N -pyridyl and pyrimidine benzamides as KCNQ2/Q3 potassium channel openers for the treatment of epilepsy

Article abstract of DOI:10.1021/ml200053x

A series of N-pyridyl benzamide KCNQ2/Q3 potassium channel openers were identified and found to be active in animal models of epilepsy and pain. The best compound 12 [ICA-027243, N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide] has an EC₅₀ of 0.38 μM and is selective for KCNQ2/Q3 channels. This compound was active in several rodent models of epilepsy and pain but upon repeated dosing had a number of unacceptable toxicities that prevented further development. On the basis of the structure-activity relationships developed around 12, a second compound, 51, [N-(2-chloro-pyrimidin-5-yl)-3,4-difluoro- benzamide, ICA-069673], was prepared and advanced into a phase 1 clinical study. Herein, we describe the structure-activity relationships that led to the identification of compound 12 and to the corresponding pyrimidine 51.

Full text of DOI:10.1021/ml200053x

LETTER
pubs.acs.org/acsmedchemlett
N-Pyridyl and Pyrimidine Benzamides as KCNQ2/Q3 Potassium
Channel Openers for the Treatment of Epilepsy
George Amato,† Rosemarie Roeloffs,‡ Greg C. Rigdon,‡ Brett Antonio,§ Theresa Mersch,§
||
Grant McNaughton-Smith,†,^ Alan D. Wickenden,§, Paul Fritch,† and Mark J. Suto*,†
Departments of †Chemistry, ‡Pharmacology, and §Biology, Icagen Inc., 4222 Emperor Boulevard, Durham, North Carolina 27702,
United States
S Supporting Information
b
ABSTRACT: A series of N-pyridyl benzamide KCNQ2/Q3 potassium channel openers were identified
and found to be active in animal models of epilepsy and pain. The best compound 12 [ICA-027243,
N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide] has an EC50 of 0.38 μM and is selective for
KCNQ2/Q3 channels. This compound was active in several rodent models of epilepsy and pain but
upon repeated dosing had a number of unacceptable toxicities that prevented further development. On
the basis of the structureꢀactivity relationships developed around 12, a second compound, 51, [N-(2-
chloro-pyrimidin-5-yl)-3,4-difluoro-benzamide, ICA-069673], was prepared and advanced into a phase
1 clinical study. Herein, we describe the structureꢀactivity relationships that led to the identification of
compound 12 and to the corresponding pyrimidine 51.
KEYWORDS: Potassium channel, KCNQ2/Q3, epilepsy
CNQ2ꢀ5 (Kv7.2ꢀ7.5) voltage-dependent potassium chan-
(>400), which included modifications to the amide (reduction
and alkylation), substitutions on both aromatic rings as well as
replacement of the substituted benzene component with hetero-
cycles. The KCNQ agonist activity was only seen with com-
pounds based directly upon the core shown in Scheme 1. The
majority of the compounds had little or no KCNQ agonist
activity (EC50 values > 10 μM).
Tables 1 and 2 contain the key pyridine derivatives prepared,
their KCNQ2/Q3 activity, and KCNQ1 activity when tested.
KCNQ114ꢀ16 is found in cardiac tissue and therefore becomes a
critical counter screen for early leads. In addition, Table 3
provides KCNQ3/Q5 data, which illustrates that compounds
can be identified thatare slightly morepotent against KCNQ2/Q3
versus KCNQ3/Q5.17 The 2-substituted-5-amino pyridines illu-
strated in Table 1 were prepared by reacting the corresponding
2-chloro-5-nitropyridine with the appropriate nucleophile fol-
lowed by catalytic reduction. The methyl and trifluoromethyl
amino pyridines were prepared from the corresponding nicotinic
acids by Curtius reaction with diphenylphosphorylazide followed
by hydrolysis of the resulting tert-butyl-carbamate.
