Synthesis of novel nicotinic ligands with multimodal action: Targeting Acetylcholine α4β2, Dopamine and Serotonin Transporters

molecules

Article

Synthesis of Novel Nicotinic Ligands with

Multimodal Action: Targeting Acetylcholine α4β2,

Dopamine and Serotonin Transporters

Juan Pablo González-Gutiérrez ¹, Hernán Armando Pessoa-Mahana ^1,*,

Patricio Ernesto Iturriaga-Vásquez ^2,3,

* ,

, Miguel Iván Reyes-Parada ^4,5

Nicolas Esteban Guerra-Díaz ¹, Martin Hodar-Salazar ², Franco Viscarra ², Pablo Paillali ²,

Gabriel Núñez-Vivanco ^6,7, Marcos Antonio Lorca-Carvajal ⁸, Jaime Mella-Raipán ⁸

and María Carolina Zúñiga ⁹

1

Departamento de Química Orgánica y Fisicoquímica, Facultad de Ciencias Químicas y Farmacéuticas,

Universidad de Chile, 8380492 Santiago, Chile; jpgonzalezg@ug.uchile.cl (J.P.G.-G.);

niko.g.diaz@gmail.com (N.E.G.-D.)

Departamento de Ciencias Químicas y Recursos Naturales, Facultad de Ingeniería y Ciencias, Universidad

2

de la Frontera, 4811230 Temuco, Chile; martinhodar@gmail.com (M.H.-S.); franco.viscarra@gmail.com (F.V.);

p.paillali01@ufromail.cl (P.P.)

Center of Excellence in Biotechnology Research Applied to the Environment, Universidad de La Frontera,

4811230 Temuco, Chile

3

4

Centro de Investigación Biomédica y Aplicada (CIBAP), Escuela de Medicina, Facultad de Ciencias Médicas,

Universidad de Santiago de Chile, 9170022 Santiago, Chile; miguel.reyes@usach.cl

Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, 3467987 Sede Talca, Chile

Centro de Bioinformática y Simulación Molecular, Universidad de Talca, 3340000 Talca, Chile;

gabioin@gmail.com

5

6

7

8

Escuela de Ingeniería Civil en Bioinformática, Universidad de Talca, Av. Lircay 3340000 Talca, Chile

Instituto de Química y Bioquímica, Facultad de Ciencias, Universidad de Valparaíso, 2360102 Valparaíso,

Chile; jaime.mella@uv.cl (J.M.-R.); marcos.lorca.c@gmail.com (M.A.L.-C.)

Departamento de Química Inorgánica and Analítica, Facultad de Ciencias Químicas y Farmacéuticas,

Universidad de Chile, 8380492 Santiago, Chile; mczuniga@ciq.uchile.cl

9

*

Correspondence: hpessoa@ciq.uchile.cl (H.A.P.-M.); patricio.iturriaga@ufrontera.cl (P.E.I.-V.);

Tel.: +(56)-9-73585686 (H.A.P.-M)

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

Received: 10 September 2019; Accepted: 20 October 2019; Published: 22 October 2019

Abstract: Nicotinic acetylcholine receptors (nAChRs), serotonin transporters (SERT) and dopamine

transporters (DAT) represent targets for the development of novel nicotinic derivatives acting

as multiligands associated with diﬀerent health conditions, such as depressive, anxiety and

addiction disorders. In the present work, a series of functionalized esters structurally related

to acetylcholine and nicotine were synthesized and pharmacologically assayed with respect to

these targets. The synthesized compounds were studied in radioligand binding assays at α4β2

nAChR, h-SERT and h-DAT. SERT experiments showed not radioligand [³H]-paroxetine displacement,

but rather an increase in the radioligand binding percentage at the central binding site was observed.

Compound 20 showed K_ivalues of 1.008

nAChR, and [³H]-paroxetine binding of 191.50% in h-SERT displacement studies, being the only

compound displaying triple aﬃnity. Compound 21 displayed K_ivalues of 0.113 0.037 M for

2 nAChR and 0.075 0.009 M for h-DAT acting as a dual ligand. Molecular docking studies on

homology models of 2 nAChR, h-DAT and h-SERT suggested potential interactions among the

compounds and agonist binding site at the 4/ 2 subunit interfaces of 2 nAChR, central binding

site of h-DAT and allosteric modulator eﬀect in h-SERT.

±

0.230

µM for h-DAT and 0.031

±

0.006 µM for α4β2

±

µ

α

4

β

±

µ

α4β

α

β

α4β

Keywords: nAChR; DAT; allosteric modulators; SERT; α4β2; allosteric modulators; aﬃnity

Molecules 2019, 24, 3808; doi:10.3390 /molecules24203808

ww w .mdpi.com /journal /molecules

Molecules 2019, 24, 3808

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1. Introduction

Nicotine (Figure 1) is the chemical substance in tobacco responsible for smoking addiction [1],

which causes major public health risks. Inhaled cigarette smoke delivers nicotine rapidly to the

brain, where it binds to nicotinic acetylcholine receptors (nAChRs) and modulates the release of

several neurotransmitters [

that function as heteropentameric ligand-gated ion channels composed of

These 2 nAChRs trigger downstream dopamine signaling in the mesolimbic system [

an area in the brain that plays an important role in pleasure and reward sensation [

nAChRs mediate many behaviors related to nicotine addiction and are the primary targets for currently

approved smoking cessation agents [ ]. However, the success of these strategies must so far be

considered limited, as the only 2 ligands currently approved for medical use are the partial

nAChR agonists Varenicline [10], a partial agonist which binds to 2 nAChRs with higher aﬃnity

but lower eﬃcacy than nicotine, and Cytisine (Figure 1) [11], both of which are used as a smoking

cessation aids [12 13]. Varenicline (Figure 1) elicits a moderate and sustained increase of dopamine

2

]. The eﬀects of nicotine are mainly mediated through speciﬁc nAChRs

4- and 2-subunits [ ].

], which is

]. The 2*

α

β

3,4

α

4β

5,6

8

7,

α4β

9

α

4

β

α4β2

α4β

,

levels in the brain reward system [14

smoking cessation attempts [16].

,15], which would elevate low dopamine levels observed during

Figure 1. Chemical structure of alkaloids S-nicotine, Cytisine and smoking cessation drug Varenicline.

A substantial proportion of all smokers have a history of depression, and among people with

depression, smoking prevalence is about twice as high as in the general population [17,18], leading to

increased morbidity and premature mortality. Earlier research raised concerns that smoking cessation

may lead to an increase in symptoms, recurrence or even emergence of depression [19].

Symptoms of depression can be induced in humans through blockade of acetylcholinesterase

(AChE) whereas antidepressant-like [20] eﬀects can be produced in animal models and some clinical

trials by limiting activity of acetylcholine (ACh, Figure 2 ) receptors [21]. Thus, ACh signaling could

contribute to the etiology of mood dysregulation. Furthermore, a vast number of articles emphasize

the signiﬁcant role of ACh on the initiation and maintenance of drug addiction due to the interactions

of the cholinergic system with other neurotransmitter systems, mainly in the ventral tegmental area

(VTA), the nucleus accumbens (NAc) and the pre-frontal cortex (PFC) [22].

Figure 2. Chemical structure of the neurotransmitters ACh, 5-HT and DA.

Molecules 2019, 24, 3808

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On the other hand, Alzheimer’s disease (AD) is a progressive neurodegenerative pathology with

severe economic and social impact [23 24]. Nowadays, drug research and development are based

,

on the cholinergic hypothesis which proposes that the selective loss of cholinergic neurons results in

a deﬁcit of ACh in speciﬁc regions of the brain (cerebral cortex and hippocampus) that mediate learning

and memory functions. Based on this hypothesis, cholinergic augmentation will improve cognition in

AD [25

as rivastigmine, donepezil, and galantamine, which avoid the hydrolysis of Ach, thereby increasing its

concentration [27 28]. However, clinical eﬃciency is limited, as available AChE inhibitors can only

,26]. Currently available treatment for patients suﬀering from AD involves AChE inhibitors such

,

ameliorate AD symptoms, and thus the search for novel compounds remains an emerging demand for

the treatment of this pathology.

AD is associated with major serotonergic alterations due to involvement of the raphe nucleus and

related projections. Additionally, both soluble and insoluble

β-Amyloid (A

β) species are associated

with impaired synaptic plasticity and dysfunctional neurotransmission in serotonergic neurons [29

,30

]

Furthermore, reductions in serotonin (5-HT, Figure 2) and its metabolite levels have been reported in

brain tissue and cerebrospinal ﬂuid in AD [31].

Studies carried out by Cirrito and Sheline [30,32] have demonstrated that activation of serotonergic

neurotransmission may be beneﬁcial in AD. The authors showed that acute administration of the

selective 5-HT reuptake inhibitor (SSRI) citalopram in mice, at doses roughly equated to those prescribed

for human patients with depression, reduced production of toxic A

with this ﬁnding, they further demonstrated that 5-HT infusion into the hippocampus of APP/PS1 mice

also reduced the A peptide in the brain. Clinical studies further support this idea, as A imaging via

β proteins in the brain. Consistent

β

positron emission tomography revealed lower cortical amyloid levels in study participants who had

taken SSRIs within the past ﬁve years versus those who had not been treated with SSRIs.

Parkinson’s disease (PD) is a chronic, progressive neurodegenerative disease characterized by

both motor and nonmotor features. The motor symptoms of PD are attributed to the loss of striatal

dopaminergic neurons, although the presence of nonmotor symptoms supports neuronal loss in

nondopaminergic areas as well [33]. Depression in PD patients is also attributed to serotonergic

dysfunction, and SSRIs are able to amend depressive symptoms of PD [34]. Furthermore, several pieces

of evidence conﬁrm the neurodegeneration in striatal and extra-striatal 5-HT pathways suggesting

that serotonergic loss plays an important role in the pathophysiology of PD [35,36]. Additionally,

both nicotinic and muscarinic AChRs have been detected in the neostriatum (caudate nucleus and

putamen), with the nAChRs activating the nigrostriatal dopamine (DA, Figure 2) release and the

mAChRs causing inhibition of dopaminergic neurons [37].

