SYNTHESIS AND ENZYME KINETICS OF TETRACYCLIC ACETYLCHOLINESTERASE
INHIBITORS
González Tanarro, C. M.* and Gütschow, M.
Pharmaceutical Chemistry, Institute of Pharmacy, University of Bonn, 53115 Bonn
140
[2a] = 0µM
S
X
H
N
N
N
1 (X=N-CH2C6H5)
3 (X=CH2)
N
N
2 (X=N-CH2C6H5)
4 (X=CH2)
N
O
(CH2)n
(CH2) n
Syntheses
1/(v - offset) (min)
0.08
100
80
0.02
60
0
0
COOEt
b
S
H
N
COOEt
c
S
N
H
N
N
N
S
H
N
S
10
2
4
6
2a (µM)
8
a) phenyl isothiocyanate, ethanol, 80 °C, 1.5 h, argon atmosphere.
b) NaOH, ethanol, methyl iodide.
c) 1,3-diaminopropane, 2-methoxyethanol, 150 °C, 24 h, autoclave.
Kinetics Results
Tab. 1: Inhibition of acetylcholinesterase by polycyclic thieno compounds.
1a
IC50 AChE (µM)
S
1.62
N
compound
3a
IC50 AChE (µM)
S
N
N
N
1b
N
N
S
2.52
N
3b
S
N
S
N
N
0.61
H
N
N
4a
N
S
S
N
N
0.92
H
N
4b
S
-0.004
140
80
60
0
0.002
0.004
1/[ASCh] (µM-1)
Fig. 2: Plot of the reciprocal rates
(average values of duplicate
experiments) vs. the reciprocal
substrate concentrations for the
inhibition of AChE by compound
2a (0-10 µM).
0.08
100
-0.002
0.1
0.06
[2b] = 0µM
[2b] = 1µM
0.08
0.04
0.02
0
0
40
2
4
6
[2b] (µM)
[2b] = 2µM
0.06
[2b] = 3µM
0.04
[2b] = 4µM
[2b] = 5µM
0.02
0
2
4
6
Fig. 3: Plot of the rates (average
values of duplicate experiments) vs.
[2b].
Nonlinear
regression
according to the equation vi = v0/
(1+[I]/IC50)+d. The insert is a
Dixon plot to show the linearity of
the plot.
-0.004
-0.002
0
0.002
0.004
1/[ASCh] (µM-1)
Fig. 4: Plot of the reciprocal rates
(average values of duplicate
experiments) vs. the reciprocal
substrate concentrations for the
inhibition of AChE by compound
2b (0-5 µM).
Methods
The inhibition of AChE from Electrophorus electricus was carried out in 0.1
M phosphate buffer, 0.1 M NaCl, pH 7.3, 6% acetonitrile, 0.35 mM 5,5´dithio-bis(2-nitrobenzoic acid) at 25 °C. Substrate of the coupled assay [3]
was 0.5 mM acetylthiocholine iodide (ASCh), and a Km value of 0.55 mM
was determined. AChE was preincubated for 15 minutes with the
compounds. 1a, 1b, 2a, 2b, 3a and 3b were measured in different
concentrations. In order to determine the type of inhibition, the
concentrations of the substrate and those of 1a, 1b, 2a and 2b were
varied as depicted in the secondary plots in Fig. 2 and 4.
N
Conclusions
>100
H
N
N
N
N
O
>100
H
N
O
O
2b
4.31
N
N
N
2a
16.8
N
[2a] = 8µM
[2a] = 10µM
10
Fig. 1: Plot of the rates (average
values of duplicate experiments) vs.
[2a].
Nonlinear
regression
according to the equation vi = v0/
(1+[I]/IC50)+d. The insert is a
Dixon plot to show the linearity of
the plot.
0
O
Scheme 1
N
[2a] = 6µM
0.1
0
0
2b (µM)
compound
[2a] = 4µM
0
N
O
8
20
N
N
6
0.2
[2a] (µM)
1/(v-offset) (min)
S
N
a
4
0
enzyme activity (%)
NH2
2
40
120
S
0.04
20
The synthesis of compound 2b is given in Scheme 1, 2a was prepared
similarly. For the preparation of the other title compounds, see [2].
N
[2a] = 2µM
0.06
1/(v - offset)
S
X
enzyme activity (%)
A wide range of evidence shows that acetylcholinesterase inhibitors can
interfere with the progression of Morbus Alzheimer. This disease is related
to the loss of cortical cholinergic neurotransmission. Furthermore,
acetylcholinesterase (AChE) was shown to accelerate the assembly of
amyloid-β-peptides into Alzheimer’s fibrils. The peripheral site of the
enzyme plays a possible role in this process [1]. In the course of our
investigations towards the synthesis and biological evaluation of polycyclic
azines, we prepared eight compounds of the depicted structural types
(a: n=2; b: n=3). These compounds were evaluated as inhibitors of AChE.
0.3
0.1
120
1/(v - offset)
Introduction
N
O
References
[1a] Sugimoto H, Yamanishi Y, Iimura Y, Kawakami Y (2000) Curr Med Chem 7:303-339; [1b]
Soreq H, Shlomo S (2001) Nature 2:294-302; [1c] Inestrosa N et al. (1996) Neuron 16:881-891.
[2a] Leistner S, Gütschow M, Vieweg H, Wagner G, Strohscheidt T, Grupe R (1988) Pharmazie
43:756-760; [2b] Ram VJ (1979) Arch Pharm 312:19-25.
[3] Ellman GL, Courtney KD, Andres V Jr, Featherstone RM (1961) Biochem Pharmacol 7:88-95.
Some of these tetracyclic compounds (1: n= 2,3 ; 2: n= 2,3) act as
inhibitors with IC50-values < 3 µM. These inhibitors contain a linear or an
angular imidazo or pyrimido moiety and a basic nitrogen of the N-benzyl
tetrahydropyridine ring. The mechanism of inhibition was analyzed with
different substrate concentrations (250 – 1000 µM). Compound 2a and 2b
interact with acetylcholinesterase in a noncompetitive manner. The same
kinetic behavior was observed for 1a and 1b, data not shown. Further
investigation are intended to elucidate the mode of enzyme-inhibitor
interaction. These studies should include inhibition kinetics with related
new compounds as well as docking experiments.