Substituted N-{3-[(1,1-dioxido-1,2-benzothiazol-3-yl)(phenyl)amino]propyl} benzamide analogs as potent Kv1.3 ion channel blockers. Part 2

Article abstract of DOI:10.1016/j.bmcl.2010.09.131

We report the synthesis and in vitro activity of a series of novel substituted N-{3-[(1,1-dioxido-1,2-benzothiazol-3-yl)(phenyl)amino]propyl} benzamide analogs. These analogs showed potent inhibitory activity against Kv1.3. Several demonstrated similar po

Full text of DOI:10.1016/j.bmcl.2010.09.131

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6989–6992
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Substituted N-{3-[(1,1-dioxido-1,2-benzothiazol-3-yl)(phenyl)amino]-
propyl}benzamide analogs as potent Kv1.3 ion channel blockers. Part 2
Curt D. Haffner a, , Stephen A. Thomson a, Yu Guo a, Kimberly Petrov a, Andrew Larkin a, Pierette Banker a,
⇑
Gregory Schaaf a, Scott Dickerson b, Jeff Gobel c, Dan Gillie d, J. Patrick Condreay d, Chuck Poole e,
Tiffany Carpenter e, John Ulrich e
a Department of Medicinal Chemistry, GlaxoSmithKline Research and Development, 5 Moore Drive, Research Triangle Park, NC 27709, United States
b Department of Computational and Structural Chemistry, GlaxoSmithKline Research and Development, 5 Moore Drive, Research Triangle Park, NC 27709, United States
c Department of Screening and Compound Profiling, GlaxoSmithKline Research and Development, 5 Moore Drive, Research Triangle Park, NC 27709, United States
d Department of Biological Reagents and Assay Development, GlaxoSmithKline Research and Development, 5 Moore Drive, Research Triangle Park, NC 27709, United States
e Department of Drug Metabolism and Pharmacokinetics, GlaxoSmithKline Research and Development, 5 Moore Drive, Research Triangle Park, NC 27709, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis and in vitro activity of a series of novel substituted N-{3-[(1,1-dioxido-1,2-
benzothiazol-3-yl)(phenyl)amino]propyl}benzamide analogs. These analogs showed potent inhibitory
activity against Kv1.3. Several demonstrated similar potency to the known Kv1.3 inhibitor PAP-1 when
tested under the IonWorks patch clamp assay conditions. Two compounds 13i and 13rr were advanced
further as potential tool compounds for in vivo validation studies.
Received 17 August 2010
Revised 23 September 2010
Accepted 24 September 2010
Available online 23 October 2010
Ó 2010 Published by Elsevier Ltd.
Keywords:
Kv1.3
1,2-Benzothiazole
Voltage gated potassium channel
Insulin resistance is a key feature found in most type-2 diabet-
ics. This resistance can exist for many years without progression to
type-2 diabetes.1 However, as the pancreatic b-cells continue to
produce insulin in response to this peripheral resistance, the de-
mand eventually far exceeds the supply ultimately leading to
hyperglycemia and full blown diabetes. It is then not surprising, gi-
ven the huge increase over the last 10–20 years in the number of
patients diagnosed with diabetes, that there continues to be a con-
certed effort to identify novel pharmacological targets in which to
treat patients afflicted with this terrible disease.2
Kv1.3 is a voltage-gated potassium channel which is a member
of the Shaker or Kv1.x channel subfamily.3 This channel has a vari-
ety of physiological functions including apoptosis, cell volume reg-
ulation and T cell stimulation.4 Recently it was reported that Kv1.3
deficient mice were protected from diet induced obesity.5 The lack
of weight gain was attributed to an increase in energy expenditure
as food intake did not vary from the control animals. These animals
also were more sensitive to insulin as they had similar glucose lev-
els to controls yet had lower circulating levels of insulin.6 Subse-
quent reports demonstrated that Kv1.3 regulated glucose uptake
through GLUT-4 translocation in both adipose and skeletal mus-
cle.7 Consistent with the link to metabolic disorders was a finding
that a variant in the promoter of the Kv1.3 gene is associated with
impaired glucose tolerance and lower insulin sensitivity in human
subjects.8
To date a number of small molecule and/or biological agents
have been discovered that inhibit a number of channels of the
Kv1.x subfamily.9 We recently reported the identification of a
dehydrosaccharin derivative 1 which showed an IC50 = 550 nM
against Kv1.310 (Fig. 1). Herein we report our continued efforts
on this chemotype which provided even more potent analogs
against this potassium channel. It was found from both a metabo-
lite ID and rat microsomal studies that N-dealkylation was a major
route by which these compounds were being metabolized (data
not shown). In vivo PK experiments revealed high clearance rates
as well. In an effort to block this process, synthetic efforts were
O
N
N
H
F
N
S
O
O
1
⇑
Corresponding author. Tel.: +1 919 483 6247; fax: +1 919 315 6964.
