Design, synthesis and pharmacological evaluation of hybrid molecules out of quinazolinimines and lipoic acid lead to highly potent and selective butyrylcholinesterase inhibitors with antioxidant properties

Article abstract of DOI:10.1016/j.bmc.2008.02.083

A set of hybrid molecules were synthesized out of lipoic acid, α,ω-diamines of different lengths serving as spacers, and cholinesterase (ChE) inhibiting [2,1-b]quinazolinimines. Depending on the length of the alkylene spacer the amide hybrids are inhibitors of acetylcholinesterase (AChE) with inhibitory activities of 0.5-4.6 μM and inhibitors of butyrylcholinesterase (BChE) with activities down to 5.7 nM, therefore greatly exceeding the inhibitory activities of the parent quinazolinimines by factors of up to 1000. Due to increasing activity at BChE with increasing length of the alkylene spacer ～100-fold selectivity toward BChE is reached with a hepta- and an octamethylene spacer. Kinetic measurements reveal competitive and reversible inhibition of both ChEs by the hybrids. Furthermore, cell viability and antioxidant activity (using the ORAC-fluorescein assay) of several hybrids were evaluated, showing cytotoxicity at concentrations from 3.7 to 10.2 μM and antioxidant properties are in the range of 0.4-0.8 Trolox equivalents (lipoic acid = 0.6).

Full text of DOI:10.1016/j.bmc.2008.02.083

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Bioorganic & Medicinal Chemistry 16 (2008) 4252–4261
Design, synthesis and pharmacological evaluation
of hybrid molecules out of quinazolinimines and lipoic acid lead
to highly potent and selective butyrylcholinesterase inhibitors
with antioxidant properties
Michael Decker,a,* Birgit Krausb and Jo¨rg Heilmannb
aLehrstuhl fu¨r Pharmazeutische/Medizinische Chemie, Institut fu¨r Pharmazie, Friedrich-Schiller-Universita¨t Jena,
Philosophenweg 14, D-07743 Jena, Germany
bLehrstuhl fu¨r Pharmazeutische Biologie, Institut fu¨r Pharmazie, Universita¨t Regensburg, Universita¨tsstraße 31,
D-93053 Regensburg, Germany
Received 7 December 2007; revised 19 February 2008; accepted 26 February 2008
Available online 29 February 2008
Abstract—A set of hybrid molecules were synthesized out of lipoic acid, a,x-diamines of different lengths serving as spacers, and
cholinesterase (ChE) inhibiting [2,1-b]quinazolinimines. Depending on the length of the alkylene spacer the amide hybrids are inhib-
itors of acetylcholinesterase (AChE) with inhibitory activities of 0.5–4.6 lM and inhibitors of butyrylcholinesterase (BChE) with
activities down to 5.7 nM, therefore greatly exceeding the inhibitory activities of the parent quinazolinimines by factors of up to
1000. Due to increasing activity at BChE with increasing length of the alkylene spacer ꢀ100-fold selectivity toward BChE is reached
with a hepta- and an octamethylene spacer. Kinetic measurements reveal competitive and reversible inhibition of both ChEs by the
hybrids. Furthermore, cell viability and antioxidant activity (using the ORAC-fluorescein assay) of several hybrids were evaluated,
showing cytotoxicity at concentrations from 3.7 to 10.2 lM and antioxidant properties are in the range of 0.4–0.8 Trolox equivalents
(lipoic acid = 0.6).
