Kv1.3-blocking 5-phenylalkoxypsoralens: A new class of immunomodulators

Article abstract of DOI:10.1124/mol.65.6.1364

The lymphocyte potassium channel Kv1.3 is widely regarded as a promising new target for immunosuppression. To identify a potent small-molecule Kv1.3 blocker, we synthesized a series of 5-phenylalkoxypsoralens and tested them by whole-cell patch clamp. The

Full text of DOI:10.1124/mol.65.6.1364

0
026-895X/04/6506-1364–1374$20.00
MOLECULAR PHARMACOLOGY
Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 65:1364–1374, 2004
Vol. 65, No. 6
3108/1151020
Printed in U.S.A.
Kv1.3-Blocking 5-Phenylalkoxypsoralens: A New Class of
Immunomodulators
Julia Vennekamp, Heike Wulff, Christine Beeton, Peter A. Calabresi, Stephan Grissmer,
¨
Wolfram Hansel, and K. George Chandy
Pharmaceutical Institute, University of Kiel, Kiel, Germany (J.V., W.H.); Department of Medical Pharmacology and Toxicology,
University of California at Davis, Davis, California (H.W.); Department of Physiology and Biophysics, University of California at
Irvine, Irvine, California (C.B., K.G.C.); Department of Pathology, Johns Hopkins Hospital, Baltimore, Maryland (P.A.C.); and
Department of Applied Physiology, University of Ulm, Ulm, Germany (S.G.)
Received November 21, 2003; accepted February 24, 2004
This article is available online at http://molpharm.aspetjournals.org
ABSTRACT
ϩ
The lymphocyte potassium channel Kv1.3 is widely regarded as nel, Kv3.1, the calcium-activated K channels (IKCa1, SK1–
a promising new target for immunosuppression. To identify a SK3, and BKCa), or the neuronal Na 1.2 channel. In a test of in
V
potent small-molecule Kv1.3 blocker, we synthesized a series vivo toxicity in rats, Psora-4 did not display any signs of acute
of 5-phenylalkoxypsoralens and tested them by whole-cell toxicity after five daily subcutaneous injections at 33 mg/kg
patch clamp. The most potent compound of this series, 5-(4- body weight. Psora-4 selectively suppressed the proliferation
phenylbutoxy)psoralen (Psora-4), blocked Kv1.3 in a use-de- of human and rat myelin-specific effector memory T cells with
pendent manner, with a Hill coefficient of 2 and an EC50 value EC50 values of 25 and 60 nM, respectively, without persistently
of 3 nM, by preferentially binding to the C-type inactivated state suppressing peripheral blood naive and central memory T cells.
of the channel. Psora-4 is the most potent small-molecule Because autoantigen-specific effector memory T cells contrib-
Kv1.3 blocker known. It exhibited 17- to 70-fold selectivity for ute to the pathogenesis of T cell-mediated autoimmune dis-
Kv1.3 over closely related Kv1-family channels (Kv1.1, Kv1.2, eases such as multiple sclerosis, Psora-4 and other Kv1.3
Kv1.4, and Kv1.7) with the exception of Kv1.5 (EC50, 7.7 nM) blockers may be useful as immunomodulators for the therapy
and showed no effect on human ether-a-go-go-related chan- of autoimmune disorders.
2
ϩ
The voltage-gated Kv1.3 channel and the Ca -activated membrane depolarization, reduced Ca
2ϩ
entry, and dimin-
2ϩ
IKCa1 channel promote and sustain Ca signaling in hu- ished cytokine production and proliferation (DeCoursey et
man T cells by hyperpolarizing the membrane and providing al., 1984; Lin et al., 1993; Ghanshani et al., 2000; Fanger et
2
ϩ
the driving force for Ca entry through voltage-independent
Ca
2
al., 2001). All quiescent human T cells express ϳ10-fold more
Kv1.3 than IKCa1 channels (ϳ300 Kv1.3 versus ϳ20 IKCa1/
cell), but activation produces distinctive channel phenotypes
2ϩ
channels (Lewis and Cahalan, 1995; Cahalan et al.,
001). Selective blockade of Kv1.3 and/or IKCa1 results in
in naive, central memory (TCM) and effector memory (TEM
)
This work was supported by grants from the National Multiple Sclerosis subsets (Wulff et al., 2003b). Naive and TCM cells require
Society (to K.G.C., P.A.C., and C.B.), the National Institutes of Health
MH59222), the Rockefeller Brothers Fund (to K.G.C.), the American Heart
Association (to H.W.), the Deutsche Forschungsgemeinschaft (Gr848/8-2; to
antigen priming in lymph nodes before trafficking to sites of
inflammation, whereas TEM cells rapidly enter inflamed tis-
(
S.G.) and the Bundesministerium f u¨ r Bildung und Forschung (iZKF Ulm, B7; sues, secrete inflammatory cytokines, and exhibit immediate
to S.G.).
effector function (Sallusto et al., 1999). Mitogenic or anti-
genic stimulation of naive and TCM cells transcriptionally
augments IKCa1 expression (ϳ500 channels/cell) while not
J.V. and H.W. contributed equally to this work.
Parts of this work have been presented previously at the Meeting of the
German Pharmaceutical Society in Halle [Arch Pharm Pharm Med Chem
2
001;334 (Suppl 2):C72], the Experimental Biology Meeting in San Diego
(
[
FASEB J 2003;17:A1051), and in the doctoral thesis of Julia Vennekamp changing Kv1.3 expression. On the other hand, TEM cells
Vennekamp J (2002) Synthese und elektrophysiologische Testung hochwirk-
up-regulate Kv1.3 but not IKCa1 during activation (ϳ1500
versus ϳ20/cell). These differences dictate the effectiveness
samer Psoralenderivate als nicht-peptidische Blocker des lymphozyt a¨ ren Kali-
umkanals Kv1.3. thesis, University of Kiel, Germany].
ABBREVIATIONS: TCM, central memory T cell subset; TEM, effector memory T cell subset; 5-MOP, 5-methoxysporalen; EAE, experimental
ϩ
autoimmune encephalomyelitis; MBP, myelin basic protein; MS, multiple sclerosis; K , voltage-gated K channel; ShK, Stichodactyla helianthus
v
toxin; HERG, human ether-a-go-go-related gene; HPLC, high-performance liquid chromatography; PBMC, peripheral blood mononuclear cells;
3
[
H]TdR, tritiated thymidine; WIN-17317–3, 1-benzyl-7-chloro-4-n-propylimino-1,4-dihydroquinoline hydrochloride; CP-339818, 1-benzyl-4-pen-
tylimino-1,4-dihydroquinoline; UK-78282, 4-[(diphenylmethoxy)methyl]-1-[3-(4-methoxyphenyl)propyl]-piperidine.