E-mail address: curt.d.haffner@gsk.com (C.D. Haffner).
Figure 1.
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.09.131

6990
C. D. Haffner et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6989–6992
F
R1 R2
R1 R2
H
N
H
N
H
N
Br
NH2
b
a
O
R1 = R2 = CH3, 2
R1 = R2 = CH3, 4
R1 = H, R2 = CH3, 5
R1 = H, R2 = CH3, 3
O
N
N
R1 R2
H
c
F
N
S
O
O
R1 = R2 = CH3, 6
R1 = H, R2= CH3, 7
Scheme 1. Reagents and conditions: (a) dioxane, PdCl2(dppf)2, dppf, NaOtBu, H2NCH2C(R1R2)CH2NH2, reflux; (b) CH2Cl2, iPr2NEt, 4-fluorobenzoyl chloride; (c) CH2Cl2,
iPr2NEt, 3-chloro-1,2-benzothiazole 1,1-dioxide.
undertaken to synthesize compounds which might possess better
potency than compound 1 and higher dose normalized AUC’s
(e.g., >500 (h kg ng/mL/mg).
Table 1
Kv1.3 inhibition data and in vitro t½’s for carboxamides 1, 6 and 7
R1
R2
Kv1.3 IC50
(lM)
Std. dev. (
lM)
Rat in vitro
a
Compd
The initial efforts to block the N-dealkylation focused on the
propyl side chain. It was felt that the N-dealkylation might be
blocked to some extent through steric hinderance. Therefore, both
the mono-methyl and gem-dimethyl analogs were synthesized.
The chemistry to generate these compounds is shown in Scheme
1. The first-step involved the palladium catalyzed coupling be-
tween bromobenzene and either 2-methyl or 2,2-dimethyl-1,3-
diaminopropane11 in the presence of sodium t-butoxide in reflux-
ing dioxane providing the amines 2 and 3. A small amount of the
biscoupled product was seen as well (ꢀ10%). The amines were then
reacted with 4-fluorobenzoyl chloride in CH2Cl2 to afford amides 4
and 5. The amides were then reacted with 3-chloro-1,2-benzothia-
zole 1,1-dioxide12 in CH2Cl2 to yield the final compounds 6 and 7.
Unexpectedly, the potency of amides 6 and 7 increased compared
to the unsubstituted amide 1. (IC50’s = 160 nM and 440 nM vs
550 nM, respectively). The in vitro t½ was measured from rat liver
microsomes and only compound 7 showed a small improvement
against the original amide 1 (Table 1). Therefore, this change by it-
self was not enough to significantly improve the in vitro PK for
these two compounds. Attempts were made to incorporate the
gem-dimethyl moiety on the carbon that was directly attached to
the aniline nitrogen, but with no success. The corresponding
t½ (min)
1
6
7
H
CH3
CH3
H
CH3
H
0.55
0.16
0.44
b
0.19
0.15
<15
<15
22
a
A brief description of the assay conditions used to determine the IC50’s can be
found in Ref. 14.
b
This compound was only run as a N = 1, however, an 11 point curve was gen-
erated which had a Z-prime P0.7.
Table 2
Kv1.3 inhibition data and in vitro t½’s for carboxamides 12a–f
Compd
R1
6-position
Kv1.3
Std. dev.
M)
Rat in vitro
t½ (min)
a
IC50
(
lM)
(l
12a
12b
12c
12d
12e
12f
N-morpholine
N-morpholine
3-CF3-Ph
3-CF3-Ph
4-pyranyl
H
F
H
F
H
F
0.47
0.34
0.56
0.70
0.70
0.53
0.24
0.11
0.28
0.31
b
30
60
<15
39
32
26
4-pyranyl
b
a
A brief description of the assay conditions used to determine the IC50’s can be
found in Ref. 14.
b
These compounds were only run as a N = 1, however, an 11 point curve was
generated for each compound which had Z-primes between 0.7 and 0.9.
a-mono-methyl compound was made, but provided no significant
improvement in the PK properties (data not shown).
