Design, synthesis and characterization of podocarpate derivatives as openers of BK channels

Article abstract of DOI:10.1016/j.bmcl.2008.08.081

We found that the podocarpic acid structure provides a new scaffold for chemical modulators of large-conductance calcium-activated K⁺ channels (BK channels). Structure-activity analysis indicates the importance of both the arrangement (i.e., location and orientation) of the carboxylic acid functionality of ring A and the hydrophobic region of ring C for expression of BK channel-opening activity.

Full text of DOI:10.1016/j.bmcl.2008.08.081

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5197–5200
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and characterization of podocarpate derivatives
as openers of BK channels
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:
We found that the podocarpic acid structure provides a new scaffold for chemical modulators of large-
conductance calcium-activated K+ channels (BK channels). Structure–activity analysis indicates the
importance of both the arrangement (i.e., location and orientation) of the carboxylic acid functionality
of ring A and the hydrophobic region of ring C for expression of BK channel-opening activity.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 15 July 2008
Revised 22 August 2008
Accepted 23 August 2008
Available online 28 August 2008
Keywords:
BK channels
Channel openers
Podocarpic acid
Large-conductance calcium-activated K+ channels (also called
maxi-K or BK channels) characteristically respond to two distinct
physiological stimuli, that is, changes in membrane voltage and
in cytosolic Ca2+ concentration, and may serve as a negative feed-
back pathway to control ionic homeostasis and cell excitability.1
BK channel opener. Moreover, our recent study15 revealed that
chemical modification of abietic acid (3), an inactive compound,
to dehydroabietic acid (4a), a resin acid derivative, resulted in
the appearance of BK channel-opening activity, and further chem-
ical modification to 12,14-dichlorodehydroabietic acid (diCl-DHAA,
4b) afforded a potent and selective BK channel opener applicable
from outside of the cell membrane. We further found that
diCl-DHAA (4b) is among the most potent synthetic activators of
BK channels, changing both the voltage and Ca2+ sensitivity of
BK channels consist of channel-forming
b-subunits (b1–b4) arranged in tetramers.2 Recent cloning studies
have revealed the presence of multiple splice variants of -sub-
a-subunits and accessory
a
units3–5 and multiple subtypes of b-subunits (b1, b2/b3 and b4).6–8
Thus, there is a large diversity of BK channels, which may be spe-
cific to tissues and organs. The BK channels are expressed in a
number of organ systems, such as smooth muscle cells, skeletal
muscle cells, neuronal cells, and secretory epithelial cells,9 and
they have important physiological roles in modulating muscle con-
traction and neuronal activities, such as synaptic transmission.10
The physiological role and widespread distribution of BK chan-
nels suggest that agents that open these channels could have pro-
found impacts on diseases such as acute stroke, epilepsy, asthma,
and bladder overactivity.11 Well-characterized BK channel openers
not only are expected to have therapeutic potential, but also
should be of assistance in understanding the function, structure
and role of BK channels.
1
R
OH
C
2
R
H
H
A
B
H
H
H
OH
H
COOH
CO H
2
CO H
2
1
2
4a: R =R =H
Maxikdiol (1)
Pimaric acid (2)
Abietic acid (3)
1
2
4b: R =R =Cl
OR
OH
OR
In our previous study,12 we discovered active terpenoid com-
pounds, including pimaric acid (2, Chart 1), which have chemical
structures similar to that of maxikdiol (1),13,14 a moderately active
H
H
H
COOH
COOH
COOH
Podocarpic acid (6a)
5(5a:R=Ph(CH ) )
R = CH , Bn, ...
2 3
3
6b-u
* 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.2008.08.081

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Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5197–5200
dealkylation of the 12-alkoxy group occurs in some cases, resulting
in recovery of the starting material 6a (e.g., 7l). As both of the
above hydrolysis conditions have synthetic limitations, we chan-
ged the methyl ester to MOM (methoxymethyl) ester, which can
be easily hydrolyzed under very mild acidic conditions. As shown
in method C, all the intermediates 8e–g, 8o–s and the final O-
substituted podocarpic acid derivatives 6e–g, 6o–s were obtained
in moderate to high yields. All the compounds prepared in this
study are compiled in Table 1.
