U.S. patent application number 14/002943 was filed with the patent office on 2014-07-03 for benzimidazole inhibitors of the sodium channel.
This patent application is currently assigned to Zalicus Pharmaceuticals Ltd.. The applicant listed for this patent is Richard Holland, Jason Lamontagne, Hassan Pajouhesh, Hossein Pajouhesh, Brendan Whelan, Lingyun Zhang. Invention is credited to Richard Holland, Jason Lamontagne, Hassan Pajouhesh, Hossein Pajouhesh, Brendan Whelan, Lingyun Zhang.
Application Number | 20140187533 14/002943 |
Document ID | / |
Family ID | 46757322 |
Filed Date | 2014-07-03 |
United States Patent
Application |
20140187533 |
Kind Code |
A1 |
Pajouhesh; Hassan ; et
al. |
July 3, 2014 |
BENZIMIDAZOLE INHIBITORS OF THE SODIUM CHANNEL
Abstract
The invention relates to compounds useful in treating conditions
associated with voltage-gated ion channel function, particularly
conditions associated with sodium channel activity. More
specifically, the invention concerns compounds (e.g., compounds
according to any of Formulas (I)-(XIII) or Compounds (1)-(236) of
Table 1) that are useful in treatment of a variety of diseases and
conditions. Formula (I) ##STR00001##
Inventors: |
Pajouhesh; Hassan; (West
Vancouver, CA) ; Holland; Richard; (Vancouver,
CA) ; Zhang; Lingyun; (Vancouver, CA) ;
Pajouhesh; Hossein; (Coquitlam, CA) ; Lamontagne;
Jason; (Burnaby, CA) ; Whelan; Brendan;
(Vancouver, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pajouhesh; Hassan
Holland; Richard
Zhang; Lingyun
Pajouhesh; Hossein
Lamontagne; Jason
Whelan; Brendan |
West Vancouver
Vancouver
Vancouver
Coquitlam
Burnaby
Vancouver |
|
CA
CA
CA
CA
CA
CA |
|
|
Assignee: |
Zalicus Pharmaceuticals
Ltd.
Vancouver
CA
|
Family ID: |
46757322 |
Appl. No.: |
14/002943 |
Filed: |
March 2, 2012 |
PCT Filed: |
March 2, 2012 |
PCT NO: |
PCT/CA12/00193 |
371 Date: |
March 17, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61448923 |
Mar 3, 2011 |
|
|
|
61448910 |
Mar 3, 2011 |
|
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Current U.S.
Class: |
514/210.18 ;
435/375; 514/210.01; 514/210.2; 514/210.21; 514/256; 514/394;
514/423; 544/333; 546/268.1; 548/306.1; 548/309.7; 548/537;
548/953 |
Current CPC
Class: |
A61P 25/00 20180101;
C07C 237/04 20130101; C07D 265/30 20130101; A61P 29/00 20180101;
C07D 205/04 20130101; C07D 401/04 20130101; C07C 237/24 20130101;
C07D 241/08 20130101; C07D 235/14 20130101; A61K 31/5377 20130101;
C07D 413/06 20130101; C07D 207/16 20130101; C07D 211/60 20130101;
C07C 237/22 20130101; C07D 401/12 20130101; C07D 231/14 20130101;
C07C 2601/14 20170501; C07C 237/06 20130101; C07D 403/04 20130101;
C07D 413/04 20130101; C07C 2601/02 20170501; C07D 403/06 20130101;
A61K 31/401 20130101; A61K 31/454 20130101 |
Class at
Publication: |
514/210.18 ;
548/306.1; 514/210.21; 546/268.1; 514/210.2; 548/953; 514/210.01;
548/537; 514/423; 548/309.7; 514/394; 544/333; 514/256;
435/375 |
International
Class: |
C07D 403/06 20060101
C07D403/06; C07D 235/14 20060101 C07D235/14; C07D 207/16 20060101
C07D207/16; C07D 401/12 20060101 C07D401/12; C07D 205/04 20060101
C07D205/04 |
Claims
1.-81. (canceled)
82. A compound having a structure according to the following
formula, ##STR00276## or a pharmaceutically acceptable salt or
solvate thereof, wherein each of R.sup.1, R.sup.2, and R.sup.3 is,
independently, H, unsubstituted C1-C6 alkyl, optionally substituted
C1-C6 haloalkyl, or halogen; m is 1 or 2; each R.sup.4 and R.sup.5
is, independently, H, optionally substituted C1-C6 alkyl, or
optionally substituted C1-C6 haloalkyl, or R.sup.4 and R.sup.5
combine to form an optionally substituted C3-C6 cycloalkyl, or
R.sup.4 and R.sup.5 combine to form an oxo (C.dbd.O) group; each of
R.sup.6 and R.sup.8 is, independently, H or optionally substituted
C1-C6 alkyl; or R.sup.6 and R.sup.8 combine to form an optionally
substituted three-to-nine membered heterocyclyl, or R.sup.6 and
R.sup.7A combine to form an optionally substituted three-to-nine
membered heterocyclyl; n is 1 or 2; each R.sup.7A and R.sup.7B is,
independently H, optionally substituted C1-C6 alkyl, or optionally
substituted C1-C6 haloalkyl; or R.sup.6 combines with R.sup.7A to
form an optionally substituted three-to-nine heterocyclyl; or an
R.sup.7A and R.sup.7B group on the same carbon combine to form an
optionally substituted C3-C6 cycloalkyl; or, when n is 2, both
R.sup.7A groups combine to form an optionally substituted C3-C6
cycloalkyl.
83.-117. (canceled)
118. The compound of claim 82, or a pharmaceutically acceptable
salt or solvate thereof, wherein one of R.sup.1, R.sup.2, and
R.sup.3 is H.
119. The compound of claim 82, or a pharmaceutically acceptable
salt or solvate thereof, wherein two of R.sup.1, R.sup.2, and
R.sup.3 are, independently, CF.sub.3, Cl, or F.
120. The compound of claim 82, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.4 and R.sup.5 are both H; or
R.sup.4 and R.sup.5 are both CH.sub.3; or R.sup.4 and R.sup.5
combine to form an optionally substituted C3-C6 cycloalkyl; or
R.sup.4 and R.sup.5 combine to form an oxo (C.dbd.O) group.
121. The compound of claim 82, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.6 combines with R.sup.7A to
form a three-to-six membered heterocyclyl ring, or wherein R.sup.6
and R.sup.8 combine to form an optionally substituted three-to-six
membered heterocyclyl.
122. The compound of claim 82, or a pharmaceutically acceptable
salt or solvate thereof, having a structure according to the
following formula, ##STR00277##
123. The compound of claim 122, or a pharmaceutically acceptable
salt or solvate thereof, having a structure: ##STR00278##
124. A compound having a structure according to the following
formula, ##STR00279## or a pharmaceutically acceptable salt or
solvate thereof, wherein each of R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 is selected, independently, from H, optionally substituted
C1-C6 alkyl, optionally substituted C1-C6 haloalkyl, optionally
substituted C6-C10 aryl, or optionally substituted 5 to 6-membered
heteroaryl, wherein at least one of R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 is halogen or optionally substituted C1-C6 haloalkyl;
R.sup.5 is H, optionally substituted C1-C6 alkyl, or optionally
substituted C1-C10 heteroalkyl; R.sup.6 is --R.sup.6A or
--CH.sub.2R.sup.6B; R.sup.6A is NH.sub.2, optionally substituted
cyclopropyl, optionally substituted azetidine, optionally
substituted cyclopentyl, optionally substituted pyrazole,
optionally substituted pyrrole, optionally substituted pyrrolidine,
optionally substituted thiazolidine, optionally substituted
thiazolidine-1,1-dioxide, optionally substituted pyrimidine,
optionally substituted C1-C10 aminoalkyl, optionally substituted
C1-C10 hydroxyalkyl, optionally substituted C1-C10 alkoxyalkyl,
optionally substituted C1-C10 haloalkyl, or optionally substituted
C1-C10 alkylsulfonyl; or R.sup.6A has a structure according to
##STR00280## wherein n is an integer between 0-4; Z.sup.1 is
CH.sub.2, NH, NCH.sub.3, or O; L.sup.1 is --CH.sub.2, --CHR.sup.4A,
--CH.sub.2C(.dbd.O), --C(.dbd.O)CH.sub.2, --CH.sub.2C(.dbd.O)NH,
--CH.sub.2C(.dbd.O)NHCH.sub.2, or --CH.sub.2NHC(.dbd.O)CH.sub.2;
each R.sup.2A and R.sup.2C, when present, is selected from OH,
N(R.sup.2B).sub.2, halogen, and unsubstituted C1-C3 alkyl, or two
R.sup.2A combine to form an oxo (.dbd.O) group, and wherein no more
than two R.sup.2A combine to form an oxo group; and each R.sup.2B
is, independently, H or unsubstituted C1-C6 alkyl; R.sup.2D is H,
OH, or NH.sub.2; and R.sup.6B is optionally substituted
cyclopropyl, optionally substituted azetidine, optionally
substituted cyclopentyl, optionally substituted pyrazole,
optionally substituted pyrrole, optionally substituted pyrrolidine,
optionally substituted thiazolidine, optionally substituted
thiazolidine-1,1-dioxide, or optionally substituted pyrimidine.
125. The compound of claim 124, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.6 comprises a --NH.sub.2
substituent.
126. The compound of claim 124, or a pharmaceutically acceptable
salt or solvate thereof, wherein R.sup.2 and R.sup.4 are both
CF.sub.3, F, or Cl.
127. A pharmaceutical composition comprising the compound of claim
82 and a pharmaceutically acceptable carrier or excipient.
128. A method to treat a disease or condition, said method
comprising administering to a subject in need of such treatment an
effective amount of the compound of claim 82.
129. The method of claim 128, wherein said condition is pain,
epilepsy, Parkinson's disease, a mood disorder, psychosis,
tinnitus, amyotropic lateral sclerosis, glaucoma, ischaemia,
spasticity disorders, obsessive compulsive disorder, restless leg
syndrome, or Tourette syndrome.
130. The method of claim 129, wherein said subject is a fasted
subject.
131. The method of claim 129, wherein said subject is a fed
subject.
132. A method of inhibiting a voltage-gated sodium channel, said
method comprising contacting a cell with the compound of claim 82.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application Nos. 61/448,923 and 61/448,910, each of which was filed
on Mar. 3, 2011, and each of which is hereby incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to compounds useful in treating
conditions associated with voltage-gated ion channel function,
particularly conditions associated with sodium channel activity.
More specifically, the invention concerns benzimdiazole compounds
that are useful in treatment numerous diseases and conditions.
BACKGROUND OF THE INVENTION
[0003] Voltage-gated sodium (Na.sub.v) channels are present in
neurons and excitable tissues where they contribute to processes
such as membrane excitability and muscle contraction (Ogata et al.,
Jpn. J. Pharmacol. (2002) 88(4) 365-77). Nine different
transmembrane .alpha.-subunits (Na.sub.v1.1-1.9) from a single
Na.sub.v1 family combine with auxiliary .beta.-subunits that modify
channel function to form functional Na.sub.v channels. Of the nine
Na.sub.v1 .alpha.-subunit isoforms, five are expressed in the
dorsal root ganglion where they are involved in setting the resting
membrane potential and the threshold for generating action
potentials, and also contribute to the upstroke as well as firing
of action potentials during sustained depolarization. In
particular, the tetrodotoxin (TTX) sensitive Na.sub.v1.7 and
TTX-insensitive Na.sub.v1.8 channel subtypes act as major
contributors to both inflammatory and neuropathic pain (Momin et
al., Curr Opin Neurobiol. 18(4):383-8, 2008; Rush et al., J.
Physiol. 579(Pt 1):1-14, 2007).
[0004] Novel allosteric modulators of voltage-gated ion channels
(e.g., sodium channels) are thus desired. Modulators may affect the
kinetics and/or the voltage potentials of, e.g., Na.sub.v1.7 and/or
Na.sub.v1.8 channels.
SUMMARY OF THE INVENTION
[0005] The invention relates to compounds useful in conditions
modulated by voltage-gated ion channels (e.g., voltage gated sodium
channels).
[0006] In a first aspect, the invention features a compound having
a structure according to the following formula,
##STR00002##
or a pharmaceutically acceptable salt or solvate thereof, where
[0007] each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is selected,
independently, from H, optionally substituted C1-C6 alkyl,
optionally substituted C1-C6 haloalkyl, optionally substituted
C6-C10 aryl, or optionally substituted 5 to 6-membered heteroaryl,
where at least one of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is
halogen or optionally substituted C1-C6 haloalkyl;
[0008] R.sup.5 is H, optionally substituted C1-C6 alkyl, or
optionally substituted C1-C10 heteroalkyl;
[0009] R.sup.6 is --R.sup.6A or --CH.sub.2R.sup.6B;
[0010] R.sup.6A is NH.sub.2, optionally substituted cyclopropyl,
optionally substituted azetidine, optionally substituted
cyclopentyl, optionally substituted pyrazole, optionally
substituted pyrrole, optionally substituted pyrrolidine, optionally
substituted thiazolidine, optionally substituted
thiazolidine-1,1-dioxide, optionally substituted pyrimidine,
optionally substituted C1-C10 aminoalkyl, optionally substituted
C1-C10 hydroxyalkyl, optionally substituted C1-C10 alkoxyalkyl,
optionally substituted C1-C10 haloalkyl, or optionally substituted
C1-C10 alkylsulfonyl; or R.sup.6A has a structure according to
##STR00003##
where
[0011] n is an integer between 0-4;
[0012] Z.sup.1 is CH.sub.2, NH, NCH.sub.3, or O;
[0013] L.sup.1 is --CH.sub.2, --CHR.sup.4A, --CH.sub.2C(.dbd.O),
--C(.dbd.O)CH.sub.2, --CH.sub.2C(.dbd.O)NH,
--CH.sub.2C(.dbd.O)NHCH.sub.2, or
--CH.sub.2NHC(.dbd.O)CH.sub.2;
[0014] each R.sup.2A and R.sup.2C, when present, is selected from
OH, N(R.sup.2B).sub.2, halogen, and unsubstituted C1-C3 alkyl, or
two R.sup.2A combine to form an oxo (.dbd.O) group, and wherein no
more than two R.sup.2A combine to form an oxo group; and
[0015] each R.sup.2B is, independently, H or unsubstituted C1-C6
alkyl;
[0016] R.sup.2D is H, OH, or NH.sub.2;
[0017] and
[0018] R.sup.6B is optionally substituted cyclopropyl, optionally
substituted azetidine, optionally substituted cyclopentyl,
optionally substituted pyrazole, optionally substituted pyrrole,
optionally substituted pyrrolidine, optionally substituted
thiazolidine, optionally substituted thiazolidine-1,1-dioxide, or
optionally substituted pyrimidine.
[0019] In some embodiments, R.sup.6A is NH.sub.2, optionally
substituted cyclopropyl, optionally substituted azetidine,
optionally substituted cyclopentyl, optionally substituted
pyrazole, optionally substituted pyrrole, optionally substituted
pyrrolidine, optionally substituted thiazolidine, optionally
substituted thiazolidine-1,1-dioxide, optionally substituted
pyrimidine, optionally substituted C1-C10 aminoalkyl, optionally
substituted C1-C10 hydroxyalkyl, optionally substituted C1-C10
alkoxyalkyl, optionally substituted C1-C10 haloalkyl, or optionally
substituted C1-C10 alkylsulfonyl.
[0020] In other embodiments, R.sup.6 has a structure according
to
##STR00004##
[0021] In some embodiments, R.sup.5 is H.
[0022] In other embodiments, R.sup.5 is optionally substituted
C1-C10 heteroalkyl.
[0023] In certain embodiments, R.sup.2 and R.sup.4 are both
CF.sub.3, F, or Cl.
[0024] In still other embodiments, R.sup.2 and R.sup.3 are both
CF.sub.3, F, or Cl.
[0025] In some embodiments, R.sup.6 is --CH.sub.2R.sup.6B, and
R.sup.6B is optionally substituted azetidine.
[0026] In certain embodiments, R.sup.6 is optionally substituted
C1-C10 aminoalkyl.
[0027] In other embodiments, the C1-C10 aminoalkyl includes an oxo
(.dbd.O) substituent, an alkoxy substituent, an N-sulfonyl group,
or any combination thereof.
[0028] In some embodiments, the compound has a structure according
to the following formula,
##STR00005##
or a pharmaceutically acceptable salt or solvate thereof, where n
is 0 or 1, and R.sup.7 is H or --C(.dbd.O)R.sup.7A, where R.sup.7A
is unsubstituted C1-C6 alkyl or optionally substituted C1-C10
aminoalkyl.
[0029] In certain embodiments, n is 0.
[0030] In other embodiments, n is 1.
[0031] In some embodiments, R.sup.7 is H or C(.dbd.O)R.sup.7A,
where R.sup.7A is unsubstituted C1-C3 alkyl or an optionally
substituted C1-C10 aminoalkyl including a terminal --NH.sub.2
group.
[0032] In further embodiments, R.sup.2 and R.sup.4 are both
CF.sub.3, F, or Cl.
[0033] In some embodiments, R.sup.2 and R.sup.3 are both CF.sub.3,
F, or Cl.
[0034] In other embodiments, R.sup.6 is optionally substituted
cyclopropyl, optionally substituted azetidine, optionally
substituted cyclopentyl, optionally substituted pyrazole,
optionally substituted pyrrole, optionally substituted pyrrolidine,
optionally substituted thiazolidine, optionally substituted
thiazolidine-1,1-dioxide, optionally substituted pyrimidine.
[0035] In still other embodiments, R.sup.6 includes a --NH.sub.2
substituent.
[0036] In some embodiments, the compound has a structure according
to the following formula,
##STR00006##
where R.sup.6B is optionally substituted azetidine, optionally
substituted cyclopentyl, optionally substituted pyrrolidine,
optionally substituted thiazolidine, optionally substituted
thiazolidine-1,1-dioxide, or optionally substituted pyrimidine.
[0037] In certain embodiments, R.sup.2 and R.sup.4 are both
CF.sub.3, F, or Cl.
[0038] In other embodiments, R.sup.2 and R.sup.3 are both CF.sub.3,
F, or Cl.
[0039] In further embodiments, R.sup.5 is H.
[0040] In still other embodiments, R.sup.6 is an optionally
substituted C1-C10 aminoalkyl group.
[0041] In some embodiments, the C1-C10 aminoalkyl includes a
terminal --NH.sub.2 group.
[0042] In other embodiments, the C1-C10 aminoalkyl includes an oxo
(.dbd.O) substituent.
[0043] In certain embodiments, R.sup.6 is
--(CH.sub.2).sub.m1(NR.sup.6C).sub.m2(C.dbd.O).sub.m3(CH.sub.2).sub.m4NR.-
sup.6DR.sup.6E or
--(CH.sub.2).sub.m1(C(CH.sub.3).sub.2).sub.m2(CH.sub.2).sub.m4NR.sup.6CR.-
sup.6D where each of m1 and m4 is, independently, an integer
between 1-6; each of m2 and m3 is, independently, 0 or 1; each of
R.sup.6C and R.sup.6E is, independently, H or unsubstituted C1-C6
alkyl; and R.sup.6D is H, unsubstituted C1-C6 alkyl, or an
N-protecting group.
[0044] In some embodiments, R.sup.6 is --(CH.sub.2).sub.m1NH.sub.2,
--CH.sub.2NHC(.dbd.O)CH.sub.2NH.sub.2,
--C(CH.sub.3).sub.2CH.sub.2NH.sub.2, --C(CH.sub.3).sub.2NH.sub.2,
and where m1 is 1, 2, or 3.
[0045] In other embodiments, R.sup.2 and R.sup.4 are both CF.sub.3,
F, or Cl.
[0046] In still other embodiments, R.sup.2 and R.sup.3 are both
CF.sub.3, F, or Cl.
[0047] In particular embodiments, one and only one of R.sup.2 or
R.sup.3 is optionally substituted phenyl.
[0048] In certain embodiments, R.sup.5 is H.
[0049] In some embodiments, R.sup.6 is optionally substituted C1-C3
haloalkyl, optionally substituted C1-C10 alkoxyalkyl, optionally
substituted C1-C10 hydroxyalkyl, or optionally substituted C1-C10
alkylsulfonyl.
[0050] In other embodiments, R.sup.6 is
--(CH.sub.2).sub.m1CF.sub.3, --(CH.sub.2).sub.m1OR.sup.6F,
--(CH.sub.2).sub.m1SO.sub.2R.sup.6G, where m1 is an integer between
1-6, R.sup.68 is H or CH.sub.3, and R.sup.6G is unsubstituted C1-C3
alkyl.
[0051] In further embodiments, R.sup.2 and R.sup.4 are both
CF.sub.3, F, or Cl.
[0052] In certain embodiments, R.sup.2 and R.sup.3 are both
CF.sub.3, F, or Cl.
[0053] In some embodiments, R.sup.5 is H.
[0054] In a second aspect, the invention features a compound having
a structure according to the following formula,
##STR00007##
or a pharmaceutically acceptable salt or solvate thereof, where
[0055] each of X.sup.1, X.sup.2, and X.sup.3 is N or CR.sup.4, and
where one and only one of X.sup.1, X.sup.2, and X.sup.3 is N;
[0056] L.sup.1 is a covalent bond, --CH.sub.2, --CHR.sup.5A,
--CH.sub.2C(.dbd.O), --C(.dbd.O)CH.sub.2, --CH.sub.2C(.dbd.O)NH,
--CH.sub.2C(.dbd.O)NHCH.sub.2, --CH.sub.2NHC(.dbd.O)CH.sub.2, or
--CH.sub.2CH.sub.2;
[0057] each of R.sup.1, R.sup.2, and R.sup.4 is, independently, H,
unsubstituted C1-C3 alkyl, optionally substituted C1-C3 haloalkyl,
or halogen;
[0058] R.sup.3 is H, optionally substituted C1-C6 alkyl, or
optionally substituted C1-C10 heteroalkyl;
[0059] R.sup.5A is selected from optionally substituted C1-C3
alkyl; and
[0060] where at least one of R.sup.1, R.sup.2, R.sup.3, and R.sup.5
is halogen or optionally substituted C1-C3 haloalkyl.
[0061] In some embodiments, L.sup.1 is CH.sub.2 or CHCF.sub.3.
[0062] In other embodiments, R.sup.3 is H.
[0063] In another aspect, the invention features a compound having
a structure according to the following formula,
##STR00008##
or a pharmaceutically acceptable salt or solvate thereof, where
[0064] n is an integer between 0-4;
[0065] R.sup.1 is selected from --CH.sub.2R.sup.3A,
--CHR.sup.4AR.sup.3A, --CH.sub.2C(.dbd.O)R.sup.3A,
--C(.dbd.O)CH.sub.2R.sup.3A, --CH.sub.2C(.dbd.O)NR.sup.4BR.sup.3A,
--CH.sub.2C(.dbd.O)NR.sup.4BCH.sub.2R.sup.3A,
--CH.sub.2NR.sup.4BC(.dbd.O)CH.sub.2R.sup.3A, --R.sup.3B,
--CH.sub.2CH.sub.2R.sup.3B, and
--CH.sub.2C(.dbd.O)NR.sup.4BCHR.sup.4CR.sup.3C;
[0066] each R.sup.2, when present, is selected from OH,
N(R.sup.2A).sub.2, halogen, and unsubstituted C1-C3 alkyl, or two
R.sup.2 combine to form an oxo (.dbd.O) group, and where no more
than two R.sup.2 combine to form an oxo group;
[0067] each R.sup.2A is, independently, H or unsubstituted C1-C6
alkyl;
[0068] R.sup.3A is selected from [0069] a benzimidazole including
at least one C-substituent selected from halogen or C1-C6 haloalkyl
or an N-substituent that is C1-C12 heteroalkyl; [0070] a pyridine
including at least one substituent selected from halogen or C1-C6
haloalkyl; and [0071] a pyrazole including at least one substituent
selected from halogen, unsubstituted C1-C3 alkyl, and C1-C6
haloalkyl;
[0072] R.sup.3B is selected from [0073] a benzimidazole including
at least one C-substituent selected from Cl, Br, I, or C1-C6
haloalkyl or an N-substituent that is a C1-C10 heteroalkyl; [0074]
a pyridine including at least one substituent selected from halogen
or C1-C6 haloalkyl; and [0075] a pyrazole including at least one
substituent selected from halogen, unsubstituted C1-C3 alkyl, and
C1-C6 haloalkyl;
[0076] R.sup.3C is optionally substituted pyridine;
[0077] R.sup.4A is optionally substituted C1-C3 alkyl;
[0078] R.sup.4B is H or optionally substituted C1-C3 alkyl;
[0079] R.sup.4C is C1-C3 haloalkyl;
[0080] Z.sup.1 is selected from CH.sub.2, O, and NR.sup.5, where
R.sup.5 is H or unsubstituted C1-C6 alkyl; and
[0081] Z.sup.2 is NH, NR.sup.6, CHR.sup.2, CR.sup.6R.sup.2, where
R.sup.6 is a covalent bond to R.sup.1.
[0082] In some embodiments, n is 0.
[0083] In other embodiments, n is 2 or 4. In further embodiments,
two R.sup.2 combine to form an oxo group. In certain embodiments,
R.sup.2 is CH.sub.3.
[0084] In some embodiments, Z.sup.1 is O, NH, CH.sub.2, or
NCH.sub.3.
[0085] In other embodiments, Z.sup.2 is N, CH, or CNH.sub.2.
[0086] The compounds described herein can have a structure
according to the following formula,
##STR00009##
or a pharmaceutically acceptable salt thereof, where
[0087] Z.sup.1 is CH.sub.2, NH, NCH.sub.3, or O;
[0088] L.sup.1 is --CH.sub.2, --CHR.sup.4A, --CH.sub.2C(.dbd.O),
--C(.dbd.O)CH.sub.2, --CH.sub.2C(.dbd.O)NH,
--CH.sub.2C(.dbd.O)NHCH.sub.2, or
--CH.sub.2NHC(.dbd.O)CH.sub.2;
[0089] R.sup.5 is H or C1-C10 heteroalkyl;
[0090] each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is,
independently, H, unsubstituted C1-C3 alkyl, optionally substituted
C1-C3 haloalkyl, or halogen, and
[0091] where at least one of R.sup.1, R.sup.2, R.sup.3, R.sup.4,
and R.sup.R5 is not H.
[0092] In some embodiments, Z.sup.1 is NH.
