The synthesis and BK channel-opening activity of N-acylaminoalkyloxime derivatives of dehydroabietic acid

Article abstract of DOI:10.1016/j.bmcl.2015.12.038

A series of N-acylaminoalkyloxime derivatives of dehydroabietic acid were synthesized and evaluated for BK channel-opening activities in an assay system of CHO-K1 cells expressing hBKα channels. The structure-activity relationship study revealed that a non-covalent interaction between the S atom of the 2-thiophene and the carbonyl O atom may contribute to conformation restriction for interaction with the ion channel. This research could guide the design and synthesis of novel abietane-based BK channel opener.

Full text of DOI:10.1016/j.bmcl.2015.12.038

Bioorganic & Medicinal Chemistry Letters 26 (2016) 283–287
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The synthesis and BK channel-opening activity
of N-acylaminoalkyloxime derivatives of dehydroabietic acid
a
a
a
b,
c
Yong-Mei Cui a, , Xin-Lan Liu , Wen-Ming Zhang , Hai-Xia Lin , Tomohiko Ohwada , Katsutoshi Ido ,
⇑
⇑
Kohei Sawada c
a Department of Chemistry, Innovative Drug Research Center, College of Sciences, Shanghai University, Shanghai 200444, China
b Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
c Tsukuba Research Laboratories, Eisai Co. Ltd, 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 N-acylaminoalkyloxime derivatives of dehydroabietic acid were synthesized and evaluated for
Received 6 September 2015
Revised 25 November 2015
Accepted 11 December 2015
Available online 12 December 2015
BK channel-opening activities in an assay system of CHO-K1 cells expressing hBKa channels. The struc-
ture-activity relationship study revealed that a non-covalent interaction between the S atom of the 2-
thiophene and the carbonyl O atom may contribute to conformation restriction for interaction with
the ion channel. This research could guide the design and synthesis of novel abietane-based BK channel
opener.
Keywords:
BK channels
Openers
Ó 2015 Elsevier Ltd. All rights reserved.
Oxime
Dehydroabietic 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 couple with other ion chan-
nels (such as Ca2+ ion channels,1,2 chloride channel,3 TRPC chan-
nels4) to serve as a negative feedback pathway controlling ionic
homeostasis, cell excitability, and neuron activity.5 BK channels
cells, and secretory epithelial cells,10 and they have important phys-
iological roles in modulating muscle contraction or neurotransmit-
ter release and hormone secretion.14
The physiological role and widespread distribution of BK chan-
nels suggest that agents that open these channels could have
profound impacts on diseases such as ischemic stroke, epilepsy,
asthma, and bladder overactivity.15 During the past few years, var-
ious classes of BK channel openers as well as their chemistry and
pharmacology have been described.16–19 Well-characterized BK
channel openers not only are expected to have therapeutic poten-
tial, but also should be of assistance in understanding the function,
structure and role of BK channels.
Our previous study showed that the dehydroabietic acid (DHAA,
1, Fig. 1) structure provides a template for chemical modulators of
BK channels.20 By introducing an oxime ether chain in position 7 of
the dehydroabietane skeleton, we obtained compounds such as
CYM04 with BK channel-opening activity comparable to that of
NS1619 and its mechanism of action has been studied.21–23 While
several interaction models23of chemical openers with BK channels
have been proposed, binding site and binding mode of chemical
openers are still unrevealed. Most probable binding sites may be
present in the transmembrane helices (TM) constituting the chan-
nel pore (such as TM5 (S5) and TM6 (S6)),24 however, the intracel-
lular domain such as S6-RCK1 linker has been proposed as a
binding site of CYM04.23 In this context, shape and contiguity of
hydrophobic moiety of the chemical BK channel openers are
consist of channel-forming
a-subunits and accessory b-subunits
(b1–b4) arranged in tetramers,6 having a voltage sensor and pore
as the membrane-spanning domain and having a cytosolic domain
containing metal-binding sites. Recently published studies on elec-
tron cryomicroscopy (cryo-EM)7 and X-ray crystallographic struc-
ture analysis8 of the BK channel have provided the first glimpse
into the assembly of the quaternary structure of this massive chan-
nel protein, corroborating the close interactions among these
domains during channel gating that were suggested by the previous
functional studies.9 Recent cloning studies have revealed the pres-
ence of multiple splice variants of
a
-subunits10–12 and multiple
subtypes of b-subunits (b1, b2/b3 and b4).13 Thus, there is a large
diversity of BK channels, which may be specific to tissues and
organs. The BK channels are expressed in a number of organ sys-
tems, such as smooth muscle cells, skeletal muscle cells, neuronal
⇑
Corresponding authors.
