Converting maslinic acid into an effective inhibitor of acylcholinesterases

Article abstract of DOI:10.1016/j.ejmech.2015.09.007

During the last decade, maslinic acid has been evaluated for many biological properties, e.g. as an anti-tumor or an anti-viral agent but also as a nutraceutical. The potential of maslinic acid and related derivatives to act as inhibitors of acetyl- or butyryl-cholinesterase was examined in this communication in more detail. Cholinesterases do still represent an interesting group of target enzymes with respect to the investigation and treatment of the Alzheimer's disease and other dementia illnesses as well. Although other triterpenoic acids have successfully been tested for their ability to act as inhibitors of cholinesterases, up to now maslinic acid has not been part of such studies.For this reason, three series of maslinic acid derivatives possessing modifications at different centers were synthesized and subjected to Ellman's assay to determine their inhibitory strength and type of inhibitory action. While parent compound maslinic acid was no inhibitor in these assays, some of the compounds exhibited an inhibition of acetylcholinesterase in the single-digit micro-molar range. Two compounds were identified as inhibitors of butyrylcholinesterase showing inhibition constants comparable to those of galantamine, a drug often used in the treatment of Alzheimer's disease. Furthermore, additional selectivity as well as cytotoxicity studies were performed underlining the potential of several derivatives and qualifying them for further investigations. Docking studies revealed that the different kinetic behavior within the same compound series may be explained by the ability of the compounds to enter the active site gorge of AChE.

Full text of DOI:10.1016/j.ejmech.2015.09.007

European Journal of Medicinal Chemistry 103 (2015) 438e445
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Converting maslinic acid into an effective inhibitor of
acylcholinesterases
Stefan Schwarz a, Anne Loesche a, Susana Dias Lucas b, Sven Sommerwerk a,
a
a
a
a, *
ꢀ
Immo Serbian , Bianka Siewert , Elke Pianowski , Rene Csuk
a
€
Bereich Organische Chemie, Martin-Luther-Universitat Halle-Wittenberg, Kurt-Mothes-Str. 2, D-06120 Halle (Saale), Germany
Instituto de Investigacao do Medicamento (iMed.ULisboa), Faculdade de Farmacia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal
b
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
During the last decade, maslinic acid has been evaluated for many biological properties, e.g. as an anti-
tumor or an anti-viral agent but also as a nutraceutical. The potential of maslinic acid and related de-
rivatives to act as inhibitors of acetyl- or butyryl-cholinesterase was examined in this communication in
more detail. Cholinesterases do still represent an interesting group of target enzymes with respect to the
investigation and treatment of the Alzheimer's disease and other dementia illnesses as well. Although
other triterpenoic acids have successfully been tested for their ability to act as inhibitors of cholines-
terases, up to now maslinic acid has not been part of such studies.
Received 20 January 2015
Received in revised form
16 March 2015
Accepted 5 September 2015
Available online 9 September 2015
Keywords:
For this reason, three series of maslinic acid derivatives possessing modifications at different centers
were synthesized and subjected to Ellman's assay to determine their inhibitory strength and type of
inhibitory action. While parent compound maslinic acid was no inhibitor in these assays, some of the
compounds exhibited an inhibition of acetylcholinesterase in the single-digit micro-molar range. Two
compounds were identified as inhibitors of butyrylcholinesterase showing inhibition constants com-
parable to those of galantamine, a drug often used in the treatment of Alzheimer's disease. Furthermore,
additional selectivity as well as cytotoxicity studies were performed underlining the potential of several
derivatives and qualifying them for further investigations. Docking studies revealed that the different
kinetic behavior within the same compound series may be explained by the ability of the compounds to
enter the active site gorge of AChE.
Maslinic acid
Augustic acid
Acetylcholinesterase
Butyrylcholinesterase
Alzheimer disease
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
better understanding as well as for finding effective treatments.
Although AD is not completely understood, several hypotheses
Many people living in the western hemisphere, and especially
those of an advanced age, are afraid of spending their last years
suffering from serious diseases, e.g. from cancer or a stroke. De-
mentia illnesses e like the Alzheimer disease (AD) e considerably
contribute to uncertainty among the population due to their impact
on cognitive abilities. Worldwide approximately 35 million in-
dividuals, i.e. one in 200, currently suffer from some kind of de-
mentia; according to some estimates within the next 30 years this
number might double [1,2]. 20 million of these persons are affected
by AD, and the prevalence of AD increases with age starting from
10% at the age of 65 to nearly 50% at 85 years [1]. Therefore, there is
a scientific and economic demand for further investigations for a
exist and are foundations or at least starting points of current
therapies. One of these theories (“amyloid hypothesis”) deals with
the neurotoxic effect of a
b-amyloid being formed by the action of
a
-, - and -secretases [3,4]. Although this is one of the most
b
g
prominent theories, there are some deficits: For instance, approx-
imately 30 per cent of healthy, middle-aged people possess equal
amounts of b-amyloid plaques being usually found in AD brains
[5,6]. Most of the therapies aiming to decrease the concentration of
the plaques, however, did not result in permanent increase of
cognitive abilities or their at least their restauration [7e10]. Other
attempts to find possible treatments focus on the investigation of
inflammatory processes, mitochondrial disorders or the
[11e13].
t-protein
Impairment in the cholinergic function, however, is of critical
importance in AD (“cholinergic hypothesis”) [1,14]. Thus, our
* Corresponding author.
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
http://dx.doi.org/10.1016/j.ejmech.2015.09.007
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

S. Schwarz et al. / European Journal of Medicinal Chemistry 103 (2015) 438e445
439
present study focusses on the neurotransmitter acetylcholine
(ACh), whose concentration is reduced during AD leading to typical
symptoms like amnesia or behavioral disorder [14e17]. The hy-
drolytic enzymes acetylcholinesterase (AChE, E.C. 3.1.1.7) and
butyrylcholinesterase (BChE, E.C. 3.1.1.8) are responsible for the
hydrolysis of ACh, thus, controlling the concentration of this
neurotransmitter in different tissues of an organism. Therein, BChE
serves as a coregulator of the cholinergic transmission, and
although it is mainly present in other parts of the body, BChE is able
to compensate a reduced AChE activity in the brain [1,18,19].
Furthermore, the AChE/BChE ratio in the brain alters from 0.2 in
normal brain to 11 during AD [14,20,21]. For this reason, both en-
zymes represent interesting targets for the development of AD
therapies or tools for a deeper insight into this disease.
The only treatments with clinical evidence [14] to AD patients
are the cholinesterase inhibitors galantamine, donepezil and riva-
stigmine. Several triterpenes have also been shown to act as in-
hibitors of AChE; this includes several hopanes [22], lanostanes [23]
and lupanes [24]. Pentacyclic triterpenoic acids and their de-
rivatives have been shown to be potent cholinesterase inhibitors in
via a four step chromatography-free synthesis starting from ole-
anolic acid as previously reported [39]. Its derivatizations were
performed using well-established reactions as described above for
the synthesis of derivatives of MA.
2.2. Biology
All compounds 1e48, including maslinic acid (MA) and augustic
acid (44), were subjected to Ellman's assays to determine their
inhibitory activity (expressed as inhibition constants Ki and Ki0) for
the enzymes AChE and BChE. The results of these measurements
are compiled in Table 1.