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
class of ChE inhibitors, which can either inhibit both
cholinesterases or are highly selective for BChE with
low micromolar or higher inhibitory activities (1,
Fig. 1).5,6 Design and synthesis of (homo-) bivalent qui-
nazolinimines led to nanomolar inhibitors, the ones with
larger sizes of the respective alicycle retained BChE
selectivity, whereas heterocycles containing a five-mem-
bered alicycle were as potent on AChE as on BChE (2,
Fig. 1).7
Different forms of dementia, which involve impairment
of cognitive functions, like Alzheimer‘s disease (AD),
are characterized by reduced levels of acetylcholine
(ACh) in the cortex and hippocampus.1 The approved
therapeutic options to AD are extremely limited to only
four acetylcholinesterase (AChE) inhibitors and the
NMDA receptor antagonist memantine. Since AChE
activity decreases progressively in certain brain regions
during the course of AD to reach 10–15% of the initial
value, while the activity of the less specific butyrylcholin-
esterase (BChE) stays the same or is increased,2–4 we
have focused on the development of BChE selective
inhibitors and identified quinazolinimines as a novel
Since the antioxidant system of elderly people loses to a
certain degree its full activity,8 and oxidative damage
was observed before the formation of AD-specific path-
ological b-amyloid plaques,9 antioxidants may be ap-
plied as neuroprotective agents in AD. Although
in vitro-data for antioxidants like vitamin E are very
promising,10,11 the outcome of clinical trials has been
ambiguous, for example, applying vitamin E as supple-
ment.12,13 Nevertheless, especially for mild cognitive
impairment (MCI) as an early stage of AD, the positive
role of antioxidants is unambiguous.14,15
Keywords: Cholinesterase inhibition; Tricyclic[2,1-b]quinazolinimines;
Butyrylcholinesterase (BChE) selectivity; Lipoic acid; Hybrids;
Antioxidants.
*
Corresponding author. Tel.: +49 3641/949817; fax: +49 3641/
949802; e-mail: m.decker@uni-jena.de
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.083

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4253
R
2
[ ]n = 1, 2, 3, 4
R = H, n = 4:
N
IC50 (AChE) = 14.4 μM
IC50 (BChE) = 76 nM
[ ]7
[ ]n = 0, 1, 2
N
N
N
N
N
N
N
[ ]n = 1, 2, 3, 4
1, e. g. n = 1, m = 4:
IC50 (AChE) = >500 μM
IC50 (BChE) = 1.1 μM
N
R = Cl, n = 1:
IC50 (AChE) = 50 nM
IC50 (BChE) = 45 nM
[ ] m = 0, 1, 2, 3
R
Figure 1. BChE selective imine-N-substituted quinazolinimines (1) containing an additional benzene ring at the imine-N and highly potent
homobivalent quinazolinimines (2).
N
O
N
N
OH
S
H
S
4:
IC50 (AChE) = 3.9 μM
IC50 (BChE) = 2.3 μM
Lipoic acid (3)
Cl
O
N
N
[ ]n = 1-6
N [ ]n = 1-6
H2N
N
N
N
N
S
H
S
H
5a-f
6a-f
Cl
Cl
Figure 2. The antioxidant and cytoprotective lipoic acid (3), an unselective monovalent quinazolinimine (4), which can be regarded as a ‘monomer’
or ‘univalent’ form of bivalent quinazolinimines (2), and target compounds 5a–f and 6a–f.
Lipoic acid (3, Fig. 2) represents such a versatile antiox-
idant,16,17 especially because it was shown that it is able
to protect neurons against amyloid-induced cell death
through the Akt signaling pathway.18 There are also
some (although limited) data for its positive effects on
disease progression in AD patients.19
showed strong activity increases,7 also the primary
amines (5a–f) yielded as synthesis intermediates were
examined, in which a simple amino-group replaces one
basic quinazolinimine moiety (Fig. 2).
