Initial SAR studies on apamin-displacing 2-aminothiazole blockers of calcium-activated small conductance potassium channels

Article abstract of DOI:10.1016/j.bmcl.2008.08.023

An initial SAR study on a series of apamin-displacing 2-aminothiazole K_Ca2 channel blockers is described. Potent inhibitors such as N-(4-methylpyridin-2-yl)-4-(pyridin-2-yl)thiazol-2-amine (13) are disclosed, and for select members of the series, the relationship between the observed activity in a thallium flux, a binding and a whole-cell electrophysiology assay is presented.

Full text of DOI:10.1016/j.bmcl.2008.08.023

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5316–5319
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Initial SAR studies on apamin-displacing 2-aminothiazole blockers
of calcium-activated small conductance potassium channels
*
Robert G. Gentles , Katherine Grant-Young, Shuanghua Hu, Yazhong Huang, 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 06492, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An initial SAR study on a series of apamin-displacing 2-aminothiazole KCa2 channel blockers is described.
Potent inhibitors such as N-(4-methylpyridin-2-yl)-4-(pyridin-2-yl)thiazol-2-amine (13) are disclosed,
and for select members of the series, the relationship between the observed activity in a thallium flux,
a binding and a whole-cell electrophysiology assay is presented.
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:
2-Aminothiazole
Small conductance potassium channel
Potassium channel blockers
Apamin
Small conductance calcium-activated potassium (KCa) channels
are expressed predominately in neuronal cells.1 They are voltage
insensitive and open in response to elevated concentrations of
intracellular calcium. Calcium ions bind to calmodulin, a protein
that is constitutively associated with the C-terminal domain of
the KCa channel. This protein modulates the state of the channel
in response to its calcium occupancy, whereby higher occupancy
is associated with opening of the channel.2–6 KCa channel opening
results in hyperpolarization of the plasma membrane7 with a con-
comitant reduction in neuronal excitability. The hyperpolarization
phenomenon (termed an afterhyperpolarization or AHP) can per-
sist for several hundreds of milliseconds,8 and significantly affects
the rate and pattern of neuronal firing.9,10
as well as schizophrenia.15 They may also prove helpful in treating
certain cognitive disorders.16,17
A limited number of compounds have been reported to be
active at KCa channels. The cyclic octapeptide apamin is a highly
selective and potent blocker with reported IC50’s between 30 pM
and 20 nM.1,18 The larger peptide Scyllatoxin is exceptionally
potent (Ki = 75 pM)19,20 and related derivatives display moderate
selectivity among the KCa channel subtypes. As shown in Figure 1
above, dequalinium (IC50 = 1.0 lM) was the first non-peptidic KCa
selective blocker identified.21 Subsequent development of this
chemotype led to the discovery of the cyclophane derivative, UCL
1684, a compound that displayed similar potency to apamin
(IC50 = 3.0 nM).22 More recently, quinoline appended diazepines23
(Ki = 140 nM) and isoquinoline analogs related to bicuculline and
N-methyl laudanosine24–28 have been reported, as well as the
non-apamin displacing 2-aminobenzimidazoles, such as NS8593
(IC50 = ꢀ500 nM).29 In this Letter, we describe a preliminary SAR
study on a series of aminothiazoles that display significant KCa
channel activity. In addition, we confirm the previously reported
activities of some of these analogs30 in alternative assay systems,
and significantly expand on reported selectivity and mechanism
of inhibition studies.
Three isoforms of KCa channels have been identified and cloned:
KCa2.1, KCa2.2 and KCa2.3. In situ hybridization, Northern blot, and
RT-PCR studies have demonstrated differential regional expression
levels of the isoforms in both rat and human brain.1,9,11,12
Correspondingly, there exists the possibility for site selective
modulation of neuronal excitability if suitable pan-KCa or sub-type
selective KCa channel modulators can be identified. Such agents
may have utility in the treatment of a number of pathological con-
ditions13 including epilepsy, depression, and Parkinson’s disease,14
The thiazole chemotype discussed here was identified from a
high-throughput screen that employed a thallium flux assay,33 in
which compounds were tested against a HEK 293 cell line recom-
binantly expressing specific KCa channel isoforms. Several chemo-
types were identified with one of the more interesting being the
2-aminothiazole derivative 1, as shown in Figure 2.
* Corresponding author.
E-mail address: robert.gentles@bms.com (R.G. Gentles).
Present address: Oblon Spivak, 1940 Duke Street, Alexandria, VA, USA.
Present address: Vanderbilt Institute of Chemical Biology and Department of
à
Pharmacology, Nashville, TN, USA.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.023

