Synthesis and biological evaluation of chalcones as inhibitors of the voltage-gated potassium channel Kv1.3

Article abstract of DOI:10.1016/j.bmcl.2008.01.099

Chalcone derivatives of the natural product khellinone were synthesised and screened for bioactivity against the voltage-gated potassium channel Kv1.3. X-ray crystallography was employed to investigate relationships between the structure and function of a

Full text of DOI:10.1016/j.bmcl.2008.01.099

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2055–2061
Synthesis and biological evaluation of chalcones as inhibitors
of the voltage-gated potassium channel Kv1.3
Julia Cianci,a,b Jonathan B. Baell,a Bernard L. Flynn,c Robert, W. Gable,d
Jorgen A. Mould,c Dharam Paulc and Andrew J. Harveya,*
aThe Walter and Eliza Hall Institute of Medical Research, Biotechnology Centre, 4 Research Avenue, La Trobe R&D Park,
Bundoora, Vic. 3086, Australia
bDepartment of Medical Biology, The University of Melbourne, Parkville, Vic. 3010, Australia
cBionomics Limited, 31 Dalgleish Street, Thebarton, SA, Australia
dSchool of Chemistry, University of Melbourne, Vic. 3010, Australia
Received 14 December 2007; revised 25 January 2008; accepted 25 January 2008
Available online 31 January 2008
Abstract—Chalcone derivatives of the natural product khellinone were synthesised and screened for bioactivity against the voltage-
gated potassium channel Kv1.3. X-ray crystallography was employed to investigate relationships between the structure and function
of a selection of the reported chalcones.
Ó 2008 Elsevier Ltd. All rights reserved.
Chalcones are a group of naturally occurring com-
pounds, found in various plant species, comprising
two aryl rings linked by an a,b-unsaturated ketone.
Chalcones have been reported with a wide variety of
biological activities, including antiparasitic,1,2 antibacte-
rial,3 antifungal,4 anticancer5,6 and antiinflammatory
activity.7 Due to this diversity of bioactivity, chalcone
could be considered a ‘privileged structure’, as described
by Evans and coworkers.8 Whilst chalcones are active
against various protein targets, modification of the priv-
ileged core could lead to novel compounds with specifi-
cally targeted inhibitory activity.
ric bulk across the single bond, forcing the chalcone to
adopt an s-trans conformation (Fig. 2). A number of re-
search groups have attempted to exploit this property of
chalcones to improve the potency of bioactive chalcones.
Antimitotic agents such as colchicine (1, Fig. 1) and
combretastatin A4 (2, Fig. 1) have served as the tem-
plate for the design of highly oxygenated chalcones with
anti-cancer activity.6,12–14 Ducki and coworkers have re-
ported on the synthesis of antimitotic a-methyl chal-
cones like 3 (Fig. 1), describing these compounds as
having up to 20-fold increase in potency over chalcones
that are unsubstituted at the a-position.12 They attribute
this increase in potency to the s-trans geometry being
favourable for activity over the s-cis conformation. Ed-
wards and coworkers have also reported on a-methyl
and a-bromo chalcones with a twofold increase in anti-
mitotic activity over unsubstituted chalcones.15 The ob-
served improvement in bioactivity may be attributed to
the enone geometry.
Enones with aromatic groups attached to the carbonyl,
as in chalcones, have a preference for the s-cis conforma-
tion (Fig. 2),11 where the carbonyl and olefin are on the
same side of the single bond joining these two groups,
in order to reduce the unfavourable interaction which
would occur in the s-trans conformation, between a b-
hydrogen and a hydrogen ortho to the carbonyl on the
aromatic ring. Alkylation at the a-position increases ste-
Amongst the chalcones with antiparasitic activ-
ity,1,2,10,16,17 Nielson and coworkers have described anti-
malarial and antileishmanial chalcones that are
analogues of Licochalcone A (4, Fig. 1).10 They deter-
mined that a-alkylation had little effect on the antiproto-
zoal activity of these chalcones, and suggest that the
enone moiety acts as a spacer between the two pharma-
Keywords: Kv1.3; Chalcones; Natural product derivatives; Ion channel
blockers; Conformational analysis; X-ray crystallography; Molecular
modelling; Electrophysiology.
*
Corresponding author. Tel.: +61 8 8354 6118; fax: +61 8 8354
6199; e-mail: aharvey@wehi.edu.au
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.099

