, p < 0.001; 56a, p < 0.0001 vs predrug

paw withdrawal in the individual group).

2.5.6. Diabetic, STZ-Induced Peripheral Neuropathy:

Inﬂuence on Heat Hyperalgesia (Hot Plate Test). In the

mouse model of painful diabetic neuropathy induced by STZ,

an overall eﬀect of treatment on the heat nociceptive threshold

was not demonstrated for both 50a and 56a in the hot plate

test (F[4,38] = 0.9254, p > 0.05, and F[4,40] = 1.589, p > 0.05,

respectively). As shown in Figure 13 (panel B), STZ slightly

increased thermal nociceptive threshold in mice but this eﬀect

compared to that of normoglycemic control did not reach

statistical signiﬁcance. In the hot plate test, neither 50a nor 56a

was eﬀective.

2.5.7. Eﬀects on Motor Coordination (Rotarod Test). In

the rotarod test, the eﬀect of the test compounds on motor

coordination of mice was assessed. Compared to the vehicle-

treated group, none of the test compounds induced motor

deﬁcits in the rotarod test.

4. METHODS

4.1. Chemistry. Commercially available reagents were purchased

from Merck, Aldrich, Acros, or ChemPur and were used without

further puriﬁcation. Solvents for reactions carried out under inert gas

(argon), such as tetrahydrofuran (THF) and DCM, were dried,

distilled, and collected under argon before use. THF was distilled

from a mixture of sodium and benzophenone, while DCM was

distilled from calcium hydride. Triethylamine (TEA) was distilled

under vacuum before use. Reactions carried out under microwave

irradiation used a Discover LabMate (CEM Corporation, USA).

Puriﬁcation of chemical compounds by column chromatography was

carried out using Sigma-Aldrich silica gel (mesh 0.063−0.200 mm) as

the stationary phase. Reactions were monitored by thin-layer

chromatography (TLC) (aluminum sheets precoated with silica gel

60 F₂₅₄(Merck)). Compounds were visualized with UV light (254

nm). Additionally, the plates were stained with a 0.5% solution of

ninhydrin in n-propanol or 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(petroleum ether (PE)/EtOAc 7:3,

v/v), S₂(PE/EtOAc 1:1, v/v), S₃(n-hexane/ethanol (EtOH)/TEA

7:2:1, v/v/v), S₄(DCM/methanol (MeOH)/NH₃9.5:0.5:0.1, v/v/v),

S₅(NH₃/MeOH/DCM/PE 9:45:120:18, v/v/v/v), S₆(DCM/

acetone (Ace) 9:1, v/v), S₇(n-hexane/ethyl acetate (EtOAc) 1:1,

v/v), S₈(DCM/MeOH, 95:5, v/v), S₉(chloroform (Chl)/Ace 9:1, v/

v), S₁₀(DCM/EtOAc 3:2, v/v) S₁₁(Chl/Ace 1:1, v/v), S₁₂(DCM/

Ace 7:3, v/v), S₁₃(25% NH₃/MeOH/DCM/PE = 640:140:100:25,

v/v/v/v). ¹H NMR and ¹³C NMR spectra were recorded on a Varian

Mercury-VX 300, with ¹H at 300.08 MHz and ¹³C at 75.46 MHz or a

JEOL ECA400II or ECX500 at magnetic ﬁeld strengths of 11.75 T

3. CONCLUSIONS

Neuropathic pain is a global public health problem and is most

frequently caused by chronic, progressive nerve disease after

surgery or trauma and viral infections in the course of diabetes

or could be induced by chemotherapy. It is worth pointing out

that painful diabetic neuropathy is a major complication of

diabetes and a cause of increased mortality. Unfortunately,

currently used drugs have limited eﬃcacy and patients remain

refractory to existing pharmacological treatment. Hence, there

is a substantial need for further development of new and

eﬀective drugs for NP therapy. Considering the above, the

present work describes SAR studies of new functionalized

amino acids as inhibitors of GATs, the biological targets in the

search for new treatment of NP. A series of 56 novel

derivatives of 3-hydroxypropanamide, 4-hydroxybutanamide,

and 4-hydroxypentamide were synthesized and evaluated

toward all four mouse GAT subtypes (mGAT1−4). On the

basis of the obtained in vitro results, we selected three

1

corresponding to H and ¹³C resonance frequencies of 500.16 and

125.77 MHz at ambient temperature (25 °C). Chemical shifts (δ) are

reported in parts per million (ppm), and coupling constants (J) are

reported in hertz (Hz). High-resolution (HR) MS was performed on

a Synapt G2-S HDMS (Waters Inc.) mass spectrometer equipped

with an electrospray ionization source and q-TOF type mass analyzer.

The instrument was controlled, and recorded data were processed

using the MassLynx v4.1 software package (Waters Inc.). Purities of

the ﬁnal compounds were determined with a Waters ACQUITY

ultraperformance liquid chromatography (UPLC) instrument

(Waters, Milford, MA, USA) coupled to a Waters TQD mass

spectrometer (ESI-tandem quadrupole).

compounds 2RS,4RS-39c (pIC₅₀(mGAT4) = 5.36

50a (pIC₅₀(mGAT2) = 5.43 0.11), and 56a

(pIC₅₀(mGAT4) = 5.04 0.04) for further investigation.

0.10),

The obtained results indicated a negligible hepatotoxic and

cytotoxic eﬀect of the tested compounds at 0.1 and 1 μM on

HepG2 and HEK-293 cells. Their safety proﬁle was also

examined in in vivo studies, where the tested compounds did

4.1.1.1. General Procedure for the Synthesis of the 3-Substituted

5-Methyldihydrofuran-2(3H)-one or Dihydrofuran-2(3H)-one De-

O

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rivatives (36, 38, 40, 42, 44, 46) (GP1). A mixture of anhydrous

K₂CO₃, corresponding amine A, C, D, E, or F (1 equiv) and TBAB

(0.18 mmol, 0.06 g, 0.01 equiv) in acetonitrile (7 mL) was stirred at 0

°C for 15 min. Then, a solution of 3-bromodihydrofuran-2(3H)-one

(34) or 3-bromo-5-methyldihydrofuran-2(3H)-one (35) (1 equiv)

was added dropwise, and stirring continued for 20 h at rt. 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.

(DCM/EtOAc = 3:2) to yield 44 (500 mg, 66%, R_f= 0.71 (S₁₀)) as a

yellow oil. Formula C₂₁H₂₄N₂O₃, FW 352.43. H NMR (300 MHz,

1

chloroform-d) δ ppm 1.34 (d, J = 6.16 Hz, 3H (CHCH₃)) 1.67 (td, J

= 12.31, 10.52 Hz, 1H (CHCH₂CH)) 2.07−2.14 (m, 1H

(CHCH₂CH)) 2.38 (s, 3H (NCH₃)) 2.85−3.03 (m, 2H

(OCH₂CH₂N)) 3.67−3.79 (m, 1H (NCH)) 4.06−4.23 (m, 1H

(CHCH₃)) 4.29−4.36 (m, 2H (OCH₂CH₂N)) 7.27−7.50 (m, 10H

(Ar)).

4.1.1.1.6. 3-[(2-{[(Diphenylmethylidene)amino]oxy}ethyl)-

(methyl)amino]oxolan-2-one (46). According to GP1 with amine

F (32) (2.95 mmol, 0.75 g, 1 equiv), 3-bromodihydrofuran-2(3H)-

one (35) (2.95 mmol, 0.52 g, 1 equiv), TBAB (0.30 mmol, 93.2 mg,

0.1 equiv), and anhydrous K₂CO₃(8.85 mmol, 1.23 g, 3 equiv) were

combined in acetonitrile (10 mL). The crude product was puriﬁed by

column chromatography over silica gel (DCM/Ace = 7:3) to yield 46

(520 mg, 52%, R_f= 0.65 (S₁)) as a yellow oil. Formula C₂₀H₂₂N₂O₃,

4.1.1.1.1. 3-{4-[Bis(3-methylthiophen-2-yl)methylidene]-

piperidin-1-yl}-5-methyloxolan-2-one (36). According to GP1 with

amine A (17) (0.86 mmol, 0.25 g, 1 equiv), 3-bromo-5-

methyldihydrofuran-2(3H)-one (34) (0.86 mmol, 0.15 g, 1 equiv),

TBAB (0.086 mmol, 27.7 mg, 0.1 equiv), and anhydrous K₂CO₃(2.58

mmol, 0.36 g, 3 equiv) were combined in acetonitrile (7 mL). The

crude product was puriﬁed by column chromatography over silica gel

(DCM/Ace = 9:1) to yield 36 (260 mg, 78%, R_f= 0.65 (S₉) as a

1

FW 338.40. H NMR (300 MHz, chloroform-d) δ ppm 2.26−2.31

1

yellow oil. Formula C₂₁H₂₅NO₂S₂, FW 387.56. H NMR (300 MHz,

(m, 1H (CHCH₂)) 2.35−2.55 (m, 4H (Me; CHCH₂)) 2.63−2.74 (m,

2H (CH₂N)) 3.49−3.56 (m, 1H (NCH)) 3.64−3.71 (m, 2H (

NOCH₂)) 4.25−4.33 (m, 1H (OCH₂)) 4.35−4.45 (m, 1H (OCH₂))

7.45−7.62 (m, 10H (Ar)).

chloroform-d) δ ppm 1.43 (d, J = 5.86 Hz, 3H) 2.11 (s, 6H) 2.32−

2.47 (m, 6H) 2.54−2.68 (m, 2H) 2.85 (dd, J = 10.84, 5.57 Hz, 2H)

3.72 (dd, J = 12.02, 8.50 Hz, 1H) 4.45 (dt, J = 10.99, 5.35 Hz, 1H)

6.77 (d, J = 5.28 Hz, 2 H) 7.13 (d, J = 4.69 Hz, 2H).

4.1.1.2. General Procedure for the Synthesis of the 4-

Hydroxypentanamide and 4-Hydroxybutanamide Derivatives

37a−c, 39a−c, 41, and 43a,b (GP2). The corresponding 3-

substituted 5-methyldihydrofuran-2(3H)-one or dihydrofuran-

2(3H)-one derivative (1 equiv), relevant 1-phenylmethanamine

hydrochloride derivative (1.5 equiv), and sodium 2-ethylhexanoate

(2.5 equiv) were dissolved in dry THF under Ar and stirred at 66 °C

for 16 h. Then, the mixture was cooled to rt, and EtOAc (5 mL/1

mmol) and 30% K₂CO₃(5 mL/1 mmol) were added. The mixture

was stirred at rt for 15 min and was extracted with EtOAc (2 × 5 mL).

The combined organic phases were dried over Na₂SO₄and

concentrated under reduced pressure. The crude product was puriﬁed

by column chromatography.

4.1.1.1.2. 3-((3-(10,11-Dihydro-5H-dibenzo[a,d][7]annulen-5-

ylidene)propyl)(methyl)amino)-5-methyldihydrofuran-2(3H)-one

(38). According to GP1 with amine C (21) (5.47 mmol, 1.44 g, 1

equiv), 3-bromo-5-methyldihydrofuran-2(3H)-one (34) (5.47 mmol,

0.76 g, 1 equiv), TBAB (1.75 mmol, 0.56 g, 0.32 equiv), and

anhydrous K₂CO₃(5.47 mmol, 0.76 g, 1 equiv) were combined in

acetonitrile (7 mL). The crude product was puriﬁed by column

chromatography over silica gel (DCM/Ace = 9:1) to yield 38 (1.88 g,

95%, R_f= 0.74 (S₁₀)) as a yellow oil. Formula C₂₄H₂₇NO₂, FW

361.49. ¹H NMR (300 MHz, chloroform-d) δ ppm 1.30−1.43 (m, 3H

(CHCH₃)) 1.61−1.88 (m, 2H (CHCH₂CH)) 2.22−2.36 (m, 5H

(NCH₃, CHCH₂CH₂)) 2.59−2.88 (m, 3H (ArCH₂CH₂Ar,

CHCH₂CH₂)) 2.96 (br s, 1H (ArCH₂CH₂Ar)) 3.28 (br s, 1H

(ArCH₂CH₂Ar)) 3.37 (d, J = 9.96 Hz, 1H (ArCH₂CH₂Ar)) 3.60−

3.70 (m, 1H (NCH)) 4.36 (dt, J = 10.99, 5.35 Hz, 1H (CHCH₃))

5.81−5.89 (m, 1H (CHCH₂CH₂)) 6.99−7.07 (m, 1H (Ar)) 7.08−

7.23 (m, 6H (Ar)) 7.24−7.31 (m, 1H (Ar)). LCMS m/z [M + H]⁺

362.20.

4.1.1.1.3. 3-[4-(9H-Fluoren-9-ylidene)piperidin-1-yl]-5-methylox-

olan-2-one (40). According to GP1 with amine D (25) (1.17 mmol,

0.29 g, 1 equiv), 3-bromo-5-methyldihydrofuran-2(3H)-one (34)

(1.17 mmol, 0.21 g, 1 equiv), TBAB (0.12 mmol, 40 mg, 0.1 equiv),

and anhydrous K₂CO₃(3.51 mmol, 0.48 g, 3 equiv) were combined in

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

chromatography over silica gel (Chl/Ace = 9:1) to yield 40 (289 mg,

71%, R_f= 0.61 (S₉)) as a yellow oil. Formula C₂₃H₂₃NO₂, FW 361.49.

¹H NMR (300 MHz, chloroform-d) δ ppm 1.30−1.41 (m, 3H) 1.98−

4.1.1.2.1. N-Benzyl-2-{4-[bis(3-methylthiophen-2-yl)-

methylidene]piperidin-1-yl}-4-hydroxypentanamide ((2RS,4RS)-

37a and 2RS,4SR)-37a). Compounds (2RS,4RS)-37a and

(2RS,4SR)-37a were prepared according to GP2 with 3-{4-[bis(3-

methylthiophen-2-yl)methylidene]piperidin-1-yl}-5-methyloxolan-2-

one (36) (0.67 mmol, 0.26 g), 1-phenylmethanamine hydrochloride

(1 mmol, 0.14 g), and sodium 2-ethylhexanoate (1.68 mmol, 0.28 g)

in 4 mL of dry THF. The obtained crude products were puriﬁed by

column chromatography over silica gel (DCM/Ace = 9:1) to yield

(2RS,4RS)-37a (180 mg, 54%, R_f= 0.56 (S₆) and (2RS,4SR)-37a (80

mg, 24%, R_f= 0.33 (S₆)) as yellow oil. ds: (2RS,4RS)-37a/(2RS,4SR)-

37a = 69:31. Formula C₂₈H₃₄N₂O₂S₂, FW 494.71. Compound

1

(2RS,4RS)-37a H NMR (300 MHz, chloroform-d) δ ppm 7.92−

8.03 (m, 1H), 7.18−7.42 (m, 5H), 7.12 (d, J = 5.27 Hz, 1H), 6.76 (d,

J = 5.28 Hz, 1H), 6.56−6.70 (m, 1H), 6.48−6.60 (m, 1H), 5.33−5.45

(m, 1H), 4.44 (s, 2H), 3.69−3.99 (m, 1H), 2.43−2.65 (m, 1H),

2.15−2.40 (m, 2H), 1.98 (ddd, J = 4.98, 9.82, 14.51 Hz, 1H), 1.71−

1.93 (m, 1H), 1.40−1.66 (m, 4H), 1.14−1.32 (m, 6H), 0.76−0.96

(m, 5H). ¹³C NMR (75 MHz, chloroform-d) δ ppm 175.6, 175.6,

170.4, 137.6, 137.5, 133.8, 129.4, 128.8, 128.8, 128.7. 127.7, 127.6,

127.5, 124.0, 71.6, 64.1, 50.5, 47.3, 47.2, 43.5, 43.3, 41.8, 31.9, 31.6,

29.6, 29.5, 25.4, 25.4, 24.5, 23.6, 22.5, 14.6, 13.9, 13.8, 11.8. HRMS-

ESI⁺m/z [M + H]⁺calcd for C₂₈H₃₄N₂O₂S₂, 495.2134; found,

2.54 (m, 6H) 2.88−2.98 (m, 4H) 3.43−3.59 (m, 1H) 4.40−4.52 (m,

1H) 7.19−7.45 (m, 6H (Ar)) 7.63−7.95 (m, 2H (Ar)).

4.1.1.1.4. 3-(4-(10,11-Dihydro-5H-dibenzo[a,d][7]annulen-5-

ylidene)piperidin-1-yl)-5-methyldihydrofuran-2(3H)-one (42). Ac-

cording to GP1 with amine E (26) (0.22 mmol, 60 mg, 1 equiv), 3-

bromo-5-methyldihydrofuran-2(3H)-one (34) (0.22 mmol, 40 mg, 1

equiv), TBAB (0.022 mmol, 7 mg, 0.1 equiv), and anhydrous K₂CO₃

(0.65 mmol, 90 mg, 3 equiv) were combined in acetonitrile (7 mL).

The crude product was puriﬁed by column chromatography over silica

gel (DCM/Ace = 7:3) to yield 42 (40 mg, 49%, R_f= 0.46 (S₁)) as a

1

495.2133. Compound (2RS,4SR)-37a H NMR (300 MHz, chloro-

form-d) δ ppm 7.21−7.41 (m, 8H), 7.13 (d, J = 5.28 Hz, 1H), 6.77

(d, J = 5.27 Hz, 1H), 4.42−4.55 (m, 2H), 3.39−3.57 (m, 1H), 2.50−

2.68 (m, 2H), 2.30 (br s, 3H), 2.08 (s, 6H), 1.64−1.90 (m, 2H),

1.09−1.35 (m, 6H), 0.76−0.92 (m, 1H). ¹³C NMR (75 MHz,

chloroform-d) δ ppm 175.5, 175.5, 170.3, 137.5, 137.4, 133.6, 129.2,

128.9, 128.9, 128.1, 127.6, 127.5, 127.4, 124.0, 71.6, 64.1, 50.4, 47.2,

47.1, 43.5, 43.2, 41.7, 31.8, 31.7, 29.7, 29.6, 25.5, 25.3, 24.6, 23.6,

22.8, 14.5, 13.8, 13.8, 11.7. HRMS-ESI⁺m/z [M + H]⁺calcd for

C₂₈H₃₄N₂O₂S₂, 495.2134; found, 495.2139.

1

yellow oil. Formula C₂₅H₂₇NO₂, FW 373.49. H NMR (300 MHz,

chloroform-d) δ ppm 1.70−1.85 (m, 2H) 2.15 (s, 4H) 2.34−2.54 (m,

4H) 2.76−2.93 (m, 4H) 3.47−3.57 (m, 1H) 3.62−3.76 (m, 3H)

4.30−4.42 (m, 1H) 7.07−7.27 (m, 8H).

