6-Hydroxy- and 6-methoxy-β-carbolines as acetyl- and butyrylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.08.067

In the course of studies directed toward the discovery of novel acetyl- and butyrylcholinesterase (AChE and BChE) inhibitors for the treatment of Alzheimer's disease, we focused on β-carbolines (BCs). 6-Oxygenated β-carboline and β-carbolinium derivatives based on the serotonin template were synthesized and tested in vitro for their ability to inhibit AChE and BChE, respectively. Particularly the carbolinium salts, which can be formed by intracerebral methylation out of the tertiary-BC prodrugs, show inhibitory activity levels reaching those of galantamine, physostigmine, and rivastigmine.

Full text of DOI:10.1016/j.bmcl.2006.08.067

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5840–5843
6-Hydroxy- and 6-methoxy-b-carbolines
as acetyl- and butyrylcholinesterase inhibitors
a
a,
b
a,
*
*
Yvonne Schott, Michael Decker, Hans Rommelspacher and Jochen Lehmann
a
Lehrstuhl f u¨ r Pharmazeutische/Medizinische Chemie, Institut f u¨ r Pharmazie, Friedrich-Schiller-Universit a¨ t Jena,
Philosophenweg 14, D-07743 Jena, Germany
b
Charit e´ -Universit a¨ tsmedizin Berlin, Institut f u¨ r Klinische Neurobiologie, Universit a¨ tskrankenhaus Benjamin Franklin,
Freie Universit a¨ t Berlin, Eschenallee 3, D-14050 Berlin, Germany
Received 18 July 2006; accepted 13 August 2006
Available online 1 September 2006
Abstract—In the course of studies directed toward the discovery of novel acetyl- and butyrylcholinesterase (AChE and BChE) inhib-
itors for the treatment of Alzheimer‘s disease, we focused on b-carbolines (BCs). 6-Oxygenated b-carboline and b-carbolinium deriv-
atives based on the serotonin template were synthesized and tested in vitro for their ability to inhibit AChE and BChE, respectively.
Particularly the carbolinium salts, which can be formed by intracerebral methylation out of the tertiary-BC prodrugs, show inhib-
itory activity levels reaching those of galantamine, physostigmine, and rivastigmine.
Ó 2006 Elsevier Ltd. All rights reserved.
The enzyme acetylcholinesterase (AChE) plays an
important role in the central nervous system. It is one
of the fastest known enzymes and catalyzes the cleavage
of acetylcholine in the synaptic cleft after depolariza-
tion. Inhibitors of acetylcholinesterase such as galanta-
mine and others are used frequently in the
tives, including quaternary salts for ChE inhibitory
activity (only AChE was tested in this in vivo assay)
and reported that some quaternary compounds—includ-
ing compound 4a—were found to be one-sixth as potent
2
as physostigmine.
1
pharmacotherapy of Alzheimer‘s disease (AD). The less
Furthermore, because endogenous BCs are considered in
the literature to induce Parkinson’s disease in non-pri-
mates, they are discussed as possible causative protox-
specific butyrylcholinesterase (BChE) has recently got
into the focus of research, because BChE concentration
stays the same or is even up-regulated while AChE is
dramatically down-regulated in the brains of patients
3
ines in idiopathic Parkinson‘s disease. Neurotoxic
b-carbolinium salts have been found in the lumbar
cerebrospinal fluid of patients suffering from Parkinson.
1
4
suffering from AD.
b-Carbolines (pyrido[3,4-b]indoles) were first found in
plants and referred to as harman alkaloids because they
occur in Peganum harmala. In the human organism they
may be formed from the condensation of the biogenic
amines tryptamine and serotonin with aldehydes or a-
keto acids, respectively. In traditional Indian medicine
the carboline-containing plant Desmodium spec.-viz. is
used to treat eye diseases and intestinal malfunctions;
the plant’s efficacy is probably due to the AChE-inhibit-
ing properties of its alkaloids. Ghosal et al. investigated
some tetrahydro- and fully aromatic carboline deriva-
It was subsequently discovered that phenylethanol-
amine-N-methyltransferase exhibits b-carboline 2N-
methyltransferase activity. If suitable non-neurotoxic
tertiary BCs could be identified, they would be bioactivat-
ed in vivo to the quaternary ChE-inhibiting BCs, that
would in turn be ‘locked’ in the brain. These compounds
could therefore be considered as target-specific prodrugs.