The activities of all the target compounds as BK channel modu-
lators were evaluated by means of automated planar array patch
clamp recording using the 64-well Population Patch Clamp (PPC)
technique.22,23 The BK channel was activated by applying a step
pulse to +100 mV from the holding potential of À90 mV to CHO-
K1 cells expressing hBK
the presence of compounds (30
a
channels, and the current amplitude in
M) was expressed as percent of
l
the drug-free control. The values represent an average of data ob-
tained from at least eight separate measurements. Stock solutions
of test compounds were prepared in DMSO at a concentration of
10 mM and diluted in buffer solution to give the desired final con-
centration (the final DMSO concentration is less than 0.4% (v/v)).
Since diCl-DHAA (4b) has already been shown to open BK chan-
nels,15,16 it was used as a positive reference agent in this study.
The structure-activity relationships illustrated in Table 1 sup-
port the potential of podocarpic acid structure as a pharmacophore
for BK channel-opening activity. Podocarpic acid itself, 6a was
found to be an effective BK channel opener, with an ionic current
increase of 207% versus the control current. For comparison, diCl-
DHAA 4b increased the current by 180%. From the results pre-
sented in Table 1, it appears that a simple alkyl ether at the
12-phenolic hydroxyl group decreased gate-opening activity (6b–
d). This may suggest that the possible H-bond donating property
of the phenol hydroxy group was important to BK channel-opening
activity. Interestingly, in the case of a propyl substituent, the
straight-chain derivative 6c showed a greater increase of ionic cur-
rent than the derivative 6d with a branched side-chain. In addition,
unsaturation of the carbon chain had no significant effect on the
activity (compound 6e vs 6c).
We also synthesized a series of aryl-group-containing deriva-
tives (6h–u). The activity in this series of compounds is highly sen-
sitive to the position and properties of the substituents. Significant
channel-opening activity was observed with 6h and 6j, which con-
tain a benzyl and a phenylpropyl group, respectively. We also
examined the effect of substitution on the phenyl ring of the ben-
zyl group of 6h. In general, substituents on the phenyl ring de-
creased the opening activity, regardless of their electronic
properties (compounds 6l-u). The para CH3-substituted derivative
6l was found to be more potent than the ortho regioisomer 6o,
which was inactive. Furthermore, the gate-opening activity de-
creased with increasing bulkiness of the aromatic alkyl group from
methyl (6l), isopropyl (6m) to tert-butyl group (compound 6n).
Introduction of an electron-withdrawing para CF3 group had little
effect on the activity (6q vs 6h). From the above results, we consid-
ered that substituents of appropriate size and spatial arrangement
on ring C of podocarpic acid derivatives may contribute to the BK
channel-modulating activity.
Figure 1. Representative conformations of 5a and 6j obtained by conformational
searching in a water environment (OPLS-AA force field, Macromodel).
the channel.16 All these BK channel openers are assumed to inter-
act with the
a subunit of the BK channels. Preliminary SAR studies
of the dehydroabietic acid derivatives (4) revealed the importance
of the carboxylic acid functionality of ring A and an appropriate
hydrophobic moiety in ring C for BK channel-opening ability.
Podocarpic acid (6a), which is readily available from the New
Zealand conifer Dacrydium cupressinum,17 is structurally closely re-
lated to dehydroabietic acid (4a). In the case of 6a, the stereochem-
istry of the carboxylic acid group is opposite to that of
dehydroabietic acid (4a), the isopropyl group is eliminated, and a
phenolic hydroxyl group is introduced onto the benzene ring.