R. G. Gentles et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5316–5319
5317
Figure 1. Examples of reported, non-peptidic blockers of KCa channels.
Figure 2. Aminothiazoles evaluated for activity at KCa channels.
This compound displayed significant potency (89% inhibition at
M) in the original screen. It was resynthesized, and its IC50 in
the thallium assay was determined to be 0.5 M. In a subsequent
[MW = 254, CLogP = 2.71, HBD = 1, HBA = 3, ACD pKa (conjugate
acid) = 3.8] held excellent promise for its advancement and utility
in investigating CNS effects of KCa channel block.
30
l
l
whole-cell electrophysiology experiment, the IC50 of 1 was found
to be in close agreement with the value obtained in the thallium
assay, as shown in Table 1. In addition to the compound’s impres-
sive activity, we anticipated that its physicochemical properties
Subsequently, we investigated the activities of the series of re-
lated aminothiazoles shown in Figure 2. These compounds are
either commercially available, or can be synthesized from readily
accessible starting materials using the methodology depicted in

5318
R. G. Gentles et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5316–5319
Table 1
progressive replacement, as shown in analogs 7 and 8. Both com-
pounds displayed very limited activity in the thallium assay. In
additional studies, the importance of the pendant amino function-
ality in 1 was explored in analogs 6 and 9, in which the amino
group was either replaced by a methylene group or simply elimi-
nated. Again, both substitutions were associated with a significant
loss of activity at the KCa channel.
The above observations raised the possibility that the activity of
1 was due to the formation of a chelate. 2-Aminothiazoles are
known ligands in a number of chelate complexes,30,34 and the idea
that the chelate may be the active species is partly supported by
reports30 showing that thiazoles and the related iron or zinc che-
lates have similar activity in a KCa2.2 rubidium flux assay. In rela-
tion to the assay systems reported here, the most likely ion that
would participate in chelate formation is Mg2+, and several com-
plexes of this type have been reported.35 Based on the SAR pre-
sented above, the most probable structure of such a complex
would be I, as shown in Figure 4, although II and III cannot be
excluded.
With the key pharmacophoric elements now established, we
next attempted to introduce additional functionality into the N-
(pyridin-2-yl) group while retaining the pyridin-2-yl group at the
4-position of the aminothiazole. The introduction of a nitrogen
atom as shown in the pyrimidin-2-yl analog 10 was again associ-
ated with a loss of activity, and we tentatively attributed this to
a reduction in the basicity of the heterocycle (ACD pKa’s: 3.8 vs
2.4). Additional attempts to introduce steric probes at either C3
or C3 in combination with C5 as in analogs 11 and 12, resulted
in a significant loss of activity.
Activities of compounds 1–13 at the KCa2.3 channel in thallium flux and electro-
physiology assays
b
Compound KCa2.3 thallium flux %
KCa2.3 thallium
KCa2.3 EP IC50
M)
Ma
flux IC50
(lM)
(l
a
inhibition at 30
l
1
2
3
4
5
6
7
8
9
10
11
12
13
14
89
45
14
14
ND
15
9
18
16
24
8
0.543 ( 0.05)
ND
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
0.059 ( 0.017)
0.000168
( 0.000136)
1.3 ( 0.2)
0.29 ( 0.05)
29
98
Apamin
0.056 ( 0.015)
0.000064 ( 0.000006)
15
Dequalinium
2.0 ( 0.6)
a
See note 38. ND, not determined.
Values are means of three experiments; standard deviation is given in
b
parentheses.
Figure 3. The majority of the analogs were prepared using the mod-
ified Hantzsch31 procedure shown in reaction A. The 2,4-di-(pyri-
din-2-yl)thiazole analog
9 was synthesized using the same
methodology employing a thioamide, as shown in B. Lastly, the
4-(pyridin-2-yl)-2-(pyridin-2-ylmethyl)thiazole derivative 6 was
synthesized using the two-step procedure shown in reactions C32
and D.
In a related study focused on the effects of substitution at the C4
vector of the N-pyridinyl moiety, we found that the N-(4-methyl-
pyridin-2-yl) derivative 13 displayed significantly improved po-
tency relative to 1. Rather than modulating the basicity of the
heterocycle, we envisage that the methyl group interacts directly
with the channel. The activity of this compound was also deter-
mined in EP experiments, and good agreement between the assays
was observed (54 nM vs 56 nM). This level of activity at the KCa2.3
channel is unprecedented in a neutral small molecule, and 13
should prove useful for KCa2 channel functional studies.
Initially, we sought to investigate the importance of the relative
disposition of the pyridine nitrogens in 1. Thus, retaining the con-
nectivity of the 4-(pyridin-2-yl)-N-2-aminothiazole moiety, the
nitrogen atom of the N-pyridinyl group was transposed as shown
in structures 2 and 3 above.
As can be seen from Table 1, both analogs lost significant activ-
ity at the KCa2.3 channel. A similar exercise was conducted, in
which the heteroatom of the pyridinyl group at the 4-position of
the thiazole ring was varied in the context of the N-(pyridin-2-yl)
moiety, as shown in compounds 4 and 5. Again, a significant loss
of activity was observed. The importance of the nitrogens in the
two terminal pyridinyl groups was further demonstrated by their
To determine the site of action of 1 and 13, we employed a Scin-
tillation Proximity Assay (SPA) to assess their ability to displace
Figure 3. Synthetic methods used for the synthesis of the 2-aminothiazoles listed in Table 1.

