Halogen substituents on the aromatic moiety of the tetracaine scaffold improve potency of cyclic nucleotide-gated channel block

Article abstract of DOI:10.1016/j.bmcl.2011.08.092

A series of new tetracaine derivatives with substituents on the aromatic ring was prepared and evaluated for block of retinal rod cyclic nucleotide-gated (CNG) channels. Aromatic substitutions had little effect starting with the basic tetracaine scaffold,

Full text of DOI:10.1016/j.bmcl.2011.08.092

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6417–6419
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Halogen substituents on the aromatic moiety of the tetracaine scaffold
improve potency of cyclic nucleotide-gated channel block
Sarah R. Kirk a, , Adriana L. Andrade b, , Kenneth Melich b, Evan P. Jackson a, Elysia Cuellar a,
Jeffrey W. Karpen b,
⇑
a Department of Chemistry, Willamette University, 900 State Street, Salem, OR 97301, USA
b Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new tetracaine derivatives with substituents on the aromatic ring was prepared and evaluated
for block of retinal rod cyclic nucleotide-gated (CNG) channels. Aromatic substitutions had little effect
starting with the basic tetracaine scaffold, but electron-withdrawing substituents significantly improved
the blocking potency of an octyl-tail derivative of tetracaine. In particular, halogen substitutions at either
the 2- or 3-position on the ring resulted in compounds that were up to eight-fold more potent than the
parent octyl-tail derivative and up to 50-fold more potent than tetracaine.
Received 17 July 2011
Accepted 19 August 2011
Available online 27 August 2011
Keywords:
Ion channels
Local anesthetics
Pore blockers
cGMP
Ó 2011 Elsevier Ltd. All rights reserved.
Retinal rods
The cyclic nucleotide-gated (CNG) channels of retinal photore-
ceptors are non-selective cation conductances that regulate the
membrane potential in response to light.1,2 Unlike related
voltage-gated potassium channels, these channels are directly
activated by the binding of cGMP, and are minimally regulated by
voltage. In photoreceptors, photons trigger a signaling cascade that
leads to a decrease in cGMP levels and closure of channels. Lately,
CNG channels have been receiving increasing attention for their role
in some forms of retinitis pigmentosa (RP), a group of inherited ret-
inal degeneration disorders.3–9 In cGMP-phosphodiesterase (pde6b)
deficient mice, a well-studied model for RP, excessive CNG channel
activation is directly involved in rod photoreceptor degeneration.10
Unfortunately, there is a dearth in pharmacological tools available
to study the role of CNG channels in more depth and to develop
therapeutics to treat certain forms of RP.
We have conducted several studies on tetracaine derivatives to
develop a potent CNG channel blocker for research and clinical
use.11–14 Tetracaine [2-(dimethylamino)ethyl 4-(butylamino)ben-
zoate] (1) is a local anesthetic that blocks CNG channels with rela-
tively high affinity.15–17 Tetracaine binds to the channel from the
intracellular side, and work on both voltage-gated sodium chan-
nels and CNG channels indicates that it interacts with the selectiv-
ity filter and pore region.18–21 From our synthetic studies, we have
found that increasing the hydrophobic character of the tail portion
of tetracaine leads to improved CNG channel block.13 Charge accu-
mulation on the head group improves block as well, but in detri-
ment to membrane permeability.11 These findings replicate what
has been determined in sodium channel mutagenesis studies, that
tetracaine’s interaction with the pore is bimodal, involving an
electrostatic interaction with negatively charged residues in the
selectivity filter and hydrophobic interactions with residues in
the pore-lining helix.18–20,22,23
In this study we sought to examine the role of tetracaine’s aro-
matic moiety in CNG channel block. Charged polyamines lacking
aromatic groups block CNG channels with much lower affinity
and permeate through the channel.24 In sodium channels, the aro-
matic moiety is thought to interact with a hydrophobic binding site
in the pore of the channel, and is responsible for high-affinity
block.25 This suggested aromatic–aromatic interaction with a phen-
ylalanine residue remains to be evaluated in CNG channels. Alterna-
tively, a cation-p interaction may be responsible for high-affinity
block in CNG channels.26–29 To assess the effect of electronic modi-
fications to the aromatic ring, we synthesized a set of aromatic
substituted derivatives of tetracaine (1) as well as a higher affinity
octyl-tail derivative (2).13,14 Substituents ranged from electron-
donating (CH3, CH3O) to electron-withdrawing (F, Cl, Br, NO2)
located meta or ortho to the ester linkage. Scheme 1 outlines the
synthesis of the eleven novel derivatives. Intermediates 5a and 7a
were synthesized from commercially available 4-fluoro-3-nitro-
benzoic acid (3a) by a nucleophilic aromatic substitution with N-
butylamine or N-octylamine.30 Intermediates 5b,c and 7b,c,e–g
were obtained by reductive amination of commercially available
⇑
Corresponding author. Tel.: +1 503 494 7463; fax: +1 503 494 4352.
E-mail address: karpenj@ohsu.edu (J.W. Karpen).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.092

