Dehydroabietic acid derivatives as a novel scaffold for large-conductance calcium-activated K+ channel openers

Article abstract of DOI:10.1016/j.bmcl.2003.08.072

We found that the dehydroabietic acid structure is a new scaffold for chemical modulators of large-conductance calcium-activated K⁺ channels (BK channels). Structure-activity relationship (SAR) studies of the dehydroabietic acid derivatives and related non-aromatic compounds such as pimaric acid revealed the importance of the carboxyl functionality and an appropriate hydrophobic moiety of the molecules for BK channel-opening ability.

Full text of DOI:10.1016/j.bmcl.2003.08.072

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3971–3974
Dehydroabietic Acid Derivatives as a Novel Scaffold for
Large-Conductance Calcium-Activated K+ Channel Openers
Tomohiko Ohwada,a,* Taro Nonomura,a Keisuke Maki,a Kazuho Sakamoto,b
Susumu Ohya,b Katsuhiko Murakib and Yuji Imaizumib
aGraduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
bGraduate School of Pharmaceutical Sciences, Nagaya City University, Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
Received 5August 2003; accepted 27 August 2003
Abstract—We found that the dehydroabietic acid structure is a new scaffold for chemical modulators of large-conductance calcium-
activated K+ channels (BK channels). Structure–activity relationship (SAR) studies of the dehydroabietic acid derivatives and
related non-aromatic compounds such as pimaric acid revealed the importance of the carboxyl functionality and an appropriate
hydrophobic moiety of the molecules for BK channel-opening ability.
# 2003 Elsevier Ltd. All rights reserved.
Large-conductance calcium-activated K+ channels (BK
channels) characteristically respond to two distinct
physiological stimuli, changes in membrane voltage and
in cytosolic Ca2+ concentration.1 The BK channels
gate-open in response to an increase in cytosolic Ca2+
concentration and membrane depolarization, resulting
in an increase of K+ efflux, which leads to rapid hyper-
polarization of the excitatory membrane and reduces
Ca2+ influx through voltage-dependent Ca2+ channels.
peripheral tissues offer rich opportunities for discover-
ing novel therapeutic agents based on BK channel
modulators, particularly openers.9,10 Chemical channel
openers are expected to quench excitatory events that
pathologically elevate the cytosolic Ca2+ and depolar-
ization of the cell membranes, and potentially have
specificity for tissues and organs of interest. Well-char-
acterized BK channel openers could be used to treat
acute stroke, epilepsy, and bladder overactivity.11 There
is some evidence for the utility of BK channel openers in
the treatment of asthma, hypertension, gastric hyper-
motility and psychoses.1
Recent cloning studies also revealed the presence of
multiple splice variants of the pore-forming a subunits 2
and multiple subtypes of b-subunits (b1, b2/b3 and
b4),3,4 which function to modulate the BK channel. The
BK channel is probably a tetramer of the a-subunit, and
in some tissues the tetramer is associated with b-sub-
units.5,6 Thus, there is significant diversity of BK chan-
nels, which may be specific to tissues and organs.2
Except for heart myocytes, the BK channels are expres-
sed in a number of organ systems, such as smooth
muscle cells, skeletal muscle cells, neuronal cells, and
secretary epithelial cells,7 and they have important
physiological roles in modulating muscle contraction
and neuronal activities,8 such as synaptic transmission.
Although many natural and artificial compounds have
been reported to show BK channel opening activity,
their activities are rather weak and nonspecific to BK
channels.12À15 Scaffolds for the molecular design of BK
channel openers are still limited. Here, we propose that
the dehydroabietic acid skeleton (3, Fig. 1) is a new
scaffold that can be used for the design of BK channel
openers.
Recently, we have found that pimaric acid (1, Fig. 1) and
related compounds have the potential to activate BK
channels at micromolar concentrations in the presence of
a physiological concentration of Ca2+ (pCa 6.5using a
Ca2+-EGTA buffer).16 First of all, we examined BK
channel activation by dehydroabietic acid 3, in compar-
ison with that of structurally related non-aromatic abi-
etic acid 2. We evaluated the effects of these compounds
These features and the widespread distribution of the
channel throughout the central nervous system and in
*Corresponding author. Fax: +81-3-5841-4735; e-mail: ohwada@
mol.f.u-tokyo.ac.jp
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.072