All the aforementioned data are consistent with the well-established notion that monoaminergic

and cholinergic neurotransmitter systems exhibit, in the central nervous system (CNS), a wide range of

functional interactions and mutual regulations [38]. They also underline the complementary role that

nAChRs and monoamines such as 5-HT and DA play in the modulation of several brain functions [39],

as well as in the physiopathology of a variety of diseases [40].

In this context, it seems attractive to search for/formulate ligands that act through simultaneous

interactions with SERT/DAT and nAChRs [41,42].

Therefore, based on the structural combination of nicotinic ligands and antidepressants, it seems

attractive to search for and design compounds that act as multi-target therapeutic ligands in α4β2

nAChRs, DAT and/or SERT for the treatment of anxiety and depressive disorders [43,44].

Based on these precedents, in the present work, we report the synthesis and biochemical evaluation

of a series of nicotine derivatives, where the N-methyl-pyrrolidine moiety of the parent drug was

connected to aromatic and alkyl aromatic esters using a polymethylenic chain of variable length (n =1, 2).

The rationale behind this design proposal is supported by pharmacological studies of our research group,

in which a series of (S)-(1-methylpyrrolidin-2-yl)methyl benzoate derivatives exhibited antagonist

activity on the

α4β2 nAChRs [4]. Thus, to extend such study, we decided to synthesize novel molecules,

which by the inclusion of aromatic moieties, might behave as multiligands, interacting simultaneously

Molecules 2019, 24, 3808

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with the α4β2 nAChRs, DAT and/or SERT. Additionally, molecular docking was performed at the

diﬀerent studied targets to rationalize the aﬃnity shown by some of our compounds.

2. Results and Discussion

2.1. Chemistry

The arylpyrrolidine ester derivatives, compounds (1–21) (Figure 3) were synthesized from the

following commercially available compounds: benzoic acid and 2-phenylacetic acid derivatives,

2,2-diphenylacetyl chloride, 2-Naphthoyl chloride,

(S)-(-)-(1-methyl-2-pyrrolidinyl)methanol,

and (±)-2-(1-methyl-2-pyrrolidinyl)ethanol.

Figure 3. Structures of the 21 synthesized compounds used in this study.

Figure 3 summarizes all the synthesized compounds, which were fully characterized

spectroscopically by FT-IR, ¹H-NMR, ¹³C-NMR and HRMS.

The substituted benzoic acids (A) were ﬁrst reacted with thionyl chloride to give the corresponding

benzoyl halides (B), which were subsequently reacted with (

and ( )-2-(1-methyl-2-pyrrolidinyl)ethanol as racemic mixture to give the corresponding ﬁnal esters

(1–12) in 61–81% yields (Supplementary Materials, Figure S1).

S)-(-)-(1-methyl-2-pyrrolidinyl)methanol

±

The same reaction was carried out with the 2-phenylacetic acid derivatives (C) to provide

the corresponding 2-arylacetylchlorides derivatives (D) (Supplementary Materials, Figure S2),

which were reacted with the commercially available (

)-2-(1-methyl-2-pyrrolidinyl)ethanol as racemic mixture, to furnish the expected esters (13

20–35% yields (Supplementary Materials, Figure S2).

Analogously the 2-Naphthoyl chloride, reacted with (

and ( )-2-(1-methyl-2-pyrrolidinyl)ethanol to give the corresponding ﬁnal esters (18

yields (Supplementary Materials, Figure S3).

Finally, the 2,2-diphenylacetyl chloride was reacted with (

and ( )-2-(1-methyl-2-pyrrolidinyl)ethanol as racemic mixture, to obtain the corresponding ﬁnal esters

(20–21) in 31–43% yields (Supplementary Materials, Figure S4).

S)-(-)-(1-methyl-2-pyrrolidinyl)methanol and

(±

–

17) in

S

)-(-)-(1-methyl-2-pyrrolidinyl) methanol

19) in 61–65%

±

–

S)-(-)-(1-methyl-2-pyrrolidinyl)methanol

±

2.2. Biological Evaluation: Binding Aﬃnities on h-DAT, h-SERT and α4β2 nAChR

To evaluate the aﬃnity (K_i) of our compounds, competitive binding assays in

α4β2 nAChR

using [³H]-cytisine as radioligand ([³H]-Cyt) was measured. Binding experiments were carried out

on whole rat brain synaptosomes. To evaluate the aﬃnity on the monoamine transporters SERT

and DAT, binding aﬃnity (K_i) was determined using [³H]-WIN 35,428 ([³H]-WIN) for h-DAT and

[³H]-paroxetine ([³H]-parox) for h-SERT as speciﬁc radioligands for the corresponding monoamine

transporters respectively. Competitive binding studies were carried out on homogenized membranes

prepared from the human clonal cell line HEK293 for h-SERT (Perkin Elmer) and CHO-K1 for h-DAT

Molecules 2019, 24, 3808

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(Perkin Elmer). The estimated K_ivalues indicate that some compounds display competitive binding

aﬃnity for h-DAT and

2 nAChR. All compounds were ineﬀective to displace [³H]-paroxetine

α4β

from h-SERT. However, an unexpected result was found with compound 20, which produced a strong

increase of aﬃnity for [³H]-paroxetine. K_ivalues for

α4β2 nACHR, h-DAT and h-SERT are shown in

Table 1.

Table 1. K_ivalues (µM) for the synthesized compounds against h-DAT, α4β2 nAChRs and

[³H]-paroxetine h-SERT expressed as binding percentage.

% of Binding

Compound

K_i(µM) h-DAT

K_i(µM) α4β2 nAChR

[³H]-Paroxetine h-SERT

Bupropion

N.E

0.370

N.E

10.0

1

2

3

4

7

86.0 ± 0.6

123.0 ± 1.2

87.5 ± 1.5

137.0 ± 1.2

100.0 ± 0.5

139.5 ± 2.0

108.0 ± 1.2

119.3 ± 2.7

92.0 ± 0.3

78.0 ± 0.6

117.0 ± 2.3

126.3 ± 1.9

191.5 ± 0.9

86.5 ± 1.5

0.023 ± 0.006

0.094 ± 0.002

0.009 ± 0.001

0.132 ± 0.038

1.788 ± 0.378

N.E

3.461 ± 0.360

0.042 ± 0.004

N.E

8

9

10

16

17

18

19

20

21

22.690 ± 7.099

3.317 ± 0.923

99.330 ± 1.411

44.240 ± 8.120

1.208 ± 0.230

0.075 ± 0.009

N.E

0.120 ± 0.037

N.E

0.023 ± 0.006

0.113 ± 0.037

The non-speciﬁc binding of [³H]-paroxetine (2 nM) in h-SERT exhibited a radioligand displacement in the presence

or absence of 25

µM of the compound under study. Fluoxetine (Flx, 25 mM) was used to deﬁne the nonspeciﬁc

ligand. The non-speciﬁc binding in the h-DAT exhibited a radioligand displacement of [³H]-WIN 35,428 at 1 nM

concentration the dissociation constant (K_d) used to estimate K_iwas 9.21 nM for [³H]-WIN 35428. In the

α4β2

nAChR displacement assays the concentration of the radio ligand was 1 nM [³H]-Cytisine and its K_d= 0.43 nM

used for the K_icalculation. The bupropion aﬃnity was just included as a comparative value and was not used in

the trial. N.E = no eﬀect at 100 µM.

Binding experiments for h-SERT show that all compounds are unable to displace [³H]-paroxetine

from its binding site, but unexpectedly, an increase of radioactivity of over 100% was found for some

values of paroxetine binding. These increases in the binding of the radioligand did not exceed 40% in

most of cases. However, compound 20 elicited an increase of 91% of the total radioactivity binding,

indicating a possible allosteric interaction of this compound, which could be responsible for the increase

of total [³H]-paroxetine bonded at the h-SERT binding site (Figure 4).

2 0 0

1 5 0

1 0 0

5 0

0

F lx

2

4

8

9

1 0

1 8

1 9

2 0

Figure 4. Graph obtained for the displacement experiments of [³H]-paroxetine for the synthesized

compounds [ 21] in the cellular background h-SERT induced to 2.5

10⁻⁵M concentration.

Fluoxetine (Flx) was used as a control for the compounds under study.

1–

×

Molecules 2019, 24, 3808

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Compounds

= 0.132 0.038

percentage binding of [³H]-paroxetine at h-SERT (123.0

presence of aromatic substituents 3-F and 3-Br would disfavor the interaction in the agonist binding

site at 2 nAChRs, but would favor the interactions at level of a putative allosteric binding site

of h-SERT. Compound with a K_iof 0.009 0.001 M on the 2 nAChRs (Figure 5 ) lacks aﬃnity

at h-SERT and h-DAT. In comparison, compounds and (not included in Table 1) did not display

2

and

4

displayed lower aﬃnity in

α

4

β

2 nAChR (K_i= 0.094

±

0.002

µ

M and K_i

±

µ

M) as compared with the unsubstituted analog 1, but induced an increase in the

±

1.2% and 137.0 1.2%), suggesting that the

±

α4β

3

±

µ

α4β

5

6

aﬃnity for any of the binding sites under consideration, suggesting that methyl substituents at C-2 or

C-4 of the aromatic ring lead to compounds with a total lack of molecular interactions at these targets.

100

2

3

10

20

50

0

-12

-10

-8

-6

-4

-2

Log [compound] M

Figure 5. Compounds eﬀects on binding of [³H]-cytisine to

mean SEM of four experiments, each one carried out in triplicate. The radioligand concentration in

all displacement studies was 1 nM.

