Structural modifications of 4-aryl-4-oxo-2-aminylbutanamides and their acetyl- and butyrylcholinesterase inhibitory activity. Investigation of AChE-ligand interactions by docking calculations and molecular dynamics simulations

Article abstract of DOI:10.1016/j.ejmech.2014.05.008

Congeneric set of thirty-eight 4-aryl-4-oxo-2-(N-aryl/cycloalkyl) butanamides has been designed, synthesized and evaluated for acetyl- and butyrylcholinesterase inhibitory activity. Structural variations included cycloalkylamino group attached to C2 posit

Full text of DOI:10.1016/j.ejmech.2014.05.008

Accepted Manuscript
Structural modifications of 4-aryl-4-oxo-2-aminylbutanamides and their acetyl- and
butyrylcholinesterase inhibitory activity. Investigation of AChE-ligand interactions by
docking calculations and molecular dynamics simulations
Maja D. Vitorović-Todorović, Catherine Koukoulitsa, Ivan O. Juranić, Ljuba M.
Mandić, Branko J. Drakulić
PII:
S0223-5234(14)00422-X
10.1016/j.ejmech.2014.05.008
EJMECH 6964
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 December 2013
Revised Date: 31 March 2014
Accepted Date: 1 May 2014
Please cite this article as: M.D. Vitorović-Todorović, C. Koukoulitsa, I.O. Juranić, L.M. Mandić,
B.J. Drakulić, Structural modifications of 4-aryl-4-oxo-2-aminylbutanamides and their acetyl- and
butyrylcholinesterase inhibitory activity. Investigation of AChE-ligand interactions by docking calculations
and molecular dynamics simulations, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.05.008.
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ACCEPTED MANUSCRIPT

Structural modifications of 4-aryl-4-oxo-2-aminylbutanamides and their acetyl- and
ACCEPTED MANUSCRIPT
butyrylcholinesterase inhibitory activity. Investigation of AChE-ligand interactions by
docking calculations and molecular dynamics simulations
a*
b
c
d
Maja D. Vitorović-Todorović , Catherine Koukoulitsa , Ivan O. Juranić , Ljuba M. Mandić ,
c
Branko J. Drakulić
a
Military Technical Institute, Ratka Resanovića 1, Belgrade, Serbia
b
Department of Chemistry, University of Athens, Panepistimiopolis-Zografou, 15771, Greece
c
Department of Chemistry-IChTM, University of Belgrade, Njegoševa 12, Belgrade, Serbia
d
Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia
Abstract
Congeneric set of thirty-eight 4-aryl-4-oxo-2-(N-aryl/cycloalkyl)butanamides has been designed,
synthesized and evaluated for acetyl- and butyrylcholinesterase inhibitory activity. Structural
variations included cycloalkylamino group attached to C2 position of butanoyl moiety, and
variation of amido moiety of molecules. Twelve compounds, mostly piperidino and imidazolo
derivatives, inhibited AChE in low micromolar range, and were inactive toward BChE. Several
N-methylpiperazino derivatives showed inhibition of BChE in low micromolar or submicromolar
concentrations, and were inactive toward AChE. Therefore, the nature of the cycloalkylamino
moiety governs the AChE/BChE selectivity profile of compounds. The most active AChE
inhibitor showed mixed-type inhibition modality, indicating its binding to free enzyme and to
enzyme-substrate complex. Thorough docking calculations of the seven most potent AChE
inhibitors from the set showed that the hydrogen bond can be formed between amide -NH-
moiety of compounds and -OH group of Tyr 124. The 10 ns unconstrained molecular dynamic
simulation of the AChE-compound 18 complex shows that this interaction is the most persistent.
This is, probably, the major anchoring point for the binding.
Keywords: 4-Aryl-4-oxo-2-aminylbutyramides; Anticholinesterase activity; Mixed type
reversible inhibitors; Docking study; Molecular dynamics
*
Corresponding author. Tel +381 11 3401 255, Fax: +381 2636061
E-mail address: mvitod@chem.bg.ac.rs (M. D. Vitorović-Todorović)

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1
. Introduction
Acetylcholinesterase (AChE) is a carboxylesterase which terminates cholinergic neuro-
transmission, by hydrolyzing neurotransmitter acetylcholine (ACh) in a synaptic cleft. Reversible
AChE inhibition is implicated in a number of disorders, including Alzheimers disease (AD) [1],
Myasthenia gravis [2], glaucoma [3], and can be used as a pretreatment against nerve agents’
intoxications [4,5]. Recently, AChE’s sister enzyme butyrylcholinesterase, also became
important as pharmacological target for AD. BChE is capable to compensate AChE catalytic
functions in synaptic cleft [6,7] and its activity significantly rises during time course in AD [8,9].
Therefore, in designing and testing of reversible inhibitors it is advisable to monitor activity
toward both enzymes.
AChE contains 20 Å deep and narrow gorge, in which five regions that are involved in the
substrate, irreversible and reversible inhibitor binding, can be distinguished (human and electric
eel AChE numbering): (1) catalytic triad residues: Ser 203, His 447, and Glu 334, [10] at the
bottom of the gorge, which directly participate in catalytic cycle, by charge relay mechanism, as
in other serine esterases; (2) oxyanion hole: is an arrangement of hydrogen bond donors which
stabilize the transient tetrahedral enzyme-substrate complex by accommodation of negatively
charged carbonyl oxygen. This region inside the active center is formed by backbone -NH-
groups of amino acid residues Gly 121, Gly 122 and Ala 204 [11,12]; (3) the ‘anionic site’(AS),
where Trp 86 is situated. This residue is conserved in all cholinesterases and it is involved in
orientation and stabilization of trimethylammonium group of AChE, by forming cation-π
interactions [13-16]; (4) acyl pocket comprises two phenylalanine residues, 295 and 297, which
interact with the substrate acyl group. They form the clamps around methyl group, and decrease
its degrees of freedom [17]; (5) peripheral anionic site (PAS) [18-20], comprises residues which
are located at the rim of the active site gorge, Tyr 72, Tyr 124, Trp 286 and Asp 74. Possible
binding sites for the reversible inhibitors comprise both AS and PAS. The so-called dimeric
(dual) inhibitors bind simultaneously to both of these sites. In BChE active site, six out of
fourteen aromatic amino acid residues in AChE (Tyr 72, Tyr 124, Trp 286, Phe 295, Phe 297 and
Tyr 337) are replaced by aliphatic ones (Asn 68, Gln 119, Ala 277, Leu 286, Val 288, and Ala
3
3
28). This causes 200 Å larger active site gorge of BChE, comparing to that of AChE.
Therefore, BChE is capable to hydrolyze wide variety of esters [21].
Recently, we reported congeneric series of the twenty 4-aryl-4-oxo-2-aminylbutanamides,
with variation of substitutents on the aroyl phenyl ring, while phenylamido moiety was retained

unchanged [22]. Alkyl substituted congeners inhibit th AChE and BChE in micromolar
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concentrations, while chloro- or methoxy- substituted compounds were proved as inactive. The
nature of cycloalkylamino moiety of molecules governed the AChE/BChE selectivity: piperidino
and imidazolo derivatives were active toward AChE, while morpholino derivatives were active
toward BChE.
In the present work, we investigated the influence of the amido moiety variation on AChE
and BChE inhibition potency and selectivity. We systematically changed the cyclic amine used
to obtain precursor aroylacrylic acid amides (3,5-dimethoxyaniline, 4-isopropylaniline and
cyclohexylamine), retaining the favorable alkyl substituents (4-i-Pr, 2,4-di-i-Pr and β-tetralinyl)
on the aroyl part of the molecule. For the Michael addition on the activated double bond, four
amines were used: piperidine, imidazole, morpholine and N-methylpiperazine. Along with this,
two more compounds were synthesized. To test the effects of the presence of highly voluminous
moiety at position C2 of the butanoic scaffold on the AChE and BChE inhibiton activity, we
synthesized addition products of 4-benzylpiperidine on the 4-(4-i-Pr-Ph)-4-oxo-2-butenoic acid
phenylamide, and of 4-(2-N,N-di-Me-aminoethyl)-1-piperazine on the 4-(4-i-Pr-Ph)-4-oxo-2-
butenoic acid 3,5-di-MeO-phenylamide. The last derivative comprises moiety that mimick
trimethylaminoethyl group of the acetylcholine. Overall, thirty-eight compounds were prepared
and their AChE and BChE inhibition activity determined. Possible interactions of the most
potent derivatives with the AChE active site residues were further investigated by docking
calculations and by molecular dynamics.
2
. Results and discussion
2
.1. Chemistry
Synthetic path to 1-38 is given in Scheme 1. Friedel-Crafts acylation of the commercially
available substituted benzenes (A) with maleic acid anhydride (B) yields aroylacrylic acids (C)
23]. Subsequently, acids were converted to acid chlorides in dry THF by phosphorous-
[
oxychloride, then in situ reacted with equimolar amounts of primary amine (3,5-
dimethoxyaniline, 4-isopropylaniline and cyclohexylamine) to give the corresponding amides
(D) [24]. Michael addition of the cyclic amines (piperidine, imidazole, morpholine, N-
methylpiperazine, 4-benzylpiperidine and 4-(2-N,N-di-Me-aminoethyl)-1-piperazine proceeded
smoothly to give target compounds 1-38.
Please put Scheme 1 here

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2
.2. Structure-activity relationships
Inhibition potency of the compounds 1-38 toward AChE and BChE, given as IC values, are
5
0
shown in Table 1, along with potency data of the previously published congeners having
unsubstituted phenylamido moiety (1a-9a) [22]. Due to limited solubility of some derivatives,
we were unable to obtain entire dose-response curve (spanning range of 20-80% of the enzyme
activity inhibition). For those compounds we reported the percent of the residual enzyme activity
at the highest concentration tested.
Please put Table 1 here
Twelve compounds, out of twenty that inhibited AChE activity, were active in low micromolar
concentrations. Most of the piperidino and imidazolo derivatives showed inhibition of AChE in
low micromolar range, while morpholino and N-methylpiperazino derivatives were inactive up to
concentrations of 15-20 µM. Clearly, the presence of bulkier substituents in position C2 of the
butanoyl moiety of molecules reduces anti-AChE activity. Similar trend was also observed with
previously synthesized compounds [22].
3
,5-Dimethoxy substitution at phenylamido moiety of the imidazolo and piperidino
derivatives, in some instances (compare 2 with 2a, and 26 with 8a) only slightly enhanced
inhibition potency, while in others (compare 13 with 4a, 14 with 5a and 25 with 7a) only slightly
diminished inhibition potency. 4-Isopropyl substituted phenylamido moiety in piperidino and
imidazolo derivatives, in most cases (6, 17, 18, 30) slightly improved anti-AChE inhibition
potency, comparing to the unsubstituted counterparts (2a, 4a, 5a and 8a). In some instances, 4-i-
Pr substitution had clearly beneficial influence on anti-AChE activity. This is especially evident
in compounds 19 and 20, in which this type of substitution turns inactive morpholino and N-
methylpiperazino derivatives into active ones, with IC values around 6.5 µM.
5
0
The change of the phenyl moiety to cyclohexyl on amido part of the piperidino and imidazolo
derivatives (9, 10, 33, 34) reduced anti-AChE activity for at least one order of magnitude. This
indicates that amido moiety of the molecules probably interacts with AChE active site residues
involving π electrons of the phenyl ring. The decrease in inhibition potency, due to the presence
of cyclohexyl moiety, was modest only for 2,4-di-i-Pr-substituted piperidino and imidazolo
derivatives 21 and 22, with IC values of 5.44 and 5.94 ꢀM, respectively.
5
0

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Nineteen derivatives influenced BChE activity (expressed as residual enzyme activity at
highest concentration tested, Table 1), but only three compounds had IC50 values in low
micromolar (16 and 24), or submicromolar concentrations (20). All three compounds bear 2,4-di-
i-Pr-substituted aroyl and N-methylpiperazino moieties. We expected that, due to a larger BChE
active site gorge, bulkier substituents on phenylamido moiety will increase potency of
compounds comparing to unsubstituted derivatives, described earlier. Surprisingly, 2,4-di-i-Pr
morpholino derivatives (15 and 19) with 3,5-dimethoxy or 4-isopropyl substitutents on
phenylamido moiety did not appear more potent, comparing to unsubstituted derivative 6a
reported in previous work (IC50 = 2.48 ꢀM). β-Tetralinyl substituted N-methylpiperazino
derivatives 28, 32 and 36, appeared moderately potent, with IC values in the range of 10-20
5
0
ꢀM. All other compounds didn’t produce significant inhibition of BChE activity.
To test the effects of the presence of highly voluminous moiety at position C2 of the butanoic
moiety of molecules on the AChE and BChE inhibiton activity, we synthesized derivative 37, by
the addition of 4-benzylpiperidine. We aimed to test whether this group will cause an increase in
inhibiton potency and whether such compound has different mode of binding to enzyme active
site (to PAS instead to AS), comparing to other derivatives. In compound 38, we introduced
dimethylaminoethyl moiety, a group that mimick trimethylaminoethyl group of ACh.
Unfortunately, both derivatives appeared inactive at the highest concentration tested (20 ꢀM).
The spectrophotometric method of Ellman was used to determine the type of inhibition and
the K for derivative 17, most active toward AChE. Compound exhibited mixed type of
i
inhibition. This behavior was indicated by intersection of double reciprocal lines in the upper left
quadrant of Lineweaver-Burk plot, shown in Figure 1 and Table S1, Supplementary information.
Generally, this type of inhibition indicates binding to free enzyme and enzyme-substrate
complex; that is, a possible binding to the site distant from the active site. The K ’s obtained were
i
0
.52 µM (binding to free enzyme) and 1.93 µM (binding to enzyme-substrate complex).
Please put Figure 1 here
2
.3. Molecular modeling
3
D-QSAR model described in previous article [22] was used to predict potency of compounds
for which we obtained IC values in this study. Fair predictivity was obtained for compounds 2,
50

