QSAR and molecular docking studies of the inhibitory activity of novel heterocyclic GABA analogues over GABA-AT

Article abstract of DOI:10.3390/molecules23112984

We have previously reported the synthesis, in vitro and in silico activities of new GABA analogues as inhibitors of the GABA-AT enzyme from Pseudomonas fluorescens, where the nitrogen atom at the γ-position is embedded in heterocyclic scaffolds. With the goal of finding more potent inhibitors, we now report the synthesis of a new set of GABA analogues with a broader variation of heterocyclic scaffolds at the γ-position such as thiazolidines, methyl-substituted piperidines, morpholine and thiomorpholine and determined their inhibitory potential over the GABA-AT enzyme from Pseudomonas fluorescens. These structural modifications led to compound 9b which showed a 73% inhibition against this enzyme. In vivo studies with PTZ-induced seizures on male CD1 mice show that compound 9b has a neuroprotective effect at a 0.50 mmole/kg dose. A QSAR study was carried out to find the molecular descriptors associated with the structural changes in the GABA scaffold to explain their inhibitory activity against GABA-AT. Employing 3D molecular descriptors allowed us to propose the GABA analogues enantiomeric active form. To evaluate the interaction with Pseudomonas fluorescens and human GABA-AT by molecular docking, the constructions of homology models was carried out. From these calculations, 9b showed a strong interaction with both GABA-AT enzymes in agreement with experimental results and the QSAR model, which indicates that bulky ligands tend to be the better inhibitors especially those with a sulfur atom on their structure.

Full text of DOI:10.3390/molecules23112984

molecules
Article
QSAR and Molecular Docking Studies of the
Inhibitory Activity of Novel Heterocyclic GABA
Analogues over GABA-AT
Josué Rodríguez-Lozada 1 , Erika Tovar-Gudiño 1, Juan Alberto Guevara-Salazar 2,
Rodrigo Said Razo-Hernández 3, Ángel Santiago 3 , Nina Pastor 3 and
Mario Fernández-Zertuche 1,
*
1
Instituto de Investigación en Ciencias Básicas y Aplicadas, Centro de Investigaciones Químicas,
Universidad Autónoma del Estado de Morelos, 62209 Cuernavaca, Morelos, Mexico;
jrl@uaem.mx (J.R.-L.); egt@uaem.mx (E.T.-G.)
Departmento de Bioquímica, Escuela Superior de Medicina, Instituto Politécnico Nacional,
11340 Cd México, Mexico; jguevaras@ipn.mx
Instituto de Investigación en Ciencias Básicas y Aplicadas, Centro de Investigación en Dinámica Celular,
Universidad Autónoma del Estado de Morelos, 62209 Cuernavaca, Morelos, Mexico;
rodrigo.razo@uaem.mx (R.S.R.-H.); jast@uaem.mx (Á.S.); nina@uaem.mx (N.P.)
Correspondence: mfz@uaem.mx; Tel.: +52-777-329-7997
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 12 September 2018; Accepted: 9 November 2018; Published: 15 November 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: We have previously reported the synthesis, in vitro and in silico activities of new GABA
analogues as inhibitors of the GABA-AT enzyme from Pseudomonas fluorescens, where the nitrogen
atom at the
inhibitors, we now report the synthesis of a new set of GABA analogues with a broader variation
of heterocyclic scaffolds at the -position such as thiazolidines, methyl-substituted piperidines,
γ-position is embedded in heterocyclic scaffolds. With the goal of finding more potent
γ
morpholine and thiomorpholine and determined their inhibitory potential over the GABA-AT enzyme
from Pseudomonas fluorescens. These structural modifications led to compound 9b which showed a
73% inhibition against this enzyme. In vivo studies with PTZ-induced seizures on male CD1 mice
show that compound 9b has a neuroprotective effect at a 0.50 mmole/kg dose. A QSAR study was
carried out to find the molecular descriptors associated with the structural changes in the GABA
scaffold to explain their inhibitory activity against GABA-AT. Employing 3D molecular descriptors
allowed us to propose the GABA analogues enantiomeric active form. To evaluate the interaction with
Pseudomonas fluorescens and human GABA-AT by molecular docking, the constructions of homology
models was carried out. From these calculations, 9b showed a strong interaction with both GABA-AT
enzymes in agreement with experimental results and the QSAR model, which indicates that bulky
ligands tend to be the better inhibitors especially those with a sulfur atom on their structure.
Keywords: heterocyclic GABA analogues; GABA-AT enzyme; QSAR; GABA-AT docking; inhibitors
1. Introduction
There are two major neurotransmitters in the regulation of neuronal activity in the mammalian
central nervous system,
inhibitory neurotransmitter and the latter an excitatory neurotransmitter [1,2
γ
-aminobutyric acid (GABA) and L-glutamic acid. The former one is an
]. GABA is synthesized via
decarboxylation of L-glutamic acid by a process catalyzed by the enzyme glutamic acid decarboxylase
(GAD) and metabolized by a transamination reaction catalyzed by the enzyme GABA-aminotransferase
(GABA-AT) [3,4].
Molecules 2018, 23, 2984; doi:10.3390/molecules23112984
www.mdpi.com/journal/molecules

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A decrease in GABA levels has been associated with a large group of psychiatric and neurological
disorders such as epilepsy [
among others.
5], Parkinson’s [6], Alzheimer’s [7] and Huntington [8] diseases
GABA levels cannot be increased by direct administration of this compound due to its low
lipophilic character, a condition that diminishes its capacity to cross the blood-brain barrier (BBB) [ ].
9
As a result, there has been an intensive research activity oriented toward the design and synthesis of
new GABA analogues with increased lipophilic character, capable of crossing the blood-brain barrier,
for the treatment of the neurological disorders listed above.
In this context, two representative GABA analogues used in the clinic are (S)-pregabalin and
(R)-baclofen. (S)-Pregabalin is an anticonvulsant, anxiolytic and analgesic drug believed to bind to the
α
2–
and peptide modulators that may be responsible for these effects [10
agonist used on the treatment of spasticity, motility improvement of patients with multiple sclerosis,
spinal cord injuries and muscle stiffness [15 16]. Due to their clinical importance, the synthesis and
biological evaluation of analogues of these compounds, have been reported in the literature where
several research groups have reported an impressive variety of modifications to their structures [17 24].
δ
protein, a voltage dependent calcium channel, reducing the release of excitatory neurotransmitters
–
14]. (R)-Baclofen is a GABAB
,
–
Another alternative to raise GABA concentration in the brain has been the design of GABA
analogues with the potential to inactivate the GABA-AT enzyme, a pyridoxal 5’-phosphate
(PLP)-dependent enzyme responsible for the degradation of this inhibitory neurotransmitter [25].
In this regard, vigabatrin (VGB) is the first GABA-AT inhibitor drug approved by the FDA for
the treatment of epilepsy. VGB is a very selective and irreversible GABA-AT inhibitor raising the
concentration of GABA on the brain [26–28].
Silverman has designed and evaluated a variety of GABA analogues as GABA-AT inhibitors [29–32]
,
including CP-115, a compound 186 times more efficient in inactivating GABA-AT than VGB and with
a high therapeutic potential for the treatment of epilepsies and cocaine addiction [33]. More recently,
the Silverman group has reported a cyclopnet-1-ene derivative 10 times more efficient than VGB as a
GABA-AT inactivator. In vivo studies show that this compound suppresses the release of dopamine
after a cocaine or nicotine dose in rats [34].
The synthesis of bicyclic analogues of GABA has been reported by Conti [35]. There are also
reports in the literature of GABA-AT inhibitors not related to the GABA framework such as sodium
valproate (VPNa), a well-known antiepileptic compound whose mode of action has been attributed to
an inhibition of the GABA-AT enzyme [36]. More recently, Siddiqui has reported the synthesis, in vitro
GABA-AT screening and anticonvulsant activity of some dihydropyrimidine carbothioamides [37].
In the design of other type of GABA-AT inhibitors via computational methods, Bansal has
designed novel GABA-AT inhibitors based on a molecular field analysis kNN-MFA 3DQSAR
model for phenyl-substituted
-phenylethylidene hydrazide GABA analogues with a variety of substituents at the phenyl ring by
QSAR techniques [40]. On the other hand, Abdulfatai has reported QSAR models for the prediction of
biological activities of quinoxaline and thiadiazoles derivatives as GABA-AT inhibitors [41 42] and
β-phenylethylidene hydrazine analogues [38,39]. Davood, identified
β
,
Jain studied the effect of halogen substitution on the docking and 3DQSAR properties of a series of
aryl-substituted thiosemicarbazones [43].
Our research group has previously reported the synthesis and biological activity of new GABA
analogues in which the nitrogen atom is part of a heterocyclic system. Specifically, the inclusion of the
nitrogen atom in a pyrrolidine or indoline ring system lead to GABA analogues such as 1–6 (Figure 1).
In this work, 3 and 4 had inhibitory activity over the GABA-AT enzyme [44].

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Figure 1. Pyrrolidine and indoline GABA analogues.
We have evaluated the effect of other structural modifications, we present in this work the
synthesis, in vitro and in silico evaluation of a new set of heterocyclic GABA analogues,
(Figure 2). These analogues maintain the GABA backbone where the nitrogen atom at the
7
,
8
and
9
γ
-position
forms part of heterocyclic ring system. The thiazolidine ring systems were designed to evaluate the
effect of a second heteroatom in a five-membered ring, piperidine and methyl substituted piperidines to
evaluate ring size and the morpholine and thiomorpholine systems to evaluate the effect of heteroatoms
in these six-membered ring systems. Piperidine-derived GABA analogues with a phenyl group at C-3
of the GABA backbone with vasoconstrictor properties have been reported in the literature [45].
Figure 2. Structures of GABA analogues 7, 8 and 9.
Analogues
7
contain the GABA framework with-no substituents at the
α
- or
β-positions. However,
analogues and
8
9
include an iso-butyl or p-Cl-phenyl substituents at the β-position in analogy to
(S)-pregabalin and (R)-baclofen in order to improve their lipophilic character. Contrary to what
happens in the case of GABA analogues with primary amine groups that allow them to react with
the PLP cofactor of the GABA-AT enzyme, the inclusion of the nitrogen atom in these non-aromatic
heterocyclic system makes them tertiary amines with higher basicity (pKb ~ 4.2). This increased
basicity favors the transfer of the acidic carboxylic proton to the nitrogen atoms generating zwitterionic
species which may facilitate their interaction in a non-covalent manner, with the GABA-AT enzyme.
We also report the in vitro inhibitory potential over the GABA-AT enzyme from Pseudomonas fluorescens
as well as docking and Quantitative Structure-Activity Relationship (QSAR) studies to explain their
GABA-AT inhibitory activity. QSAR analysis was used to propose which enantiomer has the greatest
probability to be the biological active form, for this reason 3D molecular descriptors were used.
Docking calculations help us to corroborate the QSAR hypothesis and study the interaction form
of GABA analogues with GABA-AT. In addition, we also carried out a molecular docking study

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over a homology modeled human GABA-AT enzyme to identify compounds as potential candidates
for future in vivo studies. All compounds comply with Lipinski’s rule of five thus predicting their
probability to become an oral drug [46].
2. Results
2.1. Chemistry
The synthesis of the GABA analogues 7 is shown in Table 1. Although compounds 7b, 7d, 7e and 7f
are commercially available and their use in synthesis has been reported in the literature [47–49], they were
prepared by our previously reported N-alkylation and a hydrolysis sequence [44]. The N-alkylation
reaction of 10a–f with methyl-4-bromobutanoate (11) under solvent free conditions, was carried out
at temperatures ranging between 65–70 ◦C affording esters 12 with moderate yields (58–67%). In a
subsequent step, esters 12 were subjected to a basic hydrolysis with lithium hydroxide in aqueous
methanol to afford analogues 7 with excellent yields
Table 1. Synthesis of analogues 7.
Compound
7a
Heterocycle
% Yield 12
% Yield 7
37
81
7b
7c
67
60
95
95
7d
7e
7f
67
52
58
95
98
86
For the synthesis of the
β-substituted analogues
8
and
9, it was required to prepare the
α
,
β-unsaturated esters 14. As described previously, this was accomplished by the N-alkylation
of heterocycles 10a–f with commercially available 13 (Table 2).

