Khellinone Derivatives as Blockers of the Voltage-Gated Potassium Channel Kv1.3: Synthesis and Immunosuppressive Activity

Article abstract of DOI:10.1021/jm030523s

The voltage-gated potassium channel Kv1.3 constitutes a promising new target for the treatment of T-cell-mediated autoimmune diseases such as multiple sclerosis. In this study, we report the discovery of two new classes of Kv1.3 blockers based on the natu

Full text of DOI:10.1021/jm030523s

2326
J . Med. Chem. 2004, 47, 2326-2336
Kh ellin on e Der iva tives a s Block er s of th e Volta ge-Ga ted P ota ssiu m Ch a n n el
Kv1.3: Syn th esis a n d Im m u n osu p p r essive Activity
J onathan B. Baell,*,† Robert W. Gable,| Andrew J . Harvey,† Nathan Toovey,† Tanja Herzog,‡
Wolfram Ha¨nsel,‡ and Heike Wulff*,§
The Walter and Eliza Hall Institute of Medical Research Biotechnology Centre, 4 Research Avenue, La Trobe R&D Park,
Bundoora 3086, Australia, School of Chemistry, University of Melbourne, Victoria 3010, Australia, Pharmaceutical Institute,
University of Kiel, 24118 Kiel, Germany, and Department of Medical Pharmacology and Toxicology, University of California,
Davis, California 95616
Received October 15, 2003
The voltage-gated potassium channel Kv1.3 constitutes a promising new target for the treatment
of T-cell-mediated autoimmune diseases such as multiple sclerosis. In this study, we report
the discovery of two new classes of Kv1.3 blockers based on the naturally occurring compound
khellinone, 5-acetyl-4,7-dimethoxy-6-hydroxybenzofuran: (1) khellinone dimers linked via the
alkylation of the 6-hydroxy groups and (2) chalcone derivatives of khellinone formed by Claisen-
Schmidt condensation of the 5-acetyl group with aryl aldehydes. In particular, the chalcone
3-(4,7-dimethoxy-6-hydroxybenzofuran-5-yl)-1-phenyl-3-oxopropene (16) and several of its
derivatives inhibited Kv1.3 with Kd values of 300-800 nM and a Hill coefficient of 2, displayed
moderate selectivity over other Kv1-family K+ channels, suppressed T-lymphocyte proliferation
at submicromolar concentrations, and showed no signs of acute toxicity in mice. Because of
their relatively low molecular weight and lipophilicity and their high affinity to Kv1.3, aryl-
substituted khellinone derivatives represent attractive lead compounds for the development
of more potent and selective Kv1.3 blocking immunosuppressants.
In tr od u ction
much effect on the normal immune response. In proof
of this concept, the Kv1.3-blocking polypeptides kaliotox-
in2 and Stichodactyla helianthus toxin (ShK)3 have been
shown to prevent and treat experimental autoimmune
encephalomyelitis in rats, an animal model for multiple
sclerosis. Examination of Kv1.3-deficient mice recently
revealed another, previously unrecognized role for Kv1.3.4
Studies conducted with these mice demonstrated that
Kv1.3 is involved in body weight regulation and that
blockade of Kv1.3 increased basal metabolic rate sug-
gesting that Kv1.3 might also be a new pharmacological
target for the treatment of obesity.
The voltage-gated potassium channel Kv1.3 is criti-
cally involved in the activation of human T cells and
has therefore long been pursued as a novel target for
immunosuppressive therapy. The recent report1 that
terminally differentiated effector memory T cells express
much higher levels of Kv1.3 and lower levels of the
calcium-activated potassium channel IKCa1 (KCa3.1)
than other T cells has considerably increased the
attractiveness of targeting Kv1.3. Autoreactive effector
memory T cells play a crucial role in the pathogenesis
of T-cell-mediated autoimmune diseases. For example,
in multiple sclerosis, T cells directed against compo-
nents of the myelin sheath of the central nervous system
were found to be predominantly effector memory T cells
and to exhibit the Kv1.3high phenotype.1 The same study
showed that this special K+ channel phenotype rendered
the proliferation of effector memory T cells highly
sensitive to inhibition by Kv1.3 blockers. Na¨ıve and
central memory T cells were only affected at 10-fold
higher concentrations of Kv1.3 blockers and could
escape Kv1.3 inhibition during subsequent stimulation
through up-regulation of the calcium-activated potas-
sium channel IKCa1. It thus seems plausible to pref-
erentially target the disease-inducing effector memory
T cell population with a selective Kv1.3 blocker without
Despite their exquisite potency and demonstrated
efficacy polypeptides have the disadvantage of requiring
parenteral administration and extensive formulation to
compensate for their short circulating half-lives. Several
groups in academia and in the pharmaceutical industry
have therefore tried to develop potent and selective
small molecule Kv1.3 blockers and have identified eight
chemically distinct classes of compounds.5,6 The best
characterized of these are the nortriterpene correolide,7,8
originally isolated from the bark of Spachea correa, and
the recently described cyclohexyl-substituted benz-
amides,9,10 both developed by scientists at Merck. How-
ever, despite extensive screening efforts, none of these
compound classes have so far rendered an analogue with
appropriate characteristics for clinical development,
suggesting the necessity to explore other templates for
the development of small molecule Kv1.3 blocker.
* To whom correspondence should be addressed. Phone: +61 3 9345
2108 Fax: +61 3 9345 2211. J .B.B.: E-mail: jbaell@wehi.edu.au.
Phone: 530 754 6135 Fax: 530 752 7710. H.W.: E-mail: hwulff@
ucdavis.edu.
Khellinone (1a ) and visnaginone (1b) in Figure 1 are
naturally occurring benzofuran derivatives isolated from
Ammi visnaga. While khellinone itself only weakly
blocks Kv1.3 with a Kd of 45 µM, we show here that it
serves as a versatile starting material for at least two
† The Walter and Eliza Hall Institute of Medical Research Biotech-
nology Centre.
| University of Melbourne.
‡ University of Kiel.
§ University of California, Davis.
10.1021/jm030523s CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/05/2004

Blockers of the Voltage-Gated Potassium Channel Kv1.3
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2327
lyst yielded the tetrahydro derivative 18. Chalcone 16
was also dimerized by the standard alkylation procedure
to give 19.
Unexpectedly, reaction of 1a with succinic dihy-
drazide resulted in the dihydrazone 29. The identity of
29 was confirmed by the reaction of khellinone with
hydrazine dihydrate. A Beilstein search indicated the
transformation to be without precedent.
Dimer 30 was made as a more soluble analogue of 2
by reducing the carbonyl groups of 2 using tetrabutyl-
ammonium borohydride in DCM as shown in Scheme
4. The known visnaginone chalcone 3113 was prepared
from visnaginone 1b, as shown in Scheme 5, while the
visnaginone dimer 32 resulted from alkylation of vis-
naginone with dibromo-p-xylene using cesium carbonate
in DMF.
F igu r e 1. Structures of khellinone 1a and visnaginone 1b.
Sch em e 1. Synthesis of Khellinone (1a ) Dimers 2-9a
Resu lts a n d Discu ssion
SAR of Kh ellin on e Dim er s. As shown in Table 1,
when khellinone (1a ) was dimerized through a p-xylene
linker, the potency of blocking Kv1.3 greatly increased
from a Kd of 45 µM for khellinone to 0.3 µM for 2. It is
tempting to speculate that this is due to the utilization
of a second binding site on the tetrameric Kv1.3 channel.
The progressive drop in activity in going from 2 to the
meta- and ortho-substituted xylene dimers 3 and 4,
respectively, is in line with this hypothesis. The change
from an aromatic linker to an aliphatic linker was not
favorable as the n-butyl-linked dimer 5 exhibited a Kd
of 3.5 µM. To help elucidate the possible binding
geometry of 2, a crystal structure was obtained from
crystals grown in acetic acid (Figure 2). The resulting
structure revealed that the poor activity of dimer 5 could
simply be due to the fact that the alkyl linker is too
short. This was supported by the observation of a higher
potency for the n-pentane- and n-hexane-linked dimers
6 and 7 with respective Kds of 0.82 µM and 0.68 µM.
Replacement of the central methylene unit in 6 with
an oxygen atom was not favorable as 9 had a Kd of 4
µM. Together, these data suggested that hydrophobicity
in the linker was a prerequisite for tighter binding. The
heptane-linked dimer 8 (Kd 3.0 µM), however, was
clearly too long and flexible. To investigate the role of
the linker itself in binding to Kv1.3, the O-benzyl
derivative 21 was synthesized and found to block Kv1.3
with a Kd of 10 µM, indicating that the presence of a
bulky and hydrophobic substituent alone considerably
increases the affinity of khellinone to the channel but
is not sufficient to reach the level of activity of the
dimers themselves. In an attempt to locate a possible
third khellinone binding site, the trimer 20 was tested,
but was found to be inactive. Since 2 was very poorly
soluble in aqueous medium (ClogSw ) -6.55) and this
was anticipated to limit its usefulness as a lead com-
pound, we synthesized the more hydrophilic poly-
(ethylene)glycol conjugate 15 (ClogSw ) -4.32). How-
ever, 15 did not exhibit any activity on Kv1.3. The
methyl ester precursor 13 and the carboxylic acid 14
displayed some activity against Kv1.3 with respective
Kd values of 3 µM and 5 µM but were still significantly
less active than the unsubstituted dimer 2. This dem-
onstrates the detrimental effect of polar substituents
and confirms what has been previously reported for the
structurally related alkoxypsoralens14 and furoquinoli-
a
Reagents: (i) Cs2CO3, DMF.
different classes of new Kv1.3 blockers that block Kv1.3
currents and inhibit anti-CD3 activated T-cell prolifera-
tion at submicromolar concentrations.