4.1.1.1.5. 3-[(2-{[(Diphenylmethylidene)amino]oxy}ethyl)-

(methyl)amino]-5-methyloxolan-2-one (44). According to GP1

with amine F (32) (2.16 mmol, 0.55 g, 1 equiv), 3-bromo-5-

methyldihydrofuran-2(3H)-one (34) (2.16 mmol, 0.38 g, 1 equiv),

TBAB (0.22 mmol, 69.5 mg, 0.1 equiv), and anhydrous K₂CO₃(6.48

mmol, 0.90 g, 3 equiv) were combined in acetonitrile (10 mL). The

crude product was puriﬁed by column chromatography over silica gel

4.1.1.2.2. 2-{4-[Bis(3-methylthiophen-2-yl)methylidene]-

piperidin-1-yl}-N-[(2-chlorophenyl)methyl]-4-hydroxypentanamide

((2RS,4RS)-37b and (2RS,4SR)-37b). Compounds (2RS,4RS)-37b

P

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and (2RS,4SR)-37b were prepared according to GP2 with 3-{4-

[bis(3-methylthiophen-2-yl)methylidene]piperidin-1-yl}-5-methylox-

olan-2-one (36) (2.12 mmol, 0.83 g), 1-(2-chlorophenyl)-

methanamine hydrochloride (3.18 mmol, 0.56 g), and sodium 2-

ethylhexanoate (5.34 mmol, 0.95 g) in 10 mL of dry THF. The

obtained crude products were puriﬁed by column chromatography

over silica gel (DCM/Ace = 9:1) to yield (2RS,4RS)-37b (533 mg,

47%, R_f= 0.58 (S₆)) and (2RS,4SR)-37b (238 mg, 21%, R_f= 0.35

(S₆)) as a yellow oil. ds: (2RS,4RS)-37b/(2RS,4SR)-37b = 69:31.

Formula C₂₈H₃₃ClN₂O₂S₂, FW 529.15. Compound (2RS,4RS)-37b:

¹H NMR (300 MHz, chloroform-d) δ ppm 7.58−7.68 (m, 1H),

7.35−7.43 (m, 2H), 7.21−7.28 (m, 2H), 7.11−7.16 (m, 2H), 6.76−

6.80 (m, 2H), 4.36−4.64 (m, 3H), 3.98−4.07 (m, 1H), 3.31−3.40

(m, 1H), 2.54 (d, J = 4.11 Hz, 4H), 2.26−2.37 (m, 4H), 2.10 (s, 6H),

1.74 (t, J = 6.16 Hz, 2H), 1.18 (d, J = 6.46 Hz, 3H). ¹³C NMR (75

MHz, chloroform-d) δ ppm 173.1, 143.1, 137.9, 135.5, 133.8, 133.7,

130.5, 129.7, 129.5, 129.3, 127.3, 123.9, 120.2, 65.5, 65.2, 51.0, 41.6,

34.2, 32.0, 22.9, 14.7. HRMS-ESI⁺m/z [M + H]⁺calcd for

C₂₈H₃₃ClN₂O₂S₂, 529.1745; found, 529.1747. Compound

= 0.74 (DCM/Ace = 7:3) as a yellow oil. Formula C₃₁H₃₅FN₂O₂, FW

486.27.¹H NMR (300 MHz, chloroform-d) δ ppm 1.18−1.34 (m, 4H

(CCH₃)) 1.59−1.79 (m, 2H (CHCH₂CH)) 2.03 (br s, 3H (NCH₃))

2.18−2.35 (m, 2H (CHCH₂CH₂)) 2.44 (br s, 2H (CHCH₂CH₂))

2.76 (br s, 1H (ArCH₂CH₂Ar)) 2.86−3.08 (m, 1H (ArCH₂CH₂Ar))

3.08−3.37 (m, 3H (ArCH₂CH₂Ar, NCH)) 3.63−3.76 (m, 1H

(CHOH)) 4.06−4.38 (m, 2H (NHCH₂)) 5.70 (br s, 1H

(CHCH₂CH₂)) 6.94 (br s, 3H (Ar)) 6.99−7.09 (m, 3H (Ar))

7.10−7.24 (m, 6H (Ar)) 7.97 (br s, 1H (NHCH₂)). ¹³C NMR (75

MHz, chloroform-d) δ ppm 24.61, 31.96, 33.69, 42.51, 54.38, 66.99,

67.48, 115.29, 115.58, 125.78, 126.13, 127.27, 127.65, 128.10, 128.42,

129.22, 129.33, 130.20, 133.86, 133.91, 139.71, 163.70, 174.81.

LCMS m/z [M + H]⁺= 487.22. HRMS-ESI⁺m/z [M + H]⁺calcd for

C₃₁H₃₅FN₂O₂, 487.2755; found, 487.2758.

4.1.1.2.5. N-(4-Chlorobenzyl)-2-((3-(10,11-dihydro-5H-dibenzo-

[a,d][7]annulen-5-ylidene)propyl)(methyl)amino)-4-hydroxypenta-

namide ((2RS,4RS)-39b and (2RS,4SR)-39b). Compounds

(2RS,4RS)-39b and (2RS,4SR)-39b were prepared according to

GP2 with 3-((3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-

ylidene)propyl)(methyl)amino)-5-methyldihydrofuran-2(3H)-one

(38) (1.66 mmol, 0.60 g), 1-(4-chlorophenyl)methanamine hydro-

chloride (2.73 mmol, 0.43 g), and sodium 2-ethylhexanoate (4.55

mmol, 0.76 g) in 4 mL of dry THF. The obtained crude products

were puriﬁed by column chromatography over silica gel (DCM/Ace =

9:1) to yield (2RS,4RS)-39a (358 mg, 41%, R_f= 0.33 (DCM/Ace =

9:1)) and (2RS,4SR)-39b (114 mg, 13%, R_f= 0.18 (DCM/Ace =

9:1)) as a yellow oil. ds: (2RS,4RS)-39a/(2RS,4SR)-39a = 76:24.

Formula C₃₁H₃₅ClN₂O₂, FW 503.08. Compound (2RS,4RS)-39b: ¹H

NMR (300 MHz, chloroform-d) δ ppm 1.20−1.27 (m, 2H

(CHCH₃)) 1.46−1.78 (m, 2H (CHCH₂CH)) 2.03 (br s, 3H

(NCH₃)) 2.20−2.33 (m, 2H (CHCH₂CH₂)) 2.42 (br s, 2H

(CHCH₂CH₂)) 2.75 (br s, 1H (ArCH₂CH₂Ar)) 2.93 (br s, 1H

(ArCH₂CH₂Ar)) 3.16 (d, J = 13.48 Hz, 1H (ArCH₂CH₂Ar)) 3.27 (br

s, 2H (ArCH₂CH₂Ar, NCH)) 3.65−3.77 (m, 1H (CHOH)) 4.06−

4.40 (m, 2H (NHCH₂)) 5.69 (br s, 1H (CHCH₂CH)) 6.93 (br s, 1H

(Ar)) 6.98−7.26 (m, 12H (Ar)) 7.99 (br s, 1H (NHCH₂)). ¹³C NMR

(75 MHz, chloroform-d) δ ppm 22.67, 24.56, 31.98, 33.70, 38.42,

42.52, 47.18, 53.83, 66.99, 67.43, 121.78, 125.79, 126.14, 127.29,

127.67, 128.05, 128.11, 128.42, 128.72, 128.97, 130.23, 136.61,

136.90, 139.68, 174.86. LCMS m/z [M + H]⁺= 503.23. HRMS-ESI⁺

m/z [M + H]⁺calcd for C₃₁H₃₅ClN₂O₂, 503.2460; found, 503.2462.

1

(2RS,4SR)-37b: H NMR (300 MHz, chloroform-d) δ ppm 7.57−

7.67 (m, 1H), 7.34−7.42 (m, 2H), 7.20−7.27 (m, 2H), 7.10−7.15

(m, 2H), 6.75−6.79 (m, 2H), 4.35−4.63 (m, 3H), 3.97−4.06 (m,

1H), 3.30−3.39 (m, 1H), 2.53 (d, J = 4.10 Hz, 4H), 2.25−2.36 (m,

4H), 2.09 (s, 6H), 1.73 (t, J = 6.15 Hz, 2H), 1.17 (d, J = 6.45 Hz,

3H). ¹³C NMR (75 MHz, chloroform-d) δ ppm 173.0, 143.1, 137.8,

135.5, 133.7, 133.6, 130.5, 129.6, 129.4, 129.2, 127.2, 123.9, 120.1,

65.4, 65.1, 51.0, 41.5, 34.2, 32.0, 22.9, 14.6. HRMS-ESI⁺m/z [M +

H]⁺calcd for C₂₈H₃₃ClN₂O₂S₂, 529.1745; found, 529.1747.

4.1.1.2.3. 2-{4-[Bis(3-methylthiophen-2-yl)methylidene]-

piperidin-1-yl}-N-[(4-ﬂuorophenyl)methyl]-4-hydroxypentanamide

((2RS,4RS)-37c and (2RS,4SR)-37c). Compounds (2RS,4RS)-37c and

(2RS,4SR)-37c were prepared according to GP2 with 3-{4-[bis(3-

methylthiophen-2-yl)methylidene]piperidin-1-yl}-5-methyloxolan-2-

one (36) (2.12 mmol, 0.83 g), 1-(4-ﬂuorophenyl)methanamine

hydrochloride (3.18 mmol, 0.51 g), and sodium 2-ethylhexanoate

(5.34 mmol, 0.95 g) in 10 mL of dry THF. The obtained crude

products were puriﬁed by column chromatography over silica gel

(DCM/Ace = 9:1) to yield (2RS,4RS)-37c (527 mg, 48%, R_f= 0.55

(DCM/Ace = 9:1)) and (2RS,4SR)-37c (230 mg, 21%, R_f= 0.37

(DCM/Ace = 9:1)) as a yellow oil. ds: (2RS,4RS)-23a/(2RS,4SR)-

37c = 70:30. Formula C₂₈H₃₃FN₂O₂S₂, FW 512.70. Compound

1

(2RS,4RS)-37c: H NMR (300 MHz, chloroform-d) δ ppm 7.50−

Compound (2RS,4SR)-39b: H NMR (300 MHz, chloroform-d) δ

7.59 (m, 1H), 7.20−7.29 (m, 2H), 7.12 (d, J = 5.29 Hz, 2H), 7.01 (s,

2H), 6.77 (d, J = 5.29 Hz, 2H), 4.28−4.54 (m, 3H), 3.97−4.08 (m,

1H), 3.34−3.43 (m, 1H), 2.50−2.66 (m, 4H), 2.24−2.38 (m, 4H),

2.09 (s, 6H), 1.71−1.81 (m, 2H), 1.13−1.21 (m, 3H). ¹³C NMR (75

MHz, chloroform-d) δ ppm 172.8, 163.8, 160.6, 143.0, 137.8, 134.3,

134.3, 133.8, 129.4, 129.4, 129.3, 124.1, 120.4, 115.8, 115.6, 65.7,

65.3, 51.1, 42.5, 34.5, 31.9, 31.7, 29.8, 23.2, 14.7. HRMS-ESI⁺m/z [M

+ H]⁺calcd for C₂₈H₃₃FN₂O₂S₂, 513.2040; found, 513.2045.

ppm 1.14 (d, J = 6.45 Hz, 3H (CHCH₃)) 1.60 (ddd, J = 13.63, 6.59,

3.22 Hz, 1H (CHCH₂CH)) 1.83 (br s, 1H (CHCH₂CH)) 2.05 (s,

3H (NCH₃)) 2.26 (br s, 2H (CHCH₂CH₂)) 2.46 (br s, 2H

(CHCH₂CH₂)) 2.75 (br s, 1H (ArCH₂CH₂Ar)) 2.88−3.04 (m, 1H

(ArCH₂CH₂Ar)) 3.17 (br s, 1H (ArCH₂CH₂Ar)) 3.35 (dd, J = 9.38,

2.93 Hz, 2H (ArCH₂CH₂Ar, NCH)) 4.04 (br s, 1H (CHOH)) 4.13−

4.40 (m, 2H (NHCH₂)) 5.74 (br s, 1H (CHCH₂CH₂)) 6.91−7.24

(m, 12H (Ar)) 7.69 (br s, 1H (NHCH₂)). ¹³C NMR (75 MHz,

chloroform-d) δ ppm 22.82, 27.86, 29.28, 31.98, 33.72, 42.46, 64.88,

125.79, 126.13, 127.26, 127.62, 128.02, 128.17, 128.45, 128.74,

1

Compound (2RS,4SR)-37c: H NMR (300 MHz, chloroform-d) δ

ppm 7.49−7.58 (m, 1H), 7.19−7.28 (m, 2H), 7.11 (d, J = 5.28 Hz,

2H), 7.00 (s, 2H), 6.76 (d, J = 5.28 Hz, 2H), 4.27−4.53 (m, 3H),

3.96−4.07 (m, 1H), 3.33−3.42 (m, 1H), 2.49−2.65 (m, 4H), 2.23−

2.37 (m, 4H), 2.08 (s, 6H), 1.70−1.80 (m, 2H), 1.12−1.20 (m, 3H).

¹³C NMR (75 MHz, chloroform-d) δ ppm 172.7, 163.7, 160.5, 143.0,

137.7, 134.2, 134.2, 133.7, 129.4, 129.4, 129.3, 124.0, 120.3, 115.7,

115.5, 65.6, 65.3, 51.0, 42.5, 34.4, 31.9, 31.7, 29.7, 23.1, 14.6. HRMS-

ESI⁺m/z [M + H]⁺calcd for C₂₈H₃₃FN₂O₂S₂, 513.2040; found,

513.2045.

4.1.1.2.4. 2-((3-(10,11-Dihydro-5H-dibenzo[a,d][7]annulen-5-

ylidene)propyl)(methyl)amino)-N-(4-ﬂuorobenzyl)-4-hydroxypen-

tanamide ((2RS,4RS)-39a). Compound (2RS,4RS)-39a was prepared

according to GP2 with 3-((3-(10,11-dihydro-5H-dibenzo[a,d][7]-

annulen-5-ylidene)propyl)(methyl)amino)-5-methyldihydrofuran-

2(3H)-one (38) (1.66 mmol, 0.60 g), 1-(4-ﬂuorophenyl)-

methanamine hydrochloride (2.49 mmol, 0.31 g) and sodium 2-

ethylhexanoate (4.15 mmol, 0.69 g) in 4 mL of dry THF. The

obtained crude product was puriﬁed by column chromatography over

silica gel (DCM/Ace = 9:1) to yield (2RS,4RS)-39a (249 mg, 31%, R_f

129.01, 130.19, 136.92, 139.77, 174.19. LCMS m/z [M + H]⁺

=

503.23. HRMS-ESI⁺m/z [M + H]⁺calcd for C₃₁H₃₅ClN₂O₂,

503.2460; found, 503.2462.

4.1.1.2.6. 2-((3-(10,11-Dihydro-5H-dibenzo[a,d][7]annulen-5-

ylidene)propyl)(methyl)amino)-4-hydroxy-N-(4-methylbenzyl)-

pentanamide ((2RS,4RS)-39c and (2RS,4SR)-39c). Compounds

(2RS,4RS)-39c and (2RS,4SR)-39c were prepared according to GP2

with 3-((3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ylidene)-

propyl)(methyl)amino)-5-methyldihydrofuran-2(3H)-one (38)

(1.66 mmol, 0.60 g), 1-(4-methylphenyl)methanamine hydrochloride

(2.73 mmol, 0.43 g), and sodium 2-ethylhexanoate (2.73 mmol, 0.43

g) in 4 mL of dry THF. The obtained crude products were puriﬁed by

column chromatography over silica gel (DCM/Ace = 9:1) to yield

(2RS,4RS)-39c (358 mg, 41%, R_f= 0.39 (DCM/Ace = 9:1)) and

(2RS,4SR)-39c (114 mg, 13%, R_f= 0.32 (DCM/Ace = 9:1)) as a

yellow oil. ds: (2RS,4RS)-39c/(2RS,4SR)-39c = 76:24. Formula

C₃₂H₃₈N₂O₂, FW 482.67. Compound (2RS,4RS)-39c: ¹H NMR (300

MHz, chloroform-d) δ ppm 1.21−1.27 (m, 3H (CHCH₃)) 1.64−1.73

Q

https://doi.org/10.1021/acschemneuro.1c00351

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

(m, 2H (CHCH₂CH)) 2.05 (s, 3H (ArCH₃)) 2.18−2.29 (m, 2H

(CHCH₂CH₂)) 2.32 (s, 3H (NCH₃)) 2.44 (br s, 2H (CHCH₂CH₂))

2.75 (br s, 1H (ArCH₂CH₂Ar)) 2.94 (br s, 1H (ArCH₂CH₂Ar))

3.12−3.39 (m, 3H (ArCH₂CH₂Ar, NCH)) 3.67−3.74 (m, 1H

(CHOH)) 4.13−4.40 (m, 2H (NHCH₂)) 5.72 (br s, 1H

(CHCH₂CH₂)) 6.91−7.24 (m, 12H (Ar)) 7.87 (br s, 1H

(NHCH₂)). ¹³C NMR (75 MHz, chloroform-d) δ ppm 21.11,

24.67, 28.04, 31.99, 33.72, 43.03, 66.90, 67.48, 125.78, 126.10, 127.61,

128.07, 128.45, 129.32, 130.17, 135.02, 136.93, 139.30, 139.79,

174.64. LCMS m/z [M + H]⁺= 483.29. HRMS-ESI⁺m/z [M + H]⁺

calcd for C₃₁H₃₅ClN₂O₂, 483.3006; found, 483.3003. Compound

(2RS,4SR)-23a: ¹H NMR (300 MHz, chloroform-d) δ ppm 1.13 (d, J

= 6.45 Hz, 3H (CHCH₃)) 1.61 (ddd, J = 14.07, 7.03, 3.52 Hz, 1H

(CHCH₂CH)) 1.79−1.89 (m, 1H (CHCH₂CH)) 2.07 (s, 3H

(ArCH₃)) 2.21−2.29 (m, 2H (CHCH₂CH₂))) 2.32 (s, 3H

(NCH₃)) 2.42−2.50 (m, 2H (CHCH₂CH₂)) 2.75 (br s, 1H

(ArCH₂CH₂Ar)) 2.94 (br s, 1H (ArCH₂CH₂Ar)) 3.20 (br s, 1H

(NCH)) 3.33 (dd, J = 9.38, 3.52 Hz, 2H (ArCH₂CH₂Ar)) 4.00−4.08

(m, 1H (CHOH)) 4.22−4.37 (m, 2H (NHCH₂)) 5.76 (t, J = 7.03

Hz, 1H (CHCH₂CH₂)) 6.94−7.24 (m, 12H (Ar)) 7.50 (br s, 1H

(NHCH₂)). ¹³C NMR (75 MHz, chloroform-d) δ ppm 21.10, 22.75,

27.95, 29.28, 31.96, 33.70, 42.94, 64.90, 125.78, 126.07, 127.16,

127.56, 127.65, 128.00, 128.11, 128.46, 129.30, 130.13, 135.19,

136.95, 139.85, 173.94. LCMS m/z [M + H]⁺= 483.29. HRMS-ESI⁺

m/z [M + H]⁺calcd for C₃₁H₃₅ClN₂O₂, 483.3006; found, 483.3003.