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J. Cianci et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2055–2061
O
O
O
O
O
O
NH
O
O
OH
O
O
O
O
OH
O
O
O
1
2
3
R'
O
O
O
R
O
O
O
HO
OH
O
O
OH
OH
O
4
5a R = OMe, R' = H
5b R = R' = OMe
5c R = R' = H
6
Figure 1. Highly oxygenated chalcones and related compounds with biological activity.6,9,10
cophore aromatic rings. A decrease in antimalarial
activity in chalcones with a-substitution has been re-
ported by Li and coworkers.2 They describe the enone
as a rigid spacer, and that conjugation is required for
activity, as reduced potency is observed with reduction
of the a,b-double bond.2
Scheme 1), which possess submicromolar blocking activ-
ity against the voltage-gated potassium channel Kv1.3.9
This ion channel is implicated in the pathogenesis of
autoimmune disorders such as multiple sclerosis, type I
diabetes mellitus and psoriasis.18–20 We have also de-
scribed the reduced derivative 6, which is equipotent
to 5a in blocking Kv1.3, thus suggesting that covalent
modification of the enone is not relevant to the Kv1.3
activity of these compounds.9
We have previously reported chalcones 5a and b (Fig. 1),
derivatives of the natural product khellinone (7,
O
Route A
O
O
O
i
ii
iv
O
O
O
O
O
O
O
OH
O
O
R
O
O
PMB
O
O
O
7
8
9: R = PMB
10: R = H
OH
iii
O
v
11
COOH
O
O
O
O
O
i
ii
vi
O
O
O
O
O
O
O
OH
O
PMB
O
R
O
O
OH
O
14: R = PMB
15: R = H
12
13
iii
Route B
O
16
Scheme 1. Reagents and conditions: (i) p-methoxybenzyl chloride, Cs2CO3, DMF, 50 °C, (8 89%, 13 98%); (ii) n-butyllithium, i-Pr2NH, THF,
DMPU, À78 °C, then MeI, À25 °C, (9 35%, 14 70%); (iii) I2, MeOH, 50 °C, (10 59%, 15 84%); (iv) 3-methoxybenzaldehyde, 3 M NaOH, MeOH,
60 °C, 52%; (v) 10% Pd/C, H2, MeOH, 86%; (vi) 3-carboxybenzaldehyde, 3 M NaOH, MeOH, 50 °C, 54%.

J. Cianci et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2055–2061
2057
In the current work, we have chosen to investigate fur-
ther the use of a chalcone group as a relatively rigid con-
formational SAR probe for the Kv1.3 blocking activity
of khellinone derivatives. We prepare an a-methyl chal-
cone derivative of khellinone to determine whether the s-
trans conformation is adopted and whether this favour-
ably modulates channel-blocking activity, relative to the
unsubstituted chalcone. In addition, we describe the
activity of khellinone chalcones in which the oxygen
substituents in the 4-, 6- or 7-position have been re-
moved and hence may be metabolically more robust
than their more highly oxygenated counterparts.21,22
tion of the benzofuran. The PMB-group was deprotec-