In summary, inhibitions constants for 24 compounds towards
AChE and for 3 compounds towards BChE were determined. Two
compounds, 18 and 19, however, were not soluble under the con-
ditions of the assay. For parent compound maslinic acid no inhi-
bition constants below 100 mM could be obtained. Hence, maslinic
acid does not significantly inhibit the cholinesterases; higher con-
centration could not be applied due to solubility reasons. Also,
augustic acid (44) is no inhibitor of AChE, but e in contrast to
maslinic acid e for BChE a mixed-type inhibition was observed
micro-molar range [25] with compounds of the a- or b-amyrin type
being most active. Ursolic acid, for instance, acts as mixed-type
inhibitor on AChE in the same magnitude as tacrine, a well-
established drug [26]. Oleanolic acid [27,28] and structurally
related compounds, e.g. taraxerol [29], echinocystic acid [30] or
glycyrrhetinic acid [31], possessed IC50 values and inhibition con-
stants Ki comparable to those of standard remedies like galant-
amine or donepezil.
Several studies an anti-tumor [32,33], anti-inflammatory [34] or
an anti-viral [35] activity of maslinic acid and derivatives have been
performed but the ability of these compounds to act as inhibitors of
cholinesterases has not been investigated so far. Thus we prepared
a series of maslinic acid derivatives differing in the substitution
pattern at positions C-2, C-3 and C-28. All of these derivatives were
screened for their ability to inhibit AChE and BChE; they were
tested employing Ellman's assay, and their inhibitory constants (Ki
and Ki0) as well as the type of inhibition was determined. Further-
more, seven representative compounds were selected and inves-
tigated for a selectivity towards others enzymes [lipase from
Candida antarctica (a serine hydrolase), papain (a sulfhydryl
enzyme) and carbonic anhydrase II (a metalloenzyme)]. Addition-
ally, some preliminary toxicity studies for selected derivatives were
performed employing murine embryonic fibroblasts (NiH 3T3) in a
photometric sulforhodamine B (SRB) assay.
(inhibition constants: Ki
¼
35.64
5.73
m
M
and
Ki0 ¼ 10.58 1.95
mM). The uncompetitive part of the mixed-type
inhibition (as expressed by Ki0) is predominant. This indicates
that augustic acid deploys its inhibitory action predominantly by
binding to the enzymeesubstrate complex rather than by binding
to the free enzyme.
The first group of compounds (the esters 1e27) were inhibitors
of AChE, with the 1-chloro-butylester 13 as the most active com-
pound of this series. This compound is a competitive inhibitor of
AChE (Ki ¼ 1.68 0.30
14e27 inhibition constants between 2.03 and 34.85
determined. Especially, those esters having alkyl groups with more
than three carbons showed Ki < 10 M. While compounds 13 and 27
mM). In comparison, for the esters 1e12 and
m
M were
m
were competitive inhibitors, all other compounds of this series gave
a mixed-type inhibition. Thus, the propyl ester 4 and the heptyl
ester 17 gave an almost non-competitive inhibition (similar Ki and
Ki0) while a mixed-type inhibition with a dominating competitive
part (Ki < Ki0) could be determined for the ethyl ester 2 or the 10-
butinyl ester 12. The 1-chloro-propyl ester 8 and the cyclohexyl
ester 20 represent examples for mixed-type inhibitors with a
dominating uncompetitive part (Ki > Ki0).
From the second group [representing 2,3-substituted maslinic
acid esters (28e31) and amides (32e43)] four compounds (29, 31,
40 and 43) exhibited an activity towards AChE, only e albeit in
moderate micro-molar magnitude. Thus, 29 and 31 showed a
2. Results and discussion
moderate mixed-type inhibition (Ki and Ki0 between 10 and 40
mM)
2.1. Chemistry
while the 2,3-diacetylated propargyl amide 40 was determined to
act as a competitive inhibitior of AChE, and the amide 32 is a
competitive inhibitor of BChE with an inhibition constant of
The first group of compounds (1e27, Scheme 1), representing
several esters of maslinic acid, could be obtained from maslinic acid
(MA) by reaction of MA with alkyl bromides in the presence of
powdered K2CO3 in dry DMF [36,37].
Ki ¼ 18.11 3.43
mM. Out of this series, two compounds (32 and 42)
were identified as inhibitors of BChE. Their inhibitory activity is
slightly lower than that of standard galantamine hydrobromide
The second group of compounds (Scheme 2) consists of
matching pairs of maslinic acid derivatives (28, 29), its methyl ester
(30, 31) and amides (32e43) possessing either free or acetylated
hydroxyl groups at positions C-2 and C-3. Acetylations were carried
out with acetic anhydride in pyridine [37] while the sulfamates 30
and 31 were obtained from the methyl ester 1 and sodium hydride
in THF, followed by the addition of sulfamoyl chloride [39]. The
synthesis of amides started from 2,3-diacetyl maslinic acid that was
allowed to react with thionyl chloride followed by the addition of
an amine [37].
(Ki ¼ 9.37 0.67
mM).
As far as the last group of compounds [consisting of augustic
acid (44) and related derivatives 44e48] is concerned, parent
augustic acid turned out to be a mixed-type inhibitor of BChE with a
dominating uncompetitive part (Ki
¼
35.64
5.73 mM,
Ki0 ¼ 10.58
1.95 mM); this compound is no inhibitor of AChE.
Compounds 45 and 46 did not show any activity for AChE or BChE,
while 2,3-dichloroacetyl-substituted 47 and 48 act as AChE in-
hibitors. Both compounds are mixed-type inhibitors in the single-
digit micro-molar range.
A third group of compounds included augustic acid (44) and
derivatives thereof. Augustic acid (44, Scheme 3) was synthesized
Another important criterion is the selectivity of the compounds
concerning one of the cholinesterases on one hand and concerning

440
S. Schwarz et al. / European Journal of Medicinal Chemistry 103 (2015) 438e445
Scheme 1. Structures of maslinic acid (MA) and esters 1e27.
Scheme 2. Structures of 2,3-substituted maslinic acid esters 28e31 and amides 32e43 (All ¼ allyl, Prg ¼ propargyl).
Scheme 3. Synthesis of augustic acid (44) and related derivatives (45e48); a) ref 38; b) acetanhydride, triethylamine, DMAP, DCM, r.t., 1 day; c) I. (COCl)2, DCM, triethylamine, DMF,
r.t., 2 h e benzylamine, triethylamine, DMAP, DCM, 0 ꢀC, 5 min; d) chloroacetic anhydride, triethylamine, DMAP, DCM, r.t., 1 day.
other enzymes on the other. Compounds showing the highest
selectivity (expressed by F ¼ Ki of AChE divided by Ki of BChE) to-
wards AChE are compounds 10 (F < 0.3), 13 (F < 0.17) and 14
(F < 0.28). Both most active BChE inhibitors 32 (F > 1.10) and 42
(F ¼ 1.30) showed a slight selectivity towards BChE.
sulforhodamine B assay employing murine embryonic fibroblasts
(NiH 3T3) [36,37]; the results of these assays are compiled in
Table 3. While parent maslinic acid showed an IC50 value of
16.6
mM, cytotoxicity decreased for some of the compounds: e.g. for
9 (IC50 ¼ 21.6
m
M) or 14 (IC50 ¼ 33.1
m
M). Moreover, the cytotoxicity
In addition, seven representative compounds were selected and
subjected to assays (Table 2) employing bovine carbonic anhydrase,
the lipase from C. antarctica and papain from Carica papaya. Each of
these enzymes represents a different mechanism in the active site
of the enzyme. All derivatives showed only a low or no inhibition at
all - even at the maximum concentration used in the assays. Only
compounds 9, 10 and 14 showed a marginal impact on these
enzymes.
of all derivatives was significantly lower than their inhibition
constants, except for the propyl ester 4 and the 1-bromopropyl
ester 9.
