Block of cyclic nucleotide-gated channels by tetracaine derivatives: Role of apolar interactions at two distinct locations

Article abstract of DOI:10.1016/j.bmcl.2007.11.069

A series of new tetracaine derivatives was synthesized to explore the effects of hydrophobic character on blockade of cyclic nucleotide-gated (CNG) channels. Increasing the hydrophobicity at either of two positions on the tetracaine scaffold, the tertiary amine or the butyl tail, yields blockers with increased potency. However, shape also plays an important role. While gradual increases in length of the butyl tail lead to increased potency, substitution of the butyl tail with branched alkyl or cyclic groups is deleterious.

Full text of DOI:10.1016/j.bmcl.2007.11.069

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 645–649
Block of cyclic nucleotide-gated channels by tetracaine
derivatives: Role of apolar interactions at two distinct locations
Timothy Strassmaier,a, Sarah R. Kirk,b, Tapasree Banerjia and Jeffrey W. Karpena,*
aDepartment of Physiology and Pharmacology, Oregon Health & Science University, Portland, OR 97239, USA
bDepartment of Chemistry, Willamette University, Salem, OR 97301, USA
Received 28 August 2007; revised 16 November 2007; accepted 19 November 2007
Available online 22 November 2007
Abstract—A series of new tetracaine derivatives was synthesized to explore the effects of hydrophobic character on blockade of cyc-
lic nucleotide-gated (CNG) channels. Increasing the hydrophobicity at either of two positions on the tetracaine scaffold, the tertiary
amine or the butyl tail, yields blockers with increased potency. However, shape also plays an important role. While gradual increases
in length of the butyl tail lead to increased potency, substitution of the butyl tail with branched alkyl or cyclic groups is deleterious.
Ó 2007 Elsevier Ltd. All rights reserved.
Cyclic nucleotide-gated (CNG) channels are Ca2+-per-
meable non-selective cation channels gated by the bind-
ing of cyclic nucleotides. Despite their unique mode of
activation, sequence analysis places CNG channels in
the voltage-gated potassium channel superfamily. Like
their K+-selective cousins, CNG channels function as
tetramers with the four subunits arranged around a cen-
tral ion conduction pathway. CNG channels are poised
to act as integrators of two important second messenger
systems: they are both cyclic nucleotide sensors and
Ca2+ sources. Indeed, in visual and olfactory sensation,
CNG channels translate light- and odorant-induced
changes in cyclic nucleotide levels into electrical signals,
and Ca2+ influx through open channels contributes to
feedback regulation (reviewed in1,2). CNG channels
are found in a wide range of tissues including brain,
heart, kidney, testis, liver, lung, skeletal muscle, adre-
nal gland, pancreas, and colon, suggestive of a broad
physiological utility.3–5 Limited progress has been
made in deducing the functions of CNG channels be-
yond their established roles in vision and olfaction.
For example, CNG channels have been implicated in
synaptic modulation6 and enterotoxin-linked resistance
to colon cancer.7 However, such studies rely on multi-
ple pharmacological agents, all of which modulate
CNG channel activity, but none of which are highly
selective for CNG channels.8 Potent and selective
agents will be invaluable pharmacological tools for
uncovering the physiological roles of CNG channels
and may lead to the development of novel therapeutic
agents.
Tetracaine, a local anesthetic, is a non-selective block-
er of CNG channels, as well as voltage-activated Na+
and Ca2+ channels. However, targeted modification of
the tetracaine scaffold has the potential to enhance po-
tency and selectivity for block of CNG channels.9
Studies of block of Na+ channels suggest a bimodal
interaction with the channel in which negatively
charged residues in the selectivity filter of the channel
interact with the positively charged amine, while
hydrophobic pore-lining residues interact with the
apolar portion of the local anesthetic.10,11 Tetracaine
likely blocks CNG channels via a similar interaction
with the pore. In fact, mutagenesis implicates a key
negatively charged residue in the selectivity filter of
CNGA1 as an important determinant for tetracaine
binding.12 Furthermore, addition of one or two posi-
tive charges to the tertiary amine of tetracaine results
in dramatic increases in the voltage dependence of
block and apparent affinity for the channel.9,13 Previ-
ous work has revealed that the apolar butyl tail of tet-
racaine is essential for high affinity block.13 In this
study, we further explore the importance of hydropho-
bic interactions by increasing the hydrophobic content
at two positions on the tetracaine scaffold.
Keywords: Ion channels; Local anesthetics; Pore blockers; cGMP;
Retinal rods; Vision.
*
Corresponding author. Tel.: +1 503 494 7463; fax: +1 503 494
4352; e-mail: karpenj@ohsu.edu
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.069