[0093] In other embodiments, n is 2 or 4.
[0094] In some embodiments, two R.sup.2A combine to form an oxo
group.
[0095] In other embodiments, R.sup.2A is CH.sub.3.
[0096] In still other embodiments, R.sup.1 and R.sup.4 are both
H.
[0097] In certain embodiments, R.sup.1 and R.sup.4 are,
independently, F, CF.sub.3, or Cl.
[0098] In some embodiments, R.sup.3 is F, Cl, or CF.sub.3.
[0099] In other embodiments, R.sup.2 is F, Cl, or CF.sub.3.
[0100] In still other embodiments, R.sup.5 is H.
[0101] In further embodiments, R.sup.5 is optionally substituted
C1-C10 hydroxyalkyl or C1-C10 aminoalkyl.
[0102] In other embodiments, L.sup.1 is CH.sub.2.
[0103] In other embodiments, the compound has a structure according
to the following formula,
##STR00010##
or a pharmaceutically acceptable salt thereof, where
[0104] Z.sup.1 is CH.sub.2, NH, NCH.sub.3, or O;
[0105] each of X.sup.1, X.sup.2, and X.sup.3 is N or CR.sup.8C, and
where one and only one of X.sup.1, X.sup.2, and X.sup.3 is N;
[0106] L.sup.1 is a covalent bond, --CH.sub.2, --CHR.sup.4A,
--CH.sub.2C(.dbd.O), --C(.dbd.O)CH.sub.2, --CH.sub.2C(.dbd.O)NH,
--CH.sub.2C(.dbd.O)NHCH.sub.2, --CH.sub.2NHC(.dbd.O)CH.sub.2, or
--CH.sub.2CH.sub.2;
[0107] each of R.sup.8A, R.sup.8B, and R.sup.8C is, independently,
H, unsubstituted C1-C3 alkyl, optionally substituted C1-C3
haloalkyl, or halogen, and [0108] where at least one of R.sup.7,
R.sup.8A, R.sup.8B, R.sup.8C, and R.sup.8D is not H.
[0109] In some embodiments, Z.sup.1 is NH.
[0110] In other embodiments, n is 2 or 4. In certain embodiments,
two R.sup.2 combine to form an oxo group.
[0111] In further embodiments, R.sup.2 is CH.sub.3.
[0112] In some embodiments, X.sup.2 is N.
[0113] In still other embodiments, at least one of R.sup.8A,
R.sup.8B, and R.sup.8C is F, Cl, or CF.sub.3.
[0114] In some embodiments, L.sup.1 is
--CH.sub.2C(.dbd.O)NHCH.sub.2 or --CH.sub.2NHC(.dbd.O)CH.sub.2.
[0115] In other embodiments, the compound has a structure according
to the following formula,
##STR00011##
or a pharmaceutically acceptable salt or solvate thereof
wherein
[0116] Z.sup.1 is CH.sub.2 or NH;
[0117] L.sup.1 is a covalent bond, --CH.sub.2, --CHR.sup.4A,
--CH.sub.2C(.dbd.O), --C(.dbd.O)CH.sub.2, --CH.sub.2C(.dbd.O)NH,
--CH.sub.2C(.dbd.O)NHCH.sub.2, --CH.sub.2C(.dbd.O)NHCHCF.sub.3--,
or --CH.sub.2NHC(.dbd.O)CH.sub.2;
[0118] n is an integer between 0-4;
[0119] each R.sup.2C, when present, is independently, OH, NH.sub.2,
NHCH.sub.3, N(CH.sub.3).sub.2, or unsubstituted C1-C3 alkyl, or two
R.sup.2C groups combine to form an oxo (.dbd.O) group, and wherein
no more than one R.sup.2B or R.sup.2C group can be OH NH.sub.2,
NHCH.sub.3, or N(CH.sub.3).sub.2;
[0120] R.sup.2D is H, OH, or NH.sub.2,
[0121] R.sup.5 is H or C1-C10 heteroalkyl;
[0122] each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is,
independently, H, unsubstituted C1-C3 alkyl, optionally substituted
C1-C3 haloalkyl or halogen, and
wherein at least one of R.sup.1, R.sup.2, R.sup.3, R.sup.4, and
R.sup.5 is not H.
[0123] In certain embodiments, Z.sup.1 is CH.sub.2.
[0124] In other embodiments, n is 0 or 1.
[0125] In certain embodiments, one of R.sup.2 and R.sup.3 is
NH.sub.2, NHCH.sub.3, or N(CH.sub.3).sub.2.
[0126] In further embodiments, R.sup.1 and R.sup.4 are both H.
[0127] In other embodiments, R.sup.1 and R.sup.4 are,
independently, F, CF.sub.3, or Cl.
[0128] In still other embodiments, R.sup.3 is F, Cl, or
CF.sub.3.
[0129] In certain embodiments, R.sup.2 is F, Cl, or CF.sub.3.
[0130] In some embodiments, R.sup.5 is H.
[0131] In certain embodiments, the compound has a structure
according to the following formula,
##STR00012##
or a pharmaceutically acceptable salt or solvate thereof, where
[0132] Z.sup.1 is CH.sub.2, NH, NCH.sub.3, or O;
[0133] L.sup.1 is a covalent bond, --CH.sub.2, --CHR.sup.4A,
--CH.sub.2C(.dbd.O), --C(.dbd.O)CH.sub.2, --CH.sub.2C(.dbd.O)NH,
--CH.sub.2C(.dbd.O)NHCH.sub.2, --CH.sub.2NHC(.dbd.O)CH.sub.2, or
--CH.sub.2CH.sub.2;
[0134] R.sup.7 is selected from H, optionally substituted C1-C6
alkyl, and optionally substituted C1-C10 heteroalkyl;
[0135] each of R.sup.8A and R.sup.8B is selected, independently,
from H, halogen, unsubstituted C1-C3 alkyl, and C1-C3 haloalkyl,
and
[0136] where at least one of R.sup.7, R.sup.8A and R.sup.8B is not
H.
[0137] In some embodiments, Z.sup.1 is N.
[0138] In other embodiments, n is 2 or 4.
[0139] In still other embodiments, two R.sup.2 combine to form an
oxo group.
[0140] In certain embodiments, R.sup.2 is CH.sub.3.
[0141] In some embodiments, R.sup.7 is unsubstituted C1-C3
alkyl.
[0142] In further embodiments, at least one of R.sup.8A and
R.sup.8B is F, Cl, or CF.sub.3.
[0143] In some embodiments, L.sup.1 is CH.sub.2.
[0144] In still another aspect, the invention features a compound
having a structure according to the following formula,
##STR00013##
or a pharmaceutically acceptable salt or solvate thereof, where
[0145] each of R.sup.1, R.sup.2, and R.sup.3 is, independently, H,
unsubstituted C1-C6 alkyl, optionally substituted C1-C6 haloalkyl,
or halogen;
[0146] m is 1 or 2;
[0147] each R.sup.4 and R.sup.5 is, independently, H, optionally
substituted C1-C6 alkyl, or optionally substituted C1-C6 haloalkyl,
or R.sup.4 and R.sup.5 combine to form an optionally substituted
C3-C6 cycloalkyl, or R.sup.4 and R.sup.5 combine to form an oxo
(C.dbd.O) group;
[0148] each of R.sup.6 and R.sup.8 is, independently, H or
optionally substituted C1-C6 alkyl; or R.sup.6 and R.sup.8 combine
to form an optionally substituted three-to-nine membered
heterocyclyl, or R.sup.6 and R.sup.7A combine to form an optionally
substituted three-to-nine membered heterocyclyl;
[0149] n is 1 or 2;
[0150] each R.sup.7A and R.sup.7B is, independently H, optionally
substituted C1-C6 alkyl, or optionally substituted C1-C6 haloalkyl;
or R.sup.6 combines with R.sup.7A to form an optionally substituted
three-to-nine heterocyclyl; or an R.sup.7A and R.sup.7B group on
the same carbon combine to form an optionally substituted C3-C6
cycloalkyl; or, when n is 2, both R.sup.7A groups combine to form
an optionally substituted C3-C6 cycloalkyl.
[0151] In some embodiments, each of R.sup.1, R.sup.2, and R.sup.3
is, independently, H, C1-C3 haloalkyl, or halogen.
[0152] In other embodiments, one of R.sup.1, R.sup.2, and R.sup.3
is H.
[0153] In still other embodiments, two of R.sup.1, R.sup.2, and
R.sup.3 are, independently, CF.sub.3, Cl, or F.
[0154] In certain embodiments, R.sup.4 and R.sup.5 are both H; or
R.sup.4 and R.sup.5 are both CH.sub.3; or R.sup.4 and R.sup.5
combine to form an optionally substituted C3-C6 cycloalkyl; or
R.sup.4 and R.sup.5 combine to form an oxo (C.dbd.O) group.
[0155] In other embodiments, R.sup.6 combines with R.sup.7A to form
a three-to-six membered heterocyclyl ring, or wherein R.sup.6 and
R.sup.8 combine to form an optionally substituted three-to-six
membered heterocyclyl.
[0156] In still other embodiments, R.sup.6 is H.
[0157] In some embodiments, R.sup.7A is H and R.sup.7B is
optionally substituted C1-C6 alkyl.
[0158] In still other embodiments, the compound has a structure
according to a formula that is
##STR00014##
[0159] In further embodiments, n is 1, and R.sup.7A and R.sup.7B
are both H, or R.sup.7A is H and R.sup.7B is optionally substituted
C1-C6 alkyl. In still other embodiments, R.sup.6 is H and R.sup.8
is optionally substituted C1-C6 alkyl, or wherein R.sup.6 and
R.sup.8 combine to form an optionally substituted five- to
six-membered heterocyclyl (e.g., an unsubstituted five- to
six-membered heterocyclyl or a five- to six-membered heterocyclyl
that includes a phenyl substituent).
[0160] In another aspect, the invention features a compound having
a structure selected from the group consisting of any of Compounds
(1)-(236) of Table 1, or a pharmaceutically acceptable salt or
solvate thereof. In some embodiments, the compound, or a
pharmaceutically acceptable salt or solvate thereof, selected from
the group consisting of
##STR00015## ##STR00016## ##STR00017## ##STR00018##
[0161] In another aspect, the invention features a pharmaceutical
composition that includes any of the compounds described herein
(e.g., a compound according to any of Formulas (I)-(XIII) or any of
Compounds (1)-(236) of Table 1) and a pharmaceutically acceptable
carrier or excipient.
[0162] In some embodiments, the pharmaceutical composition is
formulated in unit dosage form (e.g., a tablet, caplet, capsule,
lozenge, film, strip, gelcap, or syrup).
[0163] In still another aspect, the invention features method to
treat a disease or condition by administering to a subject in need
of such treatment an effective amount of any of the compounds
described herein (e.g., a compound according to any of Formulas
(I)-(XIII) or any of Compounds (1)-(236) of Table 1), or a
pharmaceutical composition thereof.
[0164] In certain embodiments, the disease or condition is pain,
epilepsy, Parkinson's disease, a mood disorder (e.g., a major
depressive disorder (e.g., atypical depression, melancholic
depression, psychotic major depression, catatonic depression,
postpartum depression, seasonal affective disorder, dysthymia, and
depressive disorder not otherwise specified (DD-NOS)), recurrent
brief depression, minor depressive disorder, or a bipolar
disorder), psychosis (e.g., schizophrenia), tinnitus, amyotropic
lateral sclerosis, glaucoma, ischaemia, spasticity disorders,
obsessive compulsive disorder, restless leg syndrome, and Tourette
syndrome.
[0165] In some embodiments, the subject is a fasted subject.
[0166] In certain embodiments, the subject is a fed subject.
[0167] In other embodiments, the condition is pain or epilepsy.
[0168] In some embodiments, the pain is inflammatory pain (e.g.,
inflammatory pain caused by rheumatoid arthritis, juvenile
idiopathic arthritis, ankylosing spondylitis, psoriatic arthritis,
inflammatory bowel disease, primary dysmenorrhea, or endometriosis)
or neuropathic pain.
[0169] In certain embodiments, the pain is chronic pain.
[0170] In further embodiments, the chronic pain is peripheral
neuropathic pain; central neuropathic pain, musculoskeletal pain,
headache, visceral pain, or mixed pain.
[0171] In some embodiments, the peripheral neuropathic pain is
post-herpetic neuralgia, diabetic neuropathic pain, neuropathic
cancer pain, HIV-associated neuropathy, erythromelalgia, failed
back-surgery syndrome, trigeminal neuralgia, or phantom limb pain;
said central neuropathic pain is multiple sclerosis related pain,
Parkinson disease related pain, post-stroke pain, post-traumatic
spinal cord injury pain, lumbosacral radiculopathy, cervical
radiculopathy, brachial radiculopathy, or pain in dementia; the
musculoskeletal pain is osteoarthritic pain and fibromyalgia
syndrome; inflammatory pain such as rheumatoid arthritis, or
endometriosis; the headache is migraine, cluster headache, tension
headache syndrome, facial pain, or headache caused by other
diseases; the visceral pain is interstitial cystitis, irritable
bowel syndrome, or chronic pelvic pain syndrome; or the mixed pain
is lower back pain, neck and shoulder pain, burning mouth syndrome,
or complex regional pain syndrome.
[0172] In other embodiments, the headache is migraine.
[0173] In certain embodiments, the pain is acute pain.
[0174] In further embodiments, the acute pain is nociceptive pain
or post-operative pain.
[0175] In another aspect, the invention features a method of
modulating a voltage-gated sodium channel, the method including
contacting a cell with any of the compounds described herein (e.g.,
a compound according to any of Formulas (I)-(XIII) or any of
Compounds (1)-(236) of Table 1).
[0176] The term "alkoxy" represents a chemical substituent of
formula --OR, where R is an optionally substituted C1-C6 alkyl
group, unless otherwise specified. In some embodiments, the alkyl
group can be substituted, e.g., the alkoxy group can have 1, 2, 3,
4, 5 or 6 substituent groups as defined herein.
[0177] The term "alkoxyalkyl" represents a heteroalkyl group, as
defined herein, that is described as an alkyl group that is
substituted with an alkoxy group. Exemplary unsubstituted
alkoxyalkyl groups include between 2 to 12 carbons. In some
embodiments, the alkyl and the alkoxy each can be further
substituted with 1, 2, 3, or 4 substituent groups as defined herein
for the respective group.
[0178] As used herein, the term "alkyl," "alkenyl" and "alkynyl"
include straight-chain, branched-chain and cyclic monovalent
substituents, as well as combinations of these, containing only C
and H when unsubstituted. Examples include methyl, ethyl, isobutyl,
cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl, and the like.
The term "cycloalkyl," as used herein, represents a monovalent
saturated or unsaturated non-aromatic cyclic alkyl group having
between three to nine carbons (e.g., a C3-C9 cycloalkyl), unless
otherwise specified, and is exemplified by cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl, and the
like. When the cycloalkyl group includes one carbon-carbon double
bond, the cycloalkyl group can be referred to as a "cycloalkenyl"
group. Exemplary cycloalkenyl groups include cyclopentenyl,
cyclohexenyl, and the like.
[0179] Typically, the alkyl, alkenyl and alkynyl groups contain
1-12 carbons (e.g., C1-C12 alkyl) or 2-12 carbons (e.g., C2-C12
alkenyl or C2-C12 alkynyl). In some embodiments, the alkyl groups
are C1-C8, C1-C6, C1-C4, C1-C3, or C1-C2 alkyl groups; or C2-C8,
C2-C6, C2-C4, or C2-C3 alkenyl or alkynyl groups. Further, any
hydrogen atom on one of these groups can be replaced with a
substituent as described herein.
[0180] Heteroalkyl, heteroalkenyl and heteroalkynyl are similarly
defined and contain at least one carbon atom but also contain one
or more O, S or N heteroatoms or combinations thereof within the
backbone residue whereby each heteroatom in the heteroalkyl,
heteroalkenyl or heteroalkynyl group replaces one carbon atom of
the alkyl, alkenyl or alkynyl group to which the heteroform
corresponds. In some embodiments, the heteroalkyl, heteroalkenyl
and heteroalkynyl groups have C at each terminus to which the group
is attached to other groups, and the heteroatom(s) present are not
located at a terminal position. As is understood in the art, these
heteroforms do not contain more than three contiguous heteroatoms.
In some embodiments, the heteroatom is O or N. The term
"heterocyclyl," as used herein represents cyclic heteroalkyl or
heteroalkenyl that is, e.g., a 3-, 4-, 5-, 6- or 7-membered ring,
unless otherwise specified, containing one, two, three, or four
heteroatoms independently selected from the group consisting of
nitrogen, oxygen, and sulfur. The 5-membered ring has zero to two
double bonds, and the 6- and 7-membered rings have zero to three
double bonds. The term "heterocyclyl" also represents a
heterocyclic compound having a bridged multicyclic structure in
which one or more carbons and/or heteroatoms bridges two
non-adjacent members of a monocyclic ring, e.g., a quinuclidinyl
group. The term "heterocyclyl" includes bicyclic, tricyclic, and
tetracyclic groups in which any of the above heterocyclic rings is
fused to one, two, or three carbocyclic rings, e.g., an aryl ring,
a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a
cyclopentene ring, or another monocyclic heterocyclic ring, such as
indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl,
benzothienyl and the like.
[0181] The designated number of carbons in heteroforms of alkyl,
alkenyl and alkynyl includes the heteroatom count. For example, if
heteroalkyl is defined as C1-C6, it will contain 1-6 C, N, O, or S
atoms such that the heteroalkyl contains at least one C atom and at
least one heteroatom, for example 1-5 carbons and 1 N atom, or 1-4
carbons and 2 N atoms. Similarly, when heteroalkyl is defined as
C1-C6 or C1-C4, it would contain 1-5 carbons or 1-3 carbons
respectively, i.e., at least one C is replaced by O, N or S.
Accordingly, when heteroalkenyl or heteroalkynyl is defined as
C2-C6 (or C2-C4), it would contain 2-6 or 2-4 C, N, O, or S atoms,
since the heteroalkenyl or heteroalkynyl contains at least one
carbon atom and at least one heteroatom, e.g. 2-5 carbons and 1 N
atom, or 2-4 carbons, and 2 O atoms. Further, heteroalkyl,
heteroalkenyl or heteroalkynyl substituents may also contain one or
more carbonyl groups. Examples of heteroalkyl, heteroalkenyl and
heteroalkynyl groups include CH.sub.2OCH.sub.3,
CH.sub.2N(CH.sub.3).sub.2, CH.sub.2OH, (CH.sub.2).sub.nNR.sub.2,
OR, COOR, CONR.sub.2, (CH.sub.2).sub.nOR,(CH.sub.2).sub.nCOR,
(CH.sub.2).sub.nCOOR, (CH.sub.2).sub.nSR, (CH.sub.2).sub.nSOR,
(CH.sub.2).sub.nSO.sub.2R.sub.2, (CH.sub.2).sub.nCONR.sub.2, NRCOR,
NRCOOR, OCONR.sub.2, OCOR and the like wherein the R group contains
at least one C and the size of the substituent is consistent with
the definition of e.g., alkyl, alkenyl, and alkynyl, as described
herein (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12).
[0182] As used herein, the terms "alkylene," "alkenylene," and
"alkynylene," or the prefix "alk" refer to divalent or trivalent
groups having a specified size, typically C1-C2, C1-C3, C1-C4,
C1-C6, or C1-C8 for the saturated groups (e.g., alkylene or alk)
and C2-C3, C2-C4, C2-C6, or C2-C8 for the unsaturated groups (e.g.,
alkenylene or alkynylene). They include straight-chain,
branched-chain and cyclic forms as well as combinations of these,
containing only C and H when unsubstituted. Because they are
divalent, they can link together two parts of a molecule, as
exemplified by X in the compounds described herein. Examples are
methylene, ethylene, propylene, cyclopropan-1,1-diyl, ethylidene,
2-butene-1,4-diyl, and the like. These groups can be substituted by
the groups typically suitable as substituents for alkyl, alkenyl
and alkynyl groups as set forth herein. Thus C.dbd.O is a C1
alkylene that is substituted by .dbd.O, for example. For example,
the term "alkaryl," as used herein, represents an aryl group, as
defined herein, attached to the parent molecular group through an
alkylene group, as defined herein, and the term "alkheteroaryl"
refers to a heteroaryl group, as defined herein, attached to the
parent molecular group through an alkylene group, as defined
herein. The alkylene and the aryl or heteroaryl group are each
optionally substituted as described herein.
[0183] Heteroalkylene, heteroalkenylene and heteroalkynylene are
similarly defined as divalent groups having a specified size,
typically C1-C3, C1-C4, C1-C6, or C1-C8 for the saturated groups
and C2-C3, C2-C4, C2-C6, or C2-C8 for the unsaturated groups. They
include straight chain, branched chain and cyclic groups as well as
combinations of these, and they further contain at least one carbon
atom but also contain one or more O, S or N heteroatoms or
combinations thereof within the backbone residue, whereby each
heteroatom in the heteroalkylene, heteroalkenylene or
heteroalkynylene group replaces one carbon atom of the alkylene,
alkenylene or alkynylene group to which the heteroform corresponds.
As is understood in the art, these heteroforms do not contain more
than three contiguous heteroatoms.
[0184] The term "alkylsulfonyl," as used herein, represents a
heteroalkyl group that is described as an optionally substituted
alkyl group, as described herein, that includes an --S(O).sub.2--
group.
[0185] The term "amino," as used herein, represents
--N(R.sup.N1).sub.2, wherein each R.sup.N1 is, independently, H,
OH, NO.sub.2, N(R.sup.N2).sub.2, SO.sub.2OR.sup.N2,
SO.sub.2R.sup.N2, SOR.sup.N2, SO.sub.2N(R.sup.N2).sub.2,
SON(R.sup.N2).sub.2, an N-protecting group, alkyl, alkenyl,
alkynyl, alkoxy, aryl, alkaryl, cycloalkyl, alkcycloalkyl,
heterocyclyl (e.g., heteroaryl), alkheterocyclyl (e.g.,
alkheteroaryl), or two R.sup.N1 combine to form a heterocyclyl or
an N-protecting group, and wherein each R.sup.N2 is, independently,
H, alkyl, or aryl. In a preferred embodiment, amino is --NH.sub.2,
or --NHR.sup.N1, wherein R.sup.N1 is, independently, OH, NO.sub.2,
NH.sub.2, NR.sup.N2.sub.2, SO.sub.2OR.sup.N2, SO.sub.2R.sup.N2,
SOR.sup.N2, SO.sub.2N(R.sup.N2).sub.2, SON(R.sup.N2).sub.2, alkyl,
or aryl, and each R.sup.N2 can be H, alkyl, or aryl. The term
"aminoalkyl," as used herein, represents a heteroalkyl group, as
defined hrein, that is described as an alkyl group, as defined
herein, substituted by an amino group, as defined herein. The alkyl
and amino each can be further substituted with 1, 2, 3, or 4
substituent groups as described herein for the respective group.
For example, the alkyl moiety may comprise an oxo (.dbd.O)
substituent.
[0186] "Aromatic" moiety or "aryl" moiety refers to any monocyclic
or fused ring bicyclic system which has the characteristics of
aromaticity in terms of electron distribution throughout the ring
system and includes a monocyclic or fused bicyclic moiety such as
phenyl or naphthyl; "heteroaromatic" or "heteroaryl" also refers to
such monocyclic or fused bicyclic ring systems containing one or
more heteroatoms selected from O, S and N. The inclusion of a
heteroatom permits inclusion of 5-membered rings to be considered
aromatic as well as 6-membered rings. Thus, typical
aromatic/heteroaromatic systems include pyridyl, pyrimidyl,
indolyl, benzimidazolyl, benzotriazolyl, isoquinolyl, quinolyl,
benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl,
oxazolyl, isoxazolyl, benzoxazolyl, benzoisoxazolyl, imidazolyl and
the like. Because tautomers are theoretically possible, phthalimido
is also considered aromatic. Typically, the ring systems contain
5-12 ring member atoms or 6-10 ring member atoms. In some
embodiments, the aromatic or heteroaromatic moiety is a 6-membered
aromatic rings system optionally containing 1-2 nitrogen atoms.
More particularly, the moiety is an optionally substituted phenyl,
pyridyl, indolyl, pyrimidyl, pyridazinyl, benzothiazolyl or
benzimidazolyl, pyrazolyl, imidazolyl, isoxazolyl, thiazolyl,
benzothiazolyl, indolyl. Even more particularly, such moiety is
phenyl, pyridyl, or pyrimidyl and even more particularly, it is
phenyl.
[0187] "O-aryl" or "O-heteroaryl" refers to aromatic or
heteroaromatic systems which are coupled to another residue through
an oxygen atom. A typical example of an O-aryl is phenoxy.
Similarly, "arylalkyl" refers to aromatic and heteroaromatic
systems which are coupled to another residue through a carbon
chain, saturated or unsaturated, typically of C1-C8, C1-C6, or more
particularly C1-C4 or C1-C3 when saturated or C2-C8, C2-C6, C2-C4,
or C2-C3 when unsaturated, including the heteroforms thereof. For
greater certainty, arylalkyl thus includes an aryl or heteroaryl
group as defined above connected to an alkyl, heteroalkyl, alkenyl,
heteroalkenyl, alkynyl or heteroalkynyl moiety also as defined
above. Typical arylalkyls would be an aryl(C6-C12)alkyl(C1-C8),
aryl(C6-C12)alkenyl(C2-C8), or aryl(C6-C12)alkynyl(C2-C8), plus the
heteroforms. A typical example is phenylmethyl, commonly referred
to as benzyl.
[0188] Halo may be any halogen atom, especially F, Cl, Br, or I,
and more particularly it is fluoro or chloro.
[0189] The term "haloalkyl," as used herein, represents an alkyl
group, as defined herein, substituted by a halogen group (i.e., F,
Cl, Br, or I). A haloalkyl may be substituted with one, two, three,
or, in the case of alkyl groups of two carbons or more, four
halogens. Haloalkyl groups include perfluoroalkyls. In some
embodiments, the haloalkyl group can be further substituted with 1,
2, 3, or 4 substituent groups as described herein for alkyl
groups.
[0190] The term "hydroxy," as used herein, represents an --OH
group.
[0191] The term "hydroxyalkyl," as used herein, represents an alkyl
group, as defined herein, substituted by one to three hydroxy
groups, with the proviso that no more than one hydroxy group may be
attached to a single carbon atom of the alkyl group, and is
exemplified by hydroxymethyl, dihydroxypropyl, and the like.