2.3. Docking studies
Molecular modeling studies were performed to evaluate the
molecular features being important for the inhibitory activity
against AChE of derivatives of MA, to gain some insights on their
mode of action and to explain their different kinetic behavior. For
A low cytotoxicity is mandatory for compounds intended for the
treatment of AD. Thus, IC50 were measured utilizing a photometric

S. Schwarz et al. / European Journal of Medicinal Chemistry 103 (2015) 438e445
441
Table 1
Inhibitory constants for galantamine hydrobromide (GH), MA and compounds 1e54 (Ki and Ki′ in mM), determined using Ellman's assay employing acetylcholinesterase (AChE)
and butyrylcholinesterase (BChE) with galantamine hydrobromide as standard. Four different substrate concentrations and four different inhibitor concentrations were used;
each experiment was performed at least in triplicate; n. sol e not soluble.
AChE
AChE
BChE
AChE
AChE
BChE
BChE
Ki
Ki0
Ki
Ki
Ki0
Ki
Ki0
GH
MA
1
2
3
4
5
6
7
0.54 0.01
>100
>50
3.49 0.71
>100
16.86 3.31
>30
9.37 0.67
>100
>50
>14
>100
>6
>30
>10
>10
>6
>80
>20
>10
>20
>10
>80
>6
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
>10
>10
>6
>4
>100
>10
>20
>40
>20
18.11 3.43
>20
>20
>14
>20
>20
>6
>20
>14
>10
2.03 0.01
6.14 0.28
6.49 1.68
>10
13.12 1.02
>40
20.53 7.47
>20
>20
>20
>14
>20
>20
9.24 0.93
7.22 0.69
69.56 0.14
4.67 0.25
15.40 1.14
19.77 0.44
38.58 3.11
>10
20.12 0.96
8.76 2.02
34.85 2.06
6.01 0.55
3.31 0.81
6.76 0.95
1.68 0.30
22.48 1.70
5.71 0.71
>10
4.78 1.63
n. sol.
n. sol.
10.02 0.47
>14
>30
13.73 0.12
3.45 0.24
24.43 0.23
13.44 3.19
11.51 0.31
32.51 2.41
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
>6
>20
9.38 0.80
14.83 1.66
8.14 1.85
>10
16.19 1.09
25.14 4.39
>30
>20
>10
6.85 0.47
6.25 0.77
>10
>6
5.11 0.75
2.67 0.20
39.44 2.71
31.50 2.53
12.43 1.58
>30
35.64 5.73
>20
>10
>6
45.44 3.43
10.58 1.95
>14
>14
>30
>20
9.36 0.18
16.26 0.40
>20
>6
site gorge for the different organisms. In addition, the homology
between EeAChE and hAChE is very high (88%) and no significant
differences are observed in the active site. Comparison of the
docked pose for the competitive inhibitor 27 (green, Fig. 1) with the
poses for the mixed-type inhibitors 12 and 20 (cyano and pink,
respectively, Fig. 1) showed inhibitor 27 entering deep into the
Table 2
Relative inhibition (in %) of the enzymes AChE, BChE, carbonic anhydrase II (from
bovine erythrocytes), lipase (from Candida antarctica) as well as papain (from Carica
papaya) by compounds 4, 9, 10, 13, 14, 17 and 25. Concentration (in mM) represents
the maximum concentration for each compound in the assay. Experiments were
performed at least in triplicate.
Compound
4
9
10
13
14
17
25
active-site gorge and performing H-
teractions with Tyr449 and the catalytic His447. This might explain
its competitive character. In addition, a 2-OH- interaction is
p and HeC]O weak in-
conc. [
mM]
3
40
10
5
40
3
3
p
AChE
BChE
bCA II
Lipase
56
0
39
16
1
18
0
51
0
6
0
0
79
0
0
0
0
79
7
1
0
0
62
0
0
0
0
43
0
0
0
0
observed with the Trp286 in the entrance region of the gorge. On
the other hand, the most favorable poses for the mixed-type in-
hibitors 12 and 20 suggest that those do not enter the active-site
0
0
0
Papain
gorge. However, for each compound two CH-
p interactions were
observed with the Trp286 at the entrance of the gorge, thus indi-
cating that the complex with these inhibitors forms an active site
gorge lid leading to mixed-type inhibitors. Nevertheless the dock-
ing results do not give a molecular insight to explain the different
competitive/uncompetitive character for mixed-type inhibitors 12
and 20, respectively.
Table 3
Cytoxicity [IC50 values in
malignant murine embryonic fibroblasts (NiH 3T3)] and inhibition constants Ki.
(in M for AChE) for selected compounds. Each experiment was performed at least
mM [36,37] from photometric SRB assays, using non-
m
in triplicate.
Compound MA
4
9
10
13
14
17
25
IC50
Ki (AChE)
16.6 13.4
>100
21.6
16.86 34.85
12.3
6.01
12.9
1.68 22.48
33.1
14.1
4.78
17.3
2.03
3. Conclusion
In this study 43 derivatives of maslinic acid as well as five
compounds derived from augustic acid were synthesized and
subjected to Ellman's assay to determine their potential as in-
hibitors of cholinesterases. While no inhibition could be detected
for parent maslinic acid, 22 of its derivatives inhibited AChE, and
two compounds were inhibitors for BChE in a micromolar range.
Augustic acid was an inhibitor of BChE inhibitor, whereas some of
its derivatives inhibited AChE. A chloro-butylester of maslinic acid
13 was the most active species of this study possessing an inhibi-
example, closely related compounds exhibited different kinetic
behavior and were shown to be inhibitors in a single digit micro-
molar concentration: Compound 27 was shown to be a competitive
inhibitor, while 12 inhibited AChE in a mixed-type manner with a
dominating competitive part (Ki < Ki0), and 20 was a mixed-type
inhibitor with a dominating uncompetitive part (Ki > Ki00). The
coordinates of the enzyme structure were obtained from the Pro-
tein Data Bank (accession code 4BDT), and the docking experiments
of the maslinic acid derivatives 12, 20 and 27 were performed using
GOLD 5.2 software [40,41]. We decided to use the final target hAChE
for the docking studies as no differences are observed in the active
tion constant of Ki ¼ 1.68
mM; it is a competitive inhibitor for AChE.
However, almost all of the active compounds were mixed-type
inhibitors, and small differences in their mode of inhibitory ac-
tion were detected, since the ratios of the inhibition constants Ki

442
S. Schwarz et al. / European Journal of Medicinal Chemistry 103 (2015) 438e445
1000, optical rotations on a PerkineElmer 341 polarimeter (1 cm
micro cell) and UVevis spectra on a PerkineElmer unit, Lambda 14.
Melting points were measured with a LEICA hot stage microscope
and are uncorrected. Elemental analysis was done on a Foss-
Heraeus Vario EL unit. TLC was performed on silica gel (Merck
5554, detection by UV absorption). Solvents were dried before use
according to usual procedures. The purity of the compounds was
shown to be >98% (by HPLC). Maslinic and augustic acid were
prepared from oleanolic acid as previously described [38]. The
following compounds were synthesized as previously published:
1e19, 28 and 32e40 [37], 20e27, 41 and 42 [36], and 30 and 31 [39].