N-[6-Chloro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-
ylidene]-1-butanamine has previously been identified as
an inhibitor of both AChE and BchE with inhibitory
activities of 3.9 lM and 2.3 lM, respectively.7 It repre-
sents a potent univalent compound in the series of qui-
nazolinimines synthesized (compounds bearing an
additional phenyl-group in the alkyl moiety (1) are more
potent, but are less suitable for coupling and functional-
ization).6 This quinazolinimine moiety was selected for
coupling in order to evaluate the influence of functional-
ization and coupling with lipoic acid on ChE inhibition
and BChE selectivity in particular. To find a reasonable
concentration range for further cellular in vitro testings
some representatives of the hybrids were tested for cyto-
toxicity in a murine microglial cell line. The antioxidant
activity of the hybrids was evaluated in the ORAC-fluo-
rescein assay.24
Recently, the potent AChE inhibitor tacrine was con-
nected with lipoic acid20 and melatonin,21 respectively,
to form hybrid molecules combining AChE inhibiting
with antioxidant and radical scavenging properties.
From the medicinal chemical point of view such hybrids
are of special interest, because these compounds do not
only preserve the pharmacological properties of their
components, but also show improved properties: on
the one hand some melatonin–tacrine hybrids exhibited
nanomolar AChE-inhibiting properties with higher
AChE selectivity maintaining their antioxidant proper-
ties.21 On the other hand also some tacrine–connected
to lipoic acid hybrids increased their activity at AChE
and—of special importance—also showed b-amyloid
aggregation-inhibiting properties,20 indicating interac-
tion with the peripheral anionic site (PAS), which accel-
erates amyloid fibril formation.22,23 Both kinds of
hybrids improved their activity toward BChE only to
a very low or no extent.20,21
2. Results and discussion
2.1. Synthesis
In order to combine BChE-inhibiting activities with
antioxidant ones, we wanted to synthesize hybrids out
of quinazolinimines and lipoic acid (6a–f, Fig. 2). Be-
cause of the fact that homobivalent quinazolinimines
N-[6-Chloro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-
ylidene]alkane-a,x-diamines could be synthesized in a
straightforward four-step synthesis (Scheme 1): 2-pyrro-

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4254
M. Decker et al. / Bioorg. Med. Chem. 16 (2008) 4252–4261
lidinone (7) was activated using Meerwein salt (triethylox-
onium tetrafluoroborate). The resulting 5-ethoxy-3,4-
dihydro-2H-pyrrole (8, the respective iminium ether)
reacted with 4-chloro-anthranilic acid to 6-chloro-2,3-
dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (9).7 This
quinazolinone was transformed into the thione (10)
using Lawesson‘s reagent.5,6 Thione (10) reacted in the
presence of mercury(II) bromide in moderate yields
with excess a,x-diamines to the primary amines (5a–f,
N-[6-chloro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-
ylidene]alkane-a,x-diamines) suitable for further cou-
pling with lipoic acid.
Although there are of course a number of species-depen-
dent differences between these enzymes and the human
ones, the enzymes used show sufficient homology in
the amino acid sequence to the human enzymes (e.g.,
88% for equine serum BChE).26
The inhibition profiles obtained reveal some very inter-
esting results (Table 1): concerning the amines, only
the primary propylene amine (5e) shows lower activities
at AChE than the n-butyl parent quinazolinimine (4)—
the heptamethylene amine (5e) shows the same activity
as compound 4. The butylene (5b), the pentylene (5c)
and the octylene (5f) compounds show activities of
0.48 lM, 0.17 lM, and 0.13 lM, respectively. The latter
two compounds exhibit ꢀ20-fold activity increases com-
pared to compound 4 at AChE.
In order to obtain hybrid molecules, the latter com-
pounds (5a–f) were connected to ( )-lipoic acid (3) after
its activation with N,N0-carbonyldiimidazole (CDI) to
yield the target amides (6a–f).
At BChE all activities measured are higher than 1 lM
apart from the ones of the propylene compound (5a).
The octamethylene compound (5f) shows the highest
activity of 12 nM. No relevant selectivity toward one
cholinesterase can be observed, the highest one (11-fold)
with the octamethylene compound (5f). No prominent
trend can be observed for the activity development apart
from the activity increase for 5f, which is the most active
compound in this series surpassing the parent com-
pound by a factor of ꢀ200 (Table 1).
