Synthesis and structure-activity relationship studies of 2-(N-substituted)-aminobenzimidazoles as potent negative gating modulators of small conductance Ca2+-activated K+ channels

Article abstract of DOI:10.1021/jm800809f

Small conductance Ca²⁺-activated K⁺ channels (SK channels) participate in the control of neuronal excitability, in the shaping of action potential firing patterns, and in the regulation of synaptic transmission. SK channel inhibitors have the potential of becoming new drugs for treatment of various psychiatric and neurological diseases such as depression, cognition impairment, and Parkinson's disease. In the present study we describe the structure-activity relationship (SAR) of a class of 2-(N-substituted)-2- aminobenzimidazoles that constitute a novel class of selective SK channel inhibitors that, in contrast to classical SK inhibitors, do not block the pore of the channel. The pore blocker apamin is not displaced by these compounds in binding studies, and they still inhibit SK channels in which the apamin binding site has been abolished by point mutations. These novel SK inhibitors shift the concentration-response curve for Ca²⁺ toward higher values and represent the first example of negative gating modulation as a mode-of-action for inhibition of SK channels. The first described compound in this class is NS8593 (14), and the most potent analogue identified in this study is the racemic compound 39 (NS11757), which reversibly inhibits SK3-mediated currents with a K_d value of 9 nM.

Full text of DOI:10.1021/jm800809f

J. Med. Chem. 2008, 51, 7625–7634
7625
Synthesis and Structure-Activity Relationship Studies of
2-(N-Substituted)-aminobenzimidazoles as Potent Negative Gating Modulators of
Small Conductance Ca2+-Activated K+ Channels
Ulrik S. Sørensen,* Dorte Strøbæk, Palle Christophersen, Charlotte Hougaard, Marianne L. Jensen, Elsebet Ø. Nielsen,
Dan Peters, and Lene Teuber†
NeuroSearch A/S, PederstrupVej 93, DK-2750 Ballerup, Denmark
ReceiVed July 4, 2008
Small conductance Ca2+-activated K+ channels (SK channels) participate in the control of neuronal
excitability, in the shaping of action potential firing patterns, and in the regulation of synaptic transmission.
SK channel inhibitors have the potential of becoming new drugs for treatment of various psychiatric and
neurological diseases such as depression, cognition impairment, and Parkinson’s disease. In the present
study we describe the structure-activity relationship (SAR) of a class of 2-(N-substituted)-2-aminobenz-
imidazoles that constitute a novel class of selective SK channel inhibitors that, in contrast to classical SK
inhibitors, do not block the pore of the channel. The pore blocker apamin is not displaced by these compounds
in binding studies, and they still inhibit SK channels in which the apamin binding site has been abolished
by point mutations. These novel SK inhibitors shift the concentration-response curve for Ca2+ toward
higher values and represent the first example of negative gating modulation as a mode-of-action for inhibition
of SK channels. The first described compound in this class is NS8593 (14), and the most potent analogue
identified in this study is the racemic compound 39 (NS11757), which reversibly inhibits SK3-mediated
currents with a Kd value of 9 nM.
Introduction
role of SK channels is the background for research aimed at
identifying compounds that are able to inhibit their function.
Such compounds should in principle increase neuronal excit-
ability and therefore hold a therapeutic potential for diseases
like depression,6 Parkinson’s disease, and cognitive and memory
disorders.1,2,7
The excitability and the patterns of action potential firing of
neurons as well as the function and plasticity of synapses are
balanced by the activity of many ion channels, including small
conductance Ca2+-activated K+ channels (SKa or KCa2
channels).1-3 SK channels belong to the class of Ca2+-activated
potassium channels (KCa) that are subdivided into three types:
big conductance KCa (BK), intermediate conductance KCa (IK)
(also named SK4), and SK channels. Whereas BK channels are
also gated by voltage, IK and SK channels are entirely activated
via binding of Ca2+ to the protein calmodulin (CaM), which is
constitutively attached to the C-terminus of each of the four
SK channel subunits.4 A recent study has demonstrated that the
concentration-response relationship for Ca2+ is influenced by
the phosphorylation state of CaM, where the protein kinase CK2
and the protein phosphatase 2A respectively decrease or increase
the apparent Ca2+-sensitivity by phosphorylation and dephos-
phorylation of CaM in the SK-CaM complex.5
Three highly homologous SK channel subunits have been
cloned: SK1, SK2, and SK3.8 In the central nervous system
(CNS), the two former types are mostly expressed in the cortex
and the hippocampus whereas SK3 channels to a larger extent
are confined to the subcortical areas, in particular in the
monoaminergic regions like substantia nigra pars compacta,
dorsal raphe, and locus coeruleus.9 The relatively high abun-
dance of SK3 channels in the substantia nigra is the basis for
the hypothesized symptomatic effect of SK blockers in Parkin-
son’s disease.1,2,7
The most potent, and highly selective, SK channel pore
blocker known is the octadecapeptide apamin, isolated from
venom of the honey bee Apis mellifera. Apamin contains two
disulfide bridges and two basic arginine residues (Arg13 and
Arg14) which have been identified as an essential requirement
for its effect on the SK channel, probably by interacting with
negative amino acids located in the outer pore mouth of the
channel.10 Similarly, the scorpion venom toxin scyllatoxin
(leiurotoxin I) contains a disulfide bridge and two basic arginine
moieties and it has, based on binding studies using [125I]apamin,
been found to interact with the apamin binding site. The
structural elements of these SK-selective neurotoxins are
mimicked in a number of small-molecule SK blockers such as
atracurium (1), tubocurarine (2), and pancuronium (3), as well
as in dequalinium (4) and the cyclic bis-quinolinium cyclophanes
UCL1684 (5) and UCL1848 (6)11,12 (Figure 1). These com-
pounds all displace [125I]apamin binding and are thus considered
as pore blockers acting at the apamin binding site. In compounds
5 and 6, the cyclic structure of apamin, formed by the disulfide
During a neuronal action potential, calcium ions enter the
cell via voltage-gated Ca2+ channels and activate SK channels
that then mediate a subsequent afterhyperpolarization (AHP).
