Benzamide derivatives as blockers of Kv1.3 ion channel

Article abstract of DOI:10.1016/S0960-894X(03)00014-3

The voltage-gated potassium channel, Kv1.3, is present in human T-lymphocytes. Blockade of Kv1.3 results in T-cell depolarization, inhibition of T-cell activation, and attenuation of immune responses in vivo. A class of benzamide Kv1.3 channel inhibitors

Full text of DOI:10.1016/S0960-894X(03)00014-3

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1161–1164
Benzamide Derivatives as Blockers of Kv1.3 Ion Channel
Shouwu Miao,a,* Jianming Bao,a,* Maria L. Garcia,b Joung L. Goulet,a
a
Xingfang J. Hong,a Gregory J. Kaczorowski,b FrankKayser, Gloria C. Koo,c
Andrew Kotliar,a William A. Schmalhofer,b Kashmira Shah,c Peter J. Sinclair,a
Robert S. Slaughter,b Marty S. Springer,c Mary Jo Staruch,c Nancy N. Tsou,a
a
FrederickWong, William H. Parsonsa and Kathleen M. Rupprechta
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Ion Channels, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
cDepartment of Immunology Research, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 22 July 2002; accepted 10 December 2002
Abstract—The voltage-gated potassium channel, Kv1.3, is present in human T-lymphocytes. Blockade of Kv1.3 results in T-cell
depolarization, inhibition of T-cell activation, and attenuation of immune responses in vivo. A class of benzamide Kv1.3 channel
inhibitors has been identified. The structure–activity relationship within this class of compounds in two functional assays, Rb_Kv
and T-cell proliferation, is presented. In in vitro assays, trans isomers display moderate selectivity for binding to Kv1.3 over other
Kv1.x channels present in human brain.
# 2003 Elsevier Science Ltd. All rights reserved.
Kv1.3 is a voltage-gated potassium channel that sets the
resting potential of human T-cells.1ꢀ3 Blockade of the
channel causes membrane depolarization,4 which
attenuates intracellular Ca2+ levels required for lym-
phocyte activation upon T-cell stimulation,5 and inhi-
bits immune responses in vivo. These findings suggest
that Kv1.3 represents a novel target for the development
of immunosuppressants. Researchers in the pharma-
ceutical industry and academia have been seeking
potent and selective inhibitors of the Kv1.3 channel that
will have efficacy in organ transplantation and other
immunologic illnesses.6ꢀ12
were found. A few carbamates of C1 alcohol of 1 were
also reported. Some of them have improved Kv1.3
activities over 1. These benzamides are remarkably sim-
ple and are selective against the Kv1 family of potas-
sium channels. More interestingly, the trans C1
carbamates displayed a modest selectivity between the
Kv1.3 and the Kv1.x channels in human brain based on
the rate of C-type inactivation of these channels.10
These promising results have led us to the further
exploration of this type of structure. Now, we wish to
give a more extensive account of this work.
A survey of SAR around the lead 1 was carried out. A
few selected compounds are presented in Scheme 1.
While the Kv1.3 activity can be maintained by changes
at the 2 position of the 2-methoxy phenyl ring (2b and
2c), substitutions at the 3 or 4 position only resulted in
loss of activity (2d–2f). Methylation of the amide NH or
replacement of the amide carbonyl with a sulfonyl
group significantly reduced the Kv1.3 activity as exem-
plified by the amide 3 and the sulfonamide 4. Removal
of the phenyl group also eliminated the activity (5).
However, the cyclohexyl ring can be modified without a
loss of Kv1.3 activity (6 and 7). With proper N1 sub-
stitutions, a piperidine ring is also tolerated (8a vs 8b).
Given these results, efforts were later focused on the C1
Previously, we have reported that benzamide 1 was a
potent Kv1.3 channel inhibitor.10 SARs at the 2-meth-
oxyphenyl ring were explored and no improvements
*Corresponding author. Fax: +1-732-594-5350; e-mail: bao_jianming
@merck.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00014-3