E-mail addresses: ymcui@shu.edu.cn (Y.-M. Cui), ohwada@mol.f.u-tokyo.ac.jp
(T. Ohwada).
http://dx.doi.org/10.1016/j.bmcl.2015.12.038
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

284
Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 26 (2016) 283–287
Cl
12
R1
H
F3C
12
N
O
1
9
C
N
14
Cl
OH
14
10
8
A
B
5
N
O
4
7
N
O
O
6
H
H
CO2H
COOH
F3C
NS1619
Figure 1. The structures of dehydroabietic acid (1), NS 1619, CYM04 and 2.
CO2H
N
H
R2
n
CYM04
1
2
Table 1
Structure and BKa-opening properties of dehydroabietate derivatives
R1
N
O
O
COOH
N
H
R2
n
Compound
R1
n
R2
Ionic current in the presence of test compound
(30 M) as % of control current (n = 8)
l
Buffer
NS1619
CYM04
7a
—
—
—
Br
Br
—
—
—
1
—
—
—
—
104.0 2.6
267.5 45.0
287.5 65.8
98.6 15.0
139.5 26.3
9a
1
CH3
9b
Br
1
141.8 17.8
9c
Br
Br
1
1
146.4 13.8
148.8 32.4
9d
9e
9f
Br
Br
Br
1
1
1
121.4 19.6
101.2 4.7
90.4 3.6
9g
9h
9i
Br
Br
Br
1
1
1
102.4 11.0
68.0 3.6
9j
118.0 40.0
9k
Br
1
136.4 33.3
9l
Br
Br
1
1
123.0 13.8
112.7 5.0
9m
9n
9o
Br
Br
1
1
107.0 7.9
151.3 20.4

Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 26 (2016) 283–287
285
Table 1 (continued)
Compound
R1
n
R2
Ionic current in the presence of test compound
(30 M) as % of control current (n = 8)
l
9p
9q
9r
Br
Br
Br
1
1
1
124.2 22.0
89.0 9.7
108.4 9.8
9s
9t
Br
Br
Br
1
1
1
203.6 33.8
267.3 16.4
127.6 18.8
9u
9v
9w
9x
9y
9z
Br
Br
Br
Br
Br
1
1
1
1
1
100.8 5.3
106.3 9.6
106.7 4.4
103.0 9.2
85.7 5.2
14
15
16
17
Br
Br
Br
H
2
4
5
1
135.7 9.8
103.9 8.7
97.2 14.8
105.7 12.8
assumed to be crucial for the binding, and therefore, for channel
opening activity.
The stereo-configuration of compound 9t was determined by X-
ray analysis25 (Fig. 2). In accordance with the starting material,
dehydroabietic acid, the two cyclohexane ring A and B bearing
chair and half-chair conformations, respectively, show a trans ring
junction, with two methyl groups of C19 and C20 in the axial posi-
tions. The molecule displays an E configuration with respect to the
N1@C7 double bond with an O3–N1–C7–C8 torsion angle of 173.6
(1)°. The oxime nitrogen atom is placed out of the aromatic ring
and the dihedral angle of \C6C7C8C9 is 19.35(2)°.
In the present Letter, we report the synthesis and SAR studies of
novel dehydroabietic derivatives bearing modified 7-N-acy-
laminoalkyl oximic chains with general structure 2, with the aim
of studying the space around the terminal group and exploring
the requirements for BK channel-opening activity. These chemical
modifications can also probe the nature (i.e., hydrophobicity/
hydrophilicity) of the binding site of the ion channel.