Ch em istr y
Dimers 2-9 of khellinone (1a ) were readily made by
alkylation of the phenolic oxygen atom with various bis-
bromo hydrocarbons using cesium carbonate in DMF
(General Procedure A; Scheme 1). To improve the
solubility characteristics of 2, a tetra(ethyleneglycol)
derivative was prepared through NBS-based bis-bromi-
nation of the aryl methyl groups of methyl 2,5-dimeth-
ylbenzoate 11 to give 12, which was used to bis-alkylate
khellinone, rendering 13 (Scheme 2). Transesterification
with tetra(ethyleneglycol) monomethyl ether using ti-
tanium isopropoxide as catalyst according to the method
of Ferezou et al.11 rendered 15, while direct ester
hydrolysis with lithium hydroxide gave carboxylic acid
analogue 14. The alkylated compounds 21, an O-benzyl
analogue of khellinone, and 20, a khellinone trimer
(Scheme 3), were made according to the general alky-
lation procedure A.
Claisen-Schmidt condensation of the 5-acetyl group
of khellinone with benzaldehyde yielded the chalcone
derivative 16 (Scheme 3). The dimer 22, the cinnamoyl
derivative 23, the thiophenes 24 and 25 and the
chalcones 26-28 were prepared analogously by reacting
khellinone with the respective aldehydes (Scheme 3).
Chalcone 16 was selectively reduced using ionic hydro-
genation12 with triethylsilane/TFA to give hydrocinna-
mophenone 17. Hydrogenation with 10% Pd/C as cata-

2328 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Sch em e 2. Synthesis of Pegylated Khellinone Dimer 15a
Baell et al.
a
Reagents: (i) H2SO4, MeOH; (ii) NBS, CCl4; (iii) Cs2CO3, 1a , DMF; (iv) LiOH, H2O; (v) Ti(OCHMe2)4, HO(CH2CH2O)4Me.
Sch em e 3. Various Functionalizations of Khellinonea
a
Reagents: (i) PhC(dO)H, NaOH, H2O; (ii) Et3SiH/TFA, DCM; (iii) H2, 10%Pd/C, EtOAc; (iv) Cs2CO3, p-BrCH2PhCH2Br, DMF; (v)
Cs2CO3, R,R′,R′′-tribromomesitylene, DMF, (vi) K2CO3, PhCH2Br, acetone; (vii) m-HC(dO)PhC(dO)H, NaOH, H2O; (viii) PhCHdCHC(dO)H,
NaOH, H2O; (ix) (substituted)thiophene-2-carboxaldehyde, NaOH, H2O; (x) (substituted)benzaldehyde, NaOH, H2O; (xi) (NH2NH2C(dO)CH2)2,
MeOH.
nones15 as Kv1.3 blockers. Removal of the 7-methoxy
group as per visnaginone dimer 32 resulted in a
significant drop in activity. Selective manipulation of
the 4- and 7-methoxy groups to higher alkoxy groups
did not lead to any dimers with improved activity (data
not shown). Likewise, functional manipulation of the
ketone group to a variety of oximes, hydrazones, alkenes
and chalcones, ketone reduction, and dimers linked
through the acetyl group by formation of symmetrical
hydrazones or chalcones did not improve activity. Some

Blockers of the Voltage-Gated Potassium Channel Kv1.3
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2329
Sch em e 4. Synthesis of Bis-alcohol 30a
thiophene-2-yl. The side-chain phenyl ring tolerated
meta-substitution with OH or OMe, or para-substitution
with Me, while the thiophene-2-yl ring tolerated a 4-Br
substituent. The 7-OMe of the benzofuran ring was also
required for optimal binding.
Na tu r e of Block . Following application of both the
khellinone dimers and the khellinone chalcones to cells
stably expressing Kv1.3 in whole-cell patch-clamp ex-
periments, blockade of Kv1.3 current increased during
consecutive depolarizing pulses and reached a steady-
state after 3-8 min, a phenomenon termed “use-
dependent inhibition” (Figure 3a). However, the two
compound classes differed markedly in their kinetics of
block and their Hill coefficients. While the dimers
exhibited Hill coefficients close to 1 consistent with a
1:1 stoichiometry of interaction between blocker and
channel protein and did not affect the inactivation
kinetics of Kv1.3 (Figure 3b, left), the chalcones exhib-
ited Hill coefficients of 2, suggesting that two molecules
of blocker interact with one Kv1.3 channel tetramer, and
considerably accelerated the inactivation of Kv1.3 (Fig-
ure 3b, right). The chalcones thus resemble the cyclo-
hexyl-substituted benzamides in their Kv1.3-blocking
behavior. This class of compounds9 also speeds up
inactivation and blocks Kv1.3 with a Hill coefficient of
2. A recent competition binding study conducted with a
radiolabeled benzamide suggests the presence of two
receptor sites for the benzamides on the Kv1.3 protein
that display positive allosteric cooperativity.16 Whether
something similar is the case for the chalcones remains
to be determined.
a
Reagents: (i) Bu4NBH4, DCM.
of these dimers are exemplified by compounds 19, 22,
29, and 30 in Scheme 3 and Scheme 4 and in Table 1.
In summary, hydrophobic linkers are required for
optimum activity of the dimeric khellinones linked via
alkylation of the 6-hydroxy groups. The optimum linker
length is around 7.2 Å as defined by the intra-atomic
distance between the two oxygen atoms at the 6-posi-
tion. This value is 7.17 Å in the crystal structure of 2
and 7.27 Å in a molecular model of 6 (generated using
Sybyl, Tripos Associates) assuming a staggered pentane
conformation. The respective intra-atomic aryl 6-C atom
distances are also similar at 9.85 Å and 9.82 Å,
respectively. Weaker activity in the longer, heptane-
linked dimer 8 may be due to its excessive flexibility.
The higher activity of 2 suggests that π-electron density
in the linkers may be helpful but this could merely be
due to greater conformational constraint of the xylene
linker. The 7-methoxy group was required for potent
activity, as was the carbonyl functionality in the 5-acetyl
group.
SAR of Kh ellin on e Ch a lcon es a t Kv1.3. In the
course of preparing khellinone dimers functionalized at
the 5-acetyl group, all monomeric precursors were also
tested for blockade of Kv1.3. Serendipitously, chalcone
16 was found to inhibit Kv1.3 with a Kd of 0.4 µM.
Selective reduction of the alkene group gave 17, which
was still quite potent (Kd 0.6 µM). Further reduction of
the furan ring rendered 18, which was a little less
potent (Kd 1.5 µM). Extension of the alkene chain in the
cinnamoyl derivative 23 was not useful and rendered
the resulting compound completely inactive. The vis-
naginone-derived chalcone 31 was not a potent blocker
of Kv1.3, indicating that the 7-OMe group in 16 is
important for binding to Kv1.3. We subsequently em-
barked on a more detailed SAR investigation with the
synthesis of several chalcones bearing various substit-
uents such as Cl, Br, F, CH3, CN, COOH, OH, OCH3,
NO2, N(C2H5) in ortho, meta, and para positions of the
styryl phenyl ring. The most potent of these are listed
in Table 1 (see Supporting Information for examples of
less potent analogues of this series). m-Hydroxy and
m-methoxy substitution rendered 26 and 27 with Kds
of 0.7 µM and 0.8 µM, respectively. Introduction of a
p-methyl group resulted in 28 with a Kd of 0.7 µM. A
number of heterocyclic chalcones were also investigated,
where the styryl phenyl ring was replaced by variously
substituted quinoline, pyrrole, pyridine, indole, or
thiophene rings (see Supporting Information for ex-
amples). Of these only the thiophenes 24 and 25 blocked
Kv1.3 with respective Kd values of 1 and 0.8 µM.
In summary, the optimal functionalization of the
acetophenone group in khellinone was as a chalcone,
formed from Claisen-Schmidt condensation with aryl
aldehydes. The preferred aryl ring was phenyl or
Although we have not mapped it in this study, we
suspect that the binding site for both the dimers and
the chalcones is located in a hydrophobic pocket on the
intracellular site of the Kv1.3 protein, most likely in the
water-filled cavity below the selectivity filter of the
channel. Both the binding sites for correolide17 and
verapamil18 have been previously identified to lie in this
region.