During the AHP, the membrane potential is moved further away
from the action potential threshold and the excitability of the
neuron is consequently reduced. This important physiological
* To whom correspondence should be addressed. Phone: (+45) 44608000.
Fax: (+45) 44608080. E-mail: uss@neurosearch.dk.
†
Present address: Nuevolution A/S, Rønnegade 8, DK-2100 Copenhagen,
Denmark.
a Abbreviations: SK, small conductance Ca2+-activated K+; BK, big
conductance Ca2+-activated K+; IK, intermediate conductance Ca2+
-
activated K+; CaM, calmodulin; CK2, casein kinase 2; AHP, afterhyper-
polarization; CNS, central nervous system; SAR, structure-activity rela-
tionship; EDCI, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide; MW,
microwave; HEK293, human embryonic kidney 293; TM, transmembrane;
nH, Hill coefficient; DMSO, dimethyl sulfoxide; THF, tetrahydrofuran; TEA,
triethylamine.
10.1021/jm800809f CCC: $40.75
2008 American Chemical Society
Published on Web 11/11/2008

7626 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Sørensen et al.
Figure 2. Examples of known positive modulators of SK channels.
biological investigations are N-methylbicuculline (7) and, in
particular, N-methyllaudanosine (8), which is without activity
on GABA-A receptors.
In summary, compounds of several structural classes are
capable of blocking the SK channels and until recently all SK
blockers described contained highly basic or permanently
charged groups that interact with the apamin binding site. This
has been the basis for several studies aiming to identify a
pharmacophore model for compounds acting at this binding
site.13-15
Regarding compounds acting instead by modulation of the
SK channel gating properties, there are several examples of
positive modulators such as 1-EBIO (9), NS309 (10), and the
SK3/SK2 subtype selective compound CyPPA (11)16 (Figure
2). These compounds exert their positive modulatory effect via
a concentration-dependent leftward shift of the concentration-
response curve for Ca2+. The compound (1H-benzoimidazol-
2-yl)-(R)-1,2,3,4-tetrahydronaphthalen-1-ylamine (NS8593, 14)
was recently published as the first example of a negative
modulator of SK channels,17 and in the present paper we
describe the SAR of this new class of potent and selective SK
inhibitors.18 They are all 2-aminobenzimidazoles and are
structurally distant from previously known SK channel blockers
by being “druglike” small molecules that do not carry permanent
charges.
Chemistry
The compounds described in this study are all 2-(N-
substituted)-aminobenzimidazoles. Such 2-aminobenzimidazoles
can be prepared by several methods of which the most frequently
used is the condensation of an N-substituted amine derivative
and a 2-chlorobenzimidazole (method A, Scheme 1). This
transformation has been carried out either by thermal19-23 or
high-pressure24 reactions or by using a CuI20 or palladium
catalyzed amination of the 2-chlorobenzimidazole.23,25-27 Simi-
lar displacement reactions have been used, though much less
regularly, on the corresponding 2-bromo-28,29 and 2-fluorobenz-
imidazoles30 as well as on the related 2-sulfonic acid31 and
2-sulfone.29,32 Other commonly used strategies for the formation
of 2-(N-substituted)-aminobenzimidazoles are the reductive
amination of an aldehyde or ketone with a 2-aminobenzimid-
azole33,34 (method B, Scheme 1) or, alternatively, via an
o-phenylenediamine derivative followed by desulfurization and
cyclization of the corresponding N-(2-aminoaryl)-N′-substituted
thiourea (method C, Scheme 1). This cyclization has been
promoted by use of HgO,35-42 MeI,43 or peptide coupling
reagents such as DCC,43 diisopropyl carbodiimide,44 or EDCI
(1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide).45-47
In the present study, we initially used the direct reaction of
commercially available amines with 2-chlorobenzimidazole (12)
(Scheme 2). An example is the synthesis of 14, which was
prepared from 12 and commercially available (R)-1,2,3,4-
tetrahydro-1-naphthylamine (13) (Scheme 2). The reaction was
Figure 1. Examples of known SK channel pore blockers.
Table 1. Inhibition of Apamin Binding in Striatal Membranes and of
Whole-Cell rSK3 Currents Induced by Reference Compoundsa
compd
apamin binding, Ki (µM)
rSK3 currents, Kd (µM)
1
2
3
4
5
6
7
8
5.9 ( 0.2
9.2 ( 1.3
16 ( 4
6.4 ( 0.4
16 ( 1
21 ( 2
0.14 ( 0.02
0.0018 ( 0.0003
0.0014 ( 0.0001
4.4 ( 0.8
1.5 ( 0.2
>50
0.18 ( 0.03
0.0027 ( 0.0004
0.00043 ( 0.00009
7.0b
2.6 ( 0.3
9
positive modulator
positive modulator
positive modulator
0.077 ( 0.007
10
11
14
>50
>50
>50
a All data are generated at NeuroSearch as described in the Experimental
Section. b IC50 value (nH ) 0.76).
bridging, was mimicked in a macrocyclic structure, resulting
in the most potent synthetic blockers known (Table 1). In recent
years, other classes of pore blockers have been described and
examples of two relatively weak but often used compounds for

SAR Studies of Aminobenzimidazoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7627
Scheme 1. General Procedures for the Preparation of
2-(N-Substituted)-aminobenzimidazoles
role of titanium(IV) isopropoxide is not fully understood, but
it can possibly act both as a Lewis acid catalyst and as a water
scavenger. An amount of 2 equiv of titanium(IV) isopropoxide
was applied, and LC-MS analysis of the reaction mixtures
showed clean and almost complete conversion into the imine
intermediate. Subsequent reaction with sodium triacetoxyboro-
hydride at room temperature gave the 2-(N-substituted)-amino-
benzimidazoles as racemic mixtures in good to excellent yield
(Table 4). This condensation reaction between 21 and the
carbonyl compound could in principle lead to more than one
product because of the presence of both an exocyclic and
endocyclic nitrogens in 21. However, we observed clean
conversion and only identified the product resulting from imine
formation at the exocyclic 2-amino group. This structure was
confirmed for 27 by comparing the products formed either via
reductive amination with 2-tetralone or via the condensation of
tetrahydro-1-naphthylamine and 2-chlorbenzimidazole (12),
1
which for both methods gave identical H NMR spectra.