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Table 2. Synthesis of conjugated esters 14.
Compound
Heterocycle
% Yield 14
10a
10b
10c
63
90
77
10d
10e
80
89
10f
80
The synthesis of the β-substituted analogues 8a–f and 9a–f are shown in Tables 3 and 4 respectively,
and involve the conjugate addition of cuprates 15 or 17. As can be seen on Table 3, the addition of
◦
cuprate
conjugated systems 14a
in yields ranging from 13 to 75%. The low yields reported in entries a, c and f are attributed
to decomposition of the corresponding heterocycles. Basic hydrolysis of esters 16, to afford the
corresponding -substituted i-butyl analogues was achieved with lithium hydroxide in aqueous
methanol; the yields in this step were from moderate to very good.
Table 4 describes the synthesis of the -aryl-substitutued analogues
generated from bromochlorobenzene under the same reaction conditions, to the
esters 14a afforded the -aryl-substituted esters 18 in moderate yields (entries b–f). In the case
7
, generated from i-butyl magnesium bromide and anhydrous CuI in ether at 0 C, to the
–f
at 0 ◦C (entries a–f), affords the corresponding i-butyl substituted esters
8
β
8
β
9
. Addition of cuprate 17
,
α,β-unsaturated
–
f
β
of the thiazolidine ring system (entry a), it was not possible to obtain the corresponding ester, as
decomposition of 14b was observed under different reaction conditions. Once again, basic hydrolysis
of esters 18, carried out with lithium hydroxide in aqueous methanol led to the
9a from moderate to very good yields. The -substituted analogues in Tables 3 and 4 were
synthesized in racemic form in order to quickly identify inhibitory activity over the GABA-AT enzyme.
β-substituted analogues
–
f
β

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Table 3. Synthesis of the β-substituted analogues 8.
Entries
a
Compound
8a
Heterocycle
% Yield 16
% Yield 8
13
77
b
c
8b
8c
52
20
90
70
d
e
f
8d
8e
8f
37
37
25
90
85
90
Table 4. Synthesis of the β-substituted analogues 9.
Entries
a
Compound
9a
Heterocycle
% Yield 18
% Yield 9
–
–
b
c
9b
9c
52
49
85
52
d
e
f
9d
9e
9f
49
55
58
68
69
70

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2.2. Enzymatic Inhibition
The enzymatic assay for GABA-AT inhibition is based on the role of this enzyme in the
degradation of GABA to succinic semialdehyde, and its subsequent transformation to succinate.
This involves the concomitant conversion of
dehydrogenase-coupled reaction [26 50 51]. This is evidenced by the absorbance change at 340 nm,
corresponding to the formation of -NADPH from
-NADP+, which is proportional to GABA-AT
activity. Figure 3 shows the inhibition of enzymatic catalytic activity of compounds 7a over the
β β-NADPH by a succinic-semialdehyde
-NADP+ to
,
,
β
β
–
f
GABA-AT enzyme from Pseudomonas fluorescens. The activity of these analogues was measured against
the positive controls VGB and VPNa at a 0.8 mM concentration. As can be seen in Figure 3, compounds
7a–f were inactive, as their catalytic inhibitory activity is lower than that of the positive controls.
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
Control VGB
VPNa
7a
7b
7c
7d
7e
7f
-10.0
-20.0
Figure 3. GABA-AT % enzymatic activity of compounds 7a–f as compared to VGB and VPNa.
Figure 4 shows the inhibition of enzymatic catalytic activity of compounds 8a–f and 9a–f over
the GABA-AT enzyme from Pseudomonas fluorescens; all compounds were tested as racemic mixtures.
Once again, the inhibitory activity of these analogues was measured against the positive controls VGB
and VPNa at a 0.8 mM concentration. As can be seen in Figure 4, analogues 8a
–e have an enzymatic
inhibitory activity much lower than the positive controls VGB and VPNa. Regarding analogues 9a–f,
the best compound is 9b; compound 9b displays a 73% inhibition over the enzyme, an inhibitory
power greater than that of the control drugs VGB and VPNa.

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90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
-10.0
-20.0
Figure 4. GABA-AT % enzymatic inhibition of compounds 8a–f and 9a–f as compared to VGB
and VPNa.
2.3. In Vivo Studies
The experiments were carried out in two different times of pretreatment with 9b and VPNa;
1 h and 4 h before administering PTZ. The results obtained in these tests consist of the analysis of
the latency at the first seizure, the number of seizures generated in the observation period and the
protection against death. In relation to the experiments carried out at 1 h, the latency of compound 9b
and VPNa with respect to the control, do not show significant difference, that is, at least at the doses
tested there is no change in the generation time of the first seizure (Figure 5).
Dose (mmole/kg)
Figure 5. Latency of compound 9b compared to VPNa at different doses (0.50 and 1.00 mmole/kg) at
1 h and 4 h of pretreatment. * p < 0.05: Significant difference comparing control group with 9b and
VPNa as a positive control group. Comparisons were made by the one-way ANOVA test Duncan’s
means analysis test.

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In the same way, the same type of results was analyzed for the test carried out with 4 h of
pretreatment, in which regarding to latency, it can be observed that there is a slight tendency to
increase the trend with compound 12b, despite that there is no statistically significant difference.
Only at a 1.00 mmole/kg dose of VPNa 4, there was a clear increase in latency (Figure 5).
On the other hand, the number of convulsions that each of the mice presented for each
experimental group were counted, with the purpose of observing the anticonvulsant activity through
the decrease in the number of them. It could be observed that at a 1.00 mmole/kg dose of 9b, the
number of seizures did not decrease, they rather increased significantly. At a 0.50 mmole/kg dose,
there was no significant difference. In the case of the doses tested with VPNa, there was no significant
difference; however, the results show a tendency towards a decrease in the number of seizures
(Figure 6). In the case of the number of seizures generated during the observation time, the results
obtained are quite interesting. First of all, the expected decreased in the number of seizures with VPNa
was observed. However, in the case of compound 9b, there is a very clear decrease in the number of
seizures at a 0.5 mmole/kg dose. On the other hand, at a 1.00 mmole/kg dose, there is no difference
with the control group, that is, compound 16b does not have a protecting effect, at least at the highest
dose against the number of seizures (Figure 6). Table 5 shows some parameters of anticonvulsive
activity of compounds 9b and VPNa in this model.
Dose (mmole/kg)
Figure 6. Tonic-clonic seizures number elicited by compound 9b compared to VPNa at different doses
(0.5 and 1.00 mmole/kg) at 1 h and 4h of pretreatment. * p < 0.05: Significant difference comparing
control group with 9b and VPNa as positive control group. Comparisons were made by the one-way
ANOVA test and Duncan’s means analysis test [52–54].
Table 5. Parameters of anticonvulsive activity of compounds 9b and VPNa on the PTZ-induced seizures
model. * p < 0.05.
Dose (mmole/kg)
Pre-Treatment Time (h)
Latency ± SEM (min)
Number Seizures ± SEM
Control
9b (0.50)
9b (1.00)
VPNa (0.50)
VPNa (1.00)
12.69 ± 2.06
10.27 ± 2.38
9.81 ± 1.67
1.22 ± 0.32
1.16 ± 0.16
3.33 ± 0.84 *
0.50 ± 0.22
0.50 ± 0.22
1
11.00 ± 4.36
11.00 ± 3.06 *
Control
9b (0.50)
9b (1.00)
VPNa (0.50)
VPNa (1.00)
4.24 ± 1.03
11.22 ± 7.89
9.79 ± 1.72
14.00
1.66 ± 0.40
0.33 ± 0.21 *
1.50 ± 0.34
0.16 ± 0.16 *
0.33 ± 0.21 *
4
22.50 ± 3.50

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Finally, regarding death protection, VPNa, at both doses does protect against the lethal action
of PTZ, whereas in the case of compound 9b, there is protection at a 0.5 mmole/kg dose; at a
1.00 mmole/kg dose, the lethal action significantly increases (Figure 7). Finally, neither VPNa nor 9b,
have a significant difference, that is, practically no animals show a lethal effect from the statistical
point of view (Table 5).
Dose (mmole/kg)
Figure 7. Percentage of death protection of compound 9b compared to VPNa at different doses
(0.50 and 1.00 mmole/kg) at 1 h and 4 h of pretreatment. * p < 0.05: Significant difference comparing
control group with 9b and VPNa as positive control group. Comparisons were made by the Fisher
Exact test.
It is very important to mention that compound 9b presents an atypical or non-dose-dependent
behavior, and this occurs especially at the highest dose (1.00 mmole/kg) for the experiments at
1 h and 4 h of pretreatment before the administration of PTZ. Regarding the number of seizures,
9b rather increases them, instead of decreasing, when the pretreatment is carried out during 1 h.
When the pretreatment is carried out during 4 h there is no significant difference with the control
group. However, at a dose of 0.50 mmole/kg there is a clear tendency to decrease the number of
seizures, this phenomenon is probably due to a biphasic response or hormesis that has been reported
in many substances with biological activity [55–59].
2.4. Computational Studies
2.4.1. Conformational and Optimization Geometry
The optimized structures of the most stable conformers of all GABA analogues, including the S
and R enantiomer of
8 and 9 series, are shown in Figure S45 (Supplementary Material). All vibrational
frequencies of these were positive ensuring that all the structures are minima in the potential
energy surface.
2.4.2. QSAR Analysis
Since one of our objectives is to understand the biological mechanism of our compounds,
knowledge of which enantiomer will display the greatest probability to be the biologically active form
was crucial for this study. Therefore, we employed geometrical and charge molecular descriptors for
the construction of the QSAR models; these descriptors are sensitive to the spatial position of the atoms

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in the molecule and allowed us to study both enantiomers of each compound. Therefore, the inclusion
of at least one of these descriptors in the final QSAR model was a requirement. The best mathematical
model that correlates the inhibition percent (Y) of GABA analogues with their molecular descriptors,
was validated by the QUIK, Q2ASYM, redundancy and overfitting rules. This expression, obtained with
the (S)-enantiomers, is the following:
Y = 1624.02548[RCI] − 394.09487[HOMT] + 0.4927[T(O..S)] + 34.48134[TI2] − 99.99144
(1)
R2 = 86.96 Q2LOO = 73.03 s = 4.54 F = 21.3
∆K = 0.003 (0.0) ∆Q = 0.075 (−0.005) RP = 0.356 (0.1) RN = −0.217 (−0.24)
Therefore, the GABA analogues molecular descriptors that are related to GABA-AT inhibition
are: the Jug RC Index and the Harmonic Oscillator Model of Aromaticity index (RCI and HOMT
respectively), which belong to the geometrical descriptors family. Additionally, in this mathematical
model, the sum of the topological distance between the oxygen and sulfur atoms (O..S) and the second
Mohar index (T(O..S) and TI2 respectively) from the topological descriptors family are included
(Table 6). In addition, the experimental GABA-AT inhibition % ( Y) and predicted by the QSAR model
(Ypred) values of GABA derivatives are displayed.
Table 6. Molecular descriptors values, experimental GABA-AT inhibition % (
Y
) and predicted (Ypred)
of GABA derivatives present in the QSAR model.
Ypred
Molecule
T(O..S)
TI2
HOMAT
RCI
Y
7a
7b
7c
7d
7e
7f
8a
8b
8c
8d
8e
8f
9b
9c
14
0
0
0
0
16
14
0
0
0
0
16
0
0
0
0
16
0
3.435
3.483
3.503
3.532
3.483
3.483
2.755
2.95
3.106
3.135
2.95
2.95
2.897
3.08
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
24.9
24.9
19.4
22.9
18.3
26.5
−3.5
0.84
5.96
2.64
6.2
16.8
73
28
8.6
7
25.35
18.62
21.27
21.8
20.67
29.65
5.54
2.06
7.11
9.2
0.06
6.31
-
0
0
0
0
0
0
5.781
5.747
5.781
5.771
5.767
0
1.404
1.408
1.404
1.404
1.404
0
27.46
9.34
5
16.05
-
9d
9e
9f
3.105
2.897
2.897
2.02
14.2
40
VPNa
In Equation (1), T(O..S) possess a positive coefficient indicating that if its value increases, the
GABA-AT inhibition will also increase. T(O..S) indicates that the presence of a sulfur atom will
increase the inhibitory activity of the GABA analogues, that is, increasing the value of T(O..S) is
directly correlated to the molecule size. Therefore, increasing the distance between these two atoms in
a compound, will increase its interactions with the GABA-AT enzyme.
As for TI2, its coefficient in Equation (1) is positive, indicating that increasing the size and spherical
shape of the molecule will increase its inhibitory activity, TI2 is calculated from the eigenvalues of the
Laplacian matrix as shown in Equation (2):
4
TI2 =
(2)
nSK· λnSK−1
where the nSK is the number of non-H atoms and λnSK−1 is the first non-zero eigenvalue.