4.1.2. (2a,3b) 2,3-Bis(chloroacetyloxy)-olean-12-en-28-oic acid
(29)
Maslinic acid (200 mg, 0.42 mmol), triethylamine (0.16 mL,
1.15 mmol) and catalytic amounts of DMAP were dissolved in dry
DCM (50 mL). A solution of chloroacetic anhydride (0.26 mg,
1.53 mmol) in dry DCM (10 mL) was slowly added, and stirring at
25 ꢀC was continued for another 30 min. The reaction was
quenched with by adding 2
N HCl and extracted with Et2O
(3 ꢁ 25 mL). The combined organic layers were washed with brine
(20 mL), filtered, dried (Na2SO4) and concentrated under dimin-
ished pressure. The crude product was subjected to column chro-
matography (silica gel, hexane/ethyl acetate, 7:3) to afford 29
(198 mg, 75%) as a colorless solid; m.p. 239e243 ꢀC; RF ¼ 0.38 (silica
gel, hexane/ethyl acetate, 7:3); [
a
]
¼ 22.52ꢀ (c ¼ 0.29, CHCl3); IR
D
(KBr):
n
¼ 3420s, 2949s, 1743s, 1695s, 1463m, 1410m, 1397m, 1309s,
1180s, 1025s, 994s, 970m, 924m, 823s, 791s, 718m, 651s, 572 s cmꢂ1
1H NMR (500 MHz, CDCl3):
¼ 5.25 (dd, J ¼ 3.4, 3.4 Hz,1H, CH (12)),
;
Fig. 1. Docked poses for compounds 12 (cyano), 20 (pink) and 27 (green) in the AChE
active site. AChE PDB code 4BDT was used for docking with GOLD 5.2 software [40].
Image prepared using MOE2013 [41]. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
d
5.17 (ddd, J ¼ 11.1, 10.8, 4.7 Hz, 1H, CH (2)), 4.85 (d, J ¼ 10.3 Hz, 1H,
CH (3)), 4.03 (s, 1H, CHa (33)), 4.02 (s, 1H, CHb (33)), 3.95 (s, 2H, CH2
(32)), 2.81 (dd, J ¼ 13.7, 4.1 Hz, 1H, CH (18)), 2.06 (dd, J ¼ 12.4,
4.7 Hz, 1H, CHa (1)), 2.01e1.92 (m, 1H, CHa (16)), 1.92e1.87 (m, 1H,
CHa (11)), 1.87e1.79 (m, 1H, CHb (11)), 1.79e1.65 (m, 2H, CHa
(22) þ CHa (15)), 1.65e1.51 (m, 5H, CH (9) þ CHa (19) þ CHb
(22) þ CHb (16) þ CHa (6)), 1.51e1.42 (m, 1H, CHa (7)), 1.39e1.28 (m,
3H, CHb (6) þ CHa (21) þ CHb (7)), 1.28e1.16 (m, 1H, CHb (21)),
1.15e1.09 (m, 5H, CHb (19) þ CHb (1) þ CH3 (27)), 1.09e1.02 (m, 4H,
CHb (15) þ CH3 (25)), 1.01e0.94 (m, 1H, CH (5)), 0.92 (s, 3H, CH3
(29)), 0.91 (s, 3H, CH3 (23)), 0.91 (s, 3H, CH3 (24)), 0.89 (s, 3H, CH3
(30)), 0.72 (s, 3H, CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3):
and Ki0 varied. Some of the compounds were mixed-type inhibitors
with a dominating competitive part (Ki < Ki0, e.g. for 2 and 12), and a
dominating uncompetitive part (Ki > Ki0) was determined for four
derivatives and, finally,
a nearly non-competitive inhibition
(Ki z Ki0, e.g. for 4 and 17) was found, as well.
Docking studies revealed that the different kinetic behavior for
very similar compounds can be explained by the ability of the
compounds to enter the active site gorge of AChE. The competitive
inhibitor 27 was able to enter deep into the gorge and to interact
with the catalytic machinery of AChE while the mixed-type in-
hibitors 12 and 20 interacted with the enzyme's Trp286 at the gorge
entrance indicating that these inhibitors may form a active-site lid
that governs the inhibiton of AChE.
d
¼ 184.6 (C]O, C28), 167.3 (C]O, C31), 167.0 (C]O, C34), 143.8
(Cquart., C13), 122.1 (HC]C, C12), 82.3 (CH, C3), 72.21 (CH, C2), 54.9
(CH, C5), 47.6 (CH, C9), 46.6 (Cquart., C17), 45.9 (CH2, C19), 43.6 (CH2,
C1), 41.7 (Cquart., C14), 41.0 (CH2, C32), 41.0 (CH, C18), 40.9 (CH2,
C33), 39.7 (Cquart., C4), 39.4 (Cquart., C8), 38.4 (Cquart., C10), 33.9 (CH2,
Selectivity and cytotoxicity studies for selected compounds
showed the potential of this compounds as to act as selective in-
hibitors for cholinesterases. Consequently, we were able to convert
maslinic acid into potent inhibitors of cholinesterases thus
disclosing a new field of application for this triterpenoic acid, and
qualifying derivatives thereof for further biological studies in the
field of dementia and AD research.
C21), 33,2 (CH3, C30), 32.5 (CH2, C22), 32.4 (CH2, C7), 30.8 (Cquart.
,
C20), 28.4 (CH3, C23), 27.7 (CH2, C15), 26.0 (CH3, C27), 23.7 (CH3,
C29), 23.5 (CH2, C11), 22.9 (CH2, C16), 18.2 (CH2, C6), 17.6 (CH3, C24),
17.2 (CH3, C26), 16.5 (CH3, C25) ppm; MS (ESI): m/z (%) ¼ 623.4
([MꢂH]ꢂ, 100), 1249.1 ([2MꢂH]ꢂ, 94); anal. calcd for C34H50Cl2O6
(625.66): C 65.27, H 8.05; found: C 65.01, H 8.14.
4.1.3. Benzyl (2a,3b) 2,3-bis(chloroacetyloxy)-olean-12-en-28-
4. Experimental part
amide (43)
To a solution of 29 (100 mg, 0.16 mmol) in dry dichloromethane
(20 mL), containing 2 drops of dry DMF and trimethylamine, at 0 ꢀC
oxalyl chloride (2 mL, 0.023 mol) was added, and stirring at room
temperature was continued for 1 h. The solvents were removed
under reduced pressure, dry THF (20 mL) added, and the mixture
was concentrated again. The residue was dissolved in dry DCM
(15 mL), and benzylamine (1.3 mL, 0.012 mol), 2 drops of triethyl-
amine and catalytic amounts of DMAP were added. After stirring at
room temperature for 15 min, the reaction was quenched by adding
4.1. Chemistry
4.1.1. General
The reagents were bought from commercial suppliers without
any further purification. NMR spectra were measured on VARIAN
Gemini 2000 or Unity 500 spectrometers at 27 ꢀC with trime-
thylsilane as an internal standard, d are given in ppm and J in Hertz.
Mass spectra were taken on a FINNIGAN LCQ instrument. IR spectra
were recorded on a PerkineElmer FT-IR spectrometer Spectrum
2
N
hydrochloric acid (10 mL) and extracted with Et2O (3 ꢁ 15 mL).

S. Schwarz et al. / European Journal of Medicinal Chemistry 103 (2015) 438e445
443
The combined organic layers were washed with brine (10 mL),
filtered, dried (MgSO4) and evaporated to dryness. Purification by
column chromatography (silica gel, hexane/ethyl acetate, 8:2) gave
43 (62 mg, 54%) as a colorless solid; m.p. 117e119 ꢀC; RF ¼ 0.38
C27), 23.7 (CH3, C29), 23.6 (CH2, C11), 22.9 (CH2, C16),18.0 (CH2, C6),
17.7 (CH3, C23), 17.4 (CH3, C26), 16.0 (CH3, C25) ppm; MS (ESI): m/z
(%) ¼ 623.4 ([MꢂH]ꢂ, 74), 1249.3 ([2MꢂH]ꢂ, 100); anal. calcd for
C
34H50Cl2O6 (625.66): C 65.27, H 8.05; found: C 65.05, H 7.87.