For the synthesis of compound 40 we had to prepare the
required tetralone, 7-methyl-3,4-dihydro-2H-naphthalen-1-one.
This intermediate was synthesized from 4-(p-tolyl)butyric acid
by conversion in neat thionyl chloride into the corresponding
acid chloride followed by an intramolecular Friedel-Crafts
acylation reaction using aluminum chloride in toluene to give
the 7-methyltetralone in 84% yield (Scheme 4). Furthermore,
structural modification of one of the synthesized 2-(N-substituted)-
benzimidazoles was carried out for the bromo-derivative 38,
which was reacted in a Suzuki-Miyaura cross-coupling reaction
with phenylboronic acid in the presence of a catalytic amount
of bis(triphenylphosphine)palladium(II) chloride and using MW
irradiation as heat source to give 48 in 79% isolated yield
(Scheme 5).
Scheme 2
Pharmacology
The medicinal chemistry described herein is based on selected
hits from a SK3 high-throughput screening campaign conducted
at NeuroSearch based on the membrane potential sensitive dye
DiBAC4(3). We initially identified the 2-aminobenzimidazole
22 as a weak SK3 inhibitor with a Kd value of 2.7 µM
determined in a whole-cell patch clamp electrophysiology
experiment using HEK293 cells stably expressing rat SK3
channels (Table 2). Further elaboration on the structure of 22
led to the corresponding 4-chlorophenyl- and 3,4-dichlorophenyl
derivatives 23 and 24, respectively, demonstrating up to 13-
fold higher potency (Table 2).
Compounds 22-24 possess a high structural flexibility due
to the three rotational bonds linking the benzimidazole and the
phenyl group. A series of conformationally constrained com-
pounds was synthesized to obtain knowledge on the optimal
relative positioning of the benzimidazole ring system and the
phenyl ring. In compounds 17, 18, and 25, the benzimidazole
and the phenyl moieties were connected by an additional ring
system that also contained the 2-amino group (Table 3), and
these compounds therefore do not have the hydrogen donating
properties of the 2-amino substituent. Both compounds 17 and
18 are nevertheless more potent than the noncyclized analogue
22, and a hydrogen bond donor in this position is therefore not
essential for inhibition of the SK3 channel. In comparison, the
N-(2-benzimidazolyl)-1,2,3,4-tetrahydroisoquinoline 25 is a very
weak inhibitor. This difference in biological activity could be
explained by 25 having a higher degree of conformational
constraint. Thus, the tetrahydroisoquinoline part of 25 can be
regarded as a linear and relatively inflexible extension of the
benzimidazole moiety, whereas the phenyl group in 17 and 18
first performed in refluxing toluene or acetonitrile which,
however, after several days of heating only gave a small amount
of product (LC-MS) together with unconverted starting materi-
als. Instead we found that heating in acetonitrile to 170 °C by
means of microwave (MW) irradiation in a sealed vial gave
almost complete conversion to the desired 2-aminobenzimida-
zole after a reaction time of 40 min. For compounds where the
required amine was not commercially available, we did in some
cases prepare the amine from the corresponding tetralone (19)
by conversion into the O-methyloxime (20) followed by
reduction to the primary amine (Scheme 3).48 In compounds
17 and 18, we prepared 2-phenylpyrrolidine (15) and 2-phenyl-
piperidine (16) from piperidine and pyrrolidine, respectively,
by conversion into the corresponding dihydropyrrole or tet-
rahydropyridine, followed by reaction with phenyllithium
(Scheme 3).49
The direct condensation of 2-aminobenzimidazole (21) and
tetralone using standard reaction conditions, tetrahydrofuran as
the solvent, a catalytic amount of acetic acid, and molecular
sieves gave low conversion to the desired imine intermediate
(according to LC-MS), and upon addition of sodium tri-
acetoxyborohydride only trace amounts of the corresponding
amine was formed. Imine formation had apparently not taken
place, and an alternative procedure was therefore investigated.
Moreover, titanium(IV) isopropoxide, which has been used as
an efficient and mild reagent in reductive aminations,50 indeed
proved very efficient in yielding the desired imines and
subsequently the amines in high isolated yields after treatment
with sodium triacetoxyborohydride (Scheme 3). The mechanistic

7628 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Sørensen et al.
Scheme 3a
a (a) N-chlorosuccinimide, Et2O, room temp; (b) phenyllithium (2.5 equiv, 1.9 M in t-Bu2O), Et2O; (c) CH3CN, 170 °C (MW heating); (d) NH2OMe·HCl,
NaOAc, MeOH, room temp; (e) BH3 ·THF, THF, 0-60 °C; (f) titanium(IV) isopropoxide, THF, room temp; (g) NaB(O2CCH3)3H, room temp.
Scheme 4a
Table 2. Functional Data on rSK3 Channels for Aminobenzimidazoles
22-24
compd
R
R′
Kd (rSK3, µM)
n
22
23
24
H
Cl
Cl
H
H
Cl
2.7 ( 0.1
0.64 ( 0.06
0.20 ( 0.05
3
3
4
a (a) SOCl2, cat. N,N-dimethylformamide; (b) AlCl3, toluene, 0 °C; (c)
(1) titanium(IV) isopropoxide, THF, room temp; (2) NaB(O2CCH3)3H, room
temp.
that the 2-substituted derivatives 29 and 30 were both less potent
than the corresponding 1-substituted analogues 26 and 27,
respectively. On the basis of the observations described above
(Table 3), it is evident that optimization of the relative
positioning of the benzimidazole and the phenyl rings is
important for the SK inhibitory properties of the compounds.