Selectivity. Of the dimers, 2 and 7 were tested for
selectivity over other Kv1-family channels. As shown
in Table 2, 2 was more than 10-fold selective for Kv1.3
over Kv1.1 and Kv1.2, whereas 7 was actually a more
potent inhibitor of Kv1.1 and Kv1.2 with Kds of 0.32 µM
and 0.25 µM, respectively. The latter pharmacological
profile was cause for concern, as blockade of Kv1.1 and
Kv1.2 is suspected to be the reason for correolide’s
gastrointestinal side-effects. When administered to
minipigs, correolide caused hyperactivity8 and also
elicited twitches in guinea-pig ileum by stimulating the
enteric nervous system and enhancing neurotransmitter
release.19 For this reason, these alkane-linked dimers
were not pursued further in the context of this project.
Compound 2, on the other hand, was more widely tested
and maintained selectivity for Kv1.3, being several
times less active on Kv1.5 and inactive on the calcium-
activated K+ channels IKCa1.
Of the khellinone chalcones 16, 26, and 27 were tested
for selectivity as shown in Table 2. While only 2.5- or
3-fold less active on Kv1.1, they exhibited 20-fold
selectivity over the cardiac potassium channels Kv1.5
and Kv1.7 and had no effect on Kv1.2 and IKCa1. This
selectivity profile renders the khellinone chalcones the

2330 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Sch em e 5. Synthesis of Visnaginone Derivatives 31 and 32a
Baell et al.
a
Reagents: (i) PhC(dO)H, NaOH, H2O; (ii) Cs2CO3, p-BrCH2PhCH2Br, DMF.
Ta ble 1. Kd Values of Khellinone Analogues for Block of Kv1.3
Current
compd
Kd (µM)
compd
Kd (µM)
ShK43
11 pMa
195a
0.1a
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
0.6b
4-AP39
1.5b
correolide44
1a
1b
2
3
4
5
6
7
8
no effectd
no effectc
10a
50a
100a
0.28a
0.56a
7.1a
no effectc
no effectd
1b
3.5a
0.8b
0.82a
0.68a
3.0a
0.7b
0.8b
0.7b
9
4.0a
70a
13
14
15
16
3.1a
2.0a
5.0a
17b
no effectc
0.4b
1.0a
Each compound was tested 2-3 times at 3-5 concentrations.
Values for the dissociation constant Kd and the Hill coefficient nH
were determined by fitting the Hill equation to the reduction of
area under the K+ current curve. Standard deviations were
a
b
between 2-10% in each case. nH ) 1; nH ) 2; c Tested at 10
d
µM; Tested at 1 µM. The Kds of the most potent peptidic Kv1.3
F igu r e 2. ORTEP illustration of dimer 2. Primed atoms (′)
are related to the unprimed atoms by a center of symmetry.
blocker, the sea anemone toxin ShK (Stichodactyla helianthus
toxin),43 of the unselective small molecule K+ channel blocker 4-AP
(4-aminopyridine)39 and of Merck’s small molecule Kv1.3 blocker
correolide44 are given for comparison.
toxicity study suggests that the khellinone chalcones 16,
26, and 27 are not acutely toxic and constitute promis-
ing lead compounds for the development of more potent
small molecule Kv1.3 blockers as immunosuppressants.
first known small molecule Kv1.3 blockers that are
selective over Kv1.2 and Kv1.5.6,20
In h ibition of P r olifer a tion . On the basis of the
promising pharmacological profile of the khellinone
chalcones, compounds 16, 26 and 27 were tested for
toxicity against J urkat E6-1 and MEL cells. Also
included were thiophenes 24 and 25. The poor aqueous
solubility of dimer 2 made it too difficult to work with,
and this compound was not investigated further in this
project. While the thiophenes 24 and 25 proved toxic at
10 µM, compounds 16, 26, and 27 were found to be
nontoxic at concentrations of 0.1, 1, 2.5, and 10 µM and
were therefore selected for T-cell proliferation assays.
As shown in Table 3, these compounds and the dimer 7
inhibited the anti-CD3 antibody-stimulated proliferation
of peripheral blood T cells stimulated with EC50 values
between 0.6 and 1 µM.
Acu te Toxicity in Vivo. To determine if the com-
pounds exhibited any unexpected acute toxic effects in
vivo, mice (n ) 5) were injected intravenously with a
single dose of 7, 16, 26, or 27 at 5 mg/kg as a bolus.
The animals appeared normal immediately after the
injection and did not exhibit any signs of acute toxicity
for 7 days afterward. The body weights in treated
animals (day 1: 18.0 g; day 7: 27.0 g) were similar to
control mice injected with the vehicle (day 1: 18.0 g;
day 7: 28.0 g). Collectively, the data from this limited
Con clu sion
Khellinone (1a ) is a weak inhibitor of the voltage-
gated potassium channel, Kv1.3, with a Kd of 45 µM.
However, being small (mw 234) and containing both a
phenolic group that can be alkylated and an acetyl
group that readily undergoes Claisen-Schmidt conden-
sation, it serves as a versatile template for the design
of new small molecule Kv1.3 blockers. In this sense, it
complies with developing notions that smaller, more
polar molecules make the most druggable templates,
because they give the medicinal chemist more room to
move during the optimization process.21 Dimerization
of 1a through a p-xylyl linker to give 2 increased affinity
for Kv1.3 150-fold. While 2 was selective for Kv1.3 over
a range of other Kv1-family channels, poor aqueous
solubility limited its further use. Dimers 6 and 7 were
found to block Kv1.1 and Kv1.2 more potently than
Kv1.3 and might thus be of interest for enhancing
impulse propagation in demyelinated neurons22 in
multiple sclerosis and diabetic neuropathy, where de-
struction of the myelin sheath uncovers normally silent
potassium channels in the Node of Ranvier23 that
contribute to the conduction failure observed following
demyelination.24 Using the dimer 7 as a template for

Blockers of the Voltage-Gated Potassium Channel Kv1.3
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2331
F igu r e 3. (a) Typical experiment showing onset of block and washout of 1 µM of compound 16. Currents were elicited by
depolarizing steps from -80 to 40 mV for 500 ms applied every 45 s (see pulse protocol above the current traces). With this pulse
protocol no “run-down” of the Kv1.3 current was observed over periods of 20-60 min. After establishing a stable control current
(left) 1 µM compound 16 was perfused over the cell. Equilibrium block was reached after 360 s (middle). Every second pulse is
shown; control current dashed for comparison. After 720 s compound 16 was washed out (right) through continuous perfusion of
Ringer solution (∼30 mL). Blocked current dashed for comparison. (b) Effect of 0.5 µM compound 2 and 1 µM compound 16 on
Kv1.3 currents. Equilibrium block is shown in both cases.
Ta ble 2. Selectivity (Kd, µM) for Kv1.3 over Other K+
fects. Fluorinated chalcones in particular have been
Channelsa
shown to exhibit antiinflammatory properties through
28
Kv1.1 Kv1.2 Kv1.3
Kv1.5
Kv1.7 IKCa1
inhibition the 5-lipoxygenase and reduction of nitric
oxide production.29 However, the pharmacophores pro-
posed in these studies differ considerably from the
pharmacophore described here as necessary for Kv1.3
inhibition. On the basis of the similar potencies with
which 16, 26, and 27 block Kv1.3 and suppress T-cell
proliferation, we therefore suggest that their antipro-
liferative effects are due to Kv1.3 inhibition. It has also
been proposed that chalcones in general should be
viewed with caution,30 because of their propensity as
R,â-unsaturated ketones to act as a Michael Acceptor
and thus possibly undergo covalent reactions with
proteins.31,32 However, the potency of the reduced
derivative 17 together with the fact that the effect of
the chalcones in electrophysiological experiments is
easily reversible by washing with Ringer solution
demonstrates that this is not the case here.
In conclusion, we have generated two new classes of
small molecule Kv1.3 blockers in this study: khellinone
dimers and khellinone chalcones. The chalcones in
particular constitute promising templates to further
improve potency and selectivity for Kv1.3. They are the
first small molecule Kv1.3 blockers that are selective
over the cardiac potassium channel Kv1.5 and the
neuronal potassium channel Kv1.2, and their small size
easily allows for further chemical modification in order
to develop a truly Kv1.3 selective small molecule as an
immunosuppressant.
ShK43 16 pM 10 nM 11 pM >100 nM 12 nM 28 nM
4-AP39 290
590
.2
195
0.28
0.68
0.4
0.7
0.8
270
1.1
nd
5.1
12
nd
nd
nd
10
50
>50
29
2
7
16
26
27
3.1
0.3
1.2
1.7
2.0
>100
0.25
>50
>50
>50
nd
>100
>100
>100
20
a
Kd values (mean of at least three experiments with standard
deviations between 2-10% in each case) for blockade of other Kv1-
family channels and the calcium-activated K+ channel IKCa1. nd:
not determined. The selectivities of ShK (Stichodactyla helianthus
toxin) and 4-AP (4-aminopyridine) are given for comparison.
Ta ble 3. Inhibition of T-Cell Proliferation
compd
EC50 (µM)
ShK44
0.04
0.3
0.8
0.6
1
correolide44
7
16
26
27
1
the development of Kv1.1/Kv1.2 inhibitors as symptom-
atic therapeutics for the therapy of demyelinating
diseases appears valid in the light that 7 did not display
any signs of acute toxicity when administered to mice.
