Novel BK channel openers containing dehydroabietic acid skeleton: Structure-activity relationship for peripheral substituents on ring C

Article abstract of DOI:10.1016/j.bmcl.2008.08.078

A series of dehydroabietic acid (DHAA, 2) derivatives was synthesized and evaluated as BK channel openers in an assay system of CHO-K1 cells expressing hBKα channels. Systematic modifications of the peripheral functionality of ring C of DHAA showed that t

Full text of DOI:10.1016/j.bmcl.2008.08.078

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5201–5205
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel BK channel openers containing dehydroabietic acid skeleton:
Structure–activity relationship for peripheral substituents on ring C
Yong-Mei Cui a, Eriko Yasutomi a, Yuko Otani a, Takashi Yoshinaga b, Katsutoshi Ido b,
Kohei Sawada b, Tomohiko Ohwada a,
*
a Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
b Tsukuba Research Laboratories, Eisai Co. Ltd, 5-1-3 Tokodai, Tukuba, Ibaraki 300-2635, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dehydroabietic acid (DHAA, 2) derivatives was synthesized and evaluated as BK channel open-
Received 15 July 2008
Revised 22 August 2008
Accepted 23 August 2008
Available online 28 August 2008
ers in an assay system of CHO-K1 cells expressing hBKa channels. Systematic modifications of the periph-
eral functionality of ring C of DHAA showed that the introduction of a nitro or (thio)urea group in ring C
greatly enhanced the BK channel-opening activity.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
BK channels
Openers
Dehydroabietic acid
SAR studies
Thiourea
Potassium (K+) channels are a widely distributed and struc-
turally diverse family of transmembrane proteins that have
emerged as important targets for therapeutic intervention in a
number of diseases.1–3 The K+ channel superfamily is divided
into a number of subfamilies based on molecular structure and
function. An important subfamily is a group of calcium-activated
potassium (Kca) channels that share a dependence on intracellu-
lar calcium ion concentration for initiating channel opening, and
these channels can be further categorized according to their bio-
physical properties.4 For example, based on the single channel
conductance, the Kca channels are classified as BK (maxi-K), IK,
and SK channels referring, respectively, to big (100–300 picosie-
mens (pS)), intermediate (25–100 pS), and small conductance
(2–25 pS) Kca channels. These channels vary in pharmacology,
distribution, and function, as well as sensitivity to voltage and
Ca2+ concentration. BK channels are of particular interest because
of their large channel conductance and their expression in a
range of excitable cell types, including neurons and smooth mus-
cle cells.5
smooth muscle, b4: brain).8 This diversity of BK channels and
the widespread distribution of these channels in tissues ranging
from central nervous system to vascular smooth muscle offer
important opportunities to develop new therapeutic agents. BK
channel openers have emerged as potential targets of drug treat-
ment for post-stroke neuroprotection, urinary incontinence, asth-
ma, and hyperactivity of smooth muscles.9,10
Recently, we have found that pimaric acid (1) and dehydroa-
bietic acid (DHAA, 2) exhibit BK channel-opening activities,
while the structurally related abietic acid (3) has weak activity
(Chart 1).11,12 Various chemical modifications have been carried
C
H
H
A
B
H
H
H
CO H
CO H
2
COOH
2
Pimaric acid (1) DHAA (2)
Abietic acid (3)
BK channels consist of channel-forming
a
-subunits and acces-
Cl
12
12
R
sory b-subunits arranged in tetramers.6 Recent cloning studies
13
C
C
14
have revealed the presence of multiple splice variants of
a-sub-
Cl
14
units and multiple subtypes of b-subunits (b1, b2/b3, and b4),7
which may be specific to tissues, organs and functions (e.g., b1:
A
B
A
B
H
H
CO H
2
CO H
2
diCl-DHAA (4)
5
* Corresponding author. Tel.: +81 3 5841 4730; fax: +81 3 5841 4735.
E-mail address: ohwada@mol.f.u-tokyo.ac.jp (T. Ohwada).
Chart 1.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.078