α4β2 nAChRs. Data points represent the

±

Compound

K_i= 1.788 ± 0.378 µM) suggesting that when the (

changed for ( )-(1-methyl-2-pyrrolidinyl)ethanol, the interaction with the agonist binding site

nAChR is remarkably disfavored (Figure 5). In addition, no aﬃnity for h-SERT and h-DAT was

observed for this derivative. Compound gave a comparative low aﬃnity for 2 nAChR (K_iof

3.461 ± 0.360 µM, Figure 5), whereas compound 10 resulted in be one of the best of this series, showing

a high aﬃnity for 2 nAChR (K_iof 0.042 0.004 M) and eliciting also an increase in the percentage

of union of [³H]-paroxetine at h-SERT (119.3

2.7%). Compounds 11 and 12 (not included in Table 1)

similarly to compounds and , did not display aﬃnity for any of the binding sites under consideration,

7

, in comparison with compound

1, showed a low aﬃnity for α4β2 nAChR

(

S)-(1-methyl-2-pyrrolidinyl)methanol moiety was

±

α4β2

9

α4β

α

4

β

±

µ

±

5

6

suggesting again that methyl substituents at C-2 or C-4 of the aromatic ring lead to a total lack of

molecular interactions.

The unsubstituted compound 16 showed

K_i= 22.690 ± 7.099 µM), whereas compound 17

was approximately 7-fold more potent at h-DAT with a K_iof 3.317

halogenation of compound 16 at C-3 and C-4 positions, favors the ligand interactions with the central

a

relatively low aﬃnity for h-DAT

with a 3,4-dichloro substitution pattern,

0.923 M. This indicates that the

(

,

±

µ

binding site of h-DAT. Compound 18 exhibited aﬃnities for

α

4

β

2 nAChR (K_i= 0.120 ± 0.037 µM) and

for h-DAT (K_i= 99.330 ± 1.411 µM), and also produced a small increase of the percent of [³H]-paroxetine

binding at h-SERT (117.0

±

2.3%). Compound 19 also produced an increase in the percent binding of

[³H]-paroxetine at h-SERT (126.3 ± 1.9%) and showed h-DAT aﬃnity (K_i= 44.240 ± 8.120 µM).

Compounds 18 and 19 were less potent than compounds 16 and 17 at h-DAT, indicating that

the incorporation of a naphthyl moiety (compounds 18 and 19) decreases the aﬃnity for this target

(Figure 6). On the other hand, the ethylene linker connecting the pyrrolidine moiety and the aryl ester

in 16 and 17 improved the aﬃnity as compared with compounds 13, 14 and 15 (not included in Table 1 )

which contained a methylene linker.

Molecules 2019, 24, 3808

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100

50

0

1 7

2 0

2 1

-10

-8

-6

-4

-2

L o g [c o m p o u n d ] M

Figure 6. Graph obtained for the displacement experiment of [³H]-WIN 35428. Data points represent

the mean SEM of four experiments, each one carried out in triplicate. The radioligand concentration

±

in all displacement studies was 1 nM.

On the other hand, compound 20 was the only compound of the series which displayed a triple

aﬃnity for the studied targets. Thus, this compound produced a marked increase in the percent of

[³H]-paroxetine binding at h-SERT (191.5

(

±

0.9%) (Figure 4), showed high aﬃnity at

K_i= 0.023 ± 0.006 µM) (Figure 5 ) and a moderate aﬃnity for h-DAT (K_i= 1.208 0.230

Compound 21 was the most potent of the series at h-DAT, exhibiting an aﬃnity at this target which

was higher than that of bupropion (K_i= 0.075 0.009 M vs. K_i= 0.370 M respectively). Interestingly,

compound 21 exhibited a similar aﬃnity at 2 nAChR (K_i= 0.113 0.037 M), which was also higher

than that of bupropion at this receptor (K_i= 10 M). Thus, 21 can be considered as the compound

α

4

β

2 nAChR

±

µM) (Figure 6 ).

±

µ

α

4

β

±

µ

with the best dual aﬃnity of the series. These biphenyl analogs (20 and 21), were the most potent

compounds in h-DAT binding experiments. However, compound 21 having both an ethylenic bridge

and a diphenyl ester moiety, contains the two pharmacophore portions for optimal binding aﬃnity on

h-DAT (Figure 6).

In an related work, Zhang et al. [12] described a series of novel ligands containing

cyclopropane- or isoxazole- side chains onto the 5-position of 1-(pyridin-3-yl)-1,4-diazepine or

2-(pyridin-3-yl)-2,5-diazabicyclo [2.2.1]heptane which aﬀorded compounds with selective partial

agonist propertiers for

4 subtype. They also found that the most potent compounds, structurally exhibited a pyridine

ring connected to a diazepine squeleton, where substituents at the 5-position of the pyridine core were

found to be beneﬁcial for the selectivity for 2 over 7 and ganglionic nAChRs. In connection with

α4β2* nAChR (K_iof 0.5-51.4 nM), displaying also excellent selectivity over the

α3β

α

4

β

α

the Zhang approaches, we carried out the synthesis of molecules bearing a pyridinyl group connected

to an ester function. However, in our case these modiﬁcations lead to unfavorable interactions at

the agonist binding site for both α4β2 and α7 nAChRs [4]. The change of the azaheterocycle ring by

a functionalized phenyl group aﬀorded the results already described.

In recent studies, we have replaced the ester function for an ether function [43], obtaining molecules

with agonist activity at α4β2* nAChR which in theory promote dopamine release and would not be

useful in nicotine addiction.

2.3. Docking Studies

To gain a better insight into the binding mode and possible interactions of the compounds into

the target binding sites, we carried out docking studies in a homology model of the agonist site of the

α4β2 nAChR, the central site of the DAT, and the best characterized allosteric site (the S2 site) of SERT,

using AutoDock 4.0.

Binding studies with

at the ACh binding site of

α

4

β

2 nAChRs indicated that only 10 compounds (

1–21) showed aﬃnity

α4β2 nAChRs, i.e the orthosteric site (values ranging from low micro to

submicromolar concentrations). Docking studies are in line with these results, mainly due to the

Molecules 2019, 24, 3808

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observed π–cation interaction with Trp 182 residue, retained in all the nicotine-derived synthetic

compounds and bupropion. By comparison with bupropion, the higher aﬃnities of some of our

compounds, could be explained by both the presence of hydrogen bond and hydrophobic interactions,

which would take place between the aromatic ring with Tyr 223 or Phe 119 residues (compounds

18 and 20) and between the quaternary nitrogen of the N-methylpyrrolidine with Tyr 126 residue

(compound ). On the other hand, compound exhibited an additional –cation interaction between

2,

4

,

1

3

π

the quaternary nitrogen of the N-methylpyrrolidine with the ester group of the Tyr 230 residue. In the

docking studies, the high-aﬃnity obtained for compound 21 (Figure 7B), could be explained due to

the presence of π–π interactions among the Phe 119 residue with both phenyl groups of compound

21, increasing the energy of the ligand-receptor complex (Figure 7B), these interactions are absent in

bupropion as was already mentioned [45].

Figure 7. Binding modes of compound 20 (A) and compound 21 (B) to the human α4β2 nAChR.

The orange-ligands denotes compounds 20 and 21. Dotted lines correspond to the diﬀerent interactions

which may take place in the ligand–receptor complexes.

The binding studies carried out in SERT that all compounds were unable to displace [³H]-paroxetine

from its binding site, but unexpectedly compound 20 exhibited an increase of the radioactivity, indicating

a possible allosteric interaction in SERT. Docking studies are in line with this result, mainly due to the

presence of an ionic bond and π-stacking interactions between the quaternary nitrogen with Glu 494

and Phe 335 residues, respectively. These interactions would take place in both compound 20 and the

S-citalopram co-crystallized with SERT (Figure 8B). Additionally, compound 20 exhibited the highest

aﬃnity in the allosteric h-SERT binding site, probably due to a new

of the phenyl group of 20 with Phe 556 and a –cation interaction between the phenyl group of 20 and

Arg 104 residue, which were not observed in the S-citalopram co-crystal (Figure 8B).

Binding studies in h-DAT indicate that only 6 compounds ( 21) showed aﬃnity values at the

central binding site of h-DAT. The results from the docking studies correlated with experimental

evidence, primarily due to the presence of a interaction between the aromatic group of compounds

18, 19 and 20 and the Tyr 548 residue, and a –cation interaction among the phenyl groups of compounds

π-stacking interaction between one

π

1

–

π–π

π

16 and 19 with the quaternary amine of Ala 550, both interactions retained for neo-nicotinic compounds

and bupropion. Compound 17 showed a double hydrogen bond interaction, among Tyr 470 residue

with the quaternary nitrogen of the N-methylpyrrolidine and the ester function of 17, with both

interactions absent in bupropion. Compound 21 showed the highest aﬃnity in relation to bupropion.

This experimental value can be supported by the docking results, on the basis of the following

interactions observed: a

π

–π

type interaction between both phenyl groups and Tyr 470 residue, a

π–π

interaction between one of the phenyl group and Tyr 551 residue, and two

π

–cation interactions

between the phenyl groups with Ser 227 and Tyr 548 residues. On the other hand, compound 21

showed a hydrogen bond interaction between the quaternary amine group of N-methylpyrrolidine

and Tyr 548 residue, and three hydrogen bond interactions between the ester function of 21 with Ala

Molecules 2019, 24, 3808

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550 and Tyr 551 residues, contributing in consequence to the thermodynamic stability of the system

(Figure 9B).

Figure 8. Binding modes of S-citalopram co-crystal (A) and compound 20 (B) to the human SERT.

The cyan ligand denotes S-citalopram and the yellow ligand denotes compound 20. Dotted lines

correspond to the diﬀerent interactions in the ligand–receptor complexes.

Figure 9. Binding modes of compounds 20 (A) and 21 (B) to the human DAT. The yellow ligand

denotes compound 20 and the cyan ligand corresponds to compound 21. Dotted lines correspond to

the diﬀerent interactions taking place in the ligand-receptor complexes.

3. Materials and Methods

3.1. Binding Protocol of [³H]-Cytisine on nAChR Synaptosomes

Preparation of the synaptosome fraction: the rats were sacriﬁced by decapitation, and their brain

removed without dissecting. Brain tissue was homogenized in 40 volumes using a buﬀer of 0.32 M

sucrose at pH 7.4, containing 1 mM EDTA, 0.1 mM PMSF, and 0.01% of cold and freshly prepared NaN_3,

through a glass homogenizer and teﬂon stem [46]. The homogenate was distributed in Eppendorf

tubes and centrifuged at 20,000 g for 30 min at 4 ^◦C to produce a synaptosome pellet (fraction P1)

which is used for the binding assay. This fraction P1 was subsequently stored at −80 ^◦C.