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For the RCI molecular descriptor, its coefficient in Equation (1) indicates that if the value of SPP
descriptor increases, GABA-AT inhibition by the GABA analogues will be also enhanced. RCI is
defined as an aromaticity index based on the idea of ring current, whose magnitude is determined
by its weakest link in the ring [60]. The weakest link is calculated as the maximum bond distance of
aromatic bonds (inversely proportional to the minimum total bond order).
HOMAT is derived from the HOMA index [61] and is calculated as follows:
Bπk
ꢀ
ꢁ
2
HOMT = Bπ − αk.
rkopt − rb
(3)
∑
∑
k
b=1
where Bπ is the total number of conjugated bonds, the sum runs over each conjugated bond type, Bπk
is the number of considered π-bond contributions of the kth conjugated bond type, rb is the actual
bond length, αk and rkopt are a numerical constant and the typical aromatic bond length referring to the
kth aromatic bond type (see values in the table above). This descriptor depends on the conjugation
degree of a molecule as well as on the total number of
π bonds. According to the coefficient sign in
equation 1 that corresponds to this descriptor, if the molecular aromaticity of the GABA derivatives
increases its inhibitory activity will be enhanced.
The experimental GABA-AT inhibition % values (
Ycal and pred) are presented in Table 7. In addition, the absolute value of the differences between each
Ycal and Ypred and , represented by the residualcal and residualpred terms respectively, are also shown.
Y), calculated and predicted by the QSAR model
(
Y
Y
Table 7. Experimental GABA-AT inhibition % (Y), calculated (Ycal) and predicted (Ypred) for the GABA
analogues in this study. The calculated and predicted residual values (residualcal and residualpred) are
also shown.
Ypred
Residualpred
Molecule
Y
Ycal
Residualcal
7a
7b
7c
7d
7e
7f
8a
8b
8c
8d
8e
8f
9b
9c
24.9
24.9
19.4
22.9
18.3
26.5
−3.5
0.84
5.96
2.64
6.2
16.8
73
28
8.6
7
-
25.35
18.62
21.27
21.8
20.67
29.65
5.54
2.06
7.11
9.2
0.06
6.31
-
27.46
9.34
5
-
−4.79
1.4
0.45
−6.28
1.87
−1.1
2.37
3.15
9.04
1.22
1.15
6.56
−6.14
−10.49
-
20.11
20.8
-
20.11
27.99
1.9
1.73
-
8.11
1.73
9.61
-
27.98
8.94
5.71
15.17
-
-
1.81
1.49
5.4
0.89
-
5.47
−4.47
−7.19
-
−0.02
0.34
−1.29
0.97
-
−0.54
0.74
−2
9d
9e
9f
14.2
40
16.05
-
1.85
-
VPNa
The molecules that form part of the test set are: 7a, 7d and 7c. From Table 7. molecules 8a and 8f
presented greater values of residualpred. Perhaps, this may be related to the fact that they are the only
two molecules of that series (pregabalin analogue type) that possess a sulfur atom in their structure.
Due to the residual value of 9b and VPNa, these compounds were considered as outliers; as their value
is more than three times the value of the standard deviation [62]. The linear correlation of Ycal vs. and
its R2 = 0.87 is shown in Figure 8.

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30
25
20
15
10
5
R² = 0.8696
0
-10
-5
0
5
10
15
20
25
30
-5
Y
Figure 8. The linear correlation of Ycal vs.Y.
The predictive ability of the mathematical model is shown in Figure 9. Since all the inhibition
values of the GABA derivatives obtained with the leave one out technique (LOO) and the molecules
that belong to the test set (color red circles) are displayed. The R2 = 0.78 value indicates that the model
has a good predictive power. The predictive ability evaluation of the QSAR model is displayed in
Table S3 of the Supplementary Material.
30
R² = 0.7807
25
20
15
10
5
0
-10
-5
0
5
10
15
20
25
30
-5
Y
Figure 9. The linear correlation of Ypred vs
.
Y
. Blue and red colored circles represent the training and
test molecules, respectively.
The two graphs in Figures 8 and 9 show the descriptive and predictive ability of the QSAR model.
In general terms, the model possesses a respectable descriptive and predictive power (based on its
R2 and Q2 values). Furthermore, the molecular descriptors in this model can help to explain the
inhibitory activity displayed by the GABA analogues, based on the properties that can be important

Molecules 2018, 23, 2984
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to the binding to GABA-AT. Since all the molecular descriptors included in the QSAR model can be
related with the interaction with GABA-AT (molecular shape, size and aromaticity character), the
docking results are important to confirm the descriptive power of our QSAR mathematical model.
2.4.3. Molecular Docking for Pseudomonas fluorescens GABA-AT Model
In order to explain the GABA-AT inhibition displayed by the GABA analogues, we carried
out a molecular docking study over the Pseudomonas fluorescens homology model. We analyzed the
molecular docking calculations by means of the scaffold structure (GABA, pregabalin and baclofen).
Figure 10 shows the molecular docking of all GABA analogues over the Pseudomonas GABA-AT model.
Figure 10 also shows the cavity shape where all the docking calculations were performed. It can
be observed that this cavity has a “Y” like shape; this shape explains why analogues
7 were less
potent. As was mentioned above, the T(O..S) and TI2 molecular descriptor, related to the molecular
size that according to our QSAR model increases the GABA-AT inhibitory activity of all analogues,
has the lower values for analogues
7 with respect to the 8 and 9 analogues. The specific molecular
interactions of each molecule with Pseudomonas GABA-AT are shown in the Supplementary Material
(Figures S46–S48).
Figure 10. Docking results over Pseudomonas fluorescens GABA-AT catalytic site. (
(magenta color mesh representation) of the GABA-AT catalytic site. ) GABA analogues
) (S)-Pregabalin analogues . ( ) (R)-Pregabalin analogues . ( ) (S)-Baclofen analogues . The best
compound (9b) is indicated by an arrow. ( ) (R)-Baclofen analogues . The best compound (9b) is
a) Cavity form
(b
7.
(
c
8
d
8
e
9
f
9
indicated by an arrow. PLP prosthetic group is showed as gray Van der Waals spheres and each protein
chain is colored in green and cyan. The compounds where the carboxylic acid moiety is oriented to
PLP are colored in obscure.
The linear form of GABA analogues
GABA-AT enzyme (Figure 10b). In fact, analogues
All these molecules bind in a similar form with their carboxylic moiety oriented to the PLP molecule,
except for . This difference in the interaction form is maybe related to the presence of the sulfur
atom and larger ring as compared to 7a, like 8f and 8f (which also possess a sulfur atom in their
7
, does not allow them to strongly interact with the cavity of
7
display the lowest interaction energies (Table S1).
7
structure) also interact in a different manner than that of the other analogues 8a–e and 9a–e molecules
(pregabalin and baclofen analogues, respectively).
For the pregabalin analogues 8a
that of the (S)-enantiomers (Figure 10c,d), where the amine group of the molecule is oriented towards
the PLP and the carboxylic group toward the outer part of the catalytic site, with the exception of 8f
–f, it can be shown that the (R)-enantiomers bind in similar way to
.

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The (S)-pregabalin analogues possess a lower interaction energy compared to their (R)-enantiomers
(Table S1).
For analogues 9 as for analogues 8, the (S)-enantiomers possess a better binding mode to GABA-AT
(supported by their interaction energy values) than that of the (R)-enantiomers (Table S1). Due to
their size and shape these molecules interact in the outer part of the catalytic site, so none of their
functional groups is close to the PLP molecule. On the other hand, 9b–9d interact in a similar way,
their carboxylic group interacting with Arg 143, their amino group interacting with Glu 213 and Tyr
157, and their p-Cl-phenyl substituent located in the hydrophobic zone of the catalytic site formed by
Ile 24, Ile 52, Val 82 and Cys 79. Figure 11 shows the interaction of (S) and (R) 9b with the enzyme.
Figure 11. Interactions between 9b enantiomers with P. fluorescens GABA-AT. (a) (S)-9b and (b) (R)-9b.
PLP prosthetic group is showed as Van der Waals spheres and each protein chain is colored in green
and cyan. Residues at 4 Å of each analogue are indicated. Hydrogen bonds are showed as orange
dashed lines.
The (S)-9b molecule binds in a better and stronger way than (R)-9b, the appropriate orientation of
their charged groups (amino and carboxylate moieties) favors hydrogen bond interactions with the
GABA-AT residues (Tyr 157 for the amine group and Arg 143 and Gln 81 for the carboxylate group).
2.4.4. Molecular Docking for Human GABA-AT Model
Since the main purpose of this work is to develop an alternative drug for clinical use that in the
future may be used as a drug for humans, we also carried out a molecular docking study over a human
GABA-AT model.
Figure 12 shows the binding mode of all the GABA analogues over the human GABA-AT, and
the cavity shape where all the docking calculations were done is also shown. It can be observed
that this cavity possesses a greater volume that the P. fluorescens model and a different shape.
Despite these differences, there is a recurring observation: molecules with a bigger size will possess
better interaction energy values. The interactions of each molecule with human GABA-AT are shown
in the Supplementary Material (Figures S49–S51).
In Figure 12b, the interaction of the GABA analogues 7a–f is shown. The carboxylic acid group of
compounds 7a 7b 7d and 7e is oriented to the outer part of the cavity (toward the solvent), and their
,
,
amino group is close the PLP molecule. On the other hand, 7c and 7f show an opposite orientation,
where the amino group is now oriented toward the solvent and the carboxylic acid group is close to
the PLP molecule.
The enantiomeric forms of analogues 8a–f interact in a similar way where their carboxylic acid
groups are oriented to a high electrostatic positive zone of the cavity formed by Lys 193, Lys 429,
Arg 412, His 196 and Arg 182. On the other hand, the amine group is oriented to the PLP molecule
interacting with the His 34 residue. The interaction energy of (S)-analogues 8 is stronger than that of

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the (R)-enantiomers. (R)-analogues 8a
,
8c and 8d interact in a different form that the other analogues
8b, 8e and 8f, orienting their amino group to the His 34 rather than to the PLP molecule.
Figure 12. Docking results over human GABA-AT catalytic site. (
representation) of the GABA-AT catalytic site. ( ) GABA analogues
) (R)-Pregabalin analogues . ( ) (S)-Baclofen analogues . ( ) (R)-Baclofen analogues 9
a
) Cavity form (magenta color mesh
. ( ) (S)-Pregabalin analogues
. PLP prosthetic
b
7
c
8.
(d
8
e
9
f
group is showed as gray Van der Waals spheres and each protein chain is colored in yellow and red.
The compounds where the carboxylic moiety is oriented to PLP are colored in obscure.
For analogues 9, their interaction with human GABA-AT was very similar with their carboxylic
acid groups oriented to the positive zone of the cavity catalytic site. In the (S)-analogues, almost all the
compounds oriented their amine group to Ile 416, and only 9b and 9c oriented their amine function
to the PLP molecule. On the other hand, all the (R)-analogues bind in a similar way, where their
p-Cl-phenyl substituent interacts with the Ile 416 residue, while their amine group is oriented to the
PLP molecule.
According to this approximation and based on its interaction energy value, the most potent
possible inhibitor will be (S)-8f (Table S2, Supplementary Material). All analogues
8 and 9 display
very good interaction energies with human GABA-AT. In addition, (S)-9c has an interaction energy
value that is one of the lowest. These results are related with the one obtained with the Pseudomonas
GABA-AT, since the ligands with greater volume and with a sulfur atom, possess the lowest interaction
energy (better inhibitors). This fact gives us a higher probability of success to transfer our results to a
human model.
3. Materials and Methods
3.1. General Information
Oxygen and/or moisture sensitive reactions were carried out in oven or flame-dried glassware
under nitrogen atmosphere. All reagents and solvents were purchased and used as received from
commercial sources or synthesized according to cited procedures. THF, Ether and toluene were dried
by distillation over sodium benzophenone ketyl. All other solvents were used after distillation at
normal pressure. Yields refer to chromatographically and spectroscopically pure compounds, unless
otherwise stated. Analytical thin layer chromatography (TLC) was performed on 0.25 mm silica gel
60-F plates and visualized by UV light (254 nm) and/or iodine vapor.
1H-NMR (400 MHz) and 13C-NMR (100 MHz) spectra were recorded on a Innova 400 MHz (9.4T)
or Mercury 200 or 400 MHz (9.4T) spectrometer (Varian, Palo Alto, CA, USA; chemical shifts (
δ) are
reported relative to the signal of Me4Si. NMR data are reported as follows: chemical shift (δ ppm),