(silica gel, hexane/ethyl acetate, 8:2); [
CHCl3); IR (KBr):
a
]
D
¼ ꢂ5.04ꢀ (c ¼ 0.28,
n
¼ 3432m, 2948s, 1744s, 1650m, 1517m, 1454s,
4.1.5. Benzyl (2b,3b) 2,3-bis(2-chloroacetyloxy)-olean-12-en-28-
amide (48)
1396w, 1309s, 1264s, 1185s, 1025m, 1001s, 751s, 699 s cmꢂ1
;
1H
NMR (500 MHz, CDCl3):
d
¼ 7.40e7.20 (m, 5H, CHa (37) þ CHb
Following the procedure given for the synthesis of 46, from 47
(110 mg, 0.18 mmol) 48 (100 mg, 80%) was obtained as a colorless
solid; m.p. 115e119 ꢀC; RF ¼ 0.30 (silica gel, hexane/ethyl acetate,
(37) þ CHa (38) þ CHb (38) þ CH (39)), 6.16 (dd, J ¼ 5.2, 5.2 Hz, 1H,
NH), 5.31 (dd, J ¼ 3.2, 3.2 Hz, 1H, CH (12)), 5.18 (ddd, J ¼ 11.2, 11.0,
4.5 Hz, 1H, CH (2)), 4.86 (d, J ¼ 10.3 Hz, 1H, CH (3)), 4.61 (dd, J ¼ 14.6,
6.1 Hz, 1H, CHa (35), 4.17 (dd, J ¼ 14.6, 4.4 Hz, 1H, CHb (35)), 4.05 (s,
1H, CHa (32)), 4.05 (s, 1H, CHb (32)), 3.96 (s, 2H, CH2 (33)), 2.57 (dd,
J ¼ 12.7, 3.3 Hz, 1H, CH (18)), 2.12e2.03 (m, 1H, CHa (1)), 2.03e1.93
(m, 1H, CHa (16)), 1.84 (m, J ¼ 8.7, 3.1 Hz, 2H, CHa (11) þ CHb (11)),
1.81e1.53 (m, 7H, CHa (19) þ CHa (22) þ CHb (16) þ CHb (22) þ CH
(9) þ CHa (15) þ CHa (6)), 1.53e1.18 (m, 5H, CHa (7) þ CHb (6) þ CHa
(21) þ CHb (7) þ CHb (21)), 1.17e1.12 (m, 4H, CHb (19) þ CH3 (27)),
1.12e1.05 (m, 2H, CHb (1) þ CHb (15)), 1.03 (s, 3H, CH3 (25)),
1.01e0.96 (m, 1H, CH (5)), 0.94 (s, 3H, CH3 (24)), 0.94 (s, 3H, CH3
(23)), 0.92 (s, 3H, CH3 (30)), 0.92 (s, 3H, CH3 (29)), 0.66 (s, 3H, CH3
8:2); [
a]
¼ 41.28ꢀ (c ¼ 0.33, CHCl3); IR (KBr):
n
¼ 2948vs, 2868m,
D
1762s,1740vs,1654s,1516s,1454m,1290s,1262s,1184s,1154s, 994m,
698 m cmꢂ1; 1H NMR (500 MHz, CDCl3):
d
¼ 7.34e7.30 (m, 2H, CH
(38)), 7.28e7.23 (m, 3H, CH (37) þ CH (39)), 6.15 (dd, J ¼ 6.1, 4.7 Hz,
1H, NH), 5.44e5.41 (m, 1H, CH (2)), 5.30 (dd, J ¼ 3.6, 3.6 Hz, 1H, CH
(12)), 4.71 (d, J ¼ 3.9 Hz, 1H, CH (3)), 4.60 (dd, J ¼ 14.7, 6.3 Hz, 1H,
CHa (35)), 4.16 (dd, J ¼ 14.7, 4.5 Hz, 1H, CHb (35)), 4.05e4.03 (m, 4H,
CH2 (32) þ CH2 (34)), 2.55 (dd, J ¼ 13.1, 4.0 Hz, 1H, CH (18)),
2.04e1.95 (m, 2H, CHa (1) þ CHa (16)), 1.92e1.78 (m, 2H, CH2 (11)),
1.79e1.70 (m, 2H, CHa (22) þ CHa (19)), 1.70e1.43 (m, 7H, CH2
(6) þ CHb (16) þ CHa (15) þ CHb (22) þ CH (9) þ CHa (7)), 1.40e1.32
(m, 2H, CHb (1) þ CHa (21)), 1.32e1.13 (m, 3H, CHb (21) þ CHb
(19) þ CHb (7)), 1.16 (s, 3H, CH3 (25)), 1.15 (s, 3H, CH3 (27)), 1.08 (s,
3H, CH3 (23)), 1.06e1.00 (m, 1H, CHb (15)), 0.99e0.95 (m, 1H, CH
(5)), 0.93 (s, 3H, CH3 (24)), 0.90 (s, 3H, CH3 (30)), 0.90 (s, 3H, CH3
(29)), 0.69 (s, 3H, CH3 (26)) ppm; 13C NMR (125 MHz, CDCl3):
(26)) ppm; 13C NMR (125 MHz, CDCl3):
d
¼ 177.9 (C]O, C28), 167.3
(C]O, C31), 167.0 (C]O, C34), 145.1 (HC]C, C13), 138.5 (CHAr, C36),
128.8 (CHAr, C38), 127.9 (CHAr, C37), 127.5 (CHAr, C39), 122.1 (HC]C,
C12), 82.3 (CH, C3), 72.2 (CH, C2), 54.9 (CH, C5), 47.5 (CH, C9), 46.7
(CH2, C19), 46.4 (Cquart., C17), 43.7 (CH2, C1), 43.7 (CH2, C35), 42.4
(CH, C18), 42.2 (Cquart., C14), 41.0 (CH2, C32), 40.9 (CH2, C33), 39.7
(Cquart., C4), 39.5 (Cquart., C8), 38.3 (Cquart., C10), 34.2 (CH2, C21), 33.1
(CH3, C30), 32.8 (CH2, C22), 32.2 (CH2, C7), 30.9 (Cquart., C20), 28.4
(CH3, C23), 27.4 (CH2, C15), 25.8 (CH3, C27), 23.8 (CH2, C16), 23.7
(CH3, C29), 23.6 (CH2, C11), 18.2 (CH2, C6), 17.6 (CH3, C24), 17.0 (CH3,
C26), 16.5 (CH3, C25) ppm; MS (ESI): m/z (%) ¼ 714.3 ([MþH]þ, 100),
736.1 ([MþNa]þ, 26), 1429.9 ([2MþH]þ, 32), 1451.3 ([2MþNa]þ, 52);
anal. calcd for C41H57Cl2NO5 (714.80): C 68.89, H 8.04, N 1.96;
found: C 68.69, H 8.15, N 1.77.
d
¼ 178.0 (C]O, C28), 167.0 (C]O, C33), 167.0 (C]O, C31), 145.2
(C]CH, C13), 138.5 (CAr, C36), 128.8 (CHAr, C38), 127.9 (CHAr, C37),
127.5 (CHAr, C39), 122.5 (HC]C, C12), 79.9 (CH, C2), 71.5 (CH, C3),
55.2 (CH, C5), 48.1 (CH, C9), 46.7 (CH2, C19), 46.5 (Cquart., C17), 43.7
(CH2, C35), 42.4 (CH, C18), 42.3 (Cquart., C14), 41.9 (CH2, C1), 41.0
(CH2, C32), 41.0 (CH2, C34), 39.6 (Cquart., C8), 37.6 (Cquart., C4), 36.6
(Cquart., C10), 34.2 (CH2, C21), 33.1 (CH3, C30), 32.8 (CH2, C22), 32.4
(CH2, C7), 30.8 (Cquart., C20), 29.1 (CH3, C24), 27.3 (CH2, C15), 25.9
(CH3, C27), 23.9 (CH2, C16), 23.7 (CH3, C29), 23.6 (CH2, C11), 18.0
(CH2, C6), 17.7 (CH3, C23), 17.1 (CH3, C26), 16.1 (CH3, C25) ppm; MS
(ESI): m/z (%) ¼ 714.3 ([MþH]þ, 100), 736.3 ([MþNa]þ, 16), 1429.1
([2MþH]þ, 52),1451.2 ([2MþNa]þ, 22); anal. calcd for C41H57Cl2NO5
(714.80): C 68.89, H 8.04, N 1.96; found: C 68.71, H 8.21, N 1.72.