Binding assay of [³H]-cytisine in

α

4

β2 nAChR cells: the rat complete brain P1 fraction (occupying

2 mg of wet tissue for each assay) was incubated in 2500

µL of 20 mM HEPES buﬀer solution at pH

7.4 containing 118 mm NaCl, 4.8 mm KCl, 2.5 mm CaCl₂, 200 mm Tris, 0.1 mm PMSF and 0.01%

NaN₃with or without the diﬀerent drugs under study at diﬀerent concentrations, in the presence of

Molecules 2019, 24, 3808

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1 nM of [³H]-cytisine (speciﬁc activity 35.8 Ci/mmol, Code NET1054025UC, Perkin-Elmer, Santiago,

Chile) in a ﬁnal volume of 250 L. Non-speciﬁc binding was determined using 2.5 mM of cytisine.

µ

Incubation was performed for 30 min at room temperature and then for 60 min at 4 ^◦C. Incubation was

stopped by rapid ﬁltration in Whatman GF/C ﬁlter preabsorbed in 0.5% polyethylenimine, which were

washed with cold and fresh working buﬀer 7 times, then dried, and adding scintillation liquid, left for

the whole night in Eppendorf tubes in order to measure later the radioactivity by liquid scintillation

spectrometry (MicroBeta 2450 microplate counter, PerkinElmer, Santiago, Chile). The data were plotted

by non-linear regression variable inhibitor-response dose (Prism 5.01, GraphPad, San Diego, CA, USA)

to obtain the IC₅₀and K_ivalues of the tested compounds using the Cheng-Prusoﬀ equation.

3.2. Protocol Binding of [³H]-Paroxetine on h-SERT Cells

The cellular background HEK293 containing 400

µL of h-SERT (Code RBHSTM400UA,

Perkin-Elmer, Santiago, Chile) was diluted in 12 Eppendorf tubes, containing a storage buﬀer solution

of Tris-HCl 50 mM (pH 7.4), EDTA 0.5 mM, MgCl₂10 mM and 10% sucrose, obtaining a ﬁnal volume

between 260 and 340 µL, which was ﬁnally stored at −80 ^◦C [46].

Each Eppendorf tube was incubated with 50 mM Tris HCl buﬀer (pH 7.4), 120 mM NaCl, 5 mM

KCl and the drugs under study using increasing concentrations, in presence of 2 nM of [³H]-paroxetine

(speciﬁc activity 23.1 Ci/mmol, Code NET86925UC, Perkin-Elmer, Santiago, Chile) with a ﬁnal volume

of 250

27 ^◦C, the incubation was stopped by rapid ﬁltration on a Whatman GF/C ﬁlter preabsorbed in 0.5%

polyethylenimine (PEI), washed with cold working buﬀer solution, 3 3mL, ﬁltered, and scintillation

µL [46]. Non-speciﬁc binding was determined using 25 mM ﬂuoxetine. After 30 min at

×

liquid was added. The radioactivity was measured by liquid scintillation spectrometry (MicroBeta

2450 microplate counter, PerkinElmer, Santiago, Chile). The data were represented in a bar graph

using 25 µM concentration for both, the compound under study and ﬂuoxetine as the non-speciﬁc

ligand. Each bar represents the mean ± S.E.M obtained in the experiments carried out in triplicates.

3.3. Protocol Binding of [³H]-WIN 35,428 on h-DAT Cells

The cellular background CHO-K1 with 400

Santiago, Chile) was diluted in 12 Eppendorf tubes, with a storage buﬀer solution of Tris-HCl 50 mM

(pH 7.4), EDTA 0.5 mM, MgCl₂10 mM and 10% sucrose, obtaining a ﬁnal volume of 260–340

µL of h-DAT (Code RBHDATM400UA, Perkin-Elmer,

µL

stored at

80 ^◦C [46]. Each Eppendorf tube was incubated in 50 mM Tris-HCl buﬀer solution (pH 7.4),

−

100 mM NaCl, the drugs under study were using increasing concentrations, with 1 nM of [³H]-WIN

35,428 (speciﬁc activity 82.6 Ci/mmol, Code NET1033025UC, Perkin-Elmer, Santiago, Chile) with a ﬁnal

volume of 250 µL. Non-speciﬁc binding was determined using 10 mM methylphenidate.

After 120 min at 4 ^◦C, the incubation was stopped by rapid ﬁltration on a Whatman GF/C ﬁlter

preabsorbed in 0.5% polyethylenimine (PEI), washed with cold working buﬀer solution, 3 x 3mL,

ﬁltered, and scintillation liquid was added. The radioactivity was measured by liquid scintillation

spectrometry (MicroBeta 2450 microplate counter, PerkinElmer, Santiago, Chile). The data were plotted

by non-linear regression variable inhibitor-response dose (Prism 5.01, GraphPad, San Diego, CA, USA)

to estimate the IC₅₀and K_ivalues for the tested compounds using the Cheng-Prusoﬀ equation.

3.4. Docking Analysis

Molecular docking of the compounds in the homology model of the α4β2, central binding site of

h-DAT and allosteric binding site of h-SERT was performed, using the Lamarckian genetic algorithm

search method with the AutoDock v4.0 software, San Diego, CA, USA [47]. The homology models

of the proteins were obtained from the Protein Data Bank (PDB: 5I73, 1UW6 and 4M48 respectively).

The receptors were kept rigid, while full ﬂexibility was allowed for the ligands to translate/rotate.

Polar hydrogens were added to the receptors and Kollman-united atom partial charges along with

atomic solvation parameters were assigned to the individual protein atoms. The three-dimensional

structures of each ligand were generated using the SPARTAN’08 program and energy minimized.

Molecules 2019, 24, 3808

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For each ligand, a rigid root and rotatable bonds were assigned automatically. The non-polar hydrogens

were removed and the partial charges from these were added to the carbons (Gasteiger charges).

The atom type for aromatic carbons was reassigned in order to use the AutoDock v4.0 aromatic carbon

grid map. Docking was carried out using 60

For the nAChR, the grid was positioned to include the full ligand binding pocket in the central part of

the 4/ 2 subunit interfaces to allow extensive sampling around residue 4W182. For the allosteric

×

60

×

60 grid points with a default spacing of 0.375 Å [48].

α

β

α

binding site of the h-SERT a radio lattice of 14 Å was deﬁned around co-crystal (S-Citalopram) and

was the only established restriction. The hydrogen bond generation between the carboxylate group

of E494 residue was conserved for the majority of the known complexes studied between h-SERT

and S-citalopram [49]. For the binding of h-DAT, the grid was positioned to include the full ligand

binding pocket in the central part of the protein to allow extensive sampling around the residue

Y548

of 150 individuals, calculated using 200 diﬀerent runs (i.e., 200 dockings). Each run had two stop

criteria, a maximum of 1.5

10⁶energy evaluations or a maximum of 50,000 generations, starting

. Within this grid, the Lamarckian genetic search algorithm was used with a population size

×

from a random position and conformation; default parameters were used for the Lamarckian genetic

algorithm search. The optimal conﬁguration and the resulting ligand–receptor complexes were further

processed using the PYMOL software, New York, NY, USA [48].

4. Synthetic Procedures

Melting points are uncorrected and were determined with a Reichert Galen III hot plate microscope.

¹H-NMR and ¹³C-RMN spectra were recorded using Bruker AMX 400 spectrometers at 400 MHz.

The ¹H-NMR and ¹³C-NMR chemical shifts were referenced with respect to residual solvent peaks

(

δ TMS = 0) and all quoted coupling constants J are J_HHbetween protons. The IR spectra were recorded

on a FT-IR Nicolet iS50 Thermo scientiﬁc and wavenumbers are reported in cm⁻¹. High resolution

mass spectra were recorded on a DSA–TOF AxION 2 TOF MS (Perkin Elmer, Shelton, CT, USA),

under positive mode, the molecular ion [M+1] is observed.

Reactions and product mixtures were routinely monitored by thin-layer chromatography (TLC)

on silica gel pre-coated F₂₅₄Merck plates and the compounds obtained were puriﬁed by column

chromatography using a CH₂Cl₂/CH₃OH (9:1) mixture as the mobile phase. Reagents and solvents

utilized were commercially available and used without further puriﬁcation.

4.1. General Procedures for The synthesis of Benzoyl Chloride and 2-Phenylacetyl Chloride Derivatives (

1–12)

Benzoic acid derivatives ( ) and 2-phenylacetic acid derivatives ( 12) (6–10 mmol, 0.5 g) were

1–6

7–

reacted with thionyl chloride (68.9 mmol, 5 mL) under nitrogen atmosphere for 24 h to aﬀord the

corresponding benzoyl chloride or 2-phenylacetyl chloride derivatives. The mixture was maintained

for 15 min. at room temperature and then concentrated under vacuum to give the corresponding crude

aroyl chloride derivatives, which were immediately used in the next reaction.

4.2. General Procedures for the Synthesis of (S)-(1-methyl-2-pyrrolidinyl)methyl Benzoate Derivatives (1–6)

and (S)-(1-methyl-2-pyrrolidinyl)methyl 2-naphtoate (18)

Benzoyl chloride derivatives (4.2 mmol, 0.5 mL) or 2-Naphthoyl chloride (4.2 mmol, 0.80 g)

were dissolved in dry diethyl ether (50 mL) and stirred at room temperature. One equivalent of

(S)-(-)-(1-methyl-2-pyrrolidinyl)methanol (2.2–4.2 mmol, 0.2–0.5 mL) in dry diethyl ether (30 mL)

was added drop by drop at room temperature. The reaction mixture was kept at room temperature

with stirring for 24 h. Then, the mixture was concentrated under vacuum to give a crude which

was re-dissolved in water adjusted to pH 8.0 and extracted with CH₂Cl₂(3

the hydrochloride salts were obtained from an isopropanol solution 7.4% HCl (p/v).