To evaluate the effect of increasing alicyclic ring size on
BChE inhibition,6,7 N-[6,7,8,9,10,11-hexahydro-13H-
azocino[2,1-b]quinazolin-13-ylidene]-1,8-octanediamine
(12) -obtained from the eight-membered-ring thione6
(11)- and its respective hybrid with lipoic acid (13) were
also synthesized (Scheme 2).
2.2. Pharmacology
Inhibitory activities of quinazolinimines bearing a pri-
mary amine moiety (5a–f, 12) and of the lipoic acid hy-
brids (6a–f, 13) were measured at AChE (E.C. 3.1.1.7,
type VI-S, from Electric Eel) and BChE (E.C. 3.1.1.8,
from equine serum) using the Ellman assay.5,6,25
Concerning the amide hybrids, activities from 0.47 to
4.6 lM were measured at AChE with activities compa-
rable to the amines (Table 1). The activity development
at BChE is very remarkable, because there is a
O
N
S
H
a
c
b
N
N
N
N
COOH
NH2
H5C2O
Cl
O
Cl
N
+
7
9
8
10
Cl
[ ]n = 1-6
NH2
+
d
H2N
O
N
N[ ]n = 1-6
N
e
[ ]n = 1-6
N
N
S
S
H
H2N
N
N
H
+ 3
6a-f
5a-f
Cl
Cl
Scheme 1. Synthesis of N-[6-chloro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-ylidene]alkane-a,x-diamines 5a–f and lipoic acid-quinazolinimine
hybrids 6a–f. Reagents and conditions: (a) 1—[(C2H5)3O]BF4, chloroform, rt, 15 h; 2—cold NaOH, CH2Cl2 extraction; (b) acetone, 10 ꢁC,
2 h ! 60 ꢁC, 4 h; (c) Lawesson‘s reagent, toluene, reflux, 12 h; (d) 3 equiv NEt3, 2.5 equiv HgBr2, toluene, reflux, 14 h; (e) 1 equiv CDI-activated
( )-lipoic acid, CH2Cl2, rt, 4 h.
S
O
N
N
a
[ ]n = 6
N[ ]n = 6
N
b
H2N
N
N
N
N
S
H
[ ]n = 6
S
N
+
+ 3
H2N
NH2
H
11
12
13
Scheme 2. Synthesis of N-[6,7,8,9,10,11-hexahydro-13H-azocino[2,1-b]quinazolin-13-ylidene]-1,8-octanediamine (12) and its ( )-lipoic acid hybrid
13. Reagents and conditions: (a) 3 equiv NEt3, 2.5 equiv HgBr2, toluene, reflux, 14 h; (b) 1 equiv CDI-activated ( )-lipoic acid, CH2Cl2, rt, 4 h.

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M. Decker et al. / Bioorg. Med. Chem. 16 (2008) 4252–4261
Table 1. AChE and BChE inhibition resultsa
4255
Compound
AChEb IC50, lM (pIC50 SEM)
BChEc IC50, lM (pIC50 SEM)
Selectivity IC50(AChE)/IC50(BChE)
Galanthamine
0.64 (6.197 0.052)
13.16 (4.881 0.061)
8.40 (5.076 0.033)
0.08
7
NH
N
7.16 (6.145 0.031)
2.29 (5.639 0.083)
1.8
N
C4H9
N
N
3.93 (5.406 0.085)
1.7
47
N
Cl
5a (n = 1)
5b (n = 2)
5c (n = 3)
5d (n = 4)
5e (n = 5)
5f (n = 6)
12
19.00 (4.721 0.139)
0.48 (6.315 0.089)
0.17 (6.780 0.063)
1.49 (5.828 0.104)
4.06 (5.391 0.216)
0.13 (6.893 0.103)
3.4 (5.474 0.065)
10.23 (4.990 0.229)
0.72 (6.142 0.198)
0.19 (6.731 0.113)
0.38 (6.416 0.184)
0.71 (6.148 0.091)
0.012 (7.926 0.052)
0.046 (7.336 0.079)
2.58 (5.589 0.149)
0.17 (6.773 0.097)
0.053 (7.275 0.087)
0.026 (7.589 0.168)
0.014 (7.840 0.052)
0.0057 (8.247 0.040)
0.023 (7.630 0.059)
1.9
0.7
0.9
3.9
5.7
10.7
73.9
1.1
6a (n = 1)
6b (n = 2)
6c (n = 3)
6d (n = 4)
6e (n = 5)
6f (n = 6)
13
2.84 (5.547 0.159)
4.58 (5.339 0.125)
4.38 (5.358 0.065)
0.74 (6.129 0.054)
1.94 (5.712 0.222)
0.468 (6.330 0.114)
<10% enzyme inhibition at 10ꢁ5 M
27.1
83.0
28.8
133.8
82
>5000
See Figure 2 and Schemes 1 and 2 for structures.