The general synthesis of the pyridine compounds involved the
reaction of a 2-substituted-5-aminopyridine with substituted
benzoic acid or acid chloride (Scheme 1). The yields ranged
from 20 to 60%. Typically, if the acid chlorides were not
commercially available, they were made from the corresponding
benzoic acids but used directly in the next step without purifica-
tion. Compounds 1ꢀ5 were commercially available. All of the
final compounds (Tables 1ꢀ3) were >97% pure as determined
by LC/MS and NMR.
K
nels are expressed at high levels in the brain, including
regions linked to seizure disorders, such as the cortex, hippo-
campus, and thalamus. They represent the molecular correlate of
the neuronal M current (IM), a noninactivating, slowly deactivat-
ing subthreshold Kþ current that opposes depolarizing current
and serves to stabilize membrane potential and control neuronal
excitability.1,2
Given that KCNQ2/Q3 channels lead to benign familial
neonatal convulsions, a rare form of neonatal epilepsy in
humans,3ꢀ6 it is hypothesized that KCNQ2/Q3-based M cur-
rents play an important role in the control of neuronal excitability
and epileptiform activity. Benign familial neonatal convulsions
are characterized by generalized seizures in early life, which
disappear within weeks or months of birth but may return later
in life. Moreover, disruption of the KCNQ2 gene in mice results
in hypersensitivity to the chemoconvulsant pentylenetetrazole
(PTZ),7 decreased seizure threshold to electric shock-induced
convulsions,8 and spontaneous seizures.9 This evidence suggests
that openers or activators of KCNQ channels should decrease
neuronal excitability, making them attractive drug targets for the
treatment of epilepsy and related disorders of neuronal
excitability.10ꢀ13
A screening program to identify KCNQ agonists resulted in
the identification of 3,4 dichloro-N-(pyridine-3-yl)benzamide
(1) as a weak agonist (EC50 = 6 μM, Figure 1, 86Rbþ assay,
see the Supporting Information). The corresponding 2-pyridyl
[2, 3,4 dichloro-N-(pyridine-2-yl)benzamide] and 4-pyridyl
[3, 3,4 dichloro-N-(pyridine-4-yl)benzamide] isomers as well as
the benzene derivative (4) were inactive (EC50 > 10 μM). The
subsequent chemistry effort focused on the N-(pyridine-3-
yl)benzamide core. A large set of derivatives were prepared
Received: February 18, 2011
Accepted: March 31, 2011
Published: March 31, 2011
r
2011 American Chemical Society
481
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LETTER
Table 2. KCNQ2/Q3 and KCNQ1 Activity of N-(6-Chlor-
opyridin-3-yl) Benzamidesa
KCNQ2/Q3
KCNQ1
compd no.
R-substituent
EC50 (μM)
IC50 (μM)
Figure 1. Benzanilide analogues.
12
16
17
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
48
49
3,4-F2
4-Cl
H
0.38
0.38
1.6
41
15
Scheme 1. General Synthesis of 2-Chlorpyridine
Benzanildesa
>100
NT
77
2-F
>10
1.1
3-F
4-F
0.98
0.39
0.32
0.26
5.5
66
3-CF3
4-CF3
4-Ph
5.8
2.9
>30
NT
>100
17
3-OCH3
3,4 (CH3)2
3,4-Cl2
0.69
0.67
0.27
0.08
0.42
>10
0.37
0.19
0.28
>10
>10
0.01
1.1
3-Cl
41
3-F, 4-Cl
15
3-CF3, 4-F
3.4
4-SO2NH2
NT
3.4
3-F, 4-CF3
a Reagents and conditions: (a) Nucleophile, MeOH. (b) Pd/C 10% H2,
DCM. (c) DPPA, TEA, t-butanol. (d) TFA, DCM. (e) (CO)2Cl2,
DCM, DMF (cat) 0ꢀ to room temperature. (f) DIEA, DCM, room
temperature.