×

30 mL). Finally,

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4.3. General Procedures for the Synthesis of (S)-(1-methyl-2-pyrrolidinyl)methyl 2-phenylacetate Derivatives

(13–15) and (S)-(1-methyl-2-pyrrolidinyl)methyl 2,2-diphenylacetate (20)

2-Phenylacetyl chloride derivatives and 2,2-diphenylacetyl chloride (2.5–4.2 mmol, 0.5 gr)

were dissolved in dry diethyl ether (50 mL) and stirred at room temperature. One equivalent

of (S)-(-)-(1-methyl-2-pyrrolidinyl)methanol (2.2–4.2 mmol, 0.2–0.5 mL) in dry diethyl ether (30 mL)

was slowly added. The reaction mixture was kept at room temperature with stirring for 24 h. Then,

the mixture was concentrated under vacuum to give a crude which was re-dissolved in water adjusted

to pH 8.0 and extracted with CH₂Cl₂(3

an isopropanol solution 7.4% HCl (p/v).

×

30 mL). Finally, the hydrochloride salts were obtained from

4.4. General Procedures for the Synthesis of (

±

)-2-(1-methyl-2-pyrrolidinyl)ethyl Benzoate Derivates (7–12) and

(±)-2-(1-methyl-2-pyrrolidinyl)ethyl 2-naphtoate (19)

Benzoyl chloride derivatives (3.7 mmol, 0.4–0.5 mL) and 2-naphthoyl chloride (3.7 mmol, 0.7 g)

were dissolved in dry diethyl ether (50 mL) and stirred at room temperature. One equivalent of

( )-2-(1-methyl-2-pyrrolidinyl)ethanol (3.7 mmol, 0.5 mL) in diethyl ether (30 mL) was added drop

±

by drop. The reaction mixture was kept at room temperature and stirred for 24 h. Then, the mixture

was concentrated under vacuum to give a crude which was re-dissolved in water, adjusted to pH 8.0

and extracted with CH₂Cl₂(3

solution 7.4% HCl (p/v).

×

30 mL). The hydrochloride salts were obtained from an isopropanol

4.5. General Procedures for the Synthesis of (±)-2-(1-methyl-2-pyrrolidinyl)ethyl 2-phenylacetate Derivatives

(16–17) and (±)-2-(1-methyl-2-pyrrolidinyl)ethyl 2,2-diphenylacetate (21)

2-Phenylacetyl chloride derivatives and 2,2-diphenylacetyl (2.2–4.1 mmol, 0.3–0.5 mL) was

dissolved in dry diethyl ether (50 mL) and stirred at room temperature. An equivalent of

( )-2-(1-methylpyrrolidin-2-yl)ethanol (2–4 mmol, 0.3–0.5 mL) in diethyl ether (30 mL) was added

±

dropwise. The reaction mixture was kept at room temperature and stirred for 24 h. Then, the mixture

was concentrated under vacuum to give a crude which was re-dissolved in water adjusted to pH 8.0

and extracted with CH₂Cl₂(3

solution 7.4% HCl (p/v).

×

30 mL). The hydrochloride salts were obtained from an isopropanol

4.6. (S)-(1-Methyl-2-pyrrolidinyl)methyl Benzoate (1)

Prepared as described in general procedure (Section 4.2). (S)-(-)-(1-methyl-2-pyrrolidinyl)methanol

(4.2 mmol, 0.42 mL) was added to a solution of benzoyl chloride (4.2 mmol, 0.49 mL); Yield 718 mg

(78%); m.p. 165.4–166.0 ^◦C; IR (cm⁻¹) 2908 (stretch. C-H aliph.), 1754 (stretching COO), 1280 (stretching

C-O), 1115 (stretching C-N), 716 (bending C-H Arom.); ¹H-NMR (400 MHz, D₂O)

δ 8.08 (d, J = 7.8 Hz,

2H), 7.75 (t, J = 7.5 Hz, 1H), 7.59 (t, J = 7.7 Hz, 2H), 4.76 (d, J = 2.9 Hz, 1H), 4.58 (dd, J = 13.1, J = 6.7

Hz, 1H), 3.95 (m, 1H), 3.78 (m, 1H), 3.29 (m, 1H), 3.08 (s, 3H), 2.45 (m, 1H), 2.26 (m, 1H), 2.12 (m, 2H);

¹³C-RMN (101 MHz, D₂O)

δ 165.3, 133.6, 129.4 (2C), 129.1, 128.7 (2C), 66.0, 62.5, 56.1, 39.5, 26.6, 21.7;

HRMS m/z calcd. for C₁₃H₁₇NO₂(M⁺), 220.1338; found, 220.1347.

4.7. (S)-(1-Methyl-2-pyrrolidinyl)methyl 3-ﬂuorobenzoate (2)

Prepared as described in general procedure (Section 4.2). (S)-(-)-(1-methyl-2-pyrrolidinyl)methanol

(4.2 mmol, 0.5 mL) was added to a solution of 3-ﬂuorobenzoyl chloride (4.2 mmol, 0.5 mL); Yield 807 mg

(81%); m.p. 110–111.9 ^◦C; IR (cm⁻¹) 2965 (stretch. C-H aliph.), 1724 (stretching COO), 1292 (stretching

C-O), 1071 (stretching C-N), 757 (bending C-H Arom.); ¹H-NMR (400 MHz, DMSO-d₆)

δ: 7.90 (d, J =

7.5 Hz, 1H), 7.79 (d, J = 7.5 Hz, 1H), 7.56 (m, 1H), 7.48 (t, J = 8.5 Hz, 1H), 4.63 (m, 2H), 3.81 (m, 1H),

3.56 (m, 1H), 3.13 (m, 1H), 2.87 (s, 3H), 2.25 (m, 1H), 2.00 (m, 2H), 1.87 (m, 1H); ¹³C-RMN (101 MHz,

DMSO-d₆)

δ: 167.9, 165.1-162.6, 132.4 (2C), 127.2, 122.7, 117.8, 68.9, 64.2, 58.8, 42.2, 27.7, 23.6; HRMS m/z

calcd. for C₁₃H₁₆FNO₂(M⁺), 238.1248; found, 238.1266.

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4.8. (S)-(1-Methyl-2-pyrrolidinyl)methyl 3-chlorobenzoate (3)

Prepared

as

described

above

in

general

procedure

(Section

4.2).

(S)-(-)-(1-methyl-2-pyrrolidinyl)methanol (4.2 mmol, 0.5 mL) was added to a solution of

3-chlorobenzoyl chloride (4.2 mmol, 0.54 mL); Yield 650 mg (61%); m.p. 120.5–121.9 ^◦C; IR (cm⁻¹) 2945

(stretch. C-H aliph.), 1737 (stretching COO), 1260 (stretching C-O), 1143 (stretching C-N), 748 (bending

C-H Arom.); ¹H-NMR (400 MHz, DMSO-d₆)

δ: 8.02 (d, J = 13.3 Hz, 2H), 7.77 (d, J = 8.0 Hz, 1H), 7.59 (t,

J = 7.8 Hz, 1H), 4.63 (m, 2H) 3.80 (m, 1H), 3.58 (m, 1H), 3.09 (d, J = 9.0 Hz, 1H), 2.83 (s, 3H), 2.24 (m, 1H),

2.05 (m, 2H), 1.92 (m, 1H); ¹³C-NMR (101 MHz, DMSO-d₆)

δ: 166.8, 135.3, 135.2 (2C), 131.9, 130.5, 129.4,

70.8, 59.7, 57.8, 41.3, 26.98, 22.95; HRMS m/z calcd. for C₁₃H₁₆ClNO₂(M⁺), 254.0948; found, 254.0967.

4.9. (S)-(1-Methyl-2-pyrrolidinyl)methyl 3-bromobenzoate (4)

Prepared

as

described

above

in

general

procedure

(Section

4.2).

(S)-(-)-(1-methyl-2-pyrrolidinyl)methanol (4.2 mmol, 0.5 mL) was added to a solution of

3-bromobenzoyl chloride (4.2 mmol, 0.55 mL); Yield 914 mg (73%); m.p. 99–101,4 ^◦C; IR (cm⁻¹) 3069

(stretch. C-H aliph.), 1744 (stretching COO), 1208 (stretching C-O), 1074 (stretching C-N), 728 (bending

C-H Arom.); ¹H-NMR (400 MHz, DMSO-d₆)

δ: 8.03 (s, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.82 (d, J = 7.9 Hz,

1H), 7.47 (t, J = 7.8 Hz 1H), 4.60 (dt, J = 12.7, 9.6 Hz, 2H), 3.82 (t, J = 6.88, 6.88 Hz, 1H), 3.58 (m, 1H),

3.15 (m, 1H), 2.90 (s, 3H), 2.23 (m, 1H), 1.94 (m, 3H); ¹³C-NMR (101 MHz, D₂O)

δ: 165.2, 135.9, 131.2,

129.5, 129.4, 127.3, 121.0, 66.4, 61.5, 56.2, 39.6, 25.1, 20.9; HRMS m/z calcd. for C₁₃H₁₆BrNO₂(M⁺),

298.0438; found, 298.0462.

4.10. (S)-(1-Methyl-2-pyrrolidinyl)methyl 2-methylbenzoate (5)

Prepared

as

described

above

in

general

procedure

(Section

4.2).