Molecules 2018, 23, 2984
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multiplicity (s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, sext = sextet
,
dd = doublet of doublets, m = multiplet or overlapping), coupling constant (Hz), integration.
Mass spectra (FAB+) were measured with a JMS700 mass spectrometer (JEOL, Peabody, MA, USA).
3.2. Chemistry
3.2.1. Methyl 4-Bromobutanoate (11)
4-Bromobutanoic acid (7.5 g, 45.2 mmole) was dissolved in methanol (50 mL), and then CH3SiCl
(11.4 mL, 90.4 mmole) was added at 0 ◦C; the mixture was stirred at this temperature for 30 min and
for 72 h at 25 ◦C. The solvent was removed in vacuuo. The resultant residue was purified by column
chromatography (silica-gel) eluting with hexanes:ethyl acetate 90:10 to afford the title compound as a
1
colorless oil (6.78 g, 83.4%). H-NMR (CDCl3):
δ
3.68 (s, 3H), 3.46 (t, J = 6.4 Hz, 2H), 2.50 (t, J = 7.2 Hz,
2H), 2.22–2.13 (m, 2H). 13C-NMR (CDCl3):
δ
173.11, 51.85, 32.80, 32.34, 27.87. HRMS (FAB+): m/z [M]+
calcd for C5H9BrO2: 179.9786, found (M + 1): 180.9786.
3.2.2. General Methodology for the Synthesis of Esters 12a–f
The corresponding amine 10a
, 1.0 equiv) at 25 ◦C, and the mixture was allowed to warm to 65–70 ◦C for 2 h. When the reaction
was complete, aqueous NaHCO3 was added, and the mixture was extracted with EtOAc (3 10 mL),
–f (1.0 equiv) was added dropwise to methyl 4-bromobutanoate
(2
×
dried over Na2SO4, filtered and removed in vacuuo. The crude product was purified by column
chromatography (silica-gel), eluting with hexanes: ethyl acetate 90:10.
1
Methyl 4-(thiazolidin-3-yl)butanoate (12a). 0.445 g (37%) as a yellow oil. H-NMR (200 MHz, CDCl3):
δ
4.00 (s, 2H), 3.63 (s, 1H), 3.02 (dd, J = 9.5, 3.3 Hz, 2H), 2.82 (dd, J = 9.3, 3.6 Hz, 1H), 2.37 (t, J = 7.4,
2H), 2.35 (t, J = 7 Hz, 2H), 1.77 (q, J = 7.2 Hz, 1H, H-4). 13C-NMR (50 MHz, CDCl3)
δ
173.94, 60.44,
58.01, 52.07, 51.62, 31.71, 29.6, 24.28. HRMS (FAB+): m/z [M]+ calcd for C8H15NO2S: 189.0823, found
(M + 1): 190.0904.
1
Methyl 4-(piperidin-1-yl) butanoate (12b). 0.37 g (67%) as a yellow oil. H-NMR (CDCl3)
δ
3.62 (s, 3H),
2.33 (sa, 4H), 2.28 (t, J = 8 Hz, 2H), 2.27 (t, J = 8 Hz, 2H), 1.77 (q, J = 8 Hz, 2H), 1.55–1.49 (m, 4H), 1.4–1.37
(m, 2H). 13C-NMR (CDCl3)
δ
174.13, 58.49, 54.54, 51.56, 32.23, 25.98, 24.48, 22.24. HRMS (FAB+): m/z
calcd for C10H19NO2: 185.1416, found (M + 1): 186.1532.
Methyl 4-(3-methylpiperidin-1-yl) butanoate (12c). 0.537 g (79%) as a yellow oil. 1H-NMR (CDCl3):
δ
3.67 (s, 3H), 2.84–2.78 (m, 2H), 2.35–2.29 (m, 4H), 1.87–1.77 (m, 3H), 1.71–1.49 (m, 6H), 0.85 (d,
J = 6.4 Hz, 3H). 13C-NMR (CDCl3)
δ
174.13, 62.08, 58.25, 54.02, 51.54, 33.14, 32.22, 31.18, 25.62, 22.30,
19.83. HRMS (FAB+): m/z calcd for C11H21NO2: 199.1572, found (M + 1): 200.1636.
1
Methyl 4-(4-methylpiperidin-1-yl)butanoate (12d). 0.45 g (67%) as a yellow oil. H-NMR (CDCl3):
δ 3.67
(s, 3H), 2.87–2.84 (m, 2H), 2.35–2.3 (m, 4H), 1.92–1.86 (m, 2H), 1.85–1.77 (m, 2H), 1.62–1.58 (m, 2H),
1.40–1.28 (m, 1H), 1.28 (m, 2H), 0.91 (d, J = 6.4 Hz, 3H). 13C-NMR (CDCl3)
δ
174.18, 58.21, 54.05,
51.61, 34.43, 32.30, 30.93, 22.47, 22.02. HRMS (FAB+): m/z calcd for C11H21NO2, 199.1572, found
(M + 1): 200.1656.
1
Methyl 4-morpholinobutanoate (12e). 0.445 g (52%) as a yellow oil. H-NMR (CDCl3):
δ 3.7 (dd, J = 4,
4 Hz, 4H), 3.68 (s, 3H), 2.43 (dd, J = 4, 4 Hz, 4H), 2.36 (t, J = 8 Hz, 4H), 1.82 (q, J = 8 Hz, 1H). 13C-NMR
(CDCl3) 173.92, 66.92, 57.97, 53.55, 51.48, 31.87, 21.73. HRMS (FAB+): m/z calcd for C9H17NO3:
187.1208, found (M + 1): 188.1308.
δ
1
Methyl 4-thiomorpholinobutanoate (12f). 0.47 g (58%) as a yellow oil. H-NMR (CDCl3):
2.64–2.62 (m, 4H), 2.6–2.58 (m, 4H), 2.31 (t, J = 8 Hz), 2.26 (t, J = 8Hz), 1.73 (q, J = 8 Hz). 13C-NMR
(CDCl3) 173.93, 58.24, 54.92, 51.47, 31.88, 27.93 (2C), 21.78. HRMS (FAB+): m/z calcd for C9H17NO2S:
203.098, found (M + 1): 204.1057.
δ 3.60 (s, 3H),
δ

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3.2.3. General Methodology for Hydrolysis of Methyl Esters 12a–f
The corresponding methyl ester 12a–f (1.0 equiv) was dissolved in MeOH (6 mL) and NaOH
(1.1 equiv) dissolved in water (2 mL) was added dropwise. The reaction was monitored until the
consumption of the starting material. At the end of the reaction, the MeOH was evaporated. Then HCl
was added to pH = 5, the mixture was extracted with EtOAc (3
and the solvent removed in vacuo.
×
5 mL), dried over Na2SO4, filtered
1
4-(Thiazolidin-3-yl)butanoic acid (7a). 0.165 g (81%) as a yellow oil. H-NMR (200 MHz, CD3OD)
(s, 2H), 3.08 (t, J= 5.9 Hz, 2H), 2.88 (t, J = 6.2 Hz, 2H), 2.54–2.41 (m, 2H), 2.29 (t, J= 7.3 Hz, 2H), 1.90–1.68
(m, 2H). 13C-NMR (50 MHz, CD3OD)
180.16, 60.29, 58.68, 53.73, 35.05, 29.94, 26.13. HRMS (FAB+):
δ 4.02
δ
m/z calcd for C7H13NO2S, 175.0667, experimental (M + 1): 176.0769.
4-(Piperidin-1-yl)butanoic acid (7b). 0.22 g (95%) as a yellow oil. 1H-NMR (CD3OD):
δ 3.3–3.12 (sa,
2H), 3.08 (t, J = 7.2 Hz, 2H), 2.74 (ddd, J = 15.5, 12, 12 Hz, 2H), 2.41 (t, J = 6.6 Hz, 2H), 2.01–1.92 (m,
2H), 1.86–1.84 (m, 4H), 1.66 (sa, 2H). 13C-NMR (CD3OD)
δ
181.22, 59.77, 54.39, 37.50, 24.73, 23.15,
21.54HRMS (FAB+): m/z calcd for C9H17NO2: 171.1259, found (M + 1): 172.1324.
1
4-(3-Methylpiperidin-1-yl)butanoic acid (7c). 0.21 g (93%) as a colorless oil. H-NMR (D2O):
δ
2.92 (m, 2H),
2.46–2.41 (m, 2H), 2.09 (td, J = 12.4, 2.6 Hz, 1H), 2.02 (t, J = 7.3 Hz, 2H), 1.81 (t, J = 11.4 Hz, 1H), 1.68–1.47
(m, 5H), 1.46–1.34 (m, 1H), 0.86–0.75 (m, 1H), 0.71 (d, J = 6.5 Hz, 3H). 13C-NMR (D2O)
δ
182.34, 59.72,
57.37, 52.70, 35.20, 31.10, 29.65, 23.62, 21.53, 18.45. HRMS (FAB+): m/z calcd for C10H19NO2: 185.1416,
found (M + 1): 186.1532.
1
4-(4-Methylpiperidin-1-yl)butanoic acid (7d). 0.14 g (80%) as a colorless oil. H-NMR (D2O):
δ 2.81 (d,
J = 11.6 Hz, 2H), 2.30–2.22 (m, 2H), 2.02–1.96 (m, 4H), 1.63–1.46 (m, 4H), 1.28–1.21 (m, 1H), 1.05–0.95
(m, 2H), 0.71 (d, J = 6.5 Hz). 13C-NMR (D2O)
δ
182.67, 57.36, 52.78, 35.41, 32.57, 29.41, 22.13, 20.84.
HRMS (FAB+): m/z calcd for C10H19NO2: 185.1416, found (M + 1): 186.1532.
4-Morpholinobutanoic acid (7e). 0.22 g (83%) of a yellow solid, m.p = 73–73.5 ◦C. 1H-NMR (CD3OD):
δ
3.73–3.66 (m, 4H), 2.46 (s, 4H), 2.36 (m, 2H), 2.17 (t, J = 7.5 Hz), 1.79 (q, J = 7.5 Hz). 13C-NMR (CD3OD)
180.87, 66.28 (2C), 58.7, 53.41 (2C), 35.61, 22.89. HRMS (FAB+): m/z calcd for C8H15NO3: 173.11,
δ
found (M + 1): 174.1171.
4-Thiomorpholinobutanoic acid (7f). 0.22 g (83%) of a yellow solid, m.p = 82–83 ◦C. 1H-NMR (CDCl3):
δ
2.77–2.70 (m, 4H), 2.66–2.65 (m, 4H), 2.41–2.37 (m, 2H), 2.14 (t, J = 7.5 Hz, 2H), 1.78 (q, J = 7.6 Hz).
13C-NMR (CDCl3)
δ
182.32, 60.37, 56.20 (2C), 36.95, 28.37 (2C), 24.14. HRMS (FAB+): m/z calcd for
C8H15NO2S: 189.08, found (M + 1): 190.1053.
3.2.4. General Procedure for the Synthesis of Esters 14a–f
Amines 10a
–
f
(1.0 equiv), N,N-diisopropylethylamine (1.5 equiv), ethyl (E)-4-bromobut-2-enoate
◦
(5
, 1.3 equiv) and CH2Cl2 (25 mL) were placed in a round bottom flask and stirred at
−
20 C for
1 h, under nitrogen. After completion of the reaction (TLC) the reaction mixture was extracted with
NaHCO3 (3 30 mL) and water (1 30 mL). The organic phase was dried over Na2SO4 and the
×
×
solvent removed under reduced pressure to yield the crude product. The crude product was purified
by silica gel column chromatography (9:1 hexanes:EtOAc).
1
Ethyl (E)-4-(thiazolidin-3-yl)but-2-enoate (14a). 1.21 g (63 %) as a yellow oil. H-NMR (CDCl3)
δ 6.93 (dt,
J = 16, 8 Hz, 1H), 6.01 (dt, J = 16, 1.8 Hz, 1H), 4.18 (q, J = 7 Hz, 2H), 4.03 (s, 2H), 3.15 (dd, J = 8, 1.8 Hz,
2H), 3.07 (t, J = 6.4 Hz, 2H), 2.87 (t, J = 6.4 Hz, 2H), 1.27 (t, J = 7 Hz, 3H). 13C-NMR (CDCl3)
δ
166.24,
144.94, 123.42, 60.59 (2C), 57.92, 53.90, 29.62, 14.34. HRMS (FAB+): m/z calcd for C8H15NO2S: 201.284,
found (M + 1): 202.0912.
1
Ethyl (E)-4-(piperidin-1-yl)but-2-enoate (14b). 0.9 g (90 %) as a yellow oil. H-NMR (CDCl3):
δ 6.95 (dt,
J = 15.7, 6.3 Hz, 1H), 5.92 (dt, J = 15.7, 1.6 Hz, 1H), 4.16 (q, J = 7.1 Hz, 2H), 3.07 (dd, J = 6.3, 1.6 Hz, 2H),