ted with iodine in methanol and 10 was converted to
the chalcone 11 by Claisen–Schmidt condensation. In
order to prepare 16, the furan of 7 was reduced via
hydrogenation to give 12. After PMB-protection of the
phenol to give 13, this compound was alkylated, afford-
ing 14 in 70% yield, double the yield compared with the
benzofuran. After PMB-deprotection, 15 was converted
to chalcone 16 by Claisen–Schmidt condensation.
We have previously reported the synthesis and bioactiv-
ity of 5c (Fig. 1), a chalcone analogue of khellinone with
the 7-methoxy group removed.9 The 4-desmethoxy and
6-deshydroxy khellinone chalcones targeted in this work
were prepared as follows. As outlined in Scheme 2, the
phenol of 7 was activated as the triflate with triflic anhy-
dride to give 17. No reduction of the triflate was ob-
served when Pd(OAc)2, HCOOH, Et3N and PPh3 in
DMF were used. On the basis of the accelerating effects
of bidentate phosphine ligands,23,24 triphenylphosphine
was replaced with 1,3-bis(diphenylphosphino)propane
(DPPP). This led to the reduction of the triflate 17 in
excellent yield to give compound 18, which was con-
verted to the chalcones 19 and 20 via Claisen–Schmidt
condensations with appropriate aldehydes.
The synthesis of a-methyl chalcones 11 and 16 is shown
in Scheme 1. Compound 16 was prepared primarily for
conformational analysis by X-ray crystallography.
Inclusion of the carboxylic acid group gave a more crys-
talline compound than with the methoxy group. The
furan was reduced for ease of synthesis, and it was as-
sumed that this modification would have no effect on
the enone geometry. The phenolic oxygen in khellinone
(7) was protected as the PMB-ether to give 8, which was
treated with LDA and iodomethane to give the a-meth-
ylated ketone 9. The poor yield in this step (35%) was
due in part to the competitive methylation at the 2-posi-
R
O
O
i
ii
iii
O
O
O
7
O
O
O
O
O
O
Tf
O
O
17
18
19: R = H
20: R = OMe
Scheme 2. Reagents and conditions: (i) Triflic anhydride, 2,4,6-collidine, DMAP, DCM, À40 °C to rt, 97%; (ii) n-Bu3N, DPPP, PdCl2(PPh3)2,
HCOOH, DMF, 80 °C, 89%; (iii) aldehyde, 3 M NaOH, MeOH, (19 48%, 20 36%).
Tf
OH
O
O
O
O
O
O
O
O
i
ii
O
O
O
O
O
O
22
23
21
O
O
R
iv
v
iii
O
O
O
OH
O
O
O
O
OH
O
24
25a (R=OH)
25b (R=CH3)
26
Scheme 3. Reagents and conditions: (i) BCl3, DCM, À78 °C to rt, 91%; (ii) Tf2O, Et3N, DCM, 0 °C to rt, 70%; (iii) PPh3, Pd(OAc)2, Et3N, HCOOH,
DMF, 70 °C, 55%; (iv) 3 M NaOH, EtOH, 90 °C, 25a 63%, 25b 7%; (v) 25b, 3-methoxybenzaldehyde, 1 M NaOH, MeOH, 0 °C to rt, 35%.