Derivatization of khellinone, through its acetyl group
as a chalcone, resulted in the more Kv1.3-selective
compounds 16, 26, and 27, which inhibited Kv1.3 with
Kd values of 0.4-0.8 µM and half-maximally suppressed
anti-CD3 antibody stimulated T-cell proliferation at
submicromolar levels. Perhaps because of their ease of
synthesis, chalcones have been widely used as templates
for various SAR studies and have been reported to
display a wide range of biological activities such as
antifungal,25 antiprotozoal,26 and antiproliferative27 ef-
Exp er im en ta l Section
Melting points were determined in open capillary tubes
using an electrothermal melting point apparatus (Model 9200)
and are uncorrected. Mass spectra (MS) were recorded on a
Kratos MS80 RFA mass spectrometer at the University of
Canterbury. Elemental analyses were performed for carbon,

2332 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Baell et al.
nitrogen, hydrogen, sulfur, and bromine at the University of
Otago microanalytical laboratory. Proton nuclear magnetic
resonance (1H NMR) and carbon nuclear magnetic resonance
(13C NMR) were performed on a Bruker Avance DRX 300 with
the solvents indicated. Chemical shifts were reported in parts
per million (ppm) on the δ scale and were referenced to the
appropriate solvent peaks: CDCl3 referenced to CHCl3 at δH
7.24 ppm and CDCl3 at δC 77.0 ppm; acetone-d6 referenced to
(CD3)(CHCD2)CO at δH 2.17 ppm and (CD3)2CO at δC 29.2 ppm.
IR spectra were obtained with a FTIR spectrophotometer,
using a diffuse reflectance accessory with a KBr background.
Spectra were run either in chloroform solution, neat, or as a
Nujol mull. Analytical thin-layer chromatography was per-
formed on Merck silica gel 60F254 aluminum-backed plates.
Flash chromatography was performed on Merck silica 60
following the guidelines given by Still et al.33 using ACS
analytical grade solvents. All moisture sensitive experiments
were performed in oven-dried glassware under an atmosphere
of nitrogen. Tetrahydrofuran was distilled from sodium ben-
zophenone ketal immediately prior to use. Anhydrous di-
methylformamide was purchased from Aldrich. Petroleum
ether describes a mixture of hexanes in the bp range 30-50
°C.
135.6, 144.0, 144.2, 144.6, 148.6, 201.7; IR (KBr) 1689; MS
(thermospray, LC/MS) m/z 575 (M + H+); Anal. (C32H30O10):
C, H.
1,4-Bis(5-a cetyl-4,7-d im eth oxyben zofu r a n -6-yloxy)bu -
ta n e (5). Khellinone 1a (236 mg, 1.0 mmol) and 1,4-dibro-
mobutane (59 µL, 0.5 mmol) were treated as described under
General Procedure A. The crude product was purified by flash
chromatography eluting with ethyl acetate/cyclohexane (3:7)
to afford 5 (221 mg, 84%) as white needles: Mp 119-120 °C;
1H NMR (CDCl3) δ: 1.87 (m, 4H), 2.50 (s, 6H), 3.95 (s, 6H),
4.06 (s, 6H), 4.09 (m, 4H), 6.83 (d, J ) 2.1 Hz, 2H), 7.54 (d, J
) 2.1 Hz, 2H); 13C NMR (CDCl3) δ: 26.6, 32.7, 61.0, 61.1, 74.7,
104.9, 116.3, 124.5, 134.4, 143.9, 144.4, 144.6, 148.6, 201.8;
IR (KBr, chloroform) 1702; MS (ES+) m/z 527 (M + H+); HRMS
(ES+) calcd C28H30O10 (M + H+) 526.1917, found 526.1912.
1,5-Bis(5-acetyl-4,7-dim eth oxyben zofu r an -6-yloxy)pen -
ta n e (6). Khellinone 1a (236 mg, 1.0 mmol) and 1,5-dibro-
mopentane (68 µL, 0.5 mmol) were treated as described in
General Procedure A. The crude product was purified by flash
chromatography eluting with ethyl acetate/cyclohexane (1:9
to 2:8) to give 6 (177 mg, 65%) as a gray oil: 1H NMR (CDCl3)
δ: 1.57 (m, 2H), 1.77 (tt, J ) 7.0, 7.0 Hz, 4H), 2.51 (s, 6H),
3.95 (s, 6H), 4.08 (m, 10H), 6.84 (d, J ) 2.1 Hz, 2H), 7.54 (d,
J ) 2.1 Hz, 2H); 13C NMR (CDCl3) δ: 22.2, 29.7, 32.7, 61.0,
61.1, 74.9, 104.9, 116.3, 124.5, 134.4, 143.9, 144.4, 144.7, 148.6,
201.8; IR (KBr, chloroform) 1705; MS (ES+) m/z 541 (M + H+);
HRMS (ES+) calcd C29H32O10 (M + H+) 541.2074, found
541.2070.
1,6-Bis(5-a cetyl-4,7-dim eth oxyben zofu r a n -6-yloxy)h ex-
a n e (7). Khellinone 1a (236 mg, 1.0 mmol) and 1,6-dibromo-
hexane (77 µL, 0.5 mmol) were treated as described under
General Procedure A. The crude product was purified by flash
chromatography eluting with ethyl acetate/cyclohexane (1:9
to 2:8) to give 7 (215 mg, 77%) as a colorless solid: Mp 89-90
°C (EtOAc/cyclohexane); 1H NMR (CDCl3) δ: 1.48 (m, 4H), 1.75
(m, 4H), 2.52 (s, 6H), 3.97 (s, 6H), 4.06 (m, 10H), 6.85 (d, J )
2.4 Hz, 2H), 7.55 (d, J ) 2.4 Hz, 2H); 13C NMR (CDCl3) δ: 25.6,
26.8, 32.7, 61.0, 61.2, 75.1, 104.6, 116.3, 124.5, 134.4, 143.9,
144.4, 144.8, 148.6, 201.8; IR (KBr, chloroform) 1702; MS (ES+)
m/z 555 (M + H+); Anal. (C30H34O10): C, H.
1,7-Bis(5-acetyl-4,7-dim eth oxyben zofu r an -6-yloxy)h ep-
ta n e (8). Khellinone 1a (236 mg, 1.0 mmol) and 1,7-dibromo-
heptane (85 µL, 0.5 mmol) were treated as described in General
Procedure A. The crude product was purified by flash chro-
matography eluting with ethyl acetate/cyclohexane (1:9) to give
8 (243 mg, 86%) as a yellow oil: 1H NMR (CDCl3) δ: 1.39 (m,
6H), 1.69 (m, 4H), 2.49 (s, 6H), 3.94 (s, 6H), 4.03 (m, 10H),
6.82 (d, J ) 2.3 Hz, 2H), 7.52 (d, J ) 2.3 Hz, 2H); 13C NMR
(CDCl3) δ: 25.7, 29.0, 30.0, 32.7, 60.9, 61.1, 75.1, 104.8, 116.2,
124.5, 134.3, 143.8, 144.4, 144.8, 148.6, 201.8; IR (film) 1705;
MS (ES+) m/z 569 (M + H+); Anal. (C31H36O10): C, H.
Gen er a l P r oced u r e A. To a suspension of the benzofuran
(0.5 mmol) and cesium carbonate (0.5 mmol) in DMF under
nitrogen was added the dibromide (0.25 mmol), and the
reaction mixture was stirred at 55 °C for 16 h. The reaction
mixture was diluted with ethyl acetate (10 mL), washed with
10% aqueous citric acid (3 × 10 mL) and brine (10 mL), dried
over MgSO4, and concentrated in vacuo. The resulting residue
was purified by flash chromatography.
Gen er a l P r oced u r e B. A mixture of the benzofuran (1.0
mmol) and the aldehyde (1.2 mmol) in 1 M NaOH in methanol/
water (2:1) was stirred at room temperature 16 h, and 10%
aqueous citric acid was added. If a solid precipitated, the solid
was collected by filtration and recrystallized. If an oil devel-
oped, the oil was extracted with ethyl acetate (2 × 10 mL),
and the pooled organics were washed with brine (10 mL), dried
over MgSO4, and concentrated in vacuo. The resulting residue
was recrystallized to give the pure chalcone.
1,4-Bis(5-a cetyl-4,7-d im eth oxyben zofu r a n -6-yloxym e-
th yl)ben zen e (2). Khellinone 1a (236 mg, 1.0 mmol) and R,R′-
dibromo-p-xylene (132 mg, 0.5 mmol) were treated as described
in General Procedure A. The crude product was recrystallized
from methanol to give 2 (170 mg, 59%) as a colorless solid:
Mp 192 °C (MeOH); 1H NMR (CDCl3) δ: 2.44 (s, 6H), 3.99 (s,
6H), 4.08 (s, 6H), 5.09 (s, 4H), 6.88 (d, J ) 2.3 Hz, 2H), 7.43
(s, 4H), 7.59 (d, J ) 2.3 Hz, 2H); 13C NMR (CDCl3) δ: 32.4,
60.8, 60.8, 76.1, 104.7, 116.3, 124.3, 128.2, 134.2, 136.7, 143.7,
144.0, 148.3, 201.6; IR (KBr, chloroform) 1701; MS (thermo-
spray, LC/MS) m/z 575 (M + H+); Anal. (C32H30O10): C, H.
