Preliminary SAR studies on non-apamin-displacing 4-(aminomethylaryl)pyrrazolopyrimidine KCa channel blockers

Article abstract of DOI:10.1016/j.bmcl.2008.08.026

An exploratory SAR study on a series of potent, non-apamin-displacing 4-(aminomethylaryl)pyrazolopyrimidine K_Ca channel blockers is described and their selectivity against K_Ca channel subtypes is reported. The most potent analog, 5-chloro-N-(thiophen-2-ylmethyl)pyrazolo[1,5-a]pyrimidin-7-amine (24) displayed sub-micromolar activity in both a thallium flux and whole-cell electrophysiology assay and did not displace apamin in a competitive binding study.

Full text of DOI:10.1016/j.bmcl.2008.08.026

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5694–5697
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Preliminary SAR studies on non-apamin-displacing
4-(aminomethylaryl)pyrrazolopyrimidine KCa channel blockers
*
Robert G. Gentles , Shuanghua Hu, Yazhong Huang, Katherine Grant-Young, Michael A. Poss,
Charles Andres , Tracey Fiedler, Ronald Knox, Nicholas Lodge, C. David Weaver à, David G. Harden
Research and Development, Bristol Myers Squibb Co., 5 Research Parkway, Wallingford, CT, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An exploratory SAR study on a series of potent, non-apamin-displacing 4-(aminomethylaryl)pyrazolopyr-
imidine KCa channel blockers is described and their selectivity against KCa channel subtypes is reported.
The most potent analog, 5-chloro-N-(thiophen-2-ylmethyl)pyrazolo[1,5-a]pyrimidin-7-amine (24) dis-
played sub-micromolar activity in both a thallium flux and whole-cell electrophysiology assay and did
not displace apamin in a competitive binding study.
Received 27 June 2008
Revised 7 August 2008
Accepted 8 August 2008
Available online 14 August 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Non-apamin displacing
Small conductance potassium channel
Pyrrazolopyrimidine
Apamin
Small conductance calcium-activated potassium (KCa) channels
are voltage-insensitive and open in response to elevated levels of
intra-cellular calcium.1 These ions mediate their effect by binding
to calmodulin, a protein that is constitutively associated with the
channel and that undergoes a structural change induced by higher
calcium occupancy.2–6 This alteration in the protein’s conformation
results in opening of the channel and an associated enflux of potas-
sium ions, and it is this current that is responsible for the afterhy-
perpolarization observed following the initial depolarization/re-
polarization phases of an action potential in neurons expressing
KCa channels.7,8
schizophrenia.15 KCa blockers may also prove helpful in the
treatment of certain cognitive disorders.16,17
To date, a limited number of non-peptidic compounds have been
reported to be active at KCa channels,18–20 with the majority of these
being thought to function by obstructing the pore of the channel.
Examples include the quinoline-appended diazepines;21 isoquino-
line analogs related to bicuculline, including N-methyl laudano-
sine;22–26 and a class of 2-aminothiazoles,27 as shown in Figure 1.
However, in a recent publication, it was reported that the 2-amino-
benzimidazole, NS8593 (IC50 = ꢀ500 nM) is a non-apamin-displac-
ing KCa channel blocker that exerts its effect at a site remote from
the pore region.28 In this letter, we report a preliminary SAR study
on an additional series of 5-chloro-N-(methylaryll)pyrazolo[1,5-
c]pyrimidin-7-amines that we demonstrate are also non-apamin-
displacing KCa channel blockers with activities similar to that re-
ported for the 2-aminobenzothiazoles. In addition, we report on
the KCa channel selectivities of the more potent members of this
class, and describe the behaviors of the compounds in whole cell
electrophysiology and apamin-displacing studies.
Such afterhyperpolarizations impact neuronal excitability and
can significantly modulate neuronal firing patterns.9,10
Three isoforms of KCa channels have been identified: KCa2.1,
KCa2.2, and KCa2.3, and their distributions in both rat and human
brain have been determined.1,9,11,12 From these studies, it is
apparent there exists the possibility for regional modulation of
neuronal excitability, and significant efforts have been directed at
identifying both pan- and sub-type-selective KCa channel blockers.
Compounds with appropriate profiles may have utility in the
treatment of diseases in which modulated neuronal firing would
be expected to be adventitious.13 These would include conditions
such as epilepsy, depression, Parkinson’s disease,14 and
As discussed in a previous letter,27 our search for novel KCa
channel blockers began with an HTS screen that employed a
thallium flux assay in which compounds were tested against a
recombinant HEK 293 cell line-expressing specific KCa channel
isoforms. This assay has been described previously,32 and is one
in which the IC50 obtained for the known KCa channel blocker
apamin has been repeatedly demonstrated to be consistent with
reported literature values. This screen resulted in the identification
of a number of hits, one of the more interesting being, N-benzyl-5-
chloropyrazolo [1,5-c]pyrimidin-7-amine (1), as shown in Figure 2.
* Corresponding author.
E-mail address: robert.gentles@bms.com (R.G. Gentles).
Oblon Spivak, 1940 Duke Street, Alexandria, VA, USA.
Vanderbilt Institute of Chemical Biology and Department of Pharmacology,
à
Nashville, TN, USA.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.026