4.1.4. (2b,3b) 2,3-Bis(2-chloroacetoxy)-olean-12-en-28-oic acid
(47)
To a solution of compound 44 (200 mg, 0.42 mmol) in dry
dichloromethane (20 mL), chloroacetic acid (0.20 mL, 1.69 mmol),
triethylamine (0.25 mL, 1.80 mmol) and DMAP (5 mg, 0.04 mmol)
were added, and stirring was continued for one day. Usual aqueous
work-up followed by chromatography (silica gel, hexane/ethyl ac-
etate, 8:2) yielded 47 (170 mg, 64%) as a colorless solid; m.p.
272e274 ꢀC; RF ¼ 0.27 (silica gel, hexane/ethyl acetate, 8:2);
4.2. Biology
4.2.1. Cell lines and culture conditions
The NiH 3T3 cells were included in this study. Cultures were
maintained as monolayer in RPMI 1640 (PAA Laboratories, Pasch-
ing, Germany) supplemented with 10% heat inactivated fetal bovine
serum (Biochrom AG, Berlin, Germany) and penicillin/streptomycin
(PAA Laboratories) at 37 ꢀC in a humidified atmosphere of 5% CO2/
95% air.
[
a]
¼ 80.95ꢀ (c ¼ 0.32, CHCl3); IR (KBr):
n
¼ 2950vs, 1764s, 1740vs,
D
1698s, 1462m, 1306s, 1288s, 1262m, 1180s, 1154s, 996 m cmꢂ1
;
1H
NMR (500 MHz, CDCl3):
d
¼ 5.46e5.43 (m, 1H, CH (2)), 5.28 (dd,
J ¼ 3.6, 3.6 Hz, 1H, CH (12)), 4.73 (d, J ¼ 3.9 Hz, 1H, CH (3)),
4.04e4.03 (m, 4H, CH2 (32) þ CH2 (34)), 2.83 (dd, J ¼ 13.7, 4.3 Hz,1H,
CH (18)), 2.04 (dd, J ¼ 15.2, 3.0 Hz, 1H, CHa (1)), 2.01e1.94 (m, 2H,
CHa (11) þ CHa (16)), 1.89e1.68 (m, 3H, CHb (11) þ CHa (22) þ CHa
(15)), 1.65e1.44 (m, 7H, CH2 (6) þ CHb (16) þ CHa (19) þ CHb
(22) þ CH (9) þ CHa (7)), 1.43e1.29 (m, 3H, CHb (7) þ CHa (21) þ CHb
(1)), 1.25e1.12 (m, 2H, CHb (21) þ CHb (19)), 1.22 (s, 3H, CH3 (25)),
1.13 (s, 3H, CH3 (27)), 1.10e1.04 (m, 1H, CHb (15)), 1.08 (s, 3H, CH3
(23)), 1.03e0.98 (m, 1H, CH (5)), 0.94 (s, 3H, CH3 (24)), 0.93 (s, 3H,
CH3 (29)), 0.90 (s, 3H, CH3 (30)), 0.77 (s, 3H, CH3 (26)) ppm; 13C NMR
4.2.2. Cytotoxicity assay
The cytotoxicity of the compounds was evaluated using the
sulforhodamine-B (SRB) (Sigma Aldrich) microculture colorimetric
assay as previously reported [36e38,42].
4.2.3. Enzymatic studies
4.2.3.1. Spectrophotometer and chemicals. A TECAN Spectra-
FluorPlus instrument working on the kinetic mode and measuring
(125 MHz, CDCl3):
d
¼ 184.3 (C]O, C28), 167.1 (C]O, C33), 167.0
the absorbance at
l
¼ 415 nm was used for the enzymatic studies.
(C]O, C31), 144.0 (C]CH, C13), 122.4 (HC]C, C12), 80.0 (CH, C2),
71.5 (CH, C3), 55.3 (CH, C5), 48.2 (CH, C9), 46.7 (Cquart., C17), 45.9
(CH2, C19), 41.9 (CH2, C1), 41.9 (Cquart., C14), 41.1 (CH, C18), 41.0 (CH2,
C32), 41.0 (CH2, C34), 39.5 (Cquart., C8), 37.6 (Cquart., C4), 36.8 (Cquart.
C10), 33.9 (CH2, C21), 33.2 (CH3, C30), 32.6 (CH2, C22), 32.6 (CH2,
C7), 30.8 (Cquart., C20), 29.2 (CH3, C24), 27.7 (CH2, C15), 26.2 (CH3,
Acetylcholinesterase (from Electrophorus electricus), Papain (from
Carica papaya), Lipase (from C. antarctica), 5,50-dithiobis-(2-
nitrobenzoic acid) (DTNB) and acetylthiocholine iodide were pur-
chased from Fluka. Butyrylcholinesterase (from equine serum),
carbonic anhydrase II (from bovine erythrocytes) as well as 4-
nitrophenyl acetate (4-NA) were purchased from Sigma, and
,

444
S. Schwarz et al. / European Journal of Medicinal Chemistry 103 (2015) 438e445
butyrylthiocholine idioide was bought from Aldrich.
using Trp86 N atom as active site center coordinate. The docking
radius was considered 15 Å from the active site center. The docking
protocol was validated by the docking of the co-crystallized in-
hibitor in 4BDT; the RMSD value between docked and crystallo-
graphic poses was 1.27 Å.
4.2.3.2. Solutions preparation. Preparation of 50 mM TriseHCl
buffer solutions: Tris(hydroxymethyl)-aminomethane (606 mg)
was dissolved in bidestilled water (100 mL) and the pH was
adjusted with HCl to 8.0 0.1 (for AChE, BChE and the lipase) and
6.2 0.1 (for papain), respectively. Buffers were freshly prepared
and stored in the refrigerator. AChE solution 2.005 U/ml: the
enzyme (271 U/mg, 0.037 mg) was dissolved in freshly prepared
buffer pH 8.0 (5 mL) containing NaN3 (0.98 mg). BChE solution
2.040 U/ml: the enzyme (7.54 U/mg, 1.353 mg) was dissolved in
freshly prepared buffer pH 8.0 (5 mL) containing NaN3 (0.98 mg).
Papain solution 3.889 U/ml: the enzyme (2.1 U/mg, 9.52 mg) was
dissolved in freshly prepared buffer pH 6.2 (5 mL). Lipase solution
2.003 U/ml: the enzyme (3.1 U/mg, 3.23 mg) was dissolved in
freshly prepared buffer pH 8.0 (5 mL). DTNB solution 3 mM: DTNB
(23.8 mg) was dissolved in freshly prepared buffer pH 8.0 (20 mL)
containing NaCl (116.8 mg) and MgCl2 (38.0 mg). ATChI solution
15 mM: ATChI (43.4 mg) was dissolved in bidestilled water (10 mL).
