Beta and gamma carboline derivatives as potential anti-Alzheimer agents: A comparison

Article abstract of DOI:10.1016/j.ejmech.2014.09.048

Nine novel β2- and 3-carboline derivatives bearing either methyl-, propargyl- or phenethyl-residues at the indole nitrogen were synthesized and tested as potential anti-Alzheimer drugs. Antagonism of recombinantly expressed NMDA receptors, inhibition of cholinesterases, and radical scavenging properties were determined for all compounds. Some were additionally tested in vivo for their ability to reverse scopolamine-induced cognitive impairment in an 8-arm radial maze experiment with rats. For the most promising candidates, the interaction with muscarinic M₁receptors was also investigated. With this set of compounds assays the influence of the scaffold itself and the substituents can be investigated separately. 5-Methyl-β3-carboline (6) was the most potent (0.25 1/4mol/100 g b.w.) compound in the in vivo test and might be a good starting point for the development of novel anti-Alzheimer drugs.

Full text of DOI:10.1016/j.ejmech.2014.09.048

European Journal of Medicinal Chemistry 87 (2014) 63e70
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Beta and gamma carboline derivatives as potential anti-Alzheimer
agents: A comparison
a
b
b
b
Robert Otto , Robert Penzis , Friedemann Gaube , Thomas Winckler ,
c
c
d
a
Dorothea Appenroth , Christian Fleck , Christian Tr a€ nkle , Jochen Lehmann ,
Christoph Enzensperger
Pharmaceutical / Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, Philosophenweg 14, D-07743 Jena, Germany
Pharmaceutical Biology, Institute of Pharmacy, Friedrich-Schiller-University, Semmelweisstr. 10, D-07743 Jena, Germany
Institute for Pharmacology and Toxicology, Friedrich-Schiller-University, Drackendorfer Str. 1, D-07747 Jena, Germany
Pharmaceutical Institute, Rheinische Friedrich-Wilhelms-University, Gerhard-Domagk-Str.3, D-53121 Bonn, Germany
Institute for Organic and Macromolecular Chemistry, Friedrich-Schiller-University, Humboldtstr. 10, D-07743 Jena, Germany
e, *
a
b
c
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 July 2014
Received in revised form
Nine novel b- and g-carboline derivatives bearing either methyl-, propargyl- or phenethyl-residues at the
indole nitrogen were synthesized and tested as potential anti-Alzheimer drugs. Antagonism of recom-
binantly expressed NMDA receptors, inhibition of cholinesterases, and radical scavenging properties
were determined for all compounds. Some were additionally tested in vivo for their ability to reverse
scopolamine-induced cognitive impairment in an 8-arm radial maze experiment with rats. For the most
12 September 2014
Accepted 13 September 2014
Available online 16 September 2014
promising candidates, the interaction with muscarinic M
of compounds assays the influence of the scaffold itself and the substituents can be investigated sepa-
rately. 5-Methyl- -carboline (6) was the most potent (0.25 mol/100 g b.w.) compound in the in vivo test
and might be a good starting point for the development of novel anti-Alzheimer drugs.
© 2014 Elsevier Masson SAS. All rights reserved.
1
receptors was also investigated. With this set
Keywords:
g
m
Structure-activity relationships
Neurological agents
Alzheimer's disease
Carboline
Neuroprotective
1. Introduction
b-Carbolines are natural products that are formed from indo-
lylethylamines or tryptophan in a Pictet-Spengler reaction. They
occur in different plants and animals including humans, and also
emerge from fermentation or during the cooking process. It is likely
Alzheimer's disease (AD) is a prevalent neurodegenerative dis-
order with multifactorial causes that requires multi-targeted
treatment. Inhibitors of acetylcholinesterase (AChE) and butyr-
ylcholinesterase (BChE) improve cholinergic signaling in the cen-
tral nervous system and are well established in the therapy of AD to
improve cognitive symptoms. AD patients may also benefit from
that
b-carbolines exert pharmacological effects, because they
represent a class of privileged chemical structures with a variety of
biological effects [2]. The naturally occurring harmine (1) (Fig. 1) is
the ancestor of the
(MAO) inhibitor used for the treatment of Parkinson's disease (PD)
[3]. More recently, 9-methyl -carboline (2) (Fig. 1) gained certain
interest as neuroprotective, neurorestorative, anti-inflammatory
and cognitive enhancing drug [4,5]. Moreover, some bivalent
b-carbolines. It was the first monoamine oxidase
2þ
reduction of pathologic glutamate-induced, Ca -mediated exci-
totoxicity by the approved N-methyl- -aspartate (NMDA) receptor
D
b
antagonist memantine. New drugs that simultaneously affect both
cholinergic transmission and reduce glutamate-induced excito-
toxicity may further improve AD treatment [1].
b
-
carboline derivatives have been shown to be highly potent dual
inhibitors of acetylcholinesterase and NMDA receptor [6,7];
therefore, they are potential candidates for the treatment of
neurodegenerative diseases. The bioavailability of the bivalent b-
carbolines investigated earlier [6,7] may be limited due to their
high molecular weight and therefore it was the aim of the present
work to gain similar bioactivities with smaller molecules by
*
Corresponding author.
