4-Aryl-4-oxo-N-phenyl-2-aminylbutyramides as acetyl- and butyrylcholinesterase inhibitors. Preparation, anticholinesterase activity, docking study, and 3D structure-activity relationship based on molecular interaction fields

Article abstract of DOI:10.1016/j.bmc.2009.12.042

Synthesis and anticholinesterase activity of 4-aryl-4-oxo-N-phenyl-2-aminylbutyramides, novel class of reversible, moderately potent cholinesterase inhibitors, are reported. Simple substituent variation on aroyl moiety changes anti-AChE activity for two orders of magnitude; also substitution and type of hetero(ali)cycle in position 2 of butanoic moiety govern AChE/BChE selectivity. The most potent compounds showed mixed-type inhibition, indicating their binding to free enzyme and enzyme-substrate complex. Alignment-independent 3D QSAR study on reported compounds, and compounds having similar potencies obtained from the literature, confirmed that alkyl substitution on aroyl moiety of molecules is requisite for inhibition activity. The presence of hydrophobic moiety at close distance from hydrogen bond acceptor has favorable influence on inhibition potency. Docking studies show that compounds probably bind in the middle of the AChE active site gorge, but are buried deeper inside BChE active site gorge, as a consequence of larger BChE gorge void.

Full text of DOI:10.1016/j.bmc.2009.12.042

Bioorganic & Medicinal Chemistry 18 (2010) 1181–1193
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
4-Aryl-4-oxo-N-phenyl-2-aminylbutyramides as acetyl- and butyrylcholinester-
ase inhibitors. Preparation, anticholinesterase activity, docking study, and 3D
structure–activity relationship based on molecular interaction fields
a,b,
b
b
c
*
´
´
´ ´ ´
, Ivan O. Juranic , Ljuba M. Mandic , Branko J. Drakulic
Maja D. Vitorovic-Todorovic
a
´
Military-Technical Institute, Ratka Resanovica 1, Belgrade, Serbia
b Faculty of Chemistry, University of Belgrade, Studentski Trg 12-16, Belgrade, Serbia
c Department of Chemistry-IChTM, University of Belgrade, Njegoševa 12, Belgrade, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2009
Revised 16 November 2009
Accepted 15 December 2009
Available online 22 December 2009
Synthesis and anticholinesterase activity of 4-aryl-4-oxo-N-phenyl-2-aminylbutyramides, novel class of
reversible, moderately potent cholinesterase inhibitors, are reported. Simple substituent variation on
aroyl moiety changes anti-AChE activity for two orders of magnitude; also substitution and type of het-
ero(ali)cycle in position 2 of butanoic moiety govern AChE/BChE selectivity. The most potent compounds
showed mixed-type inhibition, indicating their binding to free enzyme and enzyme–substrate complex.
Alignment-independent 3D QSAR study on reported compounds, and compounds having similar poten-
cies obtained from the literature, confirmed that alkyl substitution on aroyl moiety of molecules is req-
uisite for inhibition activity. The presence of hydrophobic moiety at close distance from hydrogen bond
acceptor has favorable influence on inhibition potency. Docking studies show that compounds probably
bind in the middle of the AChE active site gorge, but are buried deeper inside BChE active site gorge, as a
consequence of larger BChE gorge void.
Keywords:
4-Aryl-4-oxo-N-phenyl-2-
aminylbutyramides
Anticholinesterase activity
Mixed-type reversible inhibitors
Alignment-independent 3D QSAR
Docking study
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
bond donors which stabilize the transient tetrahedral enzyme–
substrate complex by accommodation of negatively charged car-
Alzheimer’s disease (AD) accounts for 50% cases of dementia in
elderly people, is a complex, progressive neurodegenerative disor-
der, followed by decrease of cognitive functions such as altered
ability of memory and learning and behavioral disturbances. The
main pathological hallmarks of the disease comprise: extracellular
formation of senile amyloid-b-peptide (Ab) plaques, the loss of
cholinergic neurons from basal forebrain and thereby decreased
levels of neurotransmitter acetylcholine (ACh) and enzymes in-
volved in its synthesis and hydrolysis: acetylcholinetransferase
(ChAT) and acetylcholinesterase (AChE). Among different strate-
gies investigated to improve cholinergic neurotransmission, the
reduction of ACh hydrolysis by AChE inhibitors is up to date the
prevalent effective AD symptomatic treatment.1
AChE contains 20 Å deep and narrow gorge, in which five re-
gions that are involved in the substrate, the irreversible and revers-
ible inhibitor binding (human and electric eel AChE numbering), can
be distinguished: (1) catalytic triad residues: Ser 203, His 447, and
Glu 334, 2 at the bottom of the gorge, which directly participate in
catalytic cycle, by charge relay mechanism, as in other serine ester-
ases,3,4 (2) oxyanion hole represents the arrangement of hydrogen
bonyl oxygen. This region inside of active center is formed by
peptide –NH– groups of amino acid residues Gly 121, Gly 122,
and Ala 204,5,6 (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 ACh,
by forming cation–
p
interactions,7–10 (4) acyl pocket comprises
two phenylalanine residues in positions 295 and 297, which inter-
acts with the substrate acyl group. They form the clamps around
acetyl methyl group of acetylcholine, and decrease its degrees of
freedom,11 (5) peripheral anionic site (PAS)12–14 comprises resi-
dues which are located on the rim of the active site gorge, Tyr
72, Tyr 124, Trp 286, and Asp 74. Possible binding sites for the
reversible inhibitors comprise AS and PAS. The so-called dimeric
(dual) inhibitors bind to both sites.
Up to now, AChE inhibition represented the pure palliative
treatment for AD. However in the past decade, another, non-cata-
lytic roles of the enzyme have been established. It was demon-
strated that AChE has a key role in the acceleration of Ab-peptide
deposition and promoting the formation of Ab-plaques.15 It is
proved that AChE inhibitors, which bind to PAS, could inhibit such
processes.16 Today, the importance of AChE’s sister enzyme, butyr-
ylcholinesterase (BChE), also rises as a pharmacological target for
AD therapy. It was found that BChE is capable to compensate AChE
* Corresponding author. Tel.: +381 11 3336738; fax: +381 11 2636061.
E-mail address: mvitod@chem.bg.ac.rs (M.D. Vitorovic´-Todorovic´).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.042

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M. D. Vitorovic-Todorovic et al. / Bioorg. Med. Chem. 18 (2010) 1181–1193
1182
catalytic functions in synaptic cleft17,18 and that its activity signif-
icantly increases, for 30–60%, during time course in AD.19,20 Of four
FDA approved AChE inhibitors, only donepezil binds simulta-
neously to active and peripheral anionic sites, but the inhibition
of Ab aggregation is modest.15 Actually, an inhibitor of AChE and
BChE that strongly interact with PAS, or with both PAS and active
site (like dimeric inhibitors), and that exhibit inhibitory effect
against AChE induced Ab aggregation, is needed.
Recently, it was shown that some
a,b-unsaturated compounds
exhibit good inhibition potency toward both AChE and BChE. Those
are chalcones21 (I), which show affinities in micromolar range and
m- and p-aminobenzoic acid maleamides (II) and maleimides (III)
(Fig. 1), which are potent nanomolar inhibitors.22–25 Some of these
derivatives act as irreversible inhibitors, probably because of Mi-
chael-type addition of nucleophilic groups present in the active
site gorge. From pharmacokinetic point of view, the presence of
Michael acceptor in drug structure could be considered as disad-
vantage, because of high reactivity, instability in physiological
conditions, and low selectivity. However, saturated analogues of
m-aminobenzoic acid maleamides show even higher inhibition
Figure 2. Novel 4-aryl-4-oxo-N-phenyl-2-aminylbutyramides, X = –CH2– (1–7) or –
O– (8–14), and dimeric analogues (21–26).
potency, compared to unsaturated ones, indicating that a,b-unsat-
uration is not necessary for anti-AChE activity.22 Therefore, the
presence of activated double bond can be used for further function-
alization, by the addition of suitably chosen nucleophiles that bear
moieties which could favorably interact with amino acid residues
inside AChE and BChE active site gorge.
sequently, the acids were converted to acid chlorides in dry THF by
phosphorous-oxychloride, then in situ reacted with equimolar
amounts of aniline to give the corresponding anilides (IV).33
Michael type addition of the corresponding amine (piperidine,
morpholine, imidazole, or 1,8-diaminooctane) to aroylacrylic acid
anilide double bond, proceeded smoothly to give target com-
pounds 1–26. Actually, Michael addition of piperazine and
diisopropylamine has also been tried. However, the expected diiso-
propylamine adduct has not been obtained, probably because of
steric reasons. Mixture of IV and 2–10-fold excess of piperazine
always yielded dimeric products, insoluble in common solvents.
Target compounds were characterized by 1H and 13C NMR spec-
troscopy and by LC-HRMS (ESI).
In continuation of our work on aroylacrylic acids and their phe-
nylamides, we decided to use the reactivity of ketovinyl moiety to-
ward common nucleophiles and to combine aroylacrylic acid
anilides (IV) with amines as nucleophiles, to obtain the corre-
sponding Michael adducts as enantiomeric mixtures.
Aroylacrylic acid anilides were chosen because they combine
amidic and a,b-unsaturated carbonyl moiety of aminobenzoic acid
maleamides, and ring A of chalcones.
As nucleophiles common scaffold of AChE inhibitors, piperidine,
morpholine, and imidazole were chosen.26–30 Also 1,8-diaminooc-
tane was used to obtain dimeric inhibitors. Inhibition potency of
the obtained compounds (1–26, Fig. 2) toward Electric Eel AChE
and horse serum BChE was tested by spectrophotometric method
of Ellman et al.,31 using acetyl- and butyrylthiocholine iodide as
substrates and tacrine as a reference standard.
2.2. Biological activity, QSAR, and molecular modeling
Inhibition potency of compounds toward AChE and BChE, ex-
pressed as IC50 values is shown in Table 1. The inhibition potency
of dimeric analogues 21–26 toward both enzymes was not deter-
mined, due to their insolubility in assay conditions. Considering
the effects of piperidine derivatives 1–7 on AChE activity, volumi-
nous alkyl substituents, 4-i-Pr, 2,4-di-i-Pr, and b-tetralinoyl, in 5–7,
decrease IC50 values for at least two orders of magnitude, in respect
to unsubstituted derivative 1, which does not show any inhibition
2. Results and discussion
2.1. Chemistry
Synthetic pathway to 1–26 is given in Scheme 1. Friedel–Crafts
acylation of the commercially available substituted benzenes (A)
with maleic acid anhydride (B) yields aroylacrylic acids (C).32 Sub-
of AChE activity at the concentration of 100 lM. Similar effects
were observed for imidazolo derivatives (15–20), except for insol-
uble 16. Derivatives 18–20, which bear voluminous alkyl groups,
exert higher potency. Due to a very low solubility of morpholino
derivatives (8–14) in water media, determination of IC50 values
was not possible, only a percent of inhibition for 11 at the highest
concentration tested is reported. Three analogues do not show any
inhibition activity at 100 lM. It is clear that the presence of mor-
pholino oxygen instead of piperidine methylene group has a nega-
tive influence on inhibition potency. The 4-MeO derivative (11)
shows 38.7% inhibition of enzyme activity at inhibitor concentra-
tion of 100 lM, and 4-i-Pr analogue (12) does not show any inhibi-
tion at the same concentration. Consequently, unlike piperidino
and imidazolo derivatives, the presence of voluminous alkyl
groups does not contribute to the inhibition potency of morpholino
derivatives. The most potent of all analogues toward AChE is 19.
Considering the effects of 1–26 on BChE activity, only one or
two compounds within each subgroup are active (Table 1). Percent
of enzyme inhibition at the highest tested inhibitor concentration
Figure 1. Chalcones (I), p- and m-aminobenzoic acid amides (II), and imides (III),
aroylacrylic acid anilides (IV).