6418
S. R. Kirk et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6417–6419
Scheme 1. R1 and R2 are defined in Tables 1 and 2; Reagents and conditions: (i) N-butylamine/N-octylamine, H2O, NaHCO3, 3 h at 150 °C; (ii) butanal/octanal, a-picoline-
borane, MeOH, 1–24 h at rt; (iii) 1,10-carbonyldiimidazole, 1,2-dimethoxyethane (glyme), 2 h at 60 °C followed by 2-(dimethylamino)ethanol, NaH, 16–24 h at rt; (iv) N-
chlorosuccinimide, acetonitrile, 24 h, reflux.
derivatives of 4-aminobenzoic acid (4b,c,e–g) with butanal or oct-
anal. The free carboxylic acid of the resulting alkylated intermedi-
ates (5 and 7) was then activated by 1,10-carbonyldiimidazole and
reacted with 2-(dimethylamino)ethanol to yield target compounds
6a–c and 8a–c,e–g.14 Compounds 6d and 8d were made from 1 and
2 using N-chlorosuccinimide, via a synthesis adapted from Lazar
et al.31
as nitro at the same position (6a) did not improve block. The
methyl group in 6c had a slightly deleterious effect on block, again
appearing to be unrelated to the electron-donating character, as
compound 6b with a methoxy group had no significant change in
blocking potency. Overall, substitutions on the aromatic ring had
only subtle effects on apparent affinity for CNG channels in this
series.
Heteromeric retinal CNG channels comprised of CNGA1 and
CNGB1 subunits were expressed in Xenopus laevis oocytes, as de-
scribed previously.14,32 CNG channel currents were elicited with
2 mM cGMP at both positive (+50 mV) and negative (ꢀ50 mV)
membrane potentials. Electrophysiological methods as well as data
analysis have been previously described in more detail.14 Potency
of CNG channel block was assessed by fitting current amplitudes
to the equation for block at a single binding site (see Tables 1
and 2). Apparent KD values at +50 and ꢀ50 mV for tetracaine (1)
and derivatives 6a–d with various substituents at the 3-position
are summarized in Table 1. Compound 6d was the only compound
in this series to have a slightly higher apparent affinity for CNG
channels than compound 1 at both membrane potentials. This ap-
peared to be unrelated to the electron-withdrawing character of Cl,
In contrast to the butyl-tail derivatives, aromatic substituents in
the octyl-tail series produced more dramatic results. Our previous
study of ester linkage substitutions led us to conclude that com-
pounds 1 and 2 bind at different locations in the CNG channel
pore,14 and the current results bear this out. Apparent KD values
at +50 and ꢀ50 mV for the octyl-tail series (compounds 2, 8a–g)
are presented in Table 2. Like compound 6d, compound 8d with
a 3-Cl substituent had a higher affinity for CNG channels than com-
pound 2; however the effect was more pronounced with an
approximately six-fold improvement compared to the 1.6-fold
improvement in compound 6d. To better understand the nature
of the interaction of compound 8d, we examined the effects of dif-
ferent halogen substituents 3-F and 3-Br (compounds 8e and f),
and position by introducing a 2-Cl substituent ortho to the ester
(compound 8g). All derivatives with halogen substituents, like
compound 8d, had superior blocking potency compared to com-
pound 2. The differences between them were small, and the
patch-to-patch variability in block did not allow us to make fine
structure–activity distinctions. Nonetheless, the derivatives in this
group were up to eight-fold more potent than compound 2 and up
to 50-fold more potent than tetracaine (1). A derivative with a
strongly electron-donating 3-methoxy substituent (8b) blocked
with roughly the same apparent affinity as 2, while a derivative
with a 3-methyl group (8c), weakly donating, gave a marginal
improvement in apparent affinity. Interestingly, the strongly elec-
tron-withdrawing nitro derivative (8a) deviated from the general
trend, blocking with a significantly lower apparent affinity than
even 1. The rationale for this result may be two-fold: (a) the nitro
group is considerably larger than any of the other derivatives
tested (Table 2, rW), and (b) while the nitro group carries no net
charge, both the nitrogen and oxygen carry formal charge which
may negatively impact binding.
Table 1
Dissociation constants for aromatic-substituted tetracaine (1) derivatives
Compounda
R
KD(+50)
(
l
M)b KD(ꢀ50)
21.8 8.6
(
l
M)b
n
rp
rW (Å)
c
d
1
H
NO2
4.9 1.8
5.5 3.2
16
7
7
7
7
0.00 1.20
0.78 2.59
ꢀ0.27 1.56
ꢀ0.17 1.72
0.23 1.75
6a
6b
6c
6d
19.6 14.4
18.3 4.4
28.1 13.4
12.8 4.1
OMe 4.3 0.8
Me
Cl
7.8 3.1
3.0 1.0
a
Depicted as the predominant protonation state at pH 7.6.
b
KD(+50) and KD(ꢀ50) are the apparent dissociation constants at +50 and ꢀ50 mV
obtained from fits of the equation IþB=I ¼ KD=ðKD þ ½BꢁÞ, where the left side is
ꢀB
current in the presence of blocker divided by current in the absence of blocker, and
This study describes very promising compounds for CNG chan-
nel research and possibly clinical usage. Compounds 8d–g have a
high apparent affinity for CNG channels in the 100–200 nM range,
but without some of the drawbacks of APPA-tetracaine,11 a deriv-
ative in which two propylamino groups in tandem were added to
[B] is blocker concentration.
c
Hammett sigma constant at the para-position, which accounts for the net
inductive and resonance effects. Positive values denote an electron-withdrawing
substituent, and negative values an electron-donating substituent.
d
Van der Waals radius; radius of a sphere that encloses the substituent.