Podocarpate derivatives have several interesting biological activi-
ties: podocarpic acid amides were reported to show liver X recep-
tor agonist activity18 and podocarpic acid esters showed cytokine
release inhibition activity.19 Methyl O-methyl-7-ketopodocarpate
was identified as a specific inhibitor of influenza A viruses in tissue
culture.20 While the carboxylic acid functionality is in an equatorial
position in dehydroabietic acid (4a), the same functionality lies in
an axial position in podocarpic acid (6a), as shown in Figure 1.
Thus, examination of podocarpic acid derivatives should give us
some indication of the structure–activity relationships of BK chan-
nel modulators and may lead to the identification of potent new BK
channel openers. Herein, we describe the synthesis and BK chan-
nel-opening activities of some podocarpic acid derivatives. Our
findings indicate that the podocarpic acid skeleton is a new scaf-
fold for BK channel modulators. In our recent work, we carried
out SAR studies of 12-alkoxy derivatives of dehydroabietic acid
(5, Chart 1) and found that these derivatives (e.g., 5a, Table 1)
showed no significant BK opening activity. In order to compare
the SAR information, we thus focused on a series of podocarpic acid
derivatives 6 bearing a 12-alkoxy group.
The three synthetic routes that were utilized to provide access
to the target compounds 6 are summarized in Scheme 1. In Method
A, podocarpic acid 6a was firstly converted to methyl ester 7a, fol-
lowed by alkylation of the 12-hydroxyl group, affording the key
intermediates 7c, 7l–n, and 7t–u. Finally, basic hydrolysis of the
ester with KOH and crown ether in MeOH under reflux gave the de-
sired 12-alkoxypodocarpic acids 6c, 6l–n, and 6t–u. However, this
hydrolysis step of methyl ester to acid proceeded very slowly,
requiring more than 3 days for completion, probably because of
severe steric hindrance between the 4-carboxylate and the 10-
methyl group in a 1,3-diaxial arrangement. According to the
literature,21 KOBut in DMSO can hydrolyze hindered esters, and
the desired acid compounds 6b, 6d and 6h–k were obtained by
using this hydrolysis condition with heating at 60 °C for 2 h (Meth-
od B). However, this condition (KOBut in DMSO) is so severe that
The carboxylic acid functionality at the C4 position of the podo-
carpic acid skeleton is also prerequisite for BK channel-opening
activity. The channel-opening activity was apparently lost in the
corresponding ester analogues (7a vs 6a, 7j vs 6j).
The apparent BK channel-opening activity of podocarpic acid
derivatives was in sharp contrast to the previous results for 12-alk-
oxy derivatives of dehydroabietic acid, which showed no signifi-
cant BK channel-opening activity (6j vs 5a, Table 1).24
Comparison of the most stable structures of 5a and 6j indicate
apparent differences in the direction and extension of the C4

Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5197–5200
5199
Table 1 (continued)
Table 1
Structure and BK
a
-opening properties of podocarpate derivatives
Compound
R1
R2
Ionic current in the presence
of test compound (30 M) as %
of control current (n = 8)
1
l
R
O
6r
6s
6t
H
H
H
138.6 7.1
OCF
CN
3
2
COOR
116.2 1.9
120.8 4.1
Compound
R1
R2
Ionic current in the presence
of test compound (30 M) as %
of control current (n = 8)
l
Buffer
4b
5a
6a
6b
—
—
—
H
—
—
—
H
H
103.8 3.3
180.4 11.9
93.2 8.4
206.9 57.4
123.7 7.7
Cl
Me
6u
7a
7j
H
109.0 4.2
118.5 4.5
116.0 4.8
OMe
6c
H
H
170.1 9.2
134.0 7.2
H
CH3
CH3
6d
6e
6f
H
H
147.9 7.4
123.8 3.1
carboxyl groups and C12 hydrophobic moieties in the two com-
pounds (Fig. 1). Therefore, the arrangement of the carboxylic acid
functionality of ring A and the hydrophobic region (ring C) is
important for a good fit to the binding sites of BK channels.