1,3-Bis(5-a cetyl-4,7-d im eth oxyben zofu r a n -6-yloxym e-
th yl)ben zen e (3). Khellinone 1a (236 mg, 1.0 mmol) and R,R′-
dibromo-m-xylene (132 mg, 0.5 mmol) were treated as de-
scribed under General Procedure A. The crude product was
recrystallized from methanol to give 3 (162 mg, 56%) as a
colorless solid: Mp 112 °C (MeOH); 1H NMR (CDCl3) δ: 2.45
(s, 6H), 3.99 (s, 6H), 4.10 (s, 6H), 5.10 (s, 4H), 6.88 (d, J ) 2.3
Hz, 2H), 7.40 (m, 3H), 7.50 (s, 1H), 7.59 (d, J ) 2.3 Hz, 2H);
13C NMR (CDCl3) δ: 32.7, 61.2, 61.2, 76.6, 105.0, 116.7, 124.7,
128.2, 128.3, 128.6, 134.6, 137.3, 144.1, 144.3, 144.6, 148.6,
201.9; IR (KBr) 1696; MS (thermospray, LC/MS) m/z 575 (M
+ H+); Anal. (C32H30O10): C, H.
Bis(5-a cet yl-4,7-d im et h oxyben zofu r a n -6-yloxyet h yl)-
eth er (9). To a suspension of 1a (236 mg, 1.0 mmol) and
anhydrous potassium carbonate (138 mg, 1.0 mmol) in DMF
(2 mL) under nitrogen was added di(2-bromoethyl) ether (63
µL, 0.5 mmol), and the reaction mixture was stirred at room
temperature for 2 d. It was diluted with ethyl acetate (10 mL),
washed with 10% aqueous citric acid (3 × 10 mL) and then
brine (10 mL), dried over MgSO4, and concentrated in vacuo.
The resulting residue was purified by flash chromatography
eluting with ethyl acetate/cyclohexane (1:9 to 3:7) to afford the
product 9 (99 mg, 37%) as a colorless oil: 1H NMR (CDCl3) δ:
2.53 (s, 6H), 3.77 (t, J ) 4.8 Hz, 4H), 3.96 (s, 6H), 4.06 (s, 6H),
4.24 (t, J ) 4.8 Hz, 6H), 6.85 (d, J ) 2.1 Hz, 2H), 7.55 (d, J )
2.1 Hz, 2H); 13C NMR (CDCl3) δ: 32.7, 61.0, 61.2, 70.0, 73.7,
104.9, 116.5, 124.5, 134.3, 144.0, 144.5, 144.8, 148.6, 201.7;
IR (film) 1689; MS (ES+) m/z 543 (M + H+); HRMS (ES+) calcd
1,2-Bis(5-a cetyl-4,7-d im eth oxyben zofu r a n -6-yloxym e-
th yl)ben zen e (4). Khellinone 1a (236 mg, 1.0 mmol) and R,R′-
dibromo-o-xylene (132 mg, 0.5 mmol) were treated as described
under General Procedure A. The crude product was recrystal-
lized from ethanol to give 4 (167 mg, 58%) as a colorless solid:
C
28H30O11 (M + H+) 543.1866, found 543.1873.
Meth yl 2,5-Dim eth ylben zoa te (11). A mixture of 2,5-
dimethylbenzoic acid (10) (1.50 g, 10 mmol), sulfuric acid (0.11
mL, 2.0 mmol), and methanol (4 mL) was heated at reflux for
4 h. The mixture was poured into water (40 mL) and extracted
with ethyl acetate (2 × 30 mL). The pooled organics were
washed with saturated sodium bicarbonate (2 × 20 mL) and
1
Mp 140 °C (EtOH); H NMR (CDCl3) δ: 2.39 (s, 6H), 3.98 (s,
6H), 4.03 (s, 6H), 5.27 (s, 4H), 6.87 (d, J ) 2.3 Hz, 2H), 7.34
(m, 2H), 7.50 (s, 2H), 7.58 (d, J ) 2.3 Hz, 2H); 13C NMR (CDCl3)
δ: 32.6, 61.1, 61.2, 73.8, 105.0, 116.7, 124.7, 128.3, 129.3, 134.7,

Blockers of the Voltage-Gated Potassium Channel Kv1.3
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2333
brine (20 mL), dried over MgSO4, and concentrated in vacuo
to give 11 (1.14 g, 70%) as a colorless oil: 1H NMR (CDCl3) δ:
2.34 (s, 3H), 2.56 (s, 3H), 3.88 (s, 3H), 7.12 (d, J ) 7.8 Hz,
1H), 7.24 (d, J ) 7.8 Hz, 1H), 7.73 (s, 1H); 1H NMR lit.34
(CDCl3) δ: 2.34 (s, 3H), 2.54 (s, 3H), 3.88 (s, 3H), 7.12 (d, J )
7.8 Hz, 1H), 7.21 (dd, J ) 1.7, 7.8 Hz, 1H), 7.70 (d, J ) 1.7 Hz,
1H,).
flash chromatography, eluting with ethyl acetate/petroleum
ether (3:2) to afford the starting material 13 (30 mg). Further
elution with ethyl acetate/petroleum ether (7:3) furnished 15
(32 mg, 50% by returned starting material) as a colorless oil:
1H NMR (CDCl3) δ: 2.49 (s, 3H), 2.49 (s, 3H), 3.35 (s, 3H),
3.51 (m, 2H), 3.58-3.68 (m, 12H), 3.81 (t, J ) 4.8 Hz, 2H),
3.99 (s, 3H), 4.00 (s, 3H), 4.03 (s, 3H), 4.11 (s, 3H), 4.44 (t, J
) 4.8 Hz, 2H), 5.12 (s, 2H), 5.56 (s, 2H), 6.88 (d, J ) 2.1 Hz,
1H), 6.90 (d, J ) 2.1 Hz, 1H), 7.59 (d, J ) 2.1 Hz, 1H), 7.61 (d,
J ) 2.1 Hz, 1H), 7.67 (d, J ) 8.1 Hz, 1H), 7.86 (d, J ) 8.1 Hz,
1H), 8.09 (s, 1H); 13C NMR (CDCl3) δ: 32.7, 32.7, 58.9, 61.1,
61.1, 61.3, 61.3, 63.5, 69.0, 70.4, 70.5, 71.8, 74.2, 76.0, 104.9,
105.0, 116.6, 116.7, 124.6, 124.6, 127.4, 127.5, 130.3, 132.3,
134.4, 134.6, 136.2, 139.9, 144.0, 144.1, 144.4, 144.5, 144.6,
148.5, 148.7, 166.4, 201.5, 201.7; IR (film) 1703, 1713; MS
(ES+) m/z 826 (M + NH4+); HRMS (ES+) calcd C42H49O16 (M
+ H+) 809.303, found 809.302.
3-(4,7-Dim et h oxy-6-h yd r oxyben zofu r a n -5-yl)-1-p h en -
yl-3-oxop r op en e (16). Khellinone 1a (236 mg, 1.0 mmol) and
benzaldehyde (152 µL, 1.5 mmol) were treated as described
in General Procedure B. The crude product was recrystallized
from methanol to give 16 (252 mg, 78%) as red needles: Mp
125-126 °C (MeOH) lit.36 129 °C (EtOH); 1H NMR (CDCl3) δ:
4.03 (s, 3H), 4.07 (s, 3H), 6.86 (d, J ) 2.3 Hz, 1H), 7.34-7.42
(m, 3H), 7.50 (d, J ) 2.3 Hz, 1H), 7.63 (m, 2H), 7.85 (m, 2H);
13C NMR (CDCl3) δ: 61.0, 61.9, 105.2, 111.8, 112.7, 127.0,
128.5, 129.0, 129.5, 130.4, 135.1, 143.4, 144.1, 150.7, 151.9,
153.2, 194.7.
3-(4,7-Dim et h oxy-6-h yd r oxyben zofu r a n -5-yl)-1-p h en -
yl-3-oxop r op a n e (17). A solution of 16 (49 mg, 0.15 mmol)
in dichloromethane (1 mL) was treated with triethylsilane (74
µL. 0.45 mmol) and trifluoroacetic acid (77 µL, 1.0 mmol) and
stirred under nitrogen for 3 h. The reaction mixture was
diluted with cyclohexane, and on concentrating, a solid pre-
cipitated. The solid was filtered to afford 17 (46 mg, 93%) as
yellow needles: Mp 112-113 °C (cyclohexane); 1H NMR
(CDCl3) δ: 3.04 (t, J ) 7.5 Hz, 2H), 3.41 (t, J ) 7.5 Hz, 2H),
4.03 (s, 3H), 4.09 (s, 3H), 6.87 (d, J ) 2.3 Hz, 1H), 7.17-7.29
(m, 5H), 7.48 (d, J ) 2.3 Hz, 1H); 13C NMR (CDCl3) δ: 30.6,
46.0, 60.6, 61.0, 105.7, 110.5, 110.8, 126.0, 128.4, 128.4, 128.9,
141.3, 143.8, 151.3, 152.1, 153.3, 206.6; IR (film) 1620, 3152;
MS (ES+) m/z 327 (M + H+); Anal. (C19H18O5): C, H.