4.1.1.2.7. N-Benzyl-2-[4-(9H-ﬂuoren-9-ylidene)piperidin-1-yl]-4-

hydroxypentanamide ((2RS,4RS)-41 and (2RS,4SR)-41). Com-

pounds (2RS,4RS)-41 and (2RS,4SR)-41 were prepared according

to GP2 with 3-[4-(9H-ﬂuoren-9-ylidene)piperidin-1-yl]-5-methylox-

olan-2-one (40) (0.59 mmol, 0.23 g), 1-phenylmethanamine

hydrochloride (0.89 mmol, 0.19 g), and sodium 2-ethylhexanoate

(1.45 mmol, 0.285 g) in 4 mL of dry THF. The obtained crude

products were puriﬁed by column chromatography over silica gel

(DCM/Ace = 9:1) to yield (2RS,4RS)-41 (150 mg, 50%, R_f= 0.48

(DCM/Ace = 9:1)) and (2RS,4SR)-41 (51 mg, 17%, R_f= 0.29

(DCM/Ace = 9:1)) as a yellow oil. ds: (2RS,4RS)-41/(2RS,4SR)-41

= 75:25. Formula C₃₀H₃₂N₂O₂, FW 452.59. Compound (2RS,4RS)-

(m, 2H), 4.47 (dd, J = 6.15, 14.95 Hz, 2H), 3.99−4.11 (m, 1H),

3.28−3.45 (m, 3H), 2.75−2.89 (m, 2H), 2.67 (dd, J = 4.40, 14.95 Hz,

2H), 2.29−2.44 (m, 6H), 1.69−1.82 (m, 2H), 1.18 (d, J = 6.45 Hz,

3H). ¹³C NMR (75 MHz, chloroform-d) δ ppm 173.0, 140.5, 140.4,

138.2, 137.9, 135.5, 133.0, 129.3, 128.8, 128.7, 127.6, 127.6, 126.9,

125.5, 65.5, 65.3, 51.9, 51.1, 43.3, 34.2, 32.4, 31.2, 22.9.

4.1.1.2.9. 2-(4-(10,11-Dihydro-5H-dibenzo[a,d][7]annulen-5-

ylidene)piperidin-1-yl)-N-(4-ﬂuorobenzyl)-4-hydroxypentanamide

((2RS,4RS)-43b and (2RS,4SR)-43b). Compounds (2RS,4RS)-43b

and (2RS,4SR)-43b were prepared according to GP2 with 3-(4-

(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ylidene)piperidin-1-

yl)-5-methyldihydrofuran-2(3H)-one (42) (0.63 mmol, 0.24 g), 1-(4-

ﬂuorophenyl)methanamine hydrochloride (0.95 mmol, 0.15 g), and

sodium 2-ethylhexanoate (1.60 mmol, 0.27 g) in 4 mL of dry THF.

The obtained crude products were puriﬁed by column chromatog-

raphy over silica gel (DCM/Ace = 9:1) to yield (2RS,4RS)-43b (120

mg, 38%, R_f= 0.54 (DCM/Ace = 9:1)) and (2RS,4SR)-43b (40 mg,

12%, R_f= 0.33 (DCM/Ace = 9:1)) as a yellow oil. ds: (2RS,4RS)-

43b/(2RS,4SR)-43b = 75:25. Formula C₃₂H₃₅FN₂O₂, FW 498.64.

1

Compound (2RS,4RS)-43b: H NMR (300 MHz, chloroform-d) δ

ppm 6.90−7.38 (m, 12H), 4.25−4.72 (m, 4H), 3.87−4.09 (m, 1H),

3.70−3.84 (m, 1H), 3.25−3.54 (m, 2H), 2.74−2.89 (m, 1H), 2.65 (d,

J = 17.58 Hz, 1H), 2.29−2.50 (m, 3H), 1.42−2.20 (m, 4H), 1.02−

1.33 (m, 5H). ¹³C NMR (75 MHz, chloroform-d) δ ppm 169.2,

163.8, 140.1, 137.9, 133.4, 129.4, 129.3, 129.3, 129.2, 128.5, 127.1,

125.5, 115.8, 115.7, 115.5, 115.4, 65.8, 65.6, 48.1, 47.3, 46.4, 44.8,

44.5, 43.3, 43.2, 42.8, 42.6, 32.4, 31.6, 25.3, 24.3, 23.9, 23.6, 23.4,

22.5, 11.8. HRMS-ESI⁺m/z [M + H]⁺calcd for C₃₂H₃₆N₂O₂F,

499.2773; found, 499.2761. Compound (2RS,4SR)-43b: ¹H NMR

(300 MHz, chloroform-d) δ ppm 6.90−7.38 (m, 12H), 4.25−4.72

(m, 4H), 3.87−4.09 (m, 1H), 3.70−3.84 (m, 1H), 3.25−3.54 (m,

2H), 2.74−2.89 (m, 1H), 2.65 (d, J = 17.58 Hz, 1H), 2.29−2.50 (m,

3H), 1.42−2.20 (m, 4H), 1.02−1.33 (m, 5H). ¹³C NMR (75 MHz,

chloroform-d) δ ppm 169.3, 163.9, 140.1, 137.8, 133.4, 129.4, 129.3,

129.3, 129.2, 128.5, 127.1, 125.4, 115.8, 115.7, 115.5, 115.4, 65.8,

65.6, 48.2, 47.3, 46.4, 44.8, 44.5, 43.4, 43.2, 42.8, 42.5, 32.4, 31.6,

25.3, 24.3, 23.9, 23.5, 23.4, 22.5, 11.7.

4.1.1.3. General Procedure for the Synthesis of the 4-

Hydroxypentanamide and 4-Hydroxybutanamide Derivatives 45

and 47a−c (GP3). These reactions were performed under an argon

atmosphere. The relevant 3-substituted 5-methyldihydrofuran-2(3H)-

one or dihydrofuran-2(3H)-one derivative (1 equiv) was heated with

the corresponding N-benzylamine (2 equiv) in dry THF at reﬂux for

48 h. When the reaction was complete, the mixture was cooled on ice,

quenched with a 1 M aqueous solution of HCl (1.5 mL), and

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.

1

41: H NMR (300 MHz, chloroform-d) δ ppm 1.17−1.31 (m, 3H)

1.69−1.89 (m, 2H) 1.96−2.57 (m, 2H) 2.70−2.87 (m, 2H) 3.08−

3.44 (m, 4H) 3.70−3.88 (m, 1H) 4.19−4.65 (m, 3H) 7.04−7.46 (m,

10H) 7.69−7.86 (m, 3H) 7.99 (br s, 1H). Compound (2RS,4SR)-41:

¹H NMR (300 MHz, chloroform-d) δ ppm 1.14−1.24 (m, 3H) 1.68−

1.88 (m, 2H) 2.38−2.54 (m, 1H) 2.67−2.89 (m, 3H) 2.95 (br s, 2H)

3.18−3.25 (m, 2H) 3.26−3.53 (m, 2H) 3.96−4.10 (m, 1H) 4.40−

4.45 (m, 1H) 4.51 (s, 1H) 7.22−7.41 (m, 11H) 7.71−7.86 (m, 3H).

4.1.1.2.8. N-Benzyl-2-(4-(10,11-dihydro-5H-dibenzo[a,d][7]-

annulen-5-ylidene)piperidin-1-yl)-4-hydroxypentanamide

((2RS,4RS)-43a and (2RS,4SR)-43a). Compounds (2RS,4RS)-43a and

(2RS,4SR)-43a were prepared according to GP2 with 3-(4-(10,11-

dihydro-5H-dibenzo[a,d][7]annulen-5-ylidene)piperidin-1-yl)-5-

methyldihydrofuran-2(3H)-one (42) (0.48 mmol, 0.18 g), 1-phenyl-

methanamine hydrochloride (0.72 mmol, 0.10 g), and sodium 2-

ethylhexanoate (1.80 mmol, 0.30 g) in 4 mL of dry THF. The

obtained crude products were puriﬁed by column chromatography

over silica gel (DCM/Ace = 9:1) to yield (2RS,4RS)-43a (120 mg,

52%, R_f= 0.48 (DCM/Ace = 9:1)) and (2RS,4SR)-43a (30 mg, 13%,

R_f= 0.29 (DCM/Ace = 9:1)) as a yellow oil. ds: (2RS,4RS)-43a/

(2RS,4SR)-43a = 80:20. Formula: C₃₂H₃₆N₂O₂, FW 480.65.

4.1.1.3.1. N-Benzyl-2-[(2-{[(diphenylmethylidene)amino]oxy}-

ethyl)(methyl)amino]-4-hydroxypentanamide ((2RS,4RS)-45 and

(2RS,4SR)-45). Compounds (2RS,4RS)-45 and (2RS,4SR)-45 were

prepared according to GP3 with 3-[(2-{[(diphenylmethylidene)-

amino]oxy}ethyl)(methyl)amino]-5-methyloxolan-2-one (44) (1.33

mmol, 0.47 g) and N-benzylamine (1.59 mmol, 0.17 g) in 10 mL of

dry THF. The obtained crude products were puriﬁed by column

chromatography over silica gel (DCM/Ace = 9:1) to yield (2RS,4RS)-

45 (309 mg, 48%, R_f= 0.48 (S₁)) and (2RS,4SR)-45 (103 mg, 16%, R_f

= 0.25 (S₁)) as a yellow oil. ds: (2RS,4RS)-45/(2RS,4SR)-45 = 75:25.

Formula C₂₈H₃₃N₃O₃, FW 459.58. Compound (2RS,4RS)-45: ¹H

NMR (300 MHz, chloroform-d) δ ppm 1.20−1.26 (m, 4H

(CHCH₃)) 1.71−1.84 (m, 2H (CHCH₂CH)) 2.24−2.30 (m, 3H

(NCH₃)) 2.77 (t, J = 5.26 Hz, 2H (OCH₂CH₂N)) 3.33−3.42 (m, 1H

(NCHCO)) 3.66−3.82 (m, 1H (OHCH)) 4.09−4.36 (m, 4H

(NHCH₂, OCH₂CH₂N)) 7.07−7.12 (m, 2H (Ar)) 7.20−7.24 (m,

3H (Ar)) 7.27−7.47 (m, 10H (Ar)) 7.84−7.92 (m, 1H (NHCH₂)).

HRMS-ESI⁺m/z [M + H]⁺calcd for C₂₈H₃₄N₃O₃, 460.2640; found,

1

Compound (2RS,4RS)-43a: H NMR (300 MHz, chloroform-d) δ

ppm 8.02 (t, J = 5.86 Hz, 1H), 7.21−7.46 (m, 5H), 6.99−7.20 (m,

8H), 4.35−4.62 (m, 2H), 3.72−3.87 (m, 1H), 3.28−3.46 (m, 3H),

2.76−2.93 (m, 2H), 2.57−2.69 (m, 2H), 2.39−2.55 (m, 3H), 2.28−

2.39 (m, 3H), 1.69−1.87 (m, 2H), 1.28 (d, J = 5.86 Hz, 3H). ¹³C

NMR (75 MHz, chloroform-d) δ ppm 174.3, 150.0, 140.4, 138.1,

138.1, 137.9, 135.7, 132.7, 129.4, 129.3, 128.8, 128.7, 127.8, 127.6,

127.5, 127.0, 126.9, 125.8, 125.5, 67.6, 67.4, 53.9, 51.8, 50.3, 43.5,

33.6, 32.5, 31.3, 31.3, 29.7, 29.3, 24.6. HRMS-ESI⁺m/z [M + H]⁺

calcd for C₃₂H₃₇N₂O₂, 481.2855; found, 481.2855. Compound

1

460.2621. Compound (2RS,4SR)-45 H NMR (300 MHz, chloro-

form-d) δ ppm 1.22−1.31 (m, 3H (CHCH₃)) 1.62−1.89 (m, 3H

(CHCH₂CH)) 1.97−2.05 (m, 2H (NCH₃)) 2.26−2.31 (m, 1H

(NCH₃)) 2.70−2.89 (m, 2H (OCH₂CH₂N)) 3.45 (dd, J = 8.72, 4.36

1

(2RS,4SR)-43a: H NMR (300 MHz, chloroform-d) δ ppm 7.42 (t,

J = 5.57 Hz, 1H), 7.24−7.39 (m, 5H), 7.06−7.16 (m, 6H), 6.99−7.06

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Hz, 1H (NCHCO)) 3.96−4.17 (m, 2H (NHCH₂)) 4.20−4.34 (m,

3H (OCH₂CH₂N)) 4.38−4.46 (m, 1H (OHCH)) 7.08−7.14 (m, 2H

(Ar)) 7.20−7.47 (m, 13H (Ar)). ¹³C NMR (126 MHz, chloroform-d)

δ ppm 170.0, 157.6, 138.4, 136.1, 133.3, 129.7, 129.1, 129.0, 128.8,

128.7, 128.4, 128.3, 128.0, 127.9, 127.6, 127.6, 127.3, 65.1, 63.9, 53.4,

43.9, 38.6, 31.5, 29.8, 23.4. HRMS-ESI⁺m/z [M + H]⁺calcd for

C₂₈H₃₄N₃O₃, 460.2595; found, 460.2591.

solution of amine F (diphenylmethanone O-(2-(methylamino)ethyl)

oxime (32)) (1.76 mmol, 0.44 g, 1 equiv) in acetonitrile (10 mL).

Then, the relevant N-benzyl-2-bromo-4-(1,3-dioxoisoindolin-2-yl)-

butanamide derivative (48a,b) (1.76 mmol, 1 equiv) was added, and

the reaction mixture was stirred at reﬂux for 24 h. Then, the

precipitate was ﬁltered, the ﬁltrate was concentrated under vacuum,

and the product was puriﬁed by column chromatography.

4.1.1.3.2. N-Benzyl-2-[(2-{[(diphenylmethylidene)amino]oxy}-

ethyl)(methyl)amino]-4-hydroxybutanamide (47a). Compound

47a was prepared according to GP3 with 3-[(2-

{[(diphenylmethylidene)amino]oxy}ethyl)(methyl)amino]oxolan-2-

one (46) (1.33 mmol, 0.45 g) and 1-phenylmethanamine (1.59 mmol,

0.17 g) in 10 mL of dry THF. The obtained crude product was

puriﬁed by column chromatography over silica gel (DCM/Ace = 7:3)

to yield 47a (332 mg, 56%, R_f= 0.50 (DCM/Ace = 7:3)). Formula

C₂₇H₃₁N₃O₃, FW 445.55. ¹H NMR (300 MHz, chloroform-d) δ ppm

1.83−1.94 (m, 2H (CH₂CH₂OH)) 2.28 (s, 3H (Me)) 2.79 (t, J =

5.39 Hz, 2H (CH₂N)) 3.28−3.35 (m, 1H (NCH)) 3.52−3.62 (m, 1H

(CH₂OH)) 3.78−3.87 (m, 1H (CH₂OH)) 4.19 (dd, J = 14.88, 6.41

Hz, 2H (CH₂O)) 4.23−4.31 (m, 2H (NHCH₂)) 7.07−7.13 (m, 2H

(Ar)) 7.18−7.47 (m, 13H (Ar)) 7.78 (t, J = 5.90 Hz, 1H (CONH)).

¹³C NMR (300 MHz, chloroform-d) δ ppm 14.20, 27.29, 38.49,

4.1.1.4.1. N-[(2-Chlorophenyl)methyl]-4-(1,3-dioxo-2,3-dihydro-

1H-isoindol-2-yl)-2-[(2-{[(diphenylmethylidene)amino]oxy}ethyl)-

(methyl)amino]butanamide (49a). According to GP4, amide 48a

(1.35 mmol, 0.59 g), amine F (diphenylmethanone O-(2-

(methylamino)ethyl) oxime (32)) (1.35 mmol, 0.34 g), KI (1.22

mmol, 0.20 g), and anhydrous K₂CO₃(3.11 mmol, 0.43 g) were

combined in acetonitrile (15 mL). The obtained crude product was

puriﬁed by column chromatography over silica gel (PE/EtOAc = 1:1)

to yield 49a (346 mg, 42%, R_f= 0.49 (PE/EtOAc (1:1)) as a yellow

1

oil. Formula C₃₅H₃₃ClN₄O₄, FW 609.11. H NMR (chloroform-d) δ

ppm 2.19 (dtd, J = 13.75, 8.06, 8.06, 6.03 Hz, 2H (CH₂CH₂NH))

2.29 (s, 3H (Me)) 2.83 (t, J = 5.51 Hz, 2H (CH₂N)) 3.26 (dd, J =

7.95, 4.87 Hz, 1H (NCH)) 3.76 (ddd, J = 14.04, 8.01, 6.41 Hz, 1H

(OCH₂)) 3.92 (ddd, J = 13.85, 8.21, 5.90 Hz, 1H (OCH₂)) 4.18−4.33

(m, 4H (NHCH₂; CH₂phthalimide)) 7.13−7.35 (m, 12H (Ar))

7.37−7.47 (m, 3H (Ar; CONH)) 7.65−7.72 (m, 2H (phthalimide))

7.79−7.85 (m, 2H (phthalimide)).