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Scheme 1. Synthesis of 1–26. Reagents: (a) AlCl3; (b) POCl3, aniline/THF; (c) NR2 (piperidine, imidazole, morpholine, octane-1,8-diamine) in CHCl3/C6H6.
Table 1
13, respectively. Generally, this type of inhibition is the result of
combination of partially competitive and non-competitive inhibi-
tions, indicating binding to free enzyme and to enzyme–substrate
complex, that is, a possible binding to site remote from the active
IC50 values of the synthesized Michael adducts (1–20) toward AChE and BChE
Compound
R
IC50 SE (
l
M)
AChE
BChE
site. The obtained Ki’s are 4.66
13 (BChE).
lM for 19 (AChE) and 3.18 lM for
1
2
3
4
5
6
7
8
9
H–
2,5-Di-Me–
4-Cl–
4-MeO–
4-i-Pr–
2,4-di-i-Pr–
b-Tetralinoyl–
H–
2,5-Di-Me–
4-Cl–
4-MeO–
4-i-Pr–
2,4-Di-i-Pr–
b-Tetralinoyl–
2,5-Di-Me–
4-Cl–
4-MeO–
4-i-Pr–
2,4-Di-i-Pr–
b-Tetralinoyl–
>100
34.81% @ 100a
17.18 0.12
Ins
>10
Insb
To gain a deeper insight into pharmacophoric pattern that gov-
erns differences in the potency of 1–20, the literature was mined
for compounds that exert activity toward Electric Eel AChE in sim-
ilar concentrations range. The AChE has anionic and peripheral
binding sites, and dual inhibitors fit simultaneously to both sites.
Such molecules usually comprise two heterocyclic moieties con-
nected with the linker of different lengths, and consequently have
large volumes. To preclude consideration of compounds that could
act as dual inhibitors, within the set obtained by the literature
search, only those having volume less than 10% larger of the most
voluminous 6 were considered for further modeling study.
Totally 38 compounds were collected, including secolycorines
(1-1 to 1-8),34 1-[bis(4-fluorophenyl)-methyl]piperazines (2-9 to
2-11),28 litebamine derivatives (3-12 to 3-21),35 imidazolyl-isoxaz-
olines (4-22 to 4-27),36 3-aryl-N-methyl-tetrahydropyridine deriv-
atives (5-28 and 5-30),37 and 5–7, 15, 17–20 (Scheme 2); spanning
>10
>40
3.47 0.04
4.86 0.03
5.64 0.02
>100
>100
Ins
7.06 0.04
39.93% @ 15
21.50% @ 15
21.55% @ 100
34.12 0.16
Ins
10
11
12
13
14
15
16
17
18
19
20
Tacrine
38.7% @ 100
>100
>100
27.22% @ 40
2.48 0.01
13.00% @ 40
42.00% @ 70
Ins
30.41 0.56
10.00% @ 30
>20
>10
>30
27.86 1.05
Ins
7.85 0.03
8.22 0.04
1.55 0.01
6.34 0.01
42.95 0.01 nM
17.00% @ 40
6.95 0.01 nM
range of potencies 0.8–240
l
M (ꢀ2.5 log(1/(IC50)) units).
a
Percent of inhibition at given concentration.
Insoluble in methanol.
b
All compounds were submitted to Pentacle, and for model
building HBD (N1), HBA (O), and hydrophobic (DRY) probe were
used. The obtained model has good statistics and predictivity
(Tables 2 and 3). Partial least square (PLS) coefficient plot obtained
shows that piperidino and morpholino analogues are more potent
than imidazolo ones. Alkyl substituted piperidino derivatives exert
higher inhibition potency, compared to unsubstituted analogue 1
(34.81% of enzyme inhibition at 100 lM). Two of morpholino ana-
logues, 9 and 13, are active. Unsubstituted morpholino derivative
(8) does not show any inhibition effect, and 4-MeO derivative
(11) shows very low inhibition at the highest concentration tested
(100 lM). Thus, alkyl substitution of morpholino analogues has
favorable influence on inhibition potency toward BChE. Because
of lower inhibition potency of imidazolo analogues toward BChE
compared to AChE, and their low solubility in assay conditions,
only the potency of 4-MeO analogue (17) was determined.
From Table 1, it is clear that generally piperidino and imidazolo
derivatives are more active toward AChE. However three morpho-
lino derivatives do not show any inhibition potency of AChE activ-
ity up to 100
lM, but five of them have some activity toward BChE
in concentrations lower than 100
lM. So, the type of heterocyclic
substitution at position 2 of butanoic moiety not only has impact
on AChE activity, but also results in AChE/BChE selectivity.
Subsequently, the spectrophotometric method of Ellman was
used to determine the type of inhibition and the Ki values of two
most active compounds, 19 and 13, toward AChE and BChE, respec-
tively. Both compounds exhibited mixed type of inhibition toward
the corresponding enzymes. This behavior was indicated by inter-
section of double reciprocal lines in the upper left quadrant
(Lineweaver–Burk plot) represented in Figures 3 and 4 for 19 and
Figure 3. Lineweaver–Burk plot of AChE (0.02 U) with substrate acetylthiocholine,
in the absence or presence of different concentrations of 19. (j) No inhibitor; (d)
1.0 lM; (N) 1.5 lM.

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M. D. Vitorovic-Todorovic et al. / Bioorg. Med. Chem. 18 (2010) 1181–1193
Table 2
PCA model
Comp.
SSx
SSxacc
VarX
VarXacc
1
2
3
4
5
53.95
17
6.19
4.46
3.26
53.95
70.95
77.14
81.60
84.86
52.55
16.58
5.80
4.22
3.11
52.55
69.13
74.93
79.16
82.27
Comp.—number of components; SSX—percentage of the X sum of squares; SSXacc
—
accumulative percentage of the X sum of squares; VarX—percentage of the X var-
iance; VarXaac—accumulative percentage of the X variance.
example, variables DRY-DRY 41, 42 that describe the presence of
two hydrophobic moieties in molecules on 13.12–13.76 Å, having
IE with DRY probe of ꢀꢁ1.4 kcal/mol (Fig. S1 in Supplementary),
are expressed for the majority of compounds. Important variables
that offer data on differences among molecules are commented
in more detail. Variable DRY-N1 260 is expressed only for 2-11,
one of the most potent of the studied molecules in the set, as
well as for 5, 6, and 19, the most potent among the prepared
compounds. This variable recognizes spatial proximity of HBA
(ꢀꢁ6.2 kcal/mol) and hydrophobic region of molecules (ꢀꢁ1.26 kcal/
mol), Figure 6. Variable O–N1 359, negatively correlated with the
potency of compounds, gives almost sharp cut-off between more
and less active compounds within the studied set. This variable is
expressed for the vast majority of compounds having potencies
Figure 4. Lineweaver–Burk plot of BChE (0.02 U) with substrate acetylthiocholine,
in the absence or presence of different concentrations of 13. (j) No inhibitor; (d)
1.0 lM; (N) 2.0 lM; (.) 3.0 lM.
with 5 latent variables is given in Figure 5. Variables expressed for
particular compound are given in Table S1 in Supplementary.
Experimental versus predicted log(1/(IC50)) values are given in Ta-
ble 4. Full analysis of the model is out of the scope of this article,
therefore the main observations that give significant information
on pharmacophoric pattern of molecules will be discussed in the
following lines. Bulk of variables having high impact on the model,
positively or negatively correlated with activity, are expressed for
the majority of 5–7, 15, 17–20 (see Table S1 in Supplementary).
This means that the reported compounds share spatial arrange-
ment of pharmacophoric points (including their interaction ener-
gies with the GRID probes used) with structurally very diverse
(dissimilar) compounds reported in the literature, having potencies
toward E. Eel AChE in a comparable range of concentrations. For
on or below 2.1 p(IC50
). Additionally, variable is expressed for the
one of less potent 17 (Fig. S2 in Supplementary). It is obvious that
the presence of HBA (N1 probe IE ꢀꢁ4.00 kcal/mol) and HBD (O
probe IE ꢀꢁ4.75 kcal/mol) in molecules, on 12.48–12.80 Å has
unfavorable influence on the potency of compounds. Matrix-like
representation of compound corelograms (Pentacle heat maps), al-
low clear observation of this difference (Fig. S3 in Supplementary).
Next, we try to find variables that differentiate 5–7, 15, 17–20
in respect to potency. Variable DRY-N1 302 (nodes distance
14.72–15.04 Å), positively correlated with potency, even not hav-
ing high impact on whole model, nicely differentiate less potent
15 from the rest of the compounds. This variable is positioned be-
Scheme 2. Structural formulae of 1–1 to 5–30.

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´
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M. D. Vitorovic-Todorovic et al. / Bioorg. Med. Chem. 18 (2010) 1181–1193
Table 3
PLS model
R2
Racc
RG-Qacc
LTO-Qacc
LOO-Qacc
2
2
2
2
Comp.
SSX
SSXacc
SDEC
SDEP
1
2
3
4
5
53.16
7.66
13.26
3.36
53.16
60.82
74.08
77.44
81.78
0.52
0.34
0.28
0.20
0.18
0.56
0.51
0.46
0.41
0.40
0.24
0.42
0.12
0.10
0.02
0.24
0.66
0.78
0.89
0.91
0.10
0.25
0.40
0.51
0.54
0.13
0.31
0.44
0.60
0.60
0.13
0.32
0.45
0.61
0.61
4.34
Comp.—number of components; SSX—percentage of the X sum of squares; SSXacc—accumulative percentage of the X sum of squares; SDEPs—standard deviation error of the
predictions; R2—coefficient of determination; R2acc—accumulative coefficient of determination; Qa2cc—accumulative squared predictive correlation coefficient (RGs-random
groups; LTO—leave two out; LOO—leave one out).
Figure 5. PLS coefficient plot for the reported model, obtained with 5 LV.
tween aroyl–alkyl substituents (DRY) and imidazolo or piperidino
N (N1) for 5, 6, 18–20, (Fig. 7) but not for the less potent 15. Addi-
tionally, variable DRY-O 230 (12.16–12.46 Å), which connects ex-
tended hydrophobic moieties on aryl ring and amido NH, is
expressed only for 5–7 and 18–20 (Fig. 8), but not for the less po-
tent 15 and 17. It should be noted that not only mere lipophilicity
of aroyl fragment, but also position of substituents differentiates
5–7, 15, 17–20 in respect to potency. In Figure S4 in Supplemen-
tary log(1/(IC50)) versus GRID DRY probe volume obtained on
ꢁ0.55 kcal/mol is shown. The most potent 19 and the less potent
15 significantly deviate from correlation, which is obvious for the
rest of the compounds. Even 15 and 19 have similar isovolumes
of the DRY probe on chosen IE, their potencies are significantly dif-
ferent. Observing the DRY isovolumes around molecules, it may be
concluded that different substitution patterns of aroyl moiety
influence their potencies.
In summary, reported analysis revealed few important points,
which will be guidance for designing of novel compounds. First,
the presence of aroyl phenyl and amidophenyl rings of molecules
fits to the two hydrophobic areas separated by ꢀ13.5 Å (variable
DRY-DRY 41) in the majority of, structurally diverse, compounds
in the set. Second, alkyl substitution on aroyl moieties of molecules
is requisite, and most favorable positions are 2 and 4. Variable
DRY-N1 302 shows that suitably chosen extended hydrophobic
moiety on aroyl phenyl ring can fine-tune the potency of com-
pounds toward E. Eel AChE, allowing prerequisite distance between
HBA and hydrophobic moiety. On the other hand, it is clear from
Table 1 that the introduction of substituents on phenyl ring turns
1.60 Å), given by variable DRY-N1 260, which can be found for
three most potent compounds (5, 6, and 19), as well as for the one
of the most potent compounds within the studied set. For 5–7, 15,
17–20, this feature is governed by nature and position of phenyl sub-
stituents on aroyl moiety, as well as by the nature of hetero(ali)cycle
on the position 2 of butanoic moiety, which result in geometry that
allows favorable spatial arrangement of pharmacophoric points (for
the description of conformers generation used in the model see Sec-
tion 4). Fourth, and this holds for the whole studied set, the presence
of HBA and HBD on spatial distance of ꢀ12.5 Å yields less potent
compounds, therefore for novel congeners alkoxy or hydroxy sub-
stituents on aroyl ring should be avoided.
To investigate possible ligand–AChE interactions, docking stud-
ies were performed to generate binding model for the most active
piperidino (5) and imidazolo (19) derivatives toward AChE. Both
enantiomers of 5 and 19 were docked to mAChE (Mus Musculus,
PDB code 2HA2). This AChE crystal structure was chosen because
it has the same conserved amino acid residues as the only available
EeAChE crystal structure has (PDB code 1C2B), but with consider-
ably better resolution. Docking studies were performed using the
latest version of AUTODOCK 4.0.1 package,38 which allows full flexibil-
ity of small ligands and of user defined amino acid residues in the
receptor. As flexible, three tyrosine residues were chosen (72, 337,
and 341). Crystallographic data14 and molecular dynamic simula-
tions39 revealed considerable mobility of these residues. Tyr 337
is located midway in the gorge and its motion could enlarge the
volume of active site gorge. Homologous residue Phe 330 in
TcAChE was also found to be flexible.40 Side-chain motion of Tyr
341 might significantly enlarge the gorge mouth and facilitate
inactive compounds (IC50 >100 lM) to moderately potent ones,
consequently change of potency for ꢀ2 orders of magnitude was
obtained. Third, the need for specific spatial arrangement of HBA
and hydrophobic moiety of molecules on close distance (1.28–
For clarity: DRY probe is complementary with hydrophobic moieties of mole-
cules, while N1 (HBD) probe is complementary with HBA moieties of molecules.