2
6
, 14, 17, 18, 21, 22, 25, 26 and 30 (r = 0.60, sd = 0.118); while potency of the compound 34
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was overestimated in some extent. Results are given in Table S2 in Supplementary Material.
Along with this, we built independent 3D QSAR model by GRIND-2 descriptors for all
compounds for which IC values toward AChE were obtained, reported in this, and in previous
5
0
study [22]. In this model we also included compounds for which we obtained residual enzyme
activity on defined concentration of the inhibitor (4, 9-12, 19, 20 and 32). For the later subset of
compounds we estimated IC values. For model building all available GRID probes were used,
5
0
DRY (hydrophobic), N1 (HBD), O (HBA) and TIP (shape probe). PLS coefficients plot for the
model obtained with 3LV are shown in Figure 2. Although we didn’t included shape probe in the
model reported in previous work, because this model contained structurally diverse set of
compounds, and good results were obtained using hydrophobic, HBD and HBA probes; DRY-
DRY, N1-N1 and DRY-O blocks, common for both models, resemble each other. DRY-DRY
block is filled with variables positively correlated with potency of compounds, implying positive
contribution of hydrophobic moieties of compounds to activity. Although variables were
expressed for all compounds in the set, higher intensity of variables for the most potent
compounds in this block can be observed, comparing to least potent ones. This implies stronger
hydrophobic interactions that most potent compounds can make with the respective biological
target. Heatmaps of DRY-DRY block are depicted in Figure S3a in Supplementary Material. It
should be noted that we also observed rough positive trend between potency of compounds and
their apolar surface areas calculated from 3D structures (data not shown). In N1-N1 block
majority of variables are negatively correlated with potency of compounds. Heatmaps of N1-N1
block also show higher intensity of variables associated with less potent compounds, comparing
to more potent ones, Figure S3b in Supplementary Material. Variable O-TIP 473 (10.0-10.4 Å),
has the highest positive impact on the overall model. It is expressed for the most active
derivatives and connects MIF’s of HBA probe (O), associated with amido -NH- group, and
MIF’s of shape (TIP) probe associated with substituents on the phenylamido ring (4-i-Pr or 3,5-
di-OMe, Figure S4 and Table S5, Supplementary Material). For compounds with unsubstituted
phenylamido moiety TIP node is associated with substituents on aroyl phenyl ring. The 5LV
PCA model explains about 55 % variability in the set (Table S6, Supplementary Material).
2
2
Statistics of the model is shown in Table 2. Reported r and q values were obtained after one
cycle of the variable selection by fractional factorial design. Experimental and calculated p(IC )
5
0
values, obtained with 3 LV, are shown in Table S5 in Supplementary Material.
Please put Figure 2 here
Please put Table 2 here

To explore possible ligand-AChE interactions, seven compounds with IC values below 5
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50
ꢀM were docked into the AChE active site. Both enantiomers of the each compound were
docked in mAChE (Mus musculus, PDB entry 2HA2), using the latest version of AutoDock 4
package [25]. Fifty binding poses were generated in each docking calculation. As the scoring
functions used for prioritization of the obtained poses are still mostly inaccurate, [26], we
decided to analyse the most favorable binding pose (according to the estimated free energy of
binding, ꢀG) along with the best pose, belonging to the most populated cluster, by LigPLot
program [27]. Detailed data with the list of all ligand-AChE interactions are given in Table 3. It
should be noted that in both types of docking poses (best ranked pose and the most populated
one), for all compounds shown in the Table 3, the hydrogen bond between amide -NH- group of
the compounds and -OH group of Tyr 124 side chain is formed. Also, in significant number of
poses, a hydrogen bond between Phe 295 backbone -NH- group and different moieties of ligands
(
phenylamido -OCH group, N3 nitrogen of imidazole ring, or aroyl carbonyl group) is formed.
3
The residues which form hydrophobic interactions with docked compounds, in majority of poses,
are His 447, Tyr 341, Phe 295, Phe 297 and Phe 338.
Please put Table 3 here
The overall description of the docking poses for all compounds is beyond the scope of this
article. In the following lines we will provide detailed discussion of the obtained docking
solutions for the three most potent derivatives, 6, 17 and 18, shown in the Figure 3. The S
enantiomers are colored cyan and the R are colored red. Surprisingly, we found that R and S
enantiomers of the each compound were docked in a mutually very similar fashion. The RMSD
differences between atomic positions of docked poses of two enantiomers were less than 2.077 Å
(
heavy atom superposition), Table S7 in Supplementary Material. In the Figure 3a and 3b the
docking solutions best ranked by the estimated free energy of binding (ꢀG), for the compounds
7 and 18, are shown. Both enantiomers of both compounds are oriented within the AChE active
1
site gorge in the similar fashion. The aroyl moieties are oriented toward the bottom of the gorge,
interacting with Trp 86 and Tyr 337 residues. Amido -NH- groups of the compounds form
hydrogen bond with the side chain -OH group of Tyr 124. Phenylamido rings of both compounds
are directed toward the entrance of the gorge (PAS) and interact with side chains of Trp 286, Phe
2
95 and Phe 297. The piperidine ring of compound 17 and the imidazole ring of compound 18
are found in the close proximity of catalytic triad residues, His 447 and Ser 203. Nitrogen N3 of
the imidazole ring of both enantiomers of compound 18 forms hydrogen bond with side chain
oxygen of Ser 203. Aroyl phenyl ring and the corresponding cycloalkylamino groups are also

found in the vicinity of oxyanion hole residues, Gly 121 a Gly 122. In the Figure 3c and 3d the
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best docking poses, that belong to the most populated cluster, for the compounds 17 and 18 are
shown. The orientation of the ligands is reversed, comparing to the energetically favored poses
(
Fig 3a and 3b). Aroyl moieties of the ligands are accommodated at the entrance of the gorge
PAS), interacting with aromatic side chains of Trp 286 and Tyr 72. Phenylamido rings of the
(
both compounds are directed toward the bottom of the AChE active site gorge and interact with
the side chains of Tyr 337, Phe 338 and Trp 86 residues. The hydrogen bond between amido -
NH- group and the side chain -OH of Tyr 124 is retained. The important difference between
energetically favorable and the most populated docking poses for compounds 17 and 18 lies in
the fact that ligands are found closer to the entrance of the gorge in the poses which belong to the
most populated cluster. The upper part of the AChE active site gorge is wider, and can easily
accommodate voluminous diisopropyl-substituted aroyl groups of 17 and 18, in a variety of
slightly different, but essentially similar conformations. Most probably because of this, the
search algorithm was able to find more similar poses belonging to the same cluster. One
important consequence of this is that cycloalkyl amino moieties of 17 and 18 are found near acyl
pocket residues Phe 295 and Phe 297, instead near catalytic triad residues His 447 and Ser 203,
as observed in the best ranked docking pose; therefore, in the most populated pose of 18, N3
nitrogen of the imidazole ring forms hydrogen bond with backbone -NH- group of Phe 295.
However, in both classes of poses, the hydrogen bond between amido -NH- group and side chain
-OH group of Tyr 124 existed.
Docking poses of the both enantiomers of compound 6 are shown in Fig. 3e. Cluster which
comprised the best ranked pose (according to the estimated ꢀG of binding) was also the most
populated one. Both enantiomers were docked almost identically into the AChE active site
gorge, and established the same type of interactions with amino acid side chains of the enzyme
(see Table 2.). The estimated free energies of binding for the both enantiomers appeared very
similar, –11.15 and –11.11 kcal/mol, for S and R, respectively. Phenylamide rings of both
enantiomers are directed toward the bottom of the AChE active site gorge, and form hydrophobic
interactions with aromatic side chains of Phe 338, Tyr 337 and Trp 86. The aroyl phenyl rings of
the both R and S enantiomers are directed toward the entrance of the gorge, and interact with Tyr
7
2, Asp 74 and Trp 286 (residues that belong to PAS). Oxygen of the aroyl carbonyl group forms
hydrogen bond with the backbone -NH- group of Phe 295. For this compound, only few poses
out of 50) were found with the aroyl phenyl ring directed toward bottom of the gorge, but with
(
considerably lower estimated free energy of binding. So, when there are no voluminous 2,4-
diisopropyl groups at aroyl moiety, which can establish important hydrophobic contact with the
amino acid residues in the active site of AChE, and give rise to the energetically most favored

(but poorly populated) docking poses (as in case of compounds 17 and 18), the most populated
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pose becomes also the energetically favored one, with phenylamido moiety directed toward
bottom of the gorge, and aroyl phenyl ring directed toward the entrance of the gorge.
Please put Figure 3 here
The docking studies provided valuable insight into possible interactions between the three
most active derivatives (6, 17 and 18) and AChE active site residues. From exhaustive analysis
of the docking poses it can be shown that there are two possible orientations of the ligands within
the AChE active site gorge. One with aroyl moiety directed toward the bottom of the gorge, and
the other with aroyl moiety directed toward the entrance of the gorge. The later one, in most of
the solutions, belonged to the most populated cluster of conformations. Majority of the
derivatives, in both classes of docking poses showed hydrogen bonding with Tyr 124 residue.
Protein flexibility was not accounted in our docking study. The knowledge on the non-
covalent contacts between enzyme and ligand over time may provide additional information
about most persistent interactions and the stability of the protein-ligand complexes. We
performed the unconstrained, 10 ns molecular dynamics simulation, starting from the most
populated docking pose of the S enantiomer of compound 18. The movie, obtained from
trajectory is given as Supplementary Material. The most important distances between AChE
active site residues and the ligand obtained from simulation are shown in Figure 4. During the
simulation, ligand moves toward the entrance of the gorge, as can be seen by a gradual increase
of the distance between the centroid defined on the indole ring of Trp 86 and the phenylamido
ring of compound 18 (4 to ~ 12 Å, Fig. 4a). After 5 ns, this distance remains stable until the end
of the simulation. We also monitored the distance between aroyl ring of compound 18 and Trp
2
86 side chain (PAS residue). A major fluctuation of the distance between centroids defined on
Trp 286 indole ring and aroyl phenyl moiety is observed during the first ns of the simulation
from 4.5 to almost 11 Å, Fig 4b, blue squares). After this time, fluctuation of the distance
(
appeared stable, with an average value of 7.5 Å. After 5 ns of simulation, the ligand rotates
around the axis which passes through centroids defined on the aroyl and on the phenylamido
rings, so the distance gradually increases. We also monitored the distance between methyl group
of ortho-i-Pr moiety and centroid defined on the indole ring of Trp 286. The profile of this
distance (Figure 4b, violet dots) is similar to aroyl-Trp 286 distance, and suggests a stable
st
th
CH···π interaction with an average distance of 4 Å, from 1 to 5 ns of the trajectory. As a result
th
th
of ligand rotation, this distance gradually increases from 5 ns to 8 ns, and after this time,
remains stable with average value of 12 Å. Docking calculations suggested a hydrogen bond

formation between backbone -NH- group of Phe 295 and the nitrogen N3 of the imidazole ring
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of compound 18. We monitored this distance during simulation (Fig. 4c, green dots). This
hydrogen bond appeared persistent only at the very beginning of the simulation, in the first 500
ps. From 1 ns to 4 ns of the simulation, the distance gradually increases. After 4 ns, the hydrogen
th
bond between these two groups is formed again, and remains stable to the end of 5 ns. After
this time, the bond breaks, as a result of ligand rotation. As a consequence of ligand rotation,
imidazole ring is directed toward Tyr 124, and hydrogen bond is formed between the N3 of
imidazole ring and the -OH group of Tyr 124 (Fig. 4c, red dots). The existence of this hydrogen
th
st
bond is also evident from 500 ps to the end of 1 ns of the MD trajectory, the same time
interval in which hydrogen bond of imidazole ring N3 and backbone -NH- group of Phe 295
cannot be observed. The distance between the side chain -OH oxygen atom of Tyr 124 and the
amido -NH- of compound 18 is shown in Fig 4d. This H-bond remains stable throughout the
rd
whole 10 ns of MD simulation. Only temporary increase of this distance appeared around the 3 ,
th
th
6
and 8 ns of the simulation. Beyond that, the hydrogen bond is stable, with overall occupancy
of 62.02 %. One additional hydrogen bond was observed, between -OH group of Tyr 72 and
th
carbonyl group of the ligand, from the 7 ns till the end of the simulation, (Fig 4e). According to
the results of this MD simulation, the most persistent, and therefore, probably, the most
important interactions between compound 18 and the AChE active site residues involve
following hydrogen bonds: persistent H-bond between Tyr 124 -OH group and amido -NH-
moiety of the ligand; a fine interplay of hydrogen bonds between imidazole N3 atom of the
compound and the Tyr 124-OH group, or the backbone -NH- group of Phe 295; as well as the
hydrogen bond between aroyl carbonyl group of compound and the Tyr 72 -OH group. Since the
ligand moves slightly toward the entrance of the gorge, and therefore lose interactions with Trp
8
6 residue, we suppose that additional, more voluminous substituents in para-position of
phenylamido moiety will increase the inhibition potency of compounds.
Please put Figure 4 here
To explore possible binding modes for the compounds active toward BChE, and to reveal
possible reasons for the selectivity of compounds toward one or another enzyme, we docked both
enantiomers of compounds 16 and 20 into BChE active site (PDB entry 1P0I). The results are
shown in the Figure 5, S enantiomers are colored cyan and R are colored red. The ligands occupy
the same spatial region of the active site, and interact with BChE amino acid residues primarily
through hydrophobic interactions. The S and R enantiomers of compound 16 have slightly
different modes of binding (Fig 5a). Aroyl phenyl ring of the S isomer of compound 16 is
pointed toward the catalytic triad residues, His 438 and Ser 198. Aroyl carbonyl group forms