(S)-(-)-(1-methyl-2-pyrrolidinyl)methanol (4.2 mmol, 0.5 mL) was added to a solution of

2-methylbenzoyl chloride (4.2 mmol, 0.55 mL); Yield 725 mg (74%); m.p. 130.5–131.6 ^◦C; IR (cm⁻¹) 2948

(stretch. C-H aliph.), 1725 (stretching COO), 1280 (stretching C-O), 1126 (stretching C-N), 757 (bending

C-H Arom.); ¹H-NMR (400 MHz, DMSO-d₆)

δ

: 7.96 (d, J = 7.7 Hz, 1H), 7.51 (t, J =7.34 Hz, 1H), 7.34 (m,

2H), 4.59 (m, 2H), 3.76 (m, 1H), 3.53 (m, 1H), 3.09 (m, 1H), 2.87 (s, 3H), 2.54 (s, 3H) 2.26 (m, 1H), 1.99 (m,

2H), 1.87 (m, 1H); ¹³C-NMR (101 MHz, DMSO-d₆)

δ: 169.3, 141.8, 134.7, 133.3, 131.8, 127.8, 127.6, 68.6,

63.7, 58.5, 42.1, 27.7, 23.5, 22.4; HRMS m/z calcd. for C₁₄H₁₉NO₂(M⁺), 234.1498; found, 234.1494.

4.11. (S)-(1-Methyl-2-pyrrolidinyl)methyl 4-methylbenzoate (6)

Prepared

)-(-)-(1-methyl-2-pyrrolidinyl)methanol (4.2 mmol, 0.5 mL) was added to a solution of

4-methylbenzoyl chloride (4.2 mmol, 0.55 mL); Yield 774 mg (79%); m.p. 158.1–159.4 ^◦C; IR (cm⁻¹

2940 (stretch. C-H aliph.), 1719 (stretching COO), 1268 (stretching C-O), 1103 (stretching C-N), 752

(bending C-H Arom.); ¹H-NMR (400 MHz, DMSO-d₆)

: 7.93 (d, J = 7.7 Hz, 2H), 7.35 (d, J = 7.8 Hz,

2H), 4.60 (m, 2H), 3.79 (m, 1H), 3.58 (m, 1H), 3.09 (m, 1H), 2.90 (s, 3H), 2.39 (s, 3H), 2.24 (m, 1H), 1.98

as

described

above

in

general

procedure

(Section

4.2).

(S

)

δ

(m, 2H), 1.84 (m, 1H); ¹³C-NMR (101 MHz, DMSO-d₆)

δ: 168.7, 146.9, 131.0 (2C), 130.9 (2C), 126.9, 68.7,

63.6, 58.5, 41.9, 27.5, 23.4, 22.3; HRMS m/z calcd. for C₁₄H₁₉NO₂(M⁺), 234.1498; found, 234.1495.

4.12. (±)-2-(1-Methyl-2-pyrrolidinyl)ethyl benzoate (7)

Prepared

as

described

above

in

general

procedure

(Section

4.4).

( )-2-(1-methyl-2-pyrrolidinyl)ethanol (3.7 mmol, 0.5 mL) was added to a solution of benzoyl chloride

±

(3.7 mmol, 0.43 mL); Yield 641 mg (74.6%); m.p. 88.6–89.1 ^◦C; IR (cm⁻¹) 2953 (stretch. C-H aliph.), 1716

(stretching COO), 1280 (stretching C-O), 1115 (stretching C-N), 711 (bending C-H Arom.); ¹H-NMR

(400 MHz, D₂O)

δ 8.04 (d, J = 7.7 Hz, 2H), 7.72 (t, J = 7.5 Hz, 1H), 7.57 (t, J = 7.5 Hz, 2H), 4.47 (m, 2H),

3.72 (m, 1H), 3.52 (m, 1H), 3.18 (m, 1H), 2.98 (s, 3H), 2.46 (m, 3H), 2.13 (dt, J = 11.16, 6.06, 6.06 Hz, 1H),

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1.87 (m, 2H); ¹³C-RMN (101 MHz, DMSO-d₆)

δ

: 165.7, 133.5, 129.5, 129.3 (2C), 128.8 (2C), 65.4, 62.0,

57.6, 38.1, 29.4, 28.8, 21.1; HRMS m/z calcd. for C₁₄H₁₉NO₂(M⁺), 234.1498; found, 234.1493.

4.13. (±)-2-(1-Methyl-2-pyrrolidinyl)ethyl 3-ﬂuorobenzoate (8)

Prepared

as

described

above

in

general

procedure

(Section

4.4).

( )-2-(1-methyl-2-pyrrolidinyl)ethanol (3.7 mmol, 0.5 mL) was added to a solution of 3-bromobenzoyl

±

chloride (3.7 mmol, 0.44 mL); Yield 636 mg (68.8%); m.p. 106.8–107.5 ^◦C; IR (cm⁻¹) 2965 (stretch. C-H

aliph.), 1721 (stretching COO), 1280 (stretching C-O), 1082 (stretching C-N), 760 (bending C-H Arom.);

¹H-NMR (400 MHz, D₂O)

δ

7.84 (d, J = 7.7 Hz, 1H), 7.72 (d, J = 9.7 Hz, 1H), 7.55 (dd, J = 13.9, J = 7.9

Hz, 1H), 7.45 (dt, J = 8.7, 1.7 Hz, 1H), 4.49 (m, 2H), 3.75 (m, 1H), 3.53 (m, 1H), 3.20 (dt, J = 11.5, 8.5 Hz,

1H), 2.99 (s, 3H), 2.51 (m, 2H), 2.13 (m, 3H), 1.91 (m, 1H); ¹³C-RMN (101 MHz, DMSO-d₆)

: 164.5,

δ

163.2-160.8, 131.9, 131.1, 125.5, 120.5, 115.8, 65.2, 62.3, 54.7, 38.1, 29.2, 28.9, 20.9; HRMS m/z calcd. for

C₁₄H₁₈FNO₂(M⁺), 252.1398; found, 252.1403.

4.14. (±)-2-(1-Methyl-2-pyrrolidinyl)ethyl 3-chlorobenzoate (9)

Prepared

as

described

above

in

general

procedure

(Section

4.4).

( )-2-(1-methyl-2-pyrrolidinyl)ethanol (3.7 mmol, 0.5 mL) was added to a solution of 3-chlorobenzoyl

±

chloride (3.7 mmol, 0.47 mL); Yield 680 mg (69.0%); m.p. 144.7–146.2 ^◦C; IR (cm⁻¹) 2916 (stretch. C-H

aliph.), 1724 (stretching COO), 1268 (stretching C-O), 1074 (stretching C-N), 750 (bending C-H Arom.);

¹H-NMR (400 MHz, D₂O)

δ

7.85 (d, J = 11.5 Hz, 2H), 7.64 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 7.9 Hz, 1H),

4.47 (m, 2H), 3.74 (m, 1H), 3.54 (m, 1H), 3.21 (m, 1H), 2.99 (s, 3H), 2.47 (m, 2H), 2.14 (m, 3H), 1.89 (m,

1H); ¹³C-NMR (101 MHz, DMSO-d₆)

δ: 164.4, 133.5, 133.3, 131.6, 130.8, 128.7, 127.9, 65.2, 62.4, 54.7, 38.1,

29.2, 28.6, 21.0; HRMS m/z calcd. for C₁₄H₁₈ClNO₂(M⁺), 268.1108; found, 268.1114.

4.15. (±)-2-(1-Methyl-2-pyrrolidinyl)ethyl 3-bromobenzoate (10)

Prepared

as

described

above

in

general

procedure

(Section

4.4).

( )-2-(1-methyl-2-pyrrolidinyl)ethanol (3.7 mmol, 0.5 mL) was added to a solution of 3-bromobenzoyl

±

chloride (3.7 mmol, 0.49 mL); Yield 690 mg (60.1%); m.p. 105.3–107.1 ^◦C; IR (cm⁻¹) 2957 (stretch. C-H

aliph.), 1716 (stretching COO), 1264 (stretching C-O), 1110 (stretching C-N), 748 (bending C-H Arom.);

¹H-NMR (400 MHz, D₂O)

δ: 8.03 (s, 1H), 7.89 (d, J = 7.9 Hz, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 7.9

Hz, 1H), 4.45 (m, 2H), 3.72 (m, 1H), 3.50 (m, 1H), 3.19 (m, 1H), 2.98 (s, 3H), 2.45 (m, 2H), 2.12 (m, 3H),

1.86 (m, 1H); ¹³C-NMR (101 MHz, DMSO-d₆)

δ: 164.3, 136.2, 131.7, 131.6, 131.1, 128.3, 121.9, 65.2, 62.4,

57.6, 38.2, 29.3, 28.7, 21.0; HRMS m/z calcd. for C₁₄H₁₈BrNO₂(M⁺), 312.0598; found, 312.0610.

4.16. (±)-2-(1-Methyl-2-pyrrolidinyl)ethyl 2-methylbenzoate (11)

Prepared

as

described

above

in

general

procedure

(Section

4.4).

( )-2-(1-methyl-2-pyrrolidinyl)ethanol (3.7 mmol, 0.5 mL) was added to a solution of 2-methylbenzoyl

±

chloride (3.7 mmol, 0.48 mL); Yield 663 mg (73.1%); m.p. 116.1–118.0 ^◦C; IR (cm⁻¹) 2965 (stretch. C-H

aliph.), 1727 (stretching COO), 1240 (stretching C-O), 1091 (stretching C-N), 740 (bending C-H Arom.);

¹H-NMR (400 MHz, D₂O)

δ: 7.86 (d, J = 7.8 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 7.36 (dd, J = 13.0, 7.4 Hz,

2H), 4.43 (m, 2H), 3.69 (dt, J = 12.3, 6.7 Hz, 1H), 3.49 (ddt, J = 13.5, 10.1, 4.3 Hz, 1H), 3.20 (dd, J = 20.0, J

= 8.5 Hz, 1H), 2.94 (s, 3H), 2.53 (s, 3H), 2.46 (m, 2H), 2.10 (m, 3H), 1.88 (m, 1H); ¹³C-NMR (101 MHz,

DMSO-d₆) δ: 166.6, 139.2, 132.24, 131.6, 130.2, 129.2, 126.0, 65.24, 61.7, 54.7, 40.1, 38.0, 29.2, 28.7, 21.1;

HRMS m/z calcd. for C₁₅H₂₁NO₂(M⁺), 248.1648; found, 248.1650.