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M. D. Vitorovic-Todorovic et al. / Bioorg. Med. Chem. 18 (2010) 1181–1193
1186
Table 4
Experimental versus predicted log(1/(IC50)) as obtained by the Pentacle model
described in the text
Comp.
log(1/(IC50)) Exp.
log(1/(IC50)) Pred.
5
6
7
15
17
18
19
20
1-1
1-2
1-3
1-4
1-5
1-6
1-7
1-8
2.460
2.313
2.249
1.555
2.105
2.085
2.810
2.198
2.539
2.078
1.975
2.575
2.523
2.055
2.001
2.264
2.672
2.917
2.945
1.658
1.840
2.142
2.108
2.186
1.801
1.972
2.167
1.565
1.208
2.699
1.142
1.157
0.795
1.678
0.618
3.094
2.909
2.909
2.384
2.361
2.305
1.864
2.081
2.217
2.454
2.277
2.131
2.145
2.296
2.250
2.208
2.182
2.159
2.237
2.620
2.842
3.106
1.618
1.732
2.091
2.061
2.052
1.980
1.843
2.127
1.393
1.480
2.535
1.334
1.057
0.817
1.658
0.770
3.055
3.163
3.083
Figure 7. Variable DRY-N1 302 (14.72–15.04 Å) shows prerequisite distance
between HBA and hydrophobic moiety. Compound 19, yellow boxes DRY probe
field, blue boxes N1 probe field.
2-9
2-10
2-11
3-12
3-13
3-14
3-15
3-16
3-17
3-18
3-19
3-20
3-21
4-22
4-23
4-24
4-25
4-26
4-27
5-28
5-29
5-30
Figure 8. Variable DRY-O 230 (12.16–12.46 Å), distance between HBD and hydro-
phobic moiety. Compound 19, yellow boxes DRY probe field, red boxes O probe
field.
bond between Tyr 337 –OH and indole –NH– group. The same
has been observed in docking studies with HuAChE.41 Docking
studies show that 19 (R) binds in the middle of AChE active site
gorge, Figure 9a. Phenylamido group is directed toward Trp 286
and forms hydrophobic contacts with its side chain, with minimal
distance of 3.74 Å. Imidazole ring is found near Phe 297 aromatic
ring (minimal distance 3.76 Å), its nitrogen atom forms hydrogen
bond with Phe 295 backbone –NH– group. Aroyl moiety is directed
toward the bottom of the gorge. Interestingly, orientation of 19 (S)
inside AChE active site gorge is reversed (Fig. 9b). Aroyl group is di-
rected toward the entrance of the gorge. Its phenyl ring forms close
hydrophobic contact to Phe 338 (4.64 Å) and Tyr 341 (3.82 Å) aro-
matic side chains. Imidazole ring is found near Ser 125 side chain
(3.86 Å) and near backbone –NH– group of Asp 74 (3.53 Å). Amido
carbonyl group forms hydrogen bond to Ser 125 –OH group
(2.04 Å). Phenylamido group is directed toward esteratic site and
forms hydrophobic contact with the indole ring of Trp 86 (3.82 Å).
Aroyl moieties of the both enantiomers of 5 form hydrophobic
contacts with aromatic sidechains of Phe 297 (3.97 Å for (R) and
3.16 for (S)), Phe 338 (3.63 Å and 3.84 Å), and Tyr 341 (3.78 Å
and 3.51 Å), Figure 9c and d. Aroyl carbonyl group of 5 (R) forms
hydrogen bond with Tyr 124 –OH group (2.04 Å), and piperidine
ring is accommodated below PAS residues (Tyr 124, Tyr 72, and
Asp 74) and forms hydrophobic contact with Leu 76 side chain
Figure 6. Variable DRY-N1 260 (1.28–1.60 Å) recognizes proximity of HBA and
hydrophobic region of molecules. Compound 5, yellow boxes DRY probe field, blue
boxes N1 probe field.
the ligand access to catalytic site. The lowest energy cluster re-
turned by AUTODOCK for each enantiomer of 5 and 19 was used for
further analysis. Best docking solutions for all docked derivatives
revealed that the positions of flexible tyrosine residues were not
significantly changed in respect to their positions in the free en-
zyme. Major change was the movement of Tyr 337 side chain to-
ward indole ring of Trp 86, and possible formation of hydrogen

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M. D. Vitorovic-Todorovic et al. / Bioorg. Med. Chem. 18 (2010) 1181–1193
Figure 9. Compounds 5(R), (a); 5(S), (b); 19(R), (c); and 19(S), (d), docked into the binding site of the AChE, highlightening the protein residues that form the main
interactions with the different structural units of the inhibitors. Hydrogen bonds are represented as black dots. Residues are colored as follows: anionic site Trp 86 and Tyr
337, orange; PAS Tyr 72, Asp 74, Tyr 124, Ser 125 violet and Trp 286 magenta; acyl binding pocket Tyr 341, Phe 297 and Phe 338 green; catalytic triad residues His 447 and Ser
203 dark blue; ligand, turquoise.
(4.70 Å). Phenylamido moiety of 5 (R) is directed toward catalytic
triad residues (His 447 and Ser 203). Piperidine ring of 5 (S) is
found near esteratic site and forms hydrophobic contact with Trp
86 (4.34 Å), while phenylamido group is directed toward Asp 74
and Ser 125.
HuAChE), 2.30 Å, while –i-Pr group forms hydrophobic contacts
with the Tyr 332 side chain (analogous to Tyr 341 in HuAChE),
3.66 Å. Piperidine ring of 5 (R) is directed toward Tyr 128
(4.14 Å), near esteratic site and interacts with Trp 82 (4.85 Å, anal-
ogous to Trp 86 in HuAChE). Aroyl carbonyl group of 5 (S) enantio-
mer forms hydrogen bond with side chain –OH group of Ser 198
(1.96 Å, while piperidine ring is directed toward Tyr 332 (3.52 Å)
and aroyl group is found near Trp 82 (3.82 Å) and Tyr 128
(4.38 Å) side chains.
Since 5 is the only compound that shows low micromolar activ-
ity toward both enzymes, we performed docking studies of 5 on
HuBChE (PDB entry: 1POI) for making a comparison with their
binding mode in AChE. Since there are less structural and molecu-
lar dynamics data for BChE we were not able to precisely decide
which residues to keep flexible during calculation, and we per-
formed classical rigid docking study. The best docked conforma-
tions obtained show that both enantiomers of 5 bind deeper
inside BChE active site gorge than they do in AChE, Figure 10 a
and b; the minimal distance between ligand and Ala 277 residue
(homologous to Trp 286 of HuAChE) is 15 Å. This is probably a con-
sequence of much wider BChE active site gorge, where six of the
fourteen AChE aromatic amino acid residues (Tyr72, Tyr 124, Trp
286, Phe 295, Phe 297, and Tyr 337) are replaced by aliphatic in
BChE (Asn 86, Gln 119, Ala 277, Leu 286, Val 288, and Ala 328). This
causes 200 Å3 larger gorge in BChE than in AChE.42 Phenylamido
groups of both enantiomers are accommodated in hydrophobic
pocket, formed of side chains of the acyl pocket residues, Leu 286
and Val 288, and the three aromatic amino acids Trp 231, Phe
329, and Phe 398. Minimal distances between phenyl ring of 5
and these residues are in range between 3.29 and 4.16 Å. Amido
carbonyl group of 5 (R) forms hydrogen bond with the side chain
–OH group of the catalytic Ser 198 (analogous to Ser 203 in
3. Conclusions
In conclusion, 4-aryl-4-oxo-N-phenyl-2-aminyl-substituted
butyramides, novel type of reversible, moderately potent cholines-
terase inhibitors, were designed, easily synthesized, and tested to-
ward AChE and BChE. Appropriate substitution on aroyl moiety
turns inactive compounds to moderately potent ones toward AChE,
that is, small structural changes produce the increase of activity for
ꢀ2 orders of magnitude. Notably, change of piperidino methylene
group by oxygen in morpholino derivatives produces inactive com-
pounds toward AChE, but some of morpholino derivatives (9 and
13) are potent toward BChE. Those derivatives are selective inhib-
itors of BChE. Compounds 19 and 13 are the most potent inhibitors
of AChE and BChE, respectively, acting in low micromolar concen-
trations, and also exert a linear mixed-type inhibition toward both
enzymes. Alignment-independent 3D-QSAR analysis on set of the
prepared and in the literature reported compounds, having compa-