O
CH3
CH3
a
b
c
NH
O
S
O
F
SO2NH2
F
SO2Cl
F
8
9
10
O
N
N
N
R1
Cl
N
H
d
S
O
F
S
F
O
O
O
11
12b,d,f
Scheme 2. Reagents and conditions: (a) 28% NH4OH, dioxane; (b) H5IO6, CH3CN, CrO3, reflux; (c) dioxane, SOCl2, reflux; (d) CH2Cl2, iPr2NEt, PhNHCH2C(CH3)- (CH3)CH2-
NHCOR1.

C. D. Haffner et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6989–6992
6991
Although the structural modifications for compounds 6 and 7
did not provide marked improvement in the in vitro rat t½’s, the
increase in potency, especially with compound 6, was noteworthy.
Another area of the molecule that was investigated to improve PK
was the 1,2-benzothiazole 1,1-dioxide ring. Therefore, the dehy-
drosaccharin ring was substituted in either the 4-, 5-, 6- or 7-posi-
tion with moieties like Cl, OMe, CH3, Br and CN (structures not
shown). However, it was found that a fluorine atom at the 6-posi-
tion in many cases improved the in vitro rat t½ values (Table 2).
The synthesis to make the fluoro-substituted compounds is shown
in Scheme 2.
The synthesis started with the commercially available sulfonyl
chloride 8 which was converted to the primary sulfonamide 9 with
28% NH4OH in dioxane. The sulfonamide was oxidatively closed
with periodic acid in CH3CN in the presence of a catalytic amount
of CrO3 at reflux13 to give 6-fluorosaccharin 10. In the next step,
compound 10 was chlorinated with SOCl2 in refluxing dioxane to
yield chloride 11. This compound was then directly reacted with
the respective anilines (synthesized as described in Scheme 1) to
generate the final compounds 12a–f.
Compounds 12b and 12d demonstrated better in vitro t½’s than
their respective hydrogen counterparts 12a and 12c. The pyranyl
derivatives 12e and 12f exhibited similar t½’s. The potency for this
set of compounds was comparable between the hydrogen contain-
ing compounds compared to their fluorinated counterparts.
The improvement or maintenance of potency for some of these
compounds in combination with some improvement in the in vitro
t½’s for amides 6, 7, 12b and 12d prompted a more thorough inves-
tigation in which compounds containing both of these features
were synthesized. Table 3 shows some of the final compounds that
were generated. In general these were made as described in
Scheme 1. In cases where the acid chloride was not available the
corresponding acid was coupled to the amine using HATU and
iPr2NEt in DMF to yield the amides which in turn were condensed
with the 3-chloro-6-fluoro-1,2-benzothiazole 1,1-dioxide 11 to
generate the final compounds.
clamp that has been reported. Within this series of compounds
several trends were observed. Firstly, it was found that compounds
13g, 13x–dd and 13jj which contained moieties that were polar
(e.g., basic amines or acids) generally were either inactive or had
lM activity. There were however, two exceptions 13h and 13kk.
Secondly, it was noted that many of the 1-substituted cycloalkyl
compounds provided potent inhibitors of Kv1.3 (cyclopropyl
through cyclohexyl). The substituents that were tolerated were hy-
droxyl, cyano, trifluoromethyl, methyl and even amino in the case
of compound 13h. Three of these groups (NH2, OH and CN) were
incorporated in an effort to improve solubility properties. The tri-
fluoromethyl substituent imparted the best potency as evidenced
Table 3
Kv1.3 inhibition data for the gem-dimethyl propyl substituted carboxamides 13a–rr
Compd R1
Kv1.3a IC50
M)
Std. dev.