hydrogen bond with Ser 198 –OH group. Phenylamido ring is found near anionic site of the
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enzyme, and forms close contact with Asn 83, while the amide bond of S enantiomer of
compound 16 is found near Trp 82. The N-methyl group on the piperazine ring forms CH···π
interaction with Trp 430 side chain. The orientation of the R enantiomer of compound 16
appeared slightly different. Aroyl phenyl ring is situated near Trp 82 residue. The N-
methylpiperazine ring is pointed toward the entrance of the gorge and does not make any
important interactions. The 3,5-dimetoxy substituted phenylamido ring is found near the Thr
1
20.
Both enantiomers of compound 20 have similar binding modes inside BChE active site (Fig.
b). Phenylamido rings are found near catalytic triad residues His 438 and Ser 198. N-
5
methylpiperazino ring makes van der Waals interactions with Tyr 332 residue. Aroyl ring is
situated in the anionic site of the enzyme, and forms hydrophobic interactions with Trp 82. The
ortho-i-Pr group of aroyl ring forms close contacts with Gly 115, Gly 116 and Gly 117
(oxyanion hole residues). Any hydrogen bonding between enantiomers of compound 20 and the
amino acid residues of BChE was not observed.
Please put Figure 5 here
The binding modes of compounds 6, 17 and 18 in the AChE active site are significantly
different from the binding modes of compounds 16 and 20 in the BChE active site. As is
mentioned in the introduction, six out of fourteen aromatic amino acid residues in AChE (Tyr 72,
Tyr 124, Trp 286, Phe 295, Phe 297 and Tyr 337) are replaced by aliphatic ones (Asn 68, Gln
3
1
19, Ala 277, Leu 286, Val 288, and Ala 328) in BChE. This causes 200 Å larger active site
gorge of BChE, comparing to that of AChE. Most probably because of this, compounds 16 and
7 are deeply buried inside of BChE active side gorge, and oriented in way that two rings of the
1
molecules are situated in the bottom of the gorge, one usually directed toward Trp 82 (anionic
site) and the other toward Ser 198 and His 438 (catalytic triad residues). In contrast to this,
compounds 6, 17 and 18 are oriented in a way that one aromatic ring interacts with AS and the
other one with PAS of AChE, with smaller cycloalkylamino moiety accommodated in the middle
of the gorge. This binding mode is probably a consequence of the less voluminous AChE active
site gorge comparing to BChE’s. It is very probable that due to bulkiness of N-methylpiperazine
ring (comparing to imidazole and piperidine ring), compounds 16 and 20 cannot be
accommodated inside AChE active site, and therefore these compounds selectively inhibit
BChE. Along with this, interactions of compounds 16 and 20 with BChE are primarily
hydrophobic by nature (only one hydrogen bond is observed for compound 16), while compound
1
8 establish few important hydrogen bonds with the Tyr 72 and the Tyr 124 residues of AChE;

as perceived from MD simulation. Tyr 72 and the Tyr 124 residues are replaced with Asn 68 and
ACCEPTED MANUSCRIPT
Gln 119 in BChE. Therefore the architecture of the BChE active site is slightly different,
comparing to AChE, and those residues cannot play the same role of hydrogen bond
donors/acceptors as corresponding residues in AChE. Moreover, compounds 6, 17, 18 are less
voluminous comparing to 16 and 20, and probably cannot establish necessary hydrophobic
contact in sufficient extent for the effective binding to the wider BChE active site gorge.
3
. Conclusions
In continuation of our work, we further investigated influence of structural modifications of 4-
aryl-4-oxo-2-aminylbutyramides on AChE and BChE inhibiton activity. We explored the
influence of the amido part of molecules on the AChE/BChE inhibition activity and selectivity,
changing the amido moiety from phenyl to 3,5-dimethoxyphenyl, 4-isopropylphenyl and
cyclohexyl and keeping the favorable alkyl substituents (2,4-di-isopropyl, 4-isopropyl and β-
tetralinyl) on the aroyl part of the molecules. For the Michael addition to the activated double
bond, piperidine, imidazole, morpholine, N-methylpiperazine, 4-benzylpiperidine and 4-(2-N,N-
di-Me-aminoethyl)-1-piperazine were used. Twelve compounds, mostly piperidino and
imidazolo derivatives, inhibited AChE in low micromolar range, and were inactive toward
BChE. Three compounds inhibited BChE in low micromolar or submicromolar concentrations.
All three compounds bear 2,4-diisopropyl substituted aroyl moiety and a N-methylpiperazine
ring at position C2 of butanoic part of molecule. As with previous set of compounds, the nature
of cycloalkylamino moiety governed the AChE/BChE selectivity of the compounds. The most
active AChE inhibitor, compound 17, exhibited mixed type reversible inhibition, indicating its
binding to free enzyme and enzyme-substrate complex. The docking study, performed with the
seven derivatives having IC values below 5 µM, showed that for all compounds a hydrogen
5
0
bond between amido -NH- group of compounds and Tyr 124 -OH group can be formed. The
unconstrained, 10 ns molecular dynamics simulation of the complex between AChE and
compound 18, showed that most persistent ligand-protein interaction is hydrogen bond between
Tyr 124 -OH group and amido -NH- moiety of compound. Few additional hydrogen bonds were
also found. Although moderately active, reported compounds will be used as templates for
further structural modifications, in order to find structural requirements needed for higher
potency.
4
. Experimental
4
.1. Chemistry

ACCEPTED MANUSCRIPT
All chemicals were purchased from Sigma-Aldrich or Merck, and were used as received. Dry
CH Cl was used for Friedel-Crafts acylations. Melting points were determined in open capillary
2
2
1
13
tubes on SMP-10 Stuart apparatus, and are uncorrected. H and C NMR spectra were recorded
in CDCl on Varian Gemini 200/50 MHz or Bruker AVANCE 500/125 MHz instruments. NMR
3
spectra of representative compounds are shown in Figure S7, Supplementary Material. Chemical
shifts are reported in parts per million (ppm) relative to tetramethylsilane (TMS) as internal
standard. Spin multiplicities are given as follows: s (singlet), d (doublet), t (triplet), m
(
multiplet), or br (broad). The HR ESI-MS spectra were recorded on Agilent Technologies 6210-
210 TOF-LC-ESI-MS instrument in positive mode. Samples were dissolved in MeOH. All
compounds prove > 98% purity, as obtained by instrumental methods of analysis.
1
4
.2. Characterization of the compounds 1-38
4
.2.1. N-(3,5-Dimethoxyphenyl)-4-(4-isopropylphenyl)-4-oxo-2-(R,S)-(1-piperidinyl)butanamide
(1).
C H N O , starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid (3,5-dimethoxy-
26
34
2
4
phenyl)amide (0.85 mmol) and a corresponding amount of piperidine, 0.179 g of 1 was obtained,
1
4
1
8.05% yield, light yellow solid, m.p. = 111-113 ºC, (toluene). H NMR (200 MHz, CDCl ) δ:
3
.28 (d, 6H, J=7.59 Hz, i-PrCH ); 1.47 (m, 2H, piperidinyl -CH -); 1.63 (m, 4H, piperidinyl -
3
2
CH -); 2.57 (t-like, 4H, J=5.16 Hz, piperidinyl -CH -); 2.93-3.04 (overlapped m, 2H, i-PrCH and
2
2
ABX); 3.66 (dd, 1H, J =6.37 Hz, J =9.40 Hz, ABX); 3.77 (s, 6H, -OCH ); 4.26 (dd, 1H,
1
,2
1,3
3
J =5.16 Hz, J =9.41 Hz, ABX); 6.22 (t-like, 1H, J =4.25 Hz, amido-p-phenyl); 6.79 (s, 1H,
1
,2
1,3
1,2
amido-o-phenyl); 6.80 (s, 1H, amido-o-phenyl); 7.33 (d, 2H, J=8.50 Hz, aroyl-m-phenyl); 7.97
1
3
(
d, 2H, J=8.50 Hz, aroyl-o-phenyl); 9.47 (s, 1H, amido -NH-). C NMR (50 MHz, CDCl ) δ:
3
2
1
4
2.38; 22.58; 23.60; 23.87; 26.71; 31.85; 34.16; 44.48; 51.00; 55.35; 96.32; 97.36; 126.65;
28.51; 134.81; 139.61; 154.54; 161.04; 170.60; 198.22. ESI-MS HR: 439.2587 (M +1), Calc.
39.2597.
4
.2.2. N-(3,5-Dimethoxyphenyl)-4-(4-isopropylphenyl)-4-oxo-2-(R,S)-(1-imidazolyl)butanamide
(2).
C H N O , starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid (3,5-dimethoxy-
24
27
3
4
phenyl)amide (0.85 mmol) and a corresponding amount of imidazole, 0.249 g of 2 was obtained,
1
6
9.55 % white solid, m.p. = 150-151 ºC (toluene). H NMR (200 MHz, CDCl ) δ: 1.24 (d, 6H,
3
J=6.71 Hz, i-PrCH ); 2.93 (m, 1H, J =7.05 Hz, J =14.11 Hz, i-Pr-CH); 3.50 (dd, 1H, J =5.64
3
1,2
1,3
1,2
Hz, J =18.11 Hz, ABX); 3.66 (s, 6H, -OCH ); 4.08 (dd, 1H, J =7.29 Hz, J =18.11 Hz,
1
,3
3
1,2
1,3

ABX); 5.66 (t, 1H, J = 6.35 H ABX); 6.18 (t like, 1H, J
ACCEPTED MANUSCRIP1,T2 =2.35 Hz, amido-p-phenyl); 6.81
z,
1
,2
(s, 1H, amido-o-phenyl); 6.82 (s, 1H, amido-o-phenyl); 7.16 (s, 1H, imidazolyl-CH-); 7.21 (s,
1
H, imidazolyl-CH-); 7.26 (d, 2H, J=8.00 Hz aroyl-m-phenyl); 7.64 (s, 1H, imidazolyl-CH-);
1
3
7
.84 (d, 2H, J=7.86, Hz aroyl-o-phenyl); 10.30 (s, 1H, amido -NH-). C NMR (50 MHz, CDCl )
3
δ: 23.49; 34.20; 41.73; 55.19; 56.75; 97.17; 98.12; 118.04; 126.83; 128.42; 129.44; 133.57;
1
37.41; 139.70; 155.60; 160.86; 166.56; 195.84. ESI-MS HR: 422.2074 (M +1), Calc. 422.2080.
4
.2.3. N-(3,5-Dimethoxyphenyl)-4-(4-isopropylphenyl)-4-oxo-2-(R,S)-(4-morpholinyl)butan-
amide (3).
C H N O , starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid (3,5-dimethoxy-
25
32
2
5
phenyl)amide (0.82 mmol) and a corresponding amount of morpholine, 0.275 g of 3 was
1
obtained, 76.01 % yield, light yellow solid, m.p. = 155-157 ºC (toluene). H NMR (200 MHz,
CDCl ) δ: 1.27 (d, 6H, J=7.46 Hz, i-PrCH ); 2.64 (t-like, 4H, J =4.00 Hz, morpholino-CH -);
3
3
1,2
2
2
.90-3.04 (overlapped m, 2H, i-PrCH and ABX); 3.64-3.86 (overlapped m, 11H, morpholino-
CH -, ABX and -OCH ); 4.27 (dd, 1H, J =5.14 Hz, J =7.14 Hz, ABX); 6.23 (t like, 1H, J
1,2
2
3
1,2
1,3
=2.82 Hz, amido-p-phenyl); 6.78 (s, 1H, amido-o-phenyl); 6.79 (s, 1H, amido-o-phenyl); 7.33 (d,
2
H, J=8.46 Hz, aroyl-m-phenyl); 7.96 (d, 2H, J=7.81 Hz, aroyl-o-phenyl); 9.22 (s, 1H, amido–
1
3
NH-). C NMR (50 MHz, CDCl ) δ: 23.54; 32.10; 34.14; 49.96; 55.30; 65.02; 67.30; 96.45;
3
9
7.46; 129.69; 128.45; 134.59; 139.34; 157.74; 161.04; 169.73; 197.74. ESI-MS HR: 441.2389
(M +1), Calc. 441.2389.
4
.2.4. N-(3,5-Dimethoxyphenyl)-4-(4-isopropylphenyl)-4-oxo-2-(R,S)-[1-(4-methylpiperazinyl)]-
butanamide (4).
C H N O , starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid (3,5-dimethoxy-
26
35
3
4
phenyl)amide (0.82 mmol) and a corresponding amount of 4-methylpiperazine, 0.159 g of 4 was
1
obtained, 42.68 % yield, white solid, m.p. = 120-122 ºC (toluene). H NMR (200 MHz, CDCl )
3
δ: 1.27 (d, 6H, J=7.36 Hz, i-PrCH ); 2.32 (s, 3H, N-CH ); 2.52 (br, 4H, piperazine-CH -); 2.67
3
3
2
(
br, 4H, piperazine-CH -); 2.93-3.08 (overlapped m, 2H, ABX and i-PrCH ); 3.63-3.77
2
3
(
overlapped signals, 7H, ABX and -OCH ); 4.34 (dd, 1H, J =5.10 Hz, J =6.86 Hz, ABX);
3 1,2 1,3
6
.23 (br, 1H, amido-p-phenyl); 6.79 (s, 1H, amido-o-phenyl); 6.80 (s, 1H, amido-o-phenyl); 7.32
(d, 2H, J=8.43, aroyl-m-phenyl); 7.96 (d, 2H, J=8.43, aroyl-o-phenyl); 9.30 (s, 1H, amido -NH-).
1
3
C NMR (50 MHz, CDCl ) δ: 23.58; 31.72; 34.16; 45.88; 55.33; 64.56; 96.28; 97.43; 126.65;
3
1
28.47; 134.70; 139.49; 154.63; 161.04; 170.04; 197.80. ESI-MS HR: 454.2707 (M +1), Calc.
54.2706.
4