S. R. Kirk et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6417–6419
6419
Table 2
Dissociation constants for aromatic-substituted compound 2 derivatives
Compounda
R1
R2
KD(+50)
(l
M)b
KD(ꢀ50)
(l
M)b
n
rp
rW (Å)
c
d
2
H
H
H
H
H
H
H
H
Cl
0.80 0.50
8.0 5.8
1.72 1.38
12.6 7.3
3.0 3.4
1.41 0.84
0.38 0.16
0.75 0.86
0.24 0.10
0.22 0.13
10
4
5
5
4
5
4
6
0.00
0.78
ꢀ0.27
ꢀ0.17
0.23
0.06
0.23
0.23
1.20
2.59
1.56
1.72
1.75
1.47
1.85
1.75
8a
8b
8c
8d
8e
8f
NO2
OMe
Me
Cl
F
Br
1.25 1.23
0.50 0.42
0.14 0.04
0.20 0.10
0.14 0.03
0.10 0.09
8g
H
a
Depicted as the predominant protonation state at pH 7.6.
KD(+50) and KD(ꢀ50) are the apparent dissociation constants at +50 and ꢀ50 mV obtained from fits of the equation IþB=I ¼ KD=ðKD þ ½BꢁÞ, where the left side is current in
b
ꢀB
the presence of blocker divided by current in the absence of blocker, and [B] is blocker concentration.
c
Hammett sigma constant at the para-position, which accounts for the net inductive and resonance effects. Positive values denote an electron-withdrawing substituent,
and negative values an electron-donating substituent.
d
Van der Waals radius; radius of a sphere that encloses the substituent.
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This work was supported by NIH Grants EY009275 and
MH071625 (J.W.K.). and NSF Grant MRI0821781 (S.R.K.). We thank
the Bioanalytical Shared Resource at OHSU for mass spectrometry
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Supplementary data
Supplementary data (synthetic chemistry procedures and 1H
NMR, 13C NMR, and ESI-MS data of compounds 6a–d and 8a–g)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2011.08.092.
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