a Values are means of at least three independent determinations.
b E.C. 3.1.1.7, type VI-S, from Electric Eel.
c E.C. 3.1.1.8, from equine serum.
continuous increase in activity from the propylene com-
pound with an activity of IC50(BChE) = 2.6 lM (6a)
and a very high activity of IC50(BChE) = 5.7 nM for
the octamethylene compound (6f) (Table 1). Therefore
a concomitant increase in selectivity toward BChE can
be observed reaching 80- and 130-fold selectivities for
the octa- and heptamethylene compounds, respectively
(Table 1). Compared to the ‘parent compound’ (4) the
octamethylene compound (6f) shows a ꢀ10-fold activity
increase toward AChE and ꢀ1000-fold increase toward
BChE.
cholinesterases were analyzed by recording substrate–
velocity curves in the presence of varying concentrations
of the inhibitor. The inhibitor concentrations selected
were slightly higher and lower than the respective IC50
value (Fig. 3).
Figure 3 shows the resulting substrate–velocity curves
for BChE and AChE, respectively, and the respective
Lineweaver–Burk plots, that is, reciprocal rates versus
reciprocal substrate concentrations for the different
inhibitor concentrations. Km values (i.e., the negative re-
ciprocal of the X intercept) differ, but in contrast the
Vmax values (i.e., the reciprocal of the Y intercept) do
not significantly change with different inhibitor concen-
trations at both cholinesterases.
Compared to the tacrine–lipoic acid hybrids prepared in
Melchiorre’s group,20 a number of relevant differences
can be observed: largely independent of spacer length
these compounds were one to two digit nanomolar
inhibitors with no or only moderate selectivity toward
BChE. The most potent compound based on 6-chloro-
tacrine was connected to lipoic acid by a propylene
spacer and it shows 40-fold AChE selectivity (lipocrine).
It is able to inhibit b-amyloid fibril formation, which
was proved by kinetic measurements showing mixed
type of inhibition by interacting with both the active site
and the PAS and also directly by a thioflavin T-based
fluorimetric assay.20
These kinetic results indicate reversible and competitive
inhibition at the active site of both AChE and BChE. In
contrast to lipocrine no interaction with the PAS of
AChE can be assumed. Also at BChE there seems to
be a reversible and competitive inhibition with the sub-
strate molecule, which means that the high affinity of the
hybrid toward BChE cannot be explained by its interac-
tion with a second binding site, which would lead to an
altered kinetic profile. To validate this assumption,
molecular modeling (docking studies at BChE) applying
this molecule would be advantageous.
In order to investigate the binding mode of the hybrid
molecules synthesized and explain the remarkable differ-
ences compared to tacrine hybrids, the most potent
octamethylene compound (6f) was selected for kinetic
measurements. The inhibition mechanisms toward both
Since very high activities at BChE could be achieved
with the hybrid molecules and a C8-spacer between
the imine-nitrogen and the amide-nitrogen, this spacer