4
.2.5.
5).
C H N O , starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid (4-isopropyl-
N-(4-Isopropylphenyl)-4-(4-isopropylphenyl)-4- 2-(R,S)-(1-piperidinyl)butanamide
ACCEPTED MANUSC Ro xI Po -T
(
27
36
2
2
phenyl)amide (0.74 mmol) and a corresponding amount of piperidine, 0.188 g of 5 was obtained,
1
5
1
2
2
9.90 % yield, light yellow solid, m.p. = 106-108 ºC (toluene). H NMR (200 MHz, CDCl ) δ:
3
.22 (d, 6H, J=7.30 Hz, i-PrCH –phenylamide); 1.27(d, 6H, J=7.30 Hz, i-PrCH -aroyl); 1.46 (m,
3
3
H, piperidinyl –CH –); 1.63 (m, 4H, piperidinyl –CH –); 2.57 (m, 4H, piperidinyl –CH –);
2
2
2
.83-3.00 (overlapped m, 3H, i-PrCH and ABX); 3.71 (dd, 1H, J =6.74 Hz, J =16.18 Hz,
1
,2
1,3
ABX); 4.26 (t, 1H, J =5.62 Hz, ABX); 7.16 (d, 2H, J=7.86 Hz, amido-m-phenyl); 7.32 (d, 2H,
1
,2
J=7.86 Hz, aroyl-m-phenyl); 7.46 (d, 2H, J=8.41, Hz amido-o-phenyl); 7.97 (d, 2H, J=7.86 Hz
1
3
aroyl-o-phenyl); 9.39 (s, 1H, amido -NH-). C NMR (50 MHz, CDCl ) δ: 23.58; 23.94; 26.69;
3
3
1
1.87; 33.49; 34.14; 51.02; 66.78; 95.70; 119.31; 126.61; 126.76; 128.49; 134.95; 135.55;
44.55; 154.38; 170.38; 198.37. ESI-MS HR: 421.2838 (M +1), Calc. 421.2855.
4
.2.6. N-(4-Isopropylphenyl)-4-(4-isopropylphenyl)-4-oxo-2-(R,S)-(1-imidazolyl)butanamide (6).
C H N O , starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid (4-isopropyl-
25
29
3
2
phenyl)amide (0.74 mmol) and a corresponding amount of imidazole, 0.251 g of 6 was obtained,
1
8
3.35 % yield, white solid, m.p. = 164-165 ºC (toluene). H NMR (200 MHz, CDCl ) δ: 1.17 (d,
3
6
H, J=7.45 Hz, i-PrCH -phenylamide); 1.24 (d, 6H, J=6.09 Hz, i-PrCH -aroyl); 2.75-3.01
3
3
(
overlapped m, 2H, i-PrCH ); 3.49 (dd, 1H, J =5.65 Hz, J =18.12 Hz, ABX); 4.10 (dd, 1H,
3 1,2 1,3
J =7.13 Hz, J =17.24 Hz, ABX); 5.65 (t, 1H, J =6.24 Hz, ABX); 7.09 (d, 2H, J=8.48 Hz,
1
,2
1,3
1,2
amido-m-phenyl); 7.14 (s, 1H, imidazolyl-CH); 7.21 (s, 1H, imidazolyl-CH); 7.27 (d, 2H,
J=8.47, aroyl-m-phenyl); 7.45 (d, 2H, J=8.48 Hz, amido-o-phenyl); 7.62 (s, 1H, imidazolyl–CH);
1
3
7
2
1
.85 (d, 2H, J=8.47, aroyl-o-phenyl); 9.97 (s, 1H, amido -NH-). C NMR (50 MHz, CDCl ) δ:
3
2.53; 23.91; 33.50; 34.21; 41.75; 56.68; 95.73; 118.02; 120.19; 126.76; 128.42; 129.55; 133.66;
35.50; 137.41; 145.31; 155.53; 166.36; 195.82. ESI-MS HR: 404.2337 (M +1), Calc. 404.2338.
4
.2.7. N-(4-Isopropylphenyl)-4-(4-isopropylphenyl)-4-oxo-2-(R,S)-(4-morpholinyl)butanamide
(7).
C H N O , starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid (4–isopropyl-
26
34
2
3
phenyl)amide (0.89 mmol) and a corresponding amount of morpholine, 7 was obtained, in
1
quantitative yield, as yellow semi-solid. H NMR (200 MHz, CDCl ) δ: 1.22 (d, 6H, J=6.69 Hz,
3
i-PrCH -phenylamide); 1.26 (d, 6H, J=7.31 Hz, i-PrCH -aroyl); 2.64 (t-like, 4H, J=4.26 Hz
3
3
morpholinyl-CH -); 2.79-3.06 (overlapped m, 3H, ABX and i-Pr-CH); 3.66-3.77 (overlapped m,
2
5
H, ABX and morpholinyl-CH -); 4.27 (dd, 1H, J =5.18 Hz, J =9.44 Hz, ABX); 7.16 (d, 2H,
2 1,2 1,3
J=8.83 Hz, amido-m-phenyl); 7.31 (d, 2H, J=7.62, aroyl-m-phenyl); 7.44 (d, 2H, J=8.22 Hz,
1
3
amido-o-phenyl); 7.96 (d, 2H, J=8.52, aroyl-o-phenyl); 9.16 (s, 1H, amido -NH-). C NMR (50

MHz, CDCl ) δ: 23.56; 23.93; 32.14; 33.47; 34.16; 50.00; 65.15; 67.31; 95.70; 119.44; 126.69;
ACCEPTED MANUSCRIPT
3
1
4
26.83; 128.49; 135.24; 144.89; 154.69; 169.55; 197.86. ESI-MS HR: 423.2642 (M +1), Calc.
23.2648.
4
.2.8.
N-(4-Isopropylphenyl)-4-(4-isopropylphenyl)-4-oxo-2-(R,S)-[1-(4-methylpiperazinyl)]-
butanamide (8).
C H N O , starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid (4-isopropyl-
27
37
3
2
phenyl)amide (0.89 mol) and a corresponding amount of 1-methylpiperazine, 0.145 g of 8 was
1
obtained, in quantitative yield, as yellow semi-solid. H NMR (200 MHz, CDCl ) δ: 1.22 (d, 6H,
3
J=7.01 Hz, i-PrCH -phenylamide); 1.26 (d, 6H, J=7.01 Hz, i-PrCH -aroyl); 2.31 (s, 3H,
3
3
piperazine-CH ); 2.87 (m, 1H, J =6.72 Hz, J =13.83 Hz, i-PrCH); 2.87 (overlapped m, 2H,
3
1,2
1,3
ABX and i-PrCH); 2.53 (br, 4H, piperazine-CH -); 2.68 (br, 4H, piperazine-CH -); 3.70 (dd, 1H,
2
2
J =7.29 Hz, J =16.34 Hz, ABX); 4.33 (t-like picks, J =7.04 Hz, ABX); 7.16 (d, 2H, J=8.30
1
,2
1,3
1,2
Hz, amido-m-phenyl); 7.31 (d, 2H, J=8.30, aroyl-m-phenyl); 7.45 (d, 2H, J=8.80 Hz, amido-o-
1
3
phenyl); 7.95 (d, 2H, J=8.05, aroyl-o-phenyl); 9.21 (s, 1H, amido -NH-). C NMR (50 MHz,
CDCl ) δ: 26.63; 23.99; 31.87; 33.55; 34.22; 45.94; 55.62; 64.80; 119.40; 126.66; 126.82;
3
1
4
28.50; 134.87; 135.41; 144.78; 154.54; 169.81; 198.01. ESI-MS HR: 436.2965 (M +1), Calc.
36.2964.
4
.2.9. N-Cyclohexyl-4-(4-isopropylphenyl)-4-oxo-2-(R,S)-(1-piperidinyl)butanamide (9).
C H N O , starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid cyclohexylamide
24
36
2
2
(0.83 mmol) and a corresponding amount of piperidine, 0.160 g of 9 was obtained, 49.74 %
1
yield, yellow semi-solid. H NMR (200 MHz, CDCl ) δ: 1.25 (d, 6H, J=6.71Hz, i-PrCH ); 1.14-
3
3
1
.45 (overlapped m, 8H, cyclohexyl-CH -, piperidinyl-CH -); 1.54-1.66 (overlapped m, 6H,
2 2
cyclohexyl-CH -, piperidinyl-CH -); 1.78-3.77 (br, 2H, cyclohexyl-CH -); 2.50 (t-like picks, 4H,
2
2
2
J=5.97 Hz, piperidinyl-CH -); 2.84 (dd, 1H, J =5.11 Hz, J =16.48 Hz, ABX); 2.95 (m, 1H, i-
2
1,2
1,3
PrCH); 3.63 (dd, 1H, J =7.39 Hz, J =16.19 Hz, ABX); 3.77 (m, 1H, cyclohexyl-CH-); 4.06
1
,2
1,3
(
dd, 1H, J =5.69 Hz, J =9.66 Hz, ABX); 7.30 (d, 2H, J=7.96Hz, aroyl-m-phenyl); 7.95 (d, 2H,
1,2 1,3
1
3
J=8.24Hz, aroyl-o-phenyl); 6.83 (d, 1H, J=13.14 Hz, amido-NH-); C NMR (50 MHz, CDCl )
3
δ: 23.58; 24.02; 24.60; 25.49; 26.60; 32.05; 32.90; 33.12; 34.14; 47.50; 50.91; 65.58; 126.56;
1
28.47; 135.26; 154.14; 171.06. ESI-MS HR: 385.2855 (M +1), Calc. 385.2842.
4
.2.10. N-Cyclohexyl-4-(4-isopropylphenyl)-4-oxo-2-(R,S)-(1-imidazolyl)butanamide (10).
C H N O , starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid cyclohexylamide
22
29
3
2
(0.83 mol) and a corresponding amount of imidazole, 0.148 g of 10 was obtained, 48.14 % yield,
1
orange semi-solid. H NMR (200 MHz, CDCl ) δ: 1.06-1.41 (m, 6H, cyclohexyl-CH -); 1.25 (d,
3
2
6
H, J=7.28Hz, i-PrCH ); 1.61 (m, 2H, cyclohexyl-CH -); 1.83 (br, 2H, cyclohexyl-CH -); 2.95
3 2 2

(
m, 1H, i-PrCH); 3.49 (dd, 1H
A
, JCC
=
E
6.
P
6 8T E
H
D
z, JM A= 1
N
7
U
.9
S
1
C
H
R
z ,I P
A
T
BX); 3.72 (m, 1H, cyclohexyl-CH-
1
,2
1,3
)
; 4.02 (dd, 1H, J =6.67 Hz, J =17.90 Hz, ABX); 5.45 (t-like picks, 1H, J =6.68 Hz, ABX);
1,2
1,3
1,2
6
.65 (d, 1H, J=7.86 Hz, amido-NH); 7.07 (s, 1H, imidazolyl-CH); 7.12 (s, 1H, imidazolyl-CH);
.30 (d, 2H, J=8.50Hz, aroyl-m-phenyl); 7.71 (s, 1H, imidazolyl-CH); 7.86 (d, 2H, J=8.49Hz,
7
1
3
aroyl-o-phenyl); C NMR (50 MHz, CDCl ) δ: 23.51; 24.60; 25.24; 32.48; 34.20; 41.61; 48.82;
3
5
3
6.39; 117.97; 126.81; 128.36; 129.31; 133.75; 137.28; 155.47; 167.34; 195.67. ESI-MS HR:
68.2336 (M +1), Calc. 368.2338.
4
.2.11. N-Cyclohexyl-4-(4-isopropylphenyl)-4-oxo-2-(R,S)-(4-morpholinyl)butanamide (11).
C H N O , starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid cyclohexylamide
23
34
2
3
(0.94 mmol) and a corresponding amount of morpholine, 0.131 g of 11 was obtained, 35.92 %
1
yield, pale orange solid, m.p. = 93-95 ºC (toluene). H NMR (200 MHz, CDCl ) δ: 1.09 (br, 6H,
3
cyclohexyl -CH -); 1.26 (d, 6H, J=11.26 Hz, i-PrCH ); 1.64 (br, 2H, cyclohexyl -CH -); 1.86
2
3
2
(
br, 2H, cyclohexyl -CH -); 2.58 (t, 4H, J =4.57 Hz, morpholino -CH -); 2.86-2.99 (overlapped
2 1,2 2
m, 2H, i-PrCH and ABX); 3.59-3.76 (overlapped m, 6H, ABX, morpholino -CH -, and
2
cyclohexyl -CH-); 4.07 (dd, 1H, J =5.10 Hz, J =7.84 Hz, ABX); 7.05 (d, 1H, J=7.84 Hz,
1
,2
1,3
1
3
amido-NH-); 7.31 (d, 2H, J=8.43, aroyl-m-phenyl); 7.94 (d, 2H, J=8.43, aroyl-o-phenyl). C
NMR (50 MHz, CDCl ) δ: 23.60; 24.66; 25.46; 32.43; 32.92; 33.21; 34.20; 47.74; 50.02; 64.97;
3
6
3
7.31; 126.67; 128.47; 135.01; 154.49; 170.20; 198.31. ESI-MS HR: 387.2639 (M +1), Calc.
87.2648.
4
.2.12. N-Cyclohexyl-4-(4-isopropylphenyl)-4-oxo-2-(R,S)-[1-(4-methylpiperazinyl)butanamide
(12).
C H N O , starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid cyclohexylamide
24
37
3
2
(0.83 mmol) and a corresponding amount of 1-methylpiperazine, 0.045 g of 12 was obtained,
1
1
1
3.46 % yield, yellow solid, m.p. = 101-103 ºC (toluene). H NMR (200 MHz, CDCl ) δ: 1.12-
3
.39 (br, 6H, cyclohexyl -CH -); 1.25 (d, 6H, J=6.97 Hz, i-PrCH ); 1.56-1.64 (br, 2H,
2
3
cyclohexyl -CH -); 1.86 (br, 2H, cyclohexyl –CH -); 2.30 (s, 3H, -N-CH ); 2.42-2.51 (br, 4H,
2
2
3
piperazine -CH -); 2.60 (d-like, 4H, piperazine -CH -); 2.83-2.99 (overlapped m, 2H, i-Pr-CH,
2
2
ABX); 3.63 (dd, 1H, J =7.94 Hz, J =16.21 Hz, ABX); 3.77 (br, 1H, cyclohexyl -CH-); 4.14
1
,2
1,3
(
dd, 1H, J =5.16 Hz, J =8.44 Hz, ABX); 7.11 (d-like, 1H, J=8.21 Hz, amido-NH); 7.30 (d,
1,2 1,3
1
3
2
H, J=8.44 Hz, aroyl-m-phenyl); 7.93 (d, 2H, J=8.44 Hz, aroyl-o-phenyl); C NMR (50 MHz,
CDCl ) δ: 23.56; 24.58; 25.42; 31.90; 32.87; 33.14; 34.12; 45.81; 47.56; 55.42; 64.51; 126.54;
3
1
28.42; 135.06; 154.25; 170.40; 198.48. ESI-MS HR: 400.2959 (M +1), Calc. 400.2964.
4
.2.13. 4-(2,4-Diisopropylphenyl)-N-(3,5-dimethoxyphenyl)-4-oxo-2-(R,S)-(1-piperidinyl)-
butanamide (13).