[0192] The term "N-protecting group," as used herein, represents
those groups intended to protect an amino group against undesirable
reactions during synthetic procedures. Commonly used N-protecting
groups are disclosed in Greene, "Protective Groups in Organic
Synthesis," 3.sup.rd Edition (John Wiley & Sons, New York,
1999), which is incorporated herein by reference. N-protecting
groups include acyl, aryloyl, or carbamyl groups such as formyl,
acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl,
2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl,
o-nitrophenoxyacetyl, .alpha.-chlorobutyryl, benzoyl,
4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral
auxiliaries such as protected or unprotected D, L or D, L-amino
acids such as alanine, leucine, phenylalanine, and the like;
sulfonyl-containing groups such as benzenesulfonyl,
p-toluenesulfonyl, and the like; carbamate forming groups such as
benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,
p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,
3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyl oxycarbonyl,
2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,
2-nitro-4,5-dimethoxybenzyloxycarbonyl,
3,4,5-trimethoxybenzyloxycarbonyl,
1-(p-biphenylyl)-1-methylethoxycarbonyl,
.alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl,
benzhydryloxy carbonyl, t-butyloxycarbonyl,
diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,
methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl,
phenoxycarbonyl, 4-nitrophenoxy carbonyl,
fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,
adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl,
and the like, alkaryl groups such as benzyl, triphenylmethyl,
benzyloxymethyl, and the like and silyl groups such as
trimethylsilyl, and the like. Preferred N-protecting groups are
formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl,
phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and
benzyloxycarbonyl (Cbz).
[0193] In general, a substituent group (e.g., alkyl, alkenyl,
alkynyl, or aryl (including all heteroforms defined above) may
itself optionally be substituted by additional substituents. The
nature of these substituents is similar to those recited with
regard to the substituents on the basic structures above. Thus,
where an embodiment of a substituent is alkyl, this alkyl may
optionally be substituted by the remaining substituents listed as
substituents where this makes chemical sense, and where this does
not undermine the size limit of alkyl per se; e.g., alkyl
substituted by alkyl or by alkenyl would simply extend the upper
limit of carbon atoms for these embodiments, and is not included.
For example, where a group is substituted, the group may be
substituted with 1, 2, 3, 4, 5, or 6 substituents. Optional
substituents include, but are not limited to: C1-C6 alkyl or
heteroaryl, C2-C6 alkenyl or heteroalkenyl, C2-C6 alkynyl or
heteroalkynyl, halogen; aryl, heteroaryl, azido(--N.sub.3), nitro
(--NO.sub.2), cyano (--CN), acyloxy(--OC(.dbd.O)R'), acyl
(--C(.dbd.O)R'), alkoxy (--OR'), amido (--NR'C(.dbd.O)R'' or
--C(.dbd.O)NRR'), amino (--NRR'), carboxylic acid (--CO.sub.2H),
carboxylic ester (--CO.sub.2R'), carbamoyl (--OC(.dbd.O)NR'R'' or
--NRC(.dbd.O)OR'), hydroxy (--OH), isocyano (--NC), sulfonate
(--S(.dbd.O).sub.2OR), sulfonamide (--S(.dbd.O).sub.2NRR' or
--NRS(.dbd.O).sub.2R'), or sulfonyl (--S(.dbd.O).sub.2R), where
each R or R.sup.1 is selected, independently, from H, C1-C6 alkyl
or heteroaryl, C2-C6 alkenyl or heteroalkenyl, 2C-6C alkynyl or
heteroalkynyl, aryl, or heteroaryl. A substituted group may have,
for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 substituents.
[0194] Typical optional substituents on aromatic or heteroaromatic
groups include independently halo, CN, NO.sub.2, CF.sub.3,
OCF.sub.3, COOR', CONR'.sub.2, OR', SR', SOR', SO.sub.2R',
NR'.sub.2, NR'(CO)R',NR'C(O)OR', NR'C(O)NR'.sub.2,
NR'SO.sub.2NR'.sub.2, or NR'SO.sub.2R', wherein each R.sup.1 is
independently H or an optionally substituted group selected from
alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,
heteroaryl, and aryl (all as defined above); or the substituent may
be an optionally substituted group selected from alkyl, alkenyl,
alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl,
heteroaryl, O-aryl, O-heteroaryl and arylalkyl.
[0195] Optional substituents on a non-aromatic group (e.g., alkyl,
alkenyl, and alkynyl groups), are typically selected from the same
list of substituents suitable for aromatic or heteroaromatic
groups, except as noted otherwise herein. A non-aromatic group may
also include a substituent selected from .dbd.O and .dbd.NOR.sup.1
where R.sup.1 is H or an optionally substituted group selected from
alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteralkynyl,
heteroaryl, and aryl (all as defined above).
[0196] The term an "effective amount" of an agent (e.g., a compound
according to any of Formulas (I)-(XIII) or any of Compounds
(1)-(236) of Table 1), as used herein, is that amount sufficient to
effect beneficial or desired results, such as clinical results,
and, as such, an "effective amount" depends upon the context in
which it is being applied. For example, in the context of
administering an agent that is a modulator of a voltage-gated ion
channel (e.g., a sodium channel such as Na.sub.v1.7 or Na.sub.v
1.8), an effective amount of an agent is, for example, an amount
sufficient to achieve a change in sodium channel activity as
compared to the response obtained without administration of the
agent.
[0197] The term "pharmaceutical composition," as used herein,
represents a composition containing a compound described herein
(e.g., a compound according to any of Formulas (I)-(XIII) or any of
Compounds (1)-(236) of Table 1) formulated with a pharmaceutically
acceptable excipient. In some embodiments, the pharmaceutical
composition is manufactured or sold with the approval of a
governmental regulatory agency as part of a therapeutic regimen for
the treatment of disease in a mammal. Pharmaceutical compositions
can be formulated, for example, for oral administration in unit
dosage form (e.g., a tablet, capsule, caplet, gelcap, or syrup);
for topical administration (e.g., as a cream, gel, lotion, or
ointment); for intravenous administration (e.g., as a sterile
solution free of particulate emboli and in a solvent system
suitable for intravenous use); or in any other formulation
described herein.
[0198] A "pharmaceutically acceptable excipient," as used herein,
refers any ingredient other than the compounds described herein
(for example, a vehicle capable of suspending or dissolving the
active compound) and having the properties of being nontoxic and
non-inflammatory in a patient. Excipients may include, for example:
antiadherents, antioxidants, binders, coatings, compression aids,
disintegrants, dyes (colors), emollients, emulsifiers, fillers
(diluents), film formers or coatings, flavors, fragrances, glidants
(flow enhancers), lubricants, preservatives, printing inks,
sorbents, suspensing or dispersing agents, sweeteners, or waters of
hydration. Exemplary excipients include, but are not limited to:
butylated hydroxytoluene (BHT), calcium carbonate, calcium
phosphate (dibasic), calcium stearate, croscarmellose, crosslinked
polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,
ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, lactose, magnesium stearate, maltitol, mannitol,
methionine, methylcellulose, methyl paraben, microcrystalline
cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone,
pregelatinized starch, propyl paraben, retinyl palmitate, shellac,
silicon dioxide, sodium carboxymethyl cellulose, sodium citrate,
sodium starch glycolate, sorbitol, starch (corn), stearic acid,
stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin
E, vitamin C, and xylitol.
[0199] The term "pharmaceutically acceptable prodrugs" as used
herein, represents those prodrugs of the compounds of the present
invention that are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of humans and animals
with undue toxicity, irritation, allergic response, and the like,
commensurate with a reasonable benefit/risk ratio, and effective
for their intended use, as well as the zwitterionic forms, where
possible, of the compounds of the invention.
[0200] The term "pharmaceutically acceptable salt," as use herein,
represents those salts of the compounds described here (e.g., a
compound according to any of Formulas (I)-(XIII) or any of
Compounds (1)-(236) of Table 1) that are, within the scope of sound
medical judgment, suitable for use in contact with the tissues of
humans and animals without undue toxicity, irritation, allergic
response and the like and are commensurate with a reasonable
benefit/risk ratio. Pharmaceutically acceptable salts are well
known in the art. For example, pharmaceutically acceptable salts
are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19,
1977 and in Pharmaceutical Salts: Properties, Selection, and Use,
(Eds. P. H. Stahl and C. G. Wermuth), Wiley-VCH, 2008. The salts
can be prepared in situ during the final isolation and purification
of the compounds described herein or separately by reacting the
free base group with a suitable organic acid.
[0201] The compounds of the invention may have ionizable groups so
as to be capable of preparation as pharmaceutically acceptable
salts. These salts may be acid addition salts involving inorganic
or organic acids or the salts may, in the case of acidic forms of
the compounds of the invention be prepared from inorganic or
organic bases. Frequently, the compounds are prepared or used as
pharmaceutically acceptable salts prepared as addition products of
pharmaceutically acceptable acids or bases. Suitable
pharmaceutically acceptable acids and bases are well-known in the
art, such as hydrochloric, sulphuric, hydrobromic, acetic, lactic,
citric, or tartaric acids for forming acid addition salts, and
potassium hydroxide, sodium hydroxide, ammonium hydroxide,
caffeine, various amines, and the like for forming basic salts.
Methods for preparation of the appropriate salts are
well-established in the art.
[0202] Representative acid addition salts include acetate, adipate,
alginate, ascorbate, aspartate, benzenesulfonate, benzoate,
bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptonate, glycerophosphate,
hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
toluenesulfonate, undecanoate, valerate salts and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium and the like, as well as
nontoxic ammonium, quaternary ammonium, and amine cations,
including, but not limited to ammonium, tetramethylammonium,
tetraethylammonium, methylamine, dimethylamine, trimethylamine,
triethylamine, ethylamine and the like.
[0203] The term "pharmaceutically acceptable solvate" as used
herein means a compound as described herein (e.g., a compound
according to any of Formulas (I)-(XIII) or any of Compounds
(1)-(236) of Table 1) where molecules of a suitable solvent are
incorporated in the crystal lattice. A suitable solvent is
physiologically tolerable at the dosage administered. For example,
solvates may be prepared by crystallization, recrystallization, or
precipitation from a solution that includes organic solvents,
water, or a mixture thereof. Examples of suitable solvents are
ethanol, water (for example, mono-, di-, and tri-hydrates),
N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),
N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC),
1,3-dimethyl-2-imidazolidinone (DMEU),
1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU),
acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl
alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water
is the solvent, the molecule is referred to as a "hydrate."
[0204] The term "prevent," as used herein, refers to prophylactic
treatment or treatment that prevents one or more symptoms or
conditions of a disease, disorder, or conditions described herein
(for example, pain (e.g., chronic or acute pain), epilepsy,
Alzheimer's disease, Parkinson's disease, cardiovascular disease,
diabetes, cancer, sleep disorders, obesity, psychosis such as
schizophrenia, overactive bladder, renal disease, neuroprotection,
addiction, and male birth control). Preventative treatment can be
initiated, for example, prior to ("pre-exposure prophylaxis") or
following ("post-exposure prophylaxis") an event that precedes the
onset of the disease, disorder, or conditions. Preventive treatment
that includes administration of a compound described herein (e.g.,
a compound according to any of Formulas (I)-(XIII) or any of
Compounds (1)-(236) of Table 1), or a pharmaceutically acceptable
salt or solvate thereof, or a pharmaceutical composition thereof,
can be acute, short-term, or chronic. The doses administered may be
varied during the course of preventative treatment.
[0205] The term "prodrug," as used herein, represents compounds
that are rapidly transformed in vivo to the parent compound of the
above formula, for example, by hydrolysis in blood. Prodrugs of the
compounds described herein may be conventional esters. Some common
esters that have been utilized as prodrugs are phenyl esters,
aliphatic (C1-C8 or C8-C24) esters, cholesterol esters,
acyloxymethyl esters, carbamates, and amino acid esters. For
example, a compound that contains an OH group may be acylated at
this position in its prodrug form. A thorough discussion is
provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery
Systems, Vol. 14 of the A.C.S. Symposium Series, Edward B. Roche,
ed., Bioreversible Carriers in Drug Design, American Pharmaceutical
Association and Pergamon Press, 1987, and Judkins et al., Synthetic
Communications 26(23):4351-4367, 1996, each of which is
incorporated herein by reference. Preferably, prodrugs of the
compounds of the present invention are suitable for use in contact
with the tissues of humans and animals with undue toxicity,
irritation, allergic response, and the like, commensurate with a
reasonable benefit/risk ratio, and effective for their intended
use.
[0206] In addition, the compounds of the invention may be coupled
through conjugation to substances designed to alter the
pharmacokinetics, for targeting, or for other reasons. Thus, the
invention further includes conjugates of these compounds. For
example, polyethylene glycol is often coupled to substances to
enhance half-life; the compounds may be coupled to liposomes
covalently or noncovalently or to other particulate carriers. They
may also be coupled to targeting agents such as antibodies or
peptidomimetics, often through linker moieties. Thus, the invention
is also directed to compounds (e.g., a compound according to any of
Formulas (I)-(XIII) or any of Compounds (1)-(236) of Table 1) when
modified so as to be included in a conjugate of this type.
[0207] As used herein, and as well understood in the art, "to
treat" a condition or "treatment" of the condition (e.g., the
conditions described herein such as pain (e.g., chronic or acute
pain), epilepsy, Alzheimer's disease, Parkinson's disease,
cardiovascular disease, diabetes, cancer, sleep disorders, obesity,
psychosis such as schizophrenia, overactive bladder, renal disease,
neuroprotection, addiction, and male birth control) is an approach
for obtaining beneficial or desired results, such as clinical
results. Beneficial or desired results can include, but are not
limited to, alleviation or amelioration of one or more symptoms or
conditions; diminishment of extent of disease, disorder, or
condition; stabilized (i.e., not worsening) state of disease,
disorder, or condition; preventing spread of disease, disorder, or
condition; delay or slowing the progress of the disease, disorder,
or condition; amelioration or palliation of the disease, disorder,
or condition; and remission (whether partial or total), whether
detectable or undetectable. "Palliating" a disease, disorder, or
condition means that the extent and/or undesirable clinical
manifestations of the disease, disorder, or condition are lessened
and/or time course of the progression is slowed or lengthened, as
compared to the extent or time course in the absence of
treatment.
[0208] The term "unit dosage form" refers to a physically discrete
unit suitable as a unitary dosage for human subjects and other
mammals, each unit containing a predetermined quantity of active
material calculated to produce the desired therapeutic effect, in
association with any suitable pharmaceutical excipient or
excipients. Exemplary, non-limiting unit dosage forms include a
tablet (e.g., a chewable tablet), caplet, capsule (e.g., a hard
capsule or a soft capsule), lozenge, film, strip, gelcap, and
syrup.
[0209] In some cases, the compounds of the invention contain one or
more chiral centers. The invention includes each of the isolated
stereoisomeric forms as well as mixtures of stereoisomers in
varying degrees of chiral purity, including racemic mixtures. It
also encompasses the various diastereomers and tautomers that can
be formed.
[0210] Compounds useful in the invention may also be isotopically
labeled compounds. Useful isotopes include hydrogen, carbon,
nitrogen, oxygen, phosphorous, fluorine, and chlorine, (e.g.,
.sup.2H, .sup.3H, .sup.13C, .sup.14C, .sup.15N, .sup.18O, .sup.17O,
.sup.31P, .sup.32P, .sup.35S, .sup.18F, and .sup.36Cl).
Isotopically labeled compounds can be prepared by synthesizing a
compound using a readily available isotopically labeled reagent in
place of a non-isotopically labeled reagent. In some embodiments,
the compound (e.g., a compound according to any of Formulas
(I)-(XIII) or any of Compounds (1)-(236) of Table 1), or a
composition that includes the compound, has the natural abundance
of each element present in the compound.
[0211] The compounds described herein (e.g., a compound according
to any of Formulas (I)-(XIII) or any of Compounds (1)-(236) of
Table 1) are also useful for the manufacture of a medicament useful
to treat conditions requiring modulation of voltage-gated ion
channel activity (e.g., sodium channel activity), and, in
particular, Na.sub.v 1.7 or Na.sub.v 1.8 channel activity.
[0212] Other features and advantages of the invention will be
apparent from the following detailed description, the drawings, and
the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0213] FIGS. 1A-1B show the modulation of ion channel activity by
the compounds described herein.
[0214] FIGS. 2A-2C and 3A-3C show data obtained in the spinal nerve
ligation (SNL) assay for select compounds of the invention.
DETAILED DESCRIPTION OF THE INVENTION
Compounds
[0215] The invention features compounds that can modulate the
activity of voltage-gated ion channels (e.g., voltage-gated sodium
channels). These compounds can also be used to treat disorders such
as pain, epilepsy, Parkinson's disease, mood disorders, psychosis
(e.g., schizophrenia), tinnitus, amyotropic lateral sclerosis,
glaucoma, ischaemia, spasticity disorders, obsessive compulsive
disorder, restless leg syndrome, and Tourette syndrome. Exemplary
compounds described herein include compounds that have a structure
according to the following formula,
##STR00019##
or a pharmaceutically acceptable salt or solvate thereof, where
[0216] each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is selected,
independently, from H, optionally substituted C1-C6 alkyl,
optionally substituted C1-C6 haloalkyl, optionally substituted
C6-C10 aryl, or optionally substituted 5 to 6-membered heteroaryl,
where at least one of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 is
halogen or optionally substituted C1-C6 haloalkyl;
[0217] R.sup.5 is H, optionally substituted C1-C6 alkyl, or
optionally substituted C1-C10 heteroalkyl;
[0218] R.sup.6 is --R.sup.6A or --CH.sub.2R.sup.6B;
[0219] R.sup.6A is NH.sub.2, optionally substituted cyclopropyl,
optionally substituted azetidine, optionally substituted
cyclopentyl, optionally substituted pyrazole, optionally
substituted pyrrole, optionally substituted pyrrolidine, optionally
substituted thiazolidine, optionally substituted
thiazolidine-1,1-dioxide, optionally substituted pyrimidine,
optionally substituted C1-C10 amino alkyl, optionally substituted
C1-C10 hydroxyalkyl, optionally substituted C1-C10 alkoxyalkyl,
optionally substituted C1-C10 haloalkyl, or optionally substituted
C1-C10 alkylsulfonyl; or R.sup.6A has a structure according to
##STR00020##
where
[0220] n is an integer between 0-4;
[0221] Z.sup.1 is CH.sub.2, NH, NCH.sub.3, or O;
[0222] L.sup.1 is --CH.sub.2, --CHR.sup.4A, --CH.sub.2C(.dbd.O),
--C(.dbd.O)CH.sub.2, --CH.sub.2C(.dbd.O)NH,
--CH.sub.2C(.dbd.O)NHCH.sub.2, or
--CH.sub.2NHC(.dbd.O)CH.sub.2;
[0223] each R.sup.2A and R.sup.2C, when present, is selected from
OH, N(R.sup.2B).sub.2, halogen, and unsubstituted C1-C3 alkyl, or
two R.sup.2A combine to form an oxo (.dbd.O) group, and wherein no
more than two R.sup.2A combine to form an oxo group; and
[0224] each R.sup.2B is, independently, H or unsubstituted C1-C6
alkyl;
[0225] R.sup.2D is H, OH, or NH.sub.2;
[0226] and
[0227] R.sup.6B is optionally substituted cyclopropyl, optionally
substituted azetidine, optionally substituted cyclopentyl,
optionally substituted pyrazole, optionally substituted pyrrole,
optionally substituted pyrrolidine, optionally substituted
thiazolidine, optionally substituted thiazolidine-1,1-dioxide, or
optionally substituted pyrimidine.
[0228] In some embodiments, R.sup.5 is H.
[0229] In other embodiments, R.sup.5 is optionally substituted
C1-C10 heteroalkyl.
[0230] In certain embodiments, R.sup.2 and R.sup.4 are both
CF.sub.3, F, or Cl.
[0231] In still other embodiments, R.sup.2 and R.sup.3 are both
CF.sub.3, F, or Cl.
[0232] In some embodiments, R.sup.6 is --CH.sub.2R.sup.6B, and
R.sup.6B is optionally substituted azetidine.
[0233] In certain embodiments, R.sup.6 is optionally substituted
C1-C10 aminoalkyl.
[0234] In other embodiments, the C1-C10 aminoalkyl includes an oxo
(.dbd.O) substituent, an alkoxy substituent, an N-sulfonyl group,
or any combination thereof.
[0235] Other embodiments (e.g., Formulas (II)-(XIII) and any of
compounds (1)-(236) of Table 1), as well as exemplary methods for
the synthesis of these compounds, are described herein.
Utility and Administration
[0236] The compounds described herein (e.g., a compound according
to any of Formulas (I)-(XIII) or any of Compounds (1)-(236) of
Table 1) are useful in the methods of the invention and, while not
bound by theory, are believed to exert their desirable effects
through their ability to modulate the activity of voltage-gated ion
channels, e.g., the activity of sodium channels such as the
Na.sub.v 1.7 and Na.sub.v1.8 channels. The compounds described
herein (e.g., a compound according to any of Formulas (I)-(XIII) or
any of Compounds (1)-(236) of Table 1) can also be used for the
treatment of certain conditions such as pain, epilepsy, migraine,
Parkinson's disease, mood disorders, schizophrenia, psychosis,
tinnitus, amyotropic lateral sclerosis, glaucoma, ischaemia,
spasticity disorders, obsessive compulsive disorder, restless leg
syndrome, and Tourette syndrome.
Modulation of Sodium Channels
[0237] There are nine Na.sub.v1 .alpha.-subunit isoforms:
Na.sub.v1.1-1.9 (see, e.g., Yu et al., Genome Biolog, 4:207, 2003).
In addition to pain, other conditions associated with
voltage-dependent sodium channel activity include seizures (e.g.,
Na.sub.v1.1), epilepsy (e.g., Na.sub.v1.2), neurodegeneration
(e.g., Na.sub.v1.1, Na.sub.v1.2), myotonia (e.g., Na.sub.v1.4),
arrhythmia (e.g., Na.sub.v1.5), and movement disorders (e.g.,
Na.sub.v1.6) as described in PCT Publication No. WO 2008/118758,
herein incorporated by reference. The expression of particular
isoforms in particular tissues can influence the therapeutic
effects of sodium channel modulators. For example, the Na.sub.v1.4
and Na.sub.v1.5 isoforms are largely found in skeletal and cardiac
myocytes (see, e.g., Gold, Exp Neurol. 210(1): 1-6, 2008).
[0238] Sodium Channel Activity and Pain
[0239] Voltage-dependent ion channels in pain-sensing neurons are
currently of great interest in developing drugs to treat pain. For
example, blocking voltage-dependent sodium channels in pain-sensing
neurons can block pain signals by interrupting initiation and
transmission of the action potential.
[0240] Studies also indicate that particular sodium channel
isoforms are predominantly expressed in peripheral sensory neurons
associated with pain sensation; for example, Na.sub.v1.7,
Na.sub.v1.8 and Na.sub.v 1.9 activity are thought to be involved in
inflammatory, and possibly neuropathic, pain (see, e.g., Cummins et
al., Pain, 131(3):243-257, 2007). The Na.sub.v1.3 isoform has also
been implicated in pain, e.g., pain associated with tissue injury
(Gold, Exp Neurol. 210(1): 1-6, 2008).
[0241] The Na.sub.v1.7 and Na.sub.v1.8 channel subtypes act as
major contributors to both inflammatory and neuropathic pain (vide
infra). Recently, mutations have been identified in the Na.sub.v1.7
channel that lead either to a gain of channel function (Dib-Hajj et
al., Brain 128:1847-1854, 2005) or more commonly to a loss of
channel function (Chatelier et al., J. Neurophisiol. 99:2241-50,
2008). These mutations underlie human heritable disorders such as
erythermalgia (Yang et al., J Med. Genet. 41(3) 171-4, 2004),
paroxysmal extreme pain disorder (Fertleman et al., Neuron. 52(5)
767-74, 2006), and congenital indifference to pain (Cox et al.,
Nature 444(7121):894-8, 2006). Behavioral studies have shown in
mice that inflammatory and acute mechanosensory pain is reduced
when Na.sub.v1.7 is knocked out in Na.sub.v1.8-positive neurons
(Nassar et al., Proc Natl Acad Sci USA. 101(34):12706-11, 2004). In
addition, siRNA of Na.sub.v1.7 attenuates inflammatory hyperalgesia
(Yeomans et al., Hum Gene Ther. 16(2) 271-7, 2005).
[0242] The Na.sub.v1.8 isoform is selectively expressed in sensory
neurons and has been identified as a target for thre treatment of
pain, e.g., chronic pain (e.g., Swanwick et al., Neurosci. Lett.
486:78-83, 2010). The role of Na.sub.v1.8 in inflammatory (Khasar
et al. Neurosci Lett. 256(1):17-20, 1998), neuropathic and
mechanical hyperalgesia (Joshi et al., Pain 123(1-2):75-82, 2006)
has also emerged using molecular techniques to knockdown Na.sub.v
1.8, which has been shown to reduce the maintenance of these
different pain states.
[0243] Lacosamide is a functionalized amino acid that has shown
effectiveness as an analgesic in several animal models of
neuropathic pain and is currently in late stages of clinical
development for epilepsy and diabetic neuropathic pain. One mode of
action that has been validated for lacosamide is inhibition of
voltage-gated sodium channel activity by selective inhibition with
the slow-inactivated conformation of the channel (Sheets et al.,
Journal of Pharmacology and Experimental Therapeutics, 326(1) 89-99
(2008)). Modulators of sodium channels, including clinically
relevant compounds, can exhibit a pronounced state-dependent
binding, where sodium channels that are rapidly and repeatedly
activated and inactivated are more readily blocked. In a simplified
scheme, voltage-gated sodium channels have four distinct states:
open, closed, fast-inactivated and slow-inactivated. Classic sodium
channel modulators, such as lidocaine, are believed to exhibit the
highest affinity for the fast-inactivated state. However,
alteration of the slow inactivated state is also clinically
relevant.
Modulation of Calcium Channels
[0244] The entry of calcium into cells through voltage-gated
calcium channels mediates a wide variety of cellular and
physiological responses, including excitation-contraction coupling,
hormone secretion and gene expression (e.g., Miller et al., Science
235:46-52 (1987); Augustine et al., Annu Rev Neurosci 10: 633-693
(1987)). In neurons, calcium channels directly affect membrane
potential and contribute to electrical properties such as
excitability, repetitive firing patterns and pacemaker activity.
