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Inhibition of cholinesterases
In the first set of compounds the methylene group of the
lead was replaced with an amino moiety (2-aminobenzimida-
zoles). Compound 16a shows the highest similarity to lead
structure 1. Although a significant loss of affinity was observed
for compound 16a, it still shows high selectivity and moderate
affinity (Ki(hCB2R)=1.9 mm; Table 1). Comparing the donor-sub-
stituted compound 16a to the unsubstituted compound 16e
(12% displacement at 10 mm, being ~30-fold less active) shows
the necessity of an electron-donating substituent. Varying the
position of the ethoxy group around the phenyl ring showed
the importance of para substitution, as a complete loss of af-
finity in case of the meta position (16b: 17% displacement at
10 mm) and a 4-fold lower affinity of the ortho-substituted
phenyl ring compound (16c: 6.6 mm) was observed (Table 1).
Exchange to different donor substituents leads to compounds
with roughly the same affinity and selectivity as for a diethyla-
mine moiety 16g (8.1 mm) or even increased affinity for the
para-phenoxy compound 16d (0.4 mm), but with lower selec-
tivity. Replacement of the aromatic with an alkyl system (cyclic
and acyclic, 16h–16m) led to decreased affinity (in the two-
digit micromolar range; Table 1). In a second set of com-
pounds, structural isomers of the 2-aminobenzimidazoles were
synthesized and evaluated. Here, the imidazopyridine 34
shows a 15-fold increased affinity (Ki(hCB2R)=127 nm) relative
to 16a, again with high selectivity over the hCB1R. In contrast,
the structural change to the 3-aminoindazole scaffold resulted
in a complete loss of affinity for hCB2R. In the third set, modifi-
cations to the alkyl chain at position 1 of the benzimidazole
core were performed, giving access to compounds 33 and 36,
which are both substituted with terminal amines instead of
the isopentyl chain. Compared with lead structure 1, both
structures show decreased affinity for hCB2R (Ki(hCB2R)=
577 nm for 33 and Ki(hCB2R)=188 nm for 36), but a 5- to 10-
fold increased affinity with respect to the 2-aminobenzimida-
zole structures (Table 1).
All target compounds were tested for their ability to inhibit
acetylcholinesterase (eeAChE, EC.3.1.1.7 from electric eel) and
butyrylcholinesterase (eqBChE, EC.3.1.1.8 from equine serum)
(Table 1). Sequence alignment had shown that both isoforms
exhibit very high sequence homology to the human enzymes
(88 and 84% sequence identity, respectively).[33]
In the initial step, the inhibitory effect of the lead structure
1 was determined at both ChEs, yielding IC50(BChE)=9.7 mm
and IC50(AChE)>100 mm (Table 1). This confirmed our initial as-
sumption that compound 1 is a suitable lead structure for
dual-acting compounds with desired selectivity, but with mod-
erate inhibitory activity at BChE and “unbalanced” affinity pro-
files. Starting from this point, the 2-aminobenzimidazole deriv-
atives 15a, 16a–16m, and 17a were synthesized. Compound
16a, being most similar to the lead structure 1, already shows
a 13-fold increased inhibitory effect and high selectivity for
BChE (IC50(BChE)=0.7 mm; Table 1); para-substitution of the
benzene ring is necessary to maintain sub-micromolar inhibito-
ry activity (cf. 16b: IC50(BChE)=9.1 mm and 16c: IC50(BChE)=
8.9 mm). Replacing the ethoxy group with other electron-do-
nating groups such as phenoxy (16d) or diethylamine (16g)
moieties, inhibition and selectivity were kept in the same
range or even slightly increased (16d IC50(BChE)=3.7 mm and
16g IC50(BChE)=0.24 mm; Table 1). Replacement of the (substi-
tuted) phenyl group with an alkyl substituent (cyclic and
linear) resulted in a decrease of inhibition. Just by adding a ter-
minal amine to the alkyl chain, inhibition could be preserved,
albeit at the cost of selectivity over AChE (16k: IC50(BChE)=
1.0 mm; Table 1). Structural isomers, i.e., using the aminoinda-
zole core, resulted in almost complete loss of inhibitory activi-
ty. Modifications of the alkyl chain at position 1 of the benzimi-
dazole core (36: IC50(BChE)=2.3 mm) led to a slightly increased
inhibitory effect (compared with first-generation lead structure
1). Taken together, apart from the inactive structural isomers,
all benzimidazoles and aminobenzimidazoles prepared inhibit
BChE in the sub-micromolar to two-digit micromolar range
with good to excellent selectivity over AChE.
Taken together, nearly all compounds prepared lack any af-
finity for hCB1R and therefore maintain selectivity with affinity
ranging from 127 nm (34) to 26 mm (31). There are two inactive
compounds: 15a and 40. The highest-affinity compound 34 is
4-fold less active than the lead structure 1. The amino benzimi-
dazoles show micromolar to sub-micromolar affinities (still
with high selectivity over hCB1R).
Radioligand binding studies at hCB1R and hCB2R
For correlating the results of the SARs obtained at BChE to
those at CB2R, the pIC50 and pKi values at each target were
plotted in the graph shown as Figure 4. Interpretation is sim-
plified by the fact that the majority of compounds still show
high selectivity over both AChE and hCB1R.
All compounds were tested for affinity to hCB1R and hCB2R in
radioligand binding studies (either HEK cells stably expressing
hCB2R, or CHO cells stably expressing hCB1R; see the Experi-
mental Section below for details). Lead structure 1 is an opti-
mized CB2R ligand from an AstraZeneca drug development
program, and we therefore regarded it as the gold standard in
terms of affinity [Ki(hCB2R)=37 nm in our tests] and selectivity
over hCB1 [24% replacement (hCB1R) of radioligand at 10 mm]
for the compounds described in this work. For compound 1 an
EC50(hCB2R) of 2.9 nm had been determined in a GTPgS
assay.[35] This value is higher than that observed in our radioli-
gand binding studies, which we had observed before. This
might be due to the different test system applied and/or the
equilibrium conditions used.[36]
Compound 31 (which is very similar to the lead structure 1,
but lacks the diethylamide moiety) shows no inhibitory effect
at both ChEs, whereas the respective amino benzimidazole
compound 15a shows a significant inhibitory effect at BChE
(IC50 =4.2 mm), The importance of the diethylamide moiety for
BChE inhibition is therefore pronounced (see Molecular Dock-
ing section below). Both compounds have lower (31:
Ki(hCB2R)=26.0 mm) or no affinity (15a) for the CB2R than lead
structure 1.
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