The oximes listed in Table 1 were synthesized according to the
pathways described in Scheme 1. The required starting ketones of
formula 3–4 were prepared as previously reported.22 Treatment of
the ketones with the appropriate O-substituted hydroxylamines S-
4 in EtOH and pyridine under heating at reflux afforded the corre-
sponding E-oxime as the sole isolated product. Deprotection of the
Boc group in the presence of trifluoroacetic acid produced key
intermediates 6a–d and 7, which reacted with appropriate acyl
chloride, followed by hydrolysis of the methyl ester with KOBut
and DMSO to yield corresponding compounds 9a–z and 14–17
(Scheme 1). The intermediate hydroxylamines S-4 were prepared
by the Mitsunobu reaction of N-hydroxyphthalimide with HO
(CH2)nCH2NHBoc using DIAD/PPh3, followed by hydrazine hydroly-
sis (Scheme 2). Compound 7a was obtained from the 12-Br-DHAA
ketone derivative 18a using similar procedures (Scheme 3).
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.26 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
a
channels, and the current amplitude in the pres-
M) was expressed as percent of the
ence of a test compound (30
l
drug-free control. The values represent an average of data obtained
from at least eight separate measurements. The results for NS1619
and CYM04 are included for comparison.
From the results presented in Table 1, the aminoethyloxime
derivative 7a (ion current = 98.6 15.0% of control at 30 lM)
showed no BK channel-opening activity. Alkyl acylation increased
the activity, as compared with the N-unsubstituted amine 7a. All
compounds 9a–d exhibited moderate BK channel-opening activity

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Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 26 (2016) 283–287
R1
R1
R1
Br
Br
Br
c
b
b
a
a
N
O
O
O
N
O
N
O
N
O
COOH
COOH
COOR
COOH
7a
COOMe
COOMe
NHBoc
NH2
NHBoc
NH2
n
n
18b
18a
3 R1 = Br
4 R1 = H
5a
5b
5c
R
R
1 = Br, n=1
6a
6b
6c
R
R
1 = Br, n=1
1 = Br, n=2
1 = Br, n=2
Scheme 3. Regents and conditions: (a) NH2OCH2CH2NHBoc, Py, EtOH, 90 °C, 97%;
R1 = Br, n=4
R1 = Br, n=4
(b) TFA, CH2Cl2, rt, 80%.
5d R1 = Br, n=5
6 R1 =H, n=1
6d R1 = Br, n=5
7 R1 =H, n=1
R1
R1
d
N
O
O
N
O
COOH
R2
COOMe
O
N
H
n
N
H
R2
n
R
1 = Br, n=1
R
1 = Br, n=1
8a-z
9a-z
10 R1 = Br, n=2
11 R1 = Br, n=4
12 R1 = Br, n=5
13 R1 =H, n=1
14 R1 = Br, n=2
15 R1 = Br, n=4
16 R1 = Br, n=5
17 R1 =H, n=1
Scheme 1. Regents and conditions: (a) NH2O(CH2)nCH2NHBoc, Py, EtOH, 90 °C, 67–
90%; (b) TFA, CH2Cl2, rt, 80–90%; (c) R2COCl, K2CO3, CH3CN, N2, rt, 15–85%; (d)
KOBut, DMSO, 21–81%.
(9a, 139.5 26.3%; 9b, 141.8 17.8%;9c, 146.4 13.8%; 9d,
148.8 32.4%).The amide derivative with phenyl group 9e showed
marginal channel-opening activity. Among the aryl-group contain-
ing derivatives 9f–n, the activity is quite sensitive to the location
and properties of the aromatic substituents. Substitution with an
electron-donating group, such as a methoxy group (9f–h) resulted
in inactivity. The ethyl derivative 9i showed blocking activity.
Among the three regioisomers 9l–n of the trifluoromethyl sub-
stituent, the ortho isomer 9l was more potent than the para and meta
CF3-substituted isomers (9m, 9n). The perfluorophenyl derivative
9k (136.4 33.3%) showed moderate BK channel-opening activity.
At the same time, we synthesized series of aromatic heterocycles
containing derivatives (9o–z). Initially, in order to find the optimal
hetero aromatic substitution, a series of 7-N-acylaminoethyl oxime
derivatives substituted with thiophene, thiazole, furan, pyrrole, pyr-
idine, etc. were prepared and evaluated. BK channel-opening activ-
ity data showed that derivatives containing five-membered
heteroaromatic rings having one sulfur atom exhibited better activ-
ities compared to those with other five- or six-membered rings (9s–
t vs 9o–r). In particular, the thiophene derivative 9t possessed the
most potent channel-opening activity, with ion current
Figure 2. X-ray crystal structure of 9t.