Calcium entry further affects neuronal functions by directly
regulating calcium-dependent ion channels and modulating the
activity of calcium-dependent enzymes such as protein kinase C and
calmodulin-dependent protein kinase II. An increase in calcium
concentration at the presynaptic nerve terminal triggers the
release of neurotransmitter, which also affects neurite outgrowth
and growth cone migration in developing neurons.
[0245] Calcium channels mediate a variety of normal physiological
functions, and are also implicated in a number of human disorders
as described herein. For example, calcium channels also have been
shown to mediate the development and maintenance of the neuronal
sensitization and hyperexcitability processes associated with
neuropathic pain, and provide attractive targets for the
development of analgesic drugs (reviewed in Vanegas et al., Pain
85: 9-18 (2000)). Native calcium channels have been classified by
their electrophysiological and pharmacological properties into T-,
L-, N-, P/Q- and R-types (reviewed in Catterall, Annu Rev Cell Dev
Biol 16: 521-555, 2000; Huguenard, Annu Rev Physiol 58: 329-348,
1996). The L-, N- and P/Q-type channels activate at more positive
potentials (high voltage-activated) and display diverse kinetics
and voltage-dependent properties (Id.). T-type channels can be
distinguished by having a more negative range of activation and
inactivation, rapid inactivation, slow deactivation, and smaller
single-channel conductances. There are three subtypes of T-type
calcium channels that have been molecularly, pharmacologically, and
elecrophysiologically identified: these subtypes have been termed
.alpha..sub.1G, .alpha..sub.1H, and .alpha..sub.1I (alternately
called Cav 3.1, Cav 3.2 and Cav 3.3 respectively).
[0246] T-type calcium channels are involved in various medical
conditions. In mice lacking the gene expressing the 3.1 subunit,
resistance to absence seizures was observed (Kim et al., Mol. Cell.
Neurosci. 18(2): 235-245 (2001)). Other studies have also
implicated the 3.2 subunit in the development of epilepsy (Su et
al., J. Neurosci. 22: 3645-3655 (2002)). There is also evidence
that some existing anticonvulsant drugs, such as ethosuximide,
function through the blockade of T-type channels (Gomora et al.,
Mol. Pharmacol. 60: 1121-1132 (2001)).
[0247] Low voltage-activated calcium channels are highly expressed
in tissues of the cardiovascular system. There is also a growing
body of evidence that suggests that T-type calcium channels are
abnormally expressed in cancerous cells and that blockade of these
channels may reduce cell proliferation in addition to inducing
apoptosis. Recent studies also show that the expression of T-type
calcium channels in breast cancer cells is proliferation state
dependent, i.e. the channels are expressed at higher levels during
the fast-replication period, and once the cells are in a
non-proliferation state, expression of this channel is minimal.
Therefore, selectively blocking calcium channel entry into
cancerous cells may be a valuable approach for preventing tumor
growth (e.g., PCT Patent Application Nos. WO 05/086971 and WO
05/77082; Taylor et al., World J. Gastroenterol. 14(32): 4984-4991
(2008); Heo et al., Biorganic & Medicinal Chemistry Letters
18:3899-3901 (2008)).
[0248] T-type calcium channels may also be involved in still other
conditions. A recent study also has shown that T-type calcium
channel antagonists inhibit high-fat diet-induced weight gain in
mice. In addition, administration of a selective T-type channel
antagonist reduced body weight and fat mass while concurrently
increasing lean muscle mass (e.g., Uebele et al., The Journal of
Clinical Investigation, 119(6):1659-1667 (2009)). T-type calcium
channels may also be involved in pain (see for example: US Patent
Publication No. 2003/0086980; PCT Publication Nos. WO 03/007953 and
WO 04/000311). In addition to cardiovascular disease, epilepsy (see
also US Patent Application No. 2006/0025397), cancer, and chronic
or acute pain, T-type calcium channels have been implicated in
diabetes (US Patent Publication No. 2003/0125269), sleep disorders
(US Patent Publication No. 2006/0003985), Parkinson's disease and
psychosis such as schizophrenia (US Patent Publication No.
2003/0087799); overactive bladder (Sui et al., British Journal of
Urology International 99(2): 436-441 (2007); US Patent Publication
No. 2004/0197825), renal disease (Hayashi et al., Journal of
Pharmacological Sciences 99: 221-227 (2005)), anxiety and
alcoholism (US Patent Publication No. 2009/0126031),
neuroprotection, and male birth control.
[0249] The modulation of ion channels by the compounds described
herein (e.g., a compound according to any of Formulas (I)-(XIII) or
any of Compounds (1)-(236) of Table 1) can be measured according to
methods known in the art (e.g., in the references provided herein).
Modulators of ion channels, e.g., voltage gated sodium and calcium
ion channels, and the medicinal chemistry or methods by which such
compounds can be identified, are also described in, for example:
Birch et al., Drug Discovery Today, 9(9):410-418 (2004); Audesirk,
"Chapter 6-Electrophysiological Analysis of Ion Channel Function,"
Neurotoxicology: Approaches and Methods, 137-156 (1995); Camerino
et al., "Chapter 4: Therapeutic Approaches to Ion Channel
Diseases," Advances in Genetics, 64:81-145 (2008); Petkov, "Chapter
16-Ion Channels," Pharmacology: Principles and Practice, 387-427
(2009); Standen et al., "Chapter 15-Patch Clamping Methods and
Analysis of Ion Channels," Principles of Medical Biology, Vol. 7,
Part 2, 355-375 (1997); Xu et al., Drug Discovery Today,
6(24):1278-1287 (2001); and Sullivan et al., Methods Mol. Biol.
114:125-133 (1999). Exemplary experimental methods are also
provided in the Examples.
Diseases and Conditions
[0250] Exemplary conditions that can be treated using the compounds
described herein include pain (e.g., chronic or acute pain),
epilepsy, Alzheimer's disease, Parkinson's disease, diabetes;
cancer; sleep disorders; obesity; mood disorders, psychosis such as
schizophrenia; overactive bladder; renal disease, neuroprotection,
and addiction. For example, the conidition can be pain (e.g.,
neuropathic pain or post-surgery pain), epilepsy, migraine,
Parkinson's disease, mood disorders, schizophrenia, psychosis,
tinnitus, amyotropic lateral sclerosis, glaucoma, ischaemia,
spasticity disorders, obsessive compulsive disorder, restless leg
syndrome, and Tourette syndrome.
[0251] Epilepsy as used herein includes but is not limited to
partial seizures such as temporal lobe epilepsy, absence seizures,
generalized seizures, and tonic/clonic seizures.
[0252] Cancer as used herein includes but is not limited to breast
carcinoma, neuroblastoma, retinoblastoma, glioma, prostate
carcinoma, esophageal carcinoma, fibrosarcoma, colorectal
carcinoma, pheochromocytoma, adrenocarcinoma, insulinoma, lung
carcinoma, melanoma, and ovarian cancer.
[0253] Acute pain as used herein includes but is not limited to
nociceptive pain and post-operative pain. Chronic pain includes but
is not limited by: peripheral neuropathic pain (e.g., post-herpetic
neuralgia, diabetic neuropathic pain, neuropathic cancer pain,
HIV-associated neuropathy, erythromelalgia, failed back-surgery
syndrome, trigeminal neuralgia, or phantom limb pain); central
neuropathic pain (e.g., multiple sclerosis related pain, Parkinson
disease related pain, post-stroke pain, post-traumatic spinal cord
injury pain, lumbosacral radiculopathy, cervical radiculopathy,
brachial radiculopathy, or pain in dementia); musculoskeletal pain
such as osteoarthritic pain and fibromyalgia syndrome; inflammatory
pain (e.g., inflammatory pain caused by rheumatoid arthritis,
juvenile idiopathic arthritis, ankylosing spondylitis, psoriatic
arthritis, inflammatory bowel disease, primary dysmenorrhea, or
endometriosis); headache such as migraine, cluster headache,
tension headache syndrome, facial pain, headache caused by other
diseases; visceral pain such as interstitial cystitis, irritable
bowel syndrome and chronic pelvic pain syndrome; and mixed pain
such as lower back pain, neck and shoulder pain, burning mouth
syndrome and complex regional pain syndrome.
[0254] In treating osteoarthritic pain, joint mobility can also
improve as the underlying chronic pain is reduced. Thus, use of
compounds of the present invention to treat osteoarthritic pain
inherently includes use of such compounds to improve joint mobility
in patients suffering from osteoarthritis.
[0255] The compounds described herein can be tested for efficacy in
any standard animal model of pain. Various models test the
sensitivity of normal animals to intense or noxious stimuli
(physiological or nociceptive pain). These tests include responses
to thermal, mechanical, or chemical stimuli. Thermal stimuli
usually involve the application of hot stimuli (typically varying
between 42-55.degree. C.) including, for example: radiant heat to
the tail (the tail flick test), radiant heat to the plantar surface
of the hindpaw (the Hargreaves test), the hotplate test, and
immersion of the hindpaw or tail into hot water. Immersion in cold
water, acetone evaporation, or cold plate tests may also be used to
test cold pain responsiveness. Tests involving mechanical stimuli
typically measure the threshold for eliciting a withdrawal reflex
of the hindpaw to graded strength monofilament von Frey hairs or to
a sustained pressure stimulus to a paw (e.g., the Ugo Basile
analgesiometer). The duration of a response to a standard pinprick
may also be measured. When using a chemical stimulus, the response
to the application or injection of a chemical irritant (e.g.,
capsaicin, mustard oil, bradykinin, ATP, formalin, acetic acid) to
the skin, muscle joints or internal organs (e.g., bladder or
peritoneum) is measured.
[0256] In addition, various tests assess pain sensitization by
measuring changes in the excitability of the peripheral or central
components of the pain neural pathway. In this regard, peripheral
sensitization (i.e., changes in the threshold and responsiveness of
high threshold nociceptors) can be induced by repeated heat stimuli
as well as the application or injection of sensitizing chemicals
(e.g., prostaglandins, bradykinin, histamine, serotonin, capsaicin,
or mustard oil). Central sensitization (i.e., changes in the
excitability of neurons in the central nervous system induced by
activity in peripheral pain fibers) can be induced by noxious
stimuli (e.g., heat), chemical stimuli (e.g., injection or
application of chemical irritants), or electrical activation of
sensory fibers.
[0257] Various pain tests developed to measure the effect of
peripheral inflammation on pain sensitivity can also be used to
study the efficacy of the compounds (Stein et al., Pharmacol.
Biochem. Behav. (1988) 31: 445-451; Woolf et al., Neurosci. (1994)
62: 327-331). Additionally, various tests assess peripheral
neuropathic pain using lesions of the peripheral nervous system.
One such example is the "axotomy pain model" (Watson, J. Physiol.
(1973) 231:41). Other similar tests include the SNL test which
involves the ligation of a spinal segmental nerve (Kim and Chung
Pain (1992) 50: 355), the Seltzer model involving partial nerve
injury (Seltzer, Pain (1990) 43: 205-18), the spared nerve injury
(SNI) model (Decosterd and Woolf, Pain (2000) 87:149), chronic
constriction injury (CCI) model (Bennett (1993) Muscle Nerve 16:
1040), tests involving toxic neuropathies such as diabetes
(streptozocin model), pyridoxine neuropathy, taxol, vincristine,
and other antineoplastic agent-induced neuropathies, tests
involving ischaemia to a nerve, peripheral neuritis models (e.g.,
CFA applied peri-neurally), models of post-herpetic neuralgia using
HSV infection, and compression models.
[0258] In all of the above tests, outcome measures may be assessed,
for example, according to behavior, electrophysiology,
neurochemistry, or imaging techniques to detect changes in neural
activity.
[0259] Exemplary models of pain are also described in the Examples
provided herein.
[0260] In addition to being able to modulate a particular
voltage-gated ion channel, e.g., a sodium channel, it may be
desirable that the compound has very low activity with respect to
the hERG channel, which is expressed in the heart: compounds that
block this channel with high potency may cause reactions which are
fatal. See, e.g., Bowlby et al., "hERG (KCNH2 or K.sub.v11.1
K.sup.+ Channels: Screening for Cardiac Arrhythmia Risk," Curr.
Drug Metab. 9(9):965-70 (2008)). Thus, for a compound that
modulates sodium channel activity, it may also be shown that the
hERG K.sup.+ channel is not inhibited or only minimally inhibited
as compared to the inhibition of the primary channel targeted.
Similarly, it may be desirable that the compound does not inhibit
cytochrome p450, an enzyme that is required for drug
detoxification. Such compounds may be particularly useful in the
methods described herein.
Pharmaceutical Compositions
[0261] For use as treatment of human and animal subjects, the
compounds of the invention can be formulated as pharmaceutical or
veterinary compositions. Depending on the subject to be treated,
the mode of administration, and the type of treatment
desired--e.g., prevention, prophylaxis, or therapy--the compounds
are formulated in ways consonant with these parameters. A summary
of such techniques is found in Remington: The Science and Practice
of Pharmacy, 21.sup.st Edition, Lippincott Williams & Wilkins,
(2005); and Encyclopedia of Pharmaceutical Technology, eds. J.
Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York,
each of which is incorporated herein by reference.
[0262] The compounds described herein (e.g., a compound according
to any of Formulas (I)-(XIII) or any of Compounds (1)-(236) of
Table 1) may be present in amounts totaling 1-95% by weight of the
total weight of the composition. The composition may be provided in
a dosage form that is suitable for intraarticular, oral, parenteral
(e.g., intravenous, intramuscular), rectal, cutaneous,
subcutaneous, topical, transdermal, sublingual, nasal, vaginal,
intravesicular, intraurethral, intrathecal, epidural, aural, or
ocular administration, or by injection, inhalation, or direct
contact with the nasal, genitourinary, gastrointesitnal,
reproductive or oral mucosa. Thus, the pharmaceutical composition
may be in the form of, e.g., tablets, capsules, pills, powders,
granulates, suspensions, emulsions, solutions, gels including
hydrogels, pastes, ointments, creams, plasters, drenches, osmotic
delivery devices, suppositories, enemas, injectables, implants,
sprays, preparations suitable for iontophoretic delivery, or
aerosols. The compositions may be formulated according to
conventional pharmaceutical practice.
[0263] In general, for use in treatment, the compounds described
herein (e.g., a compound according to any of Formulas (I)-(XIII) or
any of Compounds (1)-(236) of Table 1) may be used alone, as
mixtures of two or more compounds or in combination with other
pharmaceuticals. An example of other pharmaceuticals to combine
with the compounds described herein (e.g., a compound according to
any of Formulas (I)-(XIII) or any of Compounds (1)-(236) of Table
1) would include pharmaceuticals for the treatment of the same
indication. For example, in the treatment of pain, a compound may
be combined with another pain relief treatment such as an NSAID, or
a compound which selectively inhibits COX-2, or an opioid, or an
adjuvant analgesic such as an antidepressant. Another example of a
potential pharmaceutical to combine with the compounds described
herein (e.g., a compound according to any of Formulas (I)-(XIII) or
any of Compounds (1)-(236) of Table 1) would include
pharmaceuticals for the treatment of different yet associated or
related symptoms or indications. Depending on the mode of
administration, the compounds will be formulated into suitable
compositions to permit facile delivery. Each compound of a
combination therapy may be formulated in a variety of ways that are
known in the art. For example, the first and second agents of the
combination therapy may be formulated together or separately.
Desirably, the first and second agents are formulated together for
the simultaneous or near simultaneous administration of the
agents.
[0264] The compounds of the invention may be prepared and used as
pharmaceutical compositions comprising an effective amount of a
compound described herein (e.g., a compound according to any of
Formulas (I)-(XIII) or any of Compounds (1)-(236) of Table 1) and a
pharmaceutically acceptable carrier or excipient, as is well known
in the art. In some embodiments, the composition includes at least
two different pharmaceutically acceptable excipients or
carriers.
[0265] Formulations may be prepared in a manner suitable for
systemic administration or topical or local administration.
Systemic formulations include those designed for injection (e.g.,
intramuscular, intravenous or subcutaneous injection) or may be
prepared for transdermal, transmucosal, or oral administration. The
formulation will generally include a diluent as well as, in some
cases, adjuvants, buffers, preservatives and the like. The
compounds can be administered also in liposomal compositions or as
microemulsions.
[0266] For injection, formulations can be prepared in conventional
forms as liquid solutions or suspensions or as solid forms suitable
for solution or suspension in liquid prior to injection or as
emulsions. Suitable excipients include, for example, water, saline,
dextrose, glycerol and the like. Such compositions may also contain
amounts of nontoxic auxiliary substances such as wetting or
emulsifying agents, pH buffering agents and the like, such as, for
example, sodium acetate, sorbitan monolaurate, and so forth.
[0267] Various sustained release systems for drugs have also been
devised. See, for example, U.S. Pat. No. 5,624,677, which is herein
incorporated by reference.
[0268] Systemic administration may also include relatively
noninvasive methods such as the use of suppositories, transdermal
patches, transmucosal delivery and intranasal administration. Oral
administration is also suitable for compounds of the invention.
Suitable forms include syrups, capsules, and tablets, as is
understood in the art.
[0269] For administration to animal or human subjects, the dosage
of the compounds of the invention may be, for example, 0.01-50
mg/kg (e.g., 0.01-15 mg/kg or 0.1-10 mg/kg). For example, the
dosage can be 10-30 mg/kg.
[0270] Each compound of a combination therapy, as described herein,
may be formulated in a variety of ways that are known in the art.
For example, the first and second agents of the combination therapy
may be formulated together or separately.
[0271] The individually or separately formulated agents can be
packaged together as a kit. Non-limiting examples include, but are
not limited to, kits that contain, e.g., two pills, a pill and a
powder, a suppository and a liquid in a vial, two topical creams,
etc. The kit can include optional components that aid in the
administration of the unit dose to patients, such as vials for
reconstituting powder forms, syringes for injection, customized IV
delivery systems, inhalers, etc. Additionally, the unit dose kit
can contain instructions for preparation and administration of the
compositions. The kit may be manufactured as a single use unit dose
for one patient, multiple uses for a particular patient (at a
constant dose or in which the individual compounds may vary in
potency as therapy progresses); or the kit may contain multiple
doses suitable for administration to multiple patients ("bulk
packaging"). The kit components may be assembled in cartons,
blister packs, bottles, tubes, and the like.
[0272] Formulations for oral use include tablets containing the
active ingredient(s) in a mixture with non-toxic pharmaceutically
acceptable excipients. These excipients may be, for example, inert
diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol,
microcrystalline cellulose, starches including potato starch,
calcium carbonate, sodium chloride, lactose, calcium phosphate,
calcium sulfate, or sodium phosphate); granulating and
disintegrating agents (e.g., cellulose derivatives including
microcrystalline cellulose, starches including potato starch,
croscarmellose sodium, alginates, or alginic acid); binding agents
(e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium
alginate, gelatin, starch, pregelatinized starch, microcrystalline
cellulose, magnesium aluminum silicate, carboxymethylcellulose
sodium, methylcellulose, hydroxypropyl methylcellulose,
ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and
lubricating agents, glidants, and antiadhesives (e.g., magnesium
stearate, zinc stearate, stearic acid, silicas, hydrogenated
vegetable oils, or talc). Other pharmaceutically acceptable
excipients can be colorants, flavoring agents, plasticizers,
humectants, buffering agents, and the like.
[0273] Two or more compounds may be mixed together in a tablet,
capsule, or other vehicle, or may be partitioned. In one example,
the first compound is contained on the inside of the tablet, and
the second compound is on the outside, such that a substantial
portion of the second compound is released prior to the release of
the first compound.
[0274] Formulations for oral use may also be provided as chewable
tablets, or as hard gelatin capsules wherein the active ingredient
is mixed with an inert solid diluent (e.g., potato starch, lactose,
microcrystalline cellulose, calcium carbonate, calcium phosphate or
kaolin), or as soft gelatin capsules wherein the active ingredient
is mixed with water or an oil medium, for example, peanut oil,
liquid paraffin, or olive oil. Powders, granulates, and pellets may
be prepared using the ingredients mentioned above under tablets and
capsules in a conventional manner using, e.g., a mixer, a fluid bed
apparatus or a spray drying equipment.
[0275] Dissolution or diffusion controlled release can be achieved
by appropriate coating of a tablet, capsule, pellet, or granulate
formulation of compounds, or by incorporating the compound into an
appropriate matrix. A controlled release coating may include one or
more of the coating substances mentioned above and/or, e.g.,
shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl
alcohol, glyceryl monostearate, glyceryl distearate, glycerol
palmitostearate, ethylcellulose, acrylic resins, dl-polylactic
acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl
acetate, vinyl pyrrolidone, polyethylene, polymethacrylate,
methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels,
1,3 butylene glycol, ethylene glycol methacrylate, and/or
polyethylene glycols. In a controlled release matrix formulation,
the matrix material may also include, e.g., hydrated
methylcellulose, carnauba wax and stearyl alcohol, carbopol 934,
silicone, glyceryl tristearate, methyl acrylate-methyl
methacrylate, polyvinyl chloride, polyethylene, and/or halogenated
fluorocarbon.
[0276] The liquid forms in which the compounds and compositions of
the present invention can be incorporated for administration orally
include aqueous solutions, suitably flavored syrups, aqueous or oil
suspensions, and flavored emulsions with edible oils such as
cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as
elixirs and similar pharmaceutical vehicles.
[0277] Generally, when administered to a human, the oral dosage of
any of the compounds of the combination of the invention will
depend on the nature of the compound, and can readily be determined
by one skilled in the art. Typically, such dosage is normally about
0.001 mg to 2000 mg per day, desirably about 1 mg to 1000 mg per
day, and more desirably about 5 mg to 500 mg per day. Dosages up to
200 mg per day may be necessary.
[0278] Administration of each drug in a combination therapy, as
described herein, can, independently, be one to four times daily
for one day to one year, and may even be for the life of the
patient. Chronic, long-term administration may be indicated.
Synthesis
[0279] The reaction scheme and Examples are intended to illustrate
the synthesis of a representative number of compounds. Accordingly,
the Examples are intended to illustrate but not to limit the
invention. Additional compounds not specifically exemplified may be
synthesized using conventional methods in combination with the
methods described herein.
EXAMPLES
Example 1
Synthesis of (4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)
methanamine (5)
##STR00021##
[0280] Synthesis of tert-butyl
(2-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-2-oxoethyl)carbamate
(3)
[0281] 3,5-Bis(trifluoromethyl)benzene-1,2-diamine (1) (3.0 g, 12.3
mol), 2-((tert-butoxycarbonyl)amino)acetic acid (2) (2.1 g, 12.3
mmol), HATU (6.4 g, 17.2 mmol), and triethylamine (TEA; 3.5 mL, 25
mmol) were stirred in dichloromethane (DCM; 50 mL) at room
temperature for 17 hours. The reaction was diluted with DCM (100
mL), washed sequentially with NH.sub.4Cl (saturated solution),
NaHCO.sub.3 (saturated solution), and brine. The organics were
separated, dried (Na.sub.2SO.sub.4), and concentrated in vacuo. The
residue was purified by automated column chromatography
(EtOAc/petroleum ether, 35/65) to give tert-butyl
(2-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-2-oxoethyl)carbamate
(3) (3.07 g, 61.7%); Confirmed by LCMS (Positive ion mode).
Synthesis of tert-Butyl
((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)carbamate
(4)
[0282] tert-Butyl
(2-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-2-oxoethyl)carbamate
(3) (1.4 g, mmol) was heated in THF/AcOH (95/5, 20 mL) using a
microwave at 140.degree. C. for 2.5 hours. The reaction was
concentrated in vacuo, taken up in EtOAc, and washed with
NaHCO.sub.3 (saturated solution) to neutralize. The organic layer
was separated, dried (Na.sub.2SO.sub.4), and concentrated in vacuo.
The residue was purified by automated column chromatography
(EtOAc/PE, 50:50) to give tert-Butyl
((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)carbamate
(4), (730 mg, 54%); confirmed by LCMS (Positive ion mode)).
Synthesis of
(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methanamine
hydrochloride (5)
[0283] tert-Butyl
((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)carbamate
(4) (730 mg, 1.9 mmol) was taken up in EtOAc, and the solution was
flushed with HCl (g) for 5 minutes. The resultant suspension was
stirred at room temperature for 25 minutes then concentrated in
vacuo. The resultant solid was dried under high vacuum for 14 hours
to give
(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methanamine
hydrochloride (5), in a quantitative manner; .sup.1H NMR (300 mHz,
CD.sub.3OD) .delta. 4.57 (s, 2H), 7.81 (s, 1H), 8.21 (s, 1H).
Example 2
Synthesis of
2-amino-N-((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)acet-
amide hydrochloride (7)
##STR00022##
[0284] Synthesis of tert-butyl
(2-(((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)amino)-2-o-
xoethyl)carbamate (6)
[0285]
(4,6-Bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-methanamine
hydrochloride (5) (1 g, 2.6 mmol), BOC-gly (2) (4.55 mg, 2.6 mmol),
HATU (1.35 g, 3.64 mmol), and TEA (0.73 mL, 5.2 mm01) were stirred
in DCM at room temperature for 14 hours. The reaction was diluted
with DCM (100 mL) and washed sequentially with NH.sub.4Cl
(saturated solution), NaHCO.sub.3 (saturated solution), and brine.
The organics were separated, dried (Na.sub.2SO.sub.4), and
concentrated in vacuo. The residue was purified by automated column
chromatography (EtOAc/PE, 50/50) to give tert-butyl
(2-(((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)amino)-2-o-
xoethyl)carbamate (6) (732 mg, 64%; Confirmed by LCMS (positive ion
mode)).
Synthesis of
2-amino-N-((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)acet-
amide hydrochloride (7)
[0286] tert-Butyl
(2-(((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)amino)-2-o-
xoethyl)carbamate (6) (732 mg, 1.66 mmol) was taken up in EtOAc,
and the solution flushed with HCl (g) for 5 minutes. The suspension
was stirred at room temperature for 20 minutes, concentrated in
vacuo, and the residue purified by reverse phase HPLC to give
2-amino-N-((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)acet-
amide hydrochloride (7); .sup.1H NMR (300 mHz, CD.sub.3OD) .delta.
3.45 (s, 2H), 5.04 (s, 2H), 8.18 (s, 1H), (8.49, S, 1H).