C H N O , starting from )-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoic acid (3,5-
ACC( EE PTED MANUSCRIPT
29
40
2
4
dimethoxyphenyl)amide (0.76 mol) and a corresponding amount of piperidine, 13 was obtained,
1
in quantitative yield, orange solid, m.p. = 99-101 ºC (toluene). H NMR: (500 MHz, CDCl ) δ:
3
1
.21-1.29 (overlapped m, 12H, i-PrCH ); 1.52 (m, 2H, piperidino -CH -); 1.66 (m, 4H, piperidino
3 2
-
CH -); 2.55 (m, 4H, piperidino -CH -); 2.87-2.96 (overlapped m, 2H, i-PrCH and ABX); 3.45-
2
2
3
.52 (overlapped m, 2H, i-PrCH and ABX); 3.77 (s, 6H, -OCH ); 4.20 (dd, 1H, J =4.69 Hz,
3 1,2
J =8.04 Hz, ABX); 6.22 (t, 1H, J=2.07 Hz, amido-p-phenyl); 6.79 (s, 2H, amido-o-phenyl);
1
,3
7
9
2
1
4
.26-7.27 (overlapped d and s, 2H, aroyl-m-phenyl); 7.63 (d, 1H, J=7.79 Hz, aroyl-o-phenyl);
1
3
.37 (s, 1H, amido -NH-). C NMR (125 MHz, CDCl ) δ: 22.85; 23.16; 23.75; 23.95; 24.42;
3
6.73; 29.13; 34.26; 35.99; 44.50; 55.35; 66.27; 96.37; 97.39; 98.68; 123.16; 124.72; 127.97;
36.51; 139.64; 148.34; 152.00; 161.05; 170.57; 203.13. ESI-MS HR: 481.3075 (M +1), Calc.
81.3066.
4
.2.14. 4-(2,4-Diisopropylphenyl)-N-(3,5-dimethoxyphenyl)-4-oxo-2-(R,S)-(1-imidazolyl)butan-
amide (14).
C H N O , starting from (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoic acid (3,5-
27
33
3
4
dimethoxyphenyl)amide (0.89 mmol) and a corresponding amount of imidazole, 0.220 g of 14
1
was obtained, 52.94 % yield, orange solid, m.p. = 103-105 ºC (toluene). H NMR (200 MHz,
CDCl ) δ: 1.11-1.26 (overlapped m, 12H, i-PrCH ); 2.91 (m, 1H, J =8.69 Hz, J =14.28 Hz, i-
3
3
1,2
1,3
Pr-CH); 3.31-3.50 (overlapped m, 2H, i-PrCH and ABX); 3.68 (s, 6H, -OCH ); 3.98 (dd, 1H,
3
J =7.45 Hz, J =18.25 Hz, ABX); 5.66 (t, 1H, J =7.19 Hz, ABX); 6.20 (br, 1H, amido-p-
1
,2
1,3
1,2
phenyl); 6.81 (s, 1H, amido-m-phenyl); 6.82 (s, 1H, amido-m-phenyl); 7.01-7.10 (overlapped
signals, 2H, aroyl-m-phenyl); 7.17 (s, 1H, imidazolyl -CH-); 7.24 (s, 1H, imidazolyl -CH-); 7.48
1
3
(
d, 1H, J=8.39, aroyl-o-phenyl); 7.70 (s, 1H, imidazolyl -CH-); 9.85 (s, 1H, amido -NH-). C
NMR (50 MHz, CDCl ) δ: 23.62; 23.96; 28.99; 34.27; 45.08; 55.24; 57.19; 97.21; 98.30; 117.97;
3
1
2
23.43; 124.92; 128.38; 128.93; 134.42; 137.41; 139.56; 148.99; 153.21; 160.92; 166.44;
00.50. ESI-MS HR: 464.2532 (M +1), Calc. 464.2549.
4
.2.15.
4-(2,4-Diisopropylphenyl)-N-(3,5-dimethoxyphenyl)-4-oxo-2-(R,S)-(4-morpholinyl)-
butanamide (15).
C H N O , starting from (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoic acid (3,5-
28
38
2
5
dimethoxyphenyl)amide (1.00 mmol) and a corresponding amount of morpholine, 0.235 g of 15
1
was obtained, 48.08 % yield, orange solid, m.p. = 126-128 ºC (toluene). H NMR (200 MHz,
CDCl ) δ: 1.23-1.29 (overlapped m, 12H, i-PrCH ); 2.64 (t-like, 4H, J =3.90 Hz, morpholino -
3
3
1,2
CH -); 2.88-2.98 (overlapped m, 2H, ABX and i-PrCH); 3.41-3.65 (overlapped m, 2H, ABX and
2
–
i-PrCH); 3.77 (overlapped picks, 10H, -OCH and morpholino-CH -); 4.24 (dd, 1H, J =3.89
3 2 1,2
Hz, J =7.24 Hz, ABX); 6.23 (br, 1H, amido-p-phenyl); 6.80 (s br, 2H, amido-o-phenyl); 7.12
1
,3

(d, 1H, J=8.08, aroyl-m-phenyl); 7.28 (s, 1H, aroyl-m-phenyl); 7.65 (d, 1H, J=7.79, aroyl-o-
ACCEPTED MANUSCRIPT
1
3
phenyl); 9.22 (s, 1H, amido -NH-). C NMR (50 MHz, CDCl ) δ: 23.69; 24.14; 24.33; 29.11;
3
3
1
4.23; 36.01; 43.72; 49.98; 55.31; 64.71; 65.66; 67.31; 96.50; 97.44; 103.18; 123.23; 124.77;
28.01; 136.19; 139.38; 148.42; 152.25; 161.08; 169.67; 179.76; 194.87; 196.56; 202.57. ESI-
MS HR: 483.2845 (M +1), Calc. 483.2859.
4
.2.16.
4-(2,4-Diisopropylphenyl)-N-(3,5-dimethoxyphenyl)-4-oxo-2-(R,S)-[1-(4-methylpiper-
azinyl)]butanamide (16).
C H N O , starting from (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoic acid (3,5-
29
41
3
4
dimethoxyphenyl)amide (1.00 mmol) and a corresponding amount of 1-methylpiperazine, 0.298
1
g of 16 was obtained, 59.37 % yield, orange solid, m.p. = 109-111 ºC (toluene). H NMR (200
MHz, CDCl ) δ: 1.23-1.29 (overlapped m, 12H, i-PrCH3); 2.33 (s, 3H, N-CH ); 2.57 (br, 4H,
3
3
piperazine -CH -); 2.67 (br, 4H, piperazine -CH -); 2.85-2.96 (overlapped m, 2H, ABX and i-
2
2
PrCH); 3.42-3.58 (overlapped m, 2H, ABX and i-PrCH); 3.78 (s, 6H, -OCH ); 4.28 (dd, 1H,
3
J =4.59 Hz, J =8.52 Hz, ABX); 6.23 (t-like, 1H, J =2.24 Hz, amido-p-phenyl); 6.79 (s, 1H,
1
,2
1,3
1,2
amido-o-phenyl); 6.80 (s, 1H, amido-o-phenyl); 7.11 (d, 1H, J=8.11, aroyl-m-phenyl); 7.27 (s,
1
3
1
H, aroyl-m-phenyl); 7.64 (d, 1H, J=8.12, aroyl-o-phenyl); 9.22 (s, 1H, amido -NH-). C NMR
50 MHz, CDCl ) δ: 23.71; 24.18; 24.31; 29.12; 34.21; 35.83; 45.85; 55.33; 55.50; 65.20; 96.32;
(
3
9
7.45; 123.31; 124.70; 128.03; 163.35; 139.49; 148.32; 152.10; 161.04; 169.96; 202.81. ESI-MS
HR: 496.3166 (M +1), Calc. 496.3170.
4
.2.17.
butanamide (17).
C H N O , starting from (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoic acid (4-isopropyl-
4-(2,4-Diisopropylphenyl)-N-(4-isopropylphenyl)-4-oxo-2-(R,S)-(1-piperidinyl)-
30
42
2
2
phenyl)amide (0.79 mmol) and a corresponding amount of piperidine, 0.160 g of 17 was
1
obtained, 43.46% yield, orange semi-solid. H NMR (200 MHz, CDCl ) δ: 1.20-1.30 (overlapped
3
d, 18H, i-PrCH ); 1.47 (br, 2H, piperidinyl -CH -); 1.63 (br, 4H, piperidinyl -CH -); 2.56 (t-like
3
2
2
picks, 4H, J=14.06 Hz, piperidinyl -CH -); 2.83-2.95 (overlapped m, 3H, ABX and i-PrCH);
2
3
.41-3.60 (overlapped m, 2H, ABX and i-PrCH); 4.21 (dd, 1H, J =8.96 Hz, J =17.91 Hz,
1,2 1,3
ABX); 7.09 (d, 1H, J=8.95 Hz, aroyl-m-phenyl); 7.17 (d, 2H, J=7.68 Hz, amido-m-phenyl); 7.27
s, 1H, aroyl-m-phenyl); 7.46 (d, 2H, J=8.96Hz, amido-o-phenyl); 7.66 (d, 1H, J=8.96 Hz, aroyl-
(
1
3
o-phenyl); 9.32 (s, 1H, amido -NH-); C NMR (50 MHz, CDCl ) δ: 23.71; 23.96; 24.18; 24.36;
3
2
1
6.69; 29.13; 33.52; 34.21; 36.13; 51.07; 66.06; 119.35; 123.19; 124.63; 126.80; 128.11; 135.61;
44.58; 148.26; 151.94; 170.35; 203.30. ESI-MS HR: 463.3322 (M +1), Calc. 463.3319.

4
.2.18.
butanamide (18).
C H N O , starting from (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoic acid (4-isopropyl-
4-(2,4-Diisopropylphenyl)-N-(4-isopropy nyl)-4-oxo-2-(R,S)-(1-imidazolyl)-
ACCEPTED MANUSCRl pI Ph eT
28
35
3
2
phenyl)amide (0.79 mmol) and a corresponding amount of imidazole, 18 was obtained, in
1
quantitative yield, orange semi-solid. H NMR(200 MHz, CDCl ) δ: 1.12-1.25 (overlapped d,
3
1
3
8H, iPrCH ); 2.83-2.92 (overlapped m, 2H, i-Pr-CH); 3.36 (t-like, 1H, J =6.71 Hz, i-Pr-CH);
3 1,2
.46 (dd, 1H, J =5.25 Hz, J =17.44 Hz, ABX); 4.00 (dd, 1H, J =6.15 Hz, J =17.89 Hz,
1
,2
1,3
1,2
1,3
ABX); 5.69 (br, 1H, ABX); 7.08 (d, 1H, J=7.71Hz, aroyl-m-phenyl); 7.12 (d, 2H, J=8.05Hz,
amido-m-phenyl); 7.17 (s, 1H, imidazolyl -CH-); 7.24 (s br, 1H, imidazolyl -CH-); 7.26 (s, 1H,
aroyl-m-phenyl); 7.44 (d, 2H, J=7.49Hz, amido-o-phenyl); 7.48 (d, 1H, J=8.05 Hz, aroyl-o-
1
3
phenyl); 7.77 (s, 1H, imidazolyl -CH-); 9.32 (s, 1H, amido -NH-); C NMR (125 MHz, CDCl )
3
δ: 23.68; 23.95; 24.07; 29.08; 33.58; 34.31; 45.12; 57.18; 117.93; 120.26; 123.51; 124.93;
1
26.81; 128.31; 134.48; 135.17; 145.53; 148.96; 153.18; 166.18; 200.44. ESI-MS HR: 446.2806
(M +1), Calc. 446.2808.
4
.2.19. 4-(2,4-Diisopropylphenyl)-N-(4-isopropylphenyl)-4-oxo-2-(R,S)-(4-morpholinyl)butan-
amide (19).
C H N O , starting from (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoicacid(4-isopropyl-
29
40
2
3
phenyl)amide (0.79 mmol) and a corresponding amount of morpholine, 19 was obtained, in
1
quantitative yield, orange semi-solid. H NMR (200 MHz, CDCl ) δ: 1.23-1.30 (overlapped d,
3
1
8H, i-PrCH ); 2.64 (t-like picks, 4H, morpholino -CH -); 2.84-2.97 (overlapped m, 3H, ABX
3 2
and i-PrCH); 3.40-3.64 (overlapped m, 2H, i-PrCH and ABX); 3.77 (br, 4H, morpholino -CH -);
2
4
7
8
.24 (dd, 1H, J =4.43 Hz, J =8.07 Hz, ABX); 7.09-7.13 (d-like signal, 1H, aroyl-m-phenyl);
1,2 1,3
.18 (d, 2H, J=8.41 Hz, J= 8.41, amido-m-phenyl); 7.28 (s, 1H, aroyl-m-phenyl); 7.46 (d, 2H, J=
.41 Hz, amido-o-phenyl); 7.65 (d, 1H, J=7.84 Hz, aroyl-o-phenyl); 9.16 (s, 1H, amido -NH-);
1
3
C NMR (50 MHz, CDCl ) δ: 23.69; 23.94; 24.18; 24.29; 29.15; 33.50; 34.21; 36.20; 50.02;
3
6
1
5.44; 67.31; 119.42; 123.25; 124.70; 126.85; 128.13; 128.98; 135.30; 136.21; 144.89; 148.35;
52.19; 169.45; 202.72. ESI-MS HR: 465.3114 (M +1), Calc. 465.3117.
4
.2.20.
4-(2,4-Diisopropylphenyl)-N-(4-isopropylphenyl)-4-oxo-2-(R,S)-[1-(4-methyl-
piperazinyl)]butanamide (20).
C H N O , starting from (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoicacid (4-isopropyl-
30
43
3
2
phenyl)amide (0.79 mmol) and a corresponding amount of 1-methylpiperazine, 20 was obtained,
1
in quantitative yield, orange semi-solid. H NMR (200 MHz, CDCl ) δ: 1.21-1.29 (overlapped d,
3
1
8H, i-PrCH ); 2.32 (s, 3H, N-CH ); 2.53 (br, 4H, piperazine -CH -); 2.67 (br, 4H, piperazine -
3 3 2
CH -); 2.80-2.97 (overlapped m, 3H, ABX and i-PrCH); 3.38-3.61 (overlapped m, 2H, ABX and
2
i-PrCH); 4.28 (dd, 1H, J =4.53 Hz, J =7.76 Hz, ABX); 7.07-7.12 (d-like signals, 1H, aroyl-m-
1
,2
1,3