(9v–y). Fused with another phenyl ring (9z) also resulted in inac-
tivity. And it is notable that the 3-thiophene derivative (9u) dis-
played reduced activity compared to compound 9t. Furthermore,
the elongation of an alkylenic spacer between the oximic group
and the amide head caused an impressive drop in activity (14 vs
9t). Further extension of the chain length gave compounds 15
and 16 with no channel-opening activity. The steric situation of
these compounds and/or the elongation of the oximic chain can
be an explanation for their inactivity.
267.3 16.4% of control at 30 lM, comparable to that of CYM04.
Inspired by the potent activity possessed by the 2-thiophene deriva-
From the results shown in Table 1, the size of the acceptable
appendant N-acylamino group seems to be limited, because other
compounds containing 2-thiophene group (such as 9v, 9w, 9x, 9y
and 9z) did not show apparent activity, whereas the simple 2-thio-
phene derivative 9t is most potent. This idea is consistent with the
apparent activity of 9s, having 2-thiazole instead of 2-thiophene,
tive, further research on thiophene motif was carried out.
Using the thiophene derivative 9t as a starting point, our initial
efforts focused on the substitution pattern on the thiophene ring.
Both electron withdrawing and donating groups were introduced
to explore the SAR. It’s interesting to note that introduction of
any group to 2-thiophene ring abolished the opening activity
O
b
a
(CH2)n
(CH2)n
HO
CH2NH2
HO
CH2NHBoc
N
O
O
(CH2)n CH2NHBoc
S-1
S-2
S-3
c
H2N
(CH2)n
CH2NHBoc
O
n = 1,2,4,5
S-4
Scheme 2. Reagents and conditions: (a) Boc2O, DCM, rt, 74–87%; (b) N-hydroxyisoindoline-1,3-dione, PPh3, DIAD, toluene, 0 °C–rt, 27–31%; (c) NH2NH2ÁH2O, MeOH, rt, 63–
77%.

Y.-M. Cui et al. / Bioorg. Med. Chem. Lett. 26 (2016) 283–287
287
and also consistent with decreased but definite activities of 2-furan
References and notes
9o and 2-pyrole 9p. From the fact that the activities of 2-thiophene
9t and 2-thiazole 9s are more potent than those of 2-furan 9o, 2-
pyrole 9p and also the 3-thiophene derivative 9u, it can be hypoth-
esized that a non-covalent interaction between the S atom of the 2-
thiophene and the carbonyl O atom contribute at least partially to
conformational restriction of the N-acylamino moiety, which may
be crucial to interaction of the ion channel.27 This study also sug-
gested that it will be more appropriate to introduce photoaffinity
functionality (in order to study the binding site) to other positions
rather than the position 7 of the dehydroabietane skeleton, which
may lead to dramatical reduction of binding ability.
1. Berkefeld, H.; Sailer, C. A.; Bildl, W.; Rohde, V.; Thumfart, J. O.; Eble, S.;
Klugbauer, N.; Reisinger, E.; Bischofberger, J.; Oliver, D.; Knaus, H. G.; Schulte,
U.; Fakler, B. Science 2006, 314, 615.
2. Loane, D. J.; Lima, P. A.; Marrion, N. V. J. Cell Sci. 2007, 120, 985.
3. Greenwood, A.; Miller, L. J.; Ohya, S.; Horowitz, B. J. Biol. Chem. 2002, 277,
22119.
4. Kim, E. Y.; Alvarez-B, C. P.; Dryer, S. E. Mol. Pharmacol. 2009, 75, 466.
5. Brayden, J. E.; Nelson, M. T. Science 1992, 256, 532.
6. Vergara, C.; Latorre, R.; Marrion, N. V.; Adelman, J. P. Curr. Opin. Neurobiol. 1998,
8, 321.
7. Wan, L.; Sigworth, S. F. J. Nature 2009, 461, 292.
8. Yuan, P.; Leonetti, M. D.; Pico, A. R.; Hsiung, Y. C.; Mackinnon, R. Science 2010,
329, 182.
9. Lee, U. S.; Cui, J. Trends Neurosci. 2010, 33, 415.
It’s noteworthy that the corresponding DHAA derivative 17
showed no channel-opening activity, indicating the importance
of the bromide functionality in ring C for the activity.