3-Cl, 4-F
22
3-F, 4-CH3
33
3-CN
NT
NT
1.9
2,3-F2
4-OC6H5
Table 1. KCNQ2/Q3 Activity of N-(6-Substituted Pyridin-3-
yl)-3,4-diflourobenzamide
3-F, 4-CN
51
3-F, 4-OCH3
3-F, 4-OEt
3.2
>100
>100
>100
1.3
0.57
0.17
0.03
0.08
0.07
1.6
3-F, 4-OiPr
3-F, 4-OC6H5
3-F, 4-(4FꢀC6H5S)
3-F, 4-(4FꢀC6H5SO2)
3-F, 4-(4FꢀC6H5CH2S)
3-F, 4-(4FꢀC6H5CH2SO2)
3-F, 4-SiPr
>100
>100
>100
>100
5.8
compd no.
Y-substituent
KCNQ2/Q3 EC50 (μM)
0.16
0.11
0.14
0.26
0.18
5
6
F
4.8
6.8
CH3
3-F, 4-SiBu
>100
54
7
SCH3
>10
>10
10
3-F, 4-SO2iBu
3-F, 4-NHCH2CH2C6H5
a NT, not tested.
8
CF3
>100
9
CN
10
11
12
13
14
15
OH
>10
>10
0.38
2.7
OCH3
other substituents at this position resulted in compounds that
were at least 10-fold less active. An exception is compound
14 (EC50 = 0.48 μM). However, there was some weak activity
with fluoro derivative (5) and the methyl derivative (6) as
well as with the cyclopropylamine and morpholine analogues
(13 and 15).
Cl
NH-cyclopropyl
1-pyrrolidine
1-morpholine
0.48
7.8
On the basis of the initial results with the pyridine isomers, we
focused on identifying the optimal substituent at the 6-position
(Y) of the N-(pyridine-3-yl) benzamide core, while holding the
benzene portion constant (R1, R2 = F, Table 1). The best
Y-substituent was chlorine (12, EC50 = 0.38 μM). All of the
Next, we turned our attention to the various substituents on
the benzene portion and how changes modulated KCNQ2/Q3
agonist activity as well as KCNQ1 antagonist activity (Table 2).
In general, substituents at the R2-position were not well
tolerated,18 and R3-substituted compounds varied in their
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LETTER
Table 3. KCNQ Activity of 2-(Chloropyrimidin-5-yl)-
benzamides
Scheme 2. Synthesis of 2-Chloropyrimidine-Substituted
Benzanildesa
compd
no.
KCNQ2/Q3
KCNQ3/Q5 KCNQ1 IC50
R1
EC50(μM)
EC50 (μM)
(μM)
50 3-F
7.2
23
14.3
14
28
9
>100
>100
78
51 3,4-F2
52 3-Cl
0.69
1.1
53 3-CF3
54 3-F,4-Cl
55 3-F,4-CH3
4.3
35
a Reagents and conditions: (a) CuCl2, t-BuONO, MeCN, 65ꢀ80 ꢀC.
(b) Fe, AcOH, EtOH, H2O, 90ꢀ100 ꢀC. (c) R1COCl, pyridine, DCM.
0.41
0.60
49
10
93
In fact, 51 was found to be 20-fold selective for KCNQ2/Q3 over
KCNQ3/Q5 (Table 3) and had no measurable activity against a
panel of cardiac ion channels (IC50 values > 30 μM for hERG,
Nav1.5, L type channels, and KCNQ1) as well as no activity on
GABA(A) gated channels at 10 μM.17 This compound was tested in
the rat maximal electroshock (MES) and the PTZ anticonvulsant
assays and had oral ED50 values of 1.5 and <1 mg/kg, respectively,
which is slightly improved over the corresponding pyridine analogue
activity with 3-chloro and trifluoromethyl containing compounds
(27 and 21) having comparable activity to 12. However, the
3-cyano and 3-methoxy compounds (34 and 24) were weakly
active. The most potent compound identified was the 3-fluoro-4-
choro derivative 28 with a KCNQ2/Q3 EC50 = 0.08 μM, but 28
was also weakly active versus KCNQ1.