1
364

Psora-4, a Small-Molecule Kv1.3 Inhibitor
1365
of Kv1.3 and IKCa1 blockers in suppressing proliferation of et al., 2000), but its micromolar affinity for Kv1.3 necessi-
naive/TCM versus TEM cells. Naive/TCM cells are initially tated further analog development.
sensitive to Kv1.3 blockade but rapidly escape Kv1.3 inhibi-
In the present study, we describe Psora-4, a 5-phenyl-
tion by up-regulating IKCa1 and become sensitive to IKCa1 alkoxypsoralen that preferentially binds to the C-type inac-
blockade. In contrast, Kv1.3 blockers persistently and po- tivated state of Kv1.3. Psora-4 blocks Kv1.3 with an EC50
tently suppress the proliferation of TEM cells. The recent value of 3 nM, making it the most potent small-molecule
discovery that myelin-reactive T cells from patients with Kv1.3 inhibitor known. Psora-4 preferentially suppresses the
multiple sclerosis (MS) are Kv1.3-dependent TEM cells (Wulff proliferation of human and rat TEM cells and does not cause
acute toxicity when administered in vivo. Psora-4 may have
use as a therapeutic in autoimmune disorders.
et al., 2003b) that arise as a consequence of repeated anti-
genic stimulation during the course of disease and contribute
to disease pathogenesis has raised interest in developing
Kv1.3 blockers for the therapy of autoimmune disorders.
Several peptide and small-molecule inhibitors of Kv1.3
have been developed over the last two decades (Chandy et al.,
Materials and Methods
Chemistry. 5-MOP was purchased from Roth (Karlsruhe, Ger-
many). 5-Hydroxypsoralen was prepared from 5-MOP through ether-
cleavage with MgI2 in anhydrous diethyl ether (Schoenberg and
Aziz, 1953). The 5-phenylalkoxypsoralens (compounds 1–9 in Fig. 1)
and the 5-cyclohexylalkoxypsoralens (compounds 10–12) were syn-
2
001; Wulff et al., 2003a). The most potent peptide inhibitor,
ShK, from the Caribbean sea anemone Stichodactyla heli-
anthus (K , 11 pM), inhibits proliferation of T cells with an
d
EM
EC50 of 400 pM. ShK and kaliotoxin, another Kv1.3 blocker, thesized starting from 5-hydroxypsoralen according to the following
prevented and reversed the symptoms of experimental auto- general method: a mixture of 5-hydroxypsoralen, anhydrous K CO ,
3
2
a catalytic amount of KI, and the respective alkylating agent were
heated to reflux in anhydrous acetone under nitrogen. After comple-
tion of the reaction, the mixture was poured into ice-cold water,
acidified with HCl, and the resulting precipitate was collected by
vacuum filtration and dissolved in CH Cl . This solution was washed
immune encephalomyelitis (EAE), an animal model for MS,
high
produced by the transfer of Kv1.3
memory cells into naive rat recipients (Beeton et al.,
001a,b). No side effects were observed. These peptides have
myelin-specific rat
2
2
2
potential therapeutic usefulness for autoimmune disorders
but have to be administered parenterally, have a short cir-
with NaOH (2%) several times, dried over Na SO , concentrated
2
4
under reduced pressure, and the residue was recrystallized. Com-
1
13
culating half-life, and their toxicity profiles remain to be pounds were characterized by melting point, IR, H and C NMR
determined.
(for numbering, see compound 1 in Fig. 1), mass spectrometry, and
Small-molecule inhibitors of Kv1.3 have chemically diverse combustion analysis. C data are given only for Psora-4 (compound
13
4
) and are available upon request for the other compounds. IR data
are also available on request.
-Benzyloxypsoralen (Compound 1). This was prepared as
described previously (Caporale and Antonello, 1958).
-(2-Phenylethoxy)psoralen (Compound 2). 5-Hydroxypsor-
structures. Tetraethylammonium and 4-aminopyridine with
millimolar potency for the channel prevented EAE in rats at
concentrations that suppressed mitogen-induced prolifera-
tion of these cells (Judge et al., 1997a,b). The first small
molecules to exhibit nanomolar potency were the iminodihy-
droquinolines (WIN-17317–3, CP-339818) and the benzyl pi-
5
5
alen (150 mg, 0.7 mmol), 2-phenylethylbromide (2.0 ml, 14.8 mmol),
and K CO3 (0.2 g) were heated in 10 ml of acetone for 5 h. The
2
peridines (UK-78282), but these were not developed further product was recrystallized from methanol/H O (80:20) as a yellowish
2
1
because of a lack of specificity for Kv1.3. Subsequent efforts solid (90 mg, 40%): m.p. ϭ 154.5°C; H NMR (CDCl ) ␦ 3.18 (t, 2 H,
3
3
3
J ϭ 6.7 Hz, 5-OCH CH C H ), 4.65 (t, 2 H, J ϭ 6.7 Hz,
-OCH CH C H ), 6.21 (d, 1 H, J ϭ 9.8 Hz, H-3), 6.84 (dd, 1 H, J ϭ
.4 Hz, J ϭ 0.9 Hz, H-4Ј), 7.14 (s, br, 1 H, H-8), 7.28 to 7.38 [m, 5 H,
-O(CH ) C H ], 7.56 (d, 1 H, J ϭ 2.4 Hz, H-5Ј), 7.94 (d, 1 H, J ϭ
.8 Hz, H-4); MS m/z 306 (M ), 106, 105 (C H ), 103, 89, 79, 78, 77
C H ), 63, 51 (C H ). Calculated for C19H14O (306.32): C, 74.50%;
led to the development of correolide and trans-N-propylcarbamoy-
loxy-4-phenyl-4[3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl]cyclo-
hexanone by Merck, dichlorophenylpyrazolopyrimidines by Bris-
tol-Myers Squibb Co., and sulfamidebenzamidoindanes by Icagen
2
2
6
5
3
3
5
2
5
9
2 2 6 5
5
3
3
2
2
6
5
ϩ
ϩ
9
8
(reviewed in Wulff et al., 2003a). None is selective and/or particu-
ϩ
ϩ
(
6 5 4 3 4
larly potent for Kv1.3, necessitating a search for better blockers.
In the early 1990s, anecdotal reports from Chile and Aus-
H, 4.61%; O, 20.89%. Found: C, 74.43%; H 4.67%.
5-(3-Phenylpropoxy)psoralen (Compound 3). 5-Hydroxypsor-
tria suggesting that tea made from Ruta graveolens allevi- alen (300 mg, 1.5 mmol), 3-phenylpropylbromide (0.3 ml, 2.0 mmol),
ated the symptoms of MS instigated a search for the phar- and K
CO (0.6 g) were heated in 15 ml of acetone for 8 h. The
2
3
product was recrystallized from ethanol/H O (80:20) as a yellowish
macologically active agent. Because extracts from R.