E-mail addresses: robert.otto@uni-jena.de (R. Otto), robert.penzis@uni-jena.de
(
(
R. Penzis), Friedemann.gaube@uni-jena.de (F. Gaube), t.winckler@uni-jena.de
T. Winckler), dorothea.appenroth@mti.uni-jena.de (D. Appenroth), christian.
fleck@mti.uni-jena.de (C. Fleck), traenkle@uni-bonn.de (C. Tr €a nkle), ch.
enzensperger@uni-jena.de (C. Enzensperger).
combining promising substituents with the
scaffold.
b- and g-carboline
http://dx.doi.org/10.1016/j.ejmech.2014.09.048
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

64
R. Otto et al. / European Journal of Medicinal Chemistry 87 (2014) 63e70
aromatized as shown in Scheme 1 by catalytic oxidation with Pd/C
6] in toluene to form 12 in 81% yield. The preparation of the
analogous tetrahydro- -carboline (13) follows a classical Fischer-
indolization by reacting 4-piperidone hydrochloride with phenyl-
hydrazine hydrochloride in ethanol, containing a few drops of
aqueous HCl [21]. Afterwards, aromatization was carried out as
[
H C
3
g
N
N
O
N
N
H
CH
3
CH
3
described for the tetrahydro-b-carboline (11) moiety to obtain 14 in
(1)
(2)
a comparable yield. To accomplish indolo-N-alkylation, the fully
aromatic carbolines 12 or 14, respectively, were deprotonated with
NaH in DMF and treated with a 1.3-fold molar excess of the
appropriate alkyl halides to form 2, 6e10 [22]. Methylation at the
pyrido-N was achieved with a 5-fold molar excess of methyl iodide
in acetone, and the resulting quaternary salts 15e20 were obtained
almost quantitatively. The tetrahydro-derivatives 21 and 22 were
Fig. 1. Some established
properties.
b-carboline structures with well-known pharmacological
g
-Carbolines represent a structurally related class where the
pyridine nitrogen is shifted by one position compared to -carbo-
lines. To our knowledge, -carbolines do not occur naturally, but
certain -carbolines are investigated as drug candidates, such as
the antihistaminic mebhydrolin (3) (Fig. 2) [8]. The -carboline
b
g
obtained by NaBH
. Pharmacology
In order to characterize and compare the synthesized
4
reduction of 19 and 20 in methanol.
g
g
3
derivative flutroline (4) (Fig. 2) was used as antipsychotic [9,10].
Serotonin 5-HT6 receptor antagonism, which is associated with
b
- and g-
Alzheimer's and Parkinson's disease, was also found for some
carboline derivatives [11]. The cognition enhancer dimeboline (5)
Fig. 2) was extensively tested on working memory and as potential
g-
carbolines in detail, we made use of various pharmacological tests
to assess neuroprotective and cognition enhancing properties:
Cholinergic signaling effects were investigated by determina-
tion of acetylcholinesterase (AChE) and butyrylcholinesterase
(
anti-Alzheimer drug [12] (Fig. 2).
In this work we focus on the potential neuroprotective and
(BChE) inhibition using the Ellman assay as described earlier [6] and
nootropic effects of a set of
regard to the scaffold itself and characteristic substitution pattern.
Relying on 9-methyl -carboline (2), we investigated the indole-
N-methylation on - and -carboline scaffold, yielding 2 and 6.
b- and g-carboline derivatives with
the effect on muscarinic M
binding experiments.
1
receptors by performing equilibrium
b
Potential neuroprotective properties of the compounds were
assessed by the widely used DPPH test which reflects their radical
scavenging effects [23,24]. Furthermore the reduction of
glutamate-induced excitotoxicity was investigated using a cell-
b
g
Secondly, we probed the effect of the propargylamine moiety found
in selegiline or rasagiline, which is itself referred to as “neuro-
protective and neurorescuing” [13] and is well-known for its per-
oxynitrite scavenging effect [14] to obtain two resulting b- and g-
congeners 7 and 8. [15,16].
2
þ
based assay of glutamate-induced, Ca -mediated excitotoxicity
in mouse fibroblast cell lines L12-G10 and L13-E6 expressing NR1-
1a/NR2A and NR1-1a/NR2B subunits of the NMDA receptor,
Finally, we used an arylethylamine residue at the indole-N
comparable to dimeboline (5) because several structure-activity
relationships (SARs) on this class revealed that a simple phe-
nethyl residue is superior to the methyl-pyridine residue on the
original dimeboline [17]. The two compounds with the phenethyl
moiety are 9 and 10, respectively.
We investigated the novel compounds in various assays useful
to characterize potential anti-Alzheimer drugs. Approved anti-
Alzheimer drugs mainly target either the glutamatergic signaling
via NMDA receptor inhibition, the cholinergic system by inhibiting
acetylcholine degradation [18], or reduce oxidative stress [19].
Therefore, we determined the potency of the new compounds to
reduce glutamate-induced, Ca -mediated excitotoxicity in cell
lines expressing NR1-1a/NR2A and NR1-1a/NR2B subunits of the
NMDA receptor. In addition, we used cell-free assays to determine
the efficacy of the new compounds as AChE and BChE inhibitors and
tested them as radical scavengers. Selected compounds were also
respectively [7].
To corroborate our in vitro experiments we also performed an
in vivo test with female Wistar rats (Han:Wist). We used a
scopolamine-induced memory deficit test in an 8 arm radial maze
which is a common model to evaluate potential anti-Alzheimer
drugs on the working memory. If rats were successfully trained to
pass the 8 arm radial maze without any error, their working
memory is impaired by scopolamine and the number of errors in-
creases. The compounds were tested for their ability to antagonize
the scopolamine-induced impairment of the working memory [25].
2þ
4. Results & discussion
Nine new indolo-N-substituted carboline derivatives were
synthesized and screened for their inhibitory effects on AChE, BChE,
NMDA receptors, M receptors as well as their radical scavenging
1
effects in a DPPH test. A preliminary screening was made for all
compounds and for active compounds, an IC50 was determined.
Suitable reference substances as well as the unsubstituted carbo-
lines 12, 14 were included. Finally, we also performed in vivo assays
with rats in an 8-arm radial maze. The results of the in vitro test are
investigated for interactions with muscarinic M
their ability to reverse scopolamine-induced memory deficits in an
1
receptors and
8
-arm radial maze model with rats.