4.17. (±)-2-(1-Methyl-2-pyrrolidinyl)ethyl 4-methylbenzoate (12)

Prepared

)-2-(1-methyl-2-pyrrolidinyl)ethanol (3.7 mmol, 0.5 mL) was added to a solution of 4-methylbenzoyl

chloride (3.7 mmol, 0.49 mL); Yield 650 mg (71.4%); m.p. 174.4–175.6 ^◦C; IR (cm⁻¹) 2961 (stretch. C-H

as

described

above

in

general

procedure

(Section

4.4).

(±

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aliph.), 1721 (stretching COO), 1284 (stretching C-O), 1118 (stretching C-N), 757 (bending C-H Arom.);

¹H-NMR (400 MHz, D₂O)

: 7.93 (d, J = 8.2 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 4.46 (m, 2H), 3.74 (m, 1H),

3.52 (m, 1H), 3.19 (dt, J = 8.7 Hz, 1H), 2.98 (s, 3H), 2.48 (m, 2H), 2.43 (s, 3H), 2.12 (m, 3H), 1.90 (m, 1H);

δ

¹³C-NMR (101 MHz, DMSO-d₆)

δ: 165.6, 143.8, 129.3 (2C), 129.3 (2C), 126.8, 65.4, 61.7, 54.7, 38.1, 29.3,

28.8, 21.1, 21.0; HRMS m/z calcd. for C₁₅H₂₁NO₂(M⁺), 248.1648; found, 248.1659.

4.18. (S)-(1-Methyl-2-pyrrolidinyl)methyl 2-(4-(triﬂuoromethyl)phenyl)acetate (13)

Prepared

as

described

above

in

general

procedure

(Section

4.3).

(S)-(-)-(1-methyl-2-pyrrolidinyl)methanol (2.45 mmol, 0.29 mL) was added to a solution of

4-triﬂuoromethylphenylacetyl chloride (2.45 mmol, 0.5 gr); Yield 205 mg (27.9%); liquid brown; IR

(cm⁻¹) 2905 (stretch. C-H aliph.), 1740 (stretching COO), 1235 (stretching C-O), 1120 (stretching C-N),

810 (bending C-H Arom.); ¹H-NMR (400 MHz, D₂O)

δ: 7.74 (d, J = 8.1 Hz, 2H), 7.53 (d, J = 8.0 Hz, 2H),

4.54 (dd, J = 12.9, J = 2.4 Hz, 1H), 4.34 (dd, J = 12.9, J = 6.6 Hz, 1H), 3.94 (m, 2H), 3.77 (dd, J = 15.6, 6.6

Hz, 1H), 3.65 (m, 1H), 3.17 (m, 1H), 2.85 (s, 3H), 2.31 (m, 1H), 2.15 (m, 1H), 1.91 (m, 2H); ¹³C-NMR (101

MHz, DMSO-d₆) δ: 171.3, 135.1, 129.4, 129.1 (2C), 127.5 (2C), 124.6, 69.3, 63.3, 58.7, 54.9, 39.7, 26.1, 21.7.

4.19. (S)-(1-Methyl-2-pyrrolidinyl)methyl 2-(2,4-dichlorophenyl)acetate (14)

Prepared as described above in general procedure

(Section

4.3).

(S)-(-)-(1-methyl-2-pyrrolidinyl)methanol (4.2 mmol, 0.5 mL) was added to a solution of

2,4-chlorophenylacetyl chloride (4.2 mmol, 0.51 gr); Yield 339 mg (26.7%); m.p. 179.1–180.5 ^◦C; IR

(cm⁻¹) 2970 (stretch. C-H aliph.), 1741 (stretching COO), 1231 (stretching C-O), 1049 (stretching C-N),

846 (bending C-H Arom.), 745 (bending C-H Arom.); ¹H-NMR (400 MHz, D₂O)

δ: 7.55 (d, J = 7.7 Hz,

1H), 7.35 (m, 2H), 4.55 (dd, J = 13.0, J = 1.9 Hz, 1H), 4.36 (dd, J = 12.9, J = 6.6 Hz, 1H), 4.03 (s, 2H), 3.75

(m, 1H), 3.62 (m, 1H), 3.18 (m, 1H), 2.85 (s, 3H), 2.31 (m, 1H), 2.14 (m, 1H), 1.92 (m, 2H); ¹³C-NMR (101

MHz, DMSO-d₆) δ: 171.2, 135.2, 134.1, 130.5, 129.3, 127.9, 127.0, 68.6, 64.0, 58.9, 52.1, 38.1, 28.6, 21.4;

HRMS m/z calcd. for C₁₄H₁₇Cl₂NO₂(M⁺), 302.0663; found, 302.0686.

4.20. (S)-(1-Methyl-2-pyrrolidinyl)methyl 2-(3,4-dichlorophenyl)acetate (15)

Prepared

as

described

above

in

general

procedure

(Section

4.3).

(S)-(-)-(1-methyl-2-pyrrolidinyl)methanol (4.2 mmol, 0.5 mL) was added to a solution of

3,4-chlorophenylacetyl chloride (4.2 mmol, 0.51 gr); Yield 339 mg (26.7%); m.p. 183.5–184.8 ^◦C; IR

(cm⁻¹) 2974 (stretch. C-H aliph.), 1739 (stretching COO), 1239 (stretching C-O), 1043 (stretching C-N),

832 (bending C-H Arom.), 741 (bending C-H Arom.); ¹H-NMR (400 MHz, D₂O)

δ: 7.48 (d, J = 7.2 Hz,

2H), 7.21 (d, J = 8.3 Hz, 1H), 4.50 (dd, J = 12.9, J = 2.5 Hz, 1H), 4.32 (dd, J = 12.9, J = 6.5 Hz, 1H), 3.80 (s,

2H), 3.71 (m, 2H), 3.20 (dt, J = 16.0, 8.2 Hz, 1H), 2.88 (s, 3H), 2.29 (m, 1H), 2.15 (m, 1H), 1.92 (m, 2H);

¹³C-NMR (101 MHz, DMSO-d₆)

δ: 170.5, 136.6, 132.1, 130.7, 130.2, 129.5, 129.1, 66.4, 63.5, 54.9, 51.4,

38.8, 28.9, 21.7; HRMS m/z calcd. for C₁₄H₁₇Cl₂NO₂(M⁺), 302.0663; found, 302.0697.

4.21. (±)-2-(-1-Methyl-2-pyrrolidinyl)ethyl 2-phenylacetate (16)

Prepared

as

described

above

in

general

procedure

(Section

4.5).

( )-2-(1-methyl-2-pyrrolidinyl)ethanol (4.1 mmol, 0.56 mL) was added to a solution of phenylacetyl

±

chloride (4.1 mmol, 0.5 gr); Yield 355 mg (35.0%); m.p. 86.7–87.4 ^◦C; IR (cm⁻¹) 2961 (stretch. C-H

aliph.), 1717 (stretching COO), 1280 (stretching C-O), 1115 (stretching C-N), 716 (bending C-H Arom.);

¹H-NMR (400 MHz, D₂O)

δ

8.04 (d, J = 7.6 Hz, 2H), 7.72 (t, J = 7.5 Hz, 1H), 7.57 (t, J = 7.8 Hz, 2H), 4.48

(m, 2H), 3.74 (m, 2H), 3.53 (td, J = 5.2, 2.5 Hz, 1H), 3.45 (m, 1H), 3.18 (dt, J = 18.6, 9.1 Hz, 1H), 2.98 (s,

3H), 2.49 (m, 2H), 2.12 (m, 3H), 1.88 (m, 1H); ¹³C-RMN (101 MHz, DMSO-d₆)

: 171.3, 135.1, 129.5,

δ

129.4, 129.1, 127.2, 124.6, 69.3, 63.3, 58.7, 39.5, 38.1, 29.3, 28.7, 21.0; HRMS m/z calcd. for C₁₅H₂₁NO₂

(M⁺), 248.1648; found, 248.1667.

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4.22. (±)-2-(-1-Methyl-2-pyrrolidinyl)ethyl 2-(3,4-dichlorophenyl)acetate (17)

Prepared

as

described

above

in

general

procedure

(Section

4.5).

( )-2-(1-methyl-2-pyrrolidinyl)ethanol (2.4 mmol, 0.33 mL) was added to a solution of

±

3,4-dichlorophenylacetyl chloride (2.4 mmol, 0.5 gr); Yield 154 mg (20.0%); m.p. 148.8–150.4

^◦C; IR (cm⁻¹) 2977 (stretch. C-H aliph.), 1732 (stretching COO), 1235 (stretching C-O), 1034 (stretching

C-N), 828 (bending C-H Arom.), 736 (bending C-H Arom.); ¹H-NMR (400 MHz, D₂O)

δ 7.41 (t, J = 5.5

Hz, 2H), 7.17 (dd, J = 8.3, J = 1.6 Hz, 1H), 4.21 (m, 2H), 3.68 (d, J = 6.6 Hz, 3H), 3.23 (m, 1H), 3.10 (d, J =

9.9 Hz, 1H), 2.86 (s, 3H), 2.30 (m, 1H), 2.20 (m, 1H), 2.03 (m, 2H), 1.90 (m, 1H), 1.71 (m, 1H); ¹³C-NMR

(101 MHz, DMSO-d₆) δ: 170.2, 135.2, 131.4, 130.5, 130.2, 129.9, 129.4, 65.1, 61.4, 54.5, 38.6, 37.9, 29.0,

28.4, 20.8; HRMS m/z calcd. for C₁₅H₁₉Cl₂NO₂(M⁺), 316.0868; found, 316.0896.

4.23. (S)-(1-Methyl-2-pyrrolidinyl)methyl 2-naphthoate (18)

Prepared

as

described

above

in

general

procedure

(Section

4.2).