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S. Miao et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1161–1164
position of the cyclohexanone ring for SAR studies. A
modification at this position would be conformationally
distinct and the position of new groups would be
defined by the relative stereochemistry at C1. Analogues
at the C1 position would also have a plane of symmetry.
Compounds in Tables 1 and 2 were synthesized accord-
ing to Scheme 2. Ketone 9 was reduced by NaBH4 in
THF at low temperature to give exclusively 10 (by H
NMR), which was further reduced by LAH to afford
11.13 Compound 11 was converted to the cis alcohol 12
by a selective N-acylation with 2-methoxy benzoyl
chloride.
1
The trans alcohol 16 was prepared from 11. The amino
group of 11 was protected with a Boc group to give 14.
The C1 hydroxy group of 14 was inverted under Mitsu-
nobu conditions to give ester 15. Solvolysis of 15, removal
of the Boc protection group, and selective
acylation afforded the alcohol 16. The relative stereo-
chemistry of 16 was established by X-ray crystallography.
Amines 13 and 17 were prepared from alcohols 12 and
16, respectively, by a three-step sequence: mesylation of
the hydroxy group with MsCl, displacement of the
mesylate with azide and reduction of the azide with H2
and Pd catalyst.
Compounds 12, 13, 16, and 17 were converted to their
p-nitrophenylcarbonates, which were reacted with dif-
ferent nucleophiles to afford the analogues in Tables 1
and 2.
Scheme 1. Rb_Kv IC50’s of some benzamides.
Table 1. Rb_Kv and T-cell assay results (IC50, nM) for carbamates
of C1 alcoholsa
The Kv1.3 activity of these compounds was measured in
the Rb_Kv assay, which measures 86Rb+ efflux from
CHO cells stably transfected with the Kv1.3 channel.9,10
Table 2. Rb_Kv and T-cell assay results (IC50, nM) for analogues of
C1 aminesa
Compd
Nu
C1
Rb_Kv
T-cell (anti-CD3)
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
NHMe
NHiPr
NHnPr
trans
—
—
—
—
—
—
—
—
—
—
—
—
cis
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
58
75
50
70
320
90
90
195
222
273
361
—
688
688
1793
1000
—
—
—
—
519
1255
892
—
1099
659
—
320
—
—
—
237
915
—
—
—
NHallyl
N(Me)allyl
NH(2-Me-allyl)
NHnBu
NH(CH2)2OH
NH(CH2)2OMe
NH(CH2)3OMe
NHPh
Compd
Nu
C1
Rb_Kv
T-cell (anti-CD3)
310
140
145
>30 mM
1.7 mM
840
120
150
130
100
130
145
120
140
95
140
130
120
245
420
535
205
230
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
OMe
Oallyl
Sme
NH(allyl)
2-Br–Ph
3-Br–Ph
4-Br–Ph
Me
Et
nPr
CH¼C(Me)2
CH¼CHMe
Ph
Bn
Phenethyl
Oallyl
NH(allyl)
N(Me)(allyl)
CH¼CHMe
Ph
trans
—
—
—
—
—
—
—
—
—
—
—
—
—
—
cis
—
—
—
—
—
—
135
242
150
150
2790
>30 uM
>30 uM
470
210
200
470
440
1560
1045
570
90
140
195
100
1057
539
618
676
—
—
—
—
—
—
—
—
—
—
—
354
430
—
NHBn
N(allyl)2
NH2
NHMe
NHiPr
NHnPr
NHallyl
N(Me)allyl
NH(2-Me-allyl)
NHnBu
NH(CH2)2OH
NH(CH2)2OMe
NH(CH2)3OMe
NHPh
NHBn
NH(CH2)2Ph
NH(CH2)3Ph
N(allyl)2
OCH2CH¼CH2
399
154
609
200
75
130
120
Bn
Phenethyl
—
aValues are means of three experiments.
aValues are means of three experiments.