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T. Ohwada et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3971–3974
Figure 1. Scaffolds for BK channel modulators in this study.
on macroscopic membrane currents in human embry-
onic kidney (HEK 293) cell lines, which stably express
high levels of both the rat BK channel a-subunit and
b1-subunit.17 Dose-dependent activation of the BK
channels by 3 was detected in electrophysiological
assays, using patch-clamp recording in the whole-cell
configuration as shown in Figure 2.
Figure 3. Superimposed structures of pimaric acid (1, green), abietic
acid (2, brown) and dehydroabietic acid (3, blue).
This is consistent with the observation that the 12- and
14-monobromo derivatives (6b and 7b) both showed
weak BK channel-opening activity (Fig. 4).21 While the
activity of the 14-bromo isomer 7b is comparable to that
of the unsubstituted aromatic 3, the activity of the
Aromatic dehydroabietic acid 3 showed distinct gate-
opening activity while the nonaromatic abietic acid 2
showed a very weak activity. The activity of 3 is slightly
weaker than that of pimaric acid 1. The superposition of
compounds 1, 2 and 3 is shown in Figure 3, based on
structure minimization with the OPLS-AA force field.18
Ring A bearing the carboxylic acid group is well super-
imposed in these three compounds, while the space filling
in the ring C moiety seems different. The aromatic ring
(ring C) of dehydroabietic acid 3 was thus selected for
structural modification, since this is synthetically feasible.
Analogues wherein the phenyl ring was haloganated were
studied (Scheme 1).19 The BK channel-opening activities
are shown in Figure 4. 12,14-Dichlorodehydroabietic
acid 4 showed a remarkable increase of the activity,
while the monochloro derivative [a mixture of 12- (5a)
and 14-chloro (5b) isomers] showed comparable activity
to that of unsubstituted 3.20
Scheme 1. Synthesis of halogenated dehydroabietic acid derivatives.
Figure 2. The concentration–response relationship for the compounds
1, 2, and 3, obtained from electrophysiological assays (whole-cell
patch clamp) using HEKBKab1. The relative amplitude of peak out-
ward current at +30 mV (Icompound/Icontrol) was determined as that in
the presence of test compounds (Icompound) versus that in the absence
(Icontrol). The amplitude in their absence was taken as the unit (dotted
line).
Figure 4. The effect of aromatic halogenation and reduction of the
carboxylic acid group. The concentration–response relationships for
compounds 3–8 obtained from electrophysiological assays (whole-cell
patch clamp) using HEKBKab1. See the caption to Figure 2.

T. Ohwada et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3971–3974
3973
12-bromo isomer 6b is slightly stronger than that of 3.
Thus, the order of activity seems to be 4>6b>3>7b in
this experiment.
Furthermore, to investigate the significance of the car-
boxylic acid functionality at the C4 position of ring A,
the BK channel-opening activities of the hydroxymethyl
derivatives (8a–c), prepared by hydride reduction of the
corresponding carboxylates (4, 6a and 7a), were studied
(Scheme 2). The channel-opening activity was appar-
ently lost in the hydroxymethyl derivatives 8a–c (Fig. 4).
This is in a sharp contrast to the case of pimaric acid 1:
the corresponding alcohol derived from the reduction of
the carboxylic acid of 1 retained BK channel-opening
activity.16 The reason for this difference in the behavior
of the alcohol functionality is not known at present. The
corresponding ester analogues were also inactive (data
not shown). Thus, the carboxylic acid functionality at
the C4 position of the dehydroabietic acid skeleton is
crucial for the BK channel-opening activity.
Figure 5. The effect of the introduction of the oxygen-containing
functional groups. The concentration–response relationship for the
compounds obtained from electrophysiological assays (whole-cell
patch clamp) using HEKBKab1. See the caption to Figure 2.
We next focused on the modification of ring B of the
dehydroabietic acid skeleton by introduction of an oxy-
gen functionality (Scheme 3). Benzylic oxidation of the
dichloro derivative 4 with CrO3 in acetic acid afforded
the 7-keto derivative 9, and hydride reduction gave the
7-alcohols (10 and 11).22 The 7b-alcohol 11 showed sig-
nificantly lower activity, as compared with the parent
compound 4 (Fig. 5). On the other hand, the a-isomer
of the alcohol 10 exhibited similar activity to that of
dehalogenated 3. The O-methylation (12) and O-acet-
ylation (13) of the a-isomer of the alcohol 10 result in
slightly increased activities as compared with the origi-
nal alcohol 10 (Scheme 3 and Fig. 5). This result exclu-
ded a significant role of hydrogen bonding of the
a-hydroxyl group, either as a hydrogen-bonding accep-
tor or a donor.
benzene ring (ring C) of 3,23 and the carboxylic acid
functionality of ring A are critical for BK channel-
opening activity. However, the molecular mechanisms
involved in the activity remain to be established.
Conclusion
The present SAR study showed that the dehydroabietic
acid structure is a new scaffold for BK channel openers.
The carboxylic acid functionality and the hydro-
phobicity of ring C (the dichlorobenzene moiety in the
case of dehydroabietic acid) and ring B (i.e., unsub-
stitution of a hydrogen-bonding moiety) caused a dra-
matic increase of the channel-opening activity. Because
the compound 4 appeared to be one of the most potent
BK channel openers ever reported, the dehydroabietic
acid scaffold will be a fundamental basis for the design
of new BK channel modulators.
These observations indicate that an appropriate hydro-
phobic region (i.e., due to the halogen atoms) in the
Acknowledgements
Scheme 2. Reduction of the carboxylate and ester to a hydroxymethyl
group at the C4 position.
We are grateful to Harima Chemicals Inc., Japan, for
kindly providing us with the starting material for the
present synthetic study.
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