5
BCs can be regarded as potential anti-AD drugs as well
as endogenous tryptamine- and serotonin-derived neu-
rotoxins. To get more insight into their pharmacological
profile, we synthesized a series of systematically varied
6
-hydroxylated and 6-methoxylated harman and nor-
harman derivatives (3, 4) (Figs. 1 and 2), including the
corresponding quaternary N-methyl-carbolinium
salts. All of these compounds are methoxylated or
Keywords: b-Carbolines; Cholinesterase inhibitors.
Corresponding authors. Tel.: +49 3641 949 803; fax: +49 3641 949
802; e-mail: j.lehmann@uni-jena.de
*
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.067

Y. Schott et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5840–5843
5841
5
4
5
4
iodide after deprotonation with sodium hydride. Meth-
ylation of the pyridine-N was achieved by having the
carbolines react directly with methyl iodide in acetone
or methanol. A mixture of acetic acid and aqueous
hydrogen bromide (1:1) was used to convert the meth-
oxy group in position 6 into a hydroxy group. Further
details are given in Scheme 1.
6
6
3
3
7
N2
7
N 2
9
9
N
H
1
8
N
H
8
1
CH3
Figure 1. Norharman and harman.
R1
R1
In an initial pharmacological evaluation, the inhibitory
activities at AChE and BChE were measured in vitro
in order to get information about their ability to influ-
ence cognitive functions. The anti-AD drug galanta-
mine, physostigmine, and all target compounds
synthesized, respectively, were tested for inhibition of
AChE and BChE activity levels using the Ellman as-
N+
N
CH3
N
N
R2
R3
R2
R3
3
a-h
4 a-h
a: R
1
= OCH
= OCH
= OH; R
= OH; R
3
; R
2
= R
= H; R
= H
= H; R = CH
3
= H
b: R
d: R
f: R
h: R
1
= OCH
= OCH
= OH; R
= OH; R
3
; R
; R
= CH
= R
2
= CH
= R = CH
; R
= CH
3
; R
3
=H
3
8
,9
c: R
e: R
1
3
; R
2
3
= CH
3
1
3
2
3
say. This colorimetric assay is based on chromophores
generated in situ and formed after enzymatic cleavage of
acetyl- and butyrylthiocholine and reaction of the result-
1
2
= R
3
1
2
3
3
= H
3
g: R
1
2
3
3
1
2
3
0
Figure 2. Target compounds.
ing thiocholine with Ellman’s reagent (5,5 -dithiobis-2-
nitrobenzoic acid).
hydroxylated, respectively, in position 6 and methylated
or non-methylated in position 9 (Fig. 2).
It may not be an advantage for a cholinesterase inhibitor
(ChEI) to be selective for AChE (like galantamine); on
the contrary, a balance between AChE and BChE may
1
6
Spengler reaction using glyoxylic acid for the norhar-
-Methoxy-tryptamine (1) was cyclized via the Pictet-
6
result in higher efficacy.
,7
ꢀ
5
man and pyruvic acid for the harman derivatives.