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M. D. Vitorovic-Todorovic et al. / Bioorg. Med. Chem. 18 (2010) 1181–1193
1188
Figure 10. Compounds 5(R), (a); and 5(S), (b), docked into the binding site of BChE highlightening the protein residues that form the main interactions with the different
structural units of the inhibitors. Hydrogen bonds are represented as dots.
rable potency toward E. Eel AChE, gives useful information for de-
sign novel, hopefully, more potent compounds. Existence of ex-
tended and properly directed hydrophobic moiety on aroyl
fragment is a requisite for potency. Docking studies showed that
5 and 19 bind, probably, in the middle of the gorge. Both enantio-
mers of 5 bind deeper inside BChE active site gorge, with binding
conformations very distant from Ala 277; probably as a conse-
quence of larger BChE gorge. Although moderately active, reported
compounds will be used as templates for further structural modi-
fications, based on the reported QSAR data. Further variation of ar-
oyl substitution, amidic portion of molecule and polymethylene
chain of dimeric analogues (21–26) is planed, in order to find
structural requirements needed for higher potency and better
solubility.
necessary (except for 17 and 20, which were recrystallized from
acetone) and Michael addition gave almost quantitative yields for
the majority of compounds. All obtained compounds were used
in biological tests as the mixtures of enantiomers (1–20) or diaste-
reomers (21–26). Reported 1H NMR shifts of the A and B protons in
ABX pattern correspond to dd centers, not real chemical shifts of
the corresponding protons, for clarity.
4.1.1.1. Phenyl-4-oxo-N-phenyl-2-(R,S)-(1-piperidinyl)butyramide
(1). Starting from (E)-4-oxo-4-phenyl-2-butenoic acid phenyla-
mide (0.7 g, 2.78 mmol) and equimolar amount of piperidine, as
described above, 0.56 g of 1 was obtained, 60.21% yield, pale
yellow solid: mp 138–140 °C. 1H NMR (200 MHz, CDCl3) d: 1.37
(br, 2H, piperidine –CH2–); 1.52 (br, 4H, piperidine –CH2–); 2.56
(br, 4H, piperidine –CH2–); 3.09 (ABX, dd, J1,2 = 3.65 Hz, J1,3
=
16.84 Hz, 1H); 3.67, (ABX, dd, J1,2 = 9.55 Hz, J1,3 = 16.85 Hz, 1H);
4.01 (ABX, dd, J1,2 = 3.37 Hz, J1,3 = 9.27 Hz, 1H); 7.00, 7.04, 7.07
(triplet-like picks, 1H, amide p-phenyl); 7.25, 7.30, 7.33 (triplet-
like picks, 2H, amide m-phenyl); 7.49–7.67 (overlapping multi-
plets, 5H, amide o-phenyl, aroyl p-phenyl, aroyl m-phenyl); 8.01,
8.04 (doublet-like picks, 2H, aroyl o-phenyl); 10.62 (s, 1H, –NH–
amide). 13C NMR (50 MHz, CDCl3) d: 24.17; 26.21; 34.64; 50.64;
65.34; 119.41; 123.36; 126.17; 128.17; 128.83; 133.25; 137.11;
139.06; 169.56; 198.96. LC ESI-MS (HR): MH+ (obsd) 337.1916;
calcd for C21H25N2O2 = 337.1921.
4. Experimental
4.1. Chemistry
Melting points were measured in open capillary tubes on a
SMP-10 Stuart apparatus and are uncorrected. Direct ESI-MS spec-
tra were recorded on Agilent Technologies 6210-1210 TOF-LC-ESI-
MS instrument in positive mode. 1H and 13C NMR spectra were re-
corded in CDCl3 on Varian Gemini 200/50 MHz or Bruker AVANCE
500/125 MHz instruments. Chemical shifts are reported in parts
per million (ppm) relative to tetramethylsilane (TMS) as the inter-
nal standard, and spin multiplicities are given as follows: s (sin-
glet), d (doublet), t (triplet), m (multiplet), or br (broad).
4.1.1.2. 4-(2,5-Dimethylphenyl)-4-oxo-N-phenyl-2-(R,S)-(1-pipe-
ridinyl)butyramide (2). Starting from (E)-4-(2,5-dimethyl-
phenyl)-4-oxo-2-butenoic acid phenylamide (0.7 g, 2.50 mmol)
and equimolar amount of piperidine, as described above, 0.75 g
of 2 was obtained, 82.42% yield, yellow semi solid. 1H NMR
(200 MHz, CDCl3) d: 1.48 (m, 2H, piperidine –CH2–); 1.65 (m, 4H,
piperidine –CH2–); 2.36 (s, 3H, m-CH3); 2.48 (s, 3H, o-CH3); 2.57
4.1.1. General method to the synthesis of 1–26
Briefly, 0.5–1.0 g of aroylacrylic acid anilide (approximately
1.5–3.0 mmol) was dissolved or suspended in 10 mL of CHCl3,
20 mL of benzene was added and after 10 min of stirring, equimo-
lar amount of the corresponding amine (piperidine, morpholine, or
imidazole) was used to obtain compounds 1–20. For compounds
21–26 half of equimolar amount of 1,8-diaminooctane was used
in order to obtain dimeric compounds. For imidazole analogues
15–20, 0.4 mL of triethylamine was added and the reaction mix-
ture was refluxed for 3 h. The reaction mixture for all derivatives
was stirred overnight at room temperature. After this the solvents
were removed by spontaneous evaporation, or under the reduced
pressure to low volume. The solid or semi-solid products were col-
lected by vacuum suction and were characterized as described be-
low. Interestingly, we found that further purification was not
(t, 4H, J1,2 = 5,07 Hz, piperidine –CH2–); 2.89 (ABX, dd, J1,2
4.49 Hz, J1,3 = 16.85 Hz, 1H); 3.55 (ABX, dd, J1,2 = 8.43 Hz, J1,3
=
=
16.85 Hz, 1H); 4.24 (ABX, dd, J1,2 = 4.49 Hz, J1,3 = 7.86 Hz, 1H);
7.04, 7.07, 7.11 (triplet-like picks, 1H, amide p-phenyl);
7.14–7.19 (m, 2H, amide m-phenyl); 7.24–7.35 (m, 2H, aroyl p-
phenyl, aroyl m-phenyl); 7.53, 7.57 (m, 3H, amide o-phenyl, aroyl
o-phenyl); 9.42 (s, 1H, –NH– amide). 13C NMR (200 MHz, CDCl3)
d 20.56; 23.89; 26.66; 34.94; 51.05; 66.02; 119.19; 123.85;
128.27; 128.91; 131.64; 131.82; 134.77; 137.81; 138.30; 170.49;
202.36. LC ESI-MS (HR): MH+ (obsd) 365.2239; calcd for
C23H29N2O2 = 365.2229.