2
1
ϩ
solid (120 mg, 25%): m.p. ϭ 109°C; H NMR (CDCl ) ␦ 2.22 (quint, 2
H, J ϭ 6.9 Hz, 5-OCH
3
graveolens blocked delayed-rectifier K currents in nodes of
3
3
ϩ
2
CH
2 2 6 5
CH C H ), 2.88 [t, 2 H, J ϭ 7.5 Hz,
Ranvier (Bohuslavizki et al., 1994), blockade of K currents,
3
5
6
-O(CH ) CH C H ], 4.46 [t, 2 H, J ϭ 6.3 Hz, 5-OCH (CH ) C H ],
.27 (d, 1 H, J ϭ 9.8 Hz, H-3), 6.85 (dd, 1 H, J ϭ 2.2 Hz, J ϭ 0.8
2
2
2
3
6
5
2
2 2
5
6
5
either in demyelinated neurons or in myelin-reactive lym-
phocytes, was thought to underlie the beneficial effects of R.
graveolens. Through extensive screening, 5-methoxypsoralen
3
Hz, H-4Ј), 7.13 (s, 1 H, H-8), 7.21 to 7.34 [m, 5 H, 5-O(CH ) C H ],
2
3
6
5
3
3
7.55 (d, 1 H, J ϭ 2.4 Hz, H-5Ј), 8.10 (d, 1 H, J ϭ 9.8 Hz, H-4); MS
ϩ
ϩ
10
ϩ
ϩ
11
(
5-MOP), a compound clinically used in the therapy of psori- m/z 320 (M ), 202 (M-C H ), 174 (202-CO ), 119 (C H ), 92, 91
9
9
ϩ
ϩ
ϩ
ϩ
asis, was identified as the major K channel-blocking prin- (C
ciple of R. graveolens (Bohuslavizki et al., 1994); it blocked
Kv1.2 channels in neurons and Kv1.3 channels in T cells.
5
tients (Bohuslavizki et al., 1993), but its phototoxic activity
precluded its use as a therapeutic for MS. We therefore
generated a nonphototoxic psoralen called H37 (Wulff et al.,
H
7
), 89, 65 (C H ), 51 (C H ), 41. Calculated for C20H O
7
5 5 4 3 16 4
(
5
320.34): C, 74.99%; H, 5.03%; O, 19.98%. Found: C, 74.20%; H,
.14%.
5
-(4-Phenylbutoxy)psoralen (Compound 4). 5-Hydroxy-
-MOP reportedly improved functional deficits in MS pa-
psoralen (300 mg, 1.5 mmol), 4-phenylbutylchloride (0.5 ml,
3
3
.0 mmol), and K CO3 (0.6 g) were heated in 15 ml of acetone for
1 h. The product was recrystallized from ethanol/H O (90:10)
2
2
1
as a white solid (109 mg, 22%): m.p. ϭ 98°C; H NMR (CDCl3)
1
998) that suppressed cytokine secretion and proliferation of ␦ 1.86 to 1.91 [m, 4 H, 5-OCH (CH ) CH C H ], 2.73 [t, 2 H,
2 2 2 2 6 5
3
3
myelin-reactive encephalitogenic rat memory T cells (Strauss
J ϭ 7.2 Hz, 5-O(CH ) CH C H ], 4.44 [t, 2 H, J ϭ 6.0 Hz,
2 3 2 6 5

1
366
Vennekamp et al.
3
3
5
13
5
-OCH (CH ) C H ], 6.27 (d, 1 H, J ϭ 9.8 Hz, H-3), 6.89 (dd, 1 H,
J ϭ 9.8 Hz, J ϭ 0.7 Hz, H-4); C NMR (CDCl ) ␦ 27.70 and 29.56
3
2
2 3
5
6
5
3
5
5
J ϭ 2.4 Hz, J ϭ 1.0 Hz, H-4Ј), 7.13 (dd, 1 H, J ϭ 0.9 Hz, J ϭ [5-OCH (CH ) CH C H ], 35.52 [5-O(CH ) CH C H ], 72.82
2
2
2
2
6
5
2
3
2
6
5
0
5
.7 Hz, H-8), 7.18 to 7.23 and 7.27 to 7.33 [2 m, 3 H and 2 H, [5-OCH (CH ) C H ], 93.95 (C-8), 105.10 (C-4Ј), 106.83 (C-4a),
2 2 3 6 5
3
-O(CH ) C H ], 7.57 (d, 1 H, J ϭ 2.4 Hz, H-5Ј), 8.12 (dd, 1 H, 112.62 (C-3), 113.35 (C-6), 126.03 (C-4“), 128.41 and 128.46 (C-2”,
2
4
6
5
Fig. 1. Chemical structures of 5-MOP and its analogs Psora-1 through Psora-12. The numbering of the psoralen system (shown for Psora-1) is in
accordance with the nomenclature most commonly used in biochemical literature.

Psora-4, a Small-Molecule Kv1.3 Inhibitor
1367
3
C-3“, C-5”, C-6“), 139.31 (C-4), 141.78 (C-1”), 144.78 (C-5Ј), 149.01 (m, 10 H, 5-OCH C H C H and H-5Ј), 8.11 (dd, 1 H, J ϭ 9.8 Hz,
2
6
4
6
5
ϩ
5
ϩ
ϩ
(
C-5), 152.74 (C-8a), 158.28 (C-7), 161.25 (C-2); MS m/z 334 (M ),
J ϭ 0.6 Hz, H-4); MS m/z 368 (M ), 168, 167 (C13H ), 166, 165,
11
ϩ
ϩ
ϩ
ϩ
ϩ
2
6
7
03, 202 (M-C10H ), 174 (202-CO ), 133 (C10H ), 92, 91 (C H ), 152, 145, 115, 89, 63, 51 (C H ). Calculated for C24H16O (368.39):
12 13 7 7 4 3 4
ϩ ϩ
5 (C H ), 51 (C H ), 44. Calculated for C21H18O4 (334.37): C, C, 78.25%; H, 4.38%; O, 17.37%. Found: C, 78.285; H, 4.34%.
5
5
4
3
5.43%; H, 5.43%; O, 19.14%. Found: C, 75.34%; H, 5.41%.
-(5-Phenylpentoxy)psoralen (Compound 5). 5-Hydroxypsor- soralen (150 mg, 0.7 mmol), cyclohexylmethylbromide (1.0 ml, 7.2
alen (300 mg, 1.5 mmol), 5-phenylpentylchloride (0.7 ml, 3.9 mmol), mmol), and K CO3 (0.2 g) were heated in 10 ml of acetone for 23 h.
5-Cyclohexylmethoxypsoralen (Compound 10). 5-Hydroxyp-
5
2
and K CO3 (0.6 g) were heated in 15 ml of acetone for 27 h. The
The product was recrystallized from ethanol/H O (80:20) as a white
2
2
1
product was recrystallized from ethanol/H O (90:10) as a white solid solid (79 mg, 36%): m.p. ϭ 153.5°C; H NMR (CDCl ) ␦ 1.13 to 1.38
2
3
1
3
(
125 mg, 24%): m.p. ϭ 94.5°C; H NMR (CDCl ) ␦ 1.53 to 1.59, 1.69 and 1.73 to 1.94 (2 m, 5 H and 6 H, 5-OCH C H ), 4.24 (d, 2 H, J ϭ
3
2
6
1
1
3
to 1.77, and 1.85 to 1.95 [3 m, 3 ϫ 2 H, 5-OCH (CH ) CH C H ], 2.67 5.9 Hz, 5-OCH C H ), 6.28 (d, 1 H, J ϭ 9.8 Hz, H-3), 6.95 (dd, 1 H,
2
2 3
2
6
5
2
6
11
3
3
3
3
5
[
t, 2,H, J ϭ 7.5 Hz, 5-O(CH ) CH C H ], 4.43 [t, 2 H, J ϭ 6.4 Hz,
J ϭ 2.4 Hz, J ϭ 0.9 Hz, H-4Ј), 7.13 (s, br, 1 H, H-8), 7.57 (d, 1 H,
ϩ
2
4
2
3
6
5
3
5
-OCH (CH ) C H ], 6.25 (d, 1 H, J ϭ 9.8 Hz, H-3), 6.91 (dd, 1,H,
J ϭ 2.4 Hz, H-5Ј), 8.17 (d, 1 H, J ϭ 9.8 Hz, H-4); MS m/z 298 (M ),
2
2 4
5
6
5
3
5
ϩ ϩ ϩ ϩ
J ϭ 2.4 Hz, J ϭ 1.0 Hz, H-4Ј), 7.12 (t, 1 H, J ϭ 0.8 Hz, H-8), 7.17 203, 202 (M-C H ), 174 (202-CO ), 97 (C H ), 69, 55 (C H ), 51
7
12
7
13
4
7
ϩ
to 7.21 and 7.25 to 7.31 [2 m, 3 H and 2 H, 5-O(CH ) C H ], 7.57 (d, (C H ), 43, 41. Calculated for C18H18O4 (298.34): C, 72.47%; H,
2
4
6
5
4
3
3
3
5
1
H, J ϭ 2.4 Hz, H-5Ј), 8.08 (dd, 1 H, J ϭ 9.8 Hz, J ϭ 0.6 Hz, H-4); 6.08%; O, 21.45%. Found: C, 72.355; H, 5.99%.