M)
Kv1.5a,b IC50
(lM)
(
l
(
l
PAP-1
13a
13b
13c
13d
13e
—
CH3
CH2CF3
NHCH3
Cyclopropyl
1-OH-1-
cyclopropyl
1-CF3-1-
cyclopropyl
1-CO2H-1-
cyclopropyl
1-NH2-1-
cyclopropyl
0.74
1.18
0.15
3.08
0.28
0.82
0.32
b
0.01
0.05
0.04
0.14
3.45
1.18
0.27
2.22
0.49
1.35
13f
13g
13h
0.08
>50
0.03
b
0.24
>50
0.53
0.02
0.79
13i
13j
13k
1-CN-1-cyclopropyl 0.35
2,2-F-1-cyclopropyl 0.14
0.12
0.01
0.03
0.54
0.32
0.37
1-CH3-1-
0.20
cyclopropyl
13l
13m
13n
1-CF3-1-cyclobutyl
1-OH-1-cyclopentyl 0.47
1-NH2-1-
0.09
0.02
0.10
b
0.32
0.73
2.64
1.51
cyclopentyl
13o
13p
1-OH-1-cyclohexyl
4,4-F-1-OH-1-
cyclohexyl
0.35
0.39
b
0.09
0.45
0.63
13q
2-
0.36
b
0.83
O
Tetrahydrofuranyl
2-Pyranyl
13r
13s
13t
13u
13v
13w
13x
0.64
1.35
0.84
0.33
1.07
0.30
2.56
b
b
b
0.03
b
0.44
0.78
0.96
0.56
1.98
0.34
1.39
N
N
H
R1
CH2-2-pyranyl
3,3-CH3-4-pyranyl
1-CN-4-pyranyl
1-OH-4-pyranyl
N-Pyrrolidinyl
N-CH3-2-
N
S
O
F
O
b
b
13
pyrrolidinyl
2-Piperdinyl
13y
5.90
b
b
b
b
b
0.02
b
0.03
b
b
b
b
b
0.37
0.02
b
5.97
2.88
>50
13.7
>50
As part of the investigation into the combined SAR around com-
pound 13, an effort was also made in many cases to improve the
solubility properties that plagued some of the earlier compounds
within this template (data not shown). The SAR in Table 3 shows
that in many cases there was little or no selectivity against
Kv1.5. Kv1.5 inhibitors have also been a target of pharmacological
interest, especially for atrial fibrillation.9k The hope was to identify
an inhibitor which exhibited at least 50–100-fold selectivity so as
to not confound any findings in future in vivo validation studies.
Compounds 13f and 13l however, did show some very modest
selectivity (3 and 3.5-fold, respectively). All of the compounds de-
scribed in Table 3 were compared to the known Kv1.3 inhibitor
PAP-1. It should be noted that the reported IC50 for PAP-1 in the lit-
erature is 2 nM by whole-cell patch clamp on L929 cells stably
expressing Kv1.3.9e All of our reported IC50’s including that of
PAP-1 were obtained using the IonWorks patch clamp assay.
PAP-1 also demonstrated a 4.7-fold selectivity window against
Kv1.5 in our hands which is lower than what has been reported
in the literature9e,f, although this could easily be an artifact of the
IonWorks patch clamp assay compared to the whole-cell patch
13z
N-CH3-2-piperdinyl 3.40
N-CH3-4-piperdinyl >50
N-CH3-3-piperdinyl 12.0
13aa
13bb
13cc
13dd
13ee
13ff
13gg
13hh
13ii
3-Piperidinyl
3-Morpholinyl
2-Furanyl
>50
1.26
0.20
0.15
>50
0.54
0.75
2.0
0.41
0.36
>50
0.29
0.90
13.9
0.52
1.32
0.35
>50
3-Furanyl
2-Benzofuranyl
3-Thiazolyl
3-Oxazolyl
13jj
13kk
13ll
N-CH3-4-imidazolyl 13.1
N-CH3-2-imidazolyl 0.55
3-Pyrrazolyl
1.15
0.23
>50
0.20
0.32
10.1
0.25
13mm 2-Pyrazinyl
13nn
13oo
13pp
13qq
13rr
3-OH-4-pyridyl
3-OMe-4-pyridyl
3-OH-2-pyridyl
2-OH-3-pyridyl
C(OH)(CH3)CF3
b
0.37
0.46
9.06
0.48
0.05
0.91
0.07
a
A brief description of the assay conditions used to determine the IC50’s can be
found in Ref. 14.
b
These compounds were only run as a N = 1, however, an 11 point curve was
generated for each compound which had Z-primes between 0.7 and 0.9.