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4.1.1.3. 4-(4-Chlorophenyl)-4-oxo-N-phenyl-2-(R,S)-(1-piperidi-
nyl)butyramide (3). Starting from (E)-4-(4-chlorolphenyl)-4-oxo-
2-butenoic acid phenylamide (1.0 g, 3.50 mmol) and equimolar
amount of piperidine, as described above, 1.2 g of 3 was obtained,
92.30% yield, white solid: mp 134–136 °C. 1H NMR (200 MHz,
CDCl3) d: 1.48 (m, 2H, piperidine –CH2–); 1.65 (m, 4H, piperidine
–CH2–); 2.57 (t, 4H, J1,2 = 4.91 Hz, piperidine –CH2–); 2.84 (ABX,
dd, J1,2 = 4.21 Hz, J1,3 = 16.29 Hz, 1H); 3.67 (ABX, dd, J1,2 = 7.86 Hz,
J1,3 = 16.01 Hz 1H); 4.26 (ABX, dd, J1,2 = 4.49 Hz, J1,3 = 8.14 Hz,
1H); 7.08 (t, 1H, J1,2 = 7,34 Hz, amide p-phenyl); 7.30 (t, 2H,
J1,2 = 7,34 Hz, amide m-phenyl); 7.44 (d, 2H, J = 8.70 Hz, aroyl m-
phenyl); 7.52 (d, 2H, J = 7.61 Hz, amide o-phenyl); 7.97 (d, 2H,
J = 8.70 Hz, aroyl o-phenyl); 9.38 (s, 1H, –NH– amide). 13C NMR
(200 MHz, CDCl3) d: 23.85; 26.62; 31.61; 51.09; 66.18; 119.22;
123.97; 128.18; 128.94; 129.71; 135.52; 137.65; 139.31; 170.33;
197.61. LC ESI-MS (HR): MH+ (obsd) 371.1530; calcd for
C21H24ClN2O2 = 371.1526.
(doublet-like picks, 2H, amide o-phenyl); 7.67 (d, 1H, J = 7.97 Hz,
aroyl o-phenyl); 9.41 (s, 1H, –NH– amide). 13C NMR (125 MHz,
CDCl3) d:23.75; 23.97; 24.22; 24.40; 26.74; 29.17; 34.25; 36.06;
66.14; 119.19; 123.84; 124.66; 128.09; 128.92; 136.50; 137.87;
148.27; 151.96; 170.50; 203.17. LC ESI-MS (HR): MH+ (obsd)
421.2902; calcd for C27H37N2O2 = 421.2855.
4.1.1.7. 4-(5,6,7,8-Tetrahydro-2-naphthalenyl)-4-oxo-N-phenyl-
2-(R,S)-(1-piperidinyl)butyramide (7). Starting from (E)-4-
(5,6,7,8-tetrahydro-2-naphthalenyl)-4-oxo-2-butenoic acid phe-
nylamide (1.0 g, 3.50 mmol) and equimolar amount of piperidine,
as described above, 1.25 g of 7 was obtained, 91.91% yield, pale
brown solid: mp 92–94 °C. 1H NMR (200 MHz, CDCl3) d: 1.49 (m,
2H, piperidine –CH2–); 1.63 (m, 4H, piperidine –CH2–); 1.79 (m,
4H, tetralinoyl –CH2–); 2.57 (t, 4H, J1,2 = 5.05 Hz, piperidine –
CH2–); 2.79 (m, 4H, tetralinoyl –CH2–); 2.96 (ABX, dd, J1,2 = 5.06,
J1,3 = 16.29 Hz, 1H); 3.68 (ABX, dd, J1,2 = 6.74, J1,3 = 16.29 Hz, 1H);
4.26 (ABX, dd, J1,2 = 5.06, J1,3 = 7.30 Hz, 1H); 7.02–7.17 (overlapping
m, 2H, aroyl-m-phenyl, and amide-p-phenyl); 7.29 (t, 2H,
J1,2 = 7.50 Hz, amide m-phenyl); 7.52, 7.56 (doublet-like picks,
2H, J = 7.84, amide o-phenyl); 7.73 (br, 2H, aroyl o-phenyl); 9.47
(s, 1H, –NH– amide). 13C NMR (50 MHz, CDCl3) d: 22.82; 23.84;
26.64; 29.48; 31.81; 50.96; 65.73; 119.11; 123.76; 125;23;
128.82; 129.22; 134.44; 137.27; 137.78; 143.00; 170.55; 198.46.
LC ESI-MS (HR): MH+ (obsd) 391.2393; calcd for C25H31N2O2 =
391.2386.
4.1.1.4. 4-(4-Metoxyphenyl)-4-oxo-N-phenyl-2-(R,S)-(1-piperid-
inyl)butyramide (4). Starting from (E)-4-(4-metoxyphenyl)-4-
oxo-2-butenoic acid phenylamide (1.0 g, 3.50 mmol) and equimo-
lar amount of piperidine, as described above, 1.15 g of 4 was
obtained, 89.84% yield, pale yellow solid: mp 112–114 °C. 1H NMR
(200 MHz, CDCl3) d: 1.47 (m, 2H, piperidine –CH2–); 1.64 (m, 4H,
piperidine –CH2–); 2.58 (t, 4H, J1,2 = 5.03 Hz, piperidine –CH2–);
2.94 (ABX, dd, J1,2 = 5.05 Hz, J1,2 = 16.85 Hz, 1H); 3.67 (ABX, dd,
J1,2 = 7.30 Hz, J1,3 = 16.29 Hz, 1H); 3.86 (s, 3H, –OCH3); 4.26 (ABX,
dd, J1,2 = 5.05 Hz, J1,3 = 7.30 Hz, 1H); 6.94 (d, 2H, J = 8.70 Hz, aroyl
m-phenyl); 7.07 (t, 1H, J1,2 = 7,27 Hz, amide p-phenyl); 7.30 (t,
2H, J1,2 = 7,30 Hz, amide m-phenyl); 7.54 (d, 2H, J = 7.35 Hz, amide
o-phenyl); 8.02 (d, 2H, J = 8.98 Hz, aroyl o-phenyl); 9.38 (s, 1H, –
NH– amide). 13C NMR (200 MHz, CDCl3) d: 23.89; 26.67; 31.57;
51.00; 55.39; 65.80; 113.67; 119.19; 123.85; 128.91; 130.06;
130.51; 137.81; 163.43; 170.64; 197.19. LC ESI-MS (HR): MH+
(obsd) 367.2032; calcd for C22H26N2O3 = 367.2022.
4.1.1.8. 4-Phenyl-4-oxo-N-phenyl-2-(R,S)-(4-morpholinyl)buty-
ramide (8). Starting from (E)-4-oxo-4-phenyl-2-butenoic acid
phenylamide (0.5 g, 2.0 mmol) and equimolar amount of morpho-
line, as described above, 0.60 g of 8 was obtained, 88.63% yield,
pale yellow solid: mp 144–146 °C. 1H NMR (200 MHz, CDCl3) d:
2.66 (t, 4H, J1,2 = 4.45 Hz, morpholine –CH2–); 3.00 (ABX, dd,
J1,2 = 4.49 Hz, J1,3 = 16.28, 1H); 3.70–3.87 (overlapping m, 5H, mor-
pholine –CH2– and ABX); 4.30 (ABX, dd, J1,2 = 4.50 Hz, J1,3 = 7.30 Hz,
1H); 7.09 (triplet-like picks, 1H, J1,2 = 7.49 Hz amide p-phenyl);
7.31 (triplet-like picks, 2H, J1,2 = 7.69 Hz, amide m-phenyl); 7.43–
7.62 (overlapping m, 5H, amide o-phenyl, aroyl m-phenyl and aroyl
p-phenyl); 8.01–8.05 (m, 1H, aroyl o-phenyl); 9.21 (s, 1H, –NH–
amide). 13C NMR (50 MHz, CDCl3) d: 32.06; 50.00; 65.26; 67.31;
119.30; 124.19; 128.23; 128.69; 129.00; 133.19; 136.88; 137.52;
169.60; 198.17. LC ESI-MS (HR): MH+ (obsd) 339.1712; calcd for
C20H23N2O3 = 339.1709.
4.1.1.5. 4-(4-Isopropylphenyl)-4-oxo-N-phenyl-2-(R,S)-(1-pipe-
ridinyl)butyramide (5). Starting from (E)-4-(4-isopropylphenyl)-
4-oxo-2-butenoic acid phenylamide (1.0 g, 3.40 mmol) and equi-
molar amount of piperidine, as described above, 1.12 g of 5 was ob-
tained, 87.50% yield, pale yellow solid: mp 90–91 °C. 1H NMR
(200 MHz, CDCl3) d: 1.27 (d, 6H, J = 6.72 Hz, i-PrCH3); 1.40–1.72
(overlapping m, 6H, piperidine –CH2–); 2.57 (t, 4H, J1,2 = 5.06 Hz,
piperidine –CH2–); 2.90–3.03 (overlapping m, 2H, ABX and i-PrCH);
3.70 (ABX, dd, J1,2 = 7.02 Hz, J1,3 = 16.57 Hz, 1H); 4.27 (ABX, dd,
J1,2 = 5.06 Hz, J1,3 = 7.02 Hz, 1H); 7.07 (t, 1H, J1,2 = 7,20 Hz, amide
p-phenyl); 7.26–7.35 (overlapping m, 4H, amide m-phenyl, and ar-
oyl m-phenyl); 7.52, 7.56 (doublet-like picks, 2H, amide o-phenyl);
8.97 (d, 2H, J = 8.98 Hz, aroyl o-phenyl); 9.46 (s, 1H, –NH– amide).
13C NMR (200 MHz, CDCl3) d: 23.58; 23.89; 26.69; 31.83; 34.14;
51.02; 65.84; 119.20; 123.85; 126.63; 128.49; 134.94; 137.81;
154.42; 170.57; 198.33. LC ESI-MS (HR): MH+ (obsd) 379.2398;
calcd for C24H31N2O2 = 379.2386.
4.1.1.9. 4-(2,5-Dimethylphenyl)-4-oxo-N-phenyl-2-(R,S)-(4-mor-
pholinyl)butyramide (9). Starting from (E)-4-(2,5-dimethyl-
phenyl)-4-oxo-2-butenoic acid phenylamide (0.5 g, 1.80 mmol)
and equimolar amount of morpholine, as described above, 0.64 g
of 9 was obtained, 89.65% yield, yellow solid: mp 106–108 °C. 1H
NMR (200 MHz, CDCl3) d: 2.37 (s, 3H, m-CH3); 2.47 (s, 3H, o-
CH3); 2.66 (t, 4H, J1,2 = 4.63 Hz, morpholine –CH2–); 2.94 (ABX,
dd, J1,2 = 4.49 Hz, J1,3 = 16.85 Hz, 1H); 3.60 (ABX, dd, J1,2 = 7.86,
J1,3 = 16.28 Hz, 1H); 3.79 (q-like picks, 4H, J1,2 = 3.86, Hz,
J1,3 = 7.91 Hz, morpholine –CH2–); 4.26 (ABX, dd, J1,2 = 5.06 Hz,
J1,2 = 7.86 Hz, 1H); 7.06–7.22 (overlapping m, 3H, amide p-phenyl,
aroyl m-phenyl and aroyl p-phenyl); 7.26–7.36 (overlapping m, 2H,
amide m-phenyl); 7.52–7.58 (overlapping m, 3H, amide o-phenyl
and aroyl o-phenyl); 9.20 (s, 1H, –NH–amide). 13C NMR (50 MHz,
CDCl3) d: 20.63; 35.09; 50.04; 65.40; 67.33; 119.30; 124.17;
129.02; 131.77; 132.09; 134.94; 137.56; 137.96; 169.64; 201.77.
LC ESI-MS (HR): MH+ (obsd) 367.2035; calcd for C22H27N2O3 =
367.2022.
4.1.1.6. 4-(2,4-Diisopropylphenyl)-4-oxo-N-phenyl-2-(R,S)-(1-
piperidinyl)butyramide (6). Starting from (E)-4-(2,4-diisopropyl-
phenyl)-4-oxo-2-butenoic acid phenylamide (0.5 g, 1.49 mmol)
and equimolar amount of piperidine, as described above, 0.62 g
of 6 was obtained, 98.89% yield, pale yellow solid: mp 95–96 °C.
1H NMR (500 MHz, CDCl3) d: 1.27 (d, 12H, J = 6.72 Hz, i-PrCH3);
1.48 (m, 2H, piperidine –CH2–); 1.65 (m, 4H, piperidine –CH2–);
2.56 (t, 4H, J1,2 = 5.07 Hz, piperidine –CH2–); 2.89–2.99 (overlap-
ping m, 2H, ABX and i-PrCH); 3.41–3.60 (overlapping m, 2H, ABX
and i-PrCH); 4.23 (ABX, dd, J1,2 = 4.38 Hz, J1,3 = 8.03 Hz, 1H); 7.04,
7.08 7.12 (triplet-like picks, 1H, amide p-phenyl); 7.27–7.36 (over-
lapping m, 4H, amide m-phenyl, and aroyl m-phenyl); 7.53, 7.57
4.1.1.10. 4-(4-Chlorophenyl)-4-oxo-N-phenyl-2-(R,S)-(4-mor-
pholinyl)butyramide (10). Starting from (E)-4-(4-chlorophenyl)-
4-oxo-2-butenoic acid phenylamide (0.5 g, 1.70 mmol) and equi-