The tetrahydronaphthyl derivative 27 turned out to be the most
potent compound with a 20-fold increase in the SK3 inhibition
compared to compound 22, and although this increase in potency
could simply reflect a positive correlation between binding site
interactions and the introduction of additional lipophilicity in
the form of aliphatic carbon skeleton, it also shows that the
structural elements of 27 are locked in a more optimal and planar
conformation.
Scheme 5
On the basis of compound 27, we subsequently prepared a
series of close analogues having different substitution pattern
of the tetrahydronaphthyl group (Table 4). First, the influence
of methyl substitution on the aliphatic part of the tetrahy-
dronaphthyl ring was explored, and it was found that the
2-substituted analogue 31 had a potency comparable to that of
compound 27. Thus, although a 2-methyl substituent introduces
a degree of sterical hindrance between the tetrahydronaphthyl
group and the benzimidazole moiety, the relative positioning
of these two groups that leads to SK channel activity seems
nevertheless not compromised when tested as a mixture of the
diastereomers. In comparison, analogue 32 contains a methyl
group in the 4-position, and here, the substituent does not
influence the free rotation of the molecule and in fact results in
an improved Kd value of 0.032 µM. Despite this increase in
potency, we did not further pursue compounds having this
4-substitution, as it does introduce an additional center of
can cover a wider space relative to the benzimidazole. On the
basis of these findings, we prepared the series of compounds
26-28 containing instead two rotational bonds between the
benzimidazole moiety and the phenyl ring. In these structures,
the 2-amino group is not included in the carbocyclic skeleton
and the phenyl group is instead annulated by an aliphatic
carbocyclic ring of varying size. When comparing the activities
of 26-28, we found that the six-membered ring system in 27
resulted in the most potent compound with a Kd value of 0.13
µM. In addition, the point of attachment between the 2-amino
group and the new bicyclic ring is of importance, as we found

SAR Studies of Aminobenzimidazoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7629
Table 3. Functional Data on rSK3 Channels for Aminobenzimidazoles
only a weak inhibitor (Kd ) 6.5 µM). Considering that
substituents in the 7-position of the tetrahydronaphthyl moiety
had been well tolerated, we were interested in studying if this
could be extended to an additional phenyl ring. This modification
was first made in the ring-fused 1,2,3,4-tetrahydrophenanthren-
4-yl derivative 47, which in fact showed a 4-fold increase in
potency compared to 27. In contrast, a phenyl group connected
via a rotational bond in compound 48 gave a substantial decrease
in activity (Kd ) 0.60 µM). Finally, structural modification into
the 4,5,6,7-tetrahydrobenzo[b]thiophenyl derivative 46 gave,
compared to the six-membered carbocyclic group in 27, a
significant decrease in activity (Kd ) 0.54 µM). In conclusion,
structural modification of 27 in the phenyl ring of the tetrahy-
dronaphthyl moiety with preferably nonpolar and large groups
leads to increased SK channel inhibition. The incorporation of
polar heteroatoms as in 43 and 45 was detrimental to activity,
and despite that there could in principle be a reduced ability of
these compounds to cross the cell membrane, it supports the
hypothesis that the compounds are in general interacting with
a mainly lipophilic region of the binding site. Also, a limit for
the space available in this region was seen for compound 48 in
which a phenyl group extending out from the 7-position resulted
in a decrease in activity.
compd
method
yield (%)
Kd (rSK3, µM)
n
17
18
25
26
27
28
29
30
A
A
A
A
A
B
A
B
38
10
94
32
61
56
30
98
1.6 ( 0.2
0.75 ( 0.03
14 ( 5
3
3
3
5
7
3
3
3
2-Tetrahydronaphthylamines are chiral compounds, and in
order to study the pharmacology of the stereoisomers, we had
used 27 as an example and prepared both enantiomers from
reaction of 2-chlorobenzimidazole (12) with either of the
stereochemically pure (R)- or (S)-tetrahydronaphthylamines.17
As can be seen in Table 5, there is a 6-fold difference in the
activity of the (R)-enantiomer (14, Kd ) 0.077 µM) and the
(S)-enantiomer (49, Kd ) 0.45 µM). However, both compounds
are still relatively potent inhibitors, and since the difference in
activity is relatively small, we decided that for the subsequent
SAR study we would base our general conclusions on the
pharmacological data obtained from the tested racemates.
0.79 ( 0.09
0.13 ( 0.02
0.59 ( 0.03
1.7 ( 0.3
2.0 ( 0.4
chirality resulting in compounds that are mixtures of the
diastereomers. A 4-chloro-substitution on the phenyl ring of 22
had, as described above, resulted in the more potent SK inhibitor
23, and when preparing the corresponding chloro-substituted
tetrahydronaphthyl analogues, we obtained 33-36, of which
compound 34, being substituted in the 7-position, was the most
potent with a Kd value of 0.017 µM. Replacement of the chlorine
atom with a fluorine gave 37 with a Kd value of 0.034 µM,
whereas the larger bromo-substituent in 38 improved the Kd
further to 0.011 µM. Preparing the 6,7-dichloro substituted
derivative 39 confirmed the effect of increased bulk and
lipophilicity in this part of the molecule, as 39 (NS11757), with
a Kd value of 0.0091 µM, together with 38 turned out to be the
most potent SK3 inhibitors found within this study. Electron-
withdrawing substituents such as chlorine and bromine clearly
enhance potency, but also electron-donating groups such as
methyl or methoxy can be equally well accommodated. Thus,
compounds 40 and 41 with either 7-methyl or 5,7-dimethyl
substitution are both active as SK inhibitors, again with the
disubstituted analogue 41 as the more potent (Kd ) 0.033 µM).