2
. Chemistry
shown in Table 1. For a better comparison, data from
b-carbolines
are shown in grey and the data from the corresponding
g-carbo-
The main focus of this study was to synthesize and compare
b
-
lines are represented in white. Derivatives with the same substi-
tution pattern are shown in sequence.
carboline (9H-pyrido[3,4-b]indole) and
g-carboline (5H-pyrido
[4,3-b]indole) derivatives with various substituents (Fig. 3). To
facilitate the discussion and comparison of these compounds, we
use the terms “pyrido-N” and “indolo-N” to describe the position
where substitutions were realized at both carboline scaffolds.
4.1. Cholinesterase-inhibitory effects
We determined both AChE and BChE inhibition, because it is
assumed that BChE inhibition may be even more important for the
treatment of AD than AChE inhibition [26], which is because AChE
expression is progressively reduced in Alzheimer's patients to reach
The
Spengler-cyclisation by reacting tryptamine hydrochloride with
paraformaldehyde [20]. The formed tetrahydro- -carboline (11) is
b-carboline scaffold could be easily obtained in a Pictet-
b

R. Otto et al. / European Journal of Medicinal Chemistry 87 (2014) 63e70
65
H C
3
CH
3
F
CH
N
3
OH
N
N
N
N
N
N
F
F
CH
3
(3)
(4)
(5)
Fig. 2. Some established
g
-carboline structures with known pharmacological properties.
Fig. 3. Comparison of the
b-carboline (9H-pyrido[3,4-b]indole) and g-carboline (5H-pyrido[4,3-b]indole).
1
0e15% compared to normal values [27]. In a previous study we
found that indolo-N-linked, homobivalent -carboline derivatives
are potent inhibitors of both AChE and BChE, while monomeric
carbolines were rather ineffective [7]. Thus, it seemed not sur-
prising that the plain -carboline 12 had no effects on cholines-
terases (AChE and BChE IC50: >100 M) (Table 1). However, the
corresponding -carboline 14 showed moderate cholinesterase
inhibition with IC50 value of 19.8 M and 1.2 M for AChE and BChE,
respectively. This considerable cholinesterase inhibitory effect of
range. The obvious shift in AChE-inhibitory potency suggests a
more pronounced effect of a permanent charge to the activity of
AChE than BChE. Compared to the “gold standard” galantamine,
compounds 19 and 20 can easily compete in terms of inhibitory
potency. Compound 19 represents the so far most potent mono-
valent carboline derivative.
b
b
-
b
m
g
m
m
4.2. Inhibition of glutamate-induced excitotoxicity
g
-
carbolines can be modulated by substitution at the indolo-N. The
bulky phenethyl residue in compound 10 leads to a 10-fold increase
of the AChE inhibition and a 2-fold increase at BChE compared to
In a previous study we found that indolo-N-linked, homo-
bivalent
b-carboline derivatives with a pyrido-N-methylated, qua-
ternary nitrogen were not only potent AChE and BChE inhibitors,
2
þ
14. In contrast, the propargyl group in 8 resulted in a loss of activity.
but also effective inhibitors of the glutamate-induced, Ca -medi-
ated excitotoxicity in cell lines expressing NR1-1a/NR2A and NR1-
The smallest substituent (2) caused the smallest effect on the ac-
tivity compared to the unsubstituted 14.
1
a/NR2B subunits of the NMDA receptor [7]. This NMDA receptor-
These findings could be well transferred to the
b-carboline
antagonistic property of the quaternary bivalent -carboline was
b
moiety with one exception. As mentioned above, 12 had no activity
whereas compound 2 showed a slight inhibition at least at the
BChE. The bulky residues in compounds 9 and 7 mediated an in-
comparable to the approved drug memantine, but, interestingly,
the activity of the compound was completely lost upon partial
hydration of the pyridine ring or abstinence of the pyrido-N-methyl
group. Similarly, we found here that only the pyrido-N-quaternary
compound 19 showed considerable activity as an NMDA receptor
crease in affinity. The propargyl substitution of
hances the inhibitory potency distinctly which is the contrary to
our findings with -carbolines.
b-carboline 12 en-
g
antagonist (Table 1). Compound 19 is the first monovalent b-car-
Overall, bulky, lipophilic residues at the indole-N led to higher
inhibitory effects and a slight prevalence for BChE over AChE could
be observed for g-carboline derivatives. Quaternization mostly in-
creases the cholinesterase-inhibitory potency, most pronounced for
compound 19, where nanomolar IC50 values were achieved. The
reason for this finding might be that a permanently charged ni-
trogen is also present in the endogenous substrate, acetylcholine.
For partially reduced derivatives 21 and 22 which have the methyl
group, but lack a permanent charge, there is a nearly complete loss
of AChE inhibition, however BChE is still inhibited in a micromolar
boline that we found active in this assay set-up. Also, we have seen
here for the first time that for carboline derivatives bivalency is not
a mandatory feature for high NMDA-receptor-inhibitory activity.
Remarkably compound 19 has an approximately 7-fold higher ac-
tivity on receptors containing the NR2B subunit than the NR2A
subunit (IC50 values 5.1
this activity was drastically reduced in the analogous
compound 20 (Table 1). Thus, compared to the previously described
indolo-N-linked, homobivalent -carboline, compound 19 has a
similar activity on NR2B-containing NMDA receptors and is
m
M vs. 34.8
mM). It is worth mentioning that
g-carboline,
b

66
R. Otto et al. / European Journal of Medicinal Chemistry 87 (2014) 63e70
NH
NH
1
1
13
14
N
H
N
H
a
b
a
b
N
N
N
12
N
H
N
H
R=
2
7
CH3
6
8
N
CH
N
R
N
R
9
10
c
c
CH
3
-
+
N
1
5
H
16
18
-
I
I
+
N
CH3 17
CH
N
R
N
R
19
20
d
d
CH3
N
22
N
21
CH3
N
R
N
R
ꢀ
ꢀ
Scheme 1. Synthesis of indolo-N-substituted carboline derivatives: Reagents and conditions: a) 10% Pd/C, toluene, 5 h, reflux; b) 1.NaH, DMF, 0 C, 1 h, 2. alkyl halide, 0 C / rt,
ꢀ
1
e24 h; c) Methyl iodide, acetone, 72 h, rt; d) NaBH
4
, methanol, 0 C / rt, 1 h.