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M. D. Vitorovic-Todorovic et al. / Bioorg. Med. Chem. 18 (2010) 1181–1193
1190
molar amount of morpholine, as described above, 0.56 g of 10 was
obtained, 88.88% yield, white solid: mp 144–148 °C. 1H NMR
(200 MHz, CDCl3) d: 2.66 (t, 4H, J1,2 = 4.61 Hz, morpholine
–CH2–); 2.90 (ABX, dd, J1,2 = 4.49 Hz, J1,3 = 16.84 Hz, 1H); 3.71
(ABX, dd, partially overlapped with morpholine –CH2–,
J1,2 = 7.86 Hz, J1,3 = 16.28 Hz); 3.79 (q-like picks, 4H, J1,2 = 4.10 Hz,
J1,3 = 8.45 Hz, morpholine –CH2–); 4.29 (ABX, dd, J1,2 = 3.93 Hz,
J1,3 = 7.87 Hz, 1H); 7.09 (t, 1H, J1,2 = 7.94 Hz, amide p-phenyl);
7.31 (t, 2H, J1,2 = 7.94 Hz, amide m-phenyl); 7.45 (d, 2H,
J = 8.45 Hz, aroyl m-phenyl); 7.50, 7.54 (doublet-like picks, 2H,
J = 7.89 Hz, amide o-phenyl); 7.98 (d, 2H, J = 8.27 Hz, aroyl o-phe-
nyl); 9.17 (s, 1H, –NH– amide). 13C NMR (50 MHz, CDCl3) d
31.79; 49.96; 65.51; 67.28; 119.31; 124.28; 128.91; 129.09;
129.62; 135.28; 137.41; 139.58; 169.46; 197.04. LC ESI-MS (HR):
MH+ (obsd) 373.1315; calcd for C20H22ClN2O3 = 373.1319.
67.35; 119.28; 123.28; 124.16; 124.76; 128.13; 129.02; 136.17;
137.61; 148.41; 152.27; 169.64; 202.65. LC ESI-MS (HR): MH+
(obsd) 423.2648; calcd for C26H35N2O3 = 423.2639.
4.1.1.14. 4-(5,6,7,8-Tetrahydro-2-naphthalenyl)-4-oxo-N-phe-
nyl-2-(R,S)-(4-morpholinyl)butyramide (14). Starting from (E)-
4-(5,6,7,8-tetrahydro-2-naphthalenyl)-4-oxo-2-butenoic acid phe-
nylamide (0.72 g, 2.40 mmol) and equimolar amount of morpho-
line, as described above, 0.84 g of 14 was obtained, 89.17% yield,
pale yellow solid: mp 113–114 °C. 1H NMR (200 MHz, CDCl3) d:
1.80 (m, 4H, tetralinoyl –CH2–); 2.65 (t, 4H, J1,2 = 4.28 Hz, morpho-
line –CH2–); 2.80 (br, 4H, tetralinoyl –CH2–); 3.00 (ABX, dd,
J1,2 = 4.99 Hz, J1,3 = 16.82 Hz, 1H); 3.65–3.79 (overlapped m, 5H,
morpholine –CH2– and ABX); 4.27 (ABX, dd, J1,2 = 5.19 Hz,
J1,3 = 7.06 Hz, 1H); 7.04–7.35 (overlapped m, 4H, amide p-phenyl,
amide m-phenyl and aroyl m-phenyl); 7.51–7.56 (m, 2H, amide
o-phenyl); 7.72–7.76 (m, 2H, aroyl o-phenyl); 9.23 (s, 1H, –NH–
amide). 13C NMR (50 MHz, CDCl3) d: 22.69; 22.82; 23.84; 23.64;
29.24; 29.48; 31.81; 50.96; 65.73; 119.11; 123.76; 123.23;
128.82; 129.05; 129.22; 134.44; 137.27; 137.78; 143.00; 170.55;
198.46. LC ESI-MS (HR): MH+ (obsd) 393.2163; calcd for
C24H29N2O3 = 393.2178.
4.1.1.11. 4-(4-Metoxyphenyl)-4-oxo-N-phenyl-2-(R,S)-(4-mor-
pholinyl)butyramide (11). Starting from (E)-4-(4-metoxyphe-
nyl)-4-oxo-2-butenoic acid phenylamide (0.4 g, 1.42 mmol) and
equimolar amount of morpholine, as described above, 0.52 g of
11 was obtained, 99.42% yield, pale yellow solid: mp 132–134 °C.
1H NMR (200 MHz, CDCl3) d: 2.66 (t, 4H, J1,2 = 4.62 Hz, morpholine
–CH2–); 2.99 (ABX, dd, J1,2 = 4.81 Hz, J1,3 = 16.65 Hz, 1H); 3.65–3.85
(overlapped m, ABX and morpholine –CH2–); 4.28 (ABX, dd,
J1,2 = 4.98 Hz, J1,3 = 7.04 Hz, 1H); 6.95 (d, 2H, J = 8.89 Hz, aroyl m-
phenyl); 7.09 (t, 1H, J1,2 = 7.30 Hz, amide p-phenyl); 7.31 (t, 2H,
J1,2 = 7.53 Hz, amide m-phenyl); 7.54 (d, 2H, J = 7.50 Hz, amide o-
phenyl); 8.02 (d, 2H, J = 8.90 Hz, aroyl o-phenyl); 9.23 (s, 1H, –
NH–amide). 13C NMR (50 MHz, CDCl3) d 31.81; 50.02; 65.18;
67.35; 113.79; 119.28; 124.14; 128.98; 129.84; 130.53; 137.58;
163.59; 169.78; 196.57. LC ESI-MS (HR): MH+ (obsd) 381.2187;
calcd for C21H25N2O4 = 381.2178.
4.1.1.15. 4-(2,5-Dimethylphenyl)-4-oxo-N-phenyl-2-(R,S)-(1-imid-
azolyl)butyramide (15). Starting from (E)-4-(2,5-dimethyl-
phenyl)-4-oxo-2-butenoic acid phenylamide (0.5 g, 1.80 mmol)
and equimolar amount of imidazole, as described above, 0.59 g of
15 was obtained, 95.16% yield, pale yellow solid: mp 156–160 °C.
1H NMR (200 MHz, CDCl3) d: 2.34 (s, 3H, -CH3); 2.37 (s, 3H, –
CH3); 3.46 (ABX, dd, J1,2 = 6.17 Hz, J1,3 = 17.97 Hz, 1H); 4.08 (ABX,
dd, J1,2 = 7.87 Hz, J1,3 = 17.98 Hz, 1H); 5.63 (ABX, triplet, not re-
solved dd, J1,2 = 7.30 Hz, 1H); 7.05–7.36 (overlapping m, 7H, amide
p-phenyl, amide m-phenyl, aroyl m-phenyl, aroyl p-phenyl, and
imidazolyl CH); 7.49–7.59 (overlapped m, 4H, amide o-phenyl, ar-
oyl o-phenyl, and imidazolyl CH); 9.92 (s, 1H, –NH– amide). 13C
NMR (50 MHz, CDCl3) d 20.83; 20.98; 44.26; 56.99; 117.95;
120.11; 124.67; 128.93; 129.51; 129.85; 132.11; 132.99; 135.48;
135.75; 136.01; 137.47; 137.83; 166.45; 199.53. LC ESI-MS (HR):
MH+ (obsd) 348.1708; calcd for C21H22N3O2 = 348.1712.
4.1.1.12. 4-(4-Isopropylphenyl)-4-oxo-N-phenyl-2-(R,S)-(4-mor-
pholinyl)butyramide (12). Starting from (E)-4-(4-isopropylphe-
nyl)-4-oxo-2-butenoic acid phenylamide (0.5 g, 1.70 mmol) and
equimolar amount of morpholine, as described above, 0.61 g of
12 was obtained, 93.85% yield, pale yellow solid: mp 135–137 °C.
1H NMR (200 MHz, CDCl3) d: 1.91 (d, 6H, J = 7.27 Hz, i-PrCH3);
2.66 (t, 4H, J1,2 = 4.49 Hz, morpholine –CH2–); 2.90–3.06 (overlap-
ping m, 2H, i-PrCH and ABX); 3.68–3.85 (overlapping m, 5H,
morpholine –CH2– and ABX); 4.30 (ABX, dd, J1,2 = 5.05 Hz,
J1,3 = 7.30 Hz, 1H); 7.09 (t, 1H, J1,2 = 7.21 Hz, amide p-phenyl);
7.26–7.35 (overlapping m, 4H, amide m-phenyl, and aroyl m-phe-
nyl); 7.54 (d, 2H, J = 7.94 Hz, amide o-phenyl); 7.98 (d, 2H,
J = 8.48 Hz, aroyl o-phenyl); 9.23 (s, 1H, –NH–amide). 13C NMR
(50 MHz, CDCl3) d: 23.62; 32.05; 34.19; 50.04; 65.24; 67.37;
119.30; 124.17; 126.74; 128.51; 129.00; 134.70; 137.58; 154.75;
169.73; 197.79. LC ESI-MS (HR): MH+ (obsd) 369.1815; calcd for
C23H29N2O3 = 369.1814.
4.1.1.16. 4-(4-Chlorophenyl)-4-oxo-N-phenyl-2-(R,S)-(1-imid-
azolyl)butyramide (16). Starting from (E)-4-(4-chlorophenyl)-4-
oxo-2-butenoic acid phenylamide (0.5 g, 1.70 mmol) and equimo-
lar amount of imidazole, as described above, 0.55 g of 16 was ob-
tained, 91.66% yield, pale yellow solid: mp 162–164 °C. 1H NMR
(500 MHz, CDCl3) d: 3.49 (ABX, dd, J1,2 = 5.67 Hz, J1,2 = 17.68 Hz,
1H); 4.11 (ABX, dd, J1,2 = 7.34 Hz, 18.01 Hz, 1H); 5.63 (ABX, triplet,
not resolved dd, J1,2 = 6.00 Hz, 1H); 7.10–7.19 (overlapped m, 2H,
amide p-phenyl and imidazolyl CH); 7.26–7.29 (overlapped m,
3H, amide-m-phenyl, and imidazolyl CH); 7.43 (d, 2H, J = 8.37 Hz,
aroyl m-phenyl); 7.50 (d, 2H, J = 7.39 Hz, amide o-phenyl); 7.67
(s, 1H, imidazolyl CH); 7.86 (d, 2H, J = 8.86 Hz, aroyl o-phenyl);
9.09 (s, 1H, –NH– amide). 13C NMR (125 MHz, CDCl3) d: 41.84;
56.83; 117.91; 120.14; 124.96; 128.32; 129.00; 129.16; 129.54;
129.81; 133.88; 134.04; 137.34; 166.18; 194.84. LC ESI-MS (HR):
MH+ (obsd) 354.1010; calcd for C19H17ClN3O2 = 354.1009.
4.1.1.13. 4-(2,4-Diisopropylphenyl)-4-oxo-N-phenyl-2-(R,S)-(4-
morpholinyl)butyramide (13). Starting from (E)-4-(2,4-diisopro-
pylphenyl)-4-oxo-2-butenoic acid phenylamide (0.5 g, 1.50 mmol)
and equimolar amount of morpholine, as described above, 0.51 g of
13 was obtained, 81.59% yield, white solid: mp 137–140 °C. 1H
NMR (200 MHz, CDCl3) d: 1.27 (d, 12H, J= 6.72 Hz, i-PrCH3); 2.65
(t, 4H, J1,2 = 4.28 Hz, morpholine –CH2–); 2.86–3.00 (overlapping
m, 2H, i-PrCH and ABX); 3.40–3.64 (overlapping m, 2H, i-PrCH
and ABX); 3.78 (q, 4H, J1,2 = 3.94 Hz, J1,3 = 7.87 Hz, morpholine –
CH2–); 4.26 (ABX, dd, J1,2 = 4.49 Hz, J1,3 = 8.42 Hz, 1H); 7.06–7.14
(overlapped m, 2H, amide p-phenyl and aroyl m-phenyl); 7.26–
7.36 (overlapped m, 3H, amide m-phenyl and aroyl m-phenyl);
7.54 (d, 2H, J = 8.39 Hz, amide o-phenyl); 7.67 (d, 1H, J = 7.88 Hz,
aroyl o-phenyl); 9.18 (s, 1H, –NH– amide). 13C NMR (50 MHz,
CDCl3) d: 23.73; 24.22; 24.31; 29.17; 34.25; 36.09; 50.03; 65.53;
4.1.1.17. 4-(4-Metoxyphenyl)-4-oxo-N-phenyl-2-(R,S)-(1-imid-
azolyl)butyramide (17). Starting from (E)-4-(4-metoxyphenyl)-
4-oxo-2-butenoic acid phenylamide (0.4 g, 1.42 mmol) and equi-
molar amount of imidazole, as described above, 0.19 g of 17 was
obtained, 38.00% yield, white solid: mp 106–107 °C. 1H NMR
(200 MHz, CDCl3) d: 3.48 (ABX, dd, J1,2 = 5.62 Hz, J1,3 = 17.98 Hz,
1H); 3.84 (s, 3H, –OCH3); 4.06 (ABX, dd, J1,2 = 7.86 Hz, J1,3
=
17.97 Hz, 1H); 5.72 (ABX, dd, J1,2 = 6.18 Hz, J1,3 = 7.87 Hz, 1H);
6.88 (d, 2H, J = 8.82 Hz, aroyl m-phenyl); 7.05 (t, 1H, J1,2 = 7.18 Hz