Also, introduction of the more polar methoxy group in the
7-position gave 42 with a potency of 0.061 µM. Regarding the
consequences of adding polarity in the molecules, we made
the finding that changing the tetrahydronaphthyl group into a
thiochromanyl moiety as in compound 44 had no effect on the
activity whereas the oxygen containing chromanyl group in 43
was detrimental to SK activity. This clearly shows that the polar
chromanyl group cannot be tolerated, whereas the corresponding
thiochromanyl analogue 44 is equally potent as the physico-
chemically similar compound 27 that contains a carbon atom
of almost identical electronegativity as that of sulfur. On the
basis of these results, it could therefore be speculated that the
activity of these compounds is partly determined by the ability
of the tetrahydronaphthyl group to bind in a hydrophobic region
of the binding site. This hypothesis was supported when testing
the corresponding pyridyl analogue 45, which turned out to be
Generally, the 2-aminobenzimidazole moiety has basic prop-
erties (pKa ) 7.54 for 2-aminobenzimidazole compared to 5.53
for benzimidazole)51 and has for this reason been used regularly
as a guanidine mimetic. Hence, there have been several
applications of 2-aminobenzimidazoles within medicinal chem-
istry, recently in a variety of areas such as antibiotics,22,46
melanin-concentrating hormone antagonists,44 histamine antag-
onists,19,31 integrin RVꢀ3 antagonists,37-39 vanilloid receptor-1
antagonists,20,21 RNA cleavers,35 and kinase inhibitors.28,43,52
To examine whether a basic 2-aminobenzimidazole group is
essential for effect in our class of SK inhibitors, the two nonbasic
benzimidazole analogues 50 and 51 were tested (Figure 3).
Compound 50 is an analogue of 22 in which the 2-amino group
has been replaced by a methylene group, whereas 51 is the
corresponding amide analogue of 23. These modifications lead
to compounds without basic properties, and in fact both
compounds turned out to be inactive (Kd >10 µM). Hence,
activity of this class of compounds seems to require an
interaction with a basic and possibly protonated nitrogen atom
on the 2-aminobenzimidazole moiety.
Compound 14 has as a representative of the tetrahydronaph-
thyl compound class been characterized in more detail with
respect to mode-of-action and pharmacology on other potassium
channels.17 Most interestingly, when tested in excised patches,
it was found that the inhibition by 14 decreased as the
intracellular [Ca2+] was increased and that the compound was
equipotent when applied from either the intracellular or the
extracellular side of the cell membrane. This reflected that the
concentration-response curve for Ca2+ was right-shifted in

7630 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Sørensen et al.
Table 4. Functional Data on rSK3 Channels for the Racemic Aminobenzimidazoles Derived from 27
Table 5. Functional Data on rSK3 Channels for the Enantiomers of 27
support this finding, we have tested 14 on an hSK3 channel
that was mutated in the apamin binding site10 and found that
14 is in fact able to modulate this apamin-insensitive channel.
As shown in Figure 4 (upper left panel), a HEK293 cell
transiently transfected with hSK3 channels was inhibited by 80%
upon application of 100 nM apamin and by 75% after applica-
tion of 300 nM 14. The mutated hSK3 channel with Gln493Ala
and Asp495Ala mutations between transmembrane segment 5
and the pore loop (Figure 4, upper right panel) displayed a
current-voltage relationship similar to the wild-type hSK3
channel but was insensitive to 100 nM apamin. In contrast, 14
inhibited the mutated channel by 45% at 300 nM. Compound
14 thus remained active on the apamin-insensitive SK3 channel,
although the Kd value of 0.43 ( 0.11 µM (n ) 10) was 4-fold
higher than found for a wild-type hSK3 channel (Kd ) 0.10 (
0.01 µM, n ) 12). As the potency of 14 is dependent on the
degree of SK3 channel activation, the decreased potency could
thus reflect an increased apparent Ca2+-sensitivity of the mutated
channels. However, when characterized in inside-out patches,
the mutated hSK3 channel was found to have an activation curve
for Ca2+ (Figure 4, middle and lower panels) similar to that of
a wild-type hSK3 channel (EC50(Ca2+) ) 0.44 and 0.40 µM,
respectively). Similar to the whole-cell experiments, the potency
of 14 was reduced 3-fold from 0.67 ( 0.1 µM (n ) 8) to 2.2
( 0.4 µM (n ) 5) when tested at a Ca2+ concentration of 500
nM. Apamin is only active when applied from the extracellular
side of the membrane and was therefore not tested on the inside-
out patches. The slightly compromised effect of 14 on SK
channels devoid of the apamin binding site could indicate that
the presence of compound, a negative gating mode-of-action
that had not been described previously. In contrast to the pore
blockers (Figure 1), compound 14 does not displace [125I]apamin
in binding studies when tested in rat striatal membranes (Ki >50
µM) as well as in HEK293 cells expressing hSK3.17 To further
Figure 3. Functional data on rSK3 channels for two nonbasic
benzimidazole derivatives 50 and 51.

SAR Studies of Aminobenzimidazoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7631
Figure 5. Inhibition of hSK3 induced by 39 is due to a shift in the
Ca2+ response curve. The upper panel shows the time course of a
whole-cell experiment where 39 (100 nM) was applied during the period
indicated by the bar. The inhibition reached steady state after ap-
proximately 5 min and was reversible upon wash. The reference blocker
bicuculline methobromide (Bic, 100 µM) was applied for 20 s during
the washing period. The current was measured every 5 s at 0 mV upon
application of voltage ramps (-120 to +30 mV). The Kd value in this
experiment was fitted to 7.8 nM. The lower panel shows the Ca2+
response curves for hSK3 obtained from inside-out patches in the
absence (open circles) and in the presence (filled circles) of 39 (0.3
µM). The symbols are the average currents (relative to the maximal
current in the patch) from five to nine experiments, and the solid lines
represent the best fit to the Hill equation. In the absence of compound
an EC50 value of 0.40 µM and Hill coefficient of 4.5 was obtained,
whereas the values were 1.81 µM and 1.7 in the presence of 39.
Figure 4. Compound 14 inhibits an apamin-insensitive hSK3 channel.