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J. Cianci et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2055–2061
Table 1. EC50 values of khellinone chalcone analogues for block of
Kv1.3
O
O
R
Compound
EC50 Jurkata (lM)
EC50 L929b (lM)
R
5a
5b
5c
11
19
20
26
—
0.4
0.8
17
—
—
—
—
0.9
—
s-cis
R = H
s-trans
R > H
3.0
2.4
2.4
1.7
Figure 2. The conformational preference of chalcones switches from s-
cis (left) to s-trans (right) if the a-substituent is bulkier than a hydrogen
atom.
a Compound EC50 values were obtained by fitting a Hill equation to
the normalised reduction of the Kv1.3 current integral at compound
concentrations ranging from 10À9 to 10À5 M. All electrophysiology
was performed using a planar patch clamp (Port-A-Patch, Nanion
Technologies Gmbh, Munich) in contrast to conventional patch
clamp.
possible that 11 could adopt an s-cis form that could
be responsible for its biological activity. However, and
in agreement with the literature, molecular modelling
indicated that the s-cis form in our a-methyl chalcones
was higher in energy than the s-trans form, in the case
of 11 by 7.4 kcal/mol, precluding the possibility of this
being the biologically active conformation.27 We then
modelled 11 in the preferred s-trans conformation using
the solid-state structure of 16 as a template and superim-
posed this onto the solid-state conformation of 5b, using
the two benzene rings as the bases for superimposition.28
As can be seen from Fig. 3C, there is a remarkable de-
gree of confluence in the spatial positioning of the two
aromatic rings for s-cis and s-trans conformations. We
propose that the olefinic portion of the enone may
merely serve as a linker for the two aryl binding groups
and that 11 could consequently still bind quite strongly
to Kv1.3 in the s-trans conformation.
b Assay conditions for patch clamp on L929, along with these results
have been previously published.9
To prepare the 4-desmethoxy analogue, the methyl ether
in the 4-position of khellin (21) was selectively cleaved
using boron trichloride, affording 22 in excellent yield
(Scheme 3). Activation to the triflate 23 and subsequent
reduction to 24 were performed as described above,
although the 4-position was reduced more readily than
the 6-position triflate, and a bidentate ligand was not re-
quired. In contrast to the khellin system, which exclu-
sively yields ketone 7 upon ring opening, hydrolysis of
24 gave mostly the carboxylic acid 25a (63%) and only
a small amount of the desired ketone 25b (7%).25 The
chalcone 26 was prepared from 25b as described above.
The EC50 values for inhibition of Kv1.3 currents were
determined using the whole cell mode with human Jur-
kat cells which endogenously express Kv1.3.26 Com-
pounds 5a–5c were previously tested under the same
assay conditions using L929 cells9 and we include these
data in Table 1 for comparison. Although we have
found that there is a reasonable degree of coherence be-
tween the results from the two assays, comparisons in
the discussion are only made between data within a cer-
tain assay in the analysis of structure–activity relation-
ships (Fig. 2).
The chalcone 5c was previously reported with an EC50 in
L929 cells of 17 lM,9 a 40-fold decrease in activity of the
chalcone 5a (Table 1), indicating that the 7-methoxy
group is important for channel-blocking activity. As this
group should have no effect on the conformation of the
chalcone, this suggests that the methoxy group could
favourably interact directly with Kv1.3. Truncations of
the 4- and 6-positions are better tolerated in the chalcone
series than removal of the 7-methoxy group. The com-
pounds where the 6-hydroxy group was removed (19
and 20) both have EC50 values of 2.4 lM, and the 4-des-
methoxy chalcone 26 has an EC50 of 1.7 lM (Table 1).
When the a-methyl chalcone 11 was tested by electro-
physiology, there was only a threefold decrease in
potency relative to the unsubstituted chalcone 5b (Table
1). We considered whether this could be explained by its
structure, so the structures of both chalcone 5b and a a-
methyl chalcone (16), a conformational model of 11,
were determined by X-ray crystallography. The struc-
ture of 5b (Fig. 3A) shows s-cis geometry of the enone
as expected, with 20° torsion through the enone
(O@CC@C), and E-arrangement of the olefin. There is
a hydrogen bond between the enone carbonyl and
ortho-hydroxy group on the benzofuran. On the other
hand, 16 exhibits s-trans geometry about the enone, as
anticipated, and the benzoic acid ring is twisted signifi-
cantly from the plane of the benzofuran, with 130° tor-
sion across the enone. We wondered whether it was
A crystal structure of the 6-deoxy chalcone 20 was ob-
tained (Fig. 4A), and compared with the structure of
chalcone 5b (Fig. 4B). The enone system of 5b is not
coplanar, with 39° of torsion between the benzofuran
and the methoxy-benzene ring (Fig. 4C). The crystal
structure of compound 20 shows that even without the
hydroxy group ortho to the carbonyl, the same conforma-
tion of the carbonyl is observed, and the torsion between
the two ring systems is 30° (Fig. 4D). Overlay of the
benzofuran rings of 5b and 20 shows that the aryl rings
are aligned similarly, and could occupy the same binding
site in the ion channel (Fig. 4B).
We have determined that khellinone chalcone com-
pounds can tolerate deletion of the 4-methoxy and
6-hydroxy groups with reasonable retention of Kv1.3-
blocking activity. On the other hand, deletion of the
Unpublished data.