4.1.1.4.2. N-[(3,4-Dichlorophenyl)methyl]-4-(1,3-dioxo-2,3-dihy-

dro-1H-isoindol-2-yl)-2-[(2-{[(diphenylmethylidene)amino]oxy}-

ethyl)(methyl)amino]butanamide (49b). According to GP4, amide

48b (1.35 mmol, 0.63 g), amine F (diphenylmethanone O-(2-

(methylamino)ethyl) oxime (32)) (1.35 mmol, 0.34 g), KI (1.22

mmol, 0.20 g), and anhydrous K₂CO₃(3.11 mmol, 0.43 g) were

combined in acetonitrile (15 mL). The obtained crude product was

puriﬁed by column chromatography over silica gel (PE/EtOAc = 1:1)

to yield 49b (317 mg, 37%, R_f= 0.47 (PE/EtOAc (1:1)) as a yellow

oil. Formula C₃₅H₃₂Cl₂N₄O₄, FW 643.57. ¹H NMR (300 MHz,

chloroform-d) δ ppm 1.84−1.97 (m, 1H (CH₂CH₂NH)) 2.14−2.23

(m, 1H (CH₂CH₂NH)) 2.32 (s, 3H (Me)) 2.80 (t, J = 5.26 Hz, 2H

(CH₂N)) 3.77 (ddd, J = 13.91, 7.76, 6.54 Hz, 1H (NCH)) 3.85−3.99

(m, 2H (OCH₂)) 3.99−4.09 (m, 2H (CH₂phthalimide)) 4.25 (t, J =

5.26 Hz, 2H (NHCH₂)) 6.91 (dd, J = 8.21, 2.05 Hz, 1H (Ar) 7.10 (d,

J = 2.05 Hz, 1H (Ar)) 7.20−7.44 (m, 11H (Ar)) 7.44−7.50 (m, 1H

(CONH)) 7.66−7.74 (m, 2H (phthalimide)) 7.78−7.86 (m, 2H

(phthalimide)).

4.1.1.5. General Procedure for the Synthesis of 2-Substituted 4-

Aminobutanamide Derivatives (50a,b) (GP5). Hydrazine hydrate (2

equiv) was added to a suspension of a 2-substituted 4-

phthalimidobutanoic acid derivative (1 equiv) in ethanol (10 mL).

The solution was stirred at 60 °C for 2 h and at rt for 5 h. Then, the

precipitate was ﬁltered and washed with DCM (5 mL). The ﬁltrate

was evaporated, and the product was extracted two times with 8 mL

of DCM. The combined organic fractions were dried over Na₂SO₄,

and the obtained product was puriﬁed by column chromatography.

4.1.1.5.1. 4-Amino-N-[(2-chlorophenyl)methyl]-2-[(2-

{[(diphenylmethylidene)amino]oxy}ethyl)(methyl)amino]-

butanamide (50a). According to GP5, N-[(2-chlorophenyl)methyl]-

4 - ( 1 , 3 - d i o x o - 2 , 3 - d i h y d r o - 1 H - i s o i n d o l - 2 - y l ) - 2 - [ ( 2 -

{[(diphenylmethylidene)amino]oxy}ethyl)(methyl)amino]-

butanamide (49a) (1 mmol, 0.61 g) and hydrazine hydrate (2 mmol,

0.11 g) were combined in ethanol (7 mL). The obtained crude

product was puriﬁed by column chromatography over silica gel

(EtOAc/MeOH = 8:2 → 25% NH₃/MeOH/DCM/PE =

9:45:120:18) to yield 50a (325 mg, 68%, R_f= 0.15 (PE/EtOAc

(1:1)) as a yellow oil. Formula C₂₇H₃₁ClN₄O₂, FW 479.01. ¹H NMR

(chloroform-d) δ ppm 1.67−1.80 (m, 2H (CH₂CH₂NH₂)) 2.28 (s,

3H (Me)) 2.67−2.78 (m, 1H (CH₂N)) 2.78−2.88 (m, 3H (CH₂NH₂;

(CH₂N)) 3.22 (dd, J = 7.82, 5.26 Hz, 1H (NCH)) 4.12−4.35 (m, 4H

(NHCH₂; CH₂O)) 7.09−7.45 (m, 14H (Ar)) 7.58 (t, J = 6.16 Hz, 1H

(CONH)). ¹³C NMR (300 MHz, chloroform-d) δ ppm 29.93, 38.57,

40.44, 40.95, 53.82, 65.23, 72.82, 126.86, 127.81, 127.81, 128.05,

128.05, 128.23, 128.23, 128.40, 128.84, 129.07, 129.07, 129.13,

129.31, 129.39, 133.17, 133.20, 136.02, 136.20, 156.96, 173.47.

43.02, 53.37, 60.40, 61.68, 67.48, 72.43, 127.21, 127.33, 127.83,

128.13, 128.30, 128.54, 128.93, 129.06, 129.49, 133.13, 136.14,

138.26, 157.15, 174.07. HRMS-ESI⁺m/z [M + H]⁺calcd for

C₂₇H₃₂N₃O₃, 446.2444; found, 446.2447.

4 . 1 . 1 . 3 . 3 . N - [ ( 2 - C h l o r o p h e n y l ) m e t h y l ] - 2 - [ ( 2 -

{[(diphenylmethylidene)amino]oxy}ethyl)(methyl)amino]-4-hy-

droxybutanamide (47b). Compound 47b was prepared according to

GP3 with 3-[(2-{[(diphenylmethylidene)amino]oxy}ethyl)(methyl)-

amino]oxolan-2-one (46) (1.33 mmol, 0.45 g) and 1-(2-

chlorophenyl)methanamine (1.59 mmol, 0.23 g) in 10 mL of dry

THF. The obtained crude product was puriﬁed by column

chromatography over silica gel (DCM/Ace = 7:3) to yield 47b

(223 mg, 35%, R_f= 0.52 (DCM/Ace = 7:3)). Formula

1

C₂₇H₃₀ClN₃O₃, FW 480.01. H NMR (300 MHz, chloroform-d) δ

ppm 1.82−1.92 (m, 2H (CH₂CH₂OH)) 2.28 (s, 3H (Me)) 2.80 (t, J

= 5.51 Hz, 2H (CH₂N)) 3.28−3.36 (m, 1H (NCH)) 3.50−3.60 (m,

1H (CH₂OH)) 3.81 (dt, J = 11.09, 4.58 Hz, 1H (CH₂OH)) 4.18−

4.34 (m, 4H (NHCH₂; CH₂O)) 7.10−7.49 (m, 14H (Ar)) 7.86 (t, J =

5.90 Hz, 1H (CONH)). ¹³C NMR (300 MHz, chloroform-d) δ ppm

27.22, 29.69, 38.54, 41.17, 53.38, 61.68, 67.31, 72.57, 126.91, 127.81,

128.08, 128.25, 128.57, 128.91, 129.02, 129.05, 129.39, 129.47,

133.09, 133.21, 135.54, 136.12, 157.18, 174.31. HRMS-ESI⁺m/z [M

+ H]⁺calcd for C₂₇H₃₁N₃O₃Cl, 480.2054; found, 480.2056.

4.1. 1. 3. 4. N-[(3, 4-Dichlorophenyl)methyl]-2-[(2-

{[(diphenylmethylidene)amino]oxy}ethyl)(methyl)amino]-4-hy-

droxybutanamide (47c). Compound 47c was prepared according to

GP3 with 3-[(2-{[(diphenylmethylidene)amino]oxy}ethyl)(methyl)-

amino]oxolan-2-one (46) (1.33 mmol, 0.45 g) and 1-(3,4-

dichlorophenyl)methanamine (1.59 mmol, 0.30 g) in 10 mL of dry

THF. The obtained crude product was puriﬁed by column

chromatography over silica gel (DCM/Ace = 7:3) to yield 47c

(369 mg, 54%, R_f= 0.49 (DCM/Ace = 7:3)). Formula

1

C₂₇H₂₉Cl₂N₃O₃, FW 514.44. H NMR (300 MHz, chloroform-d) δ

ppm 1.84−1.92 (m, 2H (CH₂CH₂OH)) 2.32 (s, 3H (Me)) 2.71−

2.81 (m, 2H (CH₂N)) 3.29−3.35 (m, 1H (NCH)) 3.54−3.64 (m, 1H

(CH₂OH)) 3.82 (dt, J = 11.09, 4.58 Hz, 1H (CH₂OH)) 3.95 (d, J =

6.16 Hz, 1H (CH₂O)) 4.01 (d, J = 6.67 Hz, 1H (CH₂O)) 4.26−4.32

(m, 2H (NHCH₂)) 6.89 (dd, J = 8.21, 2.05 Hz, 1H (Ar)) 7.08 (d, J =

2.05 Hz, 1H (Ar)) 7.23−7.46 (m, 11H (Ar)) 7.86 (t, J = 6.28 Hz, 1H

(CONH)). ¹³C NMR (300 MHz, chloroform-d) δ ppm 14.20, 27.23,

38.42, 41.81, 53.33, 61.62, 67.25, 71.99, 126.63, 127.86, 128.17,

128.36, 129.05, 129.15, 129.21, 129.59, 130.36, 131.03, 132.36,

133.08, 136.09, 138.73, 157.13, 174.45. HRMS-ESI⁺m/z [M + H]⁺

calcd for C₂₇H₃₀N₃O₃Cl₂, 514.1664; found, 514.1666.

4.1.1.4. General Procedure for the Synthesis of N-Benzyl-4-(1,3-

dioxoisoindolin-2-yl)-2-(4-(diphenylmethylene)piperidin-1-yl)-

butanamide derivatives (49a,b) (GP4). Anhydrous K₂CO₃(4.40

mmol, 0.61 g, 2.5 equiv) and KI (1.7 mmol, 0.29 g) were added to a

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4.1.1.5.2. 4-Amino-N-[(3,4-dichlorophenyl)methyl]-2-[(2-

{[(diphenylmethylidene)amino]oxy}ethyl)(methyl)amino]-

butanamide (50b). According to GP5, N-(3,4-dichlorobenzyl)-4-

(1,3-dioxoisoindolin-2-yl)-2-((2-(((diphenylmethylene)amino)oxy)-

ethyl)(methyl)amino)butanamide (49b) (1 mmol, 0.64 g) and

hydrazine hydrate (2 mmol, 0.11 g) were combined in ethanol (7

mL). The obtained crude product was puriﬁed by column

chromatography over silica gel (EtOAc/MeOH = 8:2 → 25%

NH₃/MeOH/DCM/PE = 9:45:120:18) to yield 50b (442 mg, 86%,

R_f= 0.14 (PE/EtOAc (1:1)) as a yellow oil. Formula C₂₇H₃₀Cl₂N₄O₂,

FW 513.46. ¹H NMR (chloroform-d) δ ppm 1.73−1.97 (m, 2H

(CH₂CH₂NH₂)) 2.31 (s, 3H (Me)) 2.73−2.81 (m, 2H (CH₂N))

2.82−2.93 (m, 2H (CH₂NH₂)) 3.26 (dd, J = 8.21, 4.87 Hz, 1H

(NCH)) 3.90 (dd, J = 15.39, 6.16 Hz, 1H (CH₂O)) 4.02 (dd, J =

15.26, 6.54 Hz, 1H (CH₂O)) 4.27 (t, J = 5.13 Hz, 2H (NHCH₂))

6.89 (dd, J = 8.34, 2.18 Hz, 1H (Ar)) 7.09 (d, J = 1.80 Hz, 1H (Ar))

7.22−7.45 (m, 11H (Ar)) 7.73 (t, J = 6.41 Hz, 1H (CONH)). ¹³C

NMR (300 MHz, chloroform-d) δ ppm 27.70, 38.41, 39.88, 41.73,

53.59, 65.69, 72.16, 126.64, 127.84, 127.84, 128.17, 128.17, 128.33,

128.33, 129.03, 129.15, 129.15, 129.54, 130.34, 130.92, 132.30,

133.13, 136.09, 138.99, 138.99, 157.04, 173.61. HRMS-ESI⁺m/z [M

+ H]⁺calcd for C₂₉H₃₁N₄O₂Cl₂, 513.1824; found, 513.1818.

4.1.1.6. General Procedure for the Synthesis of 2-Substituted 4-

Acetamidobutanamide Derivatives (51a,b) (GP6). A mixture of

acetic acid (2 equiv) and N,N′-dicyclohexylcarbodiimide (DCC) (2

equiv) in 5 mL of DCM was stirred at 0 °C for 10 min. Then, a 4-

aminobutanamide derivative (1 equiv) and 4-dimethylaminopyridine

(DMAP) (2 equiv) were added to the reaction mixture and stirring

continued for 20 h at room temperature. The obtained N,N-

dicyclohexylurea (DCU) was ﬁltered, the ﬁltrate was evaporated, and

the product was puriﬁed by column chromatography over silica gel

(S₁₁(Chl/Ace 1:1)).

amide 53a−e (1 equiv), and TBAB (0.1 equiv) were dissolved in dry

DMF (for 55a−e, 56a−e dry DCM was used), and DIPEA (1 equiv)

was then added. After stirring at reﬂux for 16 h, extraction with DCM

and deionized water was performed. The organic phase was washed

with saturated NaHCO₃and dried with anhydrous Na₂SO₄. The

solvent was evaporated, and the product was puriﬁed by column

chromatography over silica gel (S₁₂DCM/Ace = 7:3).

4.1.1.7.1. N-Benzyl-2-{4-[bis(3-methylthiophen-2-yl)-

methylidene]piperidin-1-yl}-3-hydroxypropanamide (54a). The

synthesis was done according to GP7 with amine A (17) (1.39

mmol, 400 mg), amide 53a (1.16 mmol, 300 mg), TBAB (0.12 mmol,

40 mg), DIPEA (1.16 mmol, 201 μL), and DMF (3 mL). The

obtained crude product was puriﬁed by column chromatography over

silica gel (DCM/Ace = 7:3) to yield 54a (260 mg, 48%, R_f= 0.33 and

1

0.51 (S₆) as a yellow oil. Formula C₂₆H₃₀N₂O₂S₂, FW: 466.66. H

NMR (300 MHz, chloroform-d) δ ppm 2.00−2.12 (m, 6 H) 2.25−

2.47 (m, 3 H) 2.67−2.82 (m, 2 H) 2.85−2.98 (m, 2 H) 3.44−3.55

(m, 1 H) 3.86−4.06 (m, 2 H) 4.47 (dd, J = 16.70, 6.15 Hz, 2 H) 5.30

(s, 1 H) 6.77 (d, J = 5.28 Hz, 2 H) 7.14 (d, J = 5.27 Hz, 1 H) 7.21−

7.39 (m, 5 H) 7.97−8.09 (m, 1 H). ¹³C NMR (75 MHz, chloroform-

d) δ ppm 171.3, 141.6, 138.0, 137.4, 133.9, 129.7, 129.5, 128.8, 128.7,

127.7, 127.5, 127.5, 124.6, 124.1, 121.0, 68.1, 58.7, 53.6, 51.3, 43.2,

31.4, 14.7. HRMS-ESI⁺m/z [M + H]⁺calcd for C₂₆H₃₀N₂O₂S₂,

467.1827; found, 467.1843.

4.1.1.7.2. 2-{4-[Bis(3-methylthiophen-2-yl)methylidene]-

piperidin-1-yl}-N-[(2-chlorophenyl)methyl]-3-hydroxypropanamide

(54b). The synthesis was done according to GP7 with amine A (17)

(1.64 mmol, 470 mg), amide 53b (1.37 mmol, 400 mg), TBAB (0.14

mmol, 44 mg), DIPEA (1.37 mmol, 237 μL), and DMF (5 mL). The

obtained crude product was puriﬁed by column chromatography over

silica gel (S₁₂) to yield 54b (315 mg, 46%, R_f= 0.16 and 0.35 (S₉)) as

1

a yellow oil. Formula C₂₆H₂₉ClN₂O₂S₂, FW: 501.10. H NMR (300

4 . 1 . 1 . 6 . 1 . N - [ ( 2 - C h l o r o p h e n y l ) m e t h y l ] - 2 - [ ( 2 -

{[(diphenylmethylidene)amino]oxy}ethyl)(methyl)amino]-4-acet-

amidobutanamide (51a). According to GP6, acetic acid (0.63 mmol,

0.037 g, 36 μL), DCC (0.63 mmol, 0.13 g), 4-aminobutanamide

(50a) (0.15 g, 0.32 mmol), and DMAP (0.63 mmol, 0.08 g) were

combined in 5 mL of DCM. Compound 51a was obtained as a yellow

oil (140 mg, 86%). R_f= 0.45 (Chl/Ace = 1:1). Formula

C₂₉H₃₃Cl₁N₄O₃, FW 521.05. ¹H NMR (chloroform-d) δ ppm

1.78−1.93 (m, 5H (CH₂CH₂NH; Me)) 2.27 (s, 3H (Me)) 2.79 (t,

J = 5.39 Hz, 2H (CH₂N)) 3.11−3.23 (m, 2H (CH₂NH)) 3.33−3.45

(m, 1H (NCH)) 4.16−4.34 (m, 4H (NHCH₂; CH₂O)) 6.94 (br s, 1H

(CONH)) 7.12−7.44 (m, 14H (Ar)) 7.75 (t, J = 6.03 Hz, 1H

(CONH)). ¹³C NMR (300 MHz, chloroform-d) δ ppm 23.25, 24.34,

38.53, 39.00, 41.10, 53.42, 66.52, 66.52, 72.54, 126.87, 127.80, 127.80,

128.06, 128.06, 128.24, 128.24, 128.54, 128.87, 128.88, 129.03,

129.03, 129.43, 129.45, 133.14, 133.21, 135.68, 136.11, 157.11,

170.30, 173.88. HRMS-ESI⁺m/z [M + H]⁺calcd for C₂₉H₃₄N₄O₃Cl,

5521.2298; found, 521.22313.

MHz, chloroform-d) δ ppm 8.58−8.69 (m, 1H), 7.28−7.38 (m, 2H),

7.10−7.23 (m, 4H), 6.77 (d, J = 5.27 Hz, 2H), 4.51 (t, J = 5.27 Hz,

2H), 3.93−4.17 (m, 3H), 3.19 (s, 2H), 2.98−3.14 (m, 2H), 2.54 (d, J

= 4.10 Hz, 4H), 2.07 (s, 6H). ¹³C NMR (75 MHz, chloroform-d) δ

ppm 168.4, 139.1, 136.8, 134.9, 134.2, 133.4, 129.7, 129.6, 129.0,

127.1, 124.4, 122.3, 67.5, 58.7, 51.1, 41.3, 30.0, 14.6. HRMS-ESI⁺m/z

[M + H]⁺calcd for C₂₆H₂₉ClN₂O₂S₂, 501.1441; found, 501.1037.