Upper panels show whole-cell current-voltage curves obtained from
HEK293 cells transiently transfected with hSK3 (left panel) or the
apamin-insensitive hSK3(Q493A;D495A) (right panel). Each panel
shows the curves before (Ctrl) as well as during application of 300
nM 14 and during application of 100 nM apamin (Apa). The middle
panel shows Ca2+-induced activation of SK channels and potency of
14 when tested on an inside-out patch pulled from HEK293 cells
transiently transfected with the apamin-insensitive hSK3(Q493A;D495A).
During the first part of the experiment, the Ca2+-sensitivity was
measured by exposing the patch to the various Ca2+-buffered solutions
indicated at the x-axis. In the second part of the experiment, the potency
of 14 was determined by application of 300 nM compound during the
period indicated by the bar (Kd in this patch ) 2.3 µM). The lower
panel depicts the Ca2+ concentration-response relationships of the
wild-typechannels(opensymbols,n)9)andoftheSK3(Q493A;D495A)
(filled symbols, n ) 4). The currents were normalized to the currents
induced by 10 µM Ca2+ and represent the mean ( SEM. The solid
lines show the fitted Hill curves yielding EC50 values and nH of 0.40
µM and 4.5, as well as 0.44 µM and 4.4 for wild-type and
SK3(Q493A;D495A), respectively.
current level upon wash-out of the compound. In order to
definitively identify 39 as a negative gating modifier, Ca2+
response curves were performed using inside-out patches in the
absence and presence of 0.3 µM 39, respectively. Figure 5
(lower panel) shows that the Ca2+ response curve is right-shifted
(EC50(Ca2+) ) 1.8 µM; nH ) 1.7) compared to the control curve
(EC50(Ca2+) ) 0.4 µM; nH ) 4.5), thus confirming the
modulator mode-of-action of this series of compounds.
Compound 39 was furthermore tested for SK subtype
selectivity. However, using inside-out patches, the compound
inhibited the human isoforms hSK1, hSK2, and hSK3 with Kd
values (n ) 5) of 0.054 ( 0.012, 0.047 ( 0.013, and 0.060 (
0.012 µM, respectively. Thus, although this compound, in
comparison to 14, was improved with respect to potency, it did
not confer subtype selectivity.
amino acids close to the pore loop are involved in mediating
the negative gating modulation.
In the present study, none of the novel 2-aminobenzimidazole
derivatives 17, 18, and 22-48 presented displaced [125I]apamin
binding to rat striatal membranes (all showing Ki > 50 µM)
and all the active compounds are therefore considered as being
gating modulators. However, the racemic compound 39, char-
acterized by having a more than 8-fold higher potency than 14
(Kd ) 0.0091 µM versus 0.077 µM), was characterized with
respect to mode-of-action as well as selectivity. Figure 5 (upper
panel) shows the time course for inhibition induced by 100 nM
39. The whole cell SK current slowly returns toward control
In conclusion, we have identified a class of potent and
selective SK channel inhibitors that act through a novel
mechanism. These compounds inhibit SK currents by shifting
the concentration-response curve for Ca2+ toward higher Ca2+
concentrations rather than blocking the pore, and they are
therefore the first negative modulators of SK channels described.
They were found to inhibit SK channels in which the apamin
binding site was abolished by point mutations, confirming that
these novel compounds do not interact with the apamin binding
site. Also, they are structurally distant from all previously

7632 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Sørensen et al.
226-230 °C; MS (ES+) m/z 264 ([M + 1]+, 100); 1H NMR
(DMSO-d6) δ 1.80-2.04 (m, 3H), 2.34-2.46 (m, 1H), 3.55-3.64
(m, 1H), 3.81-3.90 (m, 1H), 5.12-5.18 (m, 1H), 6.78-6.92 (m,
2H), 7.07-7.13 (m, 2H), 7.18-7.34 (m, 5H), 11.1 (s, 1H). Anal.
(C17H17N3) C, H, N.
described small-molecule blockers of SK channels. They do not
contain permanent charges and can be described as “druglike”
small molecules with physiochemical properties that favor
penetration of the blood-brain barrier. Indeed, compound 14
(3 and 10 mg/kg intravenously) is able to affect firing rate and
firing pattern of dopaminergic neurons in ViVo53 in C57Bl/6
mice, and we are currently studying further the potential of this
class of compounds as a tool for identifying CNS effects of SK
inhibition.
2-(2-Phenylpiperidin-1-yl)-1H-benzoimidazole (18). Method A
was used (2-phenylpiperidin was prepared from literature49 pro-
cedure) and purification was by preparative LC-MS to give the
title compound as the free base: 80 mg (solid, 10%); mp 198-200
1
°C; MS (ES+) m/z 278 ([M + 1]+, 100); H NMR (DMSO-d6) δ
We have described the SAR leading from (1H-benzoimidazol-
2-yl)benzylamine (22) to the conformationally restricted 1,2,3,4-
tetrahydronaphthylamine 27. Further increase in biological
activity was obtained by modification of 27 with large and
preferably nonpolar groups on the phenyl ring of the tetrahy-
dronaphthyl moiety. In contrast, the introduction of the polar
heteroatoms nitrogen or oxygen in compounds 43 and 45 was
detrimental to activity. Adding an additional fused phenyl ring
in 47 was well tolerated, whereas the phenyl group extending
out from the 7-position in 48 resulted in a loss of activity.
Finally, we have found that inhibition of the SK channel activity
by this class of compounds requires that the basic properties of
the 2-aminobenzimidazole moiety are maintained. Among the
compounds tested, the racemic 1,2,3,4-tetrahydronaphthylamine
analogues 38 and 39 were identified as the most potent SK
inhibitors with Kd values of 0.011 and 0.0091 µM, respectively.
1.26-1.41 (m, 1H), 1.50-1.71 (m, 3H), 1.89-2.03 (m, 1H),
2.32-2.42 (m, 1H), 3.06-3.17 (m, 1H), 4.07-4.16 (m, 1H),
5.48-5.55 (m, 1H), 6.81-6.97 (m, 2H), 7.08-7.19 (m, 2H),
7.19-7.39 (m, 5H), 11.3 (s, 1H). Anal. (C18H19N3) H, N. C: calcd,
77.95; found, 77.50.