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T. Strassmaier et al. / Bioorg. Med. Chem. Lett. 18 (2008) 645–649
Scheme 1. Reagents and conditions: (a) 0.05 mol % Pd(OAc)2, 0.05% L (Josiphos ligand), R1NH2 (1.2 equiv), LiHMDS (2.4 equiv), DME, 100 °C
(38–88%); (b) N,N0-carbonyldiimidazole, R22NCH2ðCH2ÞnOH, NaH, DME, 60 °C (51–93%).
Figure 1. Blockade of retinal rod CNG channels by tetracaine derivatives: representative current traces from two different patches (A and B) showing
outward currents at +40 mV. Channels were activated by 1 mM cGMP as indicated by the black bars, and tetracaine analogues were applied in
addition as indicated by the stippled bars. The dashed lines indicate the zero current levels.
A panel of tetracaine derivatives was prepared in order
to systematically increase the hydrophobicity of the apo-
lar tail and tertiary amine regions according to Scheme
1. To study the effects of both chain length and branch-
ing, the tail region was modified in a two-step process.
The reaction of 4-chlorobenzoic acid and an appropriate
alkylamine was catalyzed with a Josiphos ligand and
Pd(II) acetate to the corresponding 4-(alkylamino)ben-
zoic acid using a synthesis adapted from Hartwig
et al.14 The resulting substituted benzoic acid derivatives

T. Strassmaier et al. / Bioorg. Med. Chem. Lett. 18 (2008) 645–649
647
Table 1. Tetracaine analogue structures and block of heteromeric CNGA1/CNGB1 channels
Compounda
Kd(40) (lM)
No. of patches
b
O
C
NH+
O
1
2
3
6.7 1.7
3.8 1.2
1.3 1.2
16
5
O
C
N
H
NH+
O
O
N
H
C
NH+
O
6
N
H
O
C
NH+
O
4
5
17
16
3
4
10
10
N
H
O
C
NH+
O
N
H
O
C
NH+
O
6
7
20
3
8
7
N
H
O
C
N+
O
1.6 0.8
N
H
O
C
NH+
NH+
O
O
8
9
8.5 2.1
1.1 0.3
10
7
N
H
O
C
N
H
O
C
H+
O
O
O
O
O
N
10
11
12
13
14
5.0 1.3
7.1 3.1
5.6 1.6
1.3 0.4
1.3 0.3
9
6
8
8
5
N
H
O
C
NH+
N
H
O
C
H+
N
N
H
O
C
H+
N
N
H
O
C
NH+
N
H
a The compounds are depicted here in what is expected to be the predominant protonation state at pH 7.6.
b Kd(40) is the apparent dissociation constant at +40 mV calculated from the equation I+B/IꢀB = Kd(40)/{Kd(40) + [B]}, where the left side is current in
the presence of blocker divided by current in the absence of blocker, and [B] is blocker concentration.