4.1.1.7.3. 2-{4-[Bis(3-methylthiophen-2-yl)methylidene]-

piperidin-1-yl}-N-[(4-ﬂuorophenyl)methyl]-3-hydroxypropanamide

(54c). The synthesis was done according to GP7 with amine A (17)

(0.70 mmol, 200 mg), amide 53c (0.58 mmol, 160 mg), TBAB (0.06

mmol, 19 mg), DIPEA (0.58 mmol, 100 μL), and DMF (3 mL). The

obtained crude product was puriﬁed by column chromatography over

silica gel (DCM/Ace = 7:3) to yield 54c (100 mg, 36%, R_f= 0.24 and

0.55 (S₁₂)) as a yellow oil. Formula C₂₆H₂₉FN₂O₂S₂, FW: 484.65. ¹H

NMR (300 MHz, chloroform-d) δ ppm 8.25−8.40 (m, 1H), 7.19−

7.33 (m, 2H), 7.13 (d, J = 4.69 Hz, 2H), 6.98 (s, 2H), 6.77 (d, J =

5.28 Hz, 2H), 4.39 (dd, J = 5.86, 11.14 Hz, 2H), 3.86−4.11 (m, 2H),

3.67−3.81 (m, 1H), 3.00−3.14 (m, 2H), 2.83−2.96 (m, 2H), 2.45 (t,

J = 5.27 Hz, 4H), 2.07 (s, 6H). ¹³C NMR (75 MHz, chloroform-d) δ

ppm 169.9, 163.7, 160.5, 140.2, 137.1, 134.0, 133.7, 133.7, 129.5,

129.3, 129.2, 124.3, 121.8, 115.7, 115.5, 67.8, 58.6, 51.2, 42.5, 30.8,

14.6. HRMS-ESI⁺m/z [M + H]⁺calcd for C₂₆H₂₉FN₂O₂S₂,

485.1733; found, 485.1728.

4.1.1.7.4. 2-((3-(5H-Dibenzo[a,d][7]annulen-5-ylidene)propyl)-

(methyl)amino)-N-benzyl-3-hydroxypropanamide (55a). The syn-

thesis was done according to GP7 with amine B (20) (1.2 mmol, 313

mg), amide 53a (1 mmol, 257 mg), TBAB (0.01 mmol, 0.32 mg),

DIPEA (1 mmol, 174 μL), DCM 5 mL. The obtained crude product

was puriﬁed by column chromatography over silica gel (DCM/Ace =

7:3) to yield 55a (131 mg, 30%, R_f= 0.65 and 0.42 (S₁₂)). ¹H NMR

(chloroform-d): δ ppm 7.61 (t. 1H, NH), 7.40−7.05 (m, 10H, Ar),

6.98−6.89 (m, 1H, ArCHCHAr), 6.88−6.63 (m, 4H, ArCH

CHAr, Ar), 5.42−5.22 (m, 1H, CH), 4.38−4.16 (m, 2H, CH₂NH),

3.89 (ddd, 1H, J = 11.1; 7.7; 4.7 Hz, CH₂′OH), 3.74 (ddd, 1H, J =

11.1; 5.2; 4.2 Hz, CHNCH₃), 3.12 (dt, 1H, J = 8.0; 4.5 Hz,

CH₂″OH), 2.71−2.38 (m, 2H, CH₂CH₂C), 2.38−2.16 (m, 2H,

CH₂CH₂C), 1.97 (d, 3H, CH₃−N). OH proton was not detected.

4.1. 1. 6. 2. N-[(3, 4-Dichlorophenyl)methyl]-2-[(2-

{[(diphenylmethylidene)amino]oxy}ethyl)(methyl)amino]-4-acet-

amidobutanamide (51b). According to GP6, acetic acid (0.63 mmol,

0.037 g, 36 μL), DCC (0.63 mmol, 0.13 g), 4-aminobutanamide

(50b) (0.16 g, 0.32 mmol), and DMAP (0.63 mmol, 0.08 g) were

combined in 5 mL of DCM. Compound 51b was obtained as a yellow

oil (173 mg, 86%). R_f= 0.47 (Chl/Ace = 1:1). Formula

1

C₂₉H₃₂Cl₂N₄O₃, FW 555.50 H NMR (chloroform-d) δ ppm 1.77−

1.89 (m, 5H (CH₂CH₂NH; Me)) 2.26 (s, 3H (Me)) 2.69 (t, J = 5.38

Hz, 2H (CH₂N)) 3.09−3.26 (m, 2H (CH₂NH)) 3.31−3.44 (m, 1H

(NCH)) 4.12−4.31 (m, 4H (NHCH₂; CH₂O)) 6.67 (br s, 1H

(CONH)) 7.09−7.45 (m, 13H (Ar)) 7.73 (t, J = 6.07 Hz, 1H

(CONH)). ¹³C NMR (300 MHz, chloroform-d) δ ppm 23.15, 24.44,

38.43, 39.10, 41.15, 53.42, 66.62, 66.68, 72.44, 126.77, 127.86, 127.99,

128.12, 128.12, 128.34, 128.34, 128.44, 128.89, 128.89, 129.03,

129.03, 129.53, 129.59, 133.14, 133.28, 135.78, 136.21, 157.31,

170.36, 173.90. HRMS-ESI⁺m/z [M + H]⁺calcd for C₂₉H₃₃N₄O₃Cl₂,

555.1930; found, 555.1919.

4.1.1.7. General Procedures for the Synthesis of 2-Substituted 3-

Hydroxypropanamide Derivatives 54a−c, 55a−f, 56a−f, 57, 58,

59a−c (GP7). The corresponding amine A−F (1.2 equiv), a relevant

T

https://doi.org/10.1021/acschemneuro.1c00351

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

¹³C NMR (chloroform-d): δ ppm 174.02 (CONH), 143.60

(ArCHCHAr), 130.91 (ArCHCHAr), 129.16 (C_Ar), 129.05,

3

128.94 (C_Ar), 128.64 (d, J_C−F= 7.4 Hz, C_Ar), 128.59(C_Dche), 128.08

(Ph₂C), 142.30 (C_Dche), 142.20 (C_Dche), 137.01(C_Dche), 136.96

(C_Dche), 134.81 (C_Ph), 133.70 (C_Dche), 133.24 (C_Dche), 131.21 (C_Dche),

131.11 (C_Dche), 130.96 (ArCHCHAr), 130.88 (ArCHCHAr),

129.50 (C_Dche), 129.42(C_Dche), 129.48 (CHCPh₂), 128.96 (C_Dche),

128.68 (C_Dche), 128.26 (C_Ph), 128.00 (C_Ph), 127.25 (C_Ph), 127.06-

(C_Ph), 126.98(C_Ph), 126.84 (C_Dche), 67.55 (CHNCH₃), 66.78

(CH₂OH), 53.26 (CH₂CH₂CH), 40.68 (CH₂NH), 38.50

(CH₃N), 27.15 (CH₂CH₂CH) HRMS-ESI⁺m/z [M + H]⁺calcd

for C₂₉H₃₀N₂O₂, 439.2386; found, 439.2374.

(C_Dche), 127,42 (C_Ar), 126.94 (C_Ar), 115.52 (C_{Ar ortoF}), 115.20 (d,

²J_C−F= 21.5 Hz, C_Ar), 67.96 (CHNCH₃), 58.65 (CH₂OH), 55.19

(CH₂CH₂CH), 42.00 (CH₂Ph), 36.92 (CH₃N), 26.61

(CH₂CH₂CH)

4.1.1.7.8. 2-((3-(5H-Dibenzo[a,d][7]annulen-5-ylidene)propyl)-

(methyl)amino)-N-(4-methylbenzyl)-3-hydroxypropanamide

(55e). The synthesis was done according to GP7 with amine B (20)

(1.2 mmol, 313 mg), amide 53e (1 mmol, 271 mg), TBAB (0.01

mmol, 0.32 mg), DIPEA (1 mmol, 174 μL), DCM 5 mL. The

obtained crude product was puriﬁed by column chromatography over

silica gel (DCM/Ace = 7:3) to yield 55a (135 mg, 30%, R_f= 0.66 and

0.42 (S₁₂)). ¹H NMR (chloroform-d): δ ppm 7.57 (t, 1H, NH),

7,43−7.11 (m, 10H, Ar), 7.07−6.95 (m, 2H, Ar), 6.91−6.79 (m, 1H,

ArCHCHAr), 6.77−6.67 (m, 1H, ArCHCHAr), 5.52−5.26 (m,

1H, CH), 4.50−4.03 (m, 2H, CH₂NH), 3.90 (ddd, 1H, J = 11.0;

8,0; 5.6 Hz, CH₂′OH), 3.74 (dt, 1H, J = 11.1; 4.5 Hz, CHNCH₃),

3.13 (td, 1H, J = 8.1; 3.8 Hz, CH₂″OH), 2.74−2.37 (m, 2H,

CH₂CH₂C), 2.32 (d, 2H, CH₂CH₂C), 2.10−1.95 (m, 6H, CH₃-

N, CH₃Ar). OH proton was not detected. ¹³C NMR (chloroform-d):

δ ppm 173,80 (CONH), 143,66 (Ph₂C), 137.07 (C_Dche), 137.03

(C_Dche), 136.97 (C_Dche), 136.78 (C_Dche), 135.28 (C_Dche), 135.26 (C_Ar-

CH₂), 134.88 (C_Ar-CH₃), 134.75 (C_Dche), 133,77 (C_Dche), 133,72-

(C_Dche), 131.19 (ArCHCHAr), 131.07 (ArCHCHAr), 129.29

(C_Dche), 128,79 (C_Dche), 128,66 (C_Dche), 128,28 (C_Dche), 128,02 (C_Ar),

127.47(C_Ar), 127.39(C_Ar), 127.03(C_Ar), 66.90 (CHNCH₃), 60.37

(CH₂OH), 58,39 (CH₂CH₂CH), 42.40 (CH₂Ar), 38,79 (CH₃N),

26.85 (CH₂CH₂CH), 21.09 (CH₃Ar).

4.1.1.7.5. 2-((3-(5H-Dibenzo[a,d][7]annulen-5-ylidene)propyl)-

(methyl)amino)-N-(2-chlorobenzyl)-3-hydroxypropanamide (55b).

The synthesis was done according to GP7 with amine B (20) (1.2

mmol, 313 mg), amide 53b (1 mmol, 290 mg), TBAB (0.01 mmol,

0.32 mg), DIPEA (1 mmol, 174 μL), DCM 5 mL. The obtained crude

product was puriﬁed by column chromatography over silica gel

(DCM/Ace = 7:3) to yield 55a (141 mg, 30%, R_f= 0.65 and 0.42

(S₁₂)). ¹H NMR (chloroform-d): δ ppm 7.66 (d, 1H, NH), 7.42−7.04

(m, 12H, Ar), 6.89−6.77 (m, 1H, ArCHCHAr), 6.69 (dd, 1H,

ArCHCHAr), 5.46 (m, 1H, CH), 4.46−4.18 (m, 2H, CH₂NH),

4.12 (q, 1H, J = 7.1 Hz, CH₂′OH), 3.91 (ddd, 1H, J = 10.2; 8.0; 2.0

Hz, CHNCH₃), 3.16 (tt, 1H, CH₂″OH), 2.78−2.44 (m, 2H,

CH₂CH₂C), 2.39−2.16 (m, 2H, CH₂CH₂C), 2.10−1.99 (m,

3H, CH₃−N) ppm. OH proton was not detected. ¹³C NMR

(chloroform-d): δ ppm 173.81 (CONH), 143.67 (Ph₂C), 142.39

(C_Ph), 135.40 (C_Ph), 137.05 (C_Dche), 137.00 (C_Dche), 134.87 (C_Dche),

134.74 (C_Dche), 133.68 (C_Dche), 131.16 (C_Dche), 131.04 (ArCH

CHAr), 131.00 (ArCHCHAr), 128.82 (C_Ph), 128.68 (C_Ph), 128.44

(C_Ph), 128.28 (C_Ph), 128.03 (C_Dche), 127.99 (C_Dche), 127.40 (C_Dche),

127.30 (C_Dche), 127.02 (CCPh₂), 126.97 (C_Dche), 126.89(C_Dche),

67.81 (CHNCH₃), 58.55(CH₂OH), 57.67 (CH₂CH₂CH), 42.73

(CH₂NH), 38.80 (CH₃N), 27.16 (CH₂CH₂CH).

4.1.1.7.9. N-Benzyl-2-((3-(10,11-dihydro-5H-dibenzo[a,d][7]-

annulen-5-ylidene)propyl)(methyl)amino)-3-hydroxypropanamide

(56a). The synthesis was done according to GP7 with amine C (21)

(1.2 mmol, 315 mg), amide 53a (1 mmol, 257 mg), TBAB (0.01

mmol, 0.32 mg), DIPEA (1 mmol, 174 μL), DCM 5 mL. The

obtained crude product was puriﬁed by column chromatography over

silica gel (DCM/Ace = 7:3) to yield 56a (132 mg, 30%, R_f= 0.70 or

0.57 (S₁₂)). ¹H NMR (chloroform-d): δ ppm 7.57 (br s, 1H, NHCO),

6.98−7.37 (m, 13H, Ar), 5.76 (t, 1H, CH), 4.37−4.48 (m, 3H,

CH₂′OH, CH₂Ar), 4.06 (t, 1H, CH-NCH₃), 3,13−3,44 (m, 3H,

ArCH₂-CH₂Ar, CH₂′OH), 2.83−3,03 (m, 2H, ArCH₂−CH₂Ar),

2.68−2.81 (m, 2H, CH₂CH₂C), 2.21−2.34 (m, 2H,

CH₂CH₂C), 2.17 (s, 3H, CH₃). OH proton was not detected.

¹³C NMR (chloroform-d): δ ppm 172.97 (CONH), 144.42 (C_Ar),

140.95 (CH), 139.75 (C_Ar), 139.25 (C_Ar), 138,09 (C_Ar), 137.02

(C_Ar), 130.04 (C_Ar), 128,64 (C_Ar), 128,47 (C_Ar), 128,33 (C_Ar), 128,11

(C_Ar), 128,04 (C_Ar), 127.57 (C_Ar), 126.07 (C_Ar), 125.76 (C_Ar), 127.19

(C_Ar) 126.07 (C_Ar), 125.76 (C_Ar), 66.92 (CHN), 59.98 (CH₂OH),

57.34 (CH₂CH₂CH), 42.87 (CH₂NH), 41.25 (CH₃N), 33.70

(ArCH₂-CH₂Ar), 32.01 (ArCH₂−CH₂Ar), 27.19 (CH₂CH₂CH).

HRMS-ESI⁺m/z [M + H]⁺calcd for C₂₉H₃₃N₂O₂, 441.2542; found,

441.2530.

4.1.1.7.6. 2-((3-(5H-Dibenzo[a,d][7]annulen-5-ylidene)propyl)-

(methyl)amino)-N-(4-chlorobenzyl)-3-hydroxypropanamide (55c).

The synthesis was done according to GP7 with amine B (20) (1.2

mmol, 313 mg), amide 53c (1 mmol, 290 mg), TBAB (0.01 mmol,

0.32 mg), DIPEA (1 mmol, 174 μL), DCM 5 mL. The obtained crude

product was puriﬁed by column chromatography over silica gel

(DCM/Ace = 7:3) to yield 55a (141 mg, 30%, R_f= 0.65 and 0.41

1

(S₁₂)). H NMR (chloroform-d): δ ppm 7.53 (dt, 1H, NH), 7.41−

7.07 (m, 9H, Ar), 6.92−6.65 (m, 5H, ArCHCHAr, Ar), 5.49−5.29

(m, 1H, CH), 4.44−4.22 (m, 2H, CH₂NH), 4.06−3.91 (m, 1H,

CH₂′OH), 3.92−3.65 (m, 1H, CHNCH₃), 3.10 (m, 1H, CH₂″OH),

2.55−2.38 (m, 2H, CH₂CH₂C), 2.31−2.13 (m, 2H, CH₂CH₂C),

2.04 (s, 3H, CH₃N) ppm. OH proton was not detected. ¹³C NMR

(chloroform-d): δ ppm 173.92 (CONH), 143.81 (Ph₂C), 143.24

(C_Dche), 142.39 (C_Dche), 137.00 (C_Dche), 136.93 (C_Dche), 134.75 (C_Ph),

133.85 (C_Dche), 133.64 (C_Dche), 133.14 (C_Dche), 133.11 (C_Dche),

131.36 (C_Ph-Cl), 131.29 (C_Ph), 130.96 (ArCHCHAr), 130.86

(ArCHCHAr), 129.00 (CHCPh₂), 128.68 (C_Ph), 128.51(C_Ph),

128.32 (C_Ph), 128.09 (C_Ph), 127.97 (C_Dche), 127.45 (C_Dche), 127.37

(C_Dche), 126.94 (C_Dche), 67.10 (CHNCH₃), 59.96 (CH₂OH), 57.35

(CH₂CH₂CH), 42.07 (CH₂NH), 41.08 (CH₃N), 26.35

(CH₂CH₂CH).