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2.37 (sa, 4H), 1.56 (q, J = 5.6 Hz, 4H), 1.44–1.38 (m, 2H), 1.25 (t, J = 7.1 Hz, 3H). 13C-NMR (CDCl3)
δ
166.39, 145.69, 123.34, 60.45, 60.25, 54.84 (2C), 26.05 (2C), 24.24, 14.39. HRMS (FAB+): m/z calcd for
C11H19NO2: 197.1416, found (M + 1): 198.1506.
1
Ethyl (E)-4-(3-methylpiperidin-1-yl)but-2-enoate (14c). 0.83 g (77%) as a yellow oil. H-NMR (CDCl3):
δ
6.96 (dt, J = 15.7, 6.3 Hz, 1H), 5.94 (dt, J = 15.7, 1.6 Hz, 1H), 4.17 (q, J = 7.2 Hz, 2H), 3.09 (dd, J = 6.3,
1.6 Hz, 2H), 2.85–2.74 (m, 2H), 1.87 (td, J = 11.3, 3.2 Hz), 1.71–1.54 (m, 5H), 1.27 (t, J = 7.2 Hz, 3H), 0.84
(d, J = 6.4 Hz, 4H). 13C-NMR (CDCl3)
δ 166.44, 145.63, 123.43, 62.22, 60.50, 60.01, 54.32, 32.90, 31.31,
25.65, 19.83, 14.42. HRMS (FAB+): m/z calcd for C12H21NO2: 211.1572, found (M + 1): 212.1651.
1
Ethyl (E)-4-(4-methylpiperidin-1-yl)but-2-enoate (14d). 0.85 g (80%) as a yellow oil. H-NMR (CDCl3)
δ
6.94 (dt, J = 15.6, 6.4 Hz, 1H), 5.92 (dt, J = 15.5, 1.6 Hz, 1H), 4.15 (q, J = 7.1 Hz, 2H), 3.08 (dd, J = 6.4
1.6 Hz, 2H), 2.82 (d, J = 11.8 Hz, 4H), 2.02–1.80 (m, 2H), 1.65–1.55 (m, 2H), 1.38–1.31 (m, 1H), 1.25
(t, J = 7.1 Hz, 3H), 0.89 (d, J = 5.7 Hz, 3H). 13C-NMR (CDCl3)
166.46, 145.77, 123.42, 60.53, 59.96,
,
δ
54.35 (2C), 34.45 (2C), 30.73, 22.06, 14.46. HRMS (FAB+): m/z calcd for C12H21NO2: 211.1572, found
(M + 1): 212.1651.
1
Ethyl (E)-4-morpholinobut-2-enoate (14e). 1.02 g (89%) as a yellow oil. H-NMR (200 MHz, CDCl3)
δ 6.91
(dt, J = 15.7, 6.2 Hz, 1H), 5.96 (dt, J = 15.7, 1.6 Hz, 1H), 4.17 (q, J = 7.1 Hz, 2H), 3.72–3.67 (m, 4H), 3.1
(dd, J = 6.2, 1.6 Hz, 2H), 2.49–2.39 (m, 4H), 1.26 (t, J = 7.1 Hz, 3H). 13C-NMR (50 MHz, CDCl3)
δ
166.05,
144.38, 123.65, 66.83 (2C), 60.38, 59.55, 53.64 (2C), 14.20. HRMS (FAB+): m/z calcd for C10H17NO3:
199.1208, found (M + 1): 200.1291.
1
Ethyl (E)-4-thiomorpholinobut-2-enoate (14f). 0.85 g (80%) as a yellow oil. H-NMR (CDCl3)
δ
6.88 (dt,
J = 15.7, 6.1 Hz, 1H), 5.94 (dt, J = 15.7, 1.7 Hz, 1H), 4.16 (q, J = 7.1 Hz, 2H), 3.11 (dd, J = 6.1, 1.7 Hz, 2H),
2.67 (m, 8H), 1.25 (t, J = 7.1 Hz, 3H). 13C-NMR (CDCl3)
δ
166.00, 144.72, 123.43, 60.30, 59.91, 54.97 (2C),
27.88 (2C), 14.15. HRMS (FAB+): m/z calcd for C12H21NO2: 215.098, found (M + 1): 216.1044.
3.2.5. General Methodology for the Conjugate Additions of 15, 17
Most of the reactions were carried out using the following procedure: CuI (2.00 equiv) was placed
in a one-neck 50 mL round-bottom flask equipped with a stirring bar and sealed with a septum.
The flask was evacuated with a vacuum pump and then purged with nitrogen, this process was
repeated three times to assure a complete nitrogen atmosphere. Anhydrous ethyl ether (25.00 mL)
was injected and the mixture stirred for 15 min and then cooled to 0 ◦C. Simultaneously, a Grignard
solution was prepared mixing finely divided Mg turnings (4.1 equiv) and catalytic amounts of I2, with
1-bromo-2-methylpropane (4.0 equiv) or 1-bromo-4-chlorobenzene (4.0 equiv) at room temperature.
The mixture was stirred for 30 min and then transferred via syringe dropwise to the copper solution at
0 ◦C, and stirred at this temperature for another 30 min period. Then, the unsaturated substrate 14a
(1.0 equiv) were added dropwise. The reaction was allowed to proceed for 24 h before being quenched
with a saturated aqueous solution of NH4Cl. The mixture was extracted with ether (3 25 mL) dryed
–f
×
over Na2SO4 and the solvent removed in vacuo to afford the crude products which were purified by
flash chromatography eluting with hexane:EtOAc (95:5). Some modifications done to this procedure
are mentioned in the specific examples below.
1
Ethyl 5-methyl-3-(thiazolidin-3-ylmethyl)hexanoate (16a). 0.023 g (5%) as a yellow oil. H-NMR (CDCl3):
δ
4.16–4.07 (m, 2H), 4.02 (q, J = 9.2 Hz, 2H), 3.04–3.0 (m, 2H), 2.90–2.82 (m, 2H), 2.44–2.36 (m, 2H), 2.27
(dd, J = 15.1, 5.6 Hz, 1H), 2.12–2.06 (m, 2H), 1.64 (m, 1H), 1.25 (t, J = 7.1 Hz, 3H), 1.22–1.08 (m, 2H), 0.89
(t, J = 6.4 Hz, 6H). 13C-NMR (CDCl3)
δ 173.54, 61.44, 60.25, 58.65, 58.14, 42.40, 38.58, 33.28, 29.88, 25.40,
23.02, 22.77, 14.42. HRMS (FAB+): m/z calcd for C13H25NO2S: 259.1606, found (M + 1): 260.1699.
1
Ethyl 5-methyl-3-(piperidin-1-ylmethyl)hexanoate (16b). 0.17 g (52%) as a yellow oil. H-NMR (CDCl3):
δ
4.05 (q, J = 7.2 Hz, 2H), 2.44–2.32 (m, 3H), 2.24–2.11 (m, 5H), 2.05–1.99 (m, 1H), 1.68–1.57 (m, 1H),
1.52–1.46 (m, 4H), 1.39–1.32 (m, 2H), 1.19 (t, J = 7.2 Hz, 3H), 1.11–0.97 (m, 2H), 0.81 (dd, J = 6.5, 5.3 Hz,