10. Chen, L.; Tian, L. J.; MacDonald, S. H.; McClaffrey, H.; Hammond, M. S. L.;
Huibant, J.; Ruth, P.; Knaus, H.; Shipston, M. J. J. Biol. Chem. 2005, 280, 33599.
11. Tian, L.; Duncan, R. R.; Hammond, M. S. J. Biol. Chem. 2005, 276, 7717.
12. 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.
13. Salkoff, L.; Butler, A.; Ferreira, G.; Santi, C.; Wei, A. Nat. Rev. Neurosci. 2006, 7,
921.
14. Sah, P.; Faber, E. S. Prog. Neurobiol. 2002, 66, 345.
15. Gribkoff, V. K.; Starrett, J. E.; Dworetzky, S. I.; Hewawasam, P.; Boissard, C. G.;
Cook, D. A. CNS Drug Rev. 2001, 7, 471.
16. Nardi, A.; Olesen, S. Curr. Med. Chem. 2008, 15, 1126.
17. Gore, V. K.; Ma, V. V.; Yin, R. Y.; Ligutti, J.; Immke, D.; Doherty, E. M.; Norman,
M. H. Bioorg. Med. Chem. Lett. 2010, 20, 3573.
18. Bukiya, A. N.; Patil, S. A.; Li, W.; Miller, D. D.; Dopico, A. M.; Bukiya, A. N.
ChemMedChem 2012, 7, 1784.
In summary, the compounds in this Letter support interest in the
dehydroabietane skeleton, with a proper N-acylaminoalkyloxime in
position 7 for positive BK channel-opening activity. The structure–
activity relationship study revealed that a non-covalent interaction
between the S atom of the 2-thiophene and the carbonyl O atom
may contribute to conformation restriction for interaction with
the ion channel and could guide the design and synthesis of novel
abietane-based BK channel opener. Further study to identify the
binding sites and investigate their efficiency on brain ischemia is
under way in the lab.
19. Zhou, Li.; Zhang, Y.-J.; Gao, L.-J.; Ye, Y.; Qi, J.-H.; Qi, Z. Eur. J. Med. Chem. 2014,
75, 301.
20. Ohwada, T.; Nonomura, T.; Maki, K.; Sakamoto, K.; Ohya, S.; Muraki, K.;
Imaizumi, Y. Bioorg. Med. Chem. Lett. 2003, 13, 3971.
21. Cui, Y.-M.; Yasutomi, E.; Otani, Y.; Yoshinaga, T.; Ido, K.; Sawada, K.; Ohwada, T.
Bioorg. Med. Chem. Lett. 2008, 18, 6386.
Acknowledgements
22. Cui, Y.-M.; Eriko, Y.; Otani, Y.; Ido, K.; Yoshinaga, T.; Sawada, K.; Ohwada, T.
Bioorg. Med. Chem. 2010, 18, 8642.
23. Gessner, G.; Cui, Y.-M.; Otani, Y.; Ohwada, T.; Soom, M.; Hoshi, T.; Helnemann,
S. T. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 3552.
The authors are grateful for support from the National Natural
Science Foundation of China (Project Nos. 81202402 and
21272154).The authors also thank Drs. H. Deng and M. Shao, The
Instrumental Analysis &Research Center of Shanghai University
for structural analysis.
24. Dai, S.; Hall, D. D.; Hell, J. W. Physiol. Rev. 2009, 89, 411.
25. Crystallographic data: crystal dimensions 0.16 mm  0.13 mm  0.08 mm;
C
27H33BrN2O4S, Mr = 561.52; trigonal space group R3, a = 9.5918(19) Å,
b = 9.6056(19) Å, c = 28.900(6) Å, V = 2662.7(9) Å3, Z = 4, Dcalc = 1.401 g cmÀ3
T = 296(2) K, 13789 total and 4729 observed [R(int) = 0.0432] reflections,
321 parameters, final [I > 2 (I)] R1 = 0.0889, wR2 = 0.2560. CCDC 1422692.
,
Supplementary data
r
26. Ido, K.; Ohwada, T.; Yasutomi, E.; Yoshinaga, T.; Arai, T.; Kato, M.; Sawada, K. J.
Pharmacol. Toxicol. Methods 2013, 67, 16.
27. Beno, B. R.; Yeung, K.-S.; Bartberger, M. D.; Pennington, L. D.; Meanwell, N. A. J.
Med. Chem. 2015, 58, 4383.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.12.
038.