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M. D. Vitorovic-Todorovic et al. / Bioorg. Med. Chem. 18 (2010) 1181–1193
amide p-phenyl); 7.14 (br, 1H, imidazolyl CH); 7.20–7.27 (over-
lapped m, 3H, amide-m-phenyl, and imidazolyl CH); 7.55 (d, 2H,
J = 7.41 Hz amide o-phenyl); 7.68 (br, 1H, imidazolyl CH); 7.88 (d,
2H, J = 8.91 Hz aroyl o-phenyl); 10.07 (s, 1H, –NH– amide). 13C
NMR (50 MHz, CDCl3) d 41.53; 55.48; 56.79; 113.91; 118.11;
120.08; 124.58; 128.87; 129.29; 130.49; 137.39; 137.87; 164.10;
166.60; 194.62. LC ESI-MS (HR): MH+ (obsd) 350.1503; calcd for
C20H20N3O3 = 350.1505.
4.1.1.21. 4-(2,5-Dimethylphenyl)-2-(R,S)-{7-[3-(R,S)-(2,5-dimeth-
ylphenyl)-3-oxo-1-phenylcarbamoyl-propylamino]-heptylami-
no}-4-oxo-N-phenyl-butyramide (21). Starting from (E)-4-
(2,5-dimethylphenyl)-4-oxo-2-butenoic acid phenylamide (0.4 g,
1.44 mmol) and a half of equimolar amount of 1,8-diaminooctane,
as described above, 0.45 g of 21 was obtained, 88.23% yield, pale
yellow semisolid. 1H NMR (200 MHz, CDCl3) d: 1.33 (br, 8H,
–CH2–); 1.52 (br, 4H, –CH2–); 2.60 (m, 2H, –CH2–); 2.71 (m, 2H,
–CH2–); 2.34 (s, 6H, m-CH3); 2.46 (s, 6H, o-CH3); 3.25 (ABX, dd,
J1,2 = 8.42 Hz, J1,3 = 17.41 Hz, 2H); 3.55 (ABX, dd, J1,2 = 3.37 Hz,
J1,3 = 17.41 Hz, 2H); 3.68 (ABX, dd, J1,2 = 3.37 Hz, J1,3 = 8.42 Hz,
2H); 7.05–7.21 (overlapped m, 6H, amide p-phenyl, aroyl p-phe-
nyl); 7.26–7.36 (overlapped m, 6H, amide m-phenyl, aroyl m-phe-
nyl); 7.53–7.61 (overlapped m, 4H, amide o-phenyl, aroyl o-
phenyl); 9.59 (s, 1H, –NH– amide). 13C NMR (50 MHz, CDCl3) d
20.83; 21.05; 27.17; 29.39; 30.22; 42.55; 48.36; 59.59; 119.24;
124.05; 128.31; 128.98; 129.62; 132.04; 132.64; 135.43; 136.66;
137.76; 171.92; 202.34. LC ESI-MS (HR): MH+ (obsd) 703.4223;
4.1.1.18. 4-(4-Isopropylphenyl)-4-oxo-N-phenyl–2-(R,S)-(1-imid-
azolyl)butyramide (18). Starting from (E)-4-(4-isopropylphenyl)-
4-oxo-2-butenoic acid phenylamide (0.5 g, 1.70 mmol) and equi-
molar amount of imidazole, as described above, 0.55 g of 18 was
obtained, 90.00% yield, pale yellow solid: mp 178 °C (decomp.).
1H NMR (500 MHz, CDCl3) d 1.25 (d, 6H, J = 7.07 Hz, i-PrCH3);
1.29 (d, 1H, J = 7.07 Hz, i-PrCH3, other conformation); 2.96 (m,
1H, i-PrCH); 3.61 (ABX, dd, J1,2 = 6.54 Hz, J1,3 = 18.17 Hz, 1H); 4.09
(ABX, dd, J1,2 = 6.90 Hz, J1,3 = 17.80 Hz, 1H); 5.76 (ABX, triplet, not
resolved dd, J1,2 = 6.54 Hz, 1H); 7.11–7.15 (overlapped m, 3H,
amide p-phenyl, imidazolyl CH); 7.23 (s, 1H, imidazolyl CH);
7.29–7.31 (overlapped m, 4H, amide m-phenyl, and aroyl m-phe-
nyl); 7.53 (d, 2H, J = 7.74 Hz, amide o-phenyl); 7.86 (d, 2H,
J = 8.42 Hz, aroyl o-phenyl); 8.77 (s, 1H, –NH– amide). 13C NMR
(125 MHz, CDCl3) d: 22.57; 34.31; 47.72; 56.99; 114.54; 118.16;
120.14; 124.94; 126.93; 128.45; 129.00; 137.24; 137.49; 155.78;
166.37; 195.56. LC ESI-MS (HR): MH+ (obsd) 362.1878; calcd for
C22H24N3O2 = 362.1869.
calcd for C43H53N4O4 = 703.4221, MH2þ (obsd) 352.2145; calcd
2
for C43H54N4O4 = 352.2136.
4.1.1.22. 4-(4-Chlorophenyl)-2-(R,S)-{7-[3-(R,S)-(4-chlorophenyl)-
3-oxo-1-phenylcarbamoyl-propylamino]-heptylamino}-4-oxo-
N-phenyl-butyramide (22). Starting from (E)-4-(4-chlorophenyl)-
4-oxo-2-butenoic acid phenylamide (0.5 g, 1.70 mmol) and a half of
equimolar amount of 1,8-diaminooctane, as described above, 0.56 g
of 22 was obtained, 91.80% yield, white solid: mp 101 °C (decomp.).
1H NMR (500 MHz, CDCl3) d: 1.31–1.36 (m, 8H, –CH2–); 1.48–1.52
(m, 4H, –CH2–); 2.55–2.60 (m, 2H, –CH2–); 2.69–2.74 (m, 2H,
–CH2–); 3.30 (ABX, dd, J1,2 = 8.44 Hz, J1,3 = 17.61 Hz, 2H); 3.58
(ABX, dd, J1,2 = 3.30 Hz, J1,3 = 17.61 Hz, 2H); 3.68 (ABX, dd,
J1,2 = 3.30 Hz, J1,3 = 8.07 Hz, 2H); 7.10 (t, 2H, J = 7.21 Hz, amide
p-phenyl); 7.32 (t, 4H, J = 7.76 Hz, amide m-phenyl); 7.44 (d, 4H,
J = 8.87 Hz, aroyl m-phenyl); 7.57 (d, 4H, J = 7.77 Hz, amide o-phe-
nyl); 7.91 (d, 4H, J = 8.87 Hz, aroyl o-phenyl); 10.23 (s, 1H, –NH–
amide). 13C NMR (125 MHz, CDCl3) d 26.91; 29.11; 29.96; 39.61;
48.11; 59.09; 119.09; 123.90; 128.05; 128.80; 129.34; 134.37;
137.42; 139.86; 171.34; 197.17. LC ESI-MS (HR): MH+ (obsd)
4.1.1.19. 4-(2,4-Diisopropylphenyl)-4-oxo-N-phenyl-2-(R,S)-(1-
imidazolyl) butyramide (19). Starting from (E)-4-(2,4-diisopro-
pylphenyl)-4-oxo-2-butenoic acid phenylamide (0.5 g, 1.50 mmol)
and equimolar amount of imidazole, as described above, 0.58 g of
19 was obtained, 96.66% yield, white solid: 170 °C (decomp.). 1H
NMR (500 MHz, CDCl3) d: 1.14 (d, 3H, J = 6.71 Hz, i-PrCH3); 1.19
(d, 3H, J = 6.71 Hz, i-PrCH3); 1.25 (d, 6H, J = 6.71 Hz, i-PrCH3);
2.99 (h, J1,2 = 7.04 Hz, J1,3 = 14.09 Hz, 1H, i-PrCH); 3.37 (h, 1H,
J1,2 = 7.04 Hz, J1,3 = 13.76 Hz, i-PrCH); 3.45 (ABX, dd, J1,2 = 6.38 Hz,
J1,3 = 17.79 Hz, 1H); 4.04, (ABX, dd, J1,2 = 7.05 Hz, J1,3 = 17.79 Hz,
1H); 5.59 (ABX, triplet, not resolved dd, J1,2 = 6.71 Hz, 1H); 7.09–
7.13 (overlapped m, 2H, amide p-phenyl, and aroyl m-phenyl);
7.15, 7.16 (doublet-like picks, 2H, imidazolyl CH); 7.25 (br, 1H
imidazolyl CH); 7.29, (t, 2H, J = 7.72 Hz, amide m-phenyl); 7.48–
7.55 (overlapped signals, 4H, aroyl m-phenyl, amide o-phenyl,
and aroyl o-phenyl); 8.95 (s, 1H, –NH– amide). 13C NMR
(125 MHz, CDCl3) d: 23.68; 24.06; 29.11; 34.33; 45.29; 57.21;
117.84; 120.16; 123.47; 124.88; 124.98; 128.31; 128.98; 130.09;
134.43; 137.42; 137.55; 149.02; 153.29; 166.36; 200.57. LC ESI-
MS (HR): MH+ (obsd) 404.2340; calcd for C25H30N3O2 = 404.2338.
715.2821; calcd for C39H43Cl2N4O4 = 715.2821; MH2þ (obsd) 388.
2
1452; calcd for C39H43Cl2N4O4 = 358.1443.
4.1.1.23. 4-(4-Metoxyphenyl)-2-(R,S)-{7-[3-(R,S)-(4-metoxyphe-
nyl)-3-oxo-1-phenylcarbamoyl-propylamino]-heptylamino}-4-
oxo-N-phenyl-butyramide (23). Starting from (E)-4-(4-metoxyl-
phenyl)-4-oxo-2-butenoic acid phenylamide (0.3 g, 1.10 mmol)
and a half of equimolar amount of 1,8-diaminooctane, as described
above, 0.38 g of 23 was obtained, 97.43% yield, pale yellow semi-
solid: mp 87–89 °C. 1H NMR (500 MHz, CDCl3) d 1.30–1.35 (m,
8H, –CH2–); 1.47–1.51 (m, 4H, –CH2–); 2.54–2.59 (m, 2H, –CH2–
); 2.69–2.74 (m, 2H, –CH2–); 3.24 (ABX, dd, J1,2 = 7.44 Hz,
J1,3 = 18.87 Hz, 2H); 3.57 (ABX, dd, J1,2 = 3.30 Hz, J1,3 = 17.24 Hz,
2H); 3.66 (ABX, dd, J1,2 = 2.93 Hz, J1,3 = 8.43 Hz, 2H); 6.92 (d, 4H,
J = 8.87 Hz, aroyl m-phenyl); 7.09 (t, 2H, J = 7.54 Hz, amide p-phe-
nyl); 7.32 (t, 4H, J = 7.54 Hz, amide m-phenyl); 7.59 (d, 4H,
J = 8.01 Hz, amide o-phenyl); 7.95 (d, 4H, J = 8.95 Hz, aroyl o-phe-
nyl); 9.64 (s, 1H, –NH– amide). 13C NMR (125 MHz, CDCl3) d:
27.17; 29.34; 30.20; 39.81; 48.44; 55.45; 59.58; 113.83; 119.23;
124.00; 128.27; 128.93; 130.48; 137.78; 163.84; 171.91; 196.97.
LC ESI-MS (HR): MH+ (obsd) 707.3804; calcd for C41H48N4O4 =
707.3809, MH22þ (obsd) 354. 1956; calcd for C39H43Cl2N4O4 =
354.1938.
4.1.1.20. 4-(5,6,7,8-Tetrahydro-2-naphthalenyl)-4-oxo-N-phenyl-
2-(R,S)-(1-imidazolyl)butyramide (20). Starting from (E)-4-(5,6,
7,8-tetrahydro-2-naphthalenyl)-4-oxo-2-butenoic acid phenyla-
mide (0.5 g, 1.80 mmol) and equimolar amount of imidazole, as
described above, 0.17 g of 20 was obtained, 25.37% yield, white
solid: mp 154–156 °C. 1H NMR (200 MHz, CDCl3) d 1.78 (br, 4H,
tetralinoyl –CH2–); 2.77 (br, 4H, tetralinoyl –CH2–); 3.48 (ABX,
dd, J1,2 = 5.62 Hz, J1,3 = 17.97 Hz, 1H); 4.09 (ABX, dd, J1,2 = 7.86 Hz,
J1,3 = 17.97 Hz, 1H); 5.66 (ABX, triplet, not resolved dd,
J1,2 = 6.17 Hz, 1H); 7.01–7.26 (overlapped m, 6H, amide p-phenyl,
amide m-phenyl, aroyl m-phenyl, and imidazolyl CH); 7.52–7.62
(overlapped m, 5H, amide o-phenyl, aroyl o-phenyl, imidazolyl
CH); 10.23 (s, 1H, –NH– amide). 13C NMR (50 MHz, CDCl3) d:
22.61; 22.76; 29.22; 29.61; 41.77; 56.72; 118.08; 120.08; 124.54;
125.12; 128.85; 129.09; 133.24; 137.39; 137.92; 144.22; 166.62;
196.13. LC ESI-MS (HR): MH+ (obsd) 374.1882; calcd for
C23H24N3O2 = 374.1869.
4.1.1.24. 4-(4-Isopropylphenyl)-2-(R,S)-{7-[3-(R,S)-(4-isopropyl-
phenyl)-3-oxo-1-phenylcarbamoyl-propylamino]-heptylamino}-
4-oxo-N-phenyl-butyramide (24). Starting from (E)-4-(4-isopro-
pylphenyl)-4-oxo-2-butenoic acid phenylamide (0.5 g, 1.70 mmol)