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T. Strassmaier et al. / Bioorg. Med. Chem. Lett. 18 (2008) 645–649
were activated with N0,N0-carbonyldiimidazole and sub-
sequently esterified using 2-N,N-dimethylaminoethanol
to yield target compounds 2–6.15 Modifications of the
head region extended the length between the tertiary
amine and the ester and/or increased the hydrocarbon
chain lengths on the tertiary amine. The head region
was modified in a single synthetic step in most cases.
A series of tertiary alkylamines were introduced via
esterification of 4-(butylamino)benzoic acid according
to the conditions outlined in step 2 of Scheme 1 to give
desired products 8–14.16
of symmetrical tetraalkyl-ammonium derivatives were
tested for block of CNG channels, significant increases
in apparent affinity were observed with the addition of
each methylene group from tetramethyl to tetrapentyl.19
In a final set of experiments, the linker length between
the tertiary amine and the ester was increased for each
version of the tertiary amine. For the dimethyl version
(1), the linker length was increased to propyl and butyl
(10 and 11). An additional diethyl amino derivative
was generated with a propyl linker in place of the ethyl
(12). Versions of 9 with propyl and butyl linkers were
also synthesized (13 and 14). Surprisingly, increasing
the distance between the tertiary amine and the ester
had no statistically significant effect on block for any
of the derivatives (Table 1). Compounds 10 and 11 were
equipotent with 1; 12 was essentially equipotent with 8;
and 13 and 14 were equipotent with 9. A lack of any ef-
fect on block, even when the linker length was doubled,
suggests plasticity in the binding site for tetracaine.
Coexpression of CNGA1 and CNGB1 in Xenopus oo-
cytes yielded heteromeric retinal rod CNG channels.
Channel blockade was assessed by applying tetracaine
analogues to inside-out excised patches in the presence
of 1 mM cGMP to fully activate the channels, as de-
picted in Figure 1 (electrophysiological methods are de-
scribed in17). Lengthening the hydrocarbon tail from
butyl (1) to hexyl (2) and octyl (3) resulted in stepwise
increases in apparent affinity (Table 1). Compounds 2
and 3 were ꢁ2- and 5-fold more potent than tetracaine,
respectively. Further, there is an apparent preference for
straight alkyl chains at the apolar end of tetracaine. For
example, while the hexyl derivative (2) was ꢁ2-fold
more potent than tetracaine, the cyclohexyl derivative
(4) was 2.5-fold less potent. The benzyl and isobutyl
derivatives (5 and 6) displayed a similar decrease in
apparent affinity relative to tetracaine (Table 1). Com-
pound 3 was far less soluble in aqueous solution than
tetracaine, requiring up to 50% methanol for a 10 mM
stock concentration. To address the concern that the en-
hanced block might be due to increased partitioning into
the membrane, we synthesized a permanently charged
quaternary amine version (7) by reaction of 3 with bro-
moethane (Scheme 2). Reduced membrane partitioning
by 7 was expected based on studies of tetracaine binding
to model membranes that demonstrate less interaction
of the charged species with the membrane relative to
the neutral form.18 Compound 7 is readily soluble in
aqueous solution and displays equal potency for retinal
rod CNG channel blockade as 3, suggesting a direct
block of the channel by both compounds (Table 1).
This study highlights two distinct locations on the tet-
racaine scaffold where hydrophobic interactions are
important for CNG channel binding. Increasing the
hydrophobic character at the tertiary amine and the
butyl tail enhanced the ability to block retinal rod
CNG channels. We explored the possibility of com-
bining increased hydrophobicity at both the tertiary
amine and the butyl tail by synthesizing a derivative
with an octyl chain substitution for the butyl tail
and two butyl chains at the tertiary amine. Poor sol-
ubility of this analogue in aqueous solution, however,
precluded its analysis by patch clamp. The effect of
increasing the length of the hydrocarbon tail from bu-
tyl to octyl is relatively small compared to the magni-
tude of the loss of apparent affinity observed when the
butyl tail is removed. Taken together with the obser-
vation that branched derivatives such as 6 are disfa-
vored, these results suggest that the butyl tail of
tetracaine may be targeted to a specific binding pocket
in the pore. When used in conjunction with site-direc-
ted mutagenesis, the new tetracaine derivatives de-
scribed here can be utilized to identify points of
specific contact with the channel pore. Overall, the re-
sults indicate that apolar groups at two locations serve
to anchor blockers that otherwise reside in the aque-
ous and ion-binding regions of the pore.
Previously, we showed that appending a butyl chain to
the tertiary amine of tetracaine, thus increasing the
hydrophobic content and generating a quaternary
amine, resulted in ꢁ2-fold increase in apparent affin-
ity.13 Here, we have increased the length of both alkyl
chains at the tertiary amine from methyl (1) to ethyl
(8), and butyl (9). While 9 was ꢁ6-fold more potent than
tetracaine, 8 was essentially equipotent with tetracaine.
These results suggest that the tertiary amine of tetra-
caine may bind at a different position in the pore than
simple tetraalkyl-ammonium derivatives. When a series
Acknowledgments
This work was supported by a grant from the National
Eye Institute (EY009275 to J.W.K.). We thank the Bio-
analytical Shared Resource at OHSU for mass spec-
trometry data, and Michael Jackson for helpful
discussions.
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Scheme 2. Reagents and condition: (a) BrCH2CH3, toluene, reflux
(70%).

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