Meth yl 2,5-Di(br om om eth yl)ben zoa te (12). To a solution
of 11 (328 mg, 2.0 mmol) in carbon tetrachloride (10 mL) was
added N-bromosuccinimide (890 mg, 5.0 mmol) and 2,2′-
azobisisobutyronitrile (16 mg, 0.1 mmol), and the reaction
mixture was heated at reflux for 4 h. The resulting suspension
was filtered, and the residue was washed with chloroform (3
× 5 mL). The pooled organics were concentrated in vacuo to
give a mixture of product and succinimide as determined by
1H NMR. The mixture was dissolved in dichloromethane (20
mL) and washed with water (2 × 10 mL). The dichloromethane
phase was dried over MgSO4 and concentrated in vacuo, and
the resulting residue was purified by flash chromatography
eluting with 2% ether in petroleum ether to furnish the product
(0.50 g, 78%) as a white solid: Mp 79.5-80 °C (MeOH); lit.35
1
81-83 °C (MeOH); H NMR (CDCl3) δ: 3.98 (s, 3H), 4.49 (s,
2H), 4.94 (s, 2H), 7.46 (d, J ) 7.8 Hz, 1H), 7.53 (dd, J ) 1.8,
7.8 Hz, 1H), 8.00 (d, J ) 1.8 Hz, 1H).
Met h yl 2,5-Bis(5-a cet yl-4,7-d im et h oxyb en zofu r a n -6-
yloxym eth yl)ben zoa te (13). Khellinone 1a (147 mg, 0.62
mmol) and 12 (100 µL, 0.31 mmol) were treated as described
in General Procedure A. Purification by flash chromatography
eluting with ethyl acetate/cyclohexane (1:4 to 2:3) afforded 13
(149 mg, 76%) as a straw-colored solid: Mp 87-89 °C; 1H NMR
(CDCl3) δ: 2.48 (s, 3H), 2.48 (s, 3H), 3.87 (s, 3H), 3.98 (s, 3H),
3.99 (s, 3H), 4.03 (s, 3H), 4.09 (s, 3H), 5.11 (s, 2H), 5.55 (s,
2H), 6.87 (d, J ) 2.7 Hz, 1H), 6.88 (d, J ) 2.7 Hz, 1H), 7.57 (d,
J ) 2.7 Hz, 1H), 7.59 (d, J ) 2.7 Hz, 1H), 7.67 (dd, J ) 1.5,
8.1 Hz, 1H), 7.85 (d, J ) 8.1 Hz, 1H), 8.07 (d, J ) 1.5 Hz, 1H);
13C NMR (CDCl3) δ: 32.7, 32.7, 51.9, 61.1, 61.1, 61.2, 61.2,
74.1, 76.0, 104.9, 105.0, 116.6, 116.7, 124.3, 124.5, 127.5, 127.7,
130.2, 132.2, 134.3, 134.5, 136.2, 139.7, 144.0, 144.1, 144.4,
144.5, 144.6, 144.6, 148.5, 148.7, 166.9, 201.5, 201.7; IR (film)
1718, 1701; MS (ES+) m/z 633 (M + H+); HRMS (ES+) calcd
C34H32O12 (M + H+) 633.1972, found 633.1981.
2,5-Bis(5-a cetyl-4,7-d im eth oxyben zofu r a n -6-yloxym e-
th yl)ben zoic Acid (14). To a solution of 13 (63 mg, 0.10
mmol) in THF (3 mL) was added 0.25 M lithium hydroxide in
methanol/water (2:1, 3 mL), and the reaction mixture was
stirred at 80 °C for 16 h. The mixture was then concentrated
to one-third volume, diluted with water (10 mL), and extracted
with ether (15 mL). The aqueous phase was cooled, acidified
to pH 4 with 1 M HCl, and extracted with ethyl acetate (2 ×
20 mL). The pooled organics were washed with brine (10 mL),
dried over MgSO4, and evaporation to afford a residue that
was purified by flash chromatography, eluting with methanol/
dichloromethane (1:25) to give 14 (26 mg, 42%) as a colorless
oil: 1H NMR (d6-acetone) δ: 2.43 (s, 3H), 2.44 (s, 3H), 4.01 (s,
3H), 4.01 (s, 3H), 4.03 (s, 3H), 4.15 (s, 3H), 5.23 (s, 2H), 5.60
(s, 2H), 7.13 (d, J ) 2.4 Hz, 1H), 7.13 (d, J ) 2.4 Hz, 1H), 7.75
(dd, J ) 1.8, 7.8 Hz, 1H), 7.86 (d, J ) 2.4 Hz, 1H), 7.86 (d, J
) 2.4 Hz, 1H), 7.91 (d, J ) 7.8 Hz, 1H), 8.18 (d, J ) 1.8 Hz,
1H); 13C NMR (d6-acetone) δ: 31.9, 31.9, 60.4, 60.5, 60.5, 60.6,
74.1, 75.6, 105.1, 105.1, 116.6, 116.7, 124.6, 124.6, 127.0, 127.4,
130.3, 131.9, 135.4, 135.6, 136.6, 139.9, 144.4, 144.5, 145.2,
149.4, 167.2, 204.1, 204.2; IR (film) 1612, 1700; MS (ES+) m/z
619 (M + H+); Anal. (C33H30O12): C, H.
2,5-Bis(5-a cetyl-4,7-d im eth oxyben zofu r a n -6-yloxym e-
th yl)ben zoic Acid , Meth oxytetr a k is(eth ylen e glycol) Es-
ter (15). To a suspension of 13 (80 mg, 0.13 mmol) in
tetrakis(ethylene glycol) monomethyl ether (720 µL, 3.80
mmol) was added titanium(IV) isopropoxide (36 µL, 0.12
mmol), and the reaction mixture was stirred under nitrogen
at 100 °C for 14 h. The reaction was quenched with 1 M HCl
(1 mL) and resulting mixture partitioned over 1 M HCl/ethyl
acetate (1:1, 120 mL). The organic phase was washed with
saturated sodium bicarbonate (60 mL), dried over MgSO4, and
concentrated in vacuo. The resulting residue was purified by
3-(2,3-Dih yd r o-4,7-d im eth oxy-6-h yd r oxyben zofu r a n -5-
yl)-1-p h en yl-3-oxop r op a n e (18). A suspension of 16 (162 mg,
0.50 mmol) and 10% Pd on carbon (60 mg) in ethyl acetate (3
mL) was treated with hydrogen at one atmosphere for 16 h.
The reaction mixture was filtered through Celite and washed
with ethyl acetate, and the filtrate was concentrated in vacuo.
The resulting solid was recrystallized from methanol to give
18 (103 mg, 63%) as pale yellow needles: Mp 113-114 °C
(methanol); 1H NMR (CDCl3) δ: 3.00 (t, J ) 7.5 Hz, 2H), 3.29
(m, 4H), 3.84 (s, 3H), 3.87 (s, 3H), 4.65 (t, J ) 8.5 Hz, 2H),
7.16-7.31 (m, 5H); 13C NMR (CDCl3) δ: 28.1, 30.7, 45.0, 59.4,
60.6, 72.9, 108.0, 108.4, 125.9, 128.4, 128.4, 141.5, 154.0, 158.6,
159.2, 204.8; IR (film) 1621; MS (ES+) m/z 329 (M + H+); Anal.
(C19H20O5): C, H.
1,4-Bis[4,7-d im et h oxy-5-(1-p h en yl-3-oxop r op en -3-yl)-
ben zofu r a n -6-yloxym eth yl]ben zen e (19). Compound 16 (34
mg, 0.10 mmol) and R,R′-dibromo-p-xylene (13 mg, 0.05 mmol)
were treated as described in General Procedure A. The crude
product was recrystallized from methanol to give 19 (25 mg,
67%) as a colorless solid: Mp 161-162 °C (methanol); 1H NMR
(CDCl3) δ: 3.93 (s, 6H), 4.05 (s, 6H), 4.97 (s, 4H), 6.89 (d, J )
2.2 Hz, 2H), 6.92 (d, J ) 15.9 Hz, 2H), 7.24 (d, J ) 15.9 Hz,
2H), 7.30-7.35 (m, 6H), 7.39-7.44 (m, 4H), 7.60 (d, J ) 2.2
Hz, 2H); 13C NMR (CDCl3) δ: 61.1, 61.2, 105.0, 116.8, 122.4,
128.3, 128.4, 128.8, 128.9, 130.4, 134.6, 136.8, 144.6, 144.9,
145.1, 145.6, 148.8, 194.3; IR (film) 1648; MS (ES+) m/z 751
(M + H+); Anal. (C46H38O10): C, H.