4.1.1.7.10. N-(2-Chlorobenzyl)-2-((3-(10.11-dihydro-5H-dibenzo-

[a,d][7]annulen-5-ylidene)propyl)(methyl)amino)-3-hydroxypropa-

namide (56b). The synthesis was done according to GP7 with amine

C (21) (1.2 mmol, 315 mg), amide 53b (1 mmol, 290 mg), TBAB

(0.01 mmol, 0.32 mg), DIPEA (1 mmol, 174 μL), DCM 5 mL. The

obtained crude product was puriﬁed by column chromatography over

silica gel (DCM/Ace = 7:3) to yield 56b (128 mg, 30%, R_f= 0.74 or

4.1.1.7.7. 2-((3-(5H-Dibenzo[a,d][7]annulen-5-ylidene)propyl)-

(methyl)amino)-N-(4-methylbenzyl)-3-hydroxypropanamide

(55d). The synthesis was done according to GP7 with amine B (20)

(1.2 mmol, 313 mg), amide 53d (1 mmol, 276 mg), TBAB (0.01

mmol, 0.32 mg), DIPEA (1 mmol, 174 μL), DCM 5 mL. The

obtained crude product was puriﬁed by column chromatography over

silica gel (DCM/Ace = 7:3) to yield 55a (136 mg, 30%, R_f= 0.66 and

1

0.55 (S₁₂)). H NMR (chloroform-d): δ ppm 7.52−7.83 (m, 1H,

CONH), 6.89−7.37 (m, 12H, Ar), 5.74 (br s, 1H, CHC), 3,75−

4.51 (m, 4H, CH₂OH, CH₂Ar), 2.84−3,06 (m, 3H, ArCH₂-CH₂Ar,

CHN), 2.66−2.81 (m, 2H, ArCH₂CH₂Ar) 2.46−264 (m, 2H,

CH₂CH₂CH), 2.21−2.37 (m, 2H, CH₂CH₂CH), 2.08−2.19

(m, 3H, CH₃) ppm. OH proton was not detected. ¹³C NMR

(chloroform-d): δ ppm 173,20 (CONH), 144.43 (C_Ar), 140.92

(CHC), 139.71 (C_Ar), 139.27 (C_Ar), 136.97(C_Ar), 136.68 (C_Ar),

130.06(C_Ar), 129.48 (C_Ar), 128,94 (C_Ar), 128,72(C_Ar), 128,41 (C_Ar),

128,16(C_Ar), 127.63(C_Ar), 127.47 (C_Ar), 126.14 (C_Ar), 125.77 (CH

C), 66.94 (CHN), 59.99 (CH₂OH), 57.35 (CH₂CH₂CH), 42.13

(CH₂NH), 41.21 (CH₃N), 33,71 (ArCH₂-CH₂Ar), 32.01 (ArCH₂-

1

0.43 (S₁₂)). H NMR (chloroform-d): δ ppm 7.62 (dt, 1H, NH),

7.44−7.12 (m, 8H, Ar), 7.08−6.63 (m, 6H, ArCHCHAr, Ar),

5.41−5.06 (m, 1H, CH), 4.32−4.15 (m, 2H, CH₂NH), 4.06−3.82

(m, 1H, CH₂′OH), 3.79−3.66 (m, 1H, CHNCH₃), 3.18−3.02 (m,

1H, CH₂″OH), 2.54−2.05 (m, 4H, CH₂CH₂C), 1.93 (s, 3H,

NCH₃). OH proton was not detected. ¹³C NMR (chloroform-d): δ

1

ppm 173.84 (CONH), 161.89 (d, J_C−F= 245.2 Hz, C_Ar-F), 143.79

(Ph₂C), 137.01 (C_Dc₄_he), 136.95 (C_Dche), 134.87 (C_Dche), 134.24

(C_Ar-CH₂), 134.17 (d, J_C−F= 4.0 Hz, C_Ar), 134.14 (C_Dche), 131.10

U

https://doi.org/10.1021/acschemneuro.1c00351

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

pubs.acs.org/chemneuro

Research Article

CH₂Ar), 27.18 (CH₂CH₂CH) HRMS-ESI⁺m/z [M + H]⁺calcd

GP7 with amine D (25) (0.49 mmol, 120 mg), amide 53a (0.40

mmol, 103 mg), TBAB (0.04 mmol, 13 mg), DIPEA (0.40 mmol, 70

μL), and DMF (2 mL). The obtained crude product was puriﬁed by

column chromatography over silica gel (DCM/Ace = 7:3) to yield 57

(55 mg, 33%, R_f= 0.33 and 0.45 (DCM/Ace 7:3)) as a yellow oil.

Formula C₂₈H₂₈N₂O_2.FW: 424.54. ¹H NMR (300 MHz, chloroform-

d) δ ppm 7.63−7.91 (m, 3H), 7.17−7.42 (m, 11H), 5.88−6.00 (m,

1H), 4.50 (d, J = 2.93 Hz, 1H), 4.42 (s, 1H), 3.82−4.05 (m, 2H),

3.67−3.83 (m, 1H), 3.18−3.59 (m, 2H), 2.76−3.04 (m, 2H), 2.57 (s,

1H), 2.38−2.49 (m, 1H), 1.20−1.50 (m, 2H).

for C₂₉H₃₂N₂O₂Cl, 475.2152; found, 475.2142

4.1.1.7.11. N-(4-Chlorobenzyl)-2-((3-(10,11-dihydro-5H-dibenzo-

[a,d][7]annulen-5-ylidene)propyl)(methyl)amino)-3-hydroxypropa-

namide (56c). The synthesis was done according to GP7 with amine

C (21) (1.2 mmol, 315 mg), amide 53c (1 mmol, 290 mg), TBAB

(0.01 mmol, 0.32 mg), DIPEA (1 mmol, 174 μL), DCM 5 mL. The

obtained crude product was puriﬁed by column chromatography over

silica gel (DCM/Ace = 7:3) to yield 55c (132 mg, 30%, R_f= 0.74 or

0.57 (S₁₂)). ¹H NMR (chloroform-d): δ ppm 7.73−7.57 (m, 1H,

CONH) 7.15−7.10 (m, 10H, Ar) 7.01−6.61 (m, 2H, Ar) 5.78 (d, 1H,

J = 8,2 Hz, CH), 4.49−4.33 (m, 2H, NHCH₂Ar), 3.96 (dd, 1H, J =

11.2; 7.6 Hz, CH₂′OH), 3.81 (dd, 1H, J = 11.2, 4.2 Hz, CH₂″OH),

3,23 (dd, 1H, J = 7.6, 4.1 Hz, CHN), 2.96−2.74 (m, 4H,

ArCH₂CH₂Ar), 2.62 (dt, 2H, J = 13.0, 6.5 Hz, CH₂CH₂CH),

2.42−2.26 (m, 2H, CH₂CH₂CH), 2.20 (s, 3H, CH₃). Proton was

not detected. ¹³C NMR (chloroform-d): δ ppm 173.75 (NHCO),

144.57(C_Ar), 140.80 (CHC),139.28 (Ar), 136.91(Ar), 135.40

(C_Ar), 133,51 (C_Ar), 128,88 (Ar), 128,47 (Ar), 128.56 (Ar), 128,41

(Ar), 128,12 (Ar), 127.60 (C_Ar), 127.21(C_Ar), 127.23 (C_Ar), 126.09

(C_Ar), 125.79 (C_Ar), 86.21 (CHC), 74.01 (CHN), 60.41 (CH₂OH),

58,22 (CH₂CH₂CH), 40.96 (NHCH₂Ar), 38.00 (CH₃N), 32.04

(ArCH₂-CH₂Ar), 28.31 (CH₂CH₂CH).

4.1.1.7.15. N-Benzyl-2-(4-(10,11-dihydro-5H-dibenzo[a,d][7]-

annulen-5-ylidene)piperidin-1-yl)-3-hydroxypropanamide (58).

The synthesis was done according to GP7 with amine E (26) (0.70

mmol, 290 mg), amide 53a (0.58 mmol, 150 mg), TBAB (0.06 mmol,

19 mg), DIPEA (0.58 mmol, 100 μL), and DMF (3 mL). The

obtained crude product was puriﬁed by column chromatography over

silica gel (DCM/Ace = 7:3) to yield 58 (105 mg, 40%, R_f= 0.22 and

0.54 (DCM/Ace 7:3)) as a yellow oil. Formula C₃₀H₃₂N₂O₂, FW:

1

452.60. H NMR (300 MHz, chloroform-d) δ ppm 7.57−7.66 (m,

1H), 7.21−7.33 (m, 5H), 7.07−7.19 (m, 6H), 7.01 (d, J = 7.03 Hz,

2H), 4.44 (d, J = 5.27 Hz, 3H), 3.21−3.43 (m, 3H), 3.06−3.18 (m,

2H), 2.77−3.02 (m, 3H), 2.49−2.72 (m, 6H).

4.1.1.7.16. N-Benzyl-2-((2-(((diphenylmethylene)amino)oxy)-

ethyl)(methyl)amino)-3-hydroxypropanamide (59a). The synthesis

was done according to GP7 with amine F (32) (1.2 mmol, 305 mg),

amide 53a (1 mmol, 257 mg), TBAB ((0.01 mmol, 0.32 mg), DIPEA

(1 mmol, 174 μL), DMF 5 mL. The obtained crude product was

puriﬁed by column chromatography over silica gel (DCM/Ace = 7:3)

to yield 59a (130 mg, 30%, R_f= 0.58 (S₁₂)) ¹H NMR (chloroform-d):

δ ppm 7.71 (s, 1H, NH), 7.49−7.11 (m, 13H, Ar), 7.12−7.00 (m, 2H,

Ar), 4.46−4.28 (m, 1H, CH₂CH₂′ON), 4.27−4.13 (m, 2H,

CH₂NH), 4.13−4.03 (m, 1H, CH₂CH₂″ON), 3,99−3.88 (m, 1H,

CH₂′OH), 3,79 (dd, 1H, CHNCH₃, J = 11.4; 4.8 Hz), 3.26 (dd, 1H,

CH₂″OH, J = 7.0; 4.7 Hz,), 2.89 (td, 2H, CH₂CH₂′ON), 2.34 (s,

3H, NCH₃). OH proton was not detected. ¹³C NMR (chloroform-d):

δ ppm 173.46 (CONH), 157.23 (Ph₂CN), 138.07 (C_Ar), 136.09

(C_Ar), 133.08 (C_Ar), 129.47(C_Ar), 129.03 (C_Ar), 128.99 (C_Ar), 128.90

(C_Ar), 128.67 (C_Ar), 128.58 (C_Ar), 128.49 (C_Ar), 128.25 (C_Ar), 128.11

(C_Ar), 128.08 (C_Ar), 127.80 (C_Ar), 127.77 (C_Ar), 127.55 (C_Ar), 127.48

(C_Ar), 127.18 (C_Ar), 72.24 (CHN), 67.38 (CH₂CH₂ON), 58.43

(CH₂OH), 53.77 (CH₂CH₂ON), 42.67 (CH₂NH), 38.87 (CH₃N)

HRMS-ESI⁺m/z [M + H]⁺calcd for C₂₆H₃₀N₃O₃, 432.2287; found,

432,2283.

4.1.1.7.12. N-(4-Fluorobenzyl)-2-((3-(10,11-dihydro-5H-dibenzo-

[a,d][7]annulen-5-ylidene)propyl)(methyl)amino)-3-hydroxypropa-

namide (56d). The synthesis was done according to GP7 with amine

C (21) (1.2 mmol, 315 mg), amide 53d (1 mmol, 276 mg), TBAB

(0.01 mmol, 0.32 mg), DIPEA (1 mmol, 174 μL), DCM 5 mL. The

obtained crude product was puriﬁed by column chromatography over

silica gel (DCM/Ace = 7:3) to yield 55d (132 mg, 30%, R_f= 0.75 or

1

0.56 (S₁₂)). H NMR (chloroform-d): δ ppm 7.76 (s, 1H, CONH),

7.29−6.82 (m, 12H, Ar), 5.71 (s, 1H, CHC), 4.41−4.18 (m, 2H,

NHCH₂Ar,), 3.96 (dd, 1H, J = 11.2, 7.7 Hz, CH₂′OH), 3,82 (dd, 1H,

CH₂′OH, J = 11.2, 4.2 Hz), 3.31 (s, 1H, CHN), 2,96 (d, 1H, J = 13,2

Hz, ArCH₂CH₂′Ar), 2.84−2.71 (m, 3H, ArCH₂′CH₂″Ar), 2.69−2.50

(m, 2H, CH₂CH₂CH), 2.39−2.22 (m, 2H, CH₂CH₂CH), 2.15

(s, 3H, CH₃). OH proton was not detected. ¹³C NMR (chloroform-

1

d): δ ppm 173.66 (NHCO), 162.06 (d, J_C−F= 245.6 Hz), 139.73

(CHC), 136.92 (C_Ar), 136.96 (C_Ar), 134.01 (C_Ar), 133,97 (d, ⁴J_C−F

= 3,3 Hz), 130.18 (C_Ar), 129.29 (C_Ar), 129.18 (C_Ar), 128,17 (C_Ar),

3

128,10 (d, J_C−F= 8,3 Hz), 127.63 (C_Ar), 126.14 (CCH), 115.45

2

(d, J_C−F= 21.6 Hz), 67.48 (CHN), 57.90 (CH₂OH), 53,20

(CH₂CH₂CH), 42.11 (NHCH₂Ar), 38.94 (CH₃N), 33.70

(ArCH₂−CH₂Ar), 32.00 (ArCH₂−CH₂Ar), 28.10, (CH₂CH₂CH).

HRMS-ESI⁺m/z [M + H]⁺calcd for C₂₉H₃₂FN₂O₂, 459.2440; found,

459.2448.

4.1.1.7.17. (2-Chlorobenzyl)-2-((2-(((diphenylmethylene)amino)-

oxy)ethyl)(methyl)amino)-3-hydroxypropanamide (59b). The syn-

thesis was done according to GP7 with amine F (32) (1.2 mmol, 305

mg), amide 53b (1 mmol, 290 mg), TBAB ((0.01 mmol, 0.32 mg),

DIPEA (1 mmol, 174 μL), DMF 5 mL. The obtained crude product

was puriﬁed by column chromatography over silica gel (DCM/Ace =

4.1.1.7.13. 2-((3-(10,11-Dihydro-5H-dibenzo[a,d][7]annulen-5-

ylidene)propyl)(methyl)amino)-3-hydroxy-N-(4-methylbenzyl)-

propanamide (56e). The synthesis was done according to GP7 with

amine C (21) (1.2 mmol, 315 mg), amide 53e (1 mmol, 271 mg),

TBAB (0.01 mmol, 0.32 mg), DIPEA (1 mmol, 174 μL), DCM 5 mL.

The obtained crude product was puriﬁed by column chromatography

over silica gel (DCM/Ace = 7:3) to yield 55e (136 mg, 30%, R_f= 0.70

or 0.57 (S₁₂)). ¹H NMR (chloroform-d): δ ppm 7.49 (s, 1H, CONH),

7.39−6.84 (m, 12H, Ar), 5.75 (t, 1H, J = 7.3 Hz, CHC), 4.35 (d,

2H, J = 6.0 Hz, NHCH₂Ar), 4.04−3.72 (m, 2H, CH₂OH), 3,47−3.11

(m, 1H, CHN), 2.98−2.91 (m, 2H, ArCH₂CH₂′Ar), 2.75 (dd, 2H, J =

12.6, 6.3 Hz, ArCH₂CH₂″Ar), 2.56 (ddd, 2H, J = 11.0, 7.6, 3.7 Hz,

CH₂CH₂CH), 2.34−2.24 (m, 5H, CH₂CH₂CH, Ar−CH₃), 2.16

(s, 3H, CH₃). OH proton was not detected. ¹³C NMR (chloroform-

d): δ ppm 172.82 (NHCO), 144.41 (C_Ar), 140.94 (CHC),

139.74(C_Ar), 139.23 (C_Ar), 137.08 (C_Ar), 137.01 (C_Ar-CH₃), 135.03

(C_Ar), 130.02 (C_Ar), 129.29 (C_Ar), 128.44 (C_Ar), 128.29(C_Ar), 128,09

(C_Ar), 128,01 (C_Ar), 127.65 (C_Ar), 127.55 (C_Ar), 127.17 (C_Ar), 126.05

(C_Ar), 125.75 (CHC), 66.90 (CHN), 59.98 (CH₂OH), 57.34

(CH₂CH₂CH), 42.64 (NHCH₂Ar), 41.25 (CH₃N), 33.69 (ArCH₂-

CH₂Ar), 32.00 (ArCH₂-CH₂Ar), 27.16 (CH₂CH₂CH), 21.08 (C_Ar-

CH₃)

1

7:3) to yield 59b (130 mg, 30%, R_f= 0.60 (S₁₂)). H NMR (300

MHz, chloroform-d) δ ppm 2.39 (s, 3 H, NCH₃) 2.89−3.00 (m, 2 H,

CH₂CH₂ON) 3.33 (dd, J = 7.62. 4.10 Hz, 1 H, CHNCH₃) 3.82

(dd, J = 11.14, 4.69 Hz, 1 H, CH′H″OH) 3.92−4.02 (m, 1 H,

CH′H″OH) 4.14−4.35 (m, 4 H, CH₂CH₂ON, NHCH₂Ar) 7.13−

7.50 (m, 14 H, Ar) 7.73 (t, J = 5.86 Hz, 1 H, NHCO). HRMS-ESI⁺

m/z [M + H]⁺calcd for C₂₆H₂₉N₃O₃Cl, 466.1897; found, 466.1900.

4.1.1.7.18. 2-((2-(((Diphenylmethylene)amino)oxy)ethyl)-

(methyl)amino)-N-(4-ﬂuorobenzyl)-3-hydroxypropanamide (59c).

The synthesis was done according to GP7 with amine F (32) (1.2

mmol, 305 mg), amide 53c (1 mmol, 276 mg), TBAB ((0.01 mmol,

0.32 mg), DIPEA (1 mmol, 174 μL), DMF 5 mL. The obtained crude

product was puriﬁed by column chromatography over silica gel

1

(DCM/Ace = 7:3) to yield 59c (130 mg, 30%, R_f= 0.57 (S₁₂)). H

NMR (300 MHz, chloroform-d) δ ppm 2.39 (d, J = 1.76 Hz, 3 H,

NCH₃) 2.84−2.99 (m, 2 H, CH₂CH₂ON) 3.32 (dt, J = 7.18, 3.74

Hz, 1 H, CHNCH₃) 3.83 (dd, J = 11.14, 4.10 Hz, 1 H, CH′H″OH)

3.92−4.03 (m,

1 H, CH′H″OH) 4.04−4.22 (m, 2 H,

CH₂CH₂ON) 4.22−4.31 (m, 2 H, NHCH₂Ar) 6.89−7.50 (m, 14

H, Ar) 7.68 (d, J = 5.27 Hz, 1 H, NHCO)). HRMS-ESI⁺m/z [M +

H]⁺calcd for C₂₆H₂₉N₃O₃F, 450,2193; found, 450,2189.

4.1.1.7.14. N-Benzyl-2-[4-(9H-ﬂuoren-9-ylidene)piperidin-1-yl]-3-

hydroxypropanamide (57). The synthesis was done according to

V

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4.1.1.7.19. N-Benzyl-2-bromopropanamide (62). The synthesis

was done according to GP8 N-benzylamine (8.8 mmol, 962 μL) with

2-bromopropanoic acid (60) (8 mmol, 724 μL), TEA (9.6 mmol,

1.24 mL), T3P (8 mmol, 4.76 mL), and DCM (32 mL) to yield 62

(1.64 g, 85%, R_f= 0.65 (S₁). Formula: C₁₀H₁₂BrNO (241.01 g/mol).

¹H NMR and ¹³C NMR consistent with literature data^57,58(300

MHz, chloroform-d) δ ppm 7.23−7.41 (m, 5H), 6.71 (br s, 1H),

4.44−4.50 (m, 2H), 4.11 (q, J = 7.18 Hz, 1H), 1.91 (d, J = 6.92 Hz,

3H).