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Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5201–5205
Table 1
Structure and BK
O
a-opening properties of 12-alkoxy DHAA derivatives
O
O
1
OR
b
a
H
H
H
CO2Me
CO2Me
CO2Me
2c
2a
2b
H
OR1
OR1
CO H
2
OH
Compound
R1
Ionic current in the presence
of test compound (30 M) as
l
c
d
e
% of control current (n = 8)
H
H
H
Buffer
4
5a
—
—
H
Me
103.8 3.3
180.4 11.9
83.0 3.8
85.9 9.2
CO2R2
CO2H
CO2Me
2d R2=Me
5a R2=H
e
6b-j
5b-j
5b
Scheme 1. Reagents and conditions: (a) AcCl, AlCl3, CS2, reflux, 80%; (b) m-CPBA,
PTSA (cat.), ClCH2CH2Cl, reflux, 69%; (c) NaHCO3, MeOH–H2O, 80%; (d) R1Br, NaH,
DMF, 0 °C–rt, 81–98%; (e) KOBut, DMSO, rt, 52–95% (except 5i, 11%).
5c
118.3 12.9
125.2 26.6
5d
5e
5f
78.7 2.2
81.8 3.3
89.5 6.7
out on DHAA, and the introduction of halogen atoms on the phe-
nyl ring, to afford 12,14-dichlorodehydroabietic acid (diCl-DHAA,
4), markedly increased the BK channel-opening activity. Also, we
found that the carboxylic acid functionality of ring A is critical
for BK channel-opening activity. All these BK channel openers
5g
are assumed to interact with the
a
-subunit,13 and the dehydro-
5h
5i
91.6 4.3
83.7 5.2
93.2 8.4
abietic acid core thus provides a template from which more po-
tent derivatives might be obtained by suitable substitution. In
this letter, we survey the BK channel-opening properties of a
series of peripherally substituted DHAA derivatives of general
structure 5, and describe some of the fundamental structure–
function relationships. The BK channel-modulating activities of
all the target compounds in this study were evaluated by means
of automated planar array patch clamp recording using the 64-
well Population Patch Clamp (PPC) technique14,15 with CHO-K1
Cl
5j
cells expressing hBK
by applying a step pulse to +100 mV from the holding potential
a channels. The BK channel was activated
of test compounds were prepared in DMSO at a concentration
of 10 mM and diluted in buffer solution to give the desired final
concentration (the final DMSO concentration is less than 0.4%(v/
v)). Since diCl-DHAA (4) has already been shown to open BK
channels,12,13 it was used as a positive reference agent in this
study.
of À90 mV. The current amplitude in the presence of compounds
(30 lM) was expressed as percent of the control recorded in the
absence of a drug. Each value represents the average of data ob-
tained in at least eight separate measurements. Stock solutions
NH2
NO2
a
NO2
+ 7b
b
+
H
H
H
H
CO2Me
CO2R
CO2Me
CO2Me
2a
7c R=Me
7b
7a
c
5k R=H
NO2
NH2
NH2
d
c
H
H
H
CO2Me
CO2Me
CO2H
7b
7d
5l
NH2
NH2
NO2
NH2
e
d
NH2
NO2
c
H
H
H
H
CO2H
CO2Me
CO2H
CO2R
5m R=H
7e R=Me
5n
2
f
7f
Scheme 2. Reagents and conditions: (a) 88% fuming HNO3, Ac2O, 7a:7b = 3:2; (b) 10% Pd-C, MeOH–THF; (c) KOH, 18-crown-6, MeOH, reflux, 52–66%; (d) Sn powder, concd
HCl, MeOH, reflux, 25–78%; (e) concd H2SO4, fuming HNO3, 0 °C–rt, 32%; (f) TMSCHN2, MeOH–toluene, rt, 81%.

Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5201–5205
5203
Table 2
Structure and BK
On the basis of the preliminary structure–activity relationships
of DHAA previously reported,12 we introduced a number of hydro-
phobic substituents at various positions on the phenyl ring (ring C)
of 2. Initially, we synthesized a series of 12-alkoxy derivatives 5a–j
as depicted in Scheme 1. Friedel–Crafts acetylation of methyl dehy-
droabietate 2a in refluxing CS2 gave the C12 acetyl derivative 2b in
80% yield, followed by Baeyer–Villiger oxidation to generate 2c,
then hydrolysis of the acetate group to afford phenol 2d as a key
intermediate.16 Alkylation of 2d with various alkyl or aryl halides
and subsequent hydrolysis of the ester using KOBut in DMSO at
rt afforded 12-alkoxy-substituted compounds 5b–j. As shown in
Table 1, 5a and all 12-alkoxy derivatives 5b–j showed no signifi-
cant channel-opening activity, except for compounds 5c and 5d,
which were only marginally active. Some of these compounds
(e.g., 5e) showed weak blocking activity. These results indicate that
an electron-donating OR substituent at the 12-position is unfavor-
able for the channel-opening activity.
We next modified ring C of DHAA by introduction of a nitro
or amino functionality as shown in Scheme 2. 12-Aminodehy-
droabietic acid 5k was obtained by mononitration of 2a, fol-
lowed by catalytic hydrogenation, then basic hydrolysis of the
ester with KOH and 18-crown-6 in methanol. Catalytic hydroge-
nation of the 14-nitro derivative (7b) under the same conditions
failed, probably because of the greater constraint at the C-14 po-
sition, but tin reduction of 7b under acidic conditions provided
the 14-amino derivative 7d, which was subjected to basic hydro-
lysis to afford 5l. Nitration of 2 with fuming nitric acid and con-
centrated sulfuric acid gave 12,14-dinitrodehydroabietic acid 5m.
Subsequent methylation and reduction of 7e with Sn–HCl, fol-
lowed by basic hydrolysis of the ester with KOH and 18-
crown-6, afforded 12,14-diaminodehydroabietic acid 5n. As
shown in Table 2, 12,14-dinitrodehydroabietic acid 5m showed
slightly stronger channel-opening activity than that of 4 (diCl-
DHAA), while the diamino derivative 5n was inactive. The 12-
and 14-monoamino derivatives (5k and 5l) also showed no
channel-opening activity. This is consistent with the previous
observation that an appropriate hydrophobic region in ring C
(i.e., the nitro functionality in the present case) is critical for
BK channel-opening activity.
a
-opening properties of dehydroabietate derivatives
R
O
NH
H
CO H
2
Compound
R
Ionic current in the presence
of test compound (30 M)
as % of control current (n = 8)
l
Buffer
4
5k
—
—
—
—
—
—
Me
103.8 3.3
180.4 11.9
88.9 3.3
5l
88.2 4.9
5m
5n
8a
199.6 16.6
110.1 4.4
96.1 4.2
8b
8c
8d
8e
8f
90.2 1.6
92.4 1.7
129.9 25.3
91.6 2.4
95.9 3.1
N
8g
8h
8i
107.1 2.5
102.8 5.5
90.0 2.4
8j
90.9 1.8
86.5 6.2
MeO
8k
Because of the low reactivity of compounds with 14-position
substituents due to steric hindrance and the importance of the
hydrophobicity of the ring C region to the BK opening activity,
we mainly focused on modification of the amino group at the
12-position (5k). Firstly, we synthesized a series of 12-N-acyl
derivatives 8a–r (Table 2). As shown in Scheme 3, direct acyla-
tion of the common intermediate 7c with various acyl chlorides
under basic conditions gave 9a–f, 9h–r, and 10, and then basic
hydrolysis of the ester using KOBut in DMSO afforded 8a–f and
8h–r. Compound 8g was prepared via aminolysis of 10 and sub-
sequent basic hydrolysis of the ester. From the results presented
in Table 2, most of the 18 compounds 8a–r tested showed no
distinct BK channel-opening activity. That is, all the alkyl acyla-
mino derivatives (8a–f, 8h) were inactive except compound 8d,
which showed weak activity. Also, the introduction of an amino
group (compound 8g), which improves aqueous solubility, had
little effect on the activity. Among the aryl-group-containing
derivatives (8i–r), the activity is quite sensitive to the location
and properties of the aromatic substituents. In the series of com-
pounds 8i, 8l, 8m, 8n, and 8q, para-substitution of a trifluoro-
methyl group on the aromatic ring (8q) resulted in increases
in the channel current, while lack of substitution (8i) or substi-
tution with an electron-donating group, such as a methoxy
group (8l) resulted in inactivity. The chloro and fluoro deriva-
tives (compounds 8m and 8n) were also inactive. Among the
three regioisomers 8o–q of the trifluoromethyl substituent, the
para isomer 8q was more potent than the ortho and meta CF3-
8l
94.1 5.9
83.4 2.1
107.9 6.7
59.4 2.5
92.5 2.3
128.4 9.9
81.8 4.9
OMe
F
8m
8n
8o
8p
8q
8r
Cl
F3C
CF3
CF3
CF
3
CF
3