ϩ ϩ ϩ
MS m/z 348 (M ), 203, 202 (M-C11H ), 174 (202-CO ), 105, 91
5-(2-Cyclohexylethoxy)psoralen (Compound 11). 5-Hy-
14
ϩ
7
ϩ
5
ϩ
3
(
(
C H ), 65 (C H ), 51 (C H ), 49, 41. Calculated for C22H20O4 droxypsoralen (300 mg, 1.5 mmol), 2-cyclohexylethylbromide (0.5 ml,
348.40): C, 75.84%; H, 5.79%, O, 18.37%. Found: C, 75.935; H, 3.3 mmol), and K CO3 (0.6 g) were heated in 15 ml of acetone for
7
5
4
2
5
.75%.
24 h. The product was recrystallized from ethanol/H O (90:10) as a
2
1
RS-5-(2-phenylpropoxy)psoralen (Compound 6). 5-Hy- white solid (263 mg, 57%): m.p. ϭ 117°C; H NMR (CDCl ) ␦ 0.95 to
3
3
droxypsoralen (300 mg, 1.5 mmol), ␤-bromocumol (0.6 ml, 3.8 mmol), 1.81 (m, 13 H, 5-OCH CH C H11), 4.49 (t, 2 H, J ϭ 6.6 Hz,
and K CO3 (0.6 g) were heated in 15 ml of acetone for 28 h. The
product was recrystallized from ethanol/H O (80:20) as a yellowish 2.4 Hz, J ϭ 1.0 Hz, H-4Ј), 7.13 (s, br, 1 H, H-8), 7,58 (d, 1 H, J ϭ 2.4
2
2 6
3
3
5-OCH CH C H ), 6.28 (d, 1 H, J ϭ 9.8 Hz, H-3), 6.95 (dd, 1 H, J ϭ
2
2 2 6 5
5
3
2
1
3
5
solid (97 mg, 20%): m.p. ϭ 141.5°C; H NMR (CDCl ) ␦ 1.47 [d, 3 H, Hz, H-5Ј), 8.15 (dd, 1 H, J ϭ 9.8 Hz, J ϭ 0.5 Hz, H-4); MS m/z 312
3
3
3
ϩ
ϩ
ϩ
ϩ
15
J ϭ 7.0 Hz, 5-OCH CH(CH )C H ], 3.34 [sext, 1 H, J ϭ 7.0 Hz, (M ), 203, 202 (M-C H ), 174 (202-CO ), 111 (C H ), 69, 67, 55
2
3
6
5
8
14
8
2
ϩ
5
-OCH CH(CH )C H ], 4.48 and 4.52 [2 dd, 2 ϫ 1 H, J ϭ 9.0 Hz, (C H ), 43, 41. Calculated for C19H20O4 (312.36): C, 73.06%; H,
2 3 6 5 AB 4 7
3
3
J
ϭ 7.0 Hz, J ϭ 6.8 Hz, 5-OCH H CH (CH )C H ], 6.17 (d, 1 6.45%; O, 20.49%. Found: C, 73.115; H, 6.52%.
BX A B X 3 6 5
AX
3
3
5
H, J ϭ 9.8 Hz, H-3), 6.84 (dd, 1 H, J ϭ 2.4 Hz, J ϭ 0.9 Hz, H-4Ј),
5-(3-Cyclohexylpropoxy)psoralen (Compound 12). 5-Hy-
5
5
7.13 (dd, 1 H, J ϭ 0.9 Hz, J ϭ 0.7 Hz, H-8), 7.28 to 7.39 [m, 5 H, droxypsoralen (300 mg, 1.5 mmol), 3-cyclohexylpropylchloride (0.9
3
5
-OCH CH(CH )C H ], 7.56 (d, 1 H, J ϭ 2.4, H-5Ј), 7.82 (dd, 1 H, ml, 5.4 mmol), and K CO (0.6 g) were heated in 15 ml of acetone for
2
3
6
5
2
3
3
5
ϩ
J ϭ 9.8 Hz, J ϭ 0.7 Hz, H-4); MS m/z 320 (M ), 203, 202 (M- 38 h. The product was recrystallized from ethanol/H O (90:10) as a
2
ϩ
ϩ
ϩ
11
ϩ
7
ϩ
5
1
C H ), 174 (202-CO ), 119 (C H ), 92, 91 (C H ), 77 (C H ), 51 white solid (223 mg, 46%): m.p. ϭ 115°C; H NMR (CDCl ) ␦ 0.87 to
9
10
9
7
6
3
ϩ
3
(
C H ), 41. Calculated for C20H16O4 (320.34): C, 74.99%; H, 5.03%; 1.93 [m, 15 H, 5-OCH (CH ) C H ], 4.43 [t, 2 H, J ϭ 6.5 Hz,
4
3
2
2
2
6
1
1
3
O, 19.98%. Found: C, 74.855; H, 5.03%.
5-OCH (CH ) C H ], 6.28 (d, 1 H, J ϭ 9.8 Hz, H-3), 6.94 (dd, 1 H,
2 2 2 6 11
3
5
5
E-5-(3-Phenyl-2-propenyloxy)psoralen (Compound 7). 5-Hy-
J ϭ 2.4 Hz, J ϭ 1.0 Hz, H-4Ј), 7.13 (t, 1 H, J ϭ 0.8 Hz, H-8), 7.58
3
3
5
droxypsoralen (300 mg, 1.5 mmol), cinnamylbromide (400 mg, 2.0 (d, 1 H, J ϭ 2.4 Hz, H-5Ј), 8.18 (dd, 1 H, J ϭ 9.8 Hz, J ϭ 0.6 Hz,
mmol), and K CO (0.6 g) were heated in 15 ml of acetone for 7 h. The
product was recrystallized from ethanol/H O (80:20) as a yellowish solid 57, 55 (C H ), 43, 41. Calculated for C20H22O4 (326.39): C, 73.60%;
ϩ
ϩ
ϩ
H-4); MS m/z 326 (M ), 203, 202 (M-C H ), 174 (202-CO ), 83, 69,
9 16
ϩ
2
3
2
4
7
1
3
(104 mg, 22%): m.p. ϭ 140°C; H NMR (CDCl ) ␦ 5.09 (dd, 2 H, J ϭ 6.0 H, 6.79%; O, 19.61%. Found: C, 73.645; H, 6.86%.