BTChI solution 15 mM: BTChI (47.6 mg) was dissolved in bidestilled
water (10 mL). 4-NA solution 6 mM: 4-NA (21.6 mg) was dissolved
in methanol (2.2 mg) and bidestilled water (17.8 mL). All solutions
were stored in Eppendorf vials in the refrigerator or freezer, if
necessary. The pure compounds were initially dissolved in DMSO,
galantamine hydrobromide (as standard for AChE and BChE) was
dissolved in bidistilled water. The final concentrations for the
enzymatic assay were obtained by diluting the stock solution with
bidistilled water. No inhibition was detected by residual DMSO
(<0.5%).
Acknowledgments
The authors like to thank Dr. Ralph Kluge for ESI-MS and Dr. Dieter
€
Strohl for the NMR measurements. The IR and optical rotations were
measured by Mrs. Jana Wiese. Many thanks are due to Dr. Thomas
Müller from the Dept. of Haematology/Oncology for providing the
cells, and to the “GründerwerkstatteBiowissenschaften” for support.
~
^
Furthermore, S.D. Lucas thanks the Fundaçao para Ciencia e Tecno-
logia (FCT) for a Postdoc research grant SFRH/BPD/64265/2009 and
for the research unit funding Pest-OE/SAL/UI4013/2014.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.09.007.
References
[1] P. Anand, B. Singh, A review on cholinesterase inhibitors for Alzheimer's
disease, Arch. Pharm. Res. 36 (2013) 375e399.
[2] C. Ballard, S. Gaulthier, A. Corbett, C. Brayne, D. Aarsland, E. Jones, Alzheimer's
disease, Lancet 377 (2011) 1019e1031.
[3] J. Hardy, D. Allsop, Amyloid deposition as the central event in the aetiology of
Alzheimer's disease, Trends Pharmacol. Sci. 12 (1991) 383e388.
[4] T. Polvikoski, R. Sulkava, M. Haltia, K. Kainulainen, A. Vuorio, A. Verkkoniemi,
€
L. Niinisto, P. Halonen, K. Kontula, Apolipoprotein E, dementia, and cortical
4.2.3.3. Enzyme assay
deposition of beta-amyloid protein, N. Engl. J. Med. 333 (1995) 1242e1247.
[5] B.E. Tomlinson, C. Blessed, M. Roth, Observations on the brains of demented
old people, J. Neurol. Sci. 11 (1970) 205e242.
[6] D.W. Dickson, H.A. Crystal, L.A. Mattiace, D.M. Masur, A.D. Blau, P. Davies, S.-
H. Yen, M.K. Aronson, Identification of normal and pathological aging in
prospectively studied nondemented elderly humans, Neurobiol. Aging 13
(1992) 179e189.
4.2.3.3.1. Cholinesterase-assay. A mixture of the DTNB solution
(125
different concentrations and one blank with water) was prepared
and incubated at 30 ꢀC for 20 min. The substrate (25
L, 4 different
mL), enzyme (25 mL) and compounds solutions (25 mL, 3
m
concentrations) was added to start the enzymatic reaction. The
¼ 415 nm) was recorded at 30 ꢀC for 30 min
[7] K. Iqbal, I. Grundke-Igbal, Alzheimer's disease,
a multifactorial disorder
absorbance data (
l
seeking multitherapies, Alzheimers Dement. 6 (2010) 420e424.
[8] S. Gilman, M. Koller, R.S. Black, L. Jenkins, S.G. Griffith, N.C. Fox, L. Eisner,
using 1 min intervals. All measurements were performed as trip-
licates. The substrate concentrations in the test were as follows:
[ATChI] ¼ [BTChI] ¼ 0.9375 mM, 0.625 mM, 0.325 mM, 0.1875 mM.
The mode of inhibition as well as Ki and Ki0 were determined using
Lineweaver-Burk plot, Dixon plot and Cornish-Bowden plot.
4.2.3.3.2. Lipase-assay. A mixture of buffer solution pH 8.0
L. Kirby, M. Boada Rovira, F. Forette, J.-M. Orgogozo, Clinical effects of A
b
immunization (AN1792) in patients with AD in an interrupted trial, Neurology
64 (2005) 1553e1562.
[9] D. Boche, J. Donald, S. Love, S. Harris, J.W. Neal, C. Holmes, J.A.R. Nicoll,
Reduction of aggregated Tau in neuronal processes but not in the cell bodies
after A
b42 immunisation in Alzheimer's disease, Acta Neuropathol. 120 (2010)
13e20.
(125
mL), enzyme (25
m
L) and compounds solutions (25
mL, 50
m
M)
L,
[10] J.O. Rinne, D.J. Brooks, M.N. Rossor, N.C. Fox, R. Bullock, W.E. Klunk,
C.A. Mathis, K. Blennow, J. Barakos, A.A. Okello, S.R.M. de Llano, E. Liu,
M. Koller, K.M. Gregg, D. Schenk, R. Black, M. Grundman, 11C-PiB PET
was prepared and incubated at 37 ꢀC for 20 min. 4-NA (25
m
0.75
bance data (
mM) was added to start the enzymatic reaction. The absor-
assessment of change in fibrillar amyloid-
disease treated with bapineuzumab:
b
load in patients with Alzheimer's
l
¼ 415 nm) was recorded at 37 ꢀC for 10 min using
a
phase 2, double-blind, placebo-
1 min intervals. All measurements were performed in duplicate.
The relative inhibition was determined as the quotient of the slopes
(compound divided by blank) of the linear ranges.
controlled, ascending-dose study, Lancet Neurol. 9 (2010) 363e372.
[11] C. Behl, Brain aging and late-onset Alzheimer's disease: many open questions,
Int. Psychogeriatr. 24 (2012) 3e9.
[12] D. Galimberti, E. Scarpini, Treatment of Alzheimer's disease: symptomatic and
disease-modifying approaches, Curr. Aging Sci. 3 (2010) 46e56.
[13] K. Iqbal, F. Liu, C.-X. Gong, Alzheimer disease therapeutics: focus on the dis-
ease and not just plaques and tangles, Biochem. Pharmacol. 88 (2014)
631e639.
[14] F. Zemek, L. Drtinova, E. Nepovimova, V. Sepsova, J. Korabecny, J. Klimes,
K. Kuca, Outcomes of Alzheimer's disease therapy with acetylcholinesterase
inhibitors and memantine, Expert Opin. Drug Saf. 13 (2014) 759e774.
[15] P.J. Whitehouse, D.L. Price, A.W. Clark, J.T. Coyle, M.R. DeLong, Alzheimer
disease: evidence for selective loss of cholinergic neurons in the nucleus
basalis, Ann. Neurol. 10 (1981) 122e126.
4.2.3.3.3. Papain-assay. The assay was performed as reported
for the lipase-Assay but using a buffer solution of pH 6.2 instead.
4.2.4. Docking studies
The 3D structure coordinates of AChE were obtained from the
Protein Data Bank, PDB code 4BDT (with 3.10 Å resolution). To
prepare the enzyme for the docking studies, the co-crystallized
inhibitor as well as crystallographic water molecules included in
the PDB files were removed. Hydrogen atoms were added, and the
protonation states were correctly assigned using the Protonate-3D
tool within the Molecular Operating Environment (MOE) 2011.13
[16] R.T. Bartus, R.L.I. Dean, B. Beer, A.S. Lippa, The cholinergic hypothesis of
geriatric memory dysfunction, Science 217 (1982) 408e414.