Example 3
Synthesis of
1-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-3-(tert-but-
yl)urea (9)
##STR00023##
[0287] Synthesis of
1-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-3-(tert-but-
yl)urea (9)
[0288]
(4,6-Bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methanamine
hydrochloride (5) (250 mg, 0.88 mmol) and TEA (0.25 mL, 1.8 mmol)
were stirred in DCM. tert-Butyl isocyanate (8) (105 .mu.L, 0.9
mmol) was added and the reaction stirred at room temperature for 1
h. The reaction was concentrated in vacuo and the residue purified
by reverse phase HPLC to give
1-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-3-(ter-
t-butyl)urea (9). Confirmed by LCMS (positive ion mode).
Example 4
Synthesis of
5-(3-chloro-4-fluorophenyl)-2-(pyrimidin-5-ylmethyl)-1H-benzo[d]imidazole
(15)
##STR00024##
[0289] Preparation of
N-(2-amino-5-bromophenyl)-2-(pyrimidin-5-yl)acetamide (12)
[0290] To a solution of 4-bromobenzene-1,2-diamine (10) (0.281 g,
1.5 mmol), 2-(pyrimidin-5-yl)acetic acid (11) (0.207 g, 1.5 mmol),
and HATU (0.741 g, 1.95 mmol) in DCM (50 ml) was added
triethylamine (0.63 ml, 4.5 mmol). The reaction mixture was stirred
at room temperature overnight. The solution was washed with
saturated sodium bicarbonate (50 ml) and brine (50 ml). The DCM
solution was dried over sodium sulfate and concentrated. The
residue was purified by automated column chromatography columned
using DCM and methanol as eluents. Yield 0.4 g, 87%. MS: m/z 306.9
(M+H.sup.+).
Preparation of
5-bromo-2-(pyrimidin-5-ylmethyl)-1H-benzo[d]imidazole (13)
[0291] A solution of
N-(2-amino-5-bromophenyl)-2-(pyrimidin-5-yl)acetamide (12) (0.4 g,
1.3 mmol) in THF (12 ml) and acetic acid (7 ml) was reacted in the
microwave at 145.degree. C. for 3 hours. The solvents were removed.
The residue was dissolved in ethyl acetate (50 ml) and washed with
saturated sodium bicarbonate (30 ml) and brine (30 ml). The ethyl
acetate solution was dried over sodium sulfate and concentrated.
The residue was purified by automated column chromatography using
DCM and methanol as eluents. Yield 0.345 g, 91%. MS: m/z 288.9
(M+H.sup.+).
Preparation of
5-(3-chloro-4-fluorophenyl)-2-(pyrimidin-5-ylmethyl)-1H-benzo[d]imidazole
(15)
[0292] Pd(dppf).sub.2Cl.sub.2.DCM (0.274 g, 0.336 mmol) was added
to a suspension of
5-bromo-2-(pyrimidin-5-ylmethyl)-1H-benzo[d]imidazole (13) (0.345
g, 1.12 mmol), 3-chloro-4-fluorobenzene boronic acid (14) (0.234 g,
1.34 mmol), and sodium carbonate (0.594 g, 5.6 mmol) in ethanol (7
ml) and toluene (7 ml). The reaction mixture was stirred at
130.degree. C. overnight. The deep brown solution was filtered
through Celite and concentrated. The residue was purified by
automated column chromatography using DCM and methanol as eluents.
Yield 0.15 g, 40%. MS: m/z 338.9 (M+H.sup.+).
Example 5
Synthesis of
(5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methanol
(21)
##STR00025##
[0293] Preparation of ethyl 2-((tert-butyldimethylsilyl)oxy)acetate
(17)
[0294] A solution of ethyl 2-hydroxyacetate (16) (3.12 g, 30 mmol)
and imidazole (2.45 g, 36 mmol) in DCM (100 ml) was added
tert-butylchlorodimethylsilane (5.43 g, 36 mmol) at 0.degree. C.
and the reaction mixture was stirred at 0.degree. C. for 1 hour.
The reaction mixture was washed with saturated sodium bicarbonate
(50 ml) and brine (50 ml). The DCM solution was dried over sodium
sulfate and concentrated. The residual (17) (6.68 g, 100% yield)
was used in the next step without further purification.
Preparation of 2-((tert-butyldimethylsilyl)oxy)acetic acid (18)
[0295] A solution of ethyl 2-((tert-butyldimethylsilyl)oxy)acetate
(17) (6.68 g, 30 mmol) in methanol (30 ml) was added 2N NaOH
solution (30 ml). The mixture was then stirred at room temperature
for 2 hours. The reaction mixture was concentrated, diluted with
water, and acidified with 2N HCl to pH 4-5. The aqueous solution
was extracted with ethyl acetate 3 times. The combined ethyl
acetate solution was washed with brine and dried over sodium
sulfate and concentrated. The residual (18) was used in the next
step without further purification. Yield 2.19 g, 38%. MS, m/z 189.1
(M-H.sup.+).
Preparation of
N-(2-amino-3,5-bis(trifluoromethyl)phenyl)-2-((isopropyldimethylsilyl)oxy-
)acetamide (19)
[0296] A solution of 3,5-bis(trifluoromethyl)benzene-1,2-diamine
(1) (1.28 g, 5.26 mmol), 2-((tert-butyldimethylsilyl)oxy)acetic
acid (18) (1 g, 5.26 mmol) and HATU (2.6 g, 6.84 mmol) in DMF (15
ml) was reacted in the microwave at 80.degree. C. for 2 hours. The
reaction mixture was diluted with ethyl acetate. The solution was
washed with saturated sodium bicarbonate and brine. The ethyl
acetate solution was dried over sodium sulfate and concentrated.
The residue (19) was purified by automated column chromatography
using petroleum ether and ethyl acetate as eluents. Yield 0.84 g,
40%. MS, m/z 403.0 (M+H.sup.+).
Preparation of
2-(((tert-butyldimethylsilyl)oxy)methyl)-5,7-bis(trifluoromethyl)-1H-benz-
o[d]imidazole (20)
[0297] A solution of
N-(2-amino-3,5-bis(trifluoromethyl)phenyl)-2-((isopropyl
dimethylsilyl)oxy)acetamide (19) (0.84 g, 2.09 mmol) in THF (14 ml)
and acetic acid (7 ml) was reacted in the microwave at 145.degree.
C. for 3 hours. The solvents were removed. The residue was
dissolved in ethyl acetate (50 ml) and washed with saturated sodium
bicarbonate (30 ml) and brine (30 ml). The ethyl acetate solution
was dried over sodium sulfate and concentrated. The residue (20)
was purified by automated column chromatography using pet ether and
ethyl acetate as eluents. Yield 0.5 g, 60%. MS, m/z 399.0
(M+H.sup.+).
Preparation of
(5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methanol
(21)
[0298] To a solution of
2-(((tert-butyldimethylsilyl)oxy)methyl)-5,7-bis(trifluoromethyl)-1H-benz-
o[d]imidazole (20) (0.5 g, 1.26 mmol) in THF (15 ml) was added 1M
TBAF solution in THF (1.7 ml, 1.7 mmol) at 0.degree. C. The
reaction mixture was stirred at room temperature overnight. The
solvent was removed. The residue was dissolved in ethyl acetate (50
ml) and washed with brine (30 ml). The ethyl acetate solution was
dried over sodium sulfate and concentrated. The residue of (34) was
purified by automated column chromatography using petroleum ether
and ethyl acetate as eluents. Yield 0.22 g, 61%. MS, m/z 284.9
(M+H.sup.+).
Example 6
Synthesis of
(1-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-1-methylur-
ea (24)
##STR00026##
[0299] Preparation of
(1-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-1-methylur-
ea (24)
[0300] A solution of
1-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-N-methylmethanamine
hydrochloride (22) (0.4 g, 1.19 mmol) and urea (23) (0.11 g, 1.83
mmol) in water (10 ml) was refluxed overnight. At this time,
crystals separated from the liquid and were then collected by
filtration. The precipitated product (24) was washed with water and
dried. Yield 0.3 g, 72%. MS, m/z 340.22 (M+H.sup.+).
Example 7
Synthesis of
(1-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-1-N-methyl-
sulfuric diamide (26)
##STR00027##
[0301] Preparation of
(1-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-1-N-methyl-
sulfuric diamide (26)
[0302] A solution of
1-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-N-methylmethanamine
hydrochloride (22) (0.2 g, 0.6 mmol), sulfamide (25) (0.16 g, 1.68
mmol) in dioxane (15 ml) was heated to reflux for 6 hours. The
reaction mixture was then cooled, and the solvent was evaporated.
The residue was dissolved in water (10 ml), and the aqueous was
extracted with ethyl acetate three times. The combined ethyl
acetate layers were combined, dried, and evaporated. The residue
(26) was purified by automated column chromatography using
petroleum ether and ethyl acetate as eluents. Yield 70%. MS, m/z
376.28 (M+H.sup.+).
Example 8
Synthesis of 4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-amine
(28)
##STR00028##
[0303] Preparation of
4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-amine (28)
[0304] To a solution of 3,5-bis(trifluoromethyl)benzene-1,2-diamine
(1) (0.40 g, 1.6 mmol) in acetonitrile (15 mL) was added a solution
of cyanogen bromide in acetonitrile (27) (5 M, 0.66 mL, 3.3 mmol).
The resultant mixture was allowed to stir at room temperature for
23 hours. At this time, the reaction was concentrated in vacuo, and
the crude product was purified by automated flash chromatography to
afford the (28); confirmed by LCMS (positive ion mode)).
Example 9
Synthesis of
2-((methylsulfonyl)methyl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imidazole
(32)
##STR00029##
[0305] Preparation of
N-(2-amino-3,5-bis(trifluoromethyl)phenyl)-2-(methylthio)acetamide
(30)
[0306] To a mixture of 3,5-bis(trifluoromethyl)benzene-1,2-diamine
(1) (0.50 g, 2.1 mmol), 2-(methylthio)acetic acid (29) (0.22 g, 2.1
mmol) and HATU (1.01 g, 2.67 mmol) in dichloromethane (15 mL) was
added triethylamine (0.9 mL, 6 mmol). The resultant solution was
stirred at room temperature for 24 hours. The reaction was then
washed with a saturated aqueous solution of sodium bicarbonate
(2.times.30 mL). The organic phase was then dried over anhydrous
sodium sulfate and concentrated in vacuo. The crude product was
purified by automated flash chromatography (2:1 hexane:ethyl
acetate) to afford the title compound (30) (0.40 g, 59%; confirmed
by LCMS (positive ion mode).) as a pale yellow oil, which
crystallized on standing. The other regioisomer was not observed
during the course of purification.
Preparation of
2-((methylthio)methyl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imidazole
(31)
[0307] To a solution of
N-(2-amino-3,5-bis(trifluoromethyl)phenyl)-2-(methylthio)acetamide
(30) (0.40 g, 1.2 mmol) in tetrahydrofuran (3 mL) was added glacial
acetic acid (2 mL). The reaction was sealed in a microwave reaction
vial and heated at 130.degree. C. for 30 minutes. The resultant
solution was concentrated in vacuo, taken up in ethyl acetate (40
mL), and washed with a saturated aqueous solution of sodium
bicarbonate (2.times.10 mL). The organic phase was then dried over
anhydrous sodium sulfate and concentrated in vacuo. The crude
product was purified by automated flash chromatography (2:1
hexane:ethyl acetate) to afford the title compound (31) (0.34 g,
89%; confirmed by LCMS (positive ion mode)) as a yellow solid.
Preparation of
2-((methylsulfonyl)methyl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imidazole
(32)
[0308] To a solution of
2-((methylthio)methyl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imidazole
(31) (0.34 g, 1.1 mmol) in dichloromethane (20 mL) was added m-CPBA
(77%, 0.73 g, 3.2 mmol) and sodium bicarbonate (0.45 g, 5.4 mmol).
The resultant mixture was stirred at room temperature for 17 hours,
at which time dichloromethane was added (30 mL). To the resultant
solution was added an aqueous solution of sodium hydroxide (2 M, 10
mL) and a saturated aqueous solution of sodium thiosulfate (10 mL).
The mixture was allowed to stir for 1 hours. The aqueous layer was
subsequently separated, acidified with hydrochloric acid (2 M, 12
mL), extracted with ethyl acetate (3.times.30 mL), and dried over
anhydrous sodium sulfate. The resultant oil was taken up in an
ethyl acetate:methanol mixture (1:1) and filtered. The filtrate was
then concentrated in vacuo, and the product was purified by
automated flash chromatography to afford the title compound (32)
(confirmed by LCMS (positive ion mode)).
Example 10
Synthesis of
N-((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)methanesulfo-
namide (34)
##STR00030##
[0309] Preparation of
N-((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)
methanesulfonamide (34)
[0310] To a solution of
(5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methanamine
hydrogen chloride (5) (0.30 g, 0.94 mmol) and DIPEA (0.3 mL, 2
mmol) in dichloromethane (25 mL) at 0.degree. C. was added
methanesulfonyl chloride (33) (0.07 mL, 0.94 mmol) dropwise via
syringe. The resultant solution was warmed to room temperature,
stirred for 72 hours, and then concentrated in vacuo. The resultant
oil was then taken up in dichloromethane (30 mL) and washed with a
saturated aqueous solution of sodium bicarbonate (20 mL) and brine
(20 mL). The organic layer was dried over anhydrous sodium sulfate
and concentrated in vacuo. The crude product was purified by
automated flash chromatography (ethyl acetate) to afford the title
compound (34) (confirmed by LCMS (positive ion mode).
Example 11
Synthesis of
(R)-2-(pyrrolidin-2-yl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imidazole
(39)
##STR00031##
[0311] Preparation of
(R)-2-(pyrrolidin-2-yl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imidazole
(39)
[0312] 3,5-bis(trifluoromethyl)benzene-1,2-diamine (1) (1.5 g, 6.14
mmol), (R)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid
(35) (1.30 g, 6.04 mmol), TEA (1.1 mL, 7.98 mmol), and HATU (3.2 g,
8.42 mmol) were dissolved in DCM (15 mL). This solution was stirred
overnight at room temperature and then concentrated in vacuo. The
residue was taken up in ethyl acetate (50 mL) and then washed
sequentially with saturated aqueous ammonium chloride (20 mL),
saturated sodium bicarbonate (20 mL), and brine (20 mL). The
organic fraction was then dried over anhydrous sodium sulfate,
filtered, and concentrated in vacuo. The residue was purified by
automated column chromatography (smooth gradient 20.fwdarw.70%
ethyl acetate:petroleum ether) to afford a mixture of the desired
isomers (36) and (37), confirmed by LCMS (positive ion mode).
[0313] The mixture of isomers was taken up in THF/AcOH (95/5) and
heated using a microwave at 140.degree. C. for 2 hours. The
reaction was concentrated in vacuo, taken up in EtOAc, and then
washed with water (30 mL), saturated aqueous sodium bicarbonate (30
mL), and brine (30 mL). The organic layer was dried over anhydrous
sodium sulfate then concentrated in vacuo. The residue was purified
by automated column chromatography (smooth gradient 20.fwdarw.70%
ethyl acetate:petroleum ether), and this initial purification was
followed by a second round of automated column chromatography
(smooth gradient 0.fwdarw.50% ethyl acetate:dichloromethane) to
afford the N-Boc-protected product (38) as clear colorless gum. The
product was taken up in HCl saturated ethyl acetate and stirred for
two hours at room temperature. The clear mixture turned milky over
time and was condensed in vacuo to give the HCl salt of the product
(39) as a white solid (1.00 g, 51% over two steps). .sup.1H NMR
(300 MHz, CD.sub.3OD) .delta. 2.24-2.40 (m, 3H), 2.67-2.75 (m, 1H),
3.52-3.59 (m, 1H), 3.63-3.70 (m, 1H), 5.17 (t, J=6 Hz, 1H), 7.86
(s, 1H), 8.25 (s, 1H).
Example 12
Synthesis of
2-(3-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)azetidin--
1-yl)-N-(1-methylcyclobutyl)acetamide (42)
##STR00032##
[0314] Preparation of
2-(3-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)azetidin--
1-yl)-N-(1-methylcyclobutyl)acetamide (42)
[0315] To a solution of
2-(azetidin-3-ylmethyl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imidazole
(40) (0.81 g, 2.5 mmol) and triethylamine (1.8 mL, 13 mmol) in
acetonitrile (50 mL) was added
2-chloro-N-(1-methylcyclobutyl)acetamide (41) (0.41 g, 2.5 mmol).
The resultant solution was stirred at 40.degree. C. for 17 hours
and concentrated in vacuo. The crude product was purified by
automated flash chromatography to afford the title compound (42).
The product was confirmed by LCMS (positive ion mode).
Example 13
Synthesis of
1-((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)piperazin-2--
one hydrochloride (47)
##STR00033##
[0316] Synthesis of tert-butyl
4-(2-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-2-oxoethyl)-3-oxopip-
erazine-1-carboxylate (45)
[0317] 2-(4-(tert-butoxycarbonyl)-2-oxopiperazin-1-yl)acetic acid
(2) (2.0 g, 7.77 mmol), 3,5 bis(trifluoromethyl)-O-phenylenediamine
(44) (2.15 g, 8.81 mmol), HOBt (1.36 g, 10.1 mmol), EDC (1.93 g,
10.1 mmol), and diisopropylethylamine (2.3 mL, 13.2 mmol) were
stirred in DMF (15 mL) at room temperature for 14 hours. The
residue was concentrated in vacuo and was taken up in EtOAc (200
ml). The reaction mixture was washed sequentially with NH.sub.4Cl
(saturated solution), NaHCO.sub.3 (saturated solution), and brine.
The organics were separated, dried (Na.sub.2SO.sub.4) and
concentrated in vacuo. The residue was purified by automated column
chromatography (EtOAc/PE) to give tert-butyl
4-(2-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-2-oxoethyl)-3-oxopip-
erazine-1-carboxylate (45) (3.5 g, 93%; confirmed by LCMS (positive
ion mode)).
Synthesis of tert-butyl
4-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-3-oxopipera-
zine-1-carboxylate (46)
[0318] tert-Butyl
4-(2-((2-amino-3,5-bis(trifluoromethyl(phenyl)amino)-2-oxoethyl)-3-oxopip-
erazine-1-carboxylate (45) (3.5 g, 7.23 mmol) was heated in
THF/AcOH (1:1, 20 mL) using a microwave at 140.degree. C. for 45
minutes. The residue was concentrated in vacuo, and the residue was
then taken up in EtOAc (150 ml). The reaction mixture was washed
sequentially with NaHCO.sub.3 (saturated solution) and brine. The
organics were separated, dried (Na.sub.2SO.sub.4), and concentrated
in vacuo. The residue was purified by automated column
chromatography (EtOAc) to give tert-butyl
4-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-3-oxopipera-
zine-1-carboxylate (46) (3.1 g, 92%; confirmed by LCMS (positive
ion mode))
Synthesis of
1-((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)piperazin-2--
one hydrochloride (47)
[0319] tert-butyl
4-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-3-oxopipera-
zine-1-carboxylate (46) (3.1 g, 6.65 mmol) was taken up in EtOAc,
and the solution was flushed with HCl (g) for 5 minutes. The
resultant suspension was stirred at room temperature for 30 minutes
then concentrated in vacuo. The resultant solid was dried under
high vacuum for 14 hours to give
1-((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)piperaz-
in-2-one hydrochloride (47) in a quantitative manner. .sup.1H NMR
(300 mHz, CD.sub.3OD) .delta. 3.70-3.75 (m, 2H), 4.00-4.10 (m, 4H),
5.10 (s, 1H), 8.15 (s, 1H), 8.2 (s, 1H), 7.86 (s, 1H).
Example 14
Synthesis of
2-(azetidin-3-ylmethyl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imidazole
(51)
##STR00034##
[0320] Preparation of tert-butyl
3-(2-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-2-oxoethyl)azetidine-
-1-carboxylate (49)
[0321] To a mixture of 3,5-bis(trifluoromethyl)benzene-1,2-diamine
(43) (3.00 g, 12.3 mmol),
2-(1-(tert-butoxycarbonyl)azetidin-3-yl)acetic acid (48) (2.64 g,
12.3 mmol), and HATU (6.08 g, 16.0 mmol) in dichloromethane (60 mL)
was added triethylamine (5.1 mL, 36.9 mmol). The resultant solution
was stirred at room temperature for 23 hours and then washed with a
saturated aqueous solution of sodium bicarbonate (2.times.70 mL).
The organic phase was then dried over anhydrous sodium sulfate and
concentrated in vacuo. The crude product was purified by automated
flash chromatography (1:1 hexane:ethyl acetate) to afford the title
compound (49) (3.46 g, 64%) as a white foam. The other regioisomer
was not observed during the course of purification. The product was
confirmed by LCMS (positive ion mode).
Preparation of tert-butyl
3-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)azetidine-1--
carboxylate (50)
[0322] To a solution of tert-butyl
3-(2-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-2-oxoethyl)azetidine-
-1-carboxylate (49) (2.54 g, 5.75 mmol) in tetrahydrofuran (15 mL)
was added glacial acetic acid (3 mL). The reaction was sealed in a
microwave reaction vial and reacted at 130.degree. C. for 45
minutes. The resultant solution was concentrated in vacuo, taken up
in ethyl acetate (80 mL), and washed with a saturated aqueous
solution of sodium bicarbonate (20 mL). The organic phase was then
dried over anhydrous sodium sulfate and concentrated in vacuo. The
crude product was purified by automated flash chromatography (1:1
hexane:ethyl acetate) to afford the title compound (50) (2.12 g,
87%) as a white solid. The product was confirmed by LCMS (positive
ion mode).
Preparation of
2-(azetidin-3-ylmethyl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imidazole
(51)
[0323] To a solution of tert-butyl
3-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)azetidine-1--
carboxylate (50) (1.06 g, 2.50 mmol) in dichloromethane (40 mL) was
added trifluoroacetic acid (2.4 mL, 31 mmol). The resultant
solution was allowed to stir at room temperature for 2.5 hours and
was then concentrated in vacuo. The resultant oil was taken up in
toluene (30 mL) then concentrated in vacuo. The crude product was
purified by automated flash chromatography (80:20:1
dichloromethane:methanol: ammonium hydroxide) to afford the title
compound (51) as a cream colored foam; .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 3.22 (quintet, 1H), 3.28 (d, 2H), 3.47 (septet,
1H), 3.99 (m, 2H), 4.16 (m, 2H), 7.64 (s, 1H), 8.00 (s, 1H). The
product was confirmed by LCMS (positive ion mode).
Example 15
Synthesis of
2-(2-oxopiperazin-1-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)acetamide
(54)
##STR00035##
[0324] Synthesis of tert-butyl
3-oxo-4-(2-oxo-2-((5-(trifluoromethyl)pyridin-2-yl)amino)ethyl)piperazine-
-1-carboxylate (53)
[0325] To a solution of 5-(trifluoromethyl)pyridin-2-amine (52)
(0.162 g, 1 .mu.mol),
2-(4-(tert-butoxycarbonyl)-2-oxopiperazin-1-yl)acetic acid (44)
(0.257 g, 1 mmol), and HATU (0.46 g, 1.3 mmol) in DMF 3 ml was
added triethylamine (0.3 ml, 3 mmol). The mixture was heated in a
microwave at 75.degree. C. for 2 hours. The reaction mixture was
diluted with ethyl acetate and washed with saturated sodium
bicarbonate aqueous solution and brine. The organic layer was dried
over sodium sulfate and concentrated. The residue was purified by
automated flash chromatography using pet ether and ethyl acetate as
eluents. Yield 0.15 g, 50%. LCMS m/z 401.9 (M+H.sup.+).
Synthesis of
2-(2-oxopiperazin-1-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)acetamide
(54)
[0326] To a solution of tert-butyl
3-oxo-4-(2-oxo-2-((5-(trifluoromethyl)pyridin-2-yl)amino)ethyl)piperazine-
-1-carboxylate (53) (0.15 g, 0.5 mmol) in ethyl acetate (3 ml) was
added saturated HCl solution in ethyl acetate (2 ml). The reaction
mixture was stirred at room temperature for 30 minutes. The
reaction mixture was then concentrated and dried in vacuo to give
2-(2-oxopiperazin-1-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)acetamide
(54) as HCl salt. Yield 0.165 g, 98%. LCMS m/z 301.9
(M+H.sup.+).
Example 16
Synthesis of
1-(2-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)ethyl)piperazin-2-
-one (61)
##STR00036##
[0327] Synthesis of tert-butyl
4-(3-ethoxy-3-oxopropyl)-3-oxopiperazine-1-carboxylate (57)
[0328] tert-Butyl 3-oxopiperazine-1-carboxylate (55) (4.0 g, 20
mmol) was stirred in dry DMF at room temperature under Ar. NaH (60%
dispersion in mineral oil) (960 mg, 24 mmol) was added, and the
reaction stirred for 30 minutes. Ethyl bromopropionate (56) (2.55
mL, 20 mmol) was added in one portion, and stirring continued for
14 hours. The reaction was partitioned between EtOAc and H.sub.2O.
The organics were separated, dried (Na.sub.2SO.sub.4), and
concentrated in vacuo to give tert-butyl
4-(3-ethoxy-3-oxopropyl)-3-oxopiperazine-1-carboxylate (57) as a
crude residue, which was used in the subsequent reaction without
additional purification. The product was confirmed with LCMS
(positive ion mode).
Synthesis of
3-(4-(tert-butoxycarbonyl)-2-oxopiperazin-1-yl)propanoic acid
(58)
[0329] tert-Butyl
4-(3-ethoxy-3-oxopropyl)-3-oxopiperazine-1-carboxylate (57); as the
crude residue from the previous step) and LiOH.H.sub.2O (1.26 g, 30
mmol) were stirred in THF/H.sub.2O/MeOH (40/40/15 mL) at room
temperature for 16 hours. The resultant solution was filtered to
remove solid precipitation. The organic solvent was removed in
vacuo, and the solution was acidified with 1M HCl. The reaction was
extracted with EtOAc (3.times.75 mL), and the organics were dried
(Na.sub.2SO.sub.4) and concentrated in vacuo to give
3-(4-(tert-butoxycarbonyl)-2-oxopiperazin-1-yl)propanoic acid (58)
(4.4 g, 81% from tert-Butyl 3-oxopiperazine-1-carboxylate (55). The
product was confirmed by LCMS (negative ion mode).