General Procedure for the Preparation of N-Substituted
2-Aminobenzimidazoles by Reductive Amination (Method B).
Synthesis of (1H-Benzoimidazol-2-yl)-(1,2,3,4-tetrahydronaph-
thalen-2-yl)amine (30). To a solution of 2-aminobenzimidazole
(21, 0.50 g, 3.76 mmol) in dry THF (10 mL) (under N2 atmosphere)
was added 1.3 equiv of ꢀ-tetralone (0.71 g, 4.88 mmol) followed
by the addition of 1.5 equiv of titanium(IV) isopropoxide (1.60 g,
5.63 mmol). The reaction mixture was stirred for 3.5 h at room
temperature followed by the addition of 2 equiv of sodium
triacetoxyborohydride (1.58 g, 7.51 mmol). After the mixture was
stirred overnight at room temperature, aqueous saturated NaHCO3
was added and the layer was extracted with EtOAc. The combined
organic phases were dried (MgSO4), filtered, and evaporated to
dryness to give the crude product which was purified by flash
chromatography (50-100% EtOAc/TEA (99:1) in hexane) to give
the title compound in 98% isolated yield (solid, 966 mg): mp
211-214 °C; MS(ES+) m/z 264 ([M + 1]+, 100); 1H NMR
(DMSO-d6) δ 1.71-1.85 (m, 1H), 2.09-2.18 (m, 1H), 2.80 (dd,
1H, J ) 9.1 and 16.3 Hz), 2.85-2.93 (m, 2H), 3.14 (dd, 1H, J )
4.8 and 16.3 Hz), 3.96-4.07 (m, 1H), 6.61-6.66 (m, 1H), 6.86
(br s, 2H), 7.07-7.19 (m, 6H), 10.6 (br s, 1H). Anal. (C17H17N3)
C, H, N.
(1H-Benzoimidazol-2-yl)(7-phenyl-1,2,3,4-tetrahydronaphtha-
len-1-yl)amine (48). Compound 38 (0.50 g, 1.46 mmol) was
dissolved in a mixture of dioxane (5 mL) and water (0.5 mL). Then
1.3 equiv of phenylboronic acid (0.23 g, 1.90 mmol), ethanol (5
mL), and 3 equiv of potassium carbonate (0.61 g, 4.3 mmol) were
added to the mixture, which was degassed with nitrogen, and 0.01
equiv of bis(triphenylphosphine)palladium(II) chloride (11 mg,
0.015 mmol) was added. The reaction mixture was heated to 120
°C in the MW oven for 100 min and cooled to room temperature.
Saturated aqueous NaHCO3 was added, and the layer was extracted
with EtOAc. The organic phases were dried (MgSO4), filtered, and
evaporated to dryness to give the crude product which was purified
by flash chromatography (10-75% EtOAc in hexane, 5%TEA) to
give 48 in 79% yield (390 mg) as a white solid: mp >250 °C; MS
(ES+) m/z 340 ([M + 1]+, 100); 1H NMR (DMSO-d6) δ 1.75-2.14
(m, 4H), 2.72-2.93 (m, 2H), 5.05-5.15 (m, 1H), 6.81-6.94 (m,
2H), 7.02-7.08 (m, 1H), 7.11-7.20 (m, 2H), 7.21-7.26 (m, 1H),
7.28-7.33 (m, 1H), 7.37-7.43 (m, 2H), 7.46-7.50 (m, 1H),
7.53-7.58 (m, 2H), 7.62-7.65 (m, 1H), 10.6 (s, 1H). Anal.
(C23H21N3) H, N. C: calcd, 81.39; found, 80.58.
Experimental Section
General Experimental Information. Chemistry. Reagents and
solvents, as well as test compounds 50 and 51, were purchased
from commercial sources and used without further purification.
Melting points were determined in an open capillary and are
uncorrected. MW irradiation was performed using an Emrys
Optimizer EXP from Biotage. Proton and carbon NMR spectra were
recorded on a 500 MHz instrument at 500 and 125 MHz,
respectively. Elemental analyses were performed at the Department
of Chemistry, University of Copenhagen, and are within (0.4 ppm
unless indicated otherwise. Crude compounds were purified by
preparative reversed-phase HPLC using a Waters autopurification
system with a Waters XTerra Prep MS C8 OBD column (30 mm
× 100 mm, 5 µm). The column was equilibrated at a flow rate of
43 mL/min with a mobile phase containing 80% solvent A (10 mM
aqueous NH4HCO3) and 20% solvent B (CH3CN). Purification was
done using a linear gradient from 20% to 95% of solvent B
(CH3CN) over 10 min for the elution of pure compounds. Analytical
HPLC analyses were performed on the same autopurification system
using the similar Waters XTerra analytical column.
General Procedure for the Preparation of N-Substituted
2-Aminobenzimidazoles from the Corresponding Amines and
2-Chlorobenzimidazole (12) (Method A). Synthesis of (1H-
Benzoimidazol-2-yl)-(R)-1,2,3,4-tetrahydronaphthalen-1-ylamine
(14, NS8593). 2-Chlorobenzimidazole (12, 5.0 g, 32.8 mmol) and
1.2 equiv of (R)-1,2,3,4-tetrahydro-1-naphthylamine (5.8 g, 39.3
mmol) were suspended in acetonitrile (5 mL) in a closed vial and
heated to 170 °C for 40 min by use of MW irradiation. After the
mixture was cooled to room temperature the precipitated solid was
filtered off and washed with acetonitrile to give the title compound
as the HCl salt (white solid, 6.42 g, 65%): mp 263-265 °C;
MS(ES+) m/z 264 ([M + 1]+, 100); optical rotation 58.7° (MeOH,
25 °C); 1H NMR (DMSO-d6) δ 1.75-1.85 (m, 1H), 1.88-1.97
(m, 2H), 2.06-2.13 (m, 1H), 2.72-2.88 (m, 2H), 5.01-5.07 (m,
1H), 7.16-7.28 (m, 5H), 7.34-7.43 (m, 3H), 9.48 (m, 1H), 12.8
(br s, 2H); 13C NMR (DMSO-d6) δ 19.7, 28.8, 30.0, 51.9, 111.7,
123.2, 126.4, 127.9, 128.8, 129.4, 130.4, 135.5, 137.6, 150.0. Anal.