3
4
3
Hz, J ϭ 1.2 Hz, 5-OCH CHCHC H ), 6.29 (d, 1 H, J ϭ 9.8 Hz, H-3),
Log P Values. Log P (log of the octanol-water partition coeffi-
2
6
5
3
3
6.45 (dt, 1 H, J ϭ 15.9 Hz, J ϭ 6.0 Hz, 5-OCH CHCHC H ), 6.76 (d, cient, a measure of hydrophobicity) values of compounds 1 through
2
6
5
3
3
br, 1 H, J ϭ 15.8 Hz, 5-OCH CHCHC H ), 6.98 (dd, 1 H, J ϭ 2.4 Hz, 12 were determined by HPLC with a Waters 1525 binary HPLC
2
6
5
5
J ϭ 0.9 Hz, H-4Ј), 7.19 (s, br, 1 H, H-8), 7.29 to 7.43 (m, 5 H,
pump (Milford, MA), a Kromasil 100 C18 column (5 ␮m, 60 ϫ 4.6
-OCH CHCHC H ), 7.61 (d, 1 H, J ϭ 2.4 Hz, H-5Ј), 8.21 (d, 1 H, J ϭ mm; EKA Chemicals Separation Products, Bohus, Sweden), and a
3
3
5
9
2
6
5
ϩ
ϩ
ϩ
.8 Hz, H-4); MS m/z 318 (M ), 202 (M-C H ), 174 (202-CO ), 118, 117 Waters 2475 multiwavelength fluorescence detector. Compounds
9
8
ϩ
ϩ
ϩ
(C H ), 116, 115, 91 (C H ), 89, 63, 51 (C H ). HRMS m/z C20H14O : were eluted with a gradient changing from 30% acetonitrile and 70%
9 9 7 7 4 3 4
1
3
calculated 318.08920, found 318.08900; C19 CH14O : calculated S o¨ rensen’s phosphate buffer, 11 mM, pH 7.4 to 78% acetonitrile and
4
3
19.09256, found 319.09240.
-Diphenylmethoxypsoralen (Compound 8). 5-Hydroxypsor- propyl-, n-butyl-, n-pentyl-, n-hexyl, n-heptyl-, and n-octylbenzene
alen (200 mg, 1.0 mmol), diphenylmethylbromide (0.4 g, 1.6 mmol), were used as standards.
22% buffer over 70 min. 4-Methylbenzaldehyde, toluene, ethyl-, n-
5
and K CO3 (0.4 g) were stirred at room temperature in 15 ml of
Cells, Cell Lines, and Clones. L929, B82, and MEL cells
2
acetone for 45 h. The product was recrystallized from acetone/H O stably expressing mKv1.1, rKv1.2, mKv1.3, mKv3.1, and hKv1.5
2
1
(
(
6
70:30) as a white solid (132 mg, 36%): m.p. ϭ 173°C; H NMR have been described previously (Grissmer et al., 1994). LTK cells
3
CDCl ) ␦ 6.29 (d, 1 H, J ϭ 9.8 Hz, H-3), 6.43 [s, 1 H, 5-OCH(C H ) ], expressing hKv1.4 were obtained from M. Tamkun (University of
3
6
5 2
3
5
5
.57 (dd, 1 H, J ϭ 2.4 Hz, J ϭ 1.0 Hz, H-4Ј), 7.18 (t, 1 H, J ϭ 0.8 Colorado, Boulder, Boulder, CO), human embryonic kidney-293
3
Hz, H-8), 7.30 to 7.41 [m, 10 H, 5-OCH(C H ) ], 7.49 (d, 1 H, J ϭ 2.4 cells expressing hSlo␣ were obtained from A. Tinker (Centre for
Hz, H-5Ј), 7.88 (dd, 1 H, J ϭ 9.8 Hz, J ϭ 0.6 Hz, H-4); MS m/z 368 Clinical Pharmacology, University College London, London, UK),
(
9
7
6
5 2
3
5
ϩ
ϩ
ϩ
ϩ
ϩ
M ), 202 (M-C13H ), 174 (202-CO ), 167 (C13H ), 118 (174–2CO ), human embryonic kidney-293 cells expressing HERG (Kv11.1)
10 11
ϩ
0, 89, 51 (C H ), 50, 49, 44. Calculated for C24H16O4 (368.39): C, were obtained from C. T. January (Department of Medicine, The
4 3
8.25%; H, 4.38%; O, 17.37%. Found: C, 78.175; H, 4.38%.
University of Wisconsin-Madison, Madison, WI), and N1E-115
5
-(4-Biphenylyl)methoxypsoralen (Compound 9). 5-Hy- neuroblastoma cells were obtained from B. Hamprecht (Physiolo-
droxypsoralen (200 mg, 1.0 mmol), biphenylmethylchloride (250 gisch-Chemisches Institut, Universit a¨ t T u¨ bingen, T u¨ bingen, Ger-
mg, 1.2 mmol), and K CO3 (0.4 g) were heated in 10 ml of acetone many). hIKCa1 (hKCa3.1), hSKCa3 (hKCa2.3), and mKv1.7 were
2
for 6 h. The product was recrystallized from acetone as a white cloned as described previously (Wulff et al., 2000; Bardien-Kruger
1
solid (120 mg, 33%): m.p. ϭ 187°C; H NMR (CDCl ) ␦ 5.49 (s, 2 H, et al., 2002) and transiently transfected into COS-7 cells using
3
3
5
-OCH C H C H ), 6.24 (d, 1 H, J ϭ 9.8 Hz, H-3), 6.97 (dd, 1 H, FuGene 6 (Roche Diagnostics, Indianapolis, IN) according to the
J ϭ 2.4 Hz, J ϭ 1.0 Hz, H-4Ј), 7.19 (s, br, 1 H, H-8), 7.34 to 7.66 manufacturer’s protocol.
2
6
4
6
5
3
5

1
368
Vennekamp et al.
The PAS T cells, a major histocompatibility complex class II-
Results
ϩ
restricted MBP-specific encephalitogenic CD4 rat T cell line, were a
kind gift from Dr. Evelyne B e´ raud (Laboratoire d’Immunologie, Fac-
ult e´ de M e´ decine, Universit e´ de la M e´ diterran e´ e, Marseille, France).
They were maintained in culture by alternating rounds of antigen-
Structure-Activity Relationship of 5-Alkoxypsor-
alens: Generation of a Potent Kv1.3 Blocker. Earlier
structure-activity relationship studies on psoralens and
induced activation and expansion in interleukin-2–containing me- related 5,7-disubstituted coumarins revealed that modifi-
ϩ
dium (Beeton et al., 2001b, 2003). Human MBP-specific CD4 mem- cations of the psoralen system or introduction of substitu-
ory T cells were generated from peripheral blood mononuclear cells ents in any positions other than the 5- or the neighboring
ϩ
(
PBMC) from a healthy volunteer according to a split-well assay 4 or 4Ј positions reduced K channel-blocking activity
described previously (Wulff et al., 2003b). Cells were stimulated 12ϫ (Wulff et al., 1998). We therefore concentrated our further
with MBP and used for the proliferation assay when they were Ͼ95%
CCR7 /CD45RA by flow cytometry.
synthetic efforts on the 5-position of the psoralen moiety.