phenyl); 7.17 (d, 2H, J=8.57
Hz, o-m-phenyl); 7.26 (s, 1H, aroyl-m-phenyl); 7.46 (d, 2H,
AC Ca mE Pi dTED MANUSCRIPT
J=8.57, amido-o-phenyl); 7.63 (d, 1H, J=8.20 Hz, aroyl-o-phenyl); 9.16 (s, 1H, amido -NH-);
1
3
C NMR (50 MHz, CDCl ) δ: 23.71; 23.96; 24.22; 24.29; 29.17; 33.50; 34.21; 36.00; 45.88;
3
5
1
5.52; 65.02; 119.39; 123.25; 124.63; 126.83; 128.18; 135.43; 136.39; 144.75; 148.24; 152.05;
69.75; 202.97. ESI-MS HR: 478.3429 (M +1), Calc. 478.3428.
4
.2.21. N-Cyclohexyl-4-(2,4-diisopropylphenyl)-4-oxo-2-(R,S)-(1-piperidinyl)butanamide (21).
C H N O , starting from (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoic acid cyclohexyl-
27
42
2
2
amide (0.88 mmol) and a corresponding amount of piperidine, 0.151 g of 21 was obtained, 40.22
1
%
yield, white solid, m.p. = 105-107 ºC (toluene). H NMR (200 MHz, CDCl ) δ: 1.09-1.46
3
(
overlapped m, 8H, cyclohexyl -CH - and piperidinyl -CH -); 1.22-1.28 (overlapped d, 12H, i-
2 2
PrCH ); 1.54-1.70 (overlapped m, 6H, piperidinyl-CH - and cyclohexyl-CH -); 1.84 (br, 2H,
3
2
2
cyclohexyl-CH -); 2.47 (t-like, 4H, J=6.97Hz, piperidinyl -CH -); 2.97 (dd, 1H, J =4.65 Hz,
2
2
1,2
J =16.27 Hz, ABX); 2.91 (m, 1H, i-PrCH); 3.35-3.53 (overlapped m, 2H, ABX and i-PrCH);
1
,3
3
.74 (m, 1H, cyclohexyl -CH-); 4.03 (dd, 1H, J =4.59 Hz, J =17.91 Hz, ABX); 7.09 (d, 1H,
1,2 1,3
J=7.83Hz, aroyl-m-phenyl); 7.24 (s, 1H, aroyl-m-phenyl); 7.66 (d, 1H, J=7.83Hz, aroyl-o-
1
3
phenyl); 6.75 (d, 1H, J=13.78 Hz, amido -NH-);. C NMR (50 MHz, CDCl ) δ: 22.31; 22.43;
3
2
5
3.71; 24.00; 24.18; 24.31; 24.58; 25.47; 26.53; 29.12; 32.88; 33.14; 34.18; 36.36; 44.43; 47.41;
0.89; 65.33; 95.73; 123.17; 124.47; 128.14; 136.70; 170.96; 203.83. ESI-MS HR: 427.3323 (M
+1), Calc. 427.3325.
4
.2.22. N-Cyclohexyl-4-(2,4-diisopropylphenyl)-4-oxo-2-(R,S)-(1-imidazolyl)butanamide (22).
C H N O , starting from (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoic acid cyclohexyl-
25
35
3
2
amide (0.88 mmol) and a corresponding amount of imidazole, 0.170 g of 22 was obtained, 47.17
1
%
yield, white solid, m.p. = 123-125 ºC (toluene). H NMR (200 MHz, CDCl ) δ: 1.04-1.35
3
(
overlapped m, 6H, cyclohexyl -CH -); 1.12-1.26 (overlapped d, 12H, i-PrCH ); 1.53-1.69 (br,
2
3
2
H, cyclohexyl -CH -); 1.83 (br, 2H, cyclohexyl -CH -); 2.91 (m, 1H, J =6.67 Hz, J =13.34
2 2 1,2 1,3
Hz, i-Pr-CH); 3.25-3.45 (overlapped m, 2H, ABX and -i-PrCH); 3.73 (m, 1H, cyclohexyl -CH-);
3
1
.96 (dd, 1H, J =6.19 Hz, J =17.98 Hz, ABX); 5.40 (t, 1H, J =6.67 Hz, ABX); 6.32 (s br,
1,2 1,3 1,2
H, amido -NH-); 7.06-7.11 (overlapped m, 2H, aroyl-m-phenyl and imidazolyl -CH-); 7.24 (d-
like, 1H, aroyl-m-phenyl); 7.46 (d, 1H, J=7.81, aroyl-o-phenyl); 7.57 (s, 1H, imidazolyl -CH-);
1
3
C NMR (50 MHz, CDCl ) δ: 26.62; 23.94; 24.04; 24.62; 25.24; 29.01; 32.48; 34.23; 45.19;
3
4
1
8.80; 56.64; 95.73; 117.73; 123.39; 124.85; 128.13; 130.09; 134.70; 137.43; 148.72; 153.01;
67.34; 200.70. ESI-MS HR: 410.2813 (M +1), Calc. 410.2802

4
.2.23. N-Cyclohexyl-4-(2,4-diisopropylphenyl)-4-oxo-2-(R,S)-(4-morpholinyl)butanamide (23).
ACCEPTED MANUSCRIPT
C H N O , starting from (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoic acid cyclohexyl-
26
40
2
3
amide (1.00 mmol) and a corresponding amount of morpholine, 0.372 g of 23 was obtained,
1
8
1
4.55 % yield, white solid, m.p. = 142-144 ºC (toluene). H NMR (200 MHz, CDCl ) δ: 1.11-
3
.41 (br, 6H, cyclohexyl -CH -); 1.23-1.29 (overlapped d, 12H, i-PrCH ); 1.65 (br, 2H,
2
3
cyclohexyl -CH -); 1.86 (br, 2H, cyclohexyl -CH -); 2.56 (t-like, 4H, J =4.40 Hz, morpholino -
2
2
1,2
CH -); 2.78-2.95 (overlapped m, 2H, ABX and i-PrCH); 3.35-3.58 (overlapped m, 2H, ABX and
2
i-PrCH); 3.72 (q-like, 4H, J =3.64 Hz, J =8.80 Hz, morpholino -CH -); 3.79 (m, 1H,
1
,2
1,3
2
cyclohexyl -CH-); 4.05 (dd, 1H, J =4.43 Hz, J =8.07 Hz, ABX); 7.07-7.12 (overlapped m,
1
,2
1,3
aroyl-m-phenyl and amido -NH-); 7.24 (br, 1H, aroyl-m-phenyl); 7.64 (d, 1H, J=7.81, aroyl-o-
1
3
phenyl); C NMR (50 MHz, CDCl ) δ: 23.71; 24.22; 24.29; 24.64; 25.46; 29.19; 32.92; 33.25;
3
3
1
4.21; 36.62; 47.67; 49.96; 64.78; 67.26; 123.26; 124.57; 128.18; 136.41; 148.12; 152.03;
70.09; 203.21. ESI-MS HR: 429.3123 (M +1), Calc. 429.3112.
4
.2.24. N-Cyclohexyl-4-(2,4-diisopropylphenyl)-4-oxo-2-(R,S)-[1-(4-methylpiperazinyl-)]butan-
amide (24).
C H N O , starting from (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-butenoic acid cyclohexyl-
27
43
3
2
amide (1.00 mmol) and a corresponding amount of 1-methylpiperazine, 0.256 g of 24 was
1
obtained, 56.47 % yield, white solid, m.p. = 96-98 ºC (toluene). H NMR (200 MHz, CDCl ) δ:
3
1
.15-1.39 (br, 6H, cyclohexyl -CH -); 1.22-1.27 (overlapped d, 12H, i-PrCH ); 1.65 (br, 2H,
2 3
cyclohexyl -CH -); 1.86 (br, 2H, cyclohexyl -CH-); 2.33 (s, 3H, piperazine-N-CH ); 2.52 (br,
2
3
4
H, piperazine -CH -); 2.60 (br, 4H, piperazine -CH -); 2.76-2.96 (overlapped m, 2H, ABX and
2 2
–
i-PrCH); 3.34-3.55 (overlapped m, 2H, ABX and i-PrCH); 3.76 (m, 1H, cyclohexyl -CH-); 4.10
(
dd, 1H, J =4.50 Hz, J =9.00 Hz, ABX); 7.04-7.11(overlapped m, aroyl-m-phenyl and
1,2 1,3
1
3
cyclohexyl-NH); 7.24 (s, 1H, aroyl-m-phenyl); 7.64 (d, 1H, J=8.04, aroyl-o-phenyl); C NMR
50 MHz, CDCl ) δ: 23.67; 24.18; 24.58; 25.42; 29.15; 32.87; 33.18; 34.16; 36.36; 45.68; 47.56;
(
3
5
4
5.32; 64.31; 123.31; 124.43; 128.14; 136.54; 147.91; 151.81; 170.26; 203.41. ESI-MS HR:
42.3436 (M +1), Calc. 442.3428.
4
.2.25. N-(3,5-Dimethoxyphenyl)-4-oxo-2-(R,S)-(1-piperidinyl)-4-(5,6,7,8-tetrahydronaphtha-
lenyl)butanamide (25).
C H N O , starting from (E)-4-oxo-4-(5,6,7,8-tetrahydronaphthalenyl)-2-butenoic acid
27
43
3
2
(3,5-dimethoxyphenyl)-amide (0.82 mmol) and a corresponding amount of piperidine, 0.239 g of
1
2
5 was obtained, 64.61% yield, pale yellow solid, m.p. = 135 ºC (toluene). H NMR (200 MHz,
CDCl ) δ: 1.48 (q-like signals, 2H, piperidinyl -CH -); 1.65 (q-like signals, 4H, piperidinyl -CH -
3
2
2
)
; 1.81 (br, 4H, tetralinoyl -CH -); 2.57 (t-like signals, 4H, piperidinyl -CH-); 2.82 (br, 4H,
2
tetralinoyl -CH -); 3.00 (dd, 1H, J = 5.06, J1,3 = 16.85, ABX); 3.68 (dd, 1H, J1,2 = 7.30, J1,3
=
2
1,2