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6H). 13C-NMR (CDCl3)
δ
173.85, 64.54, 60.04, 55.21 (2C), 43.00, 39.25, 30.97, 26.23 (2C), 25.52, 24.69,
22.95 (2C), 14.40. HRMS (FAB+): m/z calcd for C15H29NO2: 255.2198, found (M + 1): 256.2267.
1
Ethyl 5-methyl-3-((3-methylpiperidin-1-yl)methyl)hexanoate (16c). 0.09 g (20%) as an amber oil. H-NMR
(CDCl3):
3H), 2.08–1.99 (m, 1H), 1.92–1.85 (m, 1H), 1.71–1.36 (m, 6H), 1.24 (t, J = 7.1 Hz, 3H), 1.17–1.02 (m, 2H),
0.88–0.81 (m, 10H). 13C-NMR (CDCl3):
173.79 (C-3), 64.31 (C-6), 64.27 (C-6), 63.69 (C-7), 62.03 (C-7),
δ 4.09 (q, J = 7.1 Hz, 2H), 2.84–2.76 (m, 1H), 2.68–2.61 (m, 1H), 2.38–2.32 (m, 1H), 2.21–2.09 (m,
δ
60.01 (C-2), 55.52 (C-12), 53.90 (C-12), 43.10 (C-13), 43.07 (C-13), 39.21 (C-4), 33.33 (C-10), 31.41 (C-8),
31.13 (C-5), 25.80 (C-11), 25.70 (C-11), 25.54 (C-14), 23.03(C-15), 22.97 (C-16), 19.83 (C-9), 19.81 (C-9),
14.40 (C-1). HRMS (FAB+): m/z calcd for C16H31NO2: 269.2355, found (M + 1): 270.2411.
1
Ethyl 5-methyl-3-((4-methylpiperidin-1-yl)methyl)hexanoate (16d). 0.164 g (37%) as a yellow oil. H-NMR
(CDCl3):
δ 4.12 (q, J = 7.2 Hz, 2H), 2.87 (d, J = 11.3 Hz, 1H), 2.71 (d, J = 11.8 Hz, 1H), 2.36 (dd, J = 5.7 Hz,
1H), 2.18–2.11 (m, 2H), 2.07 (m, 1H), 1.96 (ddd, J = 11.5„ 11.5, 2.5 Hz), 1.74 (ddd, J = 11.6, 2.4 Hz, 1H),
1.64 (m, J = 6.4 Hz, 1H), 1.57–1.53 (m, 2H), 1.26 (t, J = 7.1 Hz, 4H), 1.20–1.03 (m, 4H), 0.91–0.85 (m, 9H).
13C-NMR (CDCl3)
δ 173.89, 64.13, 60.06, 55.56, 53.67, 42.98, 39.27, 34.78, 34.52, 31.05, 31.03, 25.44, 22.95
(2C), 22.13, 14.41. HRMS (FAB+): m/z calcd for C16H31NO2: 269.2355, found (M + 1): 270.2429.
1
Ethyl 5-methyl-3-(morpholinomethyl)hexanoate (16e). 0.166 g (37%) as a yellow oil. H-NMR (CDCl3):
δ
4.13 (q, J = 7.2 Hz, 2H), 3.71–3.59 (m, 4H), 2.50 (m, 2H), 2.34 (m, 3H), 2.26–2.16 (m, 3H), 2.13–2.06 (m,
1H), 1.71–1.59 (m, 1H), 1.26 (t, J = 7.1 Hz, 3H), 1.20–1.06 (m, 2H), 0.89 (d, J = 6.3 Hz, 6H). 13C-NMR
(CDCl3):
δ 173.71, 67.23 (2C), 64.25, 60.21, 54.16 (2C), 42.82, 39.09, 30.42, 25.41, 22.96, 22.86, 14.40.
HRMS (FAB+): m/z calcd for C14H27NO3: 257.1991, found (M + 1): 258. 2077.
1
Ethyl 5-methyl-3-(thiomorpholinomethyl)hexanoate (16f). 0.11 g (25%) as a yellow oil. H-NMR (CDCl3):
δ
4.17–4.07 (m, 2H), 2.78–2.71 (m, 2H), 2.63–2.53 (m, 6H), 2.32–2.24 (m, 2H), 2.2–2.13 (m, 2H), 2.04 (dd,
J = 12.2, 9.0 Hz, 1H), 1.69–1.57 (m, 1H), 1.27 (t, J = 7.2 Hz, 3H), 1.16–1.03 (m, 2H), 0.88 (d, J = 6.2 Hz,
6H). 13C-NMR (CDCl3):
δ
173.76, 64.60, 60.22, 55.75, 42.82, 39.15, 30.87, 28.18, 25.47, 23.03, 22.91, 14.46.
HRMS (FAB+): m/z calcd for C14H27NO3S: 273.1762, found (M + 1): 274.1858.
1
Ethyl 3-(4-chlorophenyl)-4-(piperidin-1-yl)butanoate (18b). 0.2 g (52%) as a yellow oil. H-NMR (CDCl3):
7.25 (d, J = 8.5 Hz, 2H) 7.13 (d, J = 8.5 Hz, 2H), 4.03 (q, J = 7.1 Hz, 2H), 3.35 (ddd, J = 11.9, 9.1, 6.0 Hz,
1H), 2.87 (dd, J = 15.5, 6.3 Hz, 1H), 2.49–2.41 (m, 3H), 2.41–2.32 (m, 2H), 2.31–2.23 (m, 2H), 1.55–1.46
(m, 4H), 1.42–1.38 (m, 2H), 1.16 (t, J = 7.1 Hz, 3H). 13C-NMR (CDCl3)
172.57, 141.72, 132.11, 128.90
δ
δ
(2C), 128.47 (2C), 65.17, 60.19, 54.87 (2C), 39.25 (2C), 26.00, 24.34, 14.12. HRMS (FAB+): m/z calcd for
C17H24ClNO2: 309.1496, found (M + 1): 310.1556.
1
Ethyl 3-(4-chlorophenyl)-4-(3-methylpiperidin-1-yl)butanoate (18c). 0.22 g (49%) as a yellow oil. H-NMR
(CDCl3):
δ 7.24 (d, J = 8.5 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H), 4.02 (q, J = 7.1 Hz, 2H), 3.42–3.32 (m, 1H),
2.86 (dd, J = 15.5, 6.3 Hz, 2H), 2.69 (d, J= 8.8 Hz, 1H), 2.49–2.39 (m, 2H), 2.35 (dd, J = 12.6, 5.7 Hz,
1H), 1.96 (ddd, J = 11.2, 11.2, 3.0 Hz, 1H), 1.75 (ddd, J = 11.4, 11.4, 2.7 Hz, 1H), 1.71–1.39 (m, 5H), 1.15
(t, J = 7.1 Hz, 3H), 0.83 (t, J = 6.2 Hz, 4H). 13C-NMR (CDCl3):
δ 172.66 (C-3), 141.66 (C-13), 132.14
(C-16), 128.90 (C-15), 128.48 (C-14), 64.93 (C-6), 64.90 (C-6), 63.35 (C-7), 61.42 (C-7), 60.25 (C-2), 55.28
(C-12), 53.40 (C-12), 39.32 (C-5), 39.24 (C-4), 32.92 (C-10), 31.14 (C-8), 30.87 (C-8), 25.52 (C-11), 25.38
(11), 19.67 (C-9), 19.63 (C-9), 14.10 (C-1). HRMS (FAB+): m/z calculado para C18H26ClNO2: 323.1652,
experimental (M + 1): 324.1734.
1
Ethyl 3-(4-chlorophenyl)-4-(4-methylpiperidin-1-yl) butanoate (18d). 0.75 g (49%) as a yellow oil. H-NMR
(CDCl3):
δ 7.24 (d, J = 8.5 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H), 4.03 (q, J = 7.1 Hz, 2H), 3.39–3.31 (m, 1H),
2.94–2.89 (d, J = 11.1 Hz, 1H), 2.85 (dd, J = 15.5, 6.3 Hz, 1H), 2.74 (d, J = 11.1 Hz, 1H), 2.50–2.41 (m, 2H),
2.36 (dd, J = 12.6, 5.7 Hz, 1H), 2.03 (ddd, J = 11.1, 11.1, 2.5 Hz, 1H), 1.83 (ddd, J = 11.1, 11.1, 2.5 Hz, 1H),
1.59–1.52 (m, 2H), 1.35–1.24 (m, 1H), 1.15 (t, J = 7.1 Hz, 6H), 0.89 (d, J = 6.4 Hz, 3H). 13C-NMR (CDCl3)
δ
172.36, 141.75, 132.06, 128.88 (2C), 128.42 (2C), 64.76, 60.04, 55.34, 53.18, 39.38, 39.18, 34.55, 34.28,
30.71, 21.90, 14.11. HRMS (FAB+): m/z calcd for C18H26ClNO2: 323.1652, found (M + 1): 324.1772.

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1
Ethyl 3-(4-chlorophenyl)-4-morpholinobutanoate (18e). 0.645 g (55 %) as a yellow oil. H-NMR (CDCl3):
7.25 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H), 4.05 (q, J = 7.1 Hz, 2H), 3.67–3.59 (m, 4H), 3.43–3.32
(m, 1H), 2.85 (dd, J = 15.5, 6.6 Hz, 1H), 2.58–2.30 (m, 7H), 1.16 (t, J = 7.1 Hz, 3H). 13C-NMR (CDCl3)
δ
δ
172.25, 141.22, 132.21, 128.82 (2C), 128.50 (2C), 66.89 (2C), 64.71, 60.20, 53.79 (2C), 39.12, 38.67, 14.09.
HRMS (FAB+): m/z calcd for C16H22ClNO3: 311.1288, found (M + 1): 312.1299.
1
Ethyl 3-(4-chlorophenyl)-4-thiomorpholinobutanoate (18f). 0.66 g (58 %) as a yellow oil. H-NMR (CDCl3):
δ
7.25 (d, J = 8.5 Hz, 1H), 7.12 (d, J = 8.4 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 3.40–3.30 (m, 1H, H-5),
2.83–2.77 (m, 3H), 2.66–2.56 (m, 6H), 2.5–2.39 (m, 3H), 1.18 (t, J = 7.1 Hz, 3H). 13C-NMR (CDCl3):
δ
172.42, 141.23, 132.29, 128.86 (2C), 128.55 (2C), 65.06, 60.34, 55.39 (2C), 39.13 (2C), 27.92 (2C), 14.14.
HRMS (FAB+): m/z calcd for C16H22ClNO2S: 327.106, found (M + 1): 328.1084.
3.2.6. General Procedure for the Hydrolysis of Esters 16a–f and 18b–f
The corresponding ethyl ester 16a–f or 18b–f (1.0 equiv) were dissolved in MeOH (6.0 mL), and
NaOH (1.1 equiv) dissolved in water (2 mL) was added dropwise. The reaction was monitored until
the consumption of the starting materials (tlc). When the reaction was complete, the solvent was
evaporated and aqueous HCl was added until the mixture reached a pH = 5. The mixture was extracted
with ethyl acetate (3 × 5 mL), dried over Na2SO4 and the solvent removed in vacuo.
1
5-Methyl-3-(thiazolidin-3-ylmethyl)hexanoic acid (8a). 0.055 g (77%) as a yellow oil. H-NMR (CD3OD):
δ
3.83 (s, 2H), 3.51–3.42 (m, 1H), 3.23 (m, 2H), 3.10–2.68 (m, 3H), 2.56–2.44 (m, 1H), 2.37–2.18 (m, 2H),
1.74–1.63 (m, 1H), 1.22–1.12 (m, 2H), 0.94–0.91 (m, 6H). 13C-NMR (CD3OD):
43.92, 39.09, 31.45, 30.39, 26.31, 23.36, 22.65.
δ
180.44, 60.81, 59.41, 51.43,
1
5-Methyl-3-(piperidin-1-ylmethyl)hexanoic acid (8b). 0.55 g (90%) as a yellow oil. H-NMR (CD3OD):
δ
3.04–2.99 (m, 1H), 2.85 (dd, J = 13.2, 10.4 Hz, 1H), 2.52 (dt, J = 16.6, 1.8 Hz, 1H), 2.35 (dd, J = 16.6,
10.3 Hz, 1H), 2.20 (m, 1H), 1.86 (s, 5H), 1.74–1.63 (m, 2H), 1.14 (dd, J = 7.2, 7.2 Hz, 2H), 0.935 (d, J = 4 Hz
3H), 0.925 (d, J = 4 Hz, 3H). 13C-NMR (CD3OD)
181.03, 65.16 (3C), 44.96, 44.16, 30.36, 26.11, 24.76
,
δ
(2C), 23.00, 22.96, 22.77. HRMS (FAB+): m/z calcd for C13H25NO2: 227.1885, found (M + 1): 228.1988.
5-Methyl-3-((3-methylpiperidin-1-yl)methyl)hexanoic acid (8c). 0.04 g (70%) as a yellow oil. 1H-NMR
(CD3OD):
δ 3.61–3.47 (m, 2H), 3.28–3.26 (m, 1H), 3.04 (d, J = 13.2 Hz, 1H), 2.94–2.85 (m, 2H), 2.74–2.59
(m, 1H), 2.53 (dd, J = 16.7 Hz, 1H), 2.36 (dd, J = 16.6, 10.2 Hz, 1H), 2.27–2.17 (m, 1H), 1.94–1.88 (m, 2H),
1.88–1.77 (m, 2H), 1.73–1.64 (m, 1H), 1.15 (dd, J = 7.2, 7.2 Hz, 2H), 1.02–0.99 (m, 3H), 0.95–0.92 (m, 6H).
13C-NMR (CD3OD): δ 180.92 (C-1), 65.05 (C-4), 59.38 (C-5), 53.18 (C-10), 44.79 (C-2), 44.70 (C-2), 44.15
(C-11), 44.13 (C-11), 31.54 (C-8), 30.37 (C-6, C-9), 30.26 (C-3), 26.11 (C-12), 23.05 (C-15), 22.99 (C-15),
22.80 (C-16), 22.77 (C-16), 18.95 (C-7).
(M + 1): 242.2106.
δ HRMS (FAB+): m/z calcd for C14H27NO2: 241.2042, found
5-Methyl-3-((4-methylpiperidin-1-yl)methyl)hexanoic acid (8d). 0.1 g (90%) as a yellow oil. 1H-NMR
(CD3OD): 3.64–3.56 (m, 2H), 3.04–3.00 (m, 1H), 2.88 (dd, J = 13.4, 10.3 Hz, 1H), 2.57–2.48 (m, 1H),
2.35 (dd, J = 16.8, 10.3 Hz, 1H), 2.25–2.14 (m, 1H), 1.91–1.82 (m, 2H), 1.68 (m, 1H), 1.53–1.48 (m, 3H),
1.14 (ddd, J = 7.3 Hz, 2H), 1.01 (d, J = 6.7 Hz, 3H), 0.93 (d, J = 6.7 Hz, 6H). 13C-NMR (CD3OD)
180.86,
δ
δ
64.63, 53.21 (2C), 44.75, 44.07, 32.56 (2C), 30.37, 28.50, 26.05, 22.99, 22.76, 21.24. HRMS (FAB+): m/z
calcd for C14H27NO2: 241.2042, found (M + 1): 242.2121.
5-Methyl-3-(morpholinomethyl)hexanoic acid (8e). 0.1 g (85%) as a yellow oil. 1H-NMR (CD3OD):
3.89–3.87 (m, 4H), 3.29–3.24 (m, 2H), 3.12–3.06 (m, 2H), 3.05–3.00 (m, 1H), 2.88 (dd, J = 13.1, 10.2 Hz),
2.54–2.48 (m, 1H), 2.41 (dd, J = 16.6, 9.6 Hz, 1H), 2.29–2.18 (m, 1H), 1.75–1.62 (m, 1H), 1.16 (dd, J = 3.9
3.9 Hz, 2H), 0.93 (d, J =4.8 Hz, 3H), 0.92 (d, J =4.8 Hz, 3H). 13C-NMR (CD3OD)
180.28, 65.80 (2C),
δ
,
δ
64.92, 53.58 (2C), 43.95 (2C), 29.94, 26.12, 23.03, 22.79. HRMS (FAB+): m/z calcd for C14H27NO2:
229.1678, found (M + 1): 230.1743.