Ϫ
Ϫ
The structures of 5-MOP and 12 compounds in which the
methyl group at the 5-position was replaced with a series
of phenylalkyl or cyclohexylalkyl substituents are shown
in Fig. 1. Psora-1 through Psora-5 constitute a series of
phenylalkoxypsoralens in which the length of the alkyl
chain linking the psoralen moiety and a side-chain phenyl
Electrophysiology. All compounds used for electrophysiological
testing were Ͼ98% pure as determined by combustion analysis. All
experiments were conducted in the whole-cell configuration of the
patch-clamp technique with a holding potential of Ϫ80 mV unless
otherwise stated. Pipette resistances averaged 2.0 M⍀, and series
resistance compensation of 80% was used when currents exceeded 2
nA. Kv1.3 currents were elicited by repeated 200-ms or 2-s pulses
from Ϫ80 to 40 mV, applied at intervals of 30 or 60 s. Kv1.3 currents
ring increases from one to five CH groups. Psora-6 has one
2
methyl group in the side chain, Psora-7 has a double bond
in the side chain, and Psora-8 and Psora-9 both have an
additional phenyl ring in the side chain. Psora-10, -11, and
12 contain aliphatic cyclohexyl rings in place of the side-
chain phenyl ring. Each compound was characterized by
2ϩ
were recorded in normal Ringer’s solution with a Ca -free pipette
solution containing 145 mM KF, 10 mM HEPES, 10 mM EGTA, and
2
-
mM MgCl , pH 7.2; osmolarity, 300 mOsM. EC50 values and Hill
2
coefficients were determined by fitting the Hill equation to the re-
1
13
duction of peak current measured at 40 mV. Kv1.1, Kv1.5, Kv1.7, melting point, IR, H and C NMR, mass spectrometry,
Kv1.4, and Kv3.1 currents were recorded with 200-ms depolarizing and combustion analysis (see Materials and Methods).
pulses to 40 mV applied every 10 s (Grissmer et al., 1994; Wulff et al.,
2
As an example, the effect of Psora-3 on Kv1.3 is shown in
000; Bardien-Kruger et al., 2002). For Kv1.2, we applied 200-ms Fig. 2A. Psora-3 blocked the current in a concentration-de-
pulses to 40 mV every 5 s to allow for the use-dependent activation pendent fashion, with an EC50 value of 6.3 nM (Fig. 2, A–C).
of the channel (Grissmer et al., 1994). HERG currents were recorded
Concentration-response curves for Psora-3 and the other
with a 2-step pulse from Ϫ80 mV first to 20 mV for 2 s and then to
Ϫ50 mV for 2 s (Zhou et al., 1998), and the reduction of both peak and
members of this series (Psora-1–Psora-5) revealed that the
length of the side-chain linker had a profound effect on the
potencies and Hill coefficients of these compounds (Fig. 2, B
tail current by the drug was determined. For measurements of in-
2ϩ
ϩ
termediate-conductance Ca -activated K channel IKCa (KCa3.1),
SKCa (KCa2.3), and BKCa (KCa1.1) currents, we used an internal
and C). Increasing the length of the linker from one to four
CH moieties significantly enhanced potency from an EC of
ϩ
2
50
pipette solution containing 145 mM K aspartate, 2 mM MgCl2, 10
3
50 nM for Psora-1 to an EC50 of 2.9 nM for Psora-4; further
lengthening of the side chain reduced potency (Fig. 2, B and
C). Psora-1 with one CH in the side chain blocked the chan-
2ϩ
mM HEPES, 10 mM K EGTA, and 8.5 mM CaCl (1 ␮M free Ca ),
2
2
pH 7.2, 290 to 310 mOsM and an external solution containing 160
ϩ
2
mM Na aspartate, 4.5 mM KCl, 2 mM CaCl , 1 mM MgCl , and 5
2
2
nel with a Hill coefficient approaching unity; whereas the
other four compounds in this series, Psora-2 through Psora-5,
displayed Hill coefficients of 2, suggesting that more than one
inhibitor molecule interacts with one Kv1.3 channel. The
mM HEPES, pH 7.4, 290 to 310 mOsM. Intermediate-conductance
2ϩ
ϩ
Ca -activated K channel and SKCa currents were elicited by
00-ms voltage ramps from Ϫ120 mV to 40 mV applied every 10 s,
and the reduction of slope conductance by drug at Ϫ80 mV was taken
2
as a measure of channel block (Wulff et al., 2000). BKCa currents biphenyl-substituted Psora-9 also blocked Kv1.3 in the single
were elicited by 200-ms voltage ramps from Ϫ80 to 80 mV applied nanomolar range (EC50, 3.5 nM) (Figs. 1 and 2C). Other
every 30 s, and channel block was measured as reduction of slope changes in the side chain did not improve potency. Psora-6
conductance at 35 mV. Na 1.2 currents from N1E-115 cells were with one methyl group in the side chain, Psora-7 with a
V
recorded with 100-ms pulses from Ϫ80 to 0 mV every 10 s with a double bond in the side chain, and Psora-8 with an additional
KCl-based pipette solution and an external solution containing 25
phenyl ring were all significantly less active than Psora-3,
mM glucose (Hirsh and Quandt, 1996). Blockade was determined as
Psora-4, Psora-5, and Psora-9. Replacement of the side-chain
phenyl ring with an aliphatic cyclohexyl ring in an analogous
reduction of the current minimum between 0.3 and 5 ms.
5
Proliferation Assays. Human PBMC were seeded at 2 ϫ 10
series of compounds (Psora-10, Psora-11, and Psora-12) re-
cells/well in RPMI 1640 culture medium in flat-bottomed 96-well
duced potency.
plates (final volume, 200 ␮l), preincubated with increasing con-
The single nanomolar EC50 values of Psora-3, Psora-4,
centrations of Psora-4 for 30 min, and then stimulated with 200
Psora-5, and Psora-9 make these four compounds the most
ng/ml soluble anti-CD3 monoclonal antibody (Biomeda, Foster
ϩ
potent known small-molecule Kv1.3 blockers; other known
nonpeptide blockers of Kv1.3 have EC50 values greater than
50 nM (for reviews, see Chandy et al., 2001; Coghlan et al.,
2001; Wulff et al., 2003a). At concentrations producing more
than 50% of inhibition, blockade by these four compounds
was nearly irreversible despite extensive washing. However,
City, CA) for 48 h. MBP-specific CD4 human memory T cells at
4
5
ϫ 10 cells/well were stimulated with 200 ng/ml anti-CD3 mono-
4
clonal antibody in the presence of 5 ϫ 10 autologous irradiated
PBMC (2500 rad). PAS T cells (2 ϫ 10 cells/well) were stimulated
in the presence of 2 ϫ 10 irradiated Lewis rat thymocytes (2500
rad) as antigen-presenting cells with 10 ␮g/ml MBP. [ H]TdR (1
4
6
3
␮
Ci/well) was added for the last 16 h. Cells were harvested onto block by the other psoralens with lower affinities was revers-
glass fiber filters, and radioactivity was measured in a ␤-scintil- ible. These results indicate that this class of compounds does
lation counter. not covalently react with the Kv1.3 protein and that the lack

Psora-4, a Small-Molecule Kv1.3 Inhibitor
1369
of reversibility of Psora-3, Psora-4, Psora-5, and Psora-9 is a a compound indicating a possible absorption problem after
result of their higher potency and lipophilicity. oral administration if any two of the following conditions
Although the four most potent compounds are lipophilic, apply: molecular weight Ͼ500, log PϾ 5, number of hydrogen
their potency for Kv1.3 did not correlate with their lipophi- bond donors (expressed as the sum of OH and NH groups)
licity (Fig. 2C). The log P values (a measure of their hydro- Ͼ5, and the number of hydrogen acceptors (sum of N and O
phobicity) (Hansch and Anderson, 1967; Leo, 2000) of these atoms) Ͼ10. Psora-4 and its analogs do not violate this rule
compounds (4.0–4.7), their molecular weights (320–360), and and should therefore be orally available.