considerably smaller, which may positively affect its bioavailability
after oral application. Yet it seems that only quaternary, perma-
also found the
carbolines.
g-carbolines are almost twice as potent as b-
nently charged
b
-carbolines are effective NMDA receptor antago-
As given in Table 2, the
g-carboline 6 represents the most potent
nists. So it remains an open question whether such compounds
would be able to cross the blood brain barrier and enter the CNS to
mediate neuroprotective effects. Although this seems unlikely, it
may also be considered that such compounds would be actively
transported to the CNS via transporter proteins. This question re-
mains to be addressed in future studies.
derivative with an active range between 0.25 and 1
m
mol per 100 g
b.w. In this test every compound seems to have an active range,
while higher and lower dosage leads to a loss of activity. Compound
6 shows on one side the lowest active dose and on the other side
the widest range of activity at all.
1
4.4. M receptor antagonism
4.3. In vivo radial maze test
Since the findings from the in vivo experiments do not correlate
The design of the in vivo study and the selection of the inves-
tigated compounds should give an overview on the influence of the
with the results of choline esterase inhibition test we investigated
whether the outcome of this radial maze test is related to musca-
substituents and the backbone scaffold. To assess the effects of
1
rinic M receptors. It was studied whether the compounds 2, 6, 9
substituents on
b
- and
g
-carbolines, we selected the six following
and 10 interfered with the specific equilibrium binding of the
muscarinic inverse agonist [ H] N-methyl-scopolaminium bromide
3
compounds 2, 6e10. The investigated dose was divided into halves
until the lowest effective concentration was found. Besides 9, all
investigated carbolines are able to compensate the scopolamine-
induced memory impairment significantly. Obviously a bulky
substituent at the indolo-N position decreases the effect, which
contradicts the structure activity relationship we observed for the
choline esterase inhibition. However, concerning the scaffold we
(NMS) at muscarinic acetylcholine receptors of the M
(Fig. 4). Changes introduced into the -carboline 2, i.e. either
switching to a -carboline structure as in 6, as well as a replace-
1
subtype
b
g
ment of the methyl group at the indolo-N in 2 by a phenethyl group
as in 9, both increased significantly and to a similar extent the
maximum inhibitory effect and the logIC0.5 concentration

R. Otto et al. / European Journal of Medicinal Chemistry 87 (2014) 63e70
67
Table 1
Cholinesterase and NMDA receptor inhibitory activities of compounds tested in this study.
Indolo-N residue (R1) Cpd Cholinesterase
NMDA receptors
NR1-1a/NR2A
AChE
BChE
M (pIC50 ± SEM)
NR1-1a/NR2B
IC50
m
Excitotoxicity [%]c at
10
m
M
50
m
M
10
m
M
50 mM
Pyridine- derivatives
H
12
>100
>100
93.7 ± 19.7
119.4 ± 25.2
88.2 ± 18.7 100.8 ± 11.9
100.6 ± 34.0 86.3 ± 16.0
75.6 ± 6.2
73.7 ± 10.3
14
19.8 (4.7 ± 0.048) 1.2 (5.9 ± 0.046)
CH3
2
6
>100
46.9 (4.7 ± 0.238)
99.8 ± 6.4
105.0 ± 10.2
90.3 ± 12.3 98.1 ± 8.4
104.5 ± 15.6 104.0 ± 12.2
77.8 ± 5.1
92.5 ± 7.6
14.7 (4.8 ± 0.036) 6.9 (5.2 ± 0.059)
7
8
2.0 (5.7 ± 0.105)
>100
7.2 (5.1 ± 0.068)
6.9 (5.2 ± 0.056)
98.1 ± 7.4
115.6 ± 11.6
101.1 ± 3.3
113.5 ± 24.1 100.3 ± 10.9
98.0 ± 21.7
110.1 ± 15.7
82.4 ± 6.5
CH
CH
9
0
4.2 (5.4 ± 0.053)
1.6 (5.8 ± 0.065)
3.8 (5.4 ± 0.111)
0.7 (6.2 ± 0.026)
87.2 ± 9.0
96.0 ± 32.5
236.2 ± 68.0 83.7 ± 18.3
137.5 ± 55.8 43.3 ± 16.7
389.4 ± 131.5
147.1 ± 114.1
1
Pyridinium - quats
H
15
6
6.7 (5.2 ± 0.467
3.2 (5.5 ± 0.373)
0.8 (6.1 ± 0.068)
0.9 (6.05 ± 0.239)
101.6 ± 5.7
103.1 ± 1.7
101.2 ± 7.7
104.6 ± 3.7
114.4 ± 8.0
110.6 ± 9.1
110.9 ± 7.1
104.0 ± 5.1
1
1
7
18
3.2 (5.5 ± 0.022)
2.7 (5.6 ± 0.039)
2.7 (5.6 ± 0.043)
5.2 (5.3 ± 0.018)
97.0 ± 22.9
92.9 ± 15.7
95.5 ± 18.6 105.0 ± 10.0 102.1 ± 9.3
92.6 ± 10.0 105.4 ± 10.2 106.2 ± 16.9
1
9
0
0.6 (6.2 ± 0.092)
5.2 (5.2 ± 0.029)
0.6 (6.2 ± 0.046)
0.9 (6.0 ± 0.010)
69.6 ± 9.4
23.4 ± 10.8 28.3 ± 10.5
IC50 ¼ 5.1 ± 2.5
93.8 ± 11.2
0.4 ± 4.1
IC50 ¼ 34.8 ± 1.8
m
M
m
M
2
80.3 ± 9.0
37.9 ± 12.2
47.4 ± 8.6
IC50 ¼ 39.6 ± 8.2
m
M
IC50 ¼ 29.4 ± 2.3
mM
N-methyl-tetra-hydro-pyridine
21
>100
7.9 (5.1 ± 0.361)
108.9 ± 12.6
94.7 ± 18.4
71.5 ± 12.2
62.5 ± 17.0
9
0.5 ± 15.8
3.0 ± 20.8
IC50 > 100
m
M
IC50 > 100
mM
22
97.3 (4.0 ± 0.372) 3.6 (5.5 ± 0.511)
114.5 ± 17.0
104.6 ± 18.4
8
IC50 > 100
m
M
IC50 > 100 mM
Memantine
Galantamine
>100
>100
IC50 ¼ 4.9 ± 2.5
m
M
IC50 ¼ 5.6 ± 2.4
mM
a,b
a,b
0.85 (6.1 ± 0.088) 11.13 (5.0 ± 0.141) 96.8 ± 7.5
93.2 ± 8.4
a
values taken from Ref. [7].