Aromatization of compounds 2a (R = H) and 2b
Test solutions with c = 10 lM of the tertiary amine
10
salts 3a–c, 3e–h, the tetrahydro-b-carboline derivatives
10
2a,b, and the quaternary salt 4d, respectively, were pre-
3
(
R = CH ) by Pd/C in boiling cumene yielded 3a and
3
3
3
c. The indole-N atom was methylated using methyl
pared and tested in triplicate. All of them showed less
H CO
3
H CO
3
HO
O
O
a
NH3
+
NH
COOH
N
H
1
R3
N
Cl
H R3
H CO
3
b
NH
N
H
R3
2
a,2c
c
HO
HO
H3CO
H3CO
H3CO
d
e
N+
N
0R3
N
CH3
N
H
N
H
N
H
4a,4c
R3
R3
3
4
e,3g1
3a,3c
f
d
H CO
3
d
N+
N+
N
CH3
CH3
N
H
N
CH3
3b,3d
N
CH3
R3
R3
R3
e,4g
b10,4d10
4
e
HO
HO
d
N+
N
CH3
N
N
CH3
4f10,4h10
R3
R3
CH3
f,3h
10
3
Scheme 1. Reagents and conditions: (a) for norharmans with R
3
= H: H
2
O, KOH, 1 h, rt; for harmans with R
3
3 2
= CH : methanol/H O (1:1), 12 h, rt;
(
b) HCl, 15–30 min, 100 °C; (c) Pd/C (10%Pd), cumene, 10–15 h, reflux; (d) acetone, MeI, 2–4 h, rt; (e) HBr/CH
f) i—NaH, DMF, 0 °C ! rt; ii—MeI.
3
COOH (1:1), 2–4 h, 130 °C;
(

5842
Y. Schott et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5840–5843
a
Table 1. AChE/BChE inhibition results of b-carbolines (R
1
, R , R
2
3
as defined in Fig. 2)
Compound
R
1
R
2
R
3
AChE inhibition (lM)
BChE inhibition (lM)
Selectivity
IC50 (pIC50 ± SEM)
IC50(pIC50 ± SEM)
IC50(BChE)/IC50(AChE)
Galantamine
Physostigmine
0.6 (6.197 ± 0.052)
0.5 (6.307 ± 0.112)
11.8 (4.929 ± 0.100)
2.2 (5.656 ± 0.073)
1.3 (5.885 ± 0.090)
0.8 (6.122 ± 0.030)
4.2 (5.377 ± 0.082)
1.8 (5.740 ± 0.050)
1.0 (6.007 ± 0.051)
1.9 (5.722 ± 0.133)
8.4 (5.076 ± 0.033)
1.2 (5.934 ± 0.084)
17.4 (4.758 ± 0.081)
17.5 (4.758 ± 0.366)
20.9 (4.679 ± 0.087)
1.2 (5.959 ± 0.062)
8.7 (5.059 ± 0.085)
32.2 (4.492 ± 0.057)
1.6 (5.789 ± 0.068)
2.7 (5.571 ± 0.051)
14.0
2.4
3
4
4
4
4
4
4
4
d
a
b
c
e
f
OCH
OCH
OCH
OCH
OH
3
3
3
3
CH
H
3
CH
H
3
3
1.5
8.0
CH
H
3
H
16.1
1.5
CH
H
H
2.1
OH
OH
CH
H
3
H
17.9
1.6
g
h
CH
CH
3
3
OH
CH
3
1.4
a
Values are means of three independent experiments.
ꢀ
5
than 50% inhibition at 10 M, indicating IC50 values
10 lM. To measure the exact IC50 values of these
compounds, which can penetrate the blood–brain barri-
er, the quaternary compounds show strong levels of
inhibitory activity. Therefore tertiary compounds might
act as pro-drugs for the quaternary BCs that are formed
in vivo in the brain, representing a novel and target-spe-
cific approach for the therapy of AD. Additional high
levels of activity at BChE might be a further advantage
of these compounds due to the increasing relevance of
>
low activity compounds would have afforded higher
concentrations which were not possible due to poor sol-
ubility in water.
In general, the quaternary b-carbolinium salts were
10
found to be potent inhibitors whose activity levels
reached those of the reference drugs (Table 1). No rela-
tionship between the inhibitory activity of the carbdini-
um salts at AChE and methylation in position 9 was
observed. Interestingly, there was also no prominent dif-
ference in inhibitory activity for 6-methoxylated com-
pared to 6-hydroxylated compounds.
1
this enzyme in later stages of AD.
Acknowledgments
Financial support for M. Decker by the ‘Fonds der
Chemischen Industrie’ (FCI) is gratefully acknowl-
edged. Appreciation is expressed to Katrin Fischer and
Petra Wiecha for laboratory assistance.