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1
5-Methyl-3-(thiomorpholinomethyl)hexanoic acid (8f). 0.067 g (90%) as a yellow oil. H-NMR (CD3OD):
δ
3.51–3.44 (m, 2H), 3.35–3.29 (m, 2H), 3.11–3.02 (m, 1H), 2.95–2.92 (m, 4H), 2.88–2.80 (m, 1H), 2.50 (d,
J = 16.6 Hz, 1H), 2.39 (dd, J = 16.6, 10.0 Hz, 1H), 2.25–2.16 (m, 1H), 1.68 (sept, J = 8 Hz, 1H), 1.14 (dd,
J = 7.2, 7.2 Hz, 2H), 0.92 (d, J = 5.2 Hz, 3H), 0.9 (d, J = 5.2 Hz, 3H). 13C-NMR (CD3OD)
δ 179.55, 64.35,
54.31 (2C), 43.16, 42.58, 28.83, 25.24 (2C), 24.71, 21.72, 21.41. HRMS (FAB+): m/z calcd for C14H27NO2:
245.1449, found (M + 1): 246.1524.
3-(4-Chlorophenyl)-4-(piperidin-1-yl)butanoic acid (9b). 0.3 g (85%) of a white solid, m.p = 146–149 ◦C.
1H-NMR (CD3OD):
δ
7.34 (d, J = 8.6 Hz, 2H), 7.27 (d, J = 8.6 Hz, 2H), 3.52 (dddd, J = 8 Hz, 8 Hz, 4 Hz,
4 Hz, 1H), 3.37 (dd, J = 14, 8 Hz, 3H), 3.11 (ddd, J = 13.0, 2.0, 1.2 Hz, 3H), 2.91 (dd, J = 16.7, 5.2 Hz, 1H),
2.63 (ddd, J = 13.6, 13, 1.2 Hz, 1H), 1.89–1.82 (m, 4H), 1.64 (sa, 2H). 13C-NMR (CD3OD)
179.28, 142.37,
δ
133.06, 130.11 (2C), 129.81 (2C), 64.71, 54.68, 45.63, 38.12, 24.62 (2C), 22.93. HRMS (FAB+): m/z calcd
for C15H20ClNO2: 281.1183, found (M + 1): 282.1287.
3-(4-Chlorophenyl)-4-(3-methylpiperidin-1-yl)butanoic acid (9c). 0.2 g (52%) as a yellow oil. 1H-NMR
(CD3OD):
δ 7.37–7.26 (m, 4H), 3.76 (d, J = 11.5 Hz, 1H), 3.68 (d, J = 11.8 Hz, 1H), 3.58–3.47 (m, 1H),
3.43–3.36 (m, 2H), 3.08 (d, J = 13.0 Hz, 1H), 2.93 (dd, J = 16.7, 10.8 Hz, 1H), 2.70–2.55 (m, 2H), 2.41
(t, J = 11.7 Hz, 1H), 1.95–1.79 (m, 4H), 1.21–1.09 (m, 1H), 1.01 (d, J = 6.7 Hz, 1H), 0.99 (d, J = 6.7 Hz,
2H). 13C-NMR (CD3OD):
δ 179.60 (C-1), 179.57 (C-1), 142.62(C-11), 134.13 (C-14), 130.23 (C-13), 129.97
(C-12), 64.91(C-4), 64.81 (C-4), 61.19 (C-5), 59.66 (C-5), 54.89 (C-10), 53.48 (C-10), 45.93 (C-2), 38.34
(C-3), 38.19 (C-3), 31.67 (C-8), 31.21 (C-6), 30.85 (C-6), 24.39 (C-9), 24.27 (C-9), 19.13 (C-7), 19.11 (C-7).
HRMS (FAB+): m/z calcd for C16H22ClNO2: 295.1339, found (M + 1): 296.1445.
3-(4-Chlorophenyl)-4-(4-methylpiperidin-1-yl)butanoic acid (9d). 0.26 g (68%) of a yellow solid,
. δ 7.34 (d, J = 8.6 Hz, 1H), 7.27 (d, J = 8.6 Hz, 1H), 3.77 (d,
m.p = 142–144 ◦C 1H-NMR (CD3OD):
J = 11.7 Hz, 1H), 3.54–3.49 (m, 1H), 3.47–3.34 (m, 2H), 3.08 (d, J = 12.9 Hz, 1H), 3.01–2.95 (m, 1H), 2.93
(dd, J = 16, 16 Hz, 1H), 2.78 (dd, J = 11.4, 11.4 Hz, 1H), 2.62 (dd, J = 16, 4 Hz,1H), 1.94–1.85 (m, 2H), 1.69
(m, 1H), 1.59–1.45 (m, 2H), 1.01 (d, J = 6.5 Hz, 3H). 13C-NMR (CD3OD)
δ 179.75, 142.61, 134.00, 130.10
(2C), 129.74 (2C), 64.59, 54.86, 53.36, 46.09, 38.25, 32.84, 32.57, 29.94, 21.22. HRMS (FAB+): m/z calcd for
C16H22ClNO2: 295.1339, found (M + 1): 296.1445
◦
3-(4-Chlorophenyl)-4-morpholinobutanoic acid (9e). 0.28 g (69%) of a yellow solid, m.p = 166–168 C.
1H-NMR (CD3OD):
δ
7.32 (d, J = 8.5 Hz, 2H), 7.25 (d, J = 8.5 Hz, 2H), 3.81–3.78 (m, 4H), 3.5–3.43 (dddd,
J = 4.8, 4.8, 4, 4 Hz, 1H), 3.08–2.98 (m, 3H), 2.94–2.81 (m, 4H), 2.57 (dd, J = 16.2, 4.5 Hz, 1H). 13C-NMR
(CD3OD) 178.35, 142.88, 133.90, 130.13 (2C), 130.00 (2C), 66.77 (2C), 65.41, 54.26 (2C), 43.60 (1C), 38.88.
δ
HRMS (FAB+): m/z calcd for C16H22ClNO2: 283.0975, found (M + 1): 284.1009.
3-(4-Chlorophenyl)-4-thiomorpholinobutanoic acid (9f). 0.288 g (70%) of a white solid, m.p = 144–146 ◦C.
1H-NMR (CD3OD):
δ
7.32 (d, J = 8 Hz, 2H), 7.25 (d, J = 8 Hz, 2H), 3.53–3.44 (m, 1H), 3.37–3.33 (m,
2H), 3.17–3.06 (m, 3H), 2.97–2.85 (m, 2H), 2.87–2.85 (m, 4H), 2.58 (dd, J = 16.3, 3.8 Hz, 1H). 13C-NMR
(CD3OD) 178.90, 142.77, 133.79, 129.92 (2C), 129.89 (2C), 65.79, 55.95 (2C), 44.32, 38.78, 27.23 (2C).
δ
HRMS (FAB+): m/z calcd for C16H22ClNO2: 299.0747, found (M + 1): 300.0811.
3.3. Enzyme Inhibitory Tests
The enzymatic reactions were carried out at final concentrations of 0.2 mM of α-ketoglutarate,
1.25 mM of
pyrophosphate buffer solution, pH = 8.6, at 25 ◦C for 30 min. Once the incubation period was over,
the -NADPH absorbance was measured at = 340 nm. The experiments were carried out at
β-NADP and 0.0209 mg/mL of GABA-AT from Pseudomonas fluorescens in a 0.1 M potassium
β
λ
max
final equimolar concentrations of 0.8 mM for compounds 7a–f, 8a–f and 9b–f, VGB and VPNa versus
0.8 mM of GABA.

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3.4. In Vivo Tests
All experiments were carried out on male CD1 mice with body weights between 20–26 g, which
were kept under the same conditions of ad libitum feeding, natural lightness-darkness cycle and a
temperature at 22 ◦C and four days of adaptation to laboratory conditions. The test compounds and
reference drug (sodium valproate; VPNa) were injected i.p. with volume injection at 1.0 mL/100 g
weight to groups between 6–9 mice [63–66] at 0.50 and 1.00 mmole/kg for 9b and VPNa. Another
group between 6–9 mice served as control, which received the vehicle (phosphate buffer, C = 0.1 M at
pH = 8.0). The assays were carried out at 1 h and 4 h of pretreatment. 1 h or 4 h after, 75 mg/kg of PTZ
were injected subcutaneously with volume injection at 1.0 mL/100 g weight. Each animal was placed
into an individual plastic cage for observation lasting 1 h. Seizures, tonic-clonic convulsions, death
and latency were recorded. At least 80% of the animals in the control group showed convulsions.
3.5. Computational Details
3.5.1. Conformational Analysis and Geometry Optimization
All the molecules in the study (in their neutral and zwitterionic form) were submitted to a
conformational analysis within the molecular mechanics level of theory employing the SYBYL force
field [67]. For geometry optimization semi-empirical calculations using the parametric method
number 3 (PM3) [68] over all the equilibrium conformers of the GABA derivatives were performed.
A further optimization within the density functional theory approach, employing the hybrid functional
B3LYP [69] and the 6–31*G basis set [70] was carried out. A frequency analysis was carried out to
corroborate that we were in a minimum in the potential energy surface. All the calculations were
performed in the SPARTAN 08 program [71]. Mulliken electrostatic partial charges were obtained to
be used further as molecular quantum descriptors.
3.5.2. Molecular Descriptors Calculations
We obtained a total of 209 molecular descriptors derived from the constitutional, topological,
geometrical, functional groups counts, molecular charge and molecular properties families of
descriptors (Table 8). The selection of these molecular descriptor families was based on finding the
structural modifications (functional groups, form, charge distribution and physicochemical properties)
over the GABA scaffold that improves its potency as GABA-AT inhibitors. For these calculations the
DRAGON 05 software was employed [72].
Table 8. Molecular descriptors obtained from DRAGON 05.
Molecular Descriptors
Type
MW, AMW, Sv, Se, Sp, Ss, Mv, Me, Mp, Ms, nAT, nSK, nBT, nBO, nBM, SCBO, ARR, nCIC,
nCIR, RBN, RBF, nAB, nH, nC, nO, nS, nCL, nX, nR06, nBnz
Constitutional
ZM1, ZM1V, ZM2, ZM2V, Qindex, SNar, HNar, GNar, Xt, Dz, Ram, Pol, LPRS, VDA, MSD,
SMTI, SMTIV, GMTI, GMTIV, Xu, SPI, W, WA, Har, Har2, QW, TI1, TI2, STN, HyDp, RHyDp,
w, ww, Rww, D/D, Wap, WhetZ, Whetm, Whetv, Whete, Whetp, J, JhetZ, Jhetm, Jhetv, Jhete, Jhetp,
MAXDN, MAXDP, DELS, TIE, S0K, S1K, S2K, S3K, PHI, BLI, PW2, PW3, PW4, PW5, PJI2,
CSI, ECC, AECC, DECC, MDDD, UNIP, CENT, VAR, BAC, Lop, ICR, D/Dr06, T(N..O),
T(N..S), T(N..Cl), T(O..O), T(O..S), T(O..Cl)
Topological
W3D, J3D, H3D, AGDD, DDI, ADDD, G1, G2, RGyr, SPAN, SPAM, SPH, ASP, FDI, PJI3,
L/Bw, SEig, HOMA, RCI, AROM, HOMT, DISPm, QXXm, QYYm, QZZm, DISPv, QXXv,
QYYv, QZZv, DISPe, QXXe, QYYe, QZZe, DISPp, QXXp, QYYp, QZZp, G(N..O), G(N..S),
G(N..Cl), G(O..O), G(O..S), G(O..Cl)
Geometrical
nCp, nCs, nCt, nCrs, nCrt, nCar, nCbH, nCb-, nRNH2, nRNR2, nROR, nRSR, nArX, nHDon,
nHAcc, C-001, C-002, C-003, C-006, C-024, C-025, C-026, H-046, H-047, H-050, H-052, O-059,
N-066, N-068, Cl-089, S-107
functional group counts
Ui, Hy, AMR, TPSA (NO), TPSA (tot), MLogP, MLogP2, ALogP, ALogP2
Molecular properties
Atomic charge
qpmax, qnmax, Qpos, Qneg, Qtot, Qmean, Q2, RPCG, RNCG, SPP, TE1, TE2, PCWTe, LDI