1
7.41, ABX); 3.77 (s, 6H, -OCH ); 4. , 1H, J = 5.06, J = 6.74, ABX); 6.22 (t-like
ACCEP T2 5E D( d dM ANUSCRIPT
3 1,2 1,3
signals, 1H, amido-p-phenyl); 6.79 (s, 1H, amido-o-phenyl); 6.80 (s, 1H, amido-o-phenyl); 7.15
(d, 1H, J = 8.99, aroyl-m-phenyl); 7.73 (m, 2H, aroyl-o-phenyl); 7.74 (m, 2H, aroyl-o-phenyl);
1
3
9
2
1
4
.48 (s, 1H, amide -NH-). C NMR (50 MHz, CDCl ) δ: 22.34; 22.54; 22.76; 22.89; 23.89;
3
6.73; 29.32; 29.55; 31.92; 44.50; 51.04; 55.33; 65.57; 96.34; 97.37; 125.30; 129.14; 129.31;
34.42; 137.39; 139.65; 143.16; 161.06; 170.71; 198.40. ESI-MS HR: 451.2594 (M +1), Calc.
51.2591.
4
.2.26.
N-(3,5-Dimethoxyphenyl)-4-oxo-2-(R,S)-(1-imidazolyl)-4-(5,6,7,8-tetrahydronaphtha-
lenyl)butanamide (26).
C H N O , starting from (E)-4-oxo-4-(5,6,7,8-tetrahydronaphthalenyl)-2-butenoic acid
25
27
3
4
(3,5-dimethoxyphenyl)amide (0.82 mmol) and a corresponding amount of imidazole, 0.279 g of
1
2
6 was obtained, 78.49 % yield, white solid, m.p. = 166 ºC. H NMR: (200 MHz, CDCl ) δ: 1.80
3
(
br, 4H, tetralinoyl -CH -); 2.78 (br, 4H, tetralinoyl -CH -); 3.48 (dd, 1H, J = 5.33, J1,3 =
2 2 1,2
1
7.69, ABX); 3.73 (s, 6H, -OCH ); 4.07 (dd, 1H, J = 7.58, J1,3 = 17.41, ABX); 5.58 (t-like
3 1,2
signals, 1H, ABX); 6.17 (sb, 1H, amido-p-phenyl); 6.76 (sb, 2H, amido-o-phenyl); 6.98 (s, 1H,
imidazolyl -CH-); 7.07 (d, 1H, J=8.43Hz, aroyl-m-phenyl); 7.17(s, 1H, imidazolyl -CH-); 7.58
1
3
(
br, 2H, aroyl-o-phenyl); 7.77 (s, 1H, imidazolyl -CH-); 9.99 (s, 1H, amido -NH-). C NMR (50
MHz, CDCl ) δ: 22.47; 22.62; 29.08; 29.46; 41.41; 55.10; 56.63; 96.95; 98.05; 117.87; 124.98;
3
1
28.80; 128.94; 129.40; 133.06; 136.94; 137.59; 139.40; 144.27; 160.75; 196.22. ESI-MS HR:
34.2084 (M +1), Calc. 434.2074.
4
4
.2.27. N-(3,5-Dimethoxyphenyl)-4-oxo-2-(R,S)-(4-morpholinyl)-4-(5,6,7,8-tetrahydronaphtha-
lenyl)butanamide (27).
C H N O , starting from (E)-4-oxo-4-(5,6,7,8-tetrahydronaphthalenyl)-2-butenoic acid
26
32
2
5
(3,5-dimethoxyphenyl)amide (1.00 mmol) and a corresponding amount of morpholine, 0.261 g
1
of 27 was obtained, 52.69 % yield, white solid, m.p. = 179-181 ºC (toluene). H NMR (200
MHz, CDCl ) δ: 1.81 (br, 4H, tetralinoyl -CH -); 2.65 (t-like, 4H, J =4.41 Hz, morpholino -
3
2
1,2
CH -); 2.81 (br, 4H, tetralinoyl -CH -); 3.04 (dd, 1H, J =4.99 Hz, J =16.75 Hz, ABX); 3.64-
2
2
1,2
1,3
3
.89 (overlapped signals, 11H, ABX, -OCH and morpholino -CH -); 4.24 (t-like, 1H, J =5.06
3 2 1,2
Hz, ABX); 6.23 (t-like, 1H, J =2.29 Hz, amido-p-phenyl); 6.78 (s, 1H, amido-o-phenyl); 6.79
1
,2
(s, 1H, amido-o-phenyl); 7.15 (d, 1H, J=9.00 Hz, aroyl-m-phenyl); 7.72 (br, 2H, aroyl-o-phenyl);
1
3
9
5
1
.22 (s, 1H, amido -NH-). C NMR (50 MHz, CDCl ) δ: 22.73; 22.87; 29.32; 29.57; 32.16;
3
0.02; 55.35; 66.02; 67.35; 95.73; 96.48; 97.46; 125.27; 129.14; 129.38; 134.21; 137.48; 139.40;
43.42; 161.08; 169.82; 197.86. ESI-MS HR: 453.2387 (M +1), Calc. 453.2389.

4
.2.28.
hydronaphthalenyl)butanamide (28).
C H N O , starting from (E)-4-oxo-4-(5,6,7,8-tetrahydronaphthalenyl)-2-butenoic acid
N-(3,5-Dimethoxyphenyl)-4-oxo-2-(R,S)-[1-(4-methylpiperazinyl)]-4-(5,6,7,8-tetra-
ACCEPTED MANUSCRIPT
27
35
3
4
(3,5-dimethoxyphenyl)amide (0.82 mmol) and a corresponding amount of 1-methylpiperazine,
1
0
.247 g of 28 was obtained, 64.42 % yield, pale yellow solid, m.p. = 106-108 ºC (toluene). H
NMR (200 MHz, CDCl ) δ: 1.81 (br, 4H, tetralinoyl -CH -); 2.34 (s, 3H, piperazine-N-CH -);
3
2
3
2
.55 (br, 4H, piperazine -CH -); 2.69 (br, 4H, piperazine -CH -); 2.81 (br, 4H, tetralinoyl -CH -
2 2 2
)
; 3.03 (dd, 1H, J =4.96 Hz, J =17.37 Hz, ABX); 3.67 (dd, 1H, J =7.14 Hz, J =17.37 Hz,
1,2 1,3 1,2 1,3
ABX); 3.77 (s, 6H, -OCH ); 4.31 (dd, 1H, J =4.96 Hz, J =9.31 Hz, ABX); 6.23 (br, 1H,
3
1,2
1,3
amido-p-phenyl); 6.79 (s, 1H, amido-o-phenyl); 6.80 (s, 1H, amido-o-phenyl); 7.14 (d, 1H,
J=8.57 Hz, aroyl-m-phenyl); 7.71-7.73 (overlapped signals, 2H, aroyl-o-phenyl); 9.29 (s, 1H,
1
3
amido -NH-); C NMR (50 MHz, CDCl ) δ: 22.73; 22.87; 29.30; 29.55; 31.86; 45.76; 55.35;
3
5
5.50; 64.51; 96.30; 97.50; 125.25; 129.14; 129.33; 134.30; 137.41; 139.52; 143.31; 161.06;
70.07; 198.04. ESI-MS HR: 466.2707 (M +1), Calc. 466.2706.
1
4
.2.29. N-(4-Isopropylphenyl)-4-oxo-2-(R,S)-(1-piperidinyl)-4-(5,6,7,8-tetrahydronaphthalenyl)-
butanamide (29).
C H N O , starting from (E)-4-oxo-4-(5,6,7,8-tetrahydronaphthalenyl)-2-butenoic acid (4-
28
36
2
2
isopropylphenyl)amide (0.86 mmol) and a corresponding amount of piperidine, 0.373 g of 29
1
was obtained, in quantitative yield, as yellow semi-solid. H NMR (200 MHz, CDCl ) δ: 1.22 (d,
3
6
H, J = 7.09, 4-i-PrCH ); 1.47 (q, 2H, J = 5.38, J = 10.75, piperidine -CH -); 1.64 (q, 4H, J
3 1,2 1,3 2 1,2
=
5.37, J1,3 = 10.75, piperidine -CH -); 1.80 (br, 4H, tetralinoyl -CH -); 2.57 (t, 4H, J = 5.07,
2 2 1,2
piperidine -CH -); 2.80 (br, 5H, tetralinoyl -CH - and 4-i-Pr-CH); 2.96 (dd, 1H, J = 5.02, J =
2
2
1,2
1,3
1
9.07, ABX); 3.68 (dd, 1H, J1,2 = 6.91, J1,3 = 16.57, ABX); 4.25 (t, 1H, J1,2 = 5.56, ABX); 7.14
d, 1H, J = 8.51, aroyl-m-phenyl); 7.16 (d, 2H, J = 8.06, amido-m-phenyl); 7.46 (d, 1H, J = 8.51,
amido-o-phenyl); 7.73 (s, 1H, aroyl-o-phenyl); 7.76 (s, 1H, aroyl-o-phenyl); 9.40 (s, 1H, amido -
(
1
3
NH-). C NMR (50 MHz, CDCl ) δ: 22.76; 22.89; 23.94; 26.69; 29.30; 29.53; 31.96; 33.49;
3
5
1
1.04; 65.71; 95.70; 119.29; 125.30; 126.76; 129.13; 134.53; 135.57; 137.34; 143.02; 144.53;
70.44; 198.59. ESI-MS HR: 433.2868 (M +1), Calc. 433.2849.
4
.2.30. N-(4-Isopropylphenyl)-4-oxo-2-(R,S)-(1-imidazolyl)-4-(5,6,7,8-tetrahydronaphthalenyl)-
butanamide (30).
C H N O , starting from (E)-4-oxo-4-(5,6,7,8-tetrahydronaphthalenyl)-2-butenoic acid (4-
26
29
3
2
isopropylphenyl)amide (0.86 mmol) and a corresponding amount of imidazole, 0.100 g of 30
1
was obtained, 28.00 % yield, as yellow solid, m.p. 92°C. H NMR (200 MHz, CDCl ) δ: 1.17 (d,
3
6
H, J1,2 = 7.31, i-PrCH ); 1.78 (br, 4H, tetralinoyl -CH -); 2.75-2.78 (5H, overlapped signals, 4-
3 2

i-Pr-CH- and tetralinoyl -CH2-
.30, J1,3 = 17.41, ABX); 5.64 (dd, 1H, J1,2 = 5.61, J1,3 = 7.30, ABX); 7.08 (d, 2H, J = 8.43,
amido-m-phenyl); 7.13 (br, 2H, imidazolyl -CH-); 7.20 (s, 1H, aroyl-m-phenyl); 7.44 (d, 2H, J =
ACCEPTED MANUSCRIPT
); 3.48 (dd, 1H, J = 5.61, J = 17.97, ABX); 4.08 (dd, 1H, J =
1,2 1,3 1,2
7
8
1
3
1
.42, amido-o-phenyl); 7.60 (br, 1H, imidazolyl -CH-); 7.62 (br, 2H, aroyl-o-phenyl); 9.95 (s,
1
3
H, amido -NH-). C NMR (50 MHz, CDCl ) δ: 22.615; 22.761; 23.908; 29.224; 29.588;
3
3.503; 41.751; 56.699; 118.057; 120.169; 125.122; 126.761; 129.091; 129.492; 133.279;
35.500; 137.394; 137.648; 144.185; 145.277; 166.452; 196.130. ESI-MS HR: 416.2342 (M +1),
Calc. 416.2333.
4
.2.31.
N-(4-Isopropylphenyl)-4-oxo-2-(R,S)-(4-morpholinyl)-4-(5,6,7,8-tetrahydronaphtha-
lenyl)butanamide (31).
C H N O , starting from (E)-4-oxo-4-(5,6,7,8-tetrahydronaphthalenyl)-2-butenoic acid (4-
27
34
2
3
isopropylphenyl)amide (0.86 mmol) and a corresponding amount of morpholine, 0.365 g of 31
1
was obtained, 97.28 % yield, as white solid, m.p. 129-131°C (toluene). H NMR (200 MHz,
CDCl ) δ: 1.22 (d, 6H, J=6.52 Hz, i-PrCH ); 1.81 (br, 4H, tetralinoyl -CH -); 2.65 (t-like, 4H,
3
3
2
J =4.53 Hz, morpholino -CH -); 2.81 (br, 5H, tetralinoyl -CH - and i-PrCH); 3.33 (dd, 1H,
1
,2
2
2
J =5.00 Hz, J =16.92 Hz, ABX); 3.65-3.77 (overlapped m, 5H, morpholino -CH - and ABX);
1
,2
1,3
2
4
.27 (dd, 1H, J =5.72 Hz, J =9.05 Hz, ABX); 7.12-7.19 (overlapped d, 3H, amido-m-phenyl
1,2 1,3
and aroyl-m-phenyl); 7.42 (d, 2H, J=8.61 Hz, amido-o-phenyl); 7.72 (br, 2H, aroyl-o-phenyl);
1
3
9
3
1
.17 (s, 1H, amido -NH-). C NMR (50 MHz, CDCl ) δ: 22.73; 22.85; 23.93; 29.28; 29.53;
3
2.21; 33.49; 50.04; 65.09; 67.33; 119.39; 125.25; 126.81; 129.34; 134.30; 135.30; 137.43;
43.31; 144.86; 169.56; 198.02. ESI-MS HR: 435.2654 (M +1), Calc. 435.2648.
4
.2.32.
N-(4-Isopropylphenyl)-4-oxo-2-(R,S)-[1-(4-methylpiperazinyl)]-4-(5,6,7,8-tetrahydro-
naphthalenyl)butanamide (32).
C H N O , starting from (E)-4-oxo-4-(5,6,7,8-tetrahydronaphthalenyl)-2-butenoic acid (4-
28
37
3
2
isopropylphenyl)amide (1.15 mmol) and a corresponding amount of 1-methylpiperazine, 0.432 g
1
of 32 was obtained, 83.83 % yield, as orange semi-solid. H NMR (200 MHz, CDCl ) δ:1.22 (d,
3
6
H, J=6.60, i-PrCH ); 1.81(br, 4H, tetralinoyl -CH -); 2.32 (s, 3H, piperazine-N-CH ); 2.52 (br,
3 2 3
4
H, piperazine -CH -); 2.68 (br, 4H, piperazine -CH -); 2.80 (br, 4H, tetralinoyl -CH -); 2.96 (t-
2
2
2
like, 1H, J =2.73, ABX); 2.96 (m, 1H, i-PrCH); 3.68 (dd, 1H, J =7.16 Hz, J =16.93 Hz,
1
,2
1,2
1,3
ABX); 4.33 (dd, 1H, J =5.16 Hz, J =7.04, ABX); 7.13 (d, 1H, J=8.11, aroyl-m-phenyl); 7.17
1
,2
1,3
(d, 2H, J=8.52, amido-m-phenyl); 7.46 (d, 2H, J=8.52, amido-o-phenyl); 7.72 (s, 1H, aroyl-o-
1
3
phenyl); 7.73 (d, 1H, J=5.41, aroyl-o-phenyl); 9.24 (s, 1H, amido -NH-). C NMR (50 MHz,
CDCl ) δ:22.76; 22.91; 23.98; 29.31; 29.57; 31.88; 33.52; 44.30; 45.92; 55.59; 64.67; 119.37;
3