determined at 25 mM.
b
c
glutamate-induced excitotoxicity; 0% and 100% is defined as values obtained in the presence of 100 mM ketamine or absence of ketamine [5].
3
necessary to induce a half maximum effect on [ H]NMS binding
5. Conclusion
(P < 0.05, one-way ANOVA with Tukey's post test). Remarkably,
applying both changes simultaneously as in 10 significantly low-
ered the bottom plateau of specific radioligand binding to zero
compared with 6 and 9 and significantly increased the logIC0.5
concentration back to a value not different from that of 2 (P < 0.05,
In our studies we probed the potential antidementive properties
of a set of carboline derivatives. Therefore we synthesized 8
carboline and -carboline derivatives, with different residues,
relying on drugs known for their neuroprotective properties.
b
-
g
one-way ANOVA with Tukey's post test). Obviously, in 10, the
position of the nitrogen and its substitution with a space
demanding phenethyl moiety favors its interaction with muscarinic
g
-
We tested these compounds for their ability to inhibit choline
esterase and NMDA receptor subtypes and tested them as radical
scavengers. Additionally we tested 6 different b- and g-couples in
M
1
receptors (Fig. 4). In the presence of 10 the slope of the inhi-
an in vivo rat model to test whether they are able to reverse
scopolamine-induced cognitive impairment and investigated their
interaction with muscarinic M1 receptors for some compounds.
The data are shown in Table 1.
For the choline esterase inhibitory effects, most compounds
show stronger effects for butyryl-choline esterase (BChE) than for
acetyl-choline esterase (AChE). This might a therapeutic advantage
for the treatment of AD, because AChE activity is strongly decreased
in AD patients whereas BChE is unaffected. Therefore it is more
promising to target BChE than AChE [27]. In some cases a clear
selectivity is present. Compounds 2, 8 and 21 show for AChE IC50
bition curve was significantly lower than unity which may point to
a deviation from competitive mass action behavior of the com-
pound (cf. Table 3). A compilation of the parameters collected in the
binding experiments is given in Table 3.
4.5. Radical scavenging ability e DPPH assay
The radical scavenging ability, where the reduction of the stable
free 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) is measured, was
determined at pH 5 to 6.5 and without pH adjustment [28,29]. The
test was carried out as a prescreening where the concentration of
the tested compounds was 4 times the concentration of DPPH. All in
all some structures showed slight activity but none could be
regarded as active, so no IC50 values were determined. The proce-
dure and the data are given in the Supporting information.
values below the detectable limit (>100
inhibitory potency is two- (compound 2) to 14-fold (compound 8)
stronger. Additionally all fully aromatic and not quaternary -car-
boline derivatives show noticeably higher BChE values compared to
their respective -carboline congeners.
mM) whereas the BChE
g
b

68
R. Otto et al. / European Journal of Medicinal Chemistry 87 (2014) 63e70
Table 2
and also did not test the quaternary derivatives (15, 16, 17, 18, 19,
0) in vivo. We also excluded 21 and 22 from our in vivo experi-
Reduction of errors in the radial maze test by compounds 2, 6e10.
2
Concentration of test compound [per 100 g b.w.] Number of errors
ments, because these tetrahydro-derivatives didn't show promising
in vitro effects.
9
10
7
8
2
6
In our in vivo rat model we also found all
g-carboline derivatives
Control (no test compound)
6.4 6.4 6.7 7.7 7.9 7.1
n.d. 7.0
are more potent than their -carboline congeners. The reversal of
b
0
0
0
1
2
.125
m
mol
mol
mol
mol
mol
scopolamine-induced memory impairment in an 8-arm radial
maze experiment is a common model for cognition enhancing
drugs. In our studies we investigated the reduction of errors
.25
m
7.5
4.0 6.0 3.7*
.5
m
m
4.7 7.3 5.3 1.9* 1.2* 2.3*
9.0 2.0* 2.4* 5.2 4.3 2.3*
m
3.3 8.9
n.d. 3.8
compared to controls and reduced the dose from 2 to 0.125 mmol
*
Significant reduction of errors compared to control (p < 0.05); n.d. not determined;
until we found the lowest effective dose. (See Table 2) In this
experiment we observed a bell-shaped relation between dose and
error reduction. This means that we have a most effective dose and
higher and lower doses are less effective. We suppose that on
higher concentrations other targets might be addressed, resulting
grey is the lowest dose which provides a significant reduction of errors.
in this effect. In the correct dose, all investigated beg-couples (9,10,
7, 8 and 2, 6) are able to reduce the scopolamine effect to some
extent. The phenethyl-
b
-carboline (9) displays the lowest efficacy,
mol. The
significant
mol concentration. For the propargyl
couples 7 and 8 and the methyl couples 2 and 8 the -carboline
derivatives are effective in lower concentrations than their -con-
geners. The methyl -derivative 6 is clearly the most potent com-
pound amongst all tested carbolines, reducing the number of errors
from 7.1 (control) to 3.7 at 0.25 mol per 100 g b.w..