(C17H17N3 ·HCl) C, H, N.
Pharmacology. Electrophysiological experiments and [125I]-
apamin binding to striatal membranes were carried out according
to previously described procedures.17 In short, the potency of
inhibition is given as Ki values when obtained from displacement
of radioactive apamin in equilibrium binding experiments. Three
independent concentration-response experiments were performed
using 3-10 concentrations. In patch clamp experiments, Kd values
obtained by single concentration kinetics experiments are used (with
n equaling the number of independent experiments) except for
bicuculline methbromide, where the potency is given as an IC50
value obtained from an equilibrium concentration response (fast
inhibition kinetics of this compound prevents accurate measurement
of blocker kinetics). Data are given as mean ( SEM. All
electrophysiological data were, unless specifically stated, obtained
2-(2-Phenylpyrrolidin-1-yl)-1H-benzoimidazole (17). Method
A was used (2-phenylpyrrolidin was prepared from literature49
procedure). Purification was by aqueous basic workup, and recrys-
tallization was from CH3CN/H2O: 150 mg (solid, 38%); mp

SAR Studies of Aminobenzimidazoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7633
(12) Galanakis, D.; Ganellin, C. R. Defining determinant molecular
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guinea-pig hepatocytes: the influence of polarizability and molecular
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Ca2+-activated K+ channel. Curr. Med. Chem. 2008, 15, 1305–1315.
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Enzyme Inhib. Med. Chem. 2005, 20, 517–523.
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P. M. Bis-quinolinium cyclophanes: toward a pharmacophore model
for the blockade of apamin-sensitive SKCa channels in sympathetic
neurons. Bioorg. Med. Chem. Lett. 2004, 14, 4231–4235.
with rat SK3 channels and using an extracellular solution containing
4 mM K+. In all whole-cell experiments the intracellular/pipette
solution contained Ca2+ buffered at 400 nM.
Molecular Biology. hSK3 was cloned from total human skeletal
muscle RNA as previously described.54 The hSK3 cDNA was
subcloned into the pNS3h vector. Mutated hSK3 was generated
using uracilated plasmid as template in a mutagenesis reaction in
which mutagenic oligonucleotides and T7 DNA polymerase were
used to introduce the mutations. An aliquot of the mutagenesis
reaction was transformed into E. coli XL1-Blue cells, and mutated
hSK3 were identified by the elimination of an Acc65I restriction
site. The sequence of the mutagenic oligonucleotide was as follows:
hSK3-AA, gtgaaaggtatcatgacgcgcaggccgtaactagtaacttt. The fidelity
of the construct was verified by sequencing. HEK293 cells were
transiently transfected using lipofectamin and standard trasnsfection
methods. Electrophysiological recordings were made 2-4 days after
transfection.
(16) Hougaard, C.; Eriksen, B. L.; Jørgensen, S.; Johansen, T. H.; Dyhring,
T.; Madsen, L. S.; Strøbæk, D.; Christophersen, P. Selective positive
modulation of the SK3 and SK2 subtypes of small conductance Ca2+
-
activated K+ channels. Br. J. Pharmacol. 2007, 151, 655–665.
(17) Strøbæk, D.; Hougaard, C.; Johansen, T. H.; Sørensen, U. S.; Nielsen,
E. Ø.; Nielsen, K. S.; Taylor, R. D. T.; Pedarzani, P.; Christophersen,
P. Inhibitory gating modulation of small conductance Ca2+-activated
K+ channels by the synthetic compound (R)-N-(benzimidazol-2-yl)-
1,2,3,4-tetrahydro-1-naphthylamine (NS8593) reduces afterhyperpo-
larizing current in hippocampal CA1 neurons. Mol. Pharmacol. 2006,
70, 1771–1782.
(18) Sørensen, U. S.; Teuber, L.; Peters, D.; Strøbæk, D.; Johansen, T. H.;
Nielsen, K. S.; Christophersen, P. Novel 2-Amino Benzimidazole
Derivatives and Their Use as Modulators of Small-Conductance
Calcium-Activated Potassium Channels. WO 2006/013210 A2, Febru-
ary 9, 2006.
(19) Rivara, M.; Zuliani, V.; Cocconcelli, G.; Morini, G.; Comini, M.;
Rivara, S.; Mor, M.; Bordi, F.; Barocelli, E.; Ballabeni, V.; Bertoni,
S.; Plazzi, P. V. Synthesis and biological evaluation of new non-
imidazole H3-receptor antagonists of the 2-aminobenzimidazole series.
Bioorg. Med. Chem. 2006, 14, 1413–1424.
(20) Ognyanov, V. I.; Balan, C.; Bannon, A. W.; Bo, Y.; Dominguez, C.;
Fotsch, C.; Gore, V. K.; Klionsky, L.; Ma, V. V.; Qian, Y.-X.; Tamir,
R.; Wang, X.; Xi, N.; Xu, S.; Zhu, D.; Gavva, N. R.; Treanor, J. J. S.;
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Acknowledgment. The authors thank Helle D. Rasmussen
and Tine Sparre for technical assistance with the chemical
syntheses. Jette Sonne, Anne S. Meincke, and Vibeke Meyland-
Smith are acknowledged for their assistance with patch clamp
experiments. Lene G. Larsen is acknowledged for technical
assistance with the construction of the apamin-insensitive hSK3,
and Susanne K. Hansen and Ulla Borberg are acknowledged
for conducting the apamin binding experiments.
1
Supporting Information Available: Experimental details, H
NMR data, and melting points for the compounds 22-29 and
31-47; table of combustion analysis data for all novel compounds,
14, 17, 18, 24-26, and 28-48. This material is available free of
charge via the Internet at http://pubs.acs.org.
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