R. G. Gentles et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5694–5697
5695
Figure 1. Previously reported small molecule blockers of KCa channels.
Figure 2. N-Methylaryl-5-chloropyrazolo[1,5-c]pyrimidin-7-amines evaluated for activity at KCa channels.

5696
R. G. Gentles et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5694–5697
After confirming the structure and purity of the screening sam-
observed with compound 23 were consistent with those observed
with the related pyridin-3-yl and pyridin-2-yl analogs (data not
shown), in that none showed any significant activity.
ple of 1, the compound’s IC50 was determined using the thallium
flux and electrophysiology assays, and both values were found to
be in good agreement, as can be seen in Table 1.
Given these results, we sought to develop an initial SAR around
1 to determine if this chemotype could be progressed as a series of
effective KCa channel blockers. To that end, we chose to first
explore the introduction of substituents at the methylene and aryl
moieties of the benzylamine functionality in 1, as shown by the
analogs depicted in Figure 2.
These compounds were prepared from commercial starting
materials employing standard methodology29–31 that was modified
slightly to better accommodate our specific array purification
protocols, as shown in Figure 3.
The process adopted involved heating solutions of 5,7-dichloro-
pyrazolo[1,5-a]pyrimidine in acetonitrile with one of a series of
benzyl- or heteroarylmethyl amines overnight at 80 °C. On cooling,
the desired products were isolated using preparative reverse-phase
HPLC.
More encouraging, however, was the result obtained with the
standard phenyl isostere, thiophene. The compound, 5-chloro-N-
(thiophen-2-ylmethyl)pyrazolo [1,5-c]pyrimidin-7-amine (24)
displayed significantly improved potency (IC50 = 390 nM) relative
to other analogs tested in our thallium assay, and this result was
supported in a related electrophysiology assay (IC50 = 580 nM).
In order to better understand the mechanism of the observed
channel block, we examined the activity of 24 in a Scintillation
Proximity Assay (SPA) to assess its ability to displace radio-labeled
[
125I]-apamin from the KCa2.3 channel.33,34 In this assay, (24) did
not compete with the labeled ligand, a finding that suggested that
this compound interacts with the channel at a site remote from
that occupied by apamin. Correspondingly, we were interested to
explore the possibility that this alternative binding site could be
exploited to identify small molecules that would display selectivity
between the different KCa channel subtypes.
The compounds so obtained were then screened in the thallium
flux assay, and the data associated with these experiments are
presented in Table 1.
From these results it is apparent that attempts to introduce
functionality at the ortho-position of the phenyl group resulted in
significant loss of activity. This seemed unrelated to the electronic
character of the substituent; electron withdrawing groups such as
those in analogs, 2, 4, and 6, resulted in approximately a 10- to 30-
fold loss in potency, as did electron-donating substituents as seen
in examples 3 and 5.
Subsequently, compounds 1 and 24 were tested in the thallium
flux assay against cell lines recombinantly expressing the KCa2.1
and KCa2.2 channel isoforms. As can be seen from the data in Table
2, these compounds displayed no significant selectivity between
the various channel subtypes. This was similar to our previous
observations with a series of apamin-displacing aminothiazole
blockers.27 Obviously, however, further work remains to be done
to fully investigate the potential of this series to identify KCa chan-