reducing the errors from 6.4 (control) to 4.7 errors at 0.5
respective phenethyl- -carboline (10) displays
reduction to 2.0 errors at 1
m
g
a
m
g
b
g
m
This effect could not be confirmed in equilibrium binding ex-
periments applying an orthosteric radioligand labelling the site of
the endogenous neurotransmitter acetylcholine in muscarinic M1
3
receptors. Instead, we found that the phenethyl-g-carboline (10)
Fig. 4. Inhibition of specific orthosteric [ H]NMS (0.2 nM) equilibrium binding by the
showed a considerably stronger reduction and inhibitory potency
1
indicated compounds at M receptors. Curves were fitted individually using Prism 5.03
3
for Windows (GraphPadTM Software, Inc., San Diego, U.S.A.) by applying a built-in
standard four parameter logistic equation. Data are the means ± S.E.M. of three to
four experiments performed as triplicate determinations. For details refer to phar-
macological procedures (see Supplementary information).
with regard to [ H]NMS specific binding compared to the effect of
the respective methyl-
6) (See Table 3). However, we also found that
more potent than their -isomers.
g-carboline.
(
g-carbolines are
b
Unfortunately, the results from the radical scavenging proper-
ties (DPPH assay) were hard to interpret and do not draw a clear
picture, neither in buffer, nor in acetic acid. Therefore we do not
want to include any of these data into this discussion.
Table 3
Parameters characterizing the interaction of the test compounds with the inverse
orthosteric agonist [ H]NMS at muscarinic M
experiments.
3
1
receptors in equilibrium binding
Taken the results together, we found that
rivatives are more potent than the -carboline derivatives in
inhibiting choline esterases. In muscarinic M receptors this holds
g-carboline de-
Cpd
[3H]NMS-equilibrium binding
b
logIC0.5
Bottom plateau
n slope
1
true for the derivatives under study containing a large substituent
at the indolo-N (9, 10) but not for the N-methylated derivative (2,
b
b
b
71 ± 4c
2
6
9
ꢁ5.77 ± 0.28
ꢁ4.82 ± 0.14
ꢁ4.92 ± 0.11
ꢁ5.80 ± 0.04
ꢁ0.78 ± 0.46
ꢁ0.80 ± 0.28
ꢁ0.99 ± 0.23
ꢁ0.75 ± 0.04
c
32 ± 7
27 ± 7c
6). Finally,
g-carboline derivatives are significantly more potent
a
10
0
than their b-carboline counterparts with respect to the effective
dose and error reduction of rats in the 8-arm radial maze experi-
ment with scopolamine pretreated rats. Our results render the g-
logIC0.5: log concentration of the test compound that reduces the specific equilib-
3
rium binding of [ H]NMS to muscarinic M
1
receptors in the absence of a test com-
pound by 50% (¼inflection point of the curve); bottom: lower plateau of the curve;
n: slope factor of the curve. The data shown are mean values ± S.E.M. of three to four
experiments carried out in triplicate.
carboline derivatives, especially derivative 6 as highly attractive
candidate for the development of novel compounds against
neurodegenerative conditions like Alzheimer's or Parkinson's dis-
ease. The neuroprotective properties deserve further investigation
regarding neuronal differentiation, outgrowth of dendrites and
expression of neurotrophic factors and are being actually investi-
gated in more detail.
a
Value is different from unity (F-test, P < 0.05).
b
logIC0.5 values were read from curves with n fixed to ꢁ1.
c
Value is significantly different from zero (F-test, P < 0.05). For further details see
Supporting information.
The influence of the substitution pattern at the indolo-N is much
harder to detect. Phenethyl- (10, 9) and propargyl (8, 7) derivatives
seem to be slightly more potent than methyl- (6, 2) or H (14, 12).
The NMDA antagonistic properties are not very pronounced for
the regular indolo-N alkylated derivatives, but for quaternary de-
6
. Experimental protocols
6.1. General information/methods
Melting points are uncorrected and were measured in open
rivatives 19 and 20
mM IC50 values are present. However, the qua-
1
capillary tubes, using a Gallenkamp melting point apparatus.
H
ternary 2,9-dimethyl-b-carbolinium ion is known for its neurotoxic
13
and C NMR spectral data were obtained from a Bruker Advance
50 spectrometer (250 and 63 MHz) and Advance 400 spectrom-
eter (400 and 100 MHz), respectively. MS data were determined by
potential by inhibiting complex 1 of the respiratory chain [30].
Therefore we didn't synthesize and test this particular compound
2

R. Otto et al. / European Journal of Medicinal Chemistry 87 (2014) 63e70
69
GC/MS, using a Hewlett Packard GCD-Plus (G1800C) apparatus (HP-
MS column; J&W Scientific) Elemental analyses were performed
Appendix A. Supplementary data
5
on a Hereaus Vario EL III apparatus (Elementar Analysensysteme
GmbH, Germany) at the Institute of Organic Chemistry, University
of Jena, Germany, and results were within (0.4% of the theoretical
values. Furthermore, all compounds were checked by TLC and
showed single spots. Taken together, both features ensure purities
ꢂ95%. TLC was performed on silica gel F254 plates (Merck). High
resolution mass spectrometry (HRMS) data were determined on a
TSQ QuantumAMspectrometer (Therma Electron Corporation).
The preparation of the following derivatives was described
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.09.048.
References
[
1] L. Frolich, J. Fox, F. Padberg, K. Maurer, H.J. Moller, H. Hampel, Targets of
antidementive therapy: drugs with a specific pharmacological mechanism of
action, Curr. Pharm. Des. 10 (2004) 223e229.
[2] K.M. Brummond, J.R. Goodell, M.G. Laporte, L. Wang, X.Q. Xie, Synthesis and in
silico screening of a library of beta-carboline-containing compounds, Beilstein
J. Org. Chem. 8 (2012) 1048e1058.
previously: Tetrahydro-
line (11) [20], -Carboline (14) [31],
Phenethyl)- -carboline (9) [32], 5-Methyl-
Methyl- -carboline (2) [5], 2-Methyl- -carbolinium iodide (16)
31], 2-Methyl- -carbolinium iodide (15) [7].