the number of hydrogen-bond donors and acceptors (donors 0,
Psora-4 Binds to the Inactivated State of the Kv1.3
acceptors 4) are well within the range postulated by the Channel. The most potent compound in the series, Psora-4,
Lipinksi “rule of five” (Lipinski et al., 1997) as optimal for was studied in more detail. In the experiment shown in Fig.
potential drug candidates. This rule generates an “alert” for 3A, we applied a 200-ms depolarizing pulse to elicit a control
Fig. 2. A, inhibition of Kv1.3 current by increasing concentrations of Psora-3 in a L929 cell line stably expressing Kv1.3. For each concentration,
equilibrium block after 12 pulses is shown. Currents were elicited by 200-ms depolarizing from Ϫ80 to 40 mV every 30 s. B, concentration-response
curves of Psora-1, Psora-2, Psora-3, Psora-4, and Psora-5 on Kv1.3 stably expressed in L929 cells (n ϭ number of CH2 groups in the side chain). The
compounds were tested three to five times at five or six concentrations. EC50 values and Hill coefficients were determined by fitting the Hill equation
to the reduction of peak current measured at 40 mV. Onset of block was slow for all compounds, and equilibrium block on average was reached after
1
0 to 12 depolarizing pulses. The table shows EC50 values (mean Ϯ S.D.), the selectivity (S) for Kv1.3 over Na 1.2, and the log P values of the
V
synthesized compounds (C). S ϭ EC50 Na 1.2/EC Kv1.3. Log P values (a measure of hydrophobicity) were determined by HPLC as described under
V
50
Materials and Methods. D, EC50 values of Psora-4 on cloned and native ion channels. Psora-4 was tested at three to five concentrations (n ϭ 3).
Blockade was reversible for all channels, and onset of block was slow (ϳ10–20 depolarizing pulses to reach equilibrium block for all channels).

1
370
Vennekamp et al.
Fig. 3. A, pulse protocol for experiments shown in B–E. A Kv1.3 control current was elicited by a 200-ms depolarizing pulse to 40 mV. Psora-4 (10 nM)
was then applied to the bath while the membrane was held at Ϫ80 mV. After 5 min, consecutive 200-ms or 2-s pulses were applied every 60 s. B,
inhibition of Kv1.3 after application of 10 nM Psora-4 during consecutive 200-ms pulses (numbers 1–12 correspond to pulse 1–12). C, inhibition of
Kv1.3 after application of 10 nM Psora-4 during consecutive 2-s pulses. D, effect of varying the pulse duration on time to reach steady-state block.
Ordinate: ratio of peak current at various times after start of the consecutive pulses versus current before drug application (n ϭ 5). E, effect of varying
ϩ
the holding potential on time to reach steady-state block. Pulse duration, 200 ms. F, effect of varying the external K concentration on sensitivity to
block by Psora-4. Steady-state currents 10 min after drug application (200-ms pulses to 40 mV, holding potential Ϫ80 mV) are shown. All experiments
in this figure were conducted on activated TEM cells because they were found to be generally more stable than L929 cells for recordings with 2-ms
pulses (C and D) and for holding at depolarized potentials (E).

Psora-4, a Small-Molecule Kv1.3 Inhibitor
1371
Kv1.3 current. Psora-4 was then perfused into the bath at a showed significantly reduced selectivity for Kv1.3 over
concentration (10 nM) that blocked 90% of Kv1.3 current Na 1.2 (Fig. 2C).
V
while the channel was in the closed state. After a five-min
Besides Kv1.3, the only other channel blocked potently by
interval to allow ample time for the lipophilic Psora-4 to Psora-4 was Kv1.5 (EC50, 7.7 nM), which is thought to un-
reach its binding site, we applied a second depolarizing pulse derlie the ultrarapid delayed rectifier (IKUR) current in the
and found it to be of equivalent amplitude to the control human atrium (Feng et al., 1997) and to contribute to atrial
current, indicating that the compound does not bind to the repolarization (Wang et al., 1993). Psora-3 (EC50, 24 Ϯ 2 nM)
closed state of the channel. Psora-4 block developed during and Psora-9 (EC50, 8.2 Ϯ 0.5 nM) also potently blocked this
subsequent depolarizing pulses, demonstrating that multiple channel. Interestingly, Psora-4 is 50-fold more potent as a
openings of the channel are required to reach steady-state blocker of Kv1.5 than the bisaryl S9947, a compound identi-
block, a phenomenon termed “use-dependent inhibition”. fied recently by Aventis (Bachmann et al., 2001). Because of
This result suggests that Psora-4 blocks a postactivation concerns about possible cardiac side effects, we injected rats
state, possibly the open and/or C-type inactivated conforma- with Psora-4 subcutaneously (33 mg/kg/day for 5 days); the
tions of the channel.
rats (n ϭ 8) seemed clinically normal (no signs of discomfort,
Three types of experiments were performed to distinguish seizures, paralysis, ataxia, or weakness) during the 5-day
between these two possibilities. First, the time to reach study and for 7 days afterward. The lack of overt toxicity in
steady-state block depended on the duration of the depolar- vivo encouraged us to evaluate Psora-4 further as an immu-
izing pulse. When depolarizing pulses of 200 ms to 40 mV nosuppressive.