´ ´
M. D. Vitorovic-Todorovic et al. / Bioorg. Med. Chem. 18 (2010) 1181–1193
1192
and a half of equimolar amount of 1,8-diaminooctane, as described
above, 0.58 g of 24 was obtained, 93.55% yield, pale yellow solid:
mp 92–94 °C. 1H NMR (200 MHz, CDCl3) d 1.26 (d, J = 6.74 Hz,
12H, i-PrCH3); 1.31 (br, 8H, –CH2–); 1.50 (br, 8H, –CH2–); 2.50–
2.75 (m, 4H, –CH2–); 3.28 (ABX, dd, J1,2 = 5.05 Hz, J1,3 = 16.56 Hz,
2H); 3.70, (ABX, dd, J1,2 = 7.02 Hz, J1,3 = 16.57 Hz, 2H); 4.27 (ABX,
dd, J1,2 = 5.06 Hz, J1,3 = 7.02 Hz, 2H);7.05, 7.09, 7.12 (triplet-like
picks, 2H, amide p-phenyl); 7.25–7.36 (overlapped m, 4H, amide
m-phenyl and aroyl m-phenyl); 7.57, 7.61 (doublet-like picks, 4H,
amide o-phenyl); 7.92 (d, 4H, J = 8.95 Hz, aroyl o-phenyl); 9.61 (s,
2H, –NH– amide). 13C NMR (50 MHz, CDCl3) d: 23.54; 27.13;
29.33; 30.17; 34.19; 39.97; 48.38; 59.41; 119.24; 126.80; 128.29;
128.44; 128.96; 134.13; 137.76; 155.22; 171.95; 198.206. LC
ESI-MS (HR): MH+ (obsd) 731.4536; calcd for C45H57N4O4 =
731.4519; MH22þ (obsd) 366.2302; calcd for C45H58N4O4 =
366.2294.
reaction took place in 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 5,50-dithiobis(2-nitrobenzoic)acid (DTNB), used to produce
yellow anion of 5-thio-2-nitrobenzoic acid in reaction with thioch-
oline released by AChE. Test compound was added to the enzyme
solution and preincubated at 25 °C for 35 min followed by the addi-
tion of DTNB (0.95 mL) and substrate (50 lL). Inhibition curves
were performed at least in triplicate. One triplicate sample without
test compound was always present to yield the 100% of AChE activ-
ity. The reaction was monitored for 3 min, and the color production
was measured at 412 nm. The reaction rates were compared, and
the percent inhibition, due to the presence of test compounds,
was calculated. IC50 values were determined from inhibition curves
(log inhibitor concentration vs percent inhibition).
The investigation of butyrylcholinesterase (BChE) inhibition
was carried out similarly, using 0.035 units of horse serum BChE
(Fluka) and 0.5 mM butyrylthiocholine iodide instead of AChE
and acetylthiocholine, in final volume of 2 mL. For determination
of inhibition type of 19 toward AChE and 13 toward BChE, the
Lineweaver–Burk plots were generated by using a fixed amount
of cholinesterase and varying amounts of the substrate (0.10–
0.20 mM, AChE and 0.11–0.33 mM, BChE) in the absence or pres-
ence of different inhibitor concentrations. The re-plot of the slopes
of the double reciprocal plots against inhibitor concentration gave
the inhibitor constant (Ki) as the intercept on the x-axis.
4.1.1.25. 4-(2,4-Diisopropylphenyl)-2-(R,S)-{7-[3-(R,S)-(4-isopro-
pylphenyl)-3-oxo-1-phenylcarbamoyl-propylamino]-heptylami-
no}-4-oxo-N-phenyl-butyramide (25). Starting from (E)-4-(2,4-
diisopropylphenyl)-4-oxo-2-butenoic acid phenylamide (0.5 g,
1.50 mmol) and a half of equimolar amount of 1,8-diaminooctane,
as described above, 0.56 g of 25 was obtained, 91.80% yield, pale
yellow semisolid: mp 69–70 °C. 1H NMR (500 MHz, CDCl3) d:
1.23–1.26 (br, 24H, i-PrCH3); 1.34 (br, 8H, –CH2–); 1.53 (br, 8H,
–CH2–); 2.56–2.76 (m, 4H,–CH2–); 3.24 (ABX, dd, J1,2 = 8.07 Hz,
J1,3 = 17.24 Hz, 2H); 3.47–3.51 (ABX, m, 2H); 3.67 (ABX, dd,
J1,2 = 3.30 Hz, J1,3 = 7.70 Hz, 2H); 7.07, 7.09, 7.10 (triplet-like picks,
2H, amide p-phenyl); 7.26 (br, 2H, aroyl m-phenyl); 7.29–7.33
(overlapped signals, 8H, aroyl m-phenyl, amide o-phenyl, and aroyl
o-phenyl); 9.54 (s, 1H, –NH– amide). 13C NMR (125 MHz, CDCl3) d:
23.68; 24.20; 27.23; 29.20; 29.43; 30.29; 34.27; 43.51 48.39;
59.75; 119.26; 13.38; 124.02; 124.87; 126.40; 128.95; 135.16;
137.78; 148.66; 152.79; 171.74; 203.48. LC ESI-MS (HR): MH+
4.3. 3D-QSAR methods
SMILES notation of all compounds used in model were con-
verted to 3D by OMEGA,43 using MMFF94s44 force field (build
and search), and five lowest energy conformations per compound
were generated. Every conformer was further minimized by semi-
empirical MO PM6 method,45 using implicit solvation (water) and
the structure of each compound having the lowest heat of forma-
tion was used for model building. Protonation state of each com-
pound was ascribed by Pentacle under pH value reported in the
original reference (8.00). Pentacle use AMANDA46 algorithm to
produce second generation alignment-independent molecular
descriptors (GRIND-2), insensitive to small to medium conforma-
tional changes, derived from GRID47 molecular interaction fields
(MIFs). Derived GRID MIF minima are encoded in variables that de-
scribe pair of nodes (IE of each node and the corresponding dis-
tance between nodes); descriptors are further processed by
means of built-in PCA/PLS (principal component analysis/partial
least squares) chemometric tool.
For model generation N1, O, and DRY probes were used, with
GRID resolution of 0.4 Å. For encoding, maximum auto and cross-
correlation were applied (MACC2). Probes cut-off was hold on de-
fault value, as well as MACC2 smoothing window and scale factor.
Validation of model was done by cross-validation using three
groups of approximately the same size in which the objects were
assigned randomly. For the final model leave two out and leave
one out cross-validation data are also reported. Due to unavailabil-
ity of the current Pentacle prerelease to show interaction energies
(IE) on a particular node, their values were estimated by GRID. DRY
probe isovolumes were extracted by BIOCUBE.48
(obsd) 815.5471; calcd for C51H69N4O4 = 815.5475, MH2þ (obsd)
2
408.2781; calcd for C51H70N4O4 = 408.2771.
4.1.1.26. 4-(5,6,7,8-Tetrahydro-2-naphthalenyl)-2-(R,S)-{7-[3-
(R,S)-(5,6,7,8-tetrahydro-2-naphthalenyl)-3-oxo-1-phenylcar-
bamoyl-propylamino]-heptylamino}-4-oxo-N-phenyl-butyra-
mide (26). Starting from (E)-4-(2,4-diisopropylphenyl)-4-oxo-2-
butenoic acid phenylamide (0.5 g, 1.80 mmol) and a half of equi-
molar amount of 1,8-diaminooctane, as described above, 0.57 g
of 26 was obtained, 83.82% yield, pale yellow semisolid. 1H NMR
(200 MHz, CDCl3) d: 1.30 (br, 8H, –CH2–); 1.49 (br, 4H, –CH2–);
1.79 (br, 8H, tetralinoyl –CH2–); 2.58, 2.68 (br, 4H, overlapped
broad multiplets, –CH2–); 2.79 (br, 8H, tetralinoyl –CH2-); 3.25
(ABX, dd, J1,2 = 8.43 Hz, J1,3 = 16.85 Hz, 2H); 3.56–4.11 (ABX, m,
4H); 7.05–7.15 (4H, overlapping multiplets, amide p-phenyl, ar-
oyl-m-phenyl); 7.25, 7.28, 7.32 (triplet-like picks, 4H, amide m-
phenyl); 7.57–7.70 (overlapping multiplets, aroyl o-phenyl, aroyl
m-phenyl); 9.61 (s, 1H, –NH– amide). 13C NMR (50 MHz, CDCl3)
d: 22.65; 22.79; 27.11; 29.24; 29.33; 29.57; 30.17; 40.02; 48.38;
59.45; 119.20; 123.99; 125.18; 128.27; 128.94; 129.45; 133.75;
137.76; 143.82; 171.99; 198.44. LC ESI-MS (HR): MH+ (obsd)
755.4536; calcd for C47H56N4O4 = 755.4535, MH22þ (obsd)
378.2302; calcd for C47H56N4O4 = 378.2298.
To examine influence of the grid resolution, as well as influence
of the alignment of compounds on the reported model, few more
models were built using: (a) different grid resolutions (0.2, 0.7,
and 1.0 Å) and (b) alignment of compounds within the each subset;
with retaining of all other parameters as for the reported model. In
this way the obtained patterns of PLS coefficient plots were slightly
different, but variables that have high influence on the each model
obtained were exactly the same as those described in the reported
model. So, it has been confirmed that method used is very robust in
respect to grid resolution and the alignment independent.
4.2. Biological studies
The inhibition potency of the prepared compounds on acetyl-
cholinesterase (AChE) activity was evaluated spectrophotometri-
cally by the method of Ellman et al.,31 using AChE from E. Eel,
type VI-S (Sigma) and acetylcholine iodide (0.28 mM) as substrates.
Four to six different concentrations, which produce 20–80% of en-
zyme activity inhibition, of the each compound were used. The

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´
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M. D. Vitorovic-Todorovic et al. / Bioorg. Med. Chem. 18 (2010) 1181–1193
14. Bourne, Y.; Taylor, P.; Radic´, Z.; Marchot, P. EMBO J. 2003, 22, 1.
15. Bartolini, M.; Bertucci, C.; Cavrini, V.; Andrisano, V. Biochem. Pharmacol. 2003,
65, 407.
4.4. Docking procedures
All docking experiments were performed on the Mus Musculus
AChE refined at 2.05 Å resolution (PDB entry: 2HA2). Truncated
residues were properly completed by means of Swiss PDB View-
er.49 Hydrogen atoms were added to the protein amino acid resi-
dues, non-polar hydrogens were merged, and Gesteiger charges
were loaded using AUTODOCK Tools.50 The lowest energy conforma-
tions of 5 (R) and 19 (R) were generated using Omega software43
and further optimized by PM6 semiempirical MO method45 as
implemented in MOPAC2009. VEGA ZZ 2.3.151 was used as GUI.
Conformations of 5 (S) and 19 (S) were generated as mirror images
of R configurations using VEGA ZZ. Both enantiomers of both com-
pounds were docked to mAChE using AUTODOCK 4.0.1 package.38
Docking was carried out by using the hybrid Lamarckian genetic
algorithm, 50 runs were performed with an initial population of
250 randomly placed individuals and maximum number of
1.0 ꢂ 107 energy evaluations. Resulting docked orientations within
RMSD of 0.5 Å were clustered together. All other parameters were
maintained at their default settings. The lowest energy cluster re-
turned by AUTODOCK for each compound was used for further analy-
sis. Docking on HuBChE is performed under the same conditions,
except that no amino acid residues were held flexible during
calculation.
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Republic of Serbia supports this work (Grants: 142010 and
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