J. Cianci et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2055–2061
2059
Figure 3. Crystal structure of (A) chalcone 5b, (B) a-methyl chalcone 16, and (C), two views of the proposed bioactive conformation of 11 (orange)
superimposed on 5b (white). For clarity, hydrogen atoms are not displayed.
Figure 4. (A) Crystal structure of chalcone 20, (B) alignment of the structures of 5 (white) and 20 (green). Structures showing torsion between
benzofuran and benzene rings in chalcones 5 (C) and 20 (D).

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J. Cianci et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2055–2061
9. Baell, J. B.; Gable, R. W.; Harvey, A. J.; Toovey, N.;
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14. Lawrence, N. J.; Rennison, D.; McGown, A. T.; Hadfield,
J. A. Bioorg. Med. Chem. Lett. 2003, 13, 3759.
7-methoxy group more significantly reduces potency.
Since this alteration is unlikely to affect conformation,
it is suggested that the 7-methoxy group in these com-
pounds could directly and favourably interact with
Kv1.3. Introduction of an a-methyl group into the en-
one, as in 11, effected conformational change to the s-
trans form, but without significant change in activity.
Despite the observed conformational change, com-
pounds 5b and 11 show comparable alignment of their
benzofuran and methoxyphenyl rings. The enone itself
does not appear to be important for binding, but rather
acts as a spacer to orient the key-binding elements.
15. Edwards, M. L.; Stemerick, D. M.; Sunkara, P. S. J. Med.
Chem. 1990, 33, 1948.
16. Zhai, L.; Chen, M.; Blom, J.; Theander, T. G.; Christen-
sen, S. B.; Kharazmi, A. J. Antimicrob. Chemother. 1999,
43, 793.
17. Zhai, L.; Blom, J.; Chen, M.; Christensen, S. B.; Khar-
azmi, A. Antimicrob. Agents Chemother. 1995, 39, 2742.
18. Azam, P.; Sankaranarayanan, A.; Homerick, D.; Griffey,
S.; Wulff, H. J. Invest. Derm. 2007, 127, 1419.
Acknowledgments
We are grateful to the Australian National Health and
Medical Research Council for financial support.
A.J.H. is a NHMRC Industry Fellow (406698).
19. Beeton, C.; Wulff, H.; Barbaria, J.; Clot-Faybesse, O.;
Pennington, M.; Bernard, D.; Cahalan, M. D.; Chandy,
K. G.; Beraud, E. Proc. Nat. Acad. Sci. U.S.A. 2001, 98,
13942.
20. Xu, J. C.; Wang, P. L.; Li, Y. Y.; Li, G. Y.; Kaczmarek, L.
K.; Wu, Y. L.; Koni, P. A.; Flavell, R. A.; Desir, G. V.
Proc. Nat. Acad. Sci. U.S.A. 2004, 101, 3112.
21. He, K.; Woolf, T. F.; Kindt, E. K.; Fielder, A. E.; Talaat,
R. E. Biochem. Pharmacol. 2001, 62, 191.
22. Spencer, J. P. E.; Kuhnle, G. G. C.; Williams, R. J.; Rice-
Evans, C. Biochem. J. 2003, 372, 173.
Supplementary data
Crystallographic data (excluding structure factors) for
the structures in this paper have been deposited with
the Cambridge Crystallographic Data Centre as supple-
mentary publication numbers CCDC 674128-674130.
Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK [fax: +44 (0)1223 336033 or e-mail: de-
1
posit@ccdc.cam.ac.uk]. H NMR data for compounds
23. Hayashi, T.; Konishi, M.; Kobori, Y.; Kumada, M.;
Higuchi, T.; Hirotsu, K. J. Am. Chem. Soc. 1984, 106, 158.
24. Saa, J. M.; Dopico, M.; Martorell, G.; Garciaraso, A. J.
Org. Chem. 1990, 55, 991.
11, 16, 19, 20 and 26 are available as Supporting Infor-
mation. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.bmcl.2008.01.099.
25. Carboxylic acid 25a was the major product isolated and can
be accountedfor as follows. Alkaline hydrolysis of 24 to give
the desired product 25b would involve attack by hydroxide
on C7to give intermediate 24a, followed byhydrolysis of the
b-carbonyl with loss of acetate. Unexpected product 25a
could arise either via (a) hydrolysis of the carbonyl of
tautomer 24b with loss of acetone, or via (b) direct attack by
hydroxide on C5 of 24 with loss of propyne. Attack of C5 in
either 24 or 24b would occur more readily than when a 4-
methoxy substituent is present due to reduced steric
hindrance and so can explain the different response to
hydrolysis of 21 and 24. We investigated the hydrolysis of a
model system that involved isolation of its diketone
intermediate prior to further hydrolysis (data not shown),
the result of which suggests that only mechanism (a)
accounts for the production of 25a.
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O
5
O
O
4
O
O
O
O
7
O
O
O
O
O
O
O
24
24a
24b
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J. Cianci et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2055–2061
2061
27. Minimisation was performed using the Tripos force-
field, with Gasteiger-Huckel charges, using default
values as implemented in sybyl7.3 (Tripos Associates,
Missouri). This included the use of a distance-depen-
dent dielectric of 1 using a non-bonded cutoff distance
were applied to the s-trans form of 11, which was built
in an identical conformation to the solid-state confor-
mation of 16. The s-trans form of 11 thus obtained was
calculated to be 7.4 kcal/mol lower in energy to the s-
cis form.
˚
of 8 A. The a-enone hydrogen atom in the solid-state
28. The above s-trans model of 11 was superimposed on the
solid-state structure of 5b, using the two benzene rings as
the templates for superimposition, resulting in a 12 atom
structure of 5b was changed to a methyl group. The
resulting compound, the s-cis form of 11, was allowed
to minimize but force constants of 5 were applied to
each of the four torsion angles in the enone in order to
maintain the s-cis conformation. The same restraints
˚
rmsd of 0.59 A and which is shown in Figure 3C (note in
Fig. 4 the mirror image of 5b is represented relative to
Fig. 3).