1,3,5-Tr is(5-a cetyl-4,7-d im eth oxyben zofu r a n -6-yloxy-
m eth yl)ben zen e (20). Khellinone 1a (71 mg, 0.3 mmol) and
R,R′,R′′-tribromomesitylene (36 mg, 0.1 mmol) were treated as
described in General Procedure A. The crude product was
purified by flash chromatography eluting with ethyl acetate/

2334 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Baell et al.
dichloromethane (1:19) to afford 20 (60 mg, 73%) as a colorless
solid: Mp 122-126 °C; 1H NMR (CDCl3) δ: 2.48 (s, 6H), 3.99
(s, 6H), 4.11 (s, 6H), 5.13 (s, 4H), 6.88 (d, J ) 2.1 Hz, 2H),
7.51 (s, 2H), 7.59 (d, J ) 2.1 Hz, 2H); 13C NMR (CDCl3) δ:
32.6, 61.1, 61.2, 76.5, 105.0, 116.7, 124.7, 127.7, 134.6, 137.6,
144.0, 144.3, 144.6, 148.6, 201.7; IR (film) 1699; MS (ES+) m/z
824 (M + H+); HRMS (ES+) calcd C45H43O15 (M + H+) 823.260,
found 823.262.
mg, 60%) as red needles: Mp 157-159 °C (methanol/water);
1H NMR (d6-acetone) δ: 3.94 (s, 3H), 4.13 (s, 3H), 6.94 (d, J )
7.8 Hz, 1H), 7.15 (d, J ) 2.1 Hz, 1H), 7.23 (m, 2H), 7.30 (dd,
J ) 7.8, 7.8 Hz, 1H), 7.69 (m, 2H), 7.78 (d, J ) 2.1 Hz, 1H),
8.55 (br s, 1H); 13C NMR (d6-acetone) δ: 60.9, 61.8, 106.5,
112.9, 113.0, 115.4, 118.6, 121.1, 128.3, 129.9, 131.0, 140.4,
144.1, 145.3, 151.3, 152.7, 153.0, 158.7, 195.4; IR (film) 1626,
3429; MS (ES+) m/z 341 (M + H+); Anal. (C19H16O6): C, H.
5-Acetyl-6-ben zyloxy-4,7-d im eth oxyben zofu r a n (21).
To a solution of 1a (5.0 g, .21 mmol) in acetone (200 mL) were
added potassium carbonate (10 g) and benzyl bromide (3.6 g,
21 mmol). The reaction mixture was heated at reflux with
vigorous stirring. After 8 h the solid was filtered and the
filtrate was evaporated to dryness. The resulting residue was
recrystallized from ethanol to give 21 (5.9 g, 85%) as a colorless
solid: Mp 88 °C; lit.37 86 °C (EtOH); 1H NMR (CDCl3) δ: 2.44
(s, 3H), 3.98 (s, 3H), 4.09 (s, 3H), 5.08 (s, 2H), 6.88 (d, J ) 2.3
Hz, 1H), 7.45-7.34 (m, 5H), 7.59 (d, J ) 2.3 Hz, 1H).
1,3-Bis[3-(4,7-d im eth oxy-6-h yd r oxyben zofu r a n -5-yl)-3-
oxop r op en -1-yl]ben zen e (22). Khellinone 1a (94 mg, 0.4
mmol) and isophthalaldehyde (27 mg, 0.2 mmol) were treated
as described in General Procedure B. The crude product was
recrystallized from THF/ethanol to give 22 (80 mg, 70%) as a
brown solid: Mp 166-170 °C (THF/ethanol); 1H NMR (CDCl3)
δ: 4.07 (s, 6H), 4.09 (s, 6H), 6.89 (d, J ) 2.1 Hz, 2H), 7.49 (t,
J ) 7.8 Hz, 1H), 7.53 (d, J ) 2.1 Hz, 2H), 7.69 (ddd, J ) 1.5,
1.5, 7.8 Hz, 2H), 7.84 (d, J ) 15.9 Hz, 2H), 7.85 (t, J ) 1.5 Hz,
1H), 7.93 (d, J ) 15.9 Hz, 2H), 12.62 (s, 2H); 13C NMR (CDCl3)
δ: 60.9, 61.9, 105.1, 111.8, 112.7, 127.9, 128.5, 129.8, 135.9,
142.2, 144.2, 150.7, 152.0, 153.1, 196.5; IR (KBr) 1630, 3134;
MS (ES+) m/z 571 (M + H+); HRMS (ES+) calcd C32H27O10 (M
+ H+) 571.1604, found 571.1599.
3-(4,7-Dim eth oxy-6-h ydr oxyben zofu r an -5-yl)-1-(3-m eth -
oxyp h en yl)-3-oxop r op en e (27). Khellinone 1a (236 mg, 1.0
mmol) and 3-methoxybenzaldehyde (122 µL, 1.0 mmol) were
treated as described in General Procedure B. The crude
product was recrystallized from dichloromethane/ethanol to
give 27 (213 mg, 60%) as deep red needles: Mp 99 °C (DCM/
1
ethanol); H NMR (CDCl3) δ: 3.84 (s, 3H), 4.02 (s, 3H), 4.07
(s, 3H), 6.86 (d, J ) 2.1 Hz, 1H), 6.95 (d, J ) 7.8 Hz, 1H), 7.14
(s, 1H), 7.23 (d, J ) 7.8 Hz, 1H), 7.32 (dd, J ) 7.8, 7.8 Hz,
1H), 7.50 (d, J ) 2.1 Hz, 1H), 7.81 (m, 2H); 13C NMR (CDCl3)
δ: 55.3, 61.0, 61.9, 105.2, 111.9, 112.7, 113.6, 116.1, 121.1,
127.3, 129.5, 129.9, 136.5, 143.3, 144.1, 150.7, 151.9, 153.2,
159.9, 194.7; IR (film) 1623, 3123; MS (ES+) m/z 355 (M + H+);
Anal. (C20H18O6): C, H.
3-(4,7-Dim eth oxy-6-h yd r oxyben zofu r a n -5-yl)-1-(4-m e-
th ylp h en yl)-3-oxop r op en e (28). Khellinone 1a (236 mg, 1.0
mmol) and 4-tolualdehyde (118 µL, 1.0 mmol) were treated as
described in General Procedure B. The crude product was
recrystallized from dichloromethane/methanol to give 28 (90
mg, 27%) as an orange powder: Mp 148 °C (DCM/methanol);
1H NMR (CDCl3) δ: 2.38 (s, 3H), 4.01 (s, 3H), 4.06 (s, 3H),
6.85 (d, J ) 2.1 Hz, 1H), 7.21 (d, J ) 7.8 Hz, 2H), 7.49 (d, J )
2.1 Hz, 1H), 7.53 (d, J ) 7.8 Hz, 2H), 7.83 (m, 2H); 13C NMR
(CDCl3) δ: 21.5, 61.0, 62.0, 105.2, 111.9, 112.8, 125.9, 128.5,
129.5, 129.7, 132.4, 141.1, 143.7, 144.1, 150.7, 151.8, 153.2,
194.7; IR (film) 1625, 3126; MS (ES+) m/z 339 (M + H+); HRMS
(ES+) calcd C20H18O5 (M + H+) 339.123, found 339.124; Anal.
(C20H18O5): C: calcd 71.0 found 69.4, H.
1-(4,7-Dim eth oxy-6-h ydr oxyben zofu r a n -5-yl)-5-p h en yl-
p en ta -2,4-d ien -1-on e (23). Khellinone 1a (236 mg, 1.0 mmol)
and cinnamaldehyde (126 µL, 1.0 mmol) were treated as
described in General Procedure B. The crude product was
recrystallized from dichloromethane/ethanol to give 23 (94 mg,
N,N′-Bis[1-(4,7-d im eth oxy-6-h yd r oxyben zofu r a n -5-yl)-
eth ylid en e-1-yl]h yd r a zin e (29). Meth od 1: A suspension
of succinic acid dihydrazide (146 mg, 1.0 mmol) and khellinone
1a (472 mg, 2.0 mmol) in methanol (2 mL) was heated at reflux
for 36 h. The reaction mixture was concentrated in vacuo, and
the resulting residue was filtered, suspended in dichlo-
romethane (25 mL), and washed with dichloromethane (3 ×
25 mL). The filtrate was purified by flash chromatography
eluting with ethyl acetate/dichloromethane (1:9) to afford 29
(167 mg, 36%) as a yellow powder. An analytical sample was
obtained by recrystallization from dichloromethane/ethanol:
1
27%) as an orange solid: Mp 123-125 °C (DCM/ethanol); H
NMR (CDCl3) δ: 4.02 (s, 3H), 4.06 (s, 3H), 6.85 (d, J ) 2.2 Hz,
1H), 7.01 (d, J ) 5.4 Hz, 2H), 7.30-7.42 (m, 7H), 7.67 (ddd, J
) 5.4, 5.4, 14.8 Hz, 1H); 13C NMR (CDCl3) δ: 60.6, 61.5, 104.9,
105.3, 111.4, 112.2, 126.9, 127.0, 128.5, 128.9, 129.1, 130.0,
135.8, 141.6, 143.7, 143.8, 150.3, 151.5, 152.9, 164.1; IR (film)
1625, 3120; MS (ES+) m/z 373 (M + Na+); HRMS (ES+) calcd
C
21H19O5 (M + H+) 351.123, found 351.122.