-Carboline (14) is a known and described compound [31], but
g
-carboline (13) [21], Tetrahydro-
-Carboline (12) [6], 9-(2-
-carboline (6) [33], 9-
b-carbo-
g
b
[3] J.R. Sanchez-Ramos, Banisterine and Parkinson's disease, Clin. Neuro-
pharmacol. 14 (1991) 391e402.
b
g
[
4] M. Gruss, D. Appenroth, A. Flubacher, C. Enzensperger, J. Bock, C. Fleck, G. Gille,
K. Braun, 9-Methyl-beta-carboline-induced cognitive enhancement is associ-
ated with elevated hippocampal dopamine levels and dendritic and synaptic
proliferation, J. Neurochem. 121 (2012) 924e931.
b
g
[
b
g
[
[
5] H. Rommelspacher, Preparation of 9-alkyl-
b-Carbolines for the Treatment of
our synthetic route via PC/C catalyzed oxidation of the respective
tetrahydro-derivative (13) was not described so far.
The analytical data of these known compounds were as
described in the given literature. All b-carboline derivatives were
prepared by a Pictet-Spengler reaction of tryptamine and para-
formaldehyde in phosphate buffer (according to reference [20]),
Neurodegenerative Illnesses, 2008. Germany.
6] Y. Schott, M. Decker, H. Rommelspacher, J. Lehmann, 6-Hydroxy- and 6-
methoxy-beta-carbolines as acetyl- and butyrylcholinesterase inhibitors,
Bioorg. Med. Chem. Lett. 16 (2006) 5840e5843.
[
7] Y. Rook, K.U. Schmidtke, F. Gaube, D. Schepmann, B. Wunsch, J. Heilmann,
J. Lehmann, T. Winckler, Bivalent beta-carbolines as potential multitarget anti-
Alzheimer agents, J. Med. Chem. 53 (2010) 3611e3617.
[8] D.A. Kharkevich, Pharmacology of diazoline, Farmakol. Toksikol. 20 (1957)
whereas the
Fischer Indole reaction of phenylhydrazine HCl and piperidin-4-one
O leading to the corresponding tetrahydro derivatives 11
g-carboline congeners were prepared according
46e51.
[9] A.A. Kurland, A. Nagaraju, T.E. Hanlon, Flutroline in the treatment of the
HCl ꢃ H
2
schizophrenic patient, J. Clin. Pharmacol. 22 (1982) 441e449.
[10] C.A. Harbert, J.J. Plattner, W.M. Welch, A. Weissman, B.K. Koe, Neuroleptic
activity in 5-aryltetrahydro-gamma-carbolines, J. Med. Chem. 23 (1980)
and 13 [21]. Dehydration of these tetrahydro-derivatives to the
respective fully aromatic derivatives 12 and 14 was accomplished
with palladium on charcoal (10%) in refluxing toluene, instead of
cumene which is commonly used for this type of reaction [34]. The
reaction time in refluxing toluene turned out to be equal to that of
cumene and evaporation of the solvent is much easier.
635e643.
[
11] A.J. Henderson, P.R. Guzzo, A. Ghosh, J. Kaur, J.M. Koo, K. Nacro, S. Panduga,
R. Pathak, B. Shimpukade, V. Tan, K. Xiang, J.D. Wierschke, M.L. Isherwood,
Epiminocyclohepta[b]indole analogs as 5-HT6 antagonists, Bioorg. Med.
Chem. Lett. 22 (2012) 1494e1498.
[
[
[
[
12] S.J. Webster, C.A. Wilson, C.H. Lee, E.G. Mohler, A.V. Terry Jr., J.J. Buccafusco,
The acute effects of dimebolin, a potential Alzheimer's disease treatment, on
working memory in rhesus monkeys, Br. J. Pharmacol. 164 (2011) 970e978.
13] M.B. Youdim, Multi target neuroprotective and neurorestorative anti-
Parkinson and anti-Alzheimer drugs ladostigil and m30 derived from rasagi-
line, Exp. Neurobiol. 22 (2013) 1e10.
14] S. Dragoni, V. Porcari, M. Travagli, D. Castagnolo, M. Valoti, Antioxidant
properties of propargylamine derivatives: assessment of their ability to
scavenge peroxynitrite, J. Pharm. Pharmacol. 58 (2006) 561e565.
15] W. Maruyama, Y. Akao, M.C. Carrillo, K. Kitani, M.B. Youdium, M. Naoi, Neu-
roprotection by propargylamines in Parkinson's disease: suppression of
apoptosis and induction of prosurvival genes, Neurotoxicol. Teratol. 24 (2002)
675e682.
6.2. Synthesis of indolo-N-alkyl-carboline derivatives (2, 6e10):
General procedure 1
A solution of 4 mmol carboline (12 or 14) and 4.4 mmol of NaH
in DMF was stirred at room temperature for 30 min. After addition
of 4.4 mmol of corresponding alkyl halide the solution was stirred
for another 4e16 h. The conversion was monitored by TLC and GC/
MS. When no further reaction was observed the solution was
poured into iced water extracted with dichloromethane and puri-
fied by column chromatography and was subsequently converted
into its HCl salt in 2-propanol with ethereal HCl.
[
[
[
16] L. Kupershmidt, T. Amit, O. Bar-Am, O. Weinreb, M.B. Youdim, Multi-target,
neuroprotective and neurorestorative M30 improves cognitive impairment
and reduces Alzheimer's-like Neuropathology and Age-related Alterations in
mice, Mol. Neurobiol. 46 (1) (2012) 217e220.
17] A.V. Ivachtchenko, E.B. Frolov, O.D. Mitkin, S.E. Tkachenko, I.M. Okun,
A.V. Khvat, Synthesis and biological activity of 5-styryl and 5-phenethyl-
substituted 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indoles, Bioorg. Med. Chem.