were applied every 60 s from a holding potential of Ϫ80 mV,
Psora-4 Preferentially Suppresses the Proliferation
7
00 s was required to reach steady-state block (Fig. 3, A, B, of TEM Cells. We compared the ability of Psora-4 to suppress
3
and D). Lengthening the pulse duration to 2 s and thus mitogen- or antigen-stimulated increases in [ H]thymidine
causing more of the channels to undergo C-type inactivation incorporation by a mixture of naive and TCM cells versus a
during the pulse shortened the time to reach steady-state population of TEM cells. Human T cells (either the naive/TCM
block to 300 s (Fig. 3, C and D). Second, the time-to-steady- mixed population or MBP-specific TEM cells) were stimulated
state block depended on the holding potential when the pulse through the T-cell receptor with a mitogenic monoclonal anti-
duration (200 ms) and the interpulse interval (60 s) were CD3 antibody and myelin-specific rat TEM cells with the
kept constant. At a holding potential of Ϫ50 mV, the propor- antigen MBP in the presence or absence of Psora-4. The
tion of channels in the C-type inactivated conformation is numbers of Kv1.3 and IKCa1 channels expressed per cell in
significantly enhanced because of prolonged recovery from resting and activated naive, TCM, and TEM subsets is shown
inactivation compared with a more negative holding poten- in Fig. 4A based on data published previously (Wulff et al.,
tial of Ϫ80 mV (Nguyen et al., 1996). Steady-state block was 2003b). The proliferation of human and rat TEM cells was
reached at ϳ300 s at Ϫ50 mV and at 700 s at Ϫ80 mV (Fig. suppressed by Psora-4 with EC50 values of 25 and 60 nM,
3
E), consistent with Psora-4 blocking the inactivated confor- respectively (Fig. 4B, graph top right), in keeping with the
ϩ
mation. Last, removal of C-type inactivation by 160 [K ]
Kv1.3 dependence of these cells. Psora-4 was 10-fold less
o
(
Cahalan et al., 1985; Levy and Deutsch, 1996) decreased the effective in suppressing the proliferation of naive/TCM cells
ϩ
potency of Psora-4 10-fold compared with 4.5 [K ] (Fig. 3F). (EC50, 600 nM) presumably because they quickly up-regu-
o
Taken together, these results indicate that Psora-4 preferen- lated IKCa1 and escaped Psora-4 inhibition (Fig. 4B, graph
tially blocks the C-type inactivated conformation of the chan- top left). Consistent with this idea, naive/TCM cells that had
nel.
been activated for 48 h to up-regulate IKCa1, rested for 12 h,
Selectivity and Lack of Toxicity of Psora-4. Psora-4 and then rechallenged for a further 48 h were completely
was tested for specificity against a panel of 11 channels (Fig. resistant to Psora-4 and sensitive to the IKCa1-specific in-
2
D). Psora-4 blocked cloned and native Kv1.3 channels in T hibitor TRAM-34 (Fig. 4B, graph bottom left). Thus, TEM
cells with equivalent potency. It blocked Kv1.1, Kv1.2, Kv1.4, cells are highly sensitive to suppression by Psora-4 because
and Kv1.7 in a reversible fashion and displayed 16- to 70-fold they up-regulate Kv1.3 and not IKCa1 during activation (Fig.
selectivity for Kv1.3 over these channels. Psora-4 was inef- 4A). Naive and TCM cells, in contrast, are initially ϳ10-fold
fective against HERG (Kv11.1), Kv3.1, IKCa1 (KCa3.1), less sensitive to Psora-4 than TEM cells and then become
SKCa3 (KCa2.3), and BKCa (KCa1.1). Psora-3 exhibited a sim- resistant to Kv1.3 blockade via up-regulation of IKCa1.
ilar selectivity profile as Psora-4 (data not shown), whereas These results corroborate our recent findings with the Kv1.3-
the more lipophilic Psora-5 and Psora-9 exhibited lower se- blocking peptide ShK (Wulff et al., 2003b) and demonstrate
lectivity for Kv1.3 over Kv1.2 (6- and 5-fold, respectively). ϩ that Psora-4 might have value as a potential new therapeutic
The parent compound of this series, 5-MOP, blocks Na
for autoimmune diseases dominated by autoreactive TEM
channels in amphibian nodes of Ranvier at high concentra- cells.
tions (Bohuslavizki et al., 1994; During et al., 2000), raising
concerns about possible neuronal toxicity. We therefore com-
Discussion
pared the blocking potencies of Psora-1 through Psora-9 on
ϩ
Kv1.3 and Na 1.2, the Na channel ␣-subunit predomi-
Using 5-MOP as a template, we have developed a series of
V
nantly expressed in mammalian unmyelinated and premy- 5-phenyl-alkoxypsoralens that inhibit Kv1.3 with single
elinated axons (Boiko et al., 2001) (Fig. 2, C and D). Although nanomolar affinity. The pharmacophore for channel block
5
-MOP was only 14-fold selective for Kv1.3 over Na 1.2, consists of a psoralen moiety that is attached through an
V
Psora-3, Psora-4, and Psora-9 exhibited Ͼ300-fold selectivity alkyl chain linker in the 5-position to a phenyl ring. The
for Kv1.3 over Na 1.2; blockade by these compounds was optimal length for the linker was found to be four CH groups
V
2
completely reversible. The cyclohexyl-substituted compounds as in Psora-4, our most potent compound (EC50, 3 nM). Re-

1
372
Vennekamp et al.
Fig. 4. A, diagram showing Kv1.3 and IKCa1 channel numbers per cell in human naive, TCM, and TEM T cells in the resting and activated state based
on our data published previously (Wulff et al., 2003b). B, effect of Psora-4 on the proliferation of different T cell subsets. Top left, Psora-4 (f) inhibits
the anti-CD3 antibody-stimulated proliferation of human peripheral blood T cells, consisting mostly of naive and TCM cells, with an EC50 of 600 nM.
Bottom left, the proliferation of prestimulated naive and TCM cells is inhibited by the IKCa1 blocker TRAM-34 (‚) with an EC50 of 250 nM but not
by Psora-4 (f). Top right, the anti-CD3 antibody-stimulated proliferation of human TEM cells (f) and the MBP-stimulated proliferation of rat memory
T cells (Ⅺ) is inhibited by Psora-4 with EC50 values of 25 and 60 nM, respectively.

Psora-4, a Small-Molecule Kv1.3 Inhibitor
1373
placement of the phenyl ring with an aliphatic cyclohexyl sponse unimpaired. Future studies will have to be performed
ring (e.g., Psora-10–Psora-12) reduced Kv1.3-blocking po- to ascertain whether Psora-4 is therapeutically effective in
ϩ
tency and selectivity over Na channels, suggesting that the chronic relapsing models of EAE that resemble human MS
phenyl ring represents a second center of ␲-electron density more closely than monophasic adoptive-transfer EAE and
in these compounds and is required for the selective and whether this compound, if appropriately formulated for oral
high-affinity interaction with Kv1.3. From these structure- administration, will benefit patients with MS.
activity relationships, we postulate that the 5-pheny-
lalkoxypsoralens bind to Kv1.3 via two ␲-␲ electron interac- Acknowledgments
tions positioned ϳ7 Å apart (the length of the butoxy linker
in Psora-4), one involving the psoralen moiety and the second
involving the side chain of the phenyl ring. The precise bind-
We thank Professor Dr. Eilhard Koppenh o¨ fer for his courage in
investigating the effects of R. graveolens and his continuing enthu-
siastic belief in psoralens as possible drugs for the treatment of MS.
ing site of Psora-4 on the Kv1.3 channel remains to be deter- We also thank Bj o¨ rn Henke for excellent technical assistance with
mined.
the chemical synthesis and Susann H a¨ uer and Inga Carstens for
Psora-4, the most potent small-molecule Kv1.3 blocker their determination of the log P values.
known, blocks the channel in a use-dependent manner by
binding to the C-type inactivated state in a fashion similar to
the other Kv1.3 blockers, including CP-339818 (Nguyen et
al., 1996), UK-78282 (Hanson et al., 1999), and correolide
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Address correspondence to: K. George Chandy, Department of Physiology
and Biophysics, Medical School, 346-D Med. Sci. I, University of California at
Irvine, Irvine, CA 92697. E-mail: gchandy@uci.edu