Synthesis of tert-butyl
4-(3-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-3-oxopropyl)-3-oxopi-
perazine-1-carboxylate (59)
[0330] 3-(4-(tert-Butoxycarbonyl)-2-oxopiperazin-1-yl)propanoic
acid (58) (1.52 g, 5.6 mmol), 3,5
bis(trifluoromethyl)-O-phenylenediamine (1) (1.36 g, 5.6 mmol),
HATU (2.91 g, 7.84 mmol) and TEA (1.56 mL, 11.8 mmol) were stirred
in DCM (50 mL) at room temperature for 14 hours. The reaction was
diluted with DCM (100 mL), washed sequentially with NH.sub.4Cl
(saturated solution), NaHCO.sub.3 (saturated solution) and brine,
the organics separated, dried (Na.sub.2SO.sub.4), and concentrated
in vacuo. The residue was purified by automated column
chromatography (100% EtOAc/PE) to give tert-butyl
4-(3-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-3-oxopropyl)-3-oxopi-
perazine-1-carboxylate (59) (2.64 g, 95%; confirmed by LCMS
(positive ion mode)).
Synthesis of
1-(2-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)ethyl)piperazin-2-
-one hydrochloride (61)
[0331] tert-Butyl
4-(3-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-3-oxopropyl)-3-oxopi-
perazine-1-carboxylate (59) (500 mg, 1.0 mmol) was heated in
THF/AcOH (95:5, 2 mL) using a microwave at 140.degree. C. for 2.5
hours. The residue was concentrated in vacuo to give crude
tert-butyl
4-(2-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)ethyl)-3-oxopiper-
azine-1-carboxylate (60). The crude material was taken up in EtOAc,
and the solution flushed with HCl (g) for 5 minutes. The suspension
was stirred at room temperature for 20 minutes and concentrated in
vacuo. The residue was purified by reverse phase HPLC to give
1-(2-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)ethyl)piperazin-2-
-one hydrochloride (61).
Example 17
Synthesis of
2-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-1-(piperazin-1-yl)e-
thanone (68)
##STR00037##
[0332] Synthesis of tert-butyl
4-(3-ethoxy-3-oxopropanoyl)piperazine-1-carboxylate (64)
[0333] tert-Butyl piperazine-1-carboxylate (62) (1 g, 5.4 mmol) and
TEA (837 .mu.L, 6.0 mmol) were stirred in DCM at room temperature.
Ethyl malonyl chloride (63) (810 .mu.L, 5.4 mmol) was added in one
portion, and the reaction stirred at room temperature for 1 hour.
The reaction was diluted with DCM and washed sequentially with
NH.sub.4Cl (saturated solution) and NaHCO.sub.3 (saturated
solution). The organics were separated, dried (Na.sub.2SO.sub.4),
and concentrated in vacuo. The residue was purified by automated
column chromatography (100% EtOAc) to give tert-butyl
4-(3-ethoxy-3-oxopropanoyl)piperazine-1-carboxylate (64) (1.24 g,
77%). The product was onfirmed by LCMS (positive ion mode).
Synthesis of
3-(4-(tert-butoxycarbonyl)piperazin-1-yl)-3-oxopropanoic acid
(65)
[0334] tert-Butyl
4-(3-ethoxy-3-oxopropanoyl)piperazine-1-carboxylate (64) (1.24 g,
2.8 mmol) and LiOH.H.sub.2O (176 mg, 4.2 mmol) was stirred in
THF/H.sub.2O/MeOH (30/30/10 mL) at room temperature for 14 hours.
The organic solvents were removed in vacuo, and the solution was
acidified with 1 M HCl and extracted with EtOAc. The organics were
separated, dried (Na.sub.2SO.sub.4), and concentrated in vacuo to
give 3-(4-(tert-butoxycarbonyl)piperazin-1-yl)-3-oxopropanoic acid
(65) in a quantitative fashion. The product was confirmed by LCMS
(negative ion mode) and was used in the subsequent reaction without
additional purification
Synthesis of tert-butyl
4-(3-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-3-oxopropanoyl)piper-
azine-1-carboxylate (66)
[0335] 3-(4-(tert-Butoxycarbonyl)piperazin-1-yl)-3-oxopropanoic
acid (65) (761 mg, 2.8 mmol),
3,5-bis(trifluoromethyl)benzene-1,2-diamine (43) (744 m g, 3.05
mmol), HATU (1.6 g, 4.27 mmol), and TEA (906 .mu.L, 6.5 mmol) were
stirred in DCM at room temperature for 14 hours. The precipitate
was removed by filtration. The filtrate was diluted with DCM and
washed sequentially with NH.sub.4Cl (saturated solution) and
NaHCO.sub.3 (saturated solution). The organics were separated,
dried (Na.sub.2SO.sub.4), and concentrated in vacuo. The residue
was purified by automated column chromatography (EtOAc/PE, 50/50)
to give tert-butyl
4-(3-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-3-oxopropanoyl)piper-
azine-1-carboxylate (66) (650 mg, 42%). The product was onfirmed by
LCMS (positive ion mode).
Synthesis of
2-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-1-(piperazin-1-yl)e-
thanone hydrochloride (68)
[0336] tert-Butyl
4-(3-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-3-oxopropanoyl)piper-
azine-1-carboxylate (66) (650 mg, 1.31 mmol) was heated in THF/AcOH
(95:5, 2 mL) using a microwave at 140.degree. C. for 2.5 hours. The
residue was concentrated in vacuo to give crude tert-butyl
4-(2-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)acetyl)piperazine-
-1-carboxylate (67). The crude was taken up in EtOAc, and the
solution was flushed with HCl (g) for 5 minutes. The suspension was
stirred at room temperature for 20 minutes and concentrated in
vacuo. The residue was purified by reverse phase HPLC to give
2-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-1-(piperazin-1-yl)e-
thanone hydrochloride (68).
Example 18
Synthesis of
1-((1-(2-hydroxy-2-methylpropyl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imid-
azol-2-yl)methyl)piperazin-2-one (30)
##STR00038##
[0337] Preparation of
1-((1-(2-hydroxy-2-methylpropyl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imid-
azol-2-yl)methyl)piperazin-2-one (72)
[0338]
tert-Butyl-4-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-3-oxop-
iperazine-1-carboxylate (69) (0.50 g, 1.25 mmol) and
N-(tert-butyl)-2-chloroacetamide (70) (0.20 g, 1.3 mmol) were
dissolved in acetone (10 mL). The resultant solution was stirred at
reflux for 48 hours, cooled to room temperature, and poured into
water (60 mL). The aqueous mixture was extracted with ethyl acetate
(3.times.50 mL). The combined organic fractions were dried with
anhydrous sodium sulfate, filtered, then condensed in vacuo to give
the Boc-protected product (71) (0.60 g, 93%), which was onfirmed by
LCMS (positive ion mode). A portion of this material (100 mg, 0.20
mmol) was taken up into ethyl acetate (20 mL). HCl gas was bubbled
through this solution for one minute. The reaction was then stirred
at room temperature for 30 minutes and concentrated in vacuo. The
resulting residue was purified by automated flash chromatography to
afford the desired free amine (72). The product was confirmed by
LCMS (positive ion mode).
Example 19
Synthesis of
1-((4,6-dichloro-1-(2-hydroxy-2-methylpropyl)-1H-benzo[d]imidazol-2-yl)me-
thyl)piperazin-2-one (33)
##STR00039##
[0339] Preparation of
1-((4,6-dichloro-1-(2-hydroxy-2-methylpropyl)-1H-benzo[d]imidazol-2-yl)me-
thyl)piperazin-2-one (75)
[0340]
tert-Butyl-4-((4,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-3-oxop-
iperazine-1-carboxylate (300 mg, 0.75 mmol) (69),
2,2-dimethyloxirane (73) (0.67 mL, 542 mg, 7.5 mmol), and potassium
carbonate (1.04 g, 7.5 mmol) were taken up in acetone (5 mL). The
reaction was heated at 110.degree. C. by microwave irradiation for
1 hour. The solution was concentrated in vacuo, and the residue
containing the Boc-protected product (74) was taken up in ethyl
acetate. HCl gas was bubbled through this solution for 1 minute.
The solution was then stirred for 30 minutes at room temperature
and concentrated in vacuo. The resulting residue was purified by
automated flash chromatography to afford the desired free amine
(75). The product was confirmed by LCMS (positive ion mode).
[0341] Following the general procedures as set forth in exemplary
synthetic procedures above, the following compounds listed in Table
1 were prepared. Mass spectrometry was employed with final
compounds and at various stages throughout the synthesis as a
confirmation of the identity of the product obtained (M+1). For the
mass spectrometric analysis, samples were prepared at an
approximate concentration of 1 .mu.g/mL in acetonitrile with 0.1%
formic acid. Samples were manually infused into an Applies
Biosystems API3000 triple quadrupole mass spectrometer and scanned
in Q1 in the range of 50 to 700 m/z.
TABLE-US-00001 TABLE 1 No. Structure Mol. Wt. Chemical Name 1
##STR00040## 323.237 2-(azetidin-3-ylmethyl)-4,6-
bis(trifluoromethyl)-1H- benzo[d]imidazole 2 ##STR00041## 256.131
2-(azetidin-3-ylmethyl)-5,6- dichloro-1H-benzo[d]imidazole 3
##STR00042## 436.395 2-(2-(azetidin-3-ylmethyl)-4,6-
bis(trifluoromethyl)-1H- benzo[d]imidazol-1-yl)-N-(tert-
butyl)acetamide 4 ##STR00043## 273.254 2-(azetidin-3-yl)-N-(2,2,2-
trifluoro-1-(52yridine-2- yl)ethyl)acetamide 5 ##STR00044## 259.228
2-(azetidin-3-yl)-N-(5- (trifluoromethyl)52yridine-2- yl)acetamide
6 ##STR00045## 369.289 2-(2-(azetidin-3-ylmethyl)-5,6-
dichloro-1H-benzo[d]imidazol- 1-yl)-N-(tert-butyl)acetamide 7
##STR00046## 273.254 2-(azetidin-3-yl)-N-((6-
(trifluoromethyl)52yridine-3- yl)methyl)acetamide 8 ##STR00047##
328.237 1-(2-(azetidin-3-ylmethyl)-5,6-
dichloro-1H-benzo[d]imidazol- 1-yl)-2-methylpropan-2-ol 9
##STR00048## 395.343 1-(2-(azetidin-3-ylmethyl)-4,6-
bis(trifluoromethyl)-1H- benzo[d]imidazol-1-yl)-2-
methylpropan-2-ol 10 ##STR00049## 323.237
2-(azetidin-3-ylmethyl)-5,6- bis(trifluoromethyl)-1H-
benzo[d]imidazole 11 ##STR00050## 365.274
1-(3-((4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)methyl)azetidin-1- yl)ethanone 12 ##STR00051## 380.288
2-amino-1-(3-((4,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)methyl)azetidin-1- yl)ethanone 13 ##STR00052## 394.315
3-amino-1-(3-((4,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)methyl)azetidin-1-yl)propan- 1-one 14 ##STR00053## 422.368
3-amino-1-(3-((4,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)methyl)azetidin-1-yl)-3- methylbutan-1-one 15 ##STR00054##
408.341 4-amino-1-(3-((4,6- bis(trifluoromethyl)-1H-
benzo[d]imidazol-2- yl)methyl)azetidin-1-yl)butan- 1-one 16
##STR00055## 326.238 N-(3,5- bis(trifluoromethyl)benzyl)
azetidine-3-carboxamide 17 ##STR00056## 340.264 (S)-N-(3,5-
bis(trifluoromethyl)benzyl) pyrrolidine-2-carboxamide 18
##STR00057## 340.264 .RTM.-N-(3,5- bis(trifluoromethyl)benzyl)
pyrrolidine-2-carboxamide 19 ##STR00058## 436.395
2-(3-((4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)methyl)azetidin-1-yl)-N- (tert-butyl)acetamide 20 ##STR00059##
448.405 2-(3-((4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)methyl)azetidin-1-yl)-N-(1- methylcyclobutyl)acetamide 21
##STR00060## 216.067 (5,6-dichloro-1H- benzo[d]imidazol-2-
yl)methanamine 22 ##STR00061## 273.119 2-amino-N-((5,6-dichloro-1H-
benzo[d]imidazol-2- yl)methyl)acetamide 23 ##STR00062## 283.173
(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2- yl)methanamine 24
##STR00063## 340.224 2-amino-N-((5,7- bis(trifluoromethyl)-1H-
benzo[d]imidazol-2- yl)methyl)acetamide 25 ##STR00064## 311.226
2-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)propan-
2-amine 26 ##STR00065## 309.21 1-(5,7-bis(trifluoromethyl)-1H-
benzo[d]imidazol-2- yl)cyclopropanamine 27 ##STR00066## 325.253
2-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-2-
methylpropan-1-amine 28 ##STR00067## 325.21
N-((5,7-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)methyl)acetamide 29 ##STR00068## 338.766
6-(3-chloro-4-fluorophenyl)-2- (pyrimidin-5-ylmethyl)-1H-
benzo[d]imidazole 30 ##STR00069## 384.355
2-(pyrimidin-5-ylmethyl)-6-(4- (2,2,2-trifluoroethoxy)phenyl)-
1H-benzo[d]imidazole 31 ##STR00070## 367.333
(S)-2-((4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)methyl)-4-methylpentan-1- amine 32 ##STR00071## 383.289
tert-butyl ((4,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)methyl)carbamate 33 ##STR00072## 297.2
2-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2- yl)ethanamine
34 ##STR00073## 325.253 1-(5,7-bis(trifluoromethyl)-1H-
benzo[d]imidazol-2-yl)-2- methylpropan-2-amine 35 ##STR00074##
311.226 3-(5,7-bis(trifluoromethyl)-1H-
benzo[d]imidazol-2-yl)propan- 1-amine 36 ##STR00075## 368.278
2-amino-N-((5,7- bis(trifluoromethyl)-1H-
benzo[d]imidazol-2-yl)methyl)- 2-methylpropanamide 37 ##STR00076##
382.304 3-amino-N-((5,7- bis(trifluoromethyl)-1H-
benzo[d]imidazol-2-yl)methyl)- 2,2-dimethylpropanamide 38
##STR00077## 283.173 (5,6-bis(trifluoromethyl)-1H-
benzo[d]imidazol-2- yl)methanamine 39 ##STR00078## 340.224
2-amino-N-((5,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)methyl)acetamide 40 ##STR00079## 397.315 tert-butyl ((5,7-
bis(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)methyl)(methyl)carbamate 41 ##STR00080## 297.2
1-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-N-
methylmethanamine 42 ##STR00081## 354.251 2-amino-N-((5,7-
bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)-
N-methylacetamide 43 ##STR00082## 368.278
N-((5,7-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)methyl)-N-methyl-2- (methylamino)acetamide 44 ##STR00083##
382.304 2-amino-N-((5,7- bis(trifluoromethyl)-1H-
benzo[d]imidazol-2-yl)methyl)- N,2-dimethylpropanamide 45
##STR00084## 325.253 N-((5,7-bis(trifluoromethyl)-
1H-benzo[d]imidazol-2- yl)methyl)-N-methylethanamine 46
##STR00085## 339.236 N-((5,7-bis(trifluoromethyl)-
1H-benzo[d]imidazol-2- yl)methyl)-N-methylacetamide 47 ##STR00086##
327.226 (S)-1-(4,6-bis(trifluoromethyl)-
1H-benzo[d]imidazol-2-yl)-2- methoxyethanamine 48 ##STR00087##
410.357 N-((5,7-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)methyl)-2-(tert-butylamino)- N-methylacetamide 49 ##STR00088##
311.226 1-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-N,N-
dimethylmethanamine 50 ##STR00089## 340.224
1-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)-
1-methylurea 51 ##STR00090## 323.237 (S)-2-(pyrrolidin-2-yl)-4,6-
bis(trifluoromethyl)-1H- benzo[d]imidazole 52 ##STR00091## 323.237
(R)-2-(pyrrolidin-2-yl)-4,6- bis(trifluoromethyl)-1H-
benzo[d]imidazole 53 ##STR00092## 309.21 2-(azetidin-3-yl)-4,6-
bis(trifluoromethyl)-1H- benzo[d]imidazole 54 ##STR00093## 323.237
(1r,3r)-3-(4,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)cyclobutanamine 55 ##STR00094## 323.237 (1s,3s)-3-(4,6-
bis(trifluoromethyl)-1H- benzo[d]imidazol-2- yl)cyclobutanamine 56
##STR00095## 376.278 N-{[4,6-bis(trifluoromethyl)-
1H-benzimidazol-2-yl]methyl}- N-methylsulfuric diamide 57
##STR00096## 375.29 N-((5,7-bis(trifluoromethyl)-
1H-benzo[d]imidazol-2- yl)methyl)-N- methylmethanesulfonamide 58
##STR00097## 455.355 (R)-2-amino-N-(2-(((4,6-
bis(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)methyl)(methyl)amino)-2- oxoethyl)-3- methoxypropanamide 59
##STR00098## 346.231 2-(pyrimidin-5-ylmethyl)-5,7-
bis(trifluoromethyl)-1H- benzo[d]imidazole 60 ##STR00099## 367.29
N-((5,7-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)methyl)pivalamide 61 ##STR00100## 381.316
N-((5,7-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)methyl)-3,3- dimethylbutanamide 62 ##STR00101## 382.304
1-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)-
3-(tert-butyl)urea 63 ##STR00102## 298.184 2-(methoxymethyl)-4,6-
bis(trifluoromethyl)-1H- benzo[d]imidazole 64 ##STR00103## 312.211
2-(2-methoxyethyl)-4,6- bis(trifluoromethyl)-1H- benzo[d]imidazole
65 ##STR00104## 336.156 2-(2,2,2-trifluoroethyl)-4,6-
bis(trifluoromethyl)-1H- benzo[d]imidazole 66 ##STR00105## 346.249
2-((methylsulfonyl)methyl)-4,6- bis(trifluoromethyl)-1H-
benzo[d]imidazole 67 ##STR00106## 332.204 2-(pyrazin-2-yl)-4,6-
bis(trifluoromethyl)-1H- benzo[d]imidazole 68 ##STR00107## 346.231
2-(pyrazin-2-ylmethyl)-4,6- bis(trifluoromethyl)-1H-
benzo[d]imidazole 69 ##STR00108## 269.147
4,6-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-amine 70
##STR00109## 284.158 (5,7-bis(trifluoromethyl)-1H-
benzo[d]imidazol-2- yl)methanol 71 ##STR00110## 345.242
2-(pyridin-3-ylmethyl)-5,7- bis(trifluoromethyl)-1H-
benzo[d]imidazole 72 ##STR00111## 345.242
2-(pyridin-4-ylmethyl)-5,7- bis(trifluoromethyl)-1H-
benzo[d]imidazole 73 ##STR00112## 424.384
(S)-3-(aminomethyl)-N-((5,7- bis(trifluoromethyl)-1H-
benzo[d]imidazol-2-yl)methyl)- 5-methylhexanamide 74 ##STR00113##
230.094 2-(5,7-dichloro-1H- benzo[d]imidazol-2- yl)ethanamine 75
##STR00114## 339.236 (3R,5S)-5-(5,7- bis(trifluoromethyl)-1H-
benzo[d]imidazol-2- yl)pyrrolidin-3-ol 76 ##STR00115## 311.226
2-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-N-
methylethanamine 77 ##STR00116## 383.289
(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl
tert-butylcarbamate 78 ##STR00117## 391.289
(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl
dimethylsulfamate 79 ##STR00118## 321.297 (5-(4-(2,2,2-
trifluoroethoxy)phenyl)-1H- benzo[d]imidazol-2- yl)methanamine 80
##STR00119## 275.709 (5-(3-chloro-4-fluorophenyl)-
1H-benzo[d]imidazol-2- yl)methanamine 81 ##STR00120## 361.263
N-((4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)methyl)methanesulfonamide
82 ##STR00121## 351.29 1-((4,6-bis(trifluoromethyl)-1H-
benzo[d]imidazol-2- yl)methyl)cyclopentanamine 83 ##STR00122##
341.275 (R)-4-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)thiazolidine 84 ##STR00123## 341.275
(R)-2-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)thiazolidine 85 ##STR00124## 341.275
(S)-2-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)thiazolidine 86 ##STR00125## 373.274
(R)-4-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)thiazolidine 1,1-dioxide 87 ##STR00126## 373.274
(R)-2-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)thiazolidine 1,1-dioxide 88 ##STR00127## 373.274
(S)-2-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)thiazolidine 1,1-dioxide 89 ##STR00128## 341.227
2-((2S,4R)-4-fluoropyrrolidin- 2-yl)-4,6-bis(trifluoromethyl)-
1H-benzo[d]imidazole 90 ##STR00129## 341.227
2-((2S,4S)-4-fluoropyrrolidin-2- yl)-4,6-bis(trifluoromethyl)-1H-
benzo[d]imidazole 91 ##STR00130## 359.218
(S)-2-(4,4-difluoropyrrolidin-2- yl)-4,6-bis(trifluoromethyl)-1H-
benzo[d]imidazole 92 ##STR00131## 353.263 2-((2R,4R)-4-
methoxypyrrolidin-2-yl)-4,6- bis(trifluoromethyl)-1H-
benzo[d]imidazole 93 ##STR00132## 323.237
(R)-2-(pyrrolidin-3-yl)-4,6- bis(trifluoromethyl)-1H-
benzo[d]imidazole 94 ##STR00133## 339.279
(S)-1-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)-N,2-
dimethylpropan-1-amine 95 ##STR00134## 353.306
(S)-1-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)-N,3-
dimethylbutan-1-amine 96 ##STR00135## 353.306 (1S,2S)-1-(4,6-
bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-N,2-
dimethylbutan-1-amine 97 ##STR00136## 197.185 2-(4,6-difluoro-1H-
benzo[d]imidazol-2- yl)ethanamine 98 ##STR00137## 223.222
(S)-4,6-difluoro-2-(pyrrolidin-2- yl)-1H-benzo[d]imidazole 99
##STR00138## 223.222 (S)-5,6-difluoro-2-(pyrrolidin-2-
yl)-1H-benzo[d]imidazole 100 ##STR00139## 223.222
(S)-4,5-difluoro-2-(pyrrolidin-2- yl)-1H-benzo[d]imidazole 101
##STR00140## 241.212 (S)-4,5,6-trifluoro-2- (pyrrolidin-2-yl)-1H-
benzo[d]imidazole 102 ##STR00141## 320.19 2-(1H-pyrazol-4-yl)-5,7-
bis(trifluoromethyl)-1H- benzo[d]imidazole 103 ##STR00142## 348.25
2-(3,5-dimethyl-1H-pyrazol-4- yl)-5,7-bis(trifluoromethyl)-1H-
benzo[d]imidazole 104 ##STR00143## 320.19 2-(1H-pyrazol-3-yl)-5,7-
bis(trifluoromethyl)-1H- benzo[d]imidazole 105 ##STR00144## 360.26
2-(3-cyclopropyl-1H-pyrazol-5- yl)-5,7-bis(trifluoromethyl)-1H-
benzo[d]imidazole 106 ##STR00145## 388.19
5,7-bis(trifluoromethyl)-2-(3- (trifluoromethyl)-1H-pyrazol-5-
yl)-1H-benzo[d]imidazole 107 ##STR00146## 319.21
2-(1H-pyrrol-3-yl)-5,7- bis(trifluoromethyl)-1H- benzo[d]imidazole
108 ##STR00147## 299.156 1-((5,6-dichloro-1H- benzo[d]imidazol-2-
yl)methyl)piperazin-2-one 109 ##STR00148## 298.264
1-((5-(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)methyl)piperazin-2-one 110 ##STR00149## 366.262
1-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)methyl)piperazin-2-one 111 ##STR00150## 394.315
1-((4,6-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)-
3,3-dimethylpiperazin-2-one 112 ##STR00151## 299.156
1-((4,6-dichloro-1H- benzo[d]imidazol-2- yl)methyl)piperazin-2-one
113 ##STR00152## 302.252 2-(2-oxopiperazin-1-yl)-N-(4-
(trifluoromethyl)66yridine-2- yl)acetamide 114 ##STR00153## 302.252
2-(2-oxopiperazin-1-yl)-N-(5- (trifluoromethyl)66yridine-2-
yl)acetamide 115 ##STR00154## 316.279 N-(2-methyl-6-
(trifluoromethyl)66yridine-3- yl)-2-(2-oxopiperazin-1- yl)acetamide
116 ##STR00155## 316.279 2-(2-oxopiperazin-1-yl)-N-((6-
(trifluoromethyl)67yridine-3- yl)methyl)acetamide 117 ##STR00156##
305.256 N-(1-methyl-3- (trifluoromethyl)-1H-pyrazol-5-
yl)-2-(2-oxopiperazin-1- yl)acetamide 118 ##STR00157## 287.281
2-(piperidin-4-yl)-N-(5- (trifluoromethyl)67yridine-2- yl)acetamide
119 ##STR00158## 299.156 4-((5,6-dichloro-1H- benzo[d]imidazol-2-
yl)methyl)piperazin-2-one 120 ##STR00159## 300.141
4-((5,6-dichloro-1H- benzo[d]imidazol-2- yl)methyl)morpholin-3-one
121 ##STR00160## 313.182 1-(2-(5,6-dichloro-1H- benzo[d]imidazol-2-