S. Miao et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1161–1164
1163
Scheme 2. (a) NaBH4, THF, ꢀ78 ꢁC, 100%; (b) LiAlH4, THF, reflux; (c) 2-methoxybenzoyl chloride, Et3N, rt, 86% for 12, two steps; 99% for 16;
(d) MsCl, Et3N, CH2Cl2; (e) NaN3, DMF; (f) H2, Pd/C, 87% for 13; 95% for 17; three steps; (g) (Boc)2O, Et3N, 70%; (h) PhCO2H, PPh3, DEAD,
72%; (i) NaOCH3, CH3OH/THF, rt; (j) HCl, 98%, two steps; (k) 4-nitrophenyl chloroformate, DMAP, CH2Cl2; (l) nucleophile, CH2Cl2.
Potent Kv1.3 compounds were evaluated in the human
T-cell assay.11
Compounds prepared from amines 13 and 17 are pre-
sented in Table 2. Like compounds in Table 1, the ste-
reochemistry at the C1 position dictates the SAR of
these compounds. Analogues from the cis amine 17 are
generally potent and insensitive to the steric bulkiness of
the side chain, while the trans compounds display an
SAR that correlates with the size of the side chains.
Compound 67 is the most potent compound in inhibit-
ing T-cell proliferation and it is 8-fold more potent than
the lead (1) in the T-cell assay.
The stereochemistry at C1 is important for the Rb_Kv
activity. Among carbamates prepared from the alcohols
12 and 16 (Table 1),14 Kv1.3 activity of the cis deriva-
tives (31–47) is insensitive to the change of the C1 side
chain, and is in a narrow range. In contrast, there is a
well-defined structure–activity relationship in the trans
series (18–30). A wide range of Rb_Kv activities was
observed in these compounds. An increase in the side
chain bulkiness causes a progressive decrease in the
inhibitory activity of these trans-isomers. Compounds
prepared from small primary amines potently blockthe
Kv1.3 channel (18–21 and 24). Carbamates from amines
with aromatic groups have reduced Rb_Kv activities (28
and 29). Side chains with an oxygen atom lead to com-
pounds with good Rb_Kv activities, but reduced T-cell
potencies (25 and 26). The discrepancy between the
Rb_Kv values and the corresponding T-cell values may
be explained by the fact that T-cell proliferation is a
distal effect of the consequence of blocking Kv1.3
channels. The T-cell activity of a compound is defined
as the effect of the compound on the Ca-dependent
pathway of T-cell activation. Therefore, the T-cell
potency of a blocker depends on the magnitude of the
stimulus, the donor variability, the effect of the com-
pound on other T-cell activation pathways, as well as,
its potency on Kv1.3. Major improvements in both the
Rb_Kv and the T-cell activities were observed in com-
pounds with small alkyl side chains. Compounds 18–21
are among the most potent analogues in both the
Rb_Kv and the T-cell proliferation assays.
With SARs of the C1 position in hand, an effort was
also devoted to modification of the 2-methoxyphenyl
ring to gain additional potency (Table 3). Introduction
of an N atom in the ring resulted in a decrease in the
Kv1.3 activity (70). Adding a Cl ortho to the methoxy
caused a sharp decrease in the potency (71). However,
Table 3. Modifications at benzylamide
Compd
Ar
70
71
72
73
74
75
76
Rb_Kv
IC50 (nM)
325
8380
7200
230
45
140
3730