Lett. 20 (2010) 78e82.
6.3. Synthesis of the pyrido-N-quaternary salts (15e20): general
procedure 2
18] N.I.o.A. (NIA), Alzheimer's Disease Medications e Fact Sheet, 2008. http://
www.bu.edu/alzresearch/files/pdf/ADEARMedicationsFactSheet3.pdf.
A 10-fold molar excess of methyl iodide was added to a solution
[19] G. Saxena, S.P. Singh, R. Agrawal, C. Nath, Effect of donepezil and tacrine on
oxidative stress in intracerebral streptozotocin-induced model of dementia in
mice, Eur. J. Pharmacol. 581 (2008) 283e289.
of the respective carboline derivative (7e10, 12 or 14) in acetone at
ꢀ
0
7
C. The mixture was vigorously stirred at room temperature for
[
20] Y.X. Li, S.R. Prakash, Synthesis of C-14-labeled novel IKK inhibitor: 2-[C-14]-N-
(6-chloro-9H-pyrido [3,4-b]indol-8-yl)-3-pyridinecarboxamide, J. Label.
Compd. Rad. 48 (2005) 323e330.
2 h. The precipitated solids were isolated by filtration, washed
2
with Et O and dried in vacuo.
[21] A. Bridoux, L. Goossens, R. Houssin, J.P. Henichart, Synthesis of 8-substituted
tetrahydro-gamma-carbolines, J. Heterocycl. Chem. 43 (2006) 571e578.
6
.4. Synthesis of the partially reduced compounds (21, 22): general
[22] R.H. Cao, Q. Chen, X.R. Hou, H.S. Chen, H.J. Guan, Y. Ma, W.L. Peng, A.L. Xu,
Synthesis, acute toxicities, and antitumor effects of novel 9-substituted beta-
carboline derivatives, Bioorg. Med. Chem. 12 (2004) 4613e4623.
procedure 3
[23] P. Molyneux, The use of the stable free radical diphenylpicrylhydrazyl (DPPH)
A stirred solution of 2 mmol quaternary carbolinium iodide (19,
for estimating antioxidant activity, J. Sci. Technol. 26 (2004) 211e219.
[24] M.S. Blois, Antioxidant determinations by the Use of a stable free radical,
ꢀ
2
0) in 50 mL of methanol at 0 C was treated with 20 mmol NaBH .
4
Nature 181 (1958) 1199e1200.
The mixture was stirred for 2e4 h at room temperature, the organic
phase was carefully evaporated, and 10 mL of water was added. The
aqueous phase was then extracted with dichloromethane. The
organic phase was dried over MgSO , evaporated, and dried in
4
vacuo.
[
25] L. Fang, D. Appenroth, M. Decker, M. Kiehntopf, A. Lupp, S. Peng, C. Fleck,
Y. Zhang, J. Lehmann, NO-donating tacrine hybrid compounds improve
scopolamine-induced cognition impairment and show less hepatotoxicity,
J. Med. Chem. 51 (2008) 7666e7669.
26] E. Giacobini, Cholinesterase inhibitors: new roles and therapeutic alternatives,
Pharm. Res. 50 (2004) 433e440.
[

70
R. Otto et al. / European Journal of Medicinal Chemistry 87 (2014) 63e70
[
27] E.K. Perry, R.H. Perry, G. Blessed, B.E. Tomlinson, Changes in brain cholines-
terases in senile dementia of Alzheimer type, Neuropathol. Appl. Neurobiol. 4
[31] G. Van Baelen, S. Hostyn, L. Dhooghe, P. Tapolcsanyi, P. Matyus, G. Lemiere,
R. Dommisse, M. Kaiser, R. Brun, P. Cos, L. Maes, G. Hajos, Z. Riedl, I. Nagy,
B.U. Maes, L. Pieters, Structure-activity relationship of antiparasitic and
cytotoxic indoloquinoline alkaloids, and their tricyclic and bicyclic analogues,
Bioorg. Med. Chem. 17 (2009) 7209e7217.
[32] G. Cerbai, G.F. Dipaco, Synthesis of Aryl- and Diaryl-alkyl derivatives of
Norharman, Farm. Sci. 18 (1963) 721e731.
[33] K. Eiter, M. Nagy, Zur Konstitution des Calycanthins .2. Die Synthese der
Abbaubase C12H10N2, Monatsh. Chem. 79 (1948) 17e21.
[34] D. Soerens, J. Sandrin, F. Ungemach, P. Mockry, G.S. Wu, E. Yamanaka,
L. Hutchins, M. DiPierro, J.M. Cook, Study of the Pictet-Spengler reaction in
aprotic media: synthesis of the p-galactosidase inhibitor, pyridindolol 1c,
J. Org. Chem. 44 (1978) 535e545.
(
1978) 273e277.
[
28] A.M. Schaible, H. Traber, V. Temml, S.M. Noha, R. Filosa, A. Peduto, C. Weinigel,
D. Barz, D. Schuster, O. Werz, Potent inhibition of human 5-lipoxygenase and
microsomal prostaglandin E-2 synthase-1 by the anti-carcinogenic and anti-
inflammatory agent embelin, Biochem. Pharmacol. 86 (2013) 476e486.
29] J. Bauer, A. Koeberle, F. Dehm, F. Pollastro, G. Appendino, H. Northoff, A. Rossi,
L. Sautebin, O. Werz, Arzanol, a prenylated heterodimeric phloroglucinyl
pyrone, inhibits eicosanoid biosynthesis and exhibits anti-inflammatory effi-
cacy in vivo, Biochem. Pharmacol. 81 (2011) 259e268.
[
[
30] J. Hamann, H. Rommelspacher, A. Storch, H. Reichmann, G. Gille, Neurotoxic
mechanisms of 2,9-dimethyl-beta-carbolinium ion in primary dopaminergic
culture, J. Neurochem. 98 (2006) 1185e1199.