yl)ethyl)piperazin-2-one 122 ##STR00161## 397.342
N-(tert-butyl)-2-(5,6-dichloro-2- (piperidin-4-ylmethyl)-1H-
benzo[d]imidazol-1- yl)acetamide 123 ##STR00162## 356.29
1-(5,6-dichloro-2-(piperidin-4- ylmethyl)-1H-
benzo[d]imidazol-1-yl)-2- methylpropan-2-ol 124 ##STR00163##
412.314 N-(tert-butyl)-2-(5,6-dichloro-2-
((2-oxopiperazin-1-yl)methyl)- 1H-benzo[d]imidazol-1- yl)acetamide
125 ##STR00164## 479.419 N-(tert-butyl)-2-(2-((2-
oxopiperazin-1-yl)methyl)-4,6- bis(trifluoromethyl)-1H-
benzo[d]imidazol-1- yl)acetamide 126 ##STR00165## 380.288
1-(2-(5,7-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-
yl)ethyl)piperazin-2-one 127 ##STR00166## 367.246
4-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)methyl)morpholin-3-one 128 ##STR00167## 367.246
4-((5,6-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)methyl)morpholin-3-one 129 ##STR00168## 438.367
1-((1-(2-hydroxy-2- methylpropyl)-4,6- bis(trifluoromethyl)-1H-
benzo[d]imidazol-2- yl)methyl)piperazin-2-one 130 ##STR00169##
344.332 2-(3,3-dimethyl-2-oxopiperazin- 1-yl)-N-(2,2,2-trifluoro-1-
(69yridine-2-yl)ethyl)acetamide 131 ##STR00170## 316.279
2-(2-oxopiperazin-1-yl)-N- (2,2,2-trifluoro-1-(69yridine-2-
yl)ethyl)acetamide 132 ##STR00171## 301.307
2-(piperidin-4-yl)-N-(2,2,2- trifluoro-1-(69yridine-2-
yl)ethyl)acetamide 133 ##STR00172## 330.306
2-(3,3-dimethyl-2-oxopiperazin- 1-yl)-N-(5-
(trifluoromethyl)69yridine-2- yl)acetamide 134 ##STR00173## 440.367
N-(tert-butyl)-2-(5,6-dichloro-2- ((3,3-dimethyl-2-oxopiperazin-
1-yl)methyl)-1H- benzo[d]imidazol-1- yl)acetamide 135 ##STR00174##
351.29 2-(piperidin-4-ylmethyl)-4,6- bis(trifluoromethyl)-1H-
benzo[d]imidazole 136 ##STR00175## 423.396
2-methyl-1-(2-(piperidin-4- ylmethyl)-4,6- bis(trifluoromethyl)-1H-
benzo[d]imidazol-1-yl)propan- 2-ol 137 ##STR00176## 464.448
N-(tert-butyl)-2-(2-(piperidin-4- ylmethyl)-4,6-
bis(trifluoromethyl)-1H- benzo[d]imidazol-1- yl)acetamide 138
##STR00177## 507.472 N-(tert-butyl)-2-(2-((3,3-
dimethyl-2-oxopiperazin-1- yl)methyl)-4,6- bis(trifluoromethyl)-1H-
benzo[d]imidazol-1- yl)acetamide 139 ##STR00178## 466.421
1-((1-(2-hydroxy-2- methylpropyl)-4,6- bis(trifluoromethyl)-1H-
benzo[d]imidazol-2-yl)methyl)- 3,3-dimethylpiperazin-2-one 140
##STR00179## 301.307 2-(piperidin-4-yl)-N-((6-
(trifluoromethyl)70yridine-3- yl)methyl)acetamide 141 ##STR00180##
344.332 2-(3,3-dimethyl-2-oxopiperazin- 1-yl)-N-((6-
(trifluoromethyl)71 yridine-3- yl)methyl)acetamide 142 ##STR00181##
351.29 2-(piperidin-4-ylmethyl)-5,6- bis(trifluoromethyl)-1H-
benzo[d]imidazole 143 ##STR00182## 399.315
1-((5,6-dichloro-1-(2-hydroxy- 2-methylpropyl)-1H-
benzo[d]imidazol-2-yl)methyl)- 3,3-dimethylpiperazin-2-one 144
##STR00183## 371.262 1-((5,6-dichloro-1-(2-hydroxy-
2-methylpropyl)-1H- benzo[d]imidazol-2- yl)methyl)piperazin-2-one
145 ##STR00184## 371.262 1-((4,6-dichloro-1-(2-hydroxy-
2-methylpropyl)-1H- benzo[d]imidazol-2- yl)methyl)piperazin-2-one
146 ##STR00185## 438.367 1-((1-(2-hydroxy-2- methylpropyl)-5,6-
bis(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)methyl)piperazin-2-one 147 ##STR00186## 412.314
N-(tert-butyl)-2-(4,6-dichloro-2- ((2-oxopiperazin-1-yl)methyl)-
1H-benzo[d]imidazol-1- yl)acetamide 148 ##STR00187## 479.419
N-(tert-butyl)-2-(2-((2- oxopiperazin-1-yl)methyl)-5,6-
bis(trifluoromethyl)-1H- benzo[d]imidazol-1- yl)acetamide 149
##STR00188## 366.262 1-((5,6-bis(trifluoromethyl)-1H-
benzo[d]imidazol-2- yl)methyl)piperazin-2-one 150 ##STR00189##
394.315 1-((5,6-bis(trifluoromethyl)-1H-
benzo[d]imidazol-2-yl)methyl)- 3,3-dimethylpiperazin-2-one 151
##STR00190## 466.421 1-((1-(2-hydroxy-2- methylpropyl)-5,6-
bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)-
3,3-dimethylpiperazin-2-one 152 ##STR00191## 507.472
N-(tert-butyl)-2-(2-((3,3- dimethyl-2-oxopiperazin-1-
yl)methyl)-5,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-1-
yl)acetamide 153 ##STR00192## 366.262
4-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)methyl)piperazin-2-one 154 ##STR00193## 327.209
1-((5,6-dichloro-1H- benzo[d]imidazol-2-yl)methyl)-
3,3-dimethylpiperazin-2-one 155 ##STR00194## 380.288
2-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-1-
(piperazin-1-yl)ethanone 156 ##STR00195## 381.273
2-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-1-
morpholinoethanone 157 ##STR00196## 379.3
2-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-1-
(piperidin-1-yl)ethanone 158 ##STR00197## 394.315
2-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-1-(4-
methylpiperazin-1-yl)ethanone 159 ##STR00198## 369.262 N-(3,5-
bis(trifluoromethyl)benzyl)-2- oxopiperazine-1-carboxamide 160
##STR00199## 302.228 1-((4,5,6,7-tetrafluoro-1H-
benzo[d]imidazol-2- yl)methyl)piperazin-2-one 161 ##STR00200##
284.237 1-((5,6,7-trifluoro-1H- benzo[d]imidazol-2-
yl)methyl)piperazin-2-one 162 ##STR00201## 284.237
1-((4,5,7-trifluoro-1H- benzo[d]imidazol-2-
yl)methyl)piperazin-2-one
163 ##STR00202## 266.247 1-((5,7-difluoro-1H- benzo[d]imidazol-2-
yl)methyl)piperazin-2-one 164 ##STR00203## 266.247
1-((4,7-difluoro-1H- benzo[d]imidazol-2- yl)methyl)piperazin-2-one
165 ##STR00204## 266.247 1-((6,7-difluoro-1H- benzo[d]imidazol-2-
yl)methyl)piperazin-2-one 166 ##STR00205## 266.247
1-((5,6-difluoro-1H- benzo[d]imidazol-2- yl)methyl)piperazin-2-one
167 ##STR00206## 352.235 3-(5,7-bis(trifluoromethyl)-1H-
benzo[d]imidazol-2- yl)piperazin-2-one 168 ##STR00207## 300.2
2-amino-N-(3,5- bis(trifluoromethyl)benzyl) acetamide 169
##STR00208## 354.291 N-(3,5- bis(trifluoromethyl)benzyl)
piperidine-4-carboxamide 170 ##STR00209## 355.279 N-(3,5-
bis(trifluoromethyl)benzyl) piperazine-1-carboxamide 171
##STR00210## 365.317 1-((5,7-bis(trifluoromethyl)-1H-
benzo[d]imidazol-2- yl)methyl)cyclohexanamine 172 ##STR00211##
408.341 (1S,2R)-2-amino-N-((5,7- bis(trifluoromethyl)-1H-
benzo[d]imidazol-2- yl)methyl) cyclohexanecarboxamide 173
##STR00212## 408.341 (1R,2S)-2-amino-N-((5,7-
bis(trifluoromethyl)-1H- benzo[d]imidazol-2- yl)methyl)
cyclohexanecarboxamide 174 ##STR00213## 408.341
(1R,2R)-2-amino-N-((5,7- bis(trifluoromethyl)-1H-
benzo[d]imidazol-2- yl)methyl) cyclohexanecarboxamide 175
##STR00214## 408.341 (1S,2S)-2-amino-N-((5,7-
bis(trifluoromethyl)-1H- benzo[d]imidazol-2- yl)methyl)
cyclohexanecarboxamide 176 ##STR00215## 298.211
1-((5,7-dichloro-1H- benzo[d]imidazol-2- yl)methyl)cyclohexanamine
177 ##STR00216## 365.317 1-(5,7-bis(trifluoromethyl)-1H-
benzo[d]imidazol-2-yl)-N- methylcyclohexanamine 178 ##STR00217##
351.29 1-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)cyclohexanamine 179 ##STR00218## 367.29
4-(5,7-bis(trifiuoromethyl)-1H- benzo[d]imidazol-2-yl)-N-
methyltetrahydro-2H-pyran-4- amine 180 ##STR00219## 379.343
1-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)-
N-methylcyclohexanamine 181 ##STR00220## 367.29
4-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)methyl)tetrahydro-2H-pyran- 4-amine 182 ##STR00221## 381.316
4-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)-
N-methyltetrahydro-2H-pyran- 4-amine 183 ##STR00222## 337.264
(R)-2-(piperidin-3-yl)-4,6- bis(trifluoromethyl)-1H-
benzo[d]imidazole 184 ##STR00223## 337.264
(S)-2-(piperidin-3-yl)-4,6- bis(trifluoromethyl)-1H-
benzo[d]imidazole 185 ##STR00224## 339.236
2-(4,6-bis(trifluoromethyl)-1H- benzo[d]imidazol-2- yl)morpholine
186 ##STR00225## 351.29 (1R,2R)-2-(5,7- bis(trifluoromethyl)-1H-
benzo[d]imidazol-2- yl)cyclohexanamine 187 ##STR00226## 351.29
(1S,2R)-2-(5,7- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-
yl)cyclohexanamine 188 ##STR00227## 351.29 (1S,2S)-2-(5,7-
bis(trifluoromethyl)-1H- benzo[d]imidazol-2- yl)cyclohexanamine 189
##STR00228## 340.264 (S)-N-(3,5- bis(trifluoromethyl)benzyl)
pyrrolidine-2-carboxamide 190 ##STR00229## 340.264 (R)-N-(3,5-
bis(trifluoromethyl)benzyl) pyrrolidine-2-carboxamide 191
##STR00230## 290.257 (S)-N-(4-fluoro-3- (trifluoromethyl)benzyl)
pyrrolidine-2-carboxamide 192 ##STR00231## 256.704
(S)-N-(3-chloro-4- fluorobenzyl)pyrrolidine-2- carboxamide 193
##STR00232## 333.281 N-(4-fluoro-3- (trifluoromethyl)benzyl)-2-(2-
oxopiperazin-1-yl)acetamide 194 ##STR00233## 340.264
(R)-N-(pyrrolidin-2-ylmethyl)- 3,5- bis(trifluoromethyl)benzamide
195 ##STR00234## 340.264 (S)-N-(pyrrolidin-2-ylmethyl)- 3,5-
bis(trifluoromethyl)benzamide 196 ##STR00235## 356.307
(S)-2-amino-N-(3,5- bis(trifluoromethyl)benzyl)-4-
methylpentanamide 197 ##STR00236## 370.333 (S)-N-(3,5-
bis(trifluoromethyl)benzyl)-4- methyl-2- (methylamino)pentanamide
198 ##STR00237## 383.289 N-(3,5- bis(trifluoromethyl)benzyl)-2-
(2-oxopiperazin-1-yl)acetamide 199 ##STR00238## 382.344
2-(1-aminocyclohexyl)-N-(3,5- bis(trifluoromethyl)benzyl) acetamide
200 ##STR00239## 416.36 (2R,5S)-N-(3,5-
bis(trifluoromethyl)benzyl)-5- phenylpyrrolidine-2- carboxamide 201
##STR00240## 314.227 3-amino-N-(3,5- bis(trifluoromethyl)benzyl)
propanamide 202 ##STR00241## 328.253 N-(3,5-
bis(trifluoromethyl)benzyl)-3- (methylamino)propanamide 203
##STR00242## 416.36 (2S,4R)-N-(3,5- bis(trifluoromethyl)benzyl)-4-
phenylpyrrolidine-2- carboxamide 204 ##STR00243## 416.36
(2S,4S)-N-(3,5- bis(trifluoromethyl)benzyl)-4- phenylpyrrolidine-2-
carboxamide 205 ##STR00244## 358.255 (2S,4S)-N-(3,5-
bis(trifluoromethyl)benzyl)-4- fluoropyrrolidine-2- carboxamide 206
##STR00245## 376.245 (S)-N-(3,5- bis(trifluoromethyl)benzyl)-4,4-
difluoropyrrolidine-2- carboxamide 207 ##STR00246## 368.317
(R)-N-(2-(3,5- bis(trifluoromethyl)phenyl)
propan-2-yl)pyrrolidine-2- carboxamide 208 ##STR00247## 368.317
(S)-N-(2-(3,5- bis(trifluoromethyl)phenyl)
propan-2-yl)pyrrolidine-2- carboxamide 209 ##STR00248## 447.396
(S)-N-(2-(3,5- bis(trifluoromethyl)phenyl) propan-2-yl)-1-
sulfamoylpyrrolidine-2- carboxamide 210 ##STR00249## 354.291
(R)-N-(3,5- bis(trifluoromethyl)benzyl)-1- methylpyrrolidine-2-
carboxamide 211 ##STR00250## 354.291 (S)-N-(3,5-
bis(trifluoromethyl)benzyl)-1- methylpyrrolidine-2- carboxamide 212
##STR00251## 382.344 (S)-N-(2-(3,5- bis(trifluoromethyl)phenyl)
propan-2-yl)piperidine-2- carboxamide 213 ##STR00252## 384.317
(R)-N-(2-(3,5- bis(trifluoromethyl)phenyl)
propan-2-yl)morpholine-3- carboxamide 214 ##STR00253## 384.317
(S)-N-(2-(3,5- bis(trifluoromethyl)phenyl)
propan-2-yl)morpholine-3- carboxamide 215 ##STR00254## 433.272
N-(2-(3,5- bis(trifluoromethyl)phenyl)
propan-2-yl)-3-(trifluoromethyl)- 1H-pyrazole-5-carboxamide 216
##STR00255## 396.37 (1S,2R)-2-amino-N-(2-(3,5-
bis(trifluoromethyl)phenyl) propan-2- yl)cyclohexanecarboxamide 217
##STR00256## 396.37 (1S,2S)-2-amino-N-(2-(3,5-
bis(trifluoromethyl)phenyl) propan-2- yl)cyclohexanecarboxamide 218
##STR00257## 396.37 (1S,2S)-2-amino-N-(2-(3,5-
bis(trifluoromethyl)phenyl) propan-2- yl)cyclohexanecarboxamide 219
##STR00258## 396.37 (S)-N-(1-(3,5- bis(trifluoromethyl)phenyl)
cyclopropyl)-4-methyl-2- (methylamino)pentanamide 220 ##STR00259##
366.301 (S)-N-(1-(3,5- bis(trifluoromethyl)phenyl)
cyclopropyl)pyrrolidine-2- carboxamide 221 ##STR00260## 366.301
(R)-N-(1-(3,5- bis(trifluoromethyl)phenyl)
cyclopropyl)pyrrolidine-2- carboxamide 222 ##STR00261## 384.292
(2R,4R)-N-(1-(3,5- bis(trifluoromethyl)phenyl)
cyclopropyl)-4-fluoropyrrolidine-2- carboxamide 223 ##STR00262##
384.292 (2R,4S)-N-(1-(3,5- bis(trifluoromethyl)phenyl)
cyclopropyl)-4-fluoropyrrolidine-2- carboxamide 224 ##STR00263##
300.2 2-amino-3-(3,5- bis(trifluoromethyl)phenyl) propanamide 225
##STR00264## 431.256 N-(1-(3,5- bis(trifluoromethyl)phenyl)
cyclopropyl)-3-(trifluoromethyl)- 1H-pyrazole-5-carboxamide 226
##STR00265## 370.333 (R)-2-amino-N-(2-(3,5-
bis(trifluoromethyl)phenyl) propan-2-yl)-3-methylbutanamide 227
##STR00266## 384.36 (R)-2-amino-N-(2-(3,5-
bis(trifluoromethyl)phenyl) propan-2-yl)-4-methylpentanamide 228
##STR00267## 386.333 (2R,3S)-2-amino-N-(2-(3,5-
bis(trifluoromethyl)phenyl)propan- 2-yl)-3-methoxybutanamide 229
##STR00268## 398.386 (R)-N-(2-(3,5- bis(trifluoromethyl)phenyl)
propan-2-yl)-4-methyl-2- (methylamino)pentanamide 230 ##STR00269##
426.44 (R)-N-(2-(3,5- bis(trifluoromethyl)phenyl)
propan-2-yl)-2-(isopropylamino)-4- methylpentanamide 231
##STR00270## 427.341 (R)-2-amino-N-(2-((1-(3,5-
bis(trifluoromethyl)phenyl) cyclopropyl)amino)-2-oxoethyl)-3-
methoxypropanamide 232 ##STR00271## 429.357
(R)-2-amino-N-(2-((2-(3,5- bis(trifluoromethyl)phenyl)
propan-2-yl)amino)-2-oxoethyl)-3- methoxypropanamide 233
##STR00272## 370.333 (R)-N-(3,5- bis(trifluoromethyl)benzyl)-4-
methyl-2- (methylamino)pentanamide 234 ##STR00273## 396.37
(R)-N-(1-(3,5- bis(trifluoromethyl)phenyl) cyclopropyl)-4-methyl-2-
(methylamino)pentanamide 235 ##STR00274## 356.307 (R)-N-(3,5-
bis(trifluoromethyl)benzyl)-3- methyl-2- (methylamino)butanamide
236 ##STR00275## 382.348 (R)-N-(2-(3,5- bis(trifluoromethyl)phenyl)
propan-2-yl)piperidine-2- carboxamide
Example 20
Ion Channel Studies
[0342] The generation of a HEK 293F cell line stably expressing
human Na.sub.v1.7 was achieved by co-transfecting human SCN9A and
human SCN1B cDNAs, subcloned into plasmid vectors and utilizing
standard transfection techniques. Clones were selected using
appropriate selection agents (0.3 mg/mL Zeocin and 0.8 mg/mL
Geneticin) and maintained in Dulbecco's Modified Eagle medium, 10%
fetal bovine serum, 1% non essential amino acids to .about.80%
confluence at 37.degree. C. in a humidified incubator with 95%
atmosphere and 5% CO.sub.2.
[0343] On the day of each experiment, cells that were grown to 80%
confluence in a T75 flask were harvested for use on PatchXpress
(Molecular Devices, CA, USA). Following a recovery period at
37.degree. C. in a humidified incubator with 95% atmosphere and 5%
CO.sub.2 in Dulbecco's Modified Eagle Medium, the media was
replaced with an external recording solution containing (in mM): 90
TEACl, 50 NaCl, 1.8 CaCl.sub.2, 1 MgCl.sub.2, 10 HEPES, 10 glucose,
adjusted to pH 7.4 with TEAOH and 300 mOsm with sucrose. The
internal recording solution contained (in mM): 129 CsF, 2
MgCl.sub.2, 11 EGTA, 10 HEPES, 6 NaCl, 3 Na.sub.2ATP adjusted to pH
7.2 with CsOH and 280 mOsm with sucrose. The automated liquid
handling facility of PatchXpress dispensed cells and added
compound.
[0344] Modulation of Na.sub.v1.7 channels by compounds was assessed
by promoting the channels into the inactivated state using a
conditioning voltage pulse of variable amplitude, followed by a
brief hyperpolarizing pulse with a subsequent depolarized voltage
step to measure the current amplitude in the presence and absence
of compound. Exemplary data are provided in FIG. 1.
Example 21
Assays
Modulation of Ion Channel Activity
[0345] The compounds described herein can also be assayed for
modulation of other voltage gated channels (e.g., other Na.sup.+
channel isoforms or Ca.sup.2+ channels such as Ca.sub.v3.2 T-type
channels). Additional methods are known in the art. Exemplary data
obtained according to these methods are also shown in FIG. 1.
[0346] Na.sub.v1.5 Assay
[0347] Inhibition of the TTX-resistant Na.sub.v1.5 sodium channel,
a key cardiac ion channel, can have profound effects on the
duration and amplitude of the cardiac action potential and can
result in arrhythmias and other heart malfunctions. To assess the
potential cardiac liability of compounds at an early stage in the
drug discovery process, a Na.sub.v1.5 sodium channel screening
assay was be performed on Molecular Device's PatchXpresS.TM.
automated electrophysiology platform. Under voltage-clamp
conditions, Na.sub.v1.5 currents were recorded from HEK cells
expressing the human Nav1.5 channel in the absence and presence of
increasing concentrations of the test compound to obtain an
IC.sub.50 value. The external recording solution contained (in mM):
90 TEACl, 50 NaCl, 1.8 CaCl, 1 MgCl.sub.2, 10 HEPES, 10 glucose,
adjusted to pH 7.4 with TEA-OH and to 300 mOsm with sucrose (if
necessary), while the internal patch pipette solution contained (in
mM): 129 CsF, 2 MgCl.sub.2, 11 EGTA, 10 HEPES, 3 Na.sub.2ATP
adjusted to pH 7.2 with CsOH and to 290 mOsm with sucrose (if
necessary). Na.sub.v1.5 channel currents were evoked using a
cardiac action potential waveform at 1 Hz, digitized at 31.25 kHz
and low-pass filtered at 12 kHz.
[0348] Voltage-Gated Ca.sup.2+ Channels
[0349] The compounds described herein can also be studied as
modulators of voltage-gated Ca.sup.2+ channels (e.g., Ca.sub.v1.2,
Ca.sub.v2.2, Ca.sub.v3.1, or Ca.sub.v3.2 channels). Exemplary
methods are described herein.
[0350] A. Patch Clamp Methods
[0351] To record currents from Ca.sub.v3.2 T-type Ca.sup.2+
channels expressed in HEK cells, the culture media can be replaced
with extracellular solution (ECS) containing (in mM): 142 CsCl, 10
D-glucose, 2 CaCl.sub.2, 1 MgCl.sub.2, 10 HEPES, pH adjusted to 7.4
with CsOH. Borosilicate glass patch pipettes, pulled on a P-97
micropipette puller (Sutter Instruments, Novato, Calif.) with
typical resistances of 2-4 MW, can be backfilled with intracellular
solution containing (in mM): 126.5 Cs-methanesulphonate, 2
MgCl.sub.2, 10 HEPES, 11 EGTA, 2 Na-ATP, pH adjusted to 7.3 CsOH.
Voltages were recorded in the whole-cell configuration at room
temperature (.about.21.degree. C.) using an Axopatch 200B
(Molecular Devices, Sunnyvale, Calif.) patch-clamp amplifier.
Recordings can be low-pass filtered at 1 kHz (-3 dB 4-pole Bessel
filter), digitized at 2 kHz with a Digidata 1322A interface
(Molecular Devices), and acquired using pClamp 9.2 (Molecular
Devices), with no leak subtraction being used. Test compounds,
prepared as 30 mM stock solutions in DMSO and diluted in
extracellular buffer, can be applied through a gravity driven
multi-barrelled array of capillaries (24 gauge) connected to
reservoirs controlled by solenoid valves. The effects of compounds
on Ca.sub.v3.2 slow and fast inactivation can then be evaluated
using different voltage protocols. The voltage dependence of fast
and slow channel inactivation can be examined using a two pulse
protocol. Data were analyzed and fitted using OriginPro v.7.5
(OriginLab, Northampton, Mass.) software.
[0352] B. High-throughput Ca.sub.v2.2/K.sub.ir2.3 T-Type
Fluorescent Assay
[0353] Cells were plated in 384-well, clear-bottom, black-walled,
poly-D-lysine coated plates (Becton Dickinson, Franklin Lake, N.J.)
2 days prior to use in the FLIPR assay. 100 .mu.L of cells
(1.4.times.10.sup.6 cell/mL) containing doxycyline (Sigma-Aldrich,
1.5 .mu.g/mL; to induce channel expression) were added to each well
using a Multidrop (Thermo Scientific, Waltham, Mass.) and were
maintained in 5% CO.sub.2 incubator at 37.degree. C. On the morning
of the assay, cells were transferred to a 5% CO.sub.2 incubator at
29.degree. C.
[0354] Cells can then be washed with a wash buffer containing (in
mM): 118 NaCl, 18.4 HEPES, 11.7 D-glucose, 2 CaCl.sub.2, 0.5
MgSO.sub.4, 4.7 KCl, 1.2 KH.sub.2PO.sub.4, pH adjusted to 7.2 with
NaOH. 4.4 .mu.M of the fluorescent indicator dye, Fluo-4
(Invitrogen), prepared in pluronic acid (Sigma-Aldrich), were
loaded into the wells and incubated for 45 minutes at 29.degree. C.
in 5% CO.sub.2. Cells were then rinsed with either a 2 mM KCl
closed-state buffer (in mM: 138.5 NaCl, 10 HEPES, 10 D-glucose, 1
CaCl.sub.2, and 2 KCl, with the pH adjusted to 7.4 with NaOH) when
performing the closed-state assay or 12.5 mM KCl inactivated-state
buffer (in mM: 128 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl.sub.2, and
12.5 mM KCl, with the pH adjusted to 7.4 with NaOH) when performing
the inactivated-state assay.
[0355] Concentration-dependent response curves were generated from
5 mM stock solutions prepared in DMSO (Sigma-Aldrich) and diluted
in either the 2 mM KCl buffer or 12.5 mM KCl buffer and incubated
for 20 minutes at 29.degree. C. in 5% CO.sub.2. Calcium entry was
evoked with an addition of 130 mM KCl stimulation buffer (in mM:
10.5 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl.sub.2, and 130 KCl, with
the pH adjusted to 7.4 with NaOH) for both the closed-state or
inactivated-state assay. A change in the Fluo-4 fluorescence signal
was assessed using FLIPR.sup.TETRA.TM. instrument (Molecular
Devices, Sunnyvale, Calif.) for 3 minutes following the elevation
of extracellular KCl using an illumination wavelength of 470-495 nm
with emissions recorded at 515-575 nm.
[0356] Concentration-dependent response curves were obtained by
comparing the fluorescence signal in the presence of compound and
fitted with a logistic function (1) to obtain the concentration
that inhibited 50% (IC.sub.50) of the RLU signal using OriginPro
v.7.5 software (OriginLab. Northampton, Mass.).
y = [ max - min 1 + ( [ drug ] IC 50 ) n H ] + min ( 1 )
##EQU00001##
[0357] To assess the quality of the FLIPR assays the Z-factor (2)
were used to quantify the suitability of the assay conditions using
the following equation:
Z = 1 - 3 SD sample + 3 SD control mean sample - mean control ( 2 )
##EQU00002##
[0358] Data were expressed as mean and standard deviation (SD).