3-(4,7-D i m e t h o x y -6-h y d r o x y b e n z o fu r a n -5-y l)-1-
(th iop h en -2-yl)-3-oxop r op en e (24). Khellinone 1a (94 mg,
0.4 mmol) and thiophene-2-carboxaldehyde (37 µL, 0.4 mmol)
were treated as described in General Procedure B. The crude
product was recrystallized from methanol to give 24 (80 mg,
61%) as an orange solid: Mp 129 °C (MeOH) lit.,38 125 °C; 1H
NMR (CDCl3) δ: 4.05 (s, 3H); 4.06 (s, 3H), 6.87 (d, J ) 2.3 Hz,
1H), 7.08 (dd, J ) 3.7, 5.0 Hz, 1H), 7.35 (d, J ) 3.7 Hz, 1H),
7.40 (d, J ) 5.0 Hz, 1H), 7.50 (d, J ) 2.3 Hz, 1H), 7.69 (d, J )
15.3 Hz, 1H), 7.98 (d, J ) 15.3 Hz, 1H).
1
Mp 167-168 °C (DCM/ethanol); H NMR (CDCl3) δ: 2.68 (s,
6H), 4.02 (s, 6H), 4.11 (s, 6H), 6.86 (d, J ) 2.1 Hz, 2H), 7.50
(d, J ) 2.1 Hz, 2H); 13C NMR (CDCl3) δ: 19.6, 60.9, 61.1, 105.0,
110.9, 112.6, 129.3, 143.4, 149.2, 149.2, 149.8, 168.3; IR (KBr)
1616, 3157; MS (ES+) m/z 469 (M + H+); Anal. (C24H24N2O8):
C, H, N. Meth od 2: A solution of hydrazide hydrate (25 µL,
0.5 mmol) and khellinone 1a (236 mg, 1.0 mmol) in methanol
was heated at reflux for 16 h. The reaction mixture was
concentrated in vacuo and was purified by flash chromatog-
raphy, eluting with ethyl acetate/dichloromethane (1:9) to
afford 29 (60 mg, 26%) as a yellow powder.
1-(4-Br om oth iop h en -2-yl)-3-(4,7-d im eth oxy-6-h yd r oxy-
ben zofu r a n -5-yl)-3-oxop r op en e (25). Khellinone 1a (236
mg, 1.0 mmol) and 4-bromo-2-thiophenecarboxaldehyde (118
µL, 1.0 mmol) were treated as described in General Procedure
B. The crude product was recrystallized from dichloromethane/
ethanol to give 25 (160 mg, 39%) as orange needles: Mp 139
1,4-Bis(4,7-d im et h oxy-5-[(1RS)-h yd r oxyet h yl)]b en zo-
fu r a n -6-yloxy)xylen e (30). A mixture of 2 (115 mg, 0.2 mmol)
and tetrabutylammonium borohydride (307 mg, 1.2 mmol) in
dichloromethane (1 mL) was stirred at room temperature for
48 h, and the reaction mixture was purified by flash chroma-
tography, eluting with dichloromethane to afford 30 (64 mg,
1
°C (DCM/ethanol); H NMR (CDCl3) δ: 4.04 (s, 3H), 4.05 (s,
3H), 6.86 (d, J ) 2.3 Hz, 1H), 7.03 (d, J ) 3.9 Hz, 1H), 7.07 (d,
J ) 3.9 Hz, 1H), 7.49 (d, J ) 2.3 Hz, 1H), 7.57 (d, J ) 15.3 Hz,
1H), 7.84 (d, J ) 15.3 Hz, 1H); 13C NMR (CDCl3) δ: 61.0, 61.7,
105.3, 111.5, 112.5 116.3, 126.2, 129.5, 131.4, 132.2, 135.1,
142.4, 144.1, 150.7, 152.0, 153.3, 193.6; IR (film) 1616; MS
(ES+) m/z 409, 411 (M + H+); Anal. (C17H13BrO5S): C, H, Br,
S.
1
55%) as a colorless solid: Mp 182-187 °C; H NMR (CDCl3)
δ: 1.52 (d, J ) 6.6 Hz, 6H), 3.83 (d, J ) 9.0 Hz, 2H), 4.04 (s,
6H), 4.09 (s, 6H), 5.19 (AB, J ) 10.8 Hz, 4H), 5.32 (dq, J )
6.9, 9.0 Hz, 2H), 7.05 (d, J ) 2.1 Hz, 2H), 7.62 (s, 4H), 7.80 (d,
J ) 2.1 Hz, 2H); 13C NMR (CDCl3) δ: 25.1, 60.8, 61.1, 65.1,
75.8, 104.8, 116.6, 124.6, 128.4, 134.5, 137.2, 144.1, 145.3,
145.6, 147.4; IR (KBr) 3550; MS (ES+) m/z 601 (M + Na+);
Anal. (C32H34O10): C, H.
3-(4,7-Dim et h oxy-6-h yd r oxyben zofu r a n -5-yl)-1-(3-h y-
d r oxyp h en yl)-3-oxop r op en e (26). Khellinone 1a (709 mg,
3.0 mmol) and 3-hydroxybenzaldehyde (440 mg, 3.6 mmol)
were treated as described in General Procedure B. The crude
product was recrystallized from methanol/water to give 26 (609
3-(6-Hyd r oxy-4-m et h oxyben zofu r a n -5-yl)-1-p h en yl-3-
oxop r op en e (31). Compound 31 was prepared according to

Blockers of the Voltage-Gated Potassium Channel Kv1.3
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2335
the General Procedure B from visnaginone 1b (103 mg, 0.50
mmol) and benzaldehyde (56 µL, 0.55 mmol) and was recrys-
tallized from ethanol (37 mg, 25%) as orange plates: Mp 100-
101 °C (EtOH) lit.36 106 °C (EtOH); 1H NMR (CDCl3) δ: 4.15
(s, 3H), 6.81 (s, 1H), 6.88 (d, J ) 2.4 Hz, 1H), 7.40-7.44 (m,
3H), 7.47 (d, J ) 2.4 Hz, 1H), 7.64 (m, 2H), 7.85 (m, 2H); 13C
NMR (CDCl3) δ: 61.1, 94.9, 105.1, 110.7, 111.2, 127.3, 128.3,
128.9, 130.3, 135.2, 142.8, 143.6, 155.9, 160.5, 162.1, 194.4.
1,4-Bis(5-a cetyl-4-m eth oxyben zofu r a n -6-yloxym eth yl)-
ben zen e (32). Compound 32 was prepared from visnaginone
1b (150 mg, 0.73 mmol) and R,R′-dibromo-p-xylene (96 mg, 0.36
mmol) according to General Procedure A. Purification by flash
chromatography eluting with ethyl acetate/cyclohexane (1:9
to 2:8) afforded the product (20 mg, 11%) as a white solid: Mp
196-197 °C; 1H NMR (CDCl3) δ: 2.51 (s, 6H), 4.05 (s, 6H),
5.10 (s, 4H), 6.80 (s, 2H), 6.86 (d, J ) 2.3 Hz, 2H), 7.40 (s,
4H), 7.49 (d, J ) 2.3 Hz, 2H); IR (film) 1609; MS (thermospray,
LC/MS) m/z 515 (M + H+); Anal. (C30H26O8): C, H
Electr op h ysiology. The effectiveness of the generated
compounds in blocking Kv1.3 was assayed on L929 cells stably
expressing mKv1.339 or on activated human T cells. All
experiments were conducted in the whole-cell configuration
of the patch-clamp technique with a holding potential of -80
mV. Currents were recorded in normal Ringer solution (160
mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM
HEPES, pH 7.4, 290-310 mOsm) with an internal pipet
solution containing 134 mM KF, 2 mM MgCl2, 10 mM HEPES,
10 mM EGTA (pH 7.2, 290-310 mOsm). If currents exceeded
2 nA 60-80% series resistance compensation was used.
Depolarizing pulses to 40 mV were applied every 45 s for 500
ms. Kv.1.1, Kv1.2, Kv1.5, Kv1.7, and IKCa1 currents were
recorded from cells either stably or transciently expressing
these currents as previously described.39-41 Kd values were
determined by fitting the reduction of area under the current
curve to the Hill equation.
mL dose of 5 mg/kg 7, 16, 26, or 27 in mammalian Ringer
solution with 1% ethanol and 2.5% bovine serum albumin. Five
control mice were injected with an equal volume of the vehicle.
Mice were observed for adverse effects immediately after
dosing, at 4 h after injection and daily for 7 days.
Ack n ow led gm en t. The authors are deeply indebted
to Prof. K. George Chandy for making it possible to
conduct the electrophysiological experiments in his
laboratory and to Prof. Ray Norton for facilitating a
fruitful collaboration. We also thank Elke Stoll for
excellent technical assistance. This research was funded
by the Western States Affiliate of AHA (H.W.) and the
National Multiple Sclerosis Society (H.W.).
Su p p or tin g In for m a tion Ava ila ble: Structures and
Kv1.3 blocking data for less active compounds. Crystal data,
atomic coordinates, anisotropic displacement parameters, and
geometrical parameters of 2. This material is available free
of charge via the Internet at http://pubs.acs.org.
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Ca lcu la tion of Aqu eou s Solu bility. Calculated aqueous
solubility utilized the General Solubility Equation:42 log Sw
)
0.5 - log Kow - 0.01(MP - 25), where Kow is the octanol-water
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2336 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
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