(S)-(-)-(1-methyl-2-pyrrolidinyl)methanol (4.2 mmol, 0.5 mL) was added to a solution of 2-naphthoyl

chloride (4.2 mmol, 0.80 gr); Yield 735 mg (65%); m.p. 125.5–127.1 ^◦C; IR (cm⁻¹) 3057 (stretch. C-H

aliph.), 1748 (stretching COO), 1260 (stretching C-O), 1123 (stretching C-N), 757 (bending C-H Arom.);

¹H-NMR (400 MHz, DMSO-d₆)

δ: 8.61 (s, 1H), 8.16 (m, 1H), 8.01 (m, 3H), 7.65 (dt, J = 13.61, 6.68, 6.68

Hz, 2H), 4.69 (m, 2H), 3.85 (m, 1H), 3.03 (m, 2H), 2.83 (s, 3H), 2.07 (m, 1H), 1.96 (m, 1H), 1.85 (m, 1H),

1.72 (m, 1H); ¹³C-NMR (101 MHz, DMSO-d₆)

δ: 167.4, 134.9, 132.2, 130.5, 129.3, 128.3, 128.2, 128.1,

127.7, 126.8, 125.2, 69.4, 59.1, 55.9, 39.5, 26.0, 21.8; HRMS m/z calcd. for C₁₇H₁₉NO₂(M⁺), 270.1498;

found, 270.1517.

4.24. (±)-2-(1-Methyl-2-pyrrolidinyl)ethyl 2-naphthoate (19)

Prepared

as

described

above

in

general

procedure

(Section

4.4).

( )-2-(1-methyl-2-pyrrolidinyl)ethanol (3.7 mmol, 0.5 mL) was added to a solution of 2-naphthoyl

±

chloride (3.7 mmol, 0.7 gr); Yield 637 mg (61.1%); m.p. 155.6–157.4 ^◦C; IR (cm⁻¹) 2948 (stretch. C-H

aliph.), 1727 (stretching COO), 1288 (stretching C-O), 1200 (stretching C-N), 781 (bending C-H Arom.);

¹H-NMR (400 MHz, D₂O)

δ

: 8.67 (s, 1H), 8.16 (d, J = 7.71 Hz, 1H), 8.03 (m, 3H), 7.66 (dt, J = 21.77, 7.17,

7.17 Hz, 2H), 4.45 (m, 2H), 3.55 (dt, J = 13.54, 6.59, 6.59 Hz, 1H), 3.46 (m, 1H), 3.01 (m, 1H), 2.82 (s, 3H),

2.33 (m, 1H), 2.18 (m, 1H), 1.99 (m, 3H), 1.79 (m, 1H); ¹³C-NMR (101 MHz, DMSO-d₆)

: 165.7, 135.1,

δ

132.1, 130.7, 129.4, 128.7, 128.4, 127.7, 127.0, 126.8, 124.8, 65.4, 62.1, 57.6, 38.1, 29.3, 28.8, 21.0; HRMS m/z

calcd. for C₁₈H₂₁NO₂(M⁺), 284.1648; found, 284.1677.

4.25. (S)-(1-Methyl-2-pyrrolidinyl)methyl 2,2-diphenylacetate (20)

Prepared

)-(-)-(1-methyl-2-pyrrolidinyl)methanol (2.2 mmol, 0.26 mL) was added to a solution of

2,2-diphenylacetyl chloride (2.2 mmol, 0.5 gr); Yield 293 mg (43.1%); m.p. 121.5–122.3 ^◦C; IR (cm⁻¹

2991 (stretch. C-H aliph.), 1742 (stretching COO), 1200 (stretching C-O), 1063 (stretching C-N), 827

(bending C-H Arom.), 736 (bending C-H Arom.); ¹H-NMR (400 MHz, D₂O)

: 7.39 (m, 10H), 5.34 (s,

as

described

above

in

general

procedure

(Section

4.3).

(S

)

δ

1H), 4.54 (dd, J = 12.9, J = 2.6 Hz, 1H), 4.36 (dd, J = 12.9, J = 7.1 Hz, 1H), 3.62 (td, J = 10.4, 8.8, 4.2 Hz,

1H), 3.52 (dt, J = 11.9, 6.2 Hz, 1H), 3.06 (dt, J = 11.2, 8.1 Hz, 1H), 2.64 (s, 3H), 2.23 (m, 1H), 2.05 (m, 1H),

1.84 (m, 1H), 1.73 (m, 1H); ¹³C-NMR (101 MHz, DMSO-d₆)

δ: 171.9, 139.0 (2C), 129.5 (4C), 129.3 (4C),

127.5 (2C), 69.9, 63.7, 54.2, 52.8, 37.2, 26.8, 20.5; HRMS m/z calcd. for C₂₀H₂₃NO₂(M⁺), 310.1761; found,

310.1795.

4.26. (±)-2-(-1-Methyl-2-pyrrolidinyl)ethyl 2,2-diphenylacetate (21)

Prepared

)-2-(1-methyl-2-pyrrolidinyl)ethanol (2.2 mmol, 0.3 mL) was added to a solution of 2,2-diphenylacetyl

chloride (2.17 mmol, 0.5 gr); Yield 217 mg (31.0%); m.p. 115.3–117.2 ^◦C; IR (cm⁻¹) 2984 (stretch. C-H

as

described

above

in

general

procedure

(Section

4.4).

(±

Molecules 2019, 24, 3808

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aliph.), 1735 (stretching COO), 1195 (stretching C-O), 1151 (stretching C-N), 744 (bending C-H Arom.),

700 (bending C-H Arom.); ¹H-NMR (400 MHz, D₂O)

: 7.28 (m, 10H), 5.16 (s, 1H), 4.23 (m, 2H), 3.57

(dt, J = 12.5, 6.6 Hz, 1H), 2.93 (m, 1H), 2.86 (m, 1H), 2.65 (s, 3H), 2.19 (m, 1H), 1.98 (m, 1H), 1.88 (m,

δ

2H), 1.79 (m, 1H), 1.53 (dq, J = 12.4, 8.5 Hz, 1H); ¹³C-NMR (101 MHz, DMSO-d₆)

δ: 171.6, 138.6 (2C),

128.3 (8C), 126.9 (2C), 65.0, 61.7, 55.5, 54.4, 37.8, 28.9, 28.3, 20.7; HRMS m/z calcd. for C₂₁H₂₅NO₂(M⁺),

324.1968; found, 324.1998.

5. Conclusions

Radioligand binding assays supported with docking studies were carried out with a series of

novel pyrrolidine ester derivatives in

on h-SERT cells gave no aﬃnity for any synthesized compounds in the central site of h-SERT [49].

However, compounds 10

18 and 19 produced an increase in the binding of [³H]-paroxetine,

α4β2 nAChR, h-DAT and h-SERT. The binding assays performed

2,

4,

8,

,

which may be interpreted as an allosteric h-SERT modulation. This anomalous behavior could be

assigned to a steric hindrance eﬀect on the radioligand, induced by an ionic bond and hydrophobic

interactions among our compounds with Glu 494 and Phe 335 residues in the allosteric binding site of

h-SERT, avoiding the radioligand leaving of the central site [49]. This is interesting since suggest that

the aforementioned compounds could increase the eﬃcacy of SSRIs if used in conjunction.

On the other hand, compound 20 displays the desired proﬁle, positioning it as the unique

compound exhibiting triple aﬃnity of the series. In SERT assays, compound 20 induced the highest

[³H]-paroxetine binding, a result supported by docking studies which revealed the interaction of both

phenyl groups with Phe 335 and Phe 556 residues on the allosteric binding site of h-SERT. In the same

context, compound 20 showed h-DAT interaction between one of the phenyl groups with Tyr 548 at the

central binding site, reinforcing its aﬃnity. Docking studies of compound 20 at α4β2 nAChR, showed

that one of the phenyl group adopted a conformation ﬁtting the space between Phe 119 and Trp 182

residues. On the other hand, the high h-DAT aﬃnity obtained for compound 21 can be explained

considering the interactions unveiled by docking studies. In this case, both phenyl groups of 21

interacted with Tyr 470 residue. Regarding α4β2 nAChR aﬃnity, the π–cation interactions with Tyr 230

residue and the hydrophobic interaction with Phe 119 residue probably favored the aﬃnity. Based on

these results, the presence of two geminal phenyl groups at the alfa-position in ester derivatives

resulted fundamental to obtain compounds with enhanced aﬃnity.

Finally, this extended study conducted to promissory results in the design and ﬁnding of novel

multi-ligands simultaneously acting at diﬀerent targets.

The rational design of multi-target compounds is far from being an easy task, dealing with the

crucial issues of selecting the right target combination, achieving a balanced activity towards them,

and excluding activity at the undesired target(s), while at the same time retaining drug-like properties.

Supplementary Materials: The following are available online, Figure S1: Synthesis of benzoate derivatives (

Figure S2: Synthesis of 2-phenylacetate derivatives (13 17), Figure S3: Synthesis of 2-naphthoate derivatives

(18–19) and Figure S4: Synthesis of 2,2-diphenylacetate derivatives (20–21).

1–12),

–

Author Contributions: Investigation, J.P.G.-G., H.A.P.-M. and P.E.I.-V.; Performed the synthesis of compounds,

J.P.G.-G. and N.E.G.-D.; Project Administration and Funding Acquisition, P.E.I.-V., H.A.P.-M. and M.I.R.-P.;

Performed the competitive binding assay of the compounds, M.H. and J.P.G.G; Analyzed the data, J.P.G.-G., P.E.I.-V.

and H.A.P.-M; Performed docking studies, G.N.-V., J.M.-R., F.V., P.P. and M.A.L.-C; Performed HRMS m/z, M.C.Z.;

Wrote the paper; J.P.G.-G, P.E.I.-V., M.I.R.-P. and H.A.P.-M. All authors read and approved the ﬁnal manuscript.

Funding: The ﬁnancial support for this research work, awarded by the National Foundation of Science and

Technology grants 1,150,615 (P.I.-V.), 1,170,662 (M.R.-P.) and 1,170,269 (H.P.-M.), is gratefully acknowledged.

Acknowledgments: The authors want to dedicate this work in memory of our colleague Vicente Castro Castillo^†.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Molecules 2019, 24, 3808

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Sample Availability: Samples of the compounds are available from the authors.

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Synthesis of novel nicotinic ligands with multimodal action: Targeting Acetylcholine α4β2, Dopamine and Serotonin Transporters

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