[0359] C. High-throughput Ca.sub.v3.1 T-Type Fluorescent Assay
[0360] Cells were plated in 384-well, clear-bottom, black-walled,
poly-D-lysine coated plates (Becton Dickinson, Franklin Lake, N.J.)
2 days prior to use in the FLIPR assay. 100 .mu.L of cells
(2.0.times.10.sup.6 cell/mL) containing doxycyline (Sigma-Aldrich,
1.5 .mu.g/mL; to induce channel expression) were added to each well
using a Multidrop (Thermo Scientific, Waltham, Mass.) and were
maintained in 5% CO.sub.2 incubator at 37.degree. C. On the morning
of the assay, cells were transferred to a 5% CO.sub.2 incubator at
29.degree. C.
[0361] Cells were washed with a wash buffer containing (in mM): 118
NaCl, 18.4 HEPES, 11.7 D-glucose, 0.05 CaCl.sub.2, 0.5 MgSO.sub.4,
1 KCl, and 1.2 KH.sub.2PO.sub.4, with the pH adjusted to 7.2 with
NaOH. 4.4 .mu.M of the fluorescent indicator dye, Fluo-4
(Invitrogen), prepared in pluronic acid (Sigma-Aldrich), were
loaded into the wells and incubated for 45 minutes at 29.degree. C.
in 5% CO.sub.2. Cells were then rinsed with the following low
Ca.sup.2+ buffer (in mM): 0.34 Na.sub.2HPO.sub.4, 4.2 NaHCO.sub.3,
0.44 KH.sub.2PO.sub.4, 0.41 MgSO.sub.4, 0.49 MgCl.sub.2-6H.sub.2O,
20 HEPES, 5.5 D-Glucose, 137 NaCl, 5.3 KCl, and 0.001 CaCl.sub.2,
with 0.1% BSA and the pH adjusted to 7.2 with NaOH.
Concentration-dependent response curves were generated from 5 mM
stock solutions prepared in DMSO (Sigma-Aldrich) and diluted in the
buffer containing low Ca.sup.2+ and incubated for 20 minutes at
29.degree. C. in 5% CO.sub.2 Calcium entry was evoked with an
addition of (in mM): 0.34 Na.sub.2HPO.sub.4, 4.2 NaHCO.sub.3, 0.44
KH.sub.2PO.sub.4, 0.41 MgSO.sub.4, 0.49 MgCl.sub.2-6H.sub.2O, 20
HEPES, 5.5 D-Glucose, 137 NaCl, 5.3 KCl, and 6 CaCl.sub.2, with
0.1% BSA and the pH adjusted to 7.2 with NaOH. A change in the
Fluo-4 fluorescence signal was assessed using FLIPR.sup.TETRAT.TM.
instrument (Molecular Devices, Sunnyvale, Calif.) for 3 minutes
following the elevation of extracellular KCl using an illumination
wavelength of 470-495 nm with emissions recorded at 515-575 nm.
[0362] Concentration-dependent response curves were obtained by
comparing the fluorescence signal in the presence of compound and
fitted with a logistic function (1) to obtain the concentration
that inhibited 50% (IC.sub.50) of the RLU signal using OriginPro
v.7.5 software (OriginLab, Northampton, Mass.).
y = [ max - min 1 + ( [ drug ] IC 50 ) n H ] + min ( 1 )
##EQU00003##
[0363] To assess the quality of the FLIPR assays, the Z-factor (2)
was used to quantify the suitability of the assay conditions using
the following equation:
Z = 1 - 3 SD sample + 3 SD control mean sample - mean control ( 2 )
##EQU00004##
[0364] Data were expressed as mean and standard deviation (SD).
[0365] D. High-throughput Ca.sub.v3.2/K.sub.ir2.3 T-Type
Fluorescent Assay
[0366] Cells were plated in 384-well, clear-bottom, black-walled,
poly-D-lysine coated plates (Becton Dickinson, Franklin Lake, N.J.)
2 days prior to use in the FLIPR assay. 100 .mu.L of cells
(1.2.times.10.sup.6 cell/mL) containing doxycyline (Sigma-Aldrich,
1.5 .mu.g/mL; to induce channel expression) were added to each well
using a Multidrop (Thermo Scientific, Waltham, Mass.) and were
maintained in 5% CO.sub.2 incubator at 37.degree. C. On the morning
of the assay, cells were transferred to a 5% CO.sub.2 incubator at
29.degree. C.
[0367] Cells were washed with a wash buffer containing (in mM): 118
NaCl, 18.4 HEPES, 11.7 D-glucose, 2 CaCl.sub.2, 0.5 MgSO.sub.4, 4.7
KCl, and 1.2 KH.sub.2PO.sub.4, with the pH adjusted to 7.2 with
NaOH. 4.4 .mu.M of the fluorescent indicator dye Fluo-4
(Invitrogen) prepared in pluronic acid (Sigma-Aldrich) were loaded
into the wells and incubated for 45 minutes at 29.degree. C. in 5%
CO.sub.2. Cells were then rinsed with either a 2 mM KCl
closed-state buffer (in mM: 138.5 NaCl, 10 HEPES, 10 D-glucose, 1
CaCl.sub.2, and 2 KCl, with the pH adjusted to 7.4 with NaOH) when
performing the closed-state assay or 7.6 mM KCl inactivated-state
buffer (in mM: 130.9 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl.sub.2,
and 7.6 mM KCl, with the pH adjusted to 7.4 with NaOH) when
performing the inactivated-state assay. Concentration-dependent
response curves were generated from 5 mM stock solutions prepared
in DMSO (Sigma-Aldrich), diluted in either the 2 mM KCl buffer or
7.6 mM KCl buffer, and incubated for 20 minutes at 29.degree. C. in
5% CO.sub.2. Calcium entry was evoked with an addition of either 12
mM KCl stimulation buffer (in mM: 128.5 NaCl, 10 HEPES, 10
D-glucose, 1 CaCl.sub.2, and 12 KCl, with the pH adjusted to 7.4
with NaOH) or 14.5 mM KCl stimulation buffer (in mM: 126 NaCl, 10
HEPES, 10 D-glucose, 1 CaCl.sub.2, and 14.5 KCl, with the pH
adjusted to 7.4 with NaOH) for the closed-state or
inactivated-state assay respectively. A change in the Fluo-4
fluorescence signal was assessed using FLIPR instrument (Molecular
Devices, Sunnyvale, Calif.) for 3 minutes following the elevation
of extracellular KCl using an illumination wavelength of 470-495 nm
with emissions recorded at 515-575 nm.
[0368] Concentration-dependent response curves were obtained by
comparing the fluorescence signal in the presence of compound and
fitted with a logistic function (1) to obtain the concentration
that inhibited 50% (IC.sub.50) of the RLU signal using OriginPro
v.7.5 software (OriginLab, Northampton, Mass.).
y = [ max - min 1 + ( [ drug ] IC 50 ) n H ] + min ( 1 )
##EQU00005##
[0369] To assess the quality of the FLIPR assays, the Z-factor (2)
was used to quantify the suitability of the assay conditions using
the following equation:
Z = 1 - 3 SD sample + 3 SD control mean sample - mean control ( 2 )
##EQU00006##
[0370] Data were expressed as mean and standard deviation (SD).
[0371] E. High-throughput Ca.sub.v1.2/K.sub.ir2.3 T-Type
Fluorescent Assay
[0372] Cells were plated in 384-well, clear-bottom, black-walled,
poly-D-lysine coated plates (Becton Dickinson, Franklin Lake, N.J.)
2 days prior to use in the FLIPR assay. 100 .mu.L of cells
(1.2.times.10.sup.6 cell/mL) containing doxycyline (Sigma-Aldrich,
1.5 .mu.g/mL; to induce channel expression) were added to each well
using a Multidrop (Thermo Scientific, Waltham, Mass.) and were
maintained in 5% CO.sub.2 incubator at 37.degree. C. On the morning
of the assay, cells were transferred to a 5% CO.sub.2 incubator at
29.degree. C.
[0373] Cells were washed with a wash buffer containing (in mM): 118
NaCl, 18.4 HEPES, 11.7 D-glucose, 2 CaCl.sub.2, 0.5 MgSO.sub.4, 4.7
KCl, and 1.2 KH.sub.2PO.sub.4, with the pH adjusted to 7.2 with
NaOH. 4.4 .mu.M of the fluorescent indicator dye Fluo-4
(Invitrogen) prepared in pluronic acid (Sigma-Aldrich) were loaded
into the wells and incubated for 45 minutes at 29.degree. C. in 5%
CO.sub.2. Cells were then rinsed with either a 2 mM KCl
closed-state buffer (in mM: 138.5 NaCl, 10 HEPES, 10 D-glucose, 1
CaCl.sub.2, and 2 KCl, with the pH adjusted to 7.4 with NaOH) when
performing the closed-state assay or 30 mM KCl inactivated-state
buffer (in mM: 110.5 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl.sub.2,
and 30 mM KCl, with the pH adjusted to 7.4 with NaOH) when
performing the inactivated-state assay. Concentration-dependent
response curves were generated from 5 mM stock solutions prepared
in DMSO (Sigma-Aldrich), diluted in either the 2 mM KCl buffer or
30 mM KCl buffer, and incubated for 20 minutes at 29.degree. C. in
5% CO.sub.2. Calcium entry was evoked with an addition of 130 mM
KCl stimulation buffer (in mM: 10.5 NaCl, 10 HEPES, 10 D-glucose, 1
CaCl.sub.2, and 130 KCl, with the pH adjusted to 7.4 with NaOH). A
change in the Fluo-4 fluorescence signal was assessed using
FLIPR.sup.TETRA.TM. instrument (Molecular Devices, Sunnyvale,
Calif.) for 3 minutes following the elevation of extracellular KCl
using an illumination wavelength of 470-495 nm with emissions
recorded at 515-575 nm.
[0374] Concentration-dependent response curves were obtained by
comparing the fluorescence signal in the presence of compound and
fitted with a logistic function (1) to obtain the concentration
that inhibited 50% (IC.sub.50) of the RLU signal using OriginPro
v.7.5 software (OriginLab, Northampton, Mass.).
y = [ max - min 1 + ( [ drug ] IC 50 ) n H ] + min ( 1 )
##EQU00007##
[0375] To assess the quality of the FLIPR assays, the Z-factor (2)
was used to quantify the suitability of the assay conditions using
the following equation:
Z = 1 - 3 SD sample + 3 SD control mean sample - mean control ( 2 )
##EQU00008##
[0376] Data were expressed as mean and standard deviation (SD).
[0377] hERG K.sup.+ Channel Activity
[0378] It may be desirable that the compound has very low activity
with respect to the hERG K.sup.+ channel, which is expressed in the
heart: compounds that block this channel with high potency may
cause reactions which are fatal. See, e.g., Bowlby et al., "hERG
(KCNH2 or K.sub.v11.1 K.sup.+ Channels: Screening for Cardiac
Arrhythmia Risk," Curr. Drug Metab. 9(9):965-70 (2008)). Thus, for
a compound that modulates, e.g., sodium channel activity, it may
also be shown that the hERG K.sup.+ channel is not inhibited or
only minimally inhibited as compared to the inhibition of the
primary channel targeted. Such compounds may be particularly useful
in the methods described herein.
[0379] Compounds were tested using a standard electrophysiological
assay (see Kiss et al., Assay & Drug Development Technologies,
1:1-2, 2003, and Bridgland-Taylor et al., Journal of
Pharmacological and Toxicological Methods, 54:189-199, 2006).
Briefly, compounds were tested at 3 .mu.M using IonWorks and the
percent inhibition of the peak of the slowly deactivating hERG tail
current was used to assess the affinity.
Pain Models
[0380] L5/L6 Spinal Nerve Ligation (SNL)--Chung Pain Model
[0381] The Spinal Nerve Ligation is an animal model representing
peripheral nerve injury generating a neuropathic pain syndrome. In
this model experimental animals develop the clinical symptoms of
tactile allodynia and hyperalgesia. L5/L6 Spinal nerve ligation
(SNL) injury was induced using the procedure of Kim and Chung (Kim
et al., Pain 50:355-363 (1992)) in male Sprague-Dawley rats
(Harlan; Indianapolis, Ind.) weighing 200 to 250 grams. An
exemplary protocol is provided below
[0382] The animals were anesthetized with isoflurane, the left L6
transverse process was removed, and the L5 and L6 spinal nerves
were tightly ligated with 6-0 silk suture. The wound was closed
with internal sutures and external tissue adhesive. Rats that
exhibit motor deficiency (such as paw-dragging) or failure to
exhibit subsequent tactile allodynia can be excluded from further
testing.
[0383] Sham control rats can undergo the same operation and
handling as the experimental animals, but without SNL.
[0384] Assessment of Mechanical Hyperalgesia
[0385] Baseline and post-treatment values for mechanical
hyperalgesia were evaluated using a digital Randall-Selitto device
(dRS; IITC Life Sciences, Woodland Hills, Calif.). Animals were
allowed to acclimate to the testing room for a minimum of 30
minutes before testing. Animals were placed in a restraint sling
that suspends the animal, leaving the hind limbs available for
testing. Paw compression threshold was measured once at each time
point for the ipsilateral and contralateral paws. The stimulus was
applied to the plantar surface of the hind paw by a dome-shaped tip
placed between the 3rd and 4th metatarsus, and pressure was applied
gradually over approximately 10 seconds. Measurements are taken
from the first observed nocifensive behavior of vocalization,
struggle or withdrawal. A cut-off value of 300 g was used to
prevent injury to the animal. The mean and standard error of the
mean (SEM) were determined for each paw for each treatment group.
Fourteen days after surgery, mechanical hyperalgesia was assessed
and rats were assigned to treatment groups based on pre-treatment
baseline values. Prior to initiating drug delivery, baseline
behavioural testing data can be obtained. At selected times after
infusion of the Test or Control Article behavioural data can then
be collected again.
[0386] Exemplary data are shown in FIGS. 2A-2C and 3A-3C for select
compounds of the invention. Additional data are presented in Tables
2-4 below.
[0387] Table 2 shows Compound (I) and Compound (41) (30 mg/kg,
p.o.) significantly decreased mechanical hyperalgesia at 2 and 4
hours after administration compared to vehicle treated animals.
Compound (24) (30 mg/kg, p.o.) had no significant effect on
mechanical hyperalgesia at any time point tested compared to
vehicle treated animals.
[0388] Table 3 shows that administration of Compound (33) (30
mg/kg, p.o.) significantly decreased mechanical hyperalgesia at 1,
2 and 4 hours after administration compared to vehicle treated
animals. Administration of Compound (56), Compound (31) or Compound
(34) (30 mg/kg, p.o.) significantly decreased mechanical
hyperalgesia 2 and 4 hours after administration compared to vehicle
treated animals. Administration of Compound (36) (30 mg/kg, p.o.)
had no significant effect on mechanical hyperalgesia at any time
point tested compared to vehicle treated animals.
TABLE-US-00002 TABLE 2 Response % Gab- Threshold (g) % Reversal
apentin Compound 1 hr 2 hr 4 hr 1 hr 2 hr 4 hr Peak Gabapentin
129.0 154.6 135.7 359. 55.3 41.0 -- (100 mg/kg; p.o.) DMA/PS80/
90.9 85.1 84.6 5.4 0.8 0.4 1.4 PEG400 (10:45:45, 2 mL/ kg, p.o.)
Compound (1) 93.8 120.6 113.3 9.2 30.2 24.4 54.7 (30 mg/kg, p.o.)
Compound (24) 84.0 118.4 116.6 0.8 27.9 26.5 50.5 (30 mg/kg, p.o.)
Compound (41) 99.6 108.3 109.0 13.8 20.7 21.3 37.5 (30 mg/kg,
p.o.)
TABLE-US-00003 TABLE 3 Response % Gab- Threshold (g) % Reversal
apentin Compound 1 hr 2 hr 4 hr 1 hr 2 hr 4 hr Peak Gabapentin
113.5 153.8 169.3 22.9 61.0 75.7 -- (100 mg/kg; p.o.) DMA/PS80/
87.8 85.5 83.2 -0.2 -2.2 -4.3 -3.7 PEG400 (10:45:45, 2 mL/ kg,
p.o.) Compound (31) 107.9 139.0 109.9 17.2 48.0 19.2 78.7 (30
mg/kg, p.o.) Compound (33) 113.6 136.9 150.8 22.4 44.7 58.1 73.3
(30 mg/kg, p.o.) Compound (34) 104.5 144.8 132.4 15.3 54.2 42.3
88.9 (30 mg/kg, p.o.) Compound (56) 110.8 131.4 140.4 19.8 40.4
49.4 66.2 (30 mg/kg, p.o.) Compound (36) 87.8 107.0 112.3 -0.5 18.6
23.8 30.5 (30 mg/kg, p.o.)
[0389] Exemplary data are also shown in FIG. 2C and FIG. 3C for
Compound (110) and in Table 4 below. Compound (110) is shown to
significantly decrease mechanical hyperalgesia at two and four
hours after administration compared to vehicle treated animals.
TABLE-US-00004 TABLE 4 Response % Gab- Threshold (g) % Reversal
apentin Compound 1 hr 2 hr 4 hr 1 hr 2 hr 4 hr Peak Gabapentin
129.0 154.6 135.7 359. 55.3 41.0 -- (100 mg/kg; p.o.) DMA/PS80/
90.9 85.1 84.6 5.4 0.8 0.4 1.4 PEG400 (10:45:45, 2 mL/ kg, p.o.)
Compound (110) 100.6 148.5 126.1 14.6 51.2 34.1 92.7 (30 mg/kg,
p.o.)
[0390] Assessment of Tactile Allodynia--Von Frey
[0391] The assessment of tactile allodynia can consist of measuring
the withdrawal threshold of the paw ipsilateral to the site of
nerve injury in response to probing with a series of calibrated von
Frey filaments (innocuous stimuli). Animals can be acclimated to
the suspended wire-mesh cages for 30 min before testing. Each von
Frey filament can be applied perpendicularly to the plantar surface
of the ligated paw of rats for 5 sec. A positive response can be
indicated by a sharp withdrawal of the paw. For rats, the first
testing filament is 4.31. Measurements can be taken before and
after administration of test articles. The paw withdrawal threshold
can be determined by the non-parametric method of Dixon (Dixon,
Ann. Rev. Pharmacol. Toxicol. 20:441-462 (1980)), in which the
stimulus was incrementally increased until a positive response was
obtained, and then decreased until a negative result was observed.
The protocol can be repeated until three changes in behaviour were
determined ("up and down" method; Chaplan et al., J. Neurosci.
Methods 53:55-63 (1994)). The 50% paw withdrawal threshold can be
determined as (10.sup.[Xf+k.delta.])/10,000, where X.sub.f=the
value of the last von Frey filament employed, k=Dixon value for the
positive/negative pattern, and .delta.=the logarithmic difference
between stimuli. The cut-off values for rats can be, for example,
no less than 0.2 g and no higher than 15 g (5.18 filament); for
mice no less than 0.03 g and no higher than 2.34 g (4.56 filament).
A significant drop of the paw withdrawal threshold compared to the
pre-treatment baseline is considered tactile allodynia. Rat SNL
tactile allodynia can be tested for the compounds described herein
at, e.g., 60 minutes comapred to baseline and post-SNL.
[0392] Assessment of Thermal Hypersensitivity--Hargreaves
[0393] The method of Hargreaves and colleagues (Hargreaves et al.,
Pain 32:77-8 (1988)) can be employed to assess paw-withdrawal
latency to a noxious thermal stimulus.
[0394] Rats may be allowed to acclimate within a Plexiglas
enclosure on a clear glass plate for 30 minutes. A radiant heat
source (e.g., halogen bulb coupled to an infrared filter) can then
be activated with a timer and focused onto the plantar surface of
the affected paw of treated rats. Paw-withdrawal latency can be
determined by a photocell that halts both lamp and timer when the
paw is withdrawn. The latency to withdrawal of the paw from the
radiant heat source can be determined prior to L5/L6 SNL, 7-14 days
after L5/L6 SNL but before drug, as well as after drug
administration. A maximal cut-off of 33 seconds is typically
employed to prevent tissue damage. Paw withdrawal latency can be
thus determined to the nearest 0.1 second. A significant drop of
the paw withdrawal latency from the baseline indicates the status
of thermal hyperalgesia. Antinociception is indicated by a reversal
of thermal hyperalgesia to the pre-treatment baseline or a
significant (p<0.05) increase in paw withdrawal latency above
this baseline. Data is converted to % anti hyperalgesia or % anti
nociception by the formula: (100.times.(test latency-baseline
latency)/(cut-off-baseline latency) where cut-off is 21 seconds for
determining anti hyperalgesia and 40 seconds for determining anti
nociception.
Epilepsy Models
[0395] 6 Hz Psychomotor Seizure Model of Partial Epilepsy
[0396] Compounds can be evaluated for the protection against
seizures induced by a 6 Hz, 0.2 ms rectangular pulse width of 3 s
duration, at a stimulus intensity of 32 mA (CC97) applied to the
cornea of male CF1 mice (20-30 g) according to procedures described
by Barton et al, "Pharmacological Characterization of the 6 Hz
Psychomotor Seizure Model of Partial Epilepsy," Epilepsy Res.
47(3):217-27 (2001). Seizures are characterised by the expression
of one or more of the following behaviours: stun, forelimb clonus,
twitching of the vibrissae and Straub-tail immediately following
electrical stimulation. Animals can be considered "protected" if,
following pre-treatment with a compound, the 6 Hz stimulus failed
to evoke a behavioural response as describe above.
[0397] GAERS (Genetic Absence Epilepsy Rats from Strasbourg)
Epilepsy Model
[0398] The GAERS (Genetic Absence Epilepsy Rats from Strasbourg) is
noted for its long and frequently recurring absence seizure
episodes. Investigators have determined, using electrophysiological
recordings from neurons within the thalamus, that the activity and
expression of the low-voltage calcium channels is significantly
increased in GAERS. Eight female GAERS rats, bred in the Ludwig
Institute for Cancer Research, were used for this study. Rats
weighed between 180 and 250 g and aged between 18 and 26 weeks at
the start of the experiment. Methods for conducting this assay are
known in the art.
Assessments of Neurological or Muscular Impairments
[0399] To assess a compound's undesirable side effects (toxicity),
animals can be monitored for overt signs of impaired neurological
or muscular function. In mice, the rotarod procedure (Dunham et
al., J. Am. Pharmacol. Assoc. 46:208-209 (1957)) is used to
disclose minimal muscular or neurological impairment (MMI). When a
mouse is placed on a rod that rotates at a speed of 6 rpm, the
animal can maintain its equilibrium for long periods of time. The
animal is considered toxic if it falls off this rotating rod three
times during a 1-min period. In addition to MMI, animals may
exhibit a circular or zigzag gait, abnormal body posture and spread
of the legs, tremors, hyperactivity, lack of exploratory behavior,
somnolence, stupor, catalepsy, loss of placing response and changes
in muscle tone.
Recordings on Lamina I/II Spinal Cord Neurons
[0400] Male Wistar rats (P6 to P9 for voltage-clamp and P15 to P18
for current-clamp recordings) can be anaesthetized through
intraperitoneal injection of Inactin (Sigma). The spinal cord can
then be rapidly dissected out and placed in an ice-cold solution
protective sucrose solution containing (in mM): 50 sucrose, 92
NaCl, 15 D-Glucose, 26 NaHCO.sub.3, 5 KCl, 1.25 NaH.sub.2PO.sub.4,
0.5 CaCl.sub.2, 7 MgSO.sub.4,1 kynurenic acid, and bubbled with 5%
CO.sub.2/95% O.sub.2. The meninges, dura, and dorsal and ventral
roots can then removed from the lumbar region of the spinal cord
under a dissecting microscope. The "cleaned" lumbar region of the
spinal cord may be glued to the vibratome stage and immediately
immersed in ice cold, bubbled, sucrose solution. For current-clamp
recordings, 300 to 350 .mu.m parasagittal slices can be cut to
preserve the dendritic arbour of lamina I neurons, while 350 to 400
.mu.m transverse slices can be prepared for voltage-clamped
Na.sub.v channel recordings. Slices may be allowed to recover for 1
hour at 35.degree. C. in Ringer solution containing (in mM): 125
NaCl, 20 D-Glucose, 26 NaHCO.sub.3, 3 KCl, 1.25 NaH.sub.2PO.sub.4,
2 CaCl.sub.2, 1 MgCl.sub.2, 1 kynurenic acid, 0.1 picrotoxin,
bubbled with 5% CO.sub.2/95% O.sub.2. The slice recovery chamber
can then returned to room temperature (20 to 22.degree. C.) for
recordings.
[0401] Neurons may be visualized using IR-DIC optics (Zeiss
Axioskop 2 FS plus, Gottingen, Germany), and neurons from lamina I
and the outer layer of lamina II can be selected based on their
location relative to the substantia gelatinosa layer. Neurons can
be patch-clamped using borosilicate glass patch pipettes with
resistances of 3 to 6 MW. Current-clamp recordings of lamina I/II
neurons in the intact slice, the external recording solution was
the above Ringer solution, while the internal patch pipette
solution contained (in mM): 140 KGluconate, 4 NaCl, 10 HEPES, 1
EGTA, 0.5 MgCl.sub.2, 4 MgATP, 0.5 Na.sub.2GTP, adjusted to pH 7.2
with 5 M KOH and to 290 mOsm with D-Mannitol (if necessary). Tonic
firing neurons can be selected for current-clamp experiments, while
phasic, delayed onset and single spike neurons may be discarded
(22). Recordings can be digitized at 50 kHz and low-pass filtered
at 2.4 kHz.
Pharmacokinetic Parameters
[0402] Preliminary exposure characteristics of the compounds can be
evaluated using, e.g., an in vivo Rat Early Pharmacokinetic (EPK)
study design to show bioavailability. For example, Male
Sprague-Dawley rats can be dosed via oral (PO) gavage in a
particular formulation. Blood samples can then be collected from
the animals at 6 timepoints out to 4 hours post-dose.
Pharmacokinetic analysis can then performed on the LC-MS/MS
measured concentrations for each timepoint of each compound.
Other Embodiments
[0403] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure come within
known or customary practice within the art to which the invention
pertains and may be applied to the essential features hereinbefore
set forth.
[0404] All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as
if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety.
* * * * *