Quaternary harmanium salts (i.e., with an additional
methylation in position 1, but not at the indole-N)
showed increased inhibitory activity levels by a factor
of three (4c to 4a and 4g to 4e). The highest activity
was found for the 6-methoxy-harmanium salt 4c with
IC50 = 0.8 lM for AChE and IC50 = 1.2 lM for
BChE.
References and notes
1
2
. Giacobini, E. In Cognitive Enhancing Drugs; Bucca fusco,
J. J., Ed.; Birkh a¨ user Verlag: Basel-Boston-Berlin, 2004; p
1
1.
. Ghosal, S.; Bhattacharya, S. K.; Mehta, R. J. Pharm. Sci.
972, 61, 808.
Regarding the selectivity profiles for the cholinesterases:
some preference for AChE was observed but selectivity
was not prominent, with the exception of the 9-methyl-
ated (indole-N-methylated) norharmanium derivatives:
the non-9-methylated harmanium salts 4a and 4c
showed mixed activity profiles, whereas compounds 4b
and 4f exhibited a considerable selectivity toward
AChE, indicating that indole-N methylation leads to
AChE selectivity. Therefore, most BCs tested show im-
proved activity profiles in the light of the decreased
1
3. Matsubara, K.; Gonda, T.; Sawada, H.; Uezono, T.;
Kobayashi, Y.; Kawamura, T.; Ohtaki, K. I.; Kimura, K.;
Akaike, A. J. Neurochem. 1998, 70, 727.
4
. Matsubara, K.; Kobayashi, S.; Kobayashi, Y.; Yamash-
ita, K.; Koide, H.; Hatta, M.; Iwamoto, K.; Tanaka, O.;
Kimura, K. Neurology 1995, 45, 2240.
5
6
7
. Gearhart, D. A.; Neafsey, E. J.; Collins, M. A. Neuro-
chem. Int. 2002, 40, 611.
. Ho, B. T.; McIsaac, W. M.; Walker, K. E.; Estevez, V.
J. Pharm. Sci. 1968, 57, 269.
. Bobbit, J. M.; Willis, J. P. J. Org. Chem. 1980, 45,
1978.
8. Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Feath-
1
AChE in AD patients.
The activity increase between class of quaternary com-
pounds, 4, with tertiary compounds 3 might be well ex-
plained by the resemblance of 4 to the natural substrate
acetylcholine.
erstone, R. M. Biochem. Pharmacol. 1961, 7, 88.
. Decker, M. Eur. J. Med. Chem. 2005, 40, 305.
9
1
0. 6-Methoxy-2,9-dimethyl-9H-b-carbolin-2-ium iodide (4b):
mp 304 °C; IR (KBr, selected lines) 3004, 1645, 1524,
In conclusion, a series of b-carbolines and b-carbolini-
um salts were synthesized and their inhibitory activity
levels at AChE and BChE were measured in vitro. All
of the carbolinium salts showed moderate to high activ-
ity levels in the ChEs reaching those of physostigmine,
galantamine, and rivastigmine (IC50 (AChE) = 48 lM;
IC50 (BChE) = 54 lM[1]). In contrast to the tertiary
ꢀ1 1
1
8
6
7
9
217, 810 cm . H NMR (DMSO-d ) d 9.59 (s, 1H, 1-H),
6
.78–8.74 (d, J = 6.6 Hz, 1H, 3-H), 8.61–8.57 (dd, J = 1.1,
.6 Hz, 1H, 4-H), 8.06–8.05 (d, J = 2.4 Hz, 1H, 5-H), 7.87–
.82 (dd, J = 0.5, 9.2 Hz, 1H, 8-H), 7.55–7.49 (dd, J = 2.6,
3
.0 Hz, 1H, 7-H), 4.47 (s, 3H, N(2)-CH ), 4.06 (s, 3H,
1
3
N(9)-CH ), 3.90 (s, 3H, OCH ). C NMR (DMSO) 154.8
3
3
(C6), 139.8 (C9a), 135.9 (C8a), 132.4 (C4), 130.6 (C4a),

Y. Schott et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5840–5843
5843
1
1
29.5 (C3), 122.8 (C1), 119.3 (C4b), 117.4 (C7), 112.3 (C8),
04.1 (C5), 55.8 (OCH ), 47.7 (N(2)-CH ), 30.3 (N(9)-
7.40–7.35 (dd, J = 2.5, 8.9 Hz, 1H, 7-H), 4.46 (s, 3H, N(2)-
CH ), 4.03 (s, 3H, OCH ). C NMR (DMSO) 152.6 (C6),
3 3
1
3
3
3
CH ). Anal. (C H IN O) C, H, N.
