5200
K. J. McClure et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5197–5201
a 150
H
H2N
SNI ED50=3mg/kg
ECG EC20=1.3 mg/kg
N
N
a
N
125
100
75
50
25
0
N
OMe O
N
12f
Scheme 2. Reagents and conditions: (a) 2-Methoxybenzoyl chloride, pyridine,
50 °C, 24 h, 72%.
improved metabolic stability profile as measured by human liver
microsomes, having a t1/2 of 235 min versus a t1/2 of 22 min for
12b. Unfortunately, this structural modification did lead to a loss
in HCN1 potency relative to 12b and 12e. In the case of the cyclo-
hexane analogs, replacement of the substituted piperazine group
with a simple piperidine ring (example 12n) led to a complete loss
of HCN1 potency, paralleling similar results in the indane series.
Not all substitution on the piperazine ring was tolerated. Attach-
ment of a phenyl ring to the terminal nitrogen led to a complete
loss of HCN1 potency (example 12p), further supporting the theory
that the presence of a basic nitrogen in this region is important,
and contributes to the potency of the series.
Other cores besides indane and cyclohexane were also investi-
gated (Table 3). With the exception of compounds 13e (compound
obtained from original screening deck) and 13f, all analogs were
prepared using the route outlined in Scheme 1. Compound 13f
was prepared by acylation of the commercially available
N-methyl-N0-aminoethyl piperazine (Scheme 2).
1
10
ZD7288 (mg/kg i.p.)
150
125
100
75
b
SNI ED50=6mg/kg
ECG EC20=25 mg/kg
50
25
As can be seen from the data in Table 3, all other cores investi-
gated offered no advantage over the cyclohexane core. The 6,
6-bicyclic system (example 13a) displayed similar potency to the
indane core, while the cyclopentane core (example 13b) resulted
in about a half log loss in activity relative to its cyclohexane coun-
terpart (example 12b, Table 2). The tetrahydropyran derivative
(example 13c) and phenyl piperidine analog (example 13d) main-
tained some HCN1 potency, but were both significantly less active
than their corresponding cyclohexane analogs (example 12d and
12e, Table 2). Removing some of the conformational constraint in
the molecule by converting the quaternary carbon atom to a ter-
tiary carbon (Example 13e) had a negative impact on HCN1/
HCN4 potency. Decreasing the conformational restriction between
the benzamide and piperazine groups further by converting the
quaternary carbon to a secondary carbon (13f), led to a complete
loss in HCN1 potency, highlighting the importance of the spatial
relationship of these two groups.
Based upon these results, compound 12m was chosen for fur-
ther characterization. Inhibition of heterologous human HCN2
and HCN3 were measured in the functional assay13 (pIC50 = 5.3
and 5.5, respectively). Experiments were performed in vivo to test
the effectiveness of this novel series of HCN1 blockers to alleviate
nerve injury-induced tactile allodynia in a spared nerve injury
(SNI) model14 and to evaluate their effects on heart rate, using
ZD-7288 for comparison. Figure 2a demonstrates the analgesic effi-
cacy of ZD-7288 (ED50 = 3 mg/kg) and the complete lack of thera-
peutic window with respect to heart rate reduction; significant
0
1
10
100
11m (mg/kg i.p.)
Figure 2. Comparison of the effects ZD-7288 (a) and 12m (b) on tactile allodynia
and bradycardia in mouse.
HCN4 displayed by some of these compounds in a VIPR assay was
substantiated by a similar or slightly greater separation in analge-
sic versus bradycardic potencies. Furthermore, the selectivity pro-
file of these compounds has allowed for testing in a SNI model that
clearly showed a contribution of the HCN1 channel to nerve in-
duced allodynia. Compound 12m represents a novel tool com-
pound for further exploration of the role of HCN subtypes in vivo.
Supplementary data
Supplementary data associated with this article can be found, in
References and notes
bradycardia is evident below the analgesic threshold (ED20
1.3 mg/kg). In contrast, the advantage of compound 12m (Table 2),
is clearly seen in Fig. 2b, where analgesic effects (ED50 = 6 mg/kg)
are substantially separated from bradycardic effects (ED20
25 mg/kg). Thus, the selective HCN1 blocker, 12m provides a ther-
apeutic window for the treatment of neuropathic pain, whereas the
nonselective blocker, ZD-7288, cannot be administered in a con-
centration that would alleviate neuropathic pain without affecting
heart rate.
=
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In summary, we disclose a series of novel and selective HCN1
blockers. The 10-fold or higher in vitro selectivity for HCN1 versus