In summary, we found that podocarpic acid structure repre-
sents a new scaffold for BK channel openers. The SAR findings indi-
cated that in the 12-alkoxy derivatives, the arrangement (location
and orientation) of the carboxylic acid functionality of ring A and
the hydrophobic region of ring C is critical for BK channel-opening
activity. Our findings provide a new basis for development of novel
BK channel openers derived from podocarpic acid.
6g
H
103.2 2.2
N
6h
6i
H
H
209.0 21.0
122.1 3.2
Method A
1
1
OH
OH
OR
OR
6j
H
H
243.8 6.6
123.8 5.6
a
b
c
6k
O
COOH
6a
COOMe
7a
COOMe
7c, 7l-n, 7t-u
COOH
6c, 6l-n, 6t-u
6l
H
H
150.9 4.6
125.1 2.3
Method B
1
1
OH
OR
OR
6m
d
b
COOMe
7a
COOH
6b, 6d, 6h-k
COOMe
7b,7d, 7h-k
6n
6o
H
H
115.9 3.1
88.9 1.9
Method C
1
1
OH
OR
OH
OR
e
g
f
6p
6q
H
H
130.1 3.2
COOMOM
8
COOMOM
8e-g, 8o-s
COOH
6a
COOH
6e-g, 6o-s
Scheme 1. Reagents and conditions: (a) TMSCHN2, MeOH, toluene, rt, 100%;
(b) R1Br, NaH, DMF, 0 °C–rt, 44–100%; (c) KOH, 18-crown-6, MeOH, reflux, 33–93%;
(d) KOBut, DMSO, 60 °C, 50–84%; (e) MOMCl, DIPEA, DMF, 0 °C–rt, 82%; (f) Cs2CO3,
R1Br, DMF, rt, 54–100%; (g) 6 N HCl, THF, 50 °C, 40–97%.
184.9 18.2
CF
3

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Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5197–5200
9. Knaus, H.-G.; Schwarzer, C.; Koch, R. O. A.; Eberhart, A.; Kaczorowski, G. J.;
Acknowledgments
Glossmann, H.; Wunder, F.; Pongs, O.; Garcia, M. L.; Sperk, G. J. Neurosci. 1996,
16, 955.
This work was supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, Sports, and
Culture 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
valuable discussions and encouragement in the late stage of this
program. We thank Dr. Yoichiro Ezaki, Arakawa Chemical Industry,
for a gift of podocarpic acid.
10. Sah, P.; Faber, E. S. L. Prog. Neurobiol. 2002, 66, 345.
11. Gribkoff, V. K.; Starrett, J. E., Jr.; Dworetzky, S. I.; Hewawasam, P.; Boissard, C. G.
J. R.; Huston, K.; Johnson, G.; Krishman, B. S.; Kinney, G. G.; Lombardo, L. A.;
Meanwell, N. A.; Molinoff, P. B.; Myers, R. A.; Moon, S. L.; Ortiz, A.; Pajor, L.;
Pieschl, R. L.; Post-Munson, D. J.; Signor, L. J.; Srinivas, N.; Taber, M. T.; Thalody,
G.; Trojnacki, J. T.; Wiener, H.; Yeleswarm, K.; Yeola, S. W. Nat. Med. 2001, 7,
471.
12. Imaizumi, Y.; Sakamoto, K.; Yamada, A.; Hotta, A.; Ohya, S.; Muraki, K.;
Uchiyama, M.; Ohwada, T. Mol. Pharmacol. 2002, 62, 836.
13. Singh, S. B.; Goetz, M. A.; Zink, D. L.; Dombrowski, A. W.; Polishook, J. D.; Garcia,
M. L.; Schemalhofer, W.; McManus, O. B.; Kaczorrowski, G. J. J. Chem. Soc.,
Perkin Trans. 1 1994, 22, 3349.
14. Kaczorowski, G. J.; Knaus, H. G.; Leonard, R. J.; McManus, O. B.; Garcia, M. L. J.
Bioenerg. Biomembr. 1996, 28, 255.