1
25.28; 126.83; 129.16; 129.33; 134.44; 135.44; 137.39; 143.20; 144.75; 168.89; 198.27. ESI-
ACCEPTED MANUSCRIPT
MS HR: 448.2966 (M +1), Calc. 448.2964.
4
.2.33. N-Cyclohexyl-4-oxo-2-(R,S)-1-piperidinyl-4-(5,6,7,8-tetrahydronaphtalenyl)butanamide
33):
C H N O , starting from (E)-4-oxo-4-(5,6,7,8-tetrahydronaphtalenyl)-2-butenoic acid
(
25
36
2
2
cyclohexylamide (0.96 mmol) and a corresponding amount of piperidine, 0.200 g of 33 was
1
obtained, 52.35 % yield, as pale yellow solid, m.p. 78° C (toluene). H NMR (500 MHz, CDCl )
3
δ: 1.19 (m, 2H, ax C2, cyclohexyl -CH-); 1.36 (m, 2H, ax C3, cyclohexyl -CH-); 1.44 (m, 1H, ax
C4, cyclohexyl -CH-); 1.55 (m, 4H, C3, piperidine -CH -); 1.60 (m, 1H, eq C4, cyclohexyl -CH-
2
)
; 1.62 (m, 2H, C4, piperidine -CH -); 1.68 (m, 2H, eq C3, cyclohexyl -CH-); 1.80 (d-like signal,
2
4
H, β-tetralinyl); 1.86 (m, 2H, eq C2, cyclohexyl -CH-); 2.49 and 2.54 (m, br, 4H, piperidine -
CH-); 2.80 (d-like signal, 4H, β-tetralinyl); 2.87 (dd, 1H, J1,2 = 5.31, J1,3 = 16.26, ABX); 3.60
dd, 1H, J1,2 = 7.19, J1,3 = 16.26, ABX); 3.75 (d-like signal, 1H, cyclohexyl -CH-); 4.06 (dd, 1H,
J = 5.32, J = 7.04, ABX); 7.13 (d, 1H, J = 8.29, aroyl-m-phenyl); 7.28 (sb, 1H, amido -NH-);
(
1
,2
1,3
1
3
7
2
5
3
4
.72 (overlapped d and s, 2H, aroyl-o-phenyl ; C NMR (125 MHz, CDCl ) δ: 22.61; 22.84;
3
2.88; 22.96; 24.06; 24.63; 25.44; 25.54; 26.65; 29.35; 29.57; 32.12; 32.94; 33.16; 44.66; 47.50;
0.95; 65.45; 125.28; 129.08; 129.21; 134.82; 137.26; 142.78; 171.08; 199.07. ESI-MS HR:
97.2866 (M +1), Calc. 397.2849.
.2.34. N-Cyclohexyl-4-oxo-2-(R,S)-1-imidazolyl-4-(5,6,7,8-tetrahidronaphtalenyl)butanamide
(34):
C H N O , starting from (E)-4-oxo-4-(5,6,7,8-tetrahydronaphtalenyl)-2-butanoic acid
23
29
3
2
cyclohexyl amide (0.96 mmol) and a corresponding amount of imidazole, 0.210 g of 34 was
1
obtained, 57.64 % yield, as white solid, m.p. 98° C (toluene). H NMR (200 MHz, CDCl ) δ:
3
1
.00-1.42 (m, 6H, 3,4-cyclohexyl -CH -); 1.48-1.73 (m, 4H, 2-cyclohexyl -CH -); 1.79 (br, 4H,
2 2
tetralinoyl -CH -); 2.78 (br, 4H, tetralinoyl -CH -); 3.47 (dd, 1H, J = 5.80, J1,3 = 17.88, ABX);
2
2
1,2
3
.61-3.83 (m, 1H, 1-cyclohexyl -CH-); 4.02 (dd, 1H, J1,2 = 8.21, J1,3 = 18.84, ABX); 5.42 (t, 1H,
J1,2 = 5.65, ABX); 6.80 (d-like signal, 1H, amido -NH-); 7.09 (d, 1H, J = 7.20, aroyl-m-phenyl);
1
3
7
.14-7.29 (m, 3H, imidazolyl -CH-); 7.62 (br, 2H, aroyl-m-phenyl). C NMR (50 MHz, CDCl )
3
δ: 22.58; 22.73; 24.60; 25.22; 29.17; 29.53; 32.47; 41.59; 48.74; 56.28; 95.70; 117.89; 125.05;
28.98; 129.40; 129.64; 133.39; 137.25; 137.57; 144.00; 167.42; 195.96. ESI-MS HR: 380.2337
M +1), Calc. 380.2332.
1
(
4
.2.35. N-Cyclohexyl-2-(R,S)-4-morpholinyl-4-oxo-4-(5,6,7,8-tetrahydro-2-naphthalenyl)butan-
amide (35):
C H N O , starting from (E)-4-oxo-4-(5,6,7,8-tetrahydronaphtalenyl)-2-butan cyclohexyl
24
34
2
3
amide (0.96 mmol) and a corresponding amount of morpholine, 0.250 g of 35 was obtained,

1
6
5.12 % yield, as white solid,
m.p. 106-108°C (toluene H
ACCEPTED MANUSC) .R IP TN MR (500 MHz, CDCl ) δ: 1.14-
3
1
.24 (m, 3H, cyclohexyl -CH -); 1.32-1.40 (m, 3H, cyclohexyl -CH -); 1.58-1.62 (m, 1H,
2
2
cyclohexyl-CH -); 1.65-1.71 (m, 3H, cyclohexyl -CH -); 1.81 (m, 4H, β-tetralinyl); 1.87 (m, 2H,
2
2
cyclohexyl -CH -); 2.58 (m, 4H, morpholinyl -CH -); 2.80 (m, 4H, β-tetralinyl); 2.92 (dd, 1H,
2
2
J1,2 = 5.16, J1,3 = 16.64, ABX); 3.63 (dd, 1H, J1,2 = 7.08, J1,3 = 16.45, ABX); 3.68 (m, 1H,
cyclohexyl -CH-); 3.74 (m, 4H, morpholinyl -CH -); 4.06 (dd, 1H, J = 5.16, J = 7.07, ABX);
2
1,2
1,3
1
3
7
.23 (m, 1H, amido -NH-); 7.70-7.71 (overlapped s and d, 2H, aroyl-o-phenyl); C NMR (125
MHz, CDCl ) δ: 22.83; 22.96; 24.69; 25.50; 29.37; 29.61; 32.52; 32.97; 33.26; 47.74; 50.07;
3
6
4.85; 67.35; 125.27; 128.20; 129.01; 129.09; 129.31; 134.59; 137.38; 143.11; 170.21; 198.46.
ESI-MS HR: 399.2640 (M +1), Calc. 399.2648.
4
.2.36. N-Cyclohexyl-2-(R,S)-[1-(4-methylpiperazinyl)]-4-oxo-4-(5,6,7,8-tetrahydronaphthalen-
yl)butanamide (36):
C H N O , starting from (E)-4-oxo-4-(5,6,7,8-tetrahydronaphtalenyl)-2-butanoic acid
25
37
3
2
cyclohexylamide (0.96 mmol) and a corresponding amount of N-methylpiperazine, 0.130 g of 36
1
was obtained, 32.79 % yield, as pale orange solid, m.p. 93-94°C (toluene). H NMR (500 MHz,
CDCl ) δ: 1.18 (m, 2H, ax C2, cyclohexyl -CH-); 1.35 (m, 2H, ax C3, cyclohexyl -CH-); 1.37 (m,
3
1
H, ax C4, cyclohexyl -CH-); 1.58 (m, 1H, eq C4, cyclohexyl -CH-); 1.67 (m, 2H, eq C3,
cyclohexyl -CH-); 1.80 (d-like signal, 4H, β-tetralinyl); 1.86 (m, 2H, eq C2, cyclohexyl -CH-);
2
2
.31 (s, 3H, piperazine-N-CH ); 2.50 (br, 4H, piperazine -CH -); 2.61 (br, 4H, piperazine -CH -);
3 2 2
.80 (d-like signal, 4H, β-tetralinoyl); 2.90 (dd, 1H, J = 4.99, J = 16.29, ABX); 3.60 (dd, 1H,
1
,2
1,3
J1,2 = 7.59, J1,3 = 16.69, ABX); 3.75 (m, 1H, cyclohexyl -CH-); 4.12 (dd, 1H, J1,2 = 5.00, J1,3
=
7
.25, ABX); 7.09 (d, 1H, J=8.67 Hz, amido -NH-); 7.12 (d, 1H, J = 9.20, aroyl-m-phenyl); 7.69
1
3
(
s, 1H, aroyl-o-phenyl); 7.70 (s, 1H, aroyl-o-phenyl); C NMR (125 MHz, CDCl ) δ: 22.83;
3
2
1
2.96; 24.67; 25.52; 29.35; 29.58; 32.15; 32.96; 33.23; 45.85; 47.64; 55.50; 125.24; 129.07;
29.23; 134.69; 137.28; 142.92; 170.43; 198.64. ESI-MS HR: 412.2951 (M +1), Calc. 412.2964.
4
.2.37. 2-(R,S)-(4-Benzylpiperidinyl)-4-(4-isopropylphenyl)-4-oxo-N-phenylbutanamide (37):
C H N O , starting from (E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid phenylamide
31
36
2
2
(0.7 mmol) and a corresponding amount of 4-benzylpiperidine, 37 was obtained, in quantitative
1
yield, as a yellow semi-solid. H NMR (500 MHz, CDCl ) δ: 1.26 (d, 6H, J=7.06 Hz, i-PrCH );
3
3
1
.41 (m, 1H, piperidine -CH-); 1.72 (t-like, 4H, J=13.63 Hz, piperidine -CH -); 2.21 (t-like, 1H,
2
J=11.68 Hz, benzyl -CH -); 2.45 (t-like, 1H, J=11.19 Hz, benzyl -CH -); 2.56 (m, 4H, piperidine
2
2
-
CH -); 2.94 (overlapped m, 2H, ABX and i-PrCH); 3.68 (dd, 1H, J =6.81 Hz, J =16.30 Hz,
2 1,2 1,3
ABX); 4.29 (dd, 1H, J =5.35 Hz, J =6.30 Hz, ABX); 7.08 (t, 1H, J=7.50 Hz, benzyl-p-CH);
1
,2
1,3
7
.13 (d, 2H, J=7.80 Hz, benzyl-m-phenyl); 7.19 (t, 1H, J=6.37 Hz, amido-p-phenyl); 7.26 (d, 2H,
J=7.50 Hz, aroyl-m-phenyl); 7.29-7.32 (overlapped m, 4H, amido-m-phenyl and benzyl-o-

phenyl); 7.53 (d, 2H, J=7.50 H amido-o-phenyl); 7.94 (d, 2H, J=8.35 Hz, aroyl-o-phenyl); 9.39
ACCEPTED MANUSCRIPT
z,
1
3
(
s, 1H, amido -NH-). C NMR (125 MHz, CDCl ) δ: 21.42; 23.64; 30.68; 31.93; 32.85; 33.11;
3
3
1
4.21; 37.18; 37.57; 43.05; 45.12; 48.01; 52.75; 65.48; 119.29; 123.95; 12591; 126.65; 128.21;
28.50; 128.94; 129.02; 134.90; 137.77; 140.24; 154.47; 170.47; 198.28. ESI-MS HR: 469.2852
(M +1), Calc. 469.2855.
4
.2.38. N-(3,5-Dimethoxyphenyl)-2-(R,S)-[4-(2-dimethylaminoethyl)-1-piperazinyl]-4-(4-
isopropylphenyl)-4-oxo-butanamide (38):
C H N O , starting from E)-4-(4-isopropylphenyl)-4-oxo-2-butenoic acid (3,5-dimethoxy-
29
42
4
4
phenyl)amide (0.57 mmol) and a corresponding amount of 4-(2-N,N-di-Me-aminoethyl)-1-
1
piperazine, 38 was obtained, in quantitative yield, as a yellow semi-solid. H NMR (500 MHz,
CDCl ) δ: 1.27 (d, 6H, J=7.33 Hz, i-PrCH ); 2.24 (s, 6H, N-(CH ) ); 2.43 (m, 2H, linker -CH -);
3
3
3 2
2
2
2
.48 (m, 2H, linker -CH -); 2.59 (br, 4H, piperazine -CH -); 2.67 (br, 4H, piperazine -CH -);
2 2 2
.97 (m, 1H, J =6.60 Hz, J =13.56 Hz, i-PrCH); 3.01 (dd, 1H, J =5.13 Hz, J =17.23 Hz,
1
,2
1,3
1,2
1,3
ABX); 3.68 (dd, 1H, J =6.96 Hz, J =17.23 Hz, ABX); 3.77 (s, 6H, -OCH ); 4.31 (dd, 1H,
1
,2
1,3
3
J =4.77 Hz, J =7.33 Hz, ABX); 6.22 (t, 1H, J=2.19 Hz, amido-p-phenyl); 6.78 (s, 2H, amido-
1
,2
1,3
o-phenyl); 7.32 (d, 2H, J=8.14, aroyl-m-phenyl); 7.94 (d, 2H, J=8.46, aroyl-o-phenyl); 9.29 (s,
1
3
1
5
1
H, amido -NH-). C NMR (125 MHz, CDCl ) δ: 23.60; 31.85; 45.61; 54.16; 55.36; 56.47;
3
6.82; 64.55; 96.36; 97.49; 126.64; 128.15; 128.45; 128.96; 134.73; 139.48; 154.59; 161.04;
70.06; 197.72. ESI-MS HR: 511.3283 (M +1), Calc. 511.3284.
4
.3. Anticholinesterase activity assay
The inhibition potency of the compounds 1-38 toward AChE was evaluated by Ellman
procedure [28], using the enzyme from electric eel, type VI-S (Sigma) and acetylthiocholine
iodide (0.28 mM) as substrate. Four to six different concentrations, which produce 20-80 % of
enzyme activity inhibition, of the each compound were used. The reaction took place in the final
volume of 2 mL of 0.1 M potassium phosphate buffer, pH 8.0, containing 0.03 units of AChE
and 0.3 mM 5,5’-dithiobis(2-nitrobenzoic)acid (DTNB), used to produce yellow anion of 5-thio-
2
-nitrobenzoic acid in reaction with thiocholine released by AChE. Test compound was added to
the enzyme solution and preincubated at 25 ºC for 35 min, followed by the addition of DTNB
0.95 mL) and substrate (50 µL). Inhibition curves were performed at least in triplicate. One
(
triplicate sample withouth test compound was always present to yield 100 % of AChE activity.
The reaction was monitored for 3 min, and the color production was measured at 412 nm. The
reaction rates were compared, and the percent of inhibition, due to the presence of test
compounds, was calculated. IC values were determined from inhibition curves (log inhibitor
5
0
concentration vs. percent inhibiton).