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C. D. Haffner et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6989–6992
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4. (a) Attali, B.; Romey, G.; Honore, E.; Schmid-Alliana, A.; Mattei, M.-G.; Lesage,
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Compd CLNDa aq sol Cmax (ng/mL)
DNAUCb
(h kg ng/mL/mg)
Kv1.3 IC50
M)
(lM)
@10 mpk po
(l
13i
13rr
304
52
900
2100
630
1630
0.35
0.25
a
Chemiluminescent nitrogen detection.
Dose normalized area under the curve.
b
6. Desir, G. V. Expert Opin. Ther. Targets 2005, 9, 571.
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Desire, G. V. Am. J. Physiol. Cell Physiol. 2006, 290, C345.
by compounds 13f and 13l. Compound 13rr which incorporated
both a hydroxyl and a trifluoromethyl moiety also generated an
inhibitor that was potent against Kv1.3 (IC50 = 250 nM). Many aro-
matic heterocycles showed IC50’s 6 750 nM, for example, 13ee,
13ff, 13hh, 13ii, 13kk, 13mm, 13oo and 13pp. There were how-
ever, some exceptions as demonstrated by compounds 13gg, 13jj,
8. Tschritter, O.; Machicao, F.; Stefan, N.; Schafer, S.; Weigert, C.; Staiger, H.;
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12. This compound was either purchased from commercial vendors or prepared
via the reaction of saccharin with thionyl chloride in refluxing dioxane in the
presence of a catalytic amount of DMF.
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14. Frozen CGE22 cells were thawed and then were transiently transduced to
express human Kv1.3 using 5% (MOI 50-75) Bacmam baculovirus .The cells
were grown adherently for 24 h, cultured, plated at 5000 cells per well and
assayed in an IonWorks PPC format. Channel blockers were detected by
depolarizing membrane potentials resulting in a shift in voltage dependence to
more positive potential. This was accomplished by performing a series of
voltage pulses from À70 mV (resting potential) to +40 mV; the maximum
channel activation and conduction potential. Psora-4, a known potent small
molecule inhibitor of Kv1.3 (IC50 = 724 nM under these assay conditions), was
used as the standard for this assay. The assay was configured to pick up both
tonic block and use-dependent inhibition of the compounds tested against
Kv1.3. Tonic block (pIC50) was calculated from the first 200-ms pulse current
amplitudes utilizing the following equation: tonic response = (1 À IPost1
(+40 mV)/IPre1(+40 mV)) Â 100. All tonic responses were then normalized to
DMSO control and Psora-4 control in the 384 PPC patch plate. Curve fitting
13ll, 13nn and 13qq which all had >1 lM potency. Several non-
aromatic heterocycles were examined as well as evidenced by
13q, 13r and 13u. These compounds showed reasonable potency
(IC50’s 6 640 nM), although as before there were some exceptions
(13s, 13t and 13v).
Many of the more potent compounds were further evaluated in
an effort to identify compounds that could be used for in vivo val-
idation studies. A balance between appropriate PK properties and
potency was the goal. Compounds 13f, 13l and 13j, although show-
ing very good potency, suffered from poor solubility and poor rat
PK (data not shown). Two compounds which did provide both
appropriate PK and solubility properties were compounds 13i
and 13rr. Table 4 shows some of the relevant data for these two
compounds. Both of these compounds provided the desired prop-
erties for further in vivo validation work.
In conclusion, we report the design and synthesis of a series of
substituted 3-amino-1,2-benzothiazole 1,1-dioxide derivatives.
Several of these compounds demonstrated similar potency com-
pared to PAP-1 when using the IonWorks patch clamp assay. Com-
pounds 13i and 13rr were identified as potential tool compounds
that could be used in future in vivo validation studies in diabetic
rodent models.
References and notes
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formula: y = ((B À A)/1 + (10^X/10^C)^D) + A, where B = max, A = min, C = IC50
,
D = slope. Use-dependence (UD30) block was calculated from UD data from
amplitudes measured at the first and 10th pulses. UD response = 100 Â [1 À
((IPost10/IPost1)/(IPre10/IPre1))]. All use-dependence responses were
normalized to DMSO control and Psora-4 control and using the following
curve fitting equation: y = ((B À A)/1 + (10^X/10^C)^D) + A, where B = max,
A = min, C = IC50, and D = slope. The Kv1.5 assay was run in a similar way with
the exceptions that Verapamil was used as the control and stably transfected
CHO K1 cells were used instead of the CGE22 cells. The maximum
concentration that compounds were tested was 50 lM.