[17] J.T. Coyle, D.L. Price, M.R. DeLong, Alzheimer's disease: a disorder of cortical
cholinergic innervation, Science 219 (1983) 1184e1190.
[18] E. Giacobini, E. Giacobini, in: Cholinesterases and Cholinesterase Inhibitors,
Martin Dunitz, London, 2000, pp. 181e226.
[19] M.-M. Mesulam, A. Guillozet, P. Shaw, A. Levey, E.G. Duysen, O. Lockridge,
Acetylcholinesterase knockouts establish central cholinergic pathways and
can use butyrylcholinesterase to hydrolyze acetylcholine, Neurosci 110 (2002)
software package [40], and energy was minimized using
a
MMFF94x force field. Molecular docking studies were performed
using the GoldScore scoring function from the GOLD 5.2 [39] soft-
ware package, and each ligand was subjected to 500 docking runs,

S. Schwarz et al. / European Journal of Medicinal Chemistry 103 (2015) 438e445
Phytomedicine 20 (2012) 84e88.
445
627e639.
[20] A. Ciro, J. Park, G. Burkhard, N. Yan, C. Geula, Biochemical differentiation of
cholinesterases from normal and Alzheimer's disease cortex, Curr. Alzheimer
Res. 9 (2012) 138e143.
[31] S. Schwarz, S.D. Lucas, S. Sommerwerk, R. Csuk, Amino derivatives of gly-
cyrrhetinic acid as potential inhibitors of cholinesterases, Bioorg. Med. Chem.
22 (2014) 3370e3378.
ꢀ~
[21] N.H. Greig, T. Utsuki, D.K. Ingram, Y. Wang, G. Pepeu, C. Scali, Q.-S. Yu,
J. Mamczarz, H.W. Holloway, T. Giordano, D. Chen, K. Furukawa,
K. Sambamurti, A. Brossi, D.K. Lahiri, Selective butyrylcholinesterase inhibi-
tion elevates brain acetylcholine, augments learning and lowers Alzheimer
beta-amyloid peptide inrodent, Proc. Natl. Acad. Sci. 102 (2005)
17213e17218.
[32] F.J. Reyes-Zurita, E.E. Rufino-Palomares, J.A. Lupianez, M. Cascante, Maslinic
acid, a natural triterpene from Olea europaea L., induces apoptosis in HT29
human colon-cancer cells via the mitochondrial apoptotic pathway, Cancer
Lett. 273 (2009) 44e54.
[33] M.E. Juan, U. Wenzel, V. Ruiz-Gutierrez, H. Daniel, J.M. Planas, Olive fruit ex-
tracts inhibit proliferation and induce apoptosis in HT-29 human colon cancer
cells, J. Nutr. 136 (2006) 2553e2557.
^
[22] G.F. Sousa, L.P. Duarte, A.F.C. Alcantara, G.D.F. Silva, S.A. Vieira-Filho, R.R. Silva,
D.M. Oliveira, J.A. Takahashi, New triterpenes from Maytenus robusta: struc-
tural elucidation based on NMR experimental data and theoretical calcula-
tions, Molecules 17 (2012) 13439e13456.
[34] C. Li, Z. Yang, C. Zhai, W. Qiu, D. Li, Z. Yi, L. Wang, J. Tang, M. Qian, J. Luo, M. Liu,
Maslinic acid potentiates the anti-tumor activity of tumor necrosis factor
alpha by inhibiting NF-kappaB signaling pathway, Mol. Cancer
9 (2010)
[23] I. Lee, B. Ahn, J. Choi, M. Hattori, B. Min, K. Bae, Selective cholinesterase in-
hibition by lanostane triterpenes from fruiting bodies of Ganoderma lucidum,
Bioorg. Med. Chem. 21 (2011) 6603e6607.
73e86.
[35] A. Parra, F. Rivas, P.E. Lopez, A. Garcia-Granados, A. Martinez, F. Albericio,
~
N. Marquez, E. Munoz, Solution- and solid-phase synthesis and anti-HIV ac-
[24] N. Riaz, M.A. Naveed, M. Saleem, B. Jabeen, M. Ashraf, S.A. Ejaz, A. Jabbar,
I. Ahmed, Cholinesterase inhibitory constituents from Ficus bengalensis,
J. Asian Nat. Prod. Res. 14 (2012) 1149e1155.
[25] S. Mehmood, N. Riaz, S.A. Nawaz, N. Afza, A. Malik, M.I Choudhary, New
butyrylcholinesterase inhibitory triterpenes from Salvia santolinifolia, Arch.
Pharm. Res. 29 (2006) 195e198.
[26] Y.-K. Chung, H.-J. Heo, E.-K. Kim, H.-K. Kim, T.-L. Huh, Y. Lim, S.-K. Kim, D.-
H. Shin, Inhibitory effect of ursolic acid purified from Origanum majorana L on
the acetylcholinesterase, Mol. Cells 11 (2001) 137e143.
tivity of maslinic acid derivatives containing amino acids and peptides, Bioorg.
Med. Chem. 17 (2009) 1139e1145.
[36] B. Siewert, E. Pianowski, A. Obernauer, R. Csuk, Towards cytotoxic and se-
lective derivatives of maslinic acid, Bioorg. Med. Chem. 22 (2014) 594e615.
[37] B. Siewert, E. Pianowski, R. Csuk, Esters and amides of maslinic acid trigger
apoptosis in human tumor cells and alter their mode of action with respect to
the substitution pattern at C-28, Eur. J. Med. Chem. 70 (2013) 259e272.
[38] S. Sommerwerk, R. Csuk, Convenient and chromatography-free partial syn-
thesis of maslinic acid and augustic acid, Tetrahedron Lett. 55 (2014)
5156e5158.
[39] S. Schwarz, S. Sommerwerk, S.D. Lucas, L. Heller, R. Csuk, Sulfamates of methyl
triterpenoates are effective and competitive inhibitors of carbonic anhydrase
II, Eur. J. Med. Chem. 86 (2014) 95e102.
[40] GOLD, Version 5.2; Cambridge Crystallographic Data Centre: Cambridge, U.K.;
www.ccdc.cam.ac.uk/products/gold_suite.
[27] B. Çulhaoglu, G. Yapar, T. Dirmenci, G. Topçu, Bioactive constituents of Salvia
chrysophylla Stapf, Nat. Prod. Res. 27 (2013) 438e447.
€
[28] A. Yilmaz, P. Caglar, T. Dirmenci, N. Goren, G. Topçu, A novel isopimarane
diterpenoid with acetylcholinesterase inhibitory activity from Nepeta sor-
gerae, an endemic species to the Nemrut mountain, Nat. Prod. Commun. 7
(2012) 693e696.
[29] J.H. Lee, K.T. Lee, J.H. Yang, N.I. Baek, D.K. Kim, Acetylcholinesterase inhibitors
from the twigs of Vaccinium oldhami Miquel, Arch. Pharm. Res. 27 (2004)
53e56.
[30] I.-H. Jung, S.-E. Jang, E.-H. Joh, J. Chung, M.J. Han, D.-H. Kim, Lancemaside A
isolated from Codonopsis lanceolata and its metabolite echinocystic acid
ameliorate scopolamine-induced memory and learning deficits in mice,
[41] MOE, Molecular Operating Environment, Chemical Computing Group, Mon-
treal, Canada, 2013. www.chemcomp.com.
[42] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd, New colorimetric cytotoxicity
assay for anticancer-drug screening, J. Natl. Cancer Inst. 82 (1990) 1107e1112.