4.1.1.7.20. N-Benzyl-2-bromobutanamide (63a). The synthesis

was done according to GP8 N-benzylamine (8.8 mmol, 962 μL) with

2-bromopropanoic acid (61) (8 mmol, 850 μL), TEA (9.6 mmol,

1.24 mL), T3P (8 mmol, 4.76 mL), and DCM (32 mL) to yield 63

(1.63g, 80%, R_f= 0.60 (S₁)). Formula: C₁₁H₁₄BrNO (255.01 g/mol).

¹H NMR and ¹³C NMR consistent with literature data.^57,58¹H NMR

4.09−4.31 (m, 4 H) 4.46 (d, J = 5.86 Hz, 1 H) 7.06−7.52 (m, 16 H)

7.57−7.70 (m, 1 H). ¹³C NMR (75 MHz, chloroform-d) δ 173.8,

159.2, 138.7, 136.1, 129.3, 129.0, 128.3, 128.1, 127.9, 127.7, 127.2,

72.3, 63.0, 53.3, 42.7, 38.4. HRMS-ESI⁺m/z [M + H]⁺calcd for

C₂₆H₃₀N₃O₂, 416.2338; found, 416.2327.

4.1.1.8.4. N-Benzyl-2-((2-(((diphenylmethylene)amino)oxy)-

ethyl)(methyl)amino)butanamide (66a). The synthesis was done

according to GP7 with amine F (32) (0.48 mmol, 122 mg), amide

63a (0.4 mmol, 102 mg), TBAB (0.04 mmol, 0.12 mg), TEA (0.4

mmol, 55 μL), DCM 0.5 mL. The obtained crude product was

puriﬁed by column chromatography over silica gel (DCM/Ace = 7:3)

1

to yield 66a (106 mg, 62%, R_f= 0.3 (S₁₃)). H NMR (300 MHz,

chloroform-d) δ ppm 0.98 (t, J = 7.33 Hz, 3 H, CH₂CH₃) 1.58−1.76

(m, 1 H, CH′H″CH₃) 1.78−1.92 (m, 1 H, CH′H″CH₃) 2.30 (s, 3 H,

NCH₃) 2.75−2.92 (m, 2 H, CH₂CH₂ON) 3.00 (t, J = 6.45 Hz, 1

H, CHNCH₃) 4.05−4.17 (m, 1 H, CH₂CH′H″ON) 4.21−4.28 (m,

2 H, NHCH₂Ar) 4.30−4.52 (m, 1 H, CH₂CH′H″ON) 7.07−7.50

(m, 16 H, Ar, NHCO). ¹³C NMR (75 MHz, chloroform-d) δ 173.33,

156.90, 138.86, 136.26, 133.22, 129.40, 129.11, 128.86, 128.66,

128.44, 128.27, 128.10, 127.83, 127.44, 127.03, 72.69, 69.51, 54.23,

42.80, 38.53, 20.67, 11.87. HRMS-ESI⁺m/z [M + H]⁺calcd for

C₂₇H₃₂N₃O₂, 430.2495; found, 430.2494.

4.1.1.9. General Procedure for the Synthesis of N-Benzyl-2-

bromopropanamide and N-Benzyl-2-bromobutanamide Deriva-

tives 62, 63a,b (GP8). Under an argon atmosphere, 2-bromopropa-

noic acid (60) or 2-bromobutanoic acid (61)(1 equiv) was dissolved

in anhydrous DCM, and n-propanephosphonic anhydride (T₃P, 50%

in EtOAc, 1 equiv) was added. Acid activation was carried out for 30

min at −17° C, and then TEA (1.2 equiv) and the appropriate N-

benzylamine (1.1 equiv) were added dropwise to the mixture. The

mixture was warmed up to room temperature and stirred for 2 h. The

solvent was evaporated, maintaining the water bath at a temperature

below 30 °C. The residue was puriﬁed by column chromatography

over silica gel (PE/EtOAc 7:3).

(300 MHz, chloroform-d) δ ppm 7.30−7.42 (m, 3H), 7.16−7.26 (m,

2H), 6.72 (br s, 1H), 4.40−4.51 (m, 2H), 4.32 (dd, J = 5.00, 7.57 Hz,

1H), 1.93−2.28 (m, 2H), 0.98−1.10 (m, 3H).

4.1.1.7.21. 2-Bromo-N-(2-chlorobenzyl)butanamide (63b). The

synthesis was done according to GP8 (2-chlorophenyl)methanamine

(8.8 mmol, 1062 μL) with 2-bromopropanoic acid (61) (8 mmol, 850

μL), TEA (9.6 mmol, 1.24 mL), T3P (8 mmol, 4.76 mL), and DCM

(32 mL) to yield 63 (1.9 g, 82%, R_f= 0.7 (S₁)). Formula:

C₁₁H₁₃BrClNO (288.98 g/mol). ¹H NMR (500 MHz, chloroform-d)

δ ppm 7.35−7.42 (m, 2H), 7.20−7.27 (m, 2H), 6.94 (br s, 1H), 4.55

(d, J = 6.30 Hz, 2H), 4.34 (dd, J = 5.01, 7.59 Hz, 1H), 2.13−2.22 (m,

1H), 2.02−2.10 (m, 1H), 0.97−1.09 (m, 3H). ¹³C NMR (126 MHz,

chloroform-d) δ ppm 168.6. 134.94, 133.63, 129.7, 129.14, 129.4,

53.6, 42.1, 29.3, 11.7

4.1.1.8. General Procedure for the Synthesis of 2-Substituted

Propanamide Derivatives 64a,b, 65, 66a,b, 67, 68. The general

procedure is corresponding to the GP7 method (general procedures

for the synthesis of 2-substituted 3-hydroxypropanamide derivatives

described in section 4.1.1.7).

4.1.1.9.1. N-(2-Chlorobenzyl)-2-((2-(((diphenylmethylene)-

amino)oxy)ethyl)(methyl)amino)butanamide (66b). The synthesis

was done according to GP7 with amine F (32) (0.48 mmol, 122 mg),

amide 63b (0.4 mmol, 115 mg), TBAB (0.04 mmol, 0.12 mg), TEA

(0.4 mmol, 55 μL), DCM 0.5 mL. The obtained crude product was

puriﬁed by column chromatography over silica gel (DCM/Ace = 7:3)

4.1.1.8.1. N-Benzyl-2-(4-(bis(3-methylthiophen-2-yl)methylene)-

piperidin-1-yl)butanamide(64a). The synthesis was done according

to GP7 with amine A (17) (0.48 mmol, 135 mg), amide 63a (0.4

mmol, 102 mg), TBAB (0.04 mmol, 0.12 mg), TEA (0.4 mmol, 55

μL), DCM 0.5 mL. The obtained crude product was puriﬁed by

column chromatography over silica gel (DCM/Ace = 7:3) to yield

59c (101 mg, 55%, R_f= 0.15 (S₁₃)). ¹H NMR (300 MHz, chloroform-

d) δ 7.23−7.43 (m, 9H), 7.12 (d, J = 4.69 Hz, 2H), 6.78 (d, J = 5.27

Hz, 2H), 5.31−5.31 (m, 1H), 5.32 (t, J = 5.57 Hz, 1H), 4.36−4.50

(m, 4H), 2.89 (t, J = 6.15 Hz, 1H), 2.46−2.69 (m, 4H), 2.24−2.33

(m, 4H), 2.10 (s, 6H), 1.67−1.81 (m, 2H), 0.89−1.03 (m, 5H). ¹³C

NMR (75 MHz, chloroform-d) δ 173.0, 168.9, 159.4, 143.6, 138.0,

133.6, 128.1, 124.8, 124.3, 123.8, 119.7, 74.5, 70.1, 51.5, 43.2, 31.9,

25.1, 21.3, 14.5, 11.3, 8.9.

1

to yield 66b (133 mg, 60%, R_f= 0.35 (S₁₃)). H NMR (300 MHz,

chloroform-d) δ ppm 0.96 (t, J = 7.33 Hz, 3 H, CH₂CH₃) 1.58−1.74

(m, 1 H, CH′H″CH₃) 1.78−1.93 (m, 1 H, CH′H″CH₃) 2.30 (s, 3 H,

NCH₃) 2.78−2.94 (m, 2 H, CH₂CH₂ON) 3.01 (t, J = 6.45 Hz, 1

H, CHNCH₃) 4.07−4.23 (m, 1 H, CH₂CH′H″ON) 4.23−4.33 (m,

2 H, NHCH₂Ar) 4.33−4.62 (m, 1 H, CH₂CH′H″ON) 7.08−7.51

(m, 15 H, Ar, NHCO). ¹³C NMR (75 MHz, chloroform-d) δ 173.53,

156.93, 140.43, 136.24, 136.12, 133.15, 130.22, 129.42, 129.38,

129.27, 129.11, 129.08, 128.84, 128.34, 128.23, 128.04, 127.81,

126.82, 72.74, 69.36, 54.29, 40.87, 38.48, 20.50, 12.02. HRMS-ESI⁺

m/z [M + H]⁺calcd for C₂₇H₃₁N₃O₂Cl, 464.2105; found, 464.2104.

4.1.1.9.2. N-Benzyl-2-((4,4-bis(3-methylthiophen-2-yl)but-3-en-

1-yl)(methyl)amino)propanamide (67). The synthesis was done

according to GP7 with amine G (33) (0.48 mmol, 133 mg), amide 62

(0.4 mmol, 96 mg), TBAB (0.04 mmol, 0.12 mg), TEA (0.4 mmol, 55

μL), DCM 0.5 mL. The obtained crude product was puriﬁed by

column chromatography over silica gel (DCM/Ace = 7:3) to yield 67

(105 mg, 60%, R_f= 0.35 (S₁₃)). ¹H NMR (300 MHz, chloroform-d) δ

ppm 1.17−1.26 (m, 3 H), 1.90 (s, 3 H), 2.00 (s, 3 H), 2.12 (s, 3 H),

2.23−2.31 (m, 2 H), 2.47−2.59 (m, 2 H), 3.22 (q, J = 6.45 Hz, 1 H),

4.29−4.52 (m, 2 H), 5.99 (t, J = 7.33 Hz, 1 H), 6.70−6.77 (m, 1 H),

6.83 (d, J = 4.69 Hz, 1 H), 7.02 (d, J = 5.28 Hz, 1 H), 7.18−7.32 (m,

6 H), 7.62 (br s, 1 H). ¹³C NMR (75 MHz, chloroform-d) δ 183.1,

131.3, 129.6, 128.5, 127.7, 127.2, 124.4, 122.6, 62.9, 54.1, 43.1, 37.8,

28.2, 14.7, 14.4, 9.6. HRMS-ESI⁺m/z [M + H]⁺calcd for

C₂₅H₃₀N₂OS₂, 439.1872; found, 439.1877.

4.1.1.8.2. 2-(4-(Bis(3-methylthiophen-2-yl)methylene)piperidin-

1-yl)-N-(2-chlorobenzyl)butanamide (64b). The synthesis was

done according to GP7 with amine A (17) (0.48 mmol, 135 mg),

amide 63b (0.4 mmol, 115 mg), TBAB (0.04 mmol, 0.12 mg), TEA

(0.4 mmol, 55 μL), DCM 0.5 mL. The obtained crude product was

puriﬁed by column chromatography over silica gel (DCM/Ace = 7:3)

1

to yield 64b (119 mg, 60%, R_f= 0.25 (S₁₃)). H NMR (300 MHz,

chloroform-d) δ ppm 0.89−1.02 (m, 3 H, CH₂CH₃) 1.67−1.78 (m, 2

H, CH₂CH₃) 2.10 (s, 6 H, tiophCH₃) 2.25−2.35 (m, 4 H, Ppd)

2.42−2.53 (m, 2 H, Ppd) 2.54−2.64 (m, 2 H, Ppd) 2.87 (t, J = 6.15

Hz, 1 H, CHNCH₃) 4.46−4.59 (m, 2 H, NHCH₂Ar) 6.73−6.81 (m,

2 H, Ar) 7.12 (d, J = 5.27 Hz, 2 H, Tiof) 7.18−7.27 (m, 3 H, Tiof,

Ar) 7.31−7.42 (m, 3 H, Ar) 7.60 (br s, 1 H, NHCO).

4.1.1.8.3. N-Benzyl-2-((2-(((diphenylmethylene)amino)oxy)-

ethyl)(methyl)amino)propanamide (65). The synthesis was done

according to GP7 with amine F (32) (0.48 mmol, 122 mg), amide 62

(0.4 mmol, 96 mg), TBAB (0.04 mmol, 0.12 mg), TEA (0.4 mmol, 55

μL), DCM 0.5 mL. The obtained crude product was puriﬁed by

column chromatography over silica gel (DCM/Ace = 7:3) to yield 65

(191 mg, 55%, R_f= 0.3 (S₁₃)). ¹H NMR (300 MHz, chloroform-d) δ

ppm 1.22 (d, J = 7.03 Hz, 3 H, COCHCH₃) 1.48−1.59 (m, 2 H,)

2.26 (s, 3 H) 2.76 (t, J = 5.57 Hz, 2 H) 3.27 (q, J = 7.03 Hz, 1 H)

4.1.1.9.3. N-Benzyl-2-((4,4-bis(3-methylthiophen-2-yl)but-3-en-

1-yl)(methyl)amino)butanamide (68). The synthesis was done

according to GP7 with amine G (33) (0.48 mmol, 133 mg), amide

63a (0.4 mmol, 105 mg), TBAB (0.04 mmol, 0.12 mg), TEA (0.4

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mmol, 55 μL), DCM 0.5 mL. The obtained crude product was

puriﬁed by column chromatography over silica gel (DCM/Ace = 7:3)

to yield 68 (90 mg, 50%, R_f= 0.35 (S₁₃)). H NMR (300 MHz,

chloroform-d) δ 7.20 (d, J = 2.34 Hz, 2H), 7.00−7.20 (m, 8H), 6.99−

7.21 (m, 8H), 6.73−6.77 (m, 2H), 5.37 (t, J = 5.28 Hz, 1H), 3.14−

3.35 (m, 1H), 2.53−3.03 (m, 3H), 2.33 (br s, 4H), 1.10−1.22 (m,

3H). COOH proton was not detected. HRMS-ESI⁺m/z [M + H]⁺

calcd for C₂₂H₂₄N₂O, 334.1870; found, 334.1805

1

chloroform-d) δ ppm 0.97 (t, J = 7.33 Hz, 3 H, CH₂CH₃) 1.56−1.87

(m, 3 H, thiopCH₃) 1.88−1.92 (m, 3 H, thiopCH₃) 1.98−2.04 (m, 3

H, NCH₃) 2.18 (s, 2 H, CH₂CH) 2.28 (q, J = 7.03 Hz, 2 H) 2.54−

2.68 (m, 2 H, CH₂CH₂CH) 2.95 (d, J = 18.17 Hz, 1 H, CHNCH₃)

4.33−4.55 (m, 2 H, NHCH₂Ar) 5.98 (t, J = 7.33 Hz, 1 H, CH)

6.74 (d, J = 5.27 Hz, 1 H, thiop) 6.83 (d, J = 5.27 Hz, 1 H, thiop)

6.98−7.08 (m, 1 H, thiop) 7.15−7.38 (m, 6 H, Ar, thiop) 7.44 (br s, 1

H, NHCO). ¹³C NMR HRMS-ESI⁺m/z [M + H]⁺calcd for

C₂₆H₃₂N₂OS₂, 453.2029; found, 453.2015

4.1.1.11.2. 2-((4,4-Bis(3-methylthiophen-2-yl)but-3-en-1-yl)-

(methyl)amino)propanoic Acid (75). Ethyl 2-((4,4-bis(3-methyl-

thiophen-2-yl)but-3-en-1-yl)(methyl)amino)propanoate (73) (113

mg, 0.3 mmol) in 2 mL 10 wt % NaOH was used, giving the oil

1

(quantitative) (105 mg). H NMR (300 MHz, chloroform-d) δ 7.23

(d, J = 5.28 Hz, 1H), 7.07 (d, J = 5.27 Hz, 1H), 6.86 (d, J = 5.28 Hz,

1H), 6.76 (d, J = 5.28 Hz, 1H), 5.98 (t, J = 6.74 Hz, 1H), 3.50 (br s,

1H), 3.11 (br s, 1H), 2.95 (d, J = 6.45 Hz, 1H), 2.49−2.68 (m, 5H),

2.00−2.05 (m, 3H), 1.97 (s, 3H), 1.39 (d, J = 4.10 Hz, 3H). COOH

proton was not detected.

4.1.1.10. General Procedure for the Synthesis of Ethyl 2-

Substituted Propanoate 71, 73 and Benzyl 2-Substituted

Butanoate 72 (GP11). The corresponding amine B or G (1.2

equiv), a relevant ester 69 or 70 (1 equiv), and K₂CO₃(3.5 equiv) in

DCM was stirred at 40 °C (oil bath) for 12 h. The mixture was

extracted with 2 M HCl and DCM. Organic phases were dried with

anhydrous Na₂SO₄and concentrated, and the residue was puriﬁed by

column chromatography over silica gel (S₁PE/EtOAc 7:3, v/v).

4.1.1.10.1. Ethyl N-(3-(5H-Dibenzo[a,d][7]annulen-5-ylidene)-

propyl)-N-methylalaninate (71). The synthesis was done according

to GP11 with amine B (20) (0.48 mmol, 125 mg), ethyl 2-

bromopropanoate (0.4 mmol, 52 μL), K₂CO₃(194 mg, 1,4 mmol),

DCM 0.5 mL. The obtained crude product was puriﬁed by column

chromatography over silica gel (DCM/Ace = 7:3) to yield 71 (101

4.2. In Vitro Activity. 4.2.1. [³H]GABA Uptake Assay. The

inhibitory activities of the synthesized compounds were determined

from [³H]GABA uptake assays with mGAT1, mGAT2, mGAT3, and

mGAT4 as described previously,^19,59and all compounds were tested

at a screening concentration of 100 μM.

4.2.2. MS Binding Assays. MS binding assays for mGAT1 were

performed as described earlier.⁴⁸Inhibition of mGAT1 binding by the

synthesized compounds was determined in MS binding assays at a

screening concentration of 100 μM and analyzed by LC−ESI-MS/

MS.