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Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5201–5205
Table 3
Structure and BK
substituted isomers (8o, 8p). Similarly, the 3,5-bis-CF3 derivative
(8r) was also inactive. Compound 8o rather acted as a BK chan-
nel blocker (ion current ratio = 59%).
a
-opening properties of 12-(thio)urea dehydroabietate derivatives
RHN
X
HN
Finally, based on the NS series of BK channel openers, such as
NS161917and NS1608,18 we introduced a urea or thiourea group
at the 12-position and synthesized compounds 11a–p (Table 3).
The two routes that were utilized to synthesize the target com-
pounds 11a–p are summarized in Scheme 4. In Route 1, conden-
sation of 7c with the corresponding isocyanates yielded urea
intermediates 12a–b and 12d–g. Hydrolysis of the ester of
12a–b, 12d–g with KOBut in DMSO gave the desired acids
11a–b and 11d–g. In Route 2, hydrolysis of the ester of 7c to
12-aminodehydroabietic acid 5k, followed by condensation with
various iso(thio)cyanates afforded the desired compounds 11c
and 11h–p.
From the results presented in Table 3, these (thio)urea deriva-
tives (11a–p) generally showed higher activity than the corre-
sponding N-acyl compounds (8a–r, Table 2). Compound 11b,
which is unsubstituted on the phenyl ring, was inactive. It appears
that introduction of an electron-withdrawing substituent such as
CF3 enhances channel-opening activity. The para-CF3 isomer 11h,
showing an activity similar to that of the 3,5-bis-CF3 isomer 11i,
was found to be more potent than both the ortho and meta CF3 iso-
mers (11c–d). Introduction of a Cl atom resulted in a dramatic in-
crease in BK channel-opening activity (11e vs 11d, 11m vs 11d).
Also, the chloro analogues were more efficacious than the corre-
sponding CF3 compounds (11j vs 11d, 11k vs 11i). However, intro-
duction of a OMe group resulted in a substantial loss of activity
(11l vs 11j). In addition, both of the thiourea derivatives 11o–p
were more active than the corresponding urea compounds 11d
and 11i. Further, compound 11p increased the ionic current by
240% of control current. This may suggest that the simple replace-
ment of the O atom of the urea functionality with its isosteric S
atom significantly affects the interaction of the compounds with
BK channels. Taking these results together, we conclude that the
presence of both a urea or thiourea element and an electron-with-
drawing aryl group is critical for the appearance of BK channel-
opening activity.
H
CO H
2
Compound
R
X
Ionic current in the presence
of test compound (30 M)
l
as % of control current (n = 8)
Buffer
—
—
—
—
103.8 3.3
4
180.4 11.9
89.9 4.7
11a
11b
O
O
72.7 4.5
F C
3
11c
11d
11e
11f
11g
11h
11i
O
O
O
O
O
O
O
O
114.2 2.7
CF3
99.9 5.9
Cl
127.9 3.9
156.3 3.0
138.6 5.0
138.3 4.7
132.1 3.6
135.7 3.6
CF3
Cl
F
F
F
CF3
CF
3
In summary, we carried out systematic modifications of the
peripheral functionality of ring C of DHAA, and the resulting struc-
ture–activity data indicated that the introduction of a nitro or
(thio)urea functionality in ring C greatly enhances BK channel-
opening activity. Further modifications of these and other lead
structures with the aim of improving the potency as well as the
specificity in vitro and the efficacy in vivo are in progress.
CF
Cl
3
11j
Cl
Cl
11k
O
151.2 4.7
R
R
O
O
NH
2
MeO
NH
NH
11l
O
O
94.4 1.0
a
b
Cl
H
CF
CO Me
2
H
3
H
CO H
2
CO Me
2
11m
167.9 5.8
7c
8a-f, 8h-r
Cl
9a-f, 9h-r
c
N
Cl
N
O
O
O
NH
NH
NH
11n
11o
11p
O
S
107.4 1.2
142.7 8.0
240.2 15.6
OMe
d
CF
b
3
H
H
H
CO H
2
CO Me
2
CO Me
2
CF
3
8g
9g
10
S
Scheme 3. Reagents and conditions: (a) appropriate acyl chloride (RCOCl), Et3N,
CH2Cl2, rt, 46–100%; (b) KOBut, DMSO, rt, 17–94%; (c) chloroacetyl chloride, Et3N,
CH2Cl2, rt, 80%; (d) piperidine, THF, rt, 89%.
CF
3

Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5201–5205
5205
Route 1
valuable discussions and encouragement in the late stage of this
program.
RHN
HN
RHN
HN
O
NH
O
2
References and notes
a
b
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H
CO Me
2
H
H
CO Me
2
CO H
2
7c
12a-b, 12d-g
11a-b, 11d-g
Route 2
RHN
HN
X
NH
NH
2
2
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c
d
H
H
H
CO Me
CO H
2
2
CO H
2
7c
5k
11c, 11h-n X=O
11o-p X=S
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Scheme 4. Reagents and conditions: (a) RNCO, CH2Cl2, rt, 83–100%; (b) KOBut,
DMSO, rt, 13–42%; (c) KOBut, DMSO, rt, 68%; (d) RNCX (X = O, S), CH2Cl2, rt, 28–97%.
Acknowledgments
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This work was supported by a Grant-in-Aid for Scientific Re-
search from the Ministry of Education, Science, Sports, and Cul-
ture of Japan. Y.-M.C. is grateful for a postdoctoral fellowship for
a foreign researcher from the Japan Society for the Promotion of
Science, and also thanks Prof. Masakatsu Shibasaki, Graduate
School of Pharmaceutical Sciences, The University of Tokyo, for
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