3
139.0 (C9a), 135.9 (C8a), 132.1 (C4), 130.3 (C1), 129.2
(C3), 122.6 (C4a), 119.6 (C7), 117.4 (C4b), 111.9 (C8),
14 15
2
9
selected lines) 3424, 2932, 1573, 1199-1053, 810, 617 cm
-Methyl-9H-b-carbolin-6-ol (3f): mp 250 °C; IR (KBr,
ꢀ1
.
106.4 (C5), 47.6 (N(2)-CH
O) C, H, N.
6-Methoxy-1,2,9-trimethyl-9H-b-carbolin-2-ium
3
), 30.2 (N(9)-CH
3
). Anal.
1
6
H NMR (DMSO-d ) d 8.93–8.92 (d, J = 0.9 Hz, 1H, 1-
(C13H13IN
2
H), 8.28–8.26 (d, J = 5.1 Hz, 1H, 3-H), 7.99–7.95 (dd,
J = 1.1, 5.2 Hz, 1H, 4-H), 7.55–7.53 (dd, J = 0.6, 2.4 Hz,
iodide
(4d): mp 319 °C; IR (KBr, selected lines) 3040, 1609,
1488, 1434, 1229, 1036, 810, 765, 624 cm . H NMR
ꢀ1
1
1
7
H, 5-H,) 7.50–7.45 (dd, J = 0.5, 8.8 Hz, 1H, 8-H), 7.17–
.11 (dd, J = 2.4, 8.8 Hz, 1H, 7-H), 3.89 (s, 3H, N(2)-
CH ). C NMR (DMSO) 151.0 (C6), 137.4 (C9a), 135.6
(DMSO-d ) d 8.60 (s, 2H, 3-H, 4-H), 8.00-7.99 (d,
6
13
3
J = 2.5 Hz, 1H, 5-H), 7.89-7.85 (d, J = 9.2 Hz, 1H, 8-H),
7.50-7.45 (dd, J = 2.6, 9.2 Hz, 1H, 7-H), 4.35 (s, 3H, N(2)-
(
C8a), 132.3 (C3), 126.6 (C1), 123.5 (C4b), 120.7 (C7),
1
CH ). HRMS Calcd for C H N O [M+H] : 199.0871
18.0 (C4a), 114.4 (C4), 110.5 (C8), 105.9 (C6), 29.2 (N(9)-
3 3 3
CH ), 4.25 (s, 3H, N(9)-CH ), 3.89 (s, 3H, OCH ), 3.22 (s,
+
13
3H, C(1)-CH3). C NMR (DMSO) 158.6 (C6), 158.0
(C9a), 154.8 (C1), 134.2 (C8a), 122.6 (C4a), 119.0 (C4),
117.4 (C3), 115.1 (C4b), 112.8 (C7), 112.5 (C8), 103.5 (C5),
3
12 11
2
1
99.0880.
-Hydroxy-2,9-dimethyl-9H-b-carbolin-2-ium iodide (4f)—
6
exemplary procedure: A solution of 10 mmol of 2a in
00 mL of cumene was treated with 2% Pd/C (10%) and
refluxed for 10–15 h under N . Thereafter the reaction
mixture was treated with 20 mL of ethanol, heated again
and the hot mixture filtered off the catalyst, washed with
55.7 (OCH
(C(1)-CH ). Anal. (C H IN O) C, H, N.