nel subtype-selective blockers.
In conclusion, we present
a series of 5-chloro-N-(aryl-
The meta-position of the aryl group in 1 was more tolerant of a
range of substitution. The chloro analog 7, and the nitro derivative
11, displayed modest losses of potency relative to the ortho-substi-
tuted derivatives. The methyl 8, trifluoromethyl 9, and
trifluoromethoxy 10 derivatives were essentially equipotent, and
again confirmed that the electronic character of the aromatic ring
did not appear to be a key determinant of potency. Interestingly,
the 3,5-dichloro analog 19 lost significant activity, in contrast to
the mono-substituted analog 7.
methyl)pyrazolo[1,5-c]pyrimidin-7-amines that are effective
KCa2 blockers and appear to act remotely from the classic apam-
in-binding site. Specific analogs disclosed are equipotent with
the only other previously reported, non-apamin-displacing KCa
channel blocker, and an exploratory SAR has been developed,
Table 1
Activities of compounds 1–24 at the
K
Ca2.3 channel in the thallium flux and
Introduction of functionality at the para-position of the pendant
phenyl group was not tolerated, as can be seen with analogs, 12–
17. All of these analogs show a significant loss of activity and this
would appear to be attributable to steric factors, as the electronic
character of the pendant moieties in these compounds is quite
diverse. Consistent with this observation is the inactivity of the
disubstituted analog, 18.
We next turned our attention to the substitution of the
methylene element of the benzyl group in 1. The introduction of
a methyl moiety was explored in the two enantiomers, 20 and
21. These compounds have the absolute stereochemistries (S)
and (R), respectively. Interestingly, it was found that 20 was inac-
tive at the KCa2.3 channel, but the (R)-isomer 21 was essentially
equipotent with the unsubstituted derivative 1. The immediate
higher homologs in this series were not examined, but a similar
dependence on appropriate stereochemistry might reasonably be
expected.
In an attempt to probe the steric constraints that might exist for
substituents at this position, we introduced the sterically more
demanding benzyl moiety, as seen in analog 24. This resulted in a
significant loss of activity relative to the original lead 1, which may
be interpreted as giving an indication of the steric limitations of
groups that can be introduced at this position. However, the
potential for further optimization of this series at this position is
apparent.
electrophysiology assays
b
Compound KCa2.3 Thallium flux %
K
IC50
Ca2.3 Thallium flux
K
(lM)
Ca2.3 EP IC50
a
inhibition at 30
lM
(lM)
1
2
3
4
5
6
7
8
113
62
75
50
74
87
113
91
80
71
96
30
46
43
62
25
23
25
43
1.1
1.60 ( 0.22)
19.1
14.6
>30
15.9
10.0
5.2
10.7
14.6
11.0
7.9
>30
>30
>30
15.4
>30
>30
>30
>30
IA
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
1.0
7.5
IA
0.35
0.000168
( 0.000136)
105
0.58 ( 0.05)
0.000064
( 0.000006)
Apamin
Lastly, we directed our attention to the use of heterocyclic
replacements of the phenyl group in 1. An initial investigation
using a series of pyridinyl analogs was unsuccessful. The results
a
See Note 35.
b
Values are means of three experiments, standard deviations are given in
parentheses. IA, inactive.

R. G. Gentles et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5694–5697
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Compound KCa2.1 Thallium flux
K
IC50
Ca2.2 Thallium flux
KCa2.3 Thallium flux
IC50 (lM)
a
a
a
IC50
(l
M)
(lM)
1
24
1.19
0.30
1.10
0.53
1.12
0.35
a
See Note 35.
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identified.
Acknowledgment
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