R. G. Gentles et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5316–5319
5319
Figure 4. Potential structures of magnesium thiazole chelates.
Table 2
125I]-apamin displacement assay
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[
Compound
KCa2.2 thallium
KCa2.3 [125I]-apamin
a
flux IC50
(
lM)
displacement IC50 (l
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13
0.543 ( 0.05)
0.059 ( 0.017)
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a
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Table 3
Selectivity of compounds 1 and 13 against the KCa channel isoforms, KCa2.1, KCa2.2,
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and KCa3.1
Compound
KCa2.1 thallium
KCa2.2 thallium
KCa3.1 thallium
flux IC50
flux IC50
(l
M)a
flux IC50
(l
M)a
(l
M)a
1
13
0.043
0.004
0.123
0.011
na
10% Inh. at 30 lM
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a
Values are the means of three experiments; see note 38.
radio-labeled [125I]-apamin from the KCa2.3 channel.36,37 Both
compounds competed off the peptide with IC50’s as shown in
Table 2. These results suggest that both may function by blocking
the pore of the channel, as is observed with apamin (see Table 3).
To assess the KCa channel selectivity of the thiazole chemotype,
selected analogs were assessed in the thallium flux assay against
cell lines recombinantly expressing the KCa2.1, KCa2.2, and KCa3.1
channels. No significant selectivity was observed, although it was
noted that both 1 and 13 displayed essentially no activity at the
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K
Ca3.1 channel.
In conclusion, we present a series of N-(pyridine-2-yl)-4-(pyri-
dine-2-yl)-2-aminothiazoles that display excellent potency as
Ca2 blockers. In binding studies, these compounds appear to inter-
K
act with the channel at the apamin binding site, and presumably
exert their effect by mechanically blocking the pore of the channel.
We speculate that the active species may be the thiazole itself, or a
metal chelate in which the thiazole functions as a ligand.
28. Scuvee-Moreau, J.; Liegeois, J.-F.; Massotte, L.; Seutin, V. J. Pharmacol. Exp. Ther.
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Acknowledgment
32. Pascaud, X. B. L.; Malen, C.; Danree, B. J. Med. Chem. 1971, 14, 244.
33. Weaver, C. D.; Harden, D.; Dworetzky, S. I.; Robertson, B.; Knox, R. J. J. Biomol.
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The authors thank Dr. Carl Bergstrom for his help in reviewing
this Letter.
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standard deviations reported.
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