In addition to halogen and alkyl substituents at R3 and R4,
we examined a series of heteroatom substituents (O, S, and N)
at these positions. From the large number of compounds
prepared, those compounds containing a fluorine substituent
on the R3-position and various heteroatom substituents at the
R4-position of the benzene ring (compounds 37ꢀ49) are
highlighted here for comparison. In general, these compounds
had KCNQ2/Q3 activity ranging from nanomolar to micro-
molar and varied considerably in their KCNQ1 (IC50) activity
as well (Table 2). For example, the R4O-phenyl derivatives 36
and 41 have EC50 values of 0.01 and 0.03 μM, respectively,
while the best R4O-alkyl derivative 40 has an EC50 of 0.17 μM.
However, the corresponding KCNQ1 IC50 values for 36 and
41 are < 2 μM, while the KCNQ1 activity for compound 40 is
> 100 μM.
12 (compound 12 rat MES ED50 = 1.5 mg/kg, rat PTZ ED50 =
2.2 mg/kg). Following a 2 mg/kg iv administration (rat) of 51, the
t1/2 was 1.2 h, clearance was 1039 mL/h/kg, the volume of
distribution based on the terminal phase (Vz) was 1804 mL/kg,
and the compound was well absorbed (63% bioavailable, Tmax
=
0.5 h). This compound was advanced to IND enabling GLP
safety studies and ultimately into a phase 1 clinical trials.
In summary, we have described the identification and structureꢀ
activity relationships for a series of N-pyridyl benzamide KCNQ2/Q3
potassium channel openers. The lead compounds 12 and 51 were
orally active in several animal models of epilepsy and had sufficient
in vitro selectivity and pharmacokinetic properties to advance to
rat toxicity studies. Compound 51 was advanced into a phase 1
clinical trial, the results of which will be reported elsewhere.
In the pyridine series, the compound with the best overall in
vitro and in vivo profile was 12.19,20 This compound was advanced
into 28 day toxicity studies in rat and cynomolgus monkeys, but
upon repeated dosing, the compound induced nonhemolytic
anemia in both species. It was determined that a metabolite derived
from 2-chloro-5-aminopyridine was the cause of the toxicity.21
Therefore, a search for a structurally diverse analogue with a similar
in vitro profile minus the toxicity was initiated.
’ ASSOCIATED CONTENT
S
Supporting Information. General methods, experimen-
b
tal details, analytical data, and in vitro and in vivo assay protocols.
This material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
The first set of analogues investigated were the corresponding
pyrimidine analogues (Table 3). The compounds were prepared
starting with the 2-amino-5-nitropyrimidine (Scheme 2), which was
converted to the 2-chloro analogue by treatment with t-butyl nitrite
followed by cupric chloride. The nitro compound was reduced using
iron in acetic acid to avoid reduction of the 2-choro substituent, and
the resulting 2-choro-5-aminopyrimidine was reacted with various
acid chlorides to provide the desired compounds (Table 3). As
shown in the table, compound 51 was the most potent pyrimidine
analogue and had an improved activity profile versus KCNQ1. For
example, comparing the pyrimidines and the pyridines (i.e., 50 vs
19, 51 vs 12, and 55 vs 33), the pyrimidines overall were slightly less
active on KCNQ2/Q3 channels but more selective for KNCQ2/Q3
over KCNQ1.
Corresponding Author
*Tel: 919-281-5504. Fax 919-941-0813. E-mail: msuto@
icagen.com.
Present Addresses
Johnson and Johnson, San Diego, CA.
^CEAMED, Tenerife, Spain.
’ ACKNOWLEDGMENT
The helpful comments on the manuscript by Doug Krafte are
appreciated, as is the assistance of Steve Werness for performing
the in vivo pharmacokinetic studies.
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LETTER
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