References and notes
15. Ohwada, T.; Nonomura, T.; Maki, K.; Sakamoto, K.; Ohya, S.; Muraki, K.;
Imaizumi, Y. Bioorg. Med. Chem. Lett. 2003, 13, 3971.
16. Sakamoto, K.; Nonomura, T.; Ohya, S.; Muraki, K.; Ohwada, T.; Imaizumi, Y. J.
Pharmacol. Exp. Ther. 2006, 316, 144.
1. Gribkoff, V. K.; Starrett, J. E., Jr.; Droretzky, S. I.. In Advances in Pharmacology;
August, J. T., Anders, M. W., Murad, F., Coyle, J. T., Eds.; Academic Press: New
York, 1997; Vol. 37, p 319.
2. Vergara, C.; Latorre, R.; Marrion, N. V.; Adelman, J. P. Curr. Opin. Neurobiol. 1998,
8, 321.
17. Cambie, R. C.; Rutledge, P. S.; Woodgate, P. D. Aust. J. Chem. 1993, 46, 1447.
18. Liu, W.; Chen, S.; Dropinski, J.; Colwell, L.; Robins, M.; Szymonifka, M.; Hayes,
N.; Sharma, N.; MacNaul, K.; Hernandez, M.; Burton, C.; Sparrow, C. P.; Menke,
J. G.; Singh, S. B. Bioorg. Med. Chem. Lett. 2005, 15, 4574.
19. He, W.; Gavai, A.; Huang, F. C.; Regan, J.; Hanney, B.; Poli, G.; Bruno, J.; Chan, W.
K.; Djuric, S. W.; Yu, K. T.; Zilberstein, A. Bioorg. Med. Chem. Lett. 1999, 9, 469.
20. Staschke, K. A.; Hatch, S. D.; Tang, J. C.; Hornback, W. J.; Munroe, J. E.; Colacino,
J. M.; Muesing, M. A. Virology 1998, 248, 264.
3. Chen, L.; Tian, L.; MacDonald, S. H.; McClafferty, H.; Hammond, M. S.; Huibant,
J. M.; Ruth, P.; Knaus, H. G.; Shipston, M. J. J. Biol. Chem. 2005, 280, 33599.
4. Tian, L.; Duncan, R. R.; Hammond, M. S.; Coghill, L. S.; Wen, H.; Rusinova, R.;
Clark, A. G.; Levitan, I. B.; Shipston, M. J. J. Biol. Chem. 2001, 276, 7717.
5. Zarei, M. M.; Eghbali, M.; Alioua, A.; Song, M.; Knaus, H. G.; Stefani, E.; Toro, L.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 10072.
6. Knaus, H.-G.; Folander, K.; Garcia-Calvo, M.; Garcia, M. L.; Kaczorowski, G. J.;
Smith, M.; Swanson, R. J. Biol. Chem. 1994, 269, 17274.
7. Tanaka, Y.; Koike, K.; Alioua, A.; Shigenobu, K.; Stefani, E.; Toro, L. J. Pharmacol.
Sci. 2004, 94, 339.
21. Chang, F. C.; Wood, N. F. Tetrahedron Lett. 1964, 40, 2969.
22. Finkel, A.; Wittel, A.; Yang, N.; Handran, S.; Hughes, J.; Costantin, J. J. Biomol.
Screen 2006, 11, 488.
23. John, V. H.; Dale, T. J.; Hollands, E. C.; Chen, M. X.; Partington, L.; Downie, D. L.;
Meadows, H. J.; Trezise, D. J. J. Biomol. Screen 2007, 12, 50.
8. Tseng-Crank, J.; Godinot, N.; Johansen, T. E.; Ahring, P. K.; Støbꢀk, D.; Mertz, R.;
Foster, C. D.; Olesen, S.-P.; Reinhart, P. H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,
9200.
24. Cui, Y.-M.; Yasutomi, E.; Otani, Y.; Yoshinaga, T.; Ido, K.; Sawada, K.; Ohwada,
T. Bioorg. Med. Chem. Lett. 2008, 19, 5201.