4.3. Molecular Modeling. 4.3.1. Docking Studies. For the

docking studies, we used models of human GAT-1, GAT-2, and GAT-

3 that were selected in our previous work.⁵¹They were built with the

SWISS-MODEL server based on the 4XP9 template from the Protein

Data Bank (PDB). For BGT-1 we decided to apply the extra model

built on the same template, according to previously described

procedure.⁵¹In this model, the nonhelical fragment of TM10

(residues 455−459) was additionally optimized using the MyLoop

class in the Modeller program. From among 100 reﬁned models, the

best one was selected according to the QMEAN score. For each type

of GABA transporter we used sequence alignment generated

automatically by SWISS-MODEL. The N- and C-termini were

omitted because of their low homology. Sodium and chloride ions

were transferred directly from the templates.

The ligand 3D structures were created in the Maestro program.

Ionization states were predicted under physiological conditions (pH

7.4) using the Epik and Marvin programs. Ligands were optimized in

the LigPrep module. All possible stereoisomers for each ligand were

generated. Models were prepared with Protein Preparation Wizard

using the default settings.

1

mg, 70%, R_f= 0.5 (S₁). H NMR (300 MHz, chloroform-d) δ ppm

7.21−7.41 (m, 8H), 6.86 (d, J = 1.17 Hz, 2H), 5.57 (ddd, J = 3.52,

6.74, 7.91 Hz, 1H), 4.07−4.21 (m, 2H), 3.26−3.43 (m, 1H), 2.45−

2.77 (m, 2H), 2.14−2.43 (m, 6H), 1.15−1.37 (m, 9H). ¹³C NMR (75

MHz, chloroform-d) δ 173.7, 142.7, 137.7, 134.3, 132.1, 131.5, 129.0,

128.3, 128.0, 127.2, 60.5, 54.4, 38.4, 27.8. HRMS-ESI⁺m/z [M + H]⁺

calcd for C₂₄H₂₈NO₂, 362.2120; found, 362.2117.

4.1.1.10.2. Benzyl N-(3-(5H-Dibenzo[a,d][7]annulen-5-ylidene)-

propyl)-N-methylalaninate (72). The synthesis was done according

to GP11 with amine B (20) (0.48 mmol, 125 mg), benzyl 2-

bromopropanoate (0.4 mmol, 67 μL), K₂CO₃(194 mg, 1,4 mmol),

DCM 0.5 mL. The obtained crude product was puriﬁed by column

chromatography over silica gel (DCM/Ace = 7:3) to yield 72 (1735

1

mg, 80%, R_f= 0.6 (S₁). H NMR (300 MHz, chloroform-d) δ 7.19−

7.42 (m, 13H), 6.86 (s, 2H), 5.49−5.62 (m, 1H), 5.09−5.16 (m, 2H),

3.33−3.49 (m, 1H), 2.46−2.74 (m, 2H), 2.13−2.41 (m, 6H), 1.27

(dd, J = 7.03, 12.89 Hz, 3H). HRMS-ESI⁺m/z [M + H]⁺calcd for

C₂₄H₃₀NO₂, 424.2277; found, 424.2274

4.1.1.10.3. Ethyl 2-((4,4-Bis(3-methylthiophen-2-yl)but-3-en-1-

yl)(methyl)amino)propanoate (73). The synthesis was done

according to GP11 with amine G (23) (0.48 mmol, 133 mg), ethyl

2-bromopropanoate (0.4 mmol, 52 μL), K₂CO₃(194 mg, 1,4 mmol),

DCM 0.5 mL. The obtained crude product was puriﬁed by column

chromatography over silica gel (DCM/Ace = 7:3) to yield 73 (106

The most active compound representatives were initially docked

into the models of each type of GABA transporter using the induced-

̈

ﬁt docking protocol available in the Schrodinger Suite. The box center

was deﬁned by 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 GATs. After selection of

the best optimized models, all studied compounds were docked into

the models using the GLIDE program and the ﬁnal conformations of

the models were selected based on ligand pose coherency. The grid

center in GLIDE was set as the centroid of the ligand from the

complex, and the inner box size was 15 Å × 15 Å × 15 Å. The

OPLS2005 force ﬁeld was applied during grid generation as well as

GLIDE and IFD docking.

1

mg, 70%, R_f= 0.5 (S₁). H NMR (300 MHz, chloroform-d) δ ppm

1.20−1.34 (m, 6 H) 2.03 (d, J = 8.79 Hz, 6 H) 2.26−2.36 (m, 5 H)

2.48−2.77 (m, 2 H) 3.37 (q, J = 7.03 Hz, 1 H) 4.15 (q, J = 7.03 Hz, 2

H) 6.06 (t, J = 7.33 Hz, 1 H) 6.75 (d, J = 5.28 Hz, 1 H) 6.84 (d, J =

5.27 Hz, 1 H) 7.04 (d, J = 5.27 Hz, 1 H) 7.20 (d, J = 5.27 Hz, 1 H).

HRMS-ESI⁺m/z [M + H]⁺calcd for C₂₀H₂₇NO₂S₂, 378.1556; found,

378.1560.

4.1.1.11. General Procedure for the Hydrolysis of Ethyl and

Benzyl 2-Substituted Butanoate 71 and 73 to 74 and 75 (GP12).

An aqueous 10 wt % NaOH (0.15 g/1.5 mL) solution was added to

compounds 71 and 73 (0.3 mmol) and stirred for 4.5 h at 35 °C. The

aqueous phase was acidiﬁed to pH = 3 with aq HC1 (1 M) and

extracted with DCM twice. The combined organic phases are dried

over anhydrous Na₂S0₄, ﬁltered, and then concentrated to give

product.

4.3.2. Molecular Dynamics. MD simulations were performed with

NAMD using the CHARMM36m 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 1-

palmitoyl-2-oleoylphosphatidylcholine (POPC) membrane and sol-

vated with TIP3P water molecules. The system size was 100 Å ×

100 Å. A water pore for each complex was generated. Sodium and

chloride ions (0.15 M NaCl) were added to provide standard

4.1.1.11.1. N-(3-(5H-Dibenzo[a,d][7]annulen-5-ylidene)propyl)-

N-methylalanine (74). The synthesis was done according to GP12

with ethyl N-(3-(5H-dibenzo[a,d][7]annulen-5-ylidene)propyl)-N-

methylalaninate (71) (108 mg, 0.3 mmol) in 2 mL of 10 wt %

1

NaOH to give the oil (quantitative) (100 mg). H NMR (300 MHz,

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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 time step of 2 fs and a

total duration of 10 ns. The intervals for both the energy and

trajectory recordings were 10 ps. The results were analyzed with the

VMD program.

diabetes, mice were intraperitoneally injected with STZ (a single

injection of STZ, 200 mg/kg) dissolved in 0.1 N citrate buﬀer. Age-

matched control mice received an equal volume of citrate buﬀer.

Blood glucose level was measured 1 day before (referred to as “day

0”) and repeatedly 1, 2, and 3 weeks after STZ injection using a blood

glucose monitoring system (Accu-Chek Active, Roche, France). Blood

samples for measurement of glucose concentration were obtained

from the tail vein of the mice. The animals were considered as

diabetic when their blood glucose concentration exceeded 300 mg/

dL,⁶⁶and only these mice (diabetic mice) were used in subsequent

pain tests.⁶⁵

4.5.1.4. Assessment of Tactile (Mechanical) Allodynia (von Frey

Test). The ability of the test compounds to attenuate tactile allodynia

caused by oxaliplatin, paclitaxel, and STZ was assessed using the von

Frey test. For this purpose, 3 h after oxaliplatin or paclitaxel and 21

days after STZ injection, the predrug paw withdrawal threshold was

measured for each mouse. Then, the test compounds were

administered, and 1 h later, the postdrug paw withdrawal threshold

was collected for each animal. This part of the experiment aimed to

establish the eﬀect of treatment on early phase (acute) pain

hypersensitivity induced by cytotoxic drugs. Additionally, in the

oxaliplatin model and the paclitaxel model, to assess the eﬀect of test

compounds on late phase tactile allodynia, 7 days later, measurements

of the predrug and postdrug paw withdrawal thresholds were made in

a similar manner to the measurements performed during early phase

neuropathy. At this stage of the experiment, there was no additional

oxaliplatin/paclitaxel administration.

An electronic von Frey unit (Bioseb, France) was used to assess the

mechanical nociceptive threshold (tactile allodynia) in mice. This

device has a single ﬂexible ﬁlament that applies increasing force (from

0 to 10 g) against the plantar surface of the hind paw of each mouse.

In the von Frey test, the paw withdrawal response of the animals

automatically turns oﬀ the stimulus, and the mechanical pressure that

evokes this response is recorded. On the day of the experiment, the

mice were placed individually in test compartments with a wire mesh

bottom and left there for 1 h of habituation. Subsequently, to obtain

baseline values, each mouse was tested 3 times alternately in each

hind paw. Then, the test compounds were administered, and 1 h later,

3 additional measurements were taken and averaged to obtain the

mean postdrug values for each mouse.⁶⁷

4.5.1.5. Assessment of Cold Nociceptive Threshold (Cold Plate

Test). A cold plate apparatus (hot/cold plate, Bioseb, France) set at

2.5 °C was used to assess the eﬀect of treatment on cold hyperalgesia

in oxaliplatin-treated mice. The cold plate test was conducted

immediately after the von Frey test. Three hours after oxaliplatin

administration, the animals were placed on a cold plate apparatus, and

predrug latencies to pain reaction (i.e., lifting, biting, shaking of hind

paws, jumping, movement deﬁcits, or writhing response) were

collected. Finally, the test compounds were injected, and 1 h later,

the postdrug latencies to pain reaction were measured. In this assay, a

cutoﬀ time of 60 s was established to avoid potential thermally

induced damage to the paw tissues, and animals not responding

within 60 s were removed from the apparatus and assigned a score of

60 s.^68,69

4.4. Hepatotoxicity and Cytotoxicity. Hepatotoxicity and

cytotoxicity were estimated according to previously described

protocols⁶⁰using the hepatoma HepG2 (ATCC HB-8065) and

human embryonic kidney HEK-293 (ATCC CRL1573) cell lines,

respectively. In brief, cells were seeded in 96-well plates at a density 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 from 0.1 to 100 μM for 72 h. The

antiproliferative drug DX was used as the reference. The CellTiter

96 AQueous nonradioactive cell proliferation assay (MTS) purchased

from Promega (Madison, WI, USA) was used for the determination of

cell viability. The absorbance at 492 nm was measured using an

EnSpire microplate reader (PerkinElmer, Waltham, MA, USA).

4.5. In Vivo Evaluation. 4.5.1. Materials and Methods.

4.5.1.1. Animals and Housing Conditions. Behavioral experiments

were carried out at the Department of Pharmacodynamics, Faculty of

Pharmacy, Jagiellonian University Medical College, Krakow. Tests

were performed between 9 a.m. and 2 p.m. All experimental in vivo

procedures were approved by the second Local Ethics Committee in

Krakow (Approval Numbers 32/2018 and 508/2021). The treatment

of animals was in full accordance with the ethical standards laid down

by both Polish and EU regulations (Directive 2010/63/EU). To

avoid potential bias in data recording, the investigators who were

involved in behavioral assays were blinded to the experimental groups.

Adult male albino Swiss (CD-1) mice weighing 18−22 g were

supplied by the Animal Breeding Farm of the Jagiellonian University

Faculty of Pharmacy. Before the in vivo tests, the mice were kept in

́

groups of 10 in standard plastic cages. Bedding material (Transwior,

Poland) was at least 2 cm deep to allow the mice to dig, and animals

were housed under controlled laboratory conditions (room temper-

ature of 22

humidity 50

2 °C, light/dark (12:12) cycle, lights on at 8 a.m.,

10%, and free access to food (Murigran, Agropol,

Poland) and tap water). Experimental groups consisted of 8−10

animals/dose. For behavioral tests, the mice were selected randomly

After completion of the assays, the mice were euthanized by cervical

dislocation.

4.5.1.2. Chemicals Used in the in Vivo Tests. Before the in vivo

tests, the test compounds were suspended in 1% Tween 80 (Baxter,

Poland). The compounds were then administered intraperitoneally.

The dose of 30 mg/kg of each compound was the starting dose, and if

activity was observed in the pain tests, a dose of 10 mg/kg was also

tested. The test compounds were administered only once daily on

days 1 and 7 of oxaliplatin- or paclitaxel-induced neuropathy. In STZ-

treated mice, test compounds were administered 21 days after STZ

injection. Control mice used in oxaliplatin and paclitaxel NP models

were injected with an appropriate amount of vehicle (0.9% saline).

Oxaliplatin was purchased from Activate Scientiﬁc GmbH (Ger-

many). Paclitaxel and STZ were purchased from Sigma-Aldrich

(Poland).

4.5.1.3. Induction of Neuropathy and NP. For pain studies,

oxaliplatin was dissolved in 5% glucose solution (Polfa Kutno,

Poland). The dose of oxaliplatin used to induce peripheral neuropathy

(10 mg/kg, intraperitoneal injection) was chosen on the basis of

previous research^61,62and available literature data.⁶³

Doses of both paclitaxel and STZ used for the induction of

neuropathy were selected based on our previous research.^64,65To

induce neuropathy, paclitaxel was used at a dose of 18 mg/kg. It was

prepared by dissolving in ethanol (100% (v/v); Polskie Odczynniki

Chemiczne, Gliwice, Poland) at 10% of the ﬁnal desired volume and

vortexed for 2 min. An equal volume of Cremophor EL (10% of the

ﬁnal volume) was then added, and the mixture was vortexed for the

next 10 min. Prior to injection, ice-cold physiological saline (80% of

the ﬁnal volume) was added to make up a ﬁnal volume and the

solution was maintained on ice during dosing.⁶⁴To induce type I

4.5.1.6. Assessment of Heat Nociceptive Threshold (Hot Plate

Test). Thermal (heat) nociceptive threshold was assessed in the hot

plate test as previously described.⁶⁵First, baseline (predrug) latencies

to pain reaction were established for each mouse. Then, the mice were

treated intraperitoneally with either the test compound or vehicle.

Sixty minutes later the animals were placed on the hot plate apparatus

again (hot/cold plate, Bioseb, France). This apparatus has an

electrically heated surface and is supplied with a temperature-

controller that maintains the temperature at 55 °C. The time until the

animal licked its hind paws or jumped was recorded by means of a

stop-watch. In this assay a cutoﬀ time was established (60 s) to avoid

tissue damage, and the mice not responding within 60 s were removed

from the apparatus and assigned a score of 60 s.

4.5.1.7. Rotarod Test. Before the rotarod test, the experimental

animals underwent 3 d of training on the rotarod apparatus (totarod

apparatus, May Commat RR0711, Turkey; rod diameter 2 cm) that

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rotated at a ﬁxed speed of 18 rotations per minute (rpm). During this

training session, the mice were placed on the rotating rod for 3 min

with an unlimited number of trials. The proper test was performed 24

h after the last training session. Sixty minutes after administration of

the test compounds or vehicle, the mice were tested on rods that

revolved at 6, 18, and 24 rpm. Motor deﬁcits in the mice were deﬁned

as their inability to remain on the rotarod apparatus for 1 min. The

results are expressed as the mean time spent on the rotarod.⁶⁸

4.5.1.8. Data Analysis. Data analysis of the in vivo results was

performed using GraphPad Prism software (version 8.0, CA, USA).

Numerical results obtained in behavioral tests are expressed as the

mean SEM. Statistical analysis was performed using the Shapiro−

Wilk normality test, followed by one-way analysis of variance

(ANOVA) and Tukey’s post hoc comparison (NP models). One-

way ANOVA and Dunnett’s post hoc comparison were used to test

diﬀerences between the drug-treated groups and the control group in

the rotarod test. A value of p < 0.05 was considered signiﬁcant.

https://pubs.acs.org/10.1021/acschemneuro.1c00351

Author Contributions

P.Z. and K.K. designed and coordinated the study and

analyzed the data. B.G., P.Z., and K.S. wrote the manuscript.

B.G., P.Z., K.M., and G.M. synthesized the compounds. G.C.H.

coordinated [³H] GABA uptake assay and MS binding assays.

K.Ł. and M.B. performed molecular modeling. G.L. performed

in vitro studies (hepatotoxicity and cytotoxicity). K.S. and A.R.

performed in vivo pharmacological evaluation (mouse models

of neuropathic pain). B.M., K.S., K.T.W., and G.C.H. corrected

the manuscript. All the authors contributed to and approved

the ﬁnal manuscript.

Funding

This study was ﬁnancially supported by the National Science

Center in Poland as part of Grant 2014/15/B/NZ7/00930.

The molecular modeling studies were ﬁnancially supported by

the National Science Center, Poland, Grant 2016/23/D/NZ7/

01172.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acschemneuro.1c00351.

■

sı

Notes

Synthesis of building blocks 17 (A), 20 (B), 21 (C), 25

(D), 26 (E), 32 (F), and 33 (G); RMSD changes over

The authors declare no competing ﬁnancial interest.

^§K.K.: deceased, June, 26, 2016.

1

the course of the molecular dynamics simulations; H

and ¹³C NMR spectra of synthesized compounds (PDF)

ABBREVIATIONS

■

GABA, γ-aminobutyric acid; CNS, central nervous system;

GABA_A, γ-aminobutyric acid type A receptor; GABA_B, γ-

aminobutyric acid type B receptor; GABA_C, γ-aminobutyric

acid type C receptor; GAT, GABA transporter protein; SLC6,

solute carrier 6 family; BGT1, betaine/GABA transporter 1;

IUPHAR, International Union of Basic and Clinical

Pharmacology; GAT1, GABA transporter 1; GAT2, GABA

transporter 2; RMSD, root-mean-square deviation; GAT3,

GABA transporter 3; NP, neuropathic pain; FDA, U.S. Food

and Drug Administration; BOC, tert-butyloxycarbonyl protect-

ing group; TEA, triethylamine; THF, tetrahydrofuran; DCM,

dichloromethane; STZ, streptozotocin

AUTHOR INFORMATION

Corresponding Author

■

Paula Zaręba − Faculty of Pharmacy, Jagiellonian University

Medical College, 30-688 Kraków, Poland; orcid.org/

0000-0002-5037-6183; Phone: +4812 6205464;

Email: paula.zareba@uj.edu.pl; Fax: +4812 6205450

Authors

Beata Gryzło − Faculty of Pharmacy, Jagiellonian University

Medical College, 30-688 Kraków, Poland

Katarzyna Malawska − Faculty of Pharmacy, Jagiellonian

University Medical College, 30-688 Kraków, Poland

Gabriela Mazur − Faculty of Pharmacy, Jagiellonian

University Medical College, 30-688 Kraków, Poland

Anna Rapacz − Faculty of Pharmacy, Jagiellonian University

Medical College, 30-688 Kraków, Poland

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■

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