Molecules 2018, 23, 2984
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3.5.3. QSAR Model Construction and Validation
Mathematical model construction was carried out with the genetic algorithms technique (GA) in
the MobyDigs 01 program [73]. All the molecular descriptors of the GABA analogues were used as the
independent variables (X) and percent inhibitory value as the dependent variable (Y). QSAR model
selection was based on its statistical parameters (R2 Q2
, , s, and F) values and that it passed the QUIK,
REDUNDANCY, OVERFITTING and QA2 SYMPTOTIC rules. The final QSAR model was validated with
a test group that consist of 20% of all the molecules in this study, these molecules were randomly
selected. This procedure was done a minimum of ten times.
The QUIK rule allows the rejection of models with high molecular descriptors co-linearity that can
lead to casual correlation [74]. This rule is based on the K multivariate correlation index that measures
the total correlation of a set of variables, defined as:
h
i
λj
1
−
∑
j
j λj
p
∑
K =
j = 1, . . . , p and 0 ≤ K ≤ 1
(4)
2(p−1)
p
where λj is the data from the joint X(n, p) matrix eigenvalues,
number of variables in the model. The total correlation in the set given by the descriptors
response KXY ) should always be greater than that measured only in the set of descriptors (K
n
is the number of objects and
p
is the
X
plus the
X); if
Y
(
KXY − KX < ∆K, the model is rejected; ∆K may have values between 0.0 and 0.05.
The goal of the OVERFITTING rule is to detect models with an excess of “bad” molecular
descriptors. This rule stipulates that: if RN < tN(ε) the model is rejected. The tN(ε) values are
calculated by the following equation:
p.ε − R
p.R
tN(ε) =
(5)
where
p
is the number of descriptors in the model and
ε
values range from 0.01 to 0.1. For this study
ε = 0.01. RN is defined by:
−
P
RN
=
Mj Mj < 0 and − 1 ≤ RN ≥ 0
(6)
(7)
∑
j=1
where Mj is defined by the equation:
Mj =
RjY
p
1
p
1
p
p − 1
−
−
≤ Mj ≥
p
RjY is the absolute value of the regression coefficient between the j-th descriptor and the response
. The parameter RN accounts for an excess of noisy or useless variables and can be thought as a
Y
measure of overfitting. It takes the maximum value equal to zero when non-noisy variables are in
the model.
As mentioned above, to test our model more rigorously in terms of the predictive aspect, an
evaluation of the estimated error was performed on all the molecules that were excluded from the
training process. To evaluate the internal predictive ability of our models, the leave one out technique
was employed (Q2LOO). This technique consists of evaluating each molecule within the training set to
determine as to how it corresponds to the validation set [75]. Each individual molecule is evaluated,
and its Y value is predicted:
ꢂ
ꢃ
2
n
yi − yˆi
∑
/i
i=1
n
Q2LOO = 1 −
(8)
i=1(yi − y)2
∑

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Another form to evaluate the predictive ability of our mathematical model is by the asymptotic
squared Q rule (Q2ASYMPTOTIC ) which states that a model is predictive if Q2LOO − Q2ASYM > ∆Q, where
∆Q values range from −0.005 up to 0.005. Q2ASYM is defined as:
ꢄ
ꢅ
2
ꢀ
ꢁ
n
Q2ASYM = 1 − 1 − R2
.
(9)
n − p´
where n is the number of objects and p´ is the number of variables in the model.
In addition, the standard deviation ( ) and Fisher test ( ) values were calculated, where the
parameter contains information about the correlation between the experimental and calculated
s
F
s
activities and the number of molecules in the study.
(
yˆ − y )2
n
∑
i
i
i=1
s =
(10)
n − 2
n
represents the number of molecules in the study, yˆi represents the experimental activity and yi is the
calculated activity from the QSAR model.
Meanwhile, parameter, F is related to the probability for the mathematical model to casually occur:
yˆ − y)2/d f
n
(
(
∑
∑
i
M
i=1
F =
(11)
yˆ − y )2/d f
n
i
i
E
i=1
d fM and d fE refer to the molecular descriptors of the model and error, respectively. Also, yˆi
,
y
and yi
refer to the experimental, average value of experimental and calculated activities, respectively. In the
mathematical model, and should have the smallest and largest possible values, respectively, to
s
F
ensure that the QSAR model is reliable [76].
3.5.4. Homology Structural Modeling and Refinement
Given that a full 3D structure of the GABA aminotransferase of human and P. fluorencens is
unavailable, we generated homology structural models. We used GABA aminotransferase structures
available in the Protein Data Bank server (PDB) as structural templates (Table 9) with the MODELLER
9.18 program [77]. According to the sequence alignment of GABA aminotransferases (Figure S1), those
of bacteria are similar amongst themselves, and the same is true for animal GABA aminotransferases
(human and wild boar).
For the model of human GABA-AT we used the crystallographic structure 4y0h of wild boar,
on account of its resolution (1.63 Å) and a higher sequence identity (~95%) to the human enzyme.
The chains A and B were used to build the monomers. Once done, the dimer was assembled based in
the same structure of 4y0h.
Table 9. GABA aminotransferase crystal structures available in PDB server.
% Identity Respect to
PDB_ID
Organism
% Identity Respect to Human
Resolution (Å)
P. fluorencens
4zsw
4y0h
4y0i
1ohv
1ohw
1sf2
1.70
1.63
1.66
2.30
2.30
2.40
1.80
Wild boar
Wild boar
Wild boar
Wild boar
Wild boar
E. coli
95.46
95.46
95.46
95.46
95.46
26.35
27.99
27.52
27.52
27.52
27.52
27.52
73.82
42.79
4ffc
M. abscessus
The model of P. fluorencens GABA-AT was performed using the 1sf2 crystallographic structure
of E. coli. We used this structure instead of the M. abscessus, due a better conservation with respect of

Molecules 2018, 23, 2984
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E. coli aminotransferase (~74% of identity). Again, the A and B chains were used to build the monomers
and the dimer was assembled according to the 1sf2 structure.
In the case of the bacterial GABA aminotransferases, both crystallographic structures of
Mycobacterium and E. coli are tetrameric structures instead of dimers as in animals; however, the
catalytic site is in the interface of each dimer instead of tetramer interface, suggesting that dimer is the
minimal catalytic form. This agrees with experimental studies suggesting that the dimer of bacteria is
functional [27]; therefore, we used the bacterial dimer in this work.
Finally, to optimize the complete structures of GABA-AT models, we used the CHARMM36
force field within CHARMM38b1 [78] to add hydrogen atoms and carry out a 100 step steepest
decent minimization fixing all heavy atoms, followed by a conjugate gradient minimization of the
full structure to remove bad contacts and clashes. These last protein conformations obtained were
employed to carry out the molecular docking calculations.
To incorporate the prosthetic group (PLP) in the homology models an alignment employing a
crystal structure that possessed the PLP was done (3r4t and 1ohw for the Pseudomonas and human
models respectively). Additionally, from this alignment the Fe2/S2 (inorganic) cluster, present in the
crystal structures of wild boar as a cofactor, was added to the human GABA-AT model.
3.5.5. Molecular Docking Calculations
Molecular docking calculations of all the GABA analogues (including R & S enantiomers
for molecules of series 2 and 3) over Pseudomonas and human GABA-AT structures, generated
in the homology modeling process, were done in Molegro Virtual Docker (MVD) 6.0 software
(Redwood, CA, USA) [79,80]. The cavities were detected by the expanded Van der Waals sphere
method. The calculations were performed over the catalytic site with a cavity volume of 137.73 Å3 and
339.46 Å3 for the Pseudomonas and human model respectively. The cavity selection was based on the
results of the high molecular similarity with the endogenous ligand. We employed the minimum energy
geometries of all the GABA derivatives in their zwitterionic form, and the Mulliken partial charges
obtained by the DFT calculations. The residues within 6 Å were set as flexible, 2000 minimization
steps for each flexible residue and 2000 steps of global minimization per run were set. The MolDock
Optimizer search function, based on an evolutionary algorithm, was used. A total of 20 runs with
a maximum of 4000 iterations using a population of 200 individuals (for the Pseudomonas model)
and 400 individuals (for the human model) per run were set. To calculate the interaction energy, we
used the scoring function Moldock Score [GRID]. The scoring function GRID was set at 0.2 Å and
the search sphere fixed as 15 Å radius. For the energy analysis of the ligand, the electrostatic internal
interactions, internal hydrogen bonds and the sp2-sp2 torsions were considered. The method was
validated by reproducing the experimental binding mode of the reference inhibitor within the 3r4t
crystal structure to validate the Pseudomonas model, and 1ohw and 1ohy to validate the human model,
with a Root Media Square Deviation (RMSD) of 1.7 Å, 1.3 Å and 1.8 Å respectively (Figures S2–S4, see
Supplementary Material).
4. Conclusions
The synthesis of a new series of GABA analogues 7, 8 and 9 substituted at the γ-position with
thiazolidine, piperidine, morpholine and thiomorpholine heterocyclic ring systems, was accomplished
by short and efficient synthetic routes. The chemical modifications achieved by the attachment of these
heterocyclic moieties to the GABA backbone, led to compounds with GABA-AT inhibitory activity.
As compared with the positive control compounds VGB or VPNa, enzymatic inhibition tests carried
out with all analogues, showed that 9b display a 73% inhibition over the GABA-AT enzyme. In vivo
studies carried out with male CD1 mice show that 9b has a dose-dependent neuroprotective effect.
In the number of seizures generated during the observation time, there is a decrease in the number of
seizures at a 0.5 mmole/kg dose. However, at a 1.00 mmole/kg dose, 9b has no protecting effect.

Molecules 2018, 23, 2984
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Our QSAR model allowed us to find the structural parameters of the GABA analogues that
enhance their inhibitory activity over GABA-AT. Molecular docking results confirmed the descriptive
ability of our QSAR model by correlating GABA derivatives molecular descriptors with the interaction
elements that are key to bind into GABA-AT. These results suggest that compound 9b is a good
candidate for further studies. Finally, these methods can be used for the design and further
development of new reversible GABA-AT competitive inhibitors.
Supplementary Materials: The supplementary materials are available online. Figures S1–S40: describe NMR
data for all compounds, Figures S41–S52: computational data, Tables S1–S3: describe computational data.
Author Contributions: J.R.-L., E.T.-G. and M.F.-Z. carried out the synthesis and characterization of all compounds;
J.A.G.-S. carried out the in vitro enzyme inhibitory activity; Á.S. and N.P. carried out the homology modeling of
the GABA-AT enzymes and docking analysis; R.S.R.-H. carried out the QSAR model and supervised all the in
silico analysis.
Funding: This research was funded by [CONACyT, México] grant number [241088].
Acknowledgments: We wish to thank CONACyT of Mexico for graduate student scholarships (266896 and 226849)
awarded to J.R.-L. and E.T.-G., and for the postdoctoral scholarship (C-291229-UAEMOR MOD.ORD./54/2017)
awarded to R.S.R.-H. The authors acknowledge the support of Laboratorio Nacional de Estructura de
Macromoléculas (LANEM) from Centro de Investigaciones Químicas, UAEM (LANEM, CONACyT 251613).
We also thank Laboratorio de Dinámica de Proteínas and the LANCAD for the computing resources used in this
work. We also thank the University of Colima and University of Guanajuato, México for allowing us access to
their computational software.
Conflicts of Interest: The authors state no competing interests.
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