1164
S. Miao et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1161–1164
wide range of activities. The trans-isomers also display
moderate selectivity towards Kv1.3 over other Kv1.x
channels in the in vitro binding assays. These data sug-
gest that the selective interaction of the trans-isomers
with Kv1.3 could be exploited for the development of
inhibitors of this channel for the treatment of immuno-
logical disorders with a limited side effect profile.
Table 4. Inhibition of binding of DiTc to Kv1.3 and Kv1.x channels
in human brain membranes (IC50, nM)a
Compd
X
Nu
C1
Kv1.3
Kv1.x
18
20
21
32
34
35
51
63
64
O
—
—
—
—
—
NH
—
—
NHMe
NHnPr
NHallyl
NHMe
NHnPr
NHallyl
NHallyl
Oallyl
trans
—
—
cis
—
5
5
33
30
112
104
76
170
120
68
References and Notes
18
99
79
140
29
72
80
1. Kaczorowski, G. J.; Koo, G. C. Perspect. Drug Discov.
Des. 1994, 2, 233.
2. Lewis, R. S.; Cahalan, M. D. Annu. Rev. Immunol. 1995,
13, 623.
3. Cahalan, M. D.; Chandy, K. G. Curr. Opin. Biotechnol.
1997, 8, 749.
4. Leonard, R. J.; Garcia, M. L.; Slaughter, R. S.; Reuben,
J. P. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 10094.
5. Lin, C. S.; Boltz, R. C.; Blake, J. T.; Nguyen, M.; Talento,
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R. S.; Garcia, M. L.; Kaczorowski, G. J.; Koo, G. C. J. Exp.
Med. 1993, 177, 637.
6. Hill, R. J.; Grant, A. M.; Volberg, W.; Faltynek, C.; Miller,
D.; Pagani, K.; Baizman, E.; Wang, S.; Guiles, J. W.; Krafte,
D. S. Mol. Pharm. 1995, 48, 98.
—
trans
cis
—
NHallyl
125
aValues are means of three experiments.
addition of either a Cl or an F atom para to the meth-
oxy group (73 and 74), or demethylation of the 2-meth-
oxy group (75), has a minimal effect on the activity. All
of these modifications decreased the Rb_Kv activity
except 74, where a small increase in potency was
observed.
7. Nguyen, A.; Kath, J. C.; Hanson, D. C.; Biggers, M. S.;
Canniff, P. C.; Donovan, C. B.; Mather, R. J.; Rauer, H.;
Aiyar, J.; Lepple-Wienhues, A.; Gutman, G. A.; Grissmer, S.;
Cahalan, M. D.; Candy, K. G. Mol. Pharm. 1996, 50, 1672.
8. Burgess, L. E.; Koch, K.; Cooper, K.; Biggers, M. S.;
Ramchandani, M.; Smitrovich, J. H.; Gilbert, E. J.; Bruns,
M. J.; Mather, R. J.; Donovan, C. B.; Hanson, D. C. Bioorg.
Med. Chem. Lett. 1997, 7, 1047.
9. Felix, J. P.; Bugianesi, R. M.; Schmalhofer, Borris, Robert.
B.; Goetz, M. A.; Hensens, O. D.; Bao, J.; Kayser, F.; Par-
sons, W. H.; Rupprecht, K.; Garcia, M. L.; Kaczorowski,
G. J.; Slaughter, R. S. Biochemistry 1999, 38, 4922.
10. Schmalhofer, W. A.; Bao, J.; McManus, O. B.; Green, B.;
Matyskiela, M.; Wunderler, D.; Bugianesi, R. M.; Felix, J. P.;
Hanner, M.; Linde-Arias, A.; Ponte, C. G.; Velasco, L.; Koo,
G.; Staruch, M. J.; Miao, S.; Parsons, W.; Rupprecht, K.;
Slaughter, R. S.; Kaczorowski, G. J.; Garcia, M. L. Biochem-
istry 2002, 41, 7781.
To test the selectivity of these benzamides against other
channels in the Kv1 family, the effect of these com-
pounds on diTC (C20–C29, ditritio-correolide) binding
to membranes derived either from cells expressing the
homomultimeric Kv1.3 channel or from human brain
tissue (Kv1.x, primarily heteromultimeric Kv1.1/Kv1.2
channels) was determined (Table 4).9 The data indicate
that the cis derivatives do not display selectivity as
inhibitors of diTC binding to Kv1.x channels. However,
the trans isomers are more potent inhibitors of diTC
binding to Kv1.3 than to the heteromultimeric Kv1.x
channels in brain. This modest selectivity may be due to
conformations unique to Kv1.3, which appear to be
related to the C-type inactivation property of the Kv1.3
channel.10,15 C-type inactivation is a process that
involves conformational changes at the outer mouth of
the channel. These conformational changes constrict the
pore and cause a blockade of the ion conduction path-
way.16 This unique interaction of the trans benzamides
with conformations of the Kv1.3 channel related to
C-type of interaction may allow development of selective
Kv1.3 blockers as immunosuppressants.
11. Koo, G. C.; Blake, J. T.; Shah, K.; Staruch, M. J.;
Dumont, F.; Wunderler, D.; Sanchez, M.; McManus, O. B.;
Fischer, P.; Boltz, R. C.; Goetz, M. A.; Baker, R.; Bao, J.;
Kayser, F.; Rupprecht, K. M.; Parsons, W. H.; Tong, X. C.;
Ita, I. E.; Pivnichny, J.; Vincent, S.; Cunningham, P.; Hora,
D., Jr.; Kaczorowski, G. J.; Springer, M. S. Cell. Immunol.
1999, 197, 99.
12. Lew, A.; Chamberlin, A. R. Bioorg. Med. Chem. Lett.
1999, 23, 3267.
13. Ketone 9 is commercially available from Acros. When
ketone 9 was directly reduced to 11 by LAH in THF at reflux,
a ratio of 11 to 1 was obtained.
14. Compounds 18, 20–21, 25–26, 28–29, 30–32, 34–35, 39–40,
42–43, and 46 have been previously disclosed in ref 10.
15. Vianna-Jorge, R.; Oliveira, C. F.; Garcia, M. L.; Kaczor-
owski, G. J.; Suarez-Kurtz, G. Br. J. Pharmacol. 2000, 131,
772.
In summary, starting from the lead 1, a series of benza-
mide Kv1.3 channel inhibitors has been prepared. Some
of these compounds show improved potencies in both
the Rb_Kv and the T-cell assays. Compounds that are
8-fold more potent in the T-cell assay and up to 4-
fold more potent in the Rb_Kv assay, respectively, than
lead 1 were identified. While the activity of the cis ana-
logues is not sensitive to the nature of the C1 sub-
stituents, the corresponding trans-isomers display a
16. Liu, Y.; Jurman, M. E.; Yellen, G. Neuron 1996, 16, 859.