3 3 3
), 45.5 (N(2)-CH ), 33.5 (N(9)-CH ), 16.3
1
3
15 17
2
2
6-Hydroxy-1,9-dimethyl-9H-b-carbolin-2-ium bromide (3h):
mp 320 °C; IR (KBr, selected lines) 3556-3412, 2932, 1621,
ꢀ1 1
1579, 1476, 1374, 1205, 813, 618 cm . H NMR (DMSO-
d6) d 9.16 (s, 1H, OH), 8.10–8.08 (d, J = 5.3 Hz, 1H, 3-H),
7.84–7.81 (dd, J = 0.5, 5.2 Hz, 1H, 4-H), 7.51–7.47 (m, 2H,
5-H, 8-H), 7.13–7.07 (dd, J = 2.4, 9.0 Hz, 1H, 7-H), 4.08
1
50 mL of hot ethanol. The solvent was removed under
reduced pressure and the residue was recrystallized from
toluene. Product 3a (5 mmol) was dissolved in DMF and
the mixture cooled to 0 °C, after which 6.75 mmol of NaH
was added. The mixture was allowed to reach room
temperature and stirred for an hour until evolution of
hydrogen stopped. The mixture was cooled to ꢀ10 °C,
1
3
(s, 3H, N(9)-CH ), 2.97 (s, 3H, C(1)-CH ). C NMR
3
(DMSO) 151.1 (C6), 141.9 (C9a), 136.7 (C1), 136.1 (C8a),
135.8 (C4), 127.2 (C3), 121.0 (C7), 118.1 (C4a), 112.9
3
3
(C4b), 110.9 (C8), 105.9 (C5), 32.2 (N(9)-CH ), 23.3 (C(1)-
5
mmol of MeI was added and the conditions were
CH ). HRMS Calcd for C H N O: M 213.1028. Found
3
13 13
2
retained for 15 min. Thereafter, the mixture was allowed
to reach rt and stirring was continued for 12 h. The
mixture was then treated with 85 mL of water and 3b was
extracted with chloroform. A mixture of 1.5 mmol of 3b
and 20 mL HBr/acetic acid (1:1) was stirred for 2–4 h at
M 213.0985.
6-Hydroxy-1,2,9-trimethyl-9H-b-carbolin-2-ium iodide (4 h):
ꢀ
1
mp 320 °C; IR (KBr, selected lines) cm 3148, 1513, 1482,
1
1338, 1217, 810, 635. H NMR (DMSO-d ) d 9.67 (s, 2H,
6
OH), 8.56–8.53 (d, J = 6.6 Hz, 1H, 3-H), 8.52–8.49 (d,
J = 6.6 Hz, 1H, 4-H), 7.76–7.72 (d, J = 9.1 Hz, 1H, 8-H),
7.65–7.64 (d, J = 2.2 Hz, 5-H), 7.36–7.31 (d, J = 2.4,
1
30 °C, then evaporated to dryness to yield 3f. Eight
millimole of MeI was added to a solution of 0.5 mmol of
f in 20 mL of acetone. The product 4f was filtered off and
dried in vacuo. mp 258 °C; IR (KBr, selected lines) 3172,
3
9.0 Hz, 1H, 7-H), 4.34 (s, 3H, N(2)-CH
N(9)-CH ), 3.19 (s, 3H, CCH ). C NMR (DMSO) 152.5
3
), 4.19 (s, 3H,
1
3
3 3
ꢀ
1 1
1
645, 1621,1518, 1362, 1211, 816, 774, 599 cm . H NMR
(C6), 141.4 (C1), 139.5 (C9a), 135.1 (C8a), 133.9 (C4),
130.8 (C3), 122.4 (C7), 119.3 (C4a), 115.1 (C4b), 112.1
(
DMSO-d ) d 9.69 (s, 1H, OH), 9.54 (s, 1H, 1-H), 8.69–
6
8
1
.67 (d, J = 6.4 Hz, 1H, 3-H), 8.55–8.52 (d, J = 6.5 Hz,
H, 4-H), 7.74 (s, 1H, 8-H), 7.70 (d, J = 1.7 Hz, 1H, 5-H),
(C8), 105.7 (C5) 45.4 (N(2)-CH
(C(1)-CH ). Anal. (C14H15BrN O) C, H, N.
3 3
), 33.4 (N(9)-CH ), 16.3
3
2