U.S. patent application number 15/475240 was filed with the patent office on 2017-07-20 for compounds for nonsense suppression, and methods for their use.
This patent application is currently assigned to PTC Therapeutics, Inc.. The applicant listed for this patent is PTC Therapeutics, Inc.. Invention is credited to Neil Almstead, Jeffrey A. Campbell, Guangming Chen, Gary M. Karp, Ellen Welch, Richard Wilde.
Application Number | 20170204073 15/475240 |
Document ID | / |
Family ID | 35517967 |
Filed Date | 2017-07-20 |
United States Patent
Application |
20170204073 |
Kind Code |
A1 |
Almstead; Neil ; et
al. |
July 20, 2017 |
COMPOUNDS FOR NONSENSE SUPPRESSION, AND METHODS FOR THEIR USE
Abstract
The present invention relates to methods, compounds, and
compositions for treating or preventing diseases associated with
nonsense mutations in an mRNA by administering the compounds or
compositions of the present invention. More particularly, the
present invention relates to methods, compounds, and compositions
for suppressing premature translation termination associated with a
nonsense mutation in an mRNA.
Inventors: |
Almstead; Neil; (Princeton,
NJ) ; Chen; Guangming; (Bridgewater, NJ) ;
Karp; Gary M.; (Princeton Junction, NJ) ; Welch;
Ellen; (Califon, NJ) ; Wilde; Richard;
(Somerville, NJ) ; Campbell; Jeffrey A.;
(Bethlehem, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PTC Therapeutics, Inc. |
South Plainfield |
NJ |
US |
|
|
Assignee: |
PTC Therapeutics, Inc.
South Plainfield
NJ
|
Family ID: |
35517967 |
Appl. No.: |
15/475240 |
Filed: |
March 31, 2017 |
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Application
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Patent Number |
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11577191 |
Mar 12, 2009 |
9611230 |
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PCT/US05/36673 |
Oct 13, 2005 |
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15475240 |
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60617653 |
Oct 13, 2004 |
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60617670 |
Oct 13, 2004 |
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60617633 |
Oct 13, 2004 |
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60617634 |
Oct 13, 2004 |
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60617655 |
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60624170 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 9/00 20180101; C07F
9/65306 20130101; C07D 401/04 20130101; C07D 417/04 20130101; A61P
3/10 20180101; A61P 25/00 20180101; A61P 29/00 20180101; A61P 37/00
20180101; A61P 37/06 20180101; C07D 263/32 20130101; A61P 5/14
20180101; A61P 25/16 20180101; A61P 43/00 20180101; C07D 233/64
20130101; C07D 403/04 20130101; A61P 19/08 20180101; A61P 25/28
20180101; C07D 261/08 20130101; A61P 1/16 20180101; C07D 413/12
20130101; A61P 21/00 20180101; A61P 37/02 20180101; C07D 251/24
20130101; A61P 9/10 20180101; C07D 271/06 20130101; C07D 277/30
20130101; A61P 19/00 20180101; A61P 13/12 20180101; C07D 285/12
20130101; A61P 35/02 20180101; C07D 333/24 20130101; A61P 3/04
20180101; A61P 7/04 20180101; C07D 249/08 20130101; C07D 271/107
20130101; C07D 231/12 20130101; A61P 19/02 20180101; C07D 413/06
20130101; C07D 413/04 20130101; C07D 307/68 20130101; A61P 3/06
20180101; A61P 11/00 20180101; C07D 271/10 20130101; A61P 7/00
20180101; A61P 35/00 20180101; C07D 405/04 20130101; C07D 409/04
20130101; A61P 27/02 20180101 |
International
Class: |
C07D 271/06 20060101
C07D271/06; C07D 413/06 20060101 C07D413/06; C07D 417/04 20060101
C07D417/04; C07D 413/04 20060101 C07D413/04 |
Claims
1.-31. (canceled)
32. A compound of Formula (1-C) or Formula (1-D): ##STR01309##
wherein: R.sub.1 is hydrogen, C.sub.1-C.sub.6 alkyl, or Na.sup.+,
or Mg.sup.2+; R.sub.3 is absent or independently selected from
halogen, hydroxy, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 alkoxy, or
nitro; W is selected from: (a) a C.sub.2-C.sub.6 alkynyl,
optionally substituted with a phenyl; (b) a C.sub.1-C.sub.8
straight chain or branched chain alkyl which is optionally
substituted with one or more of the following independently
selected groups: a C.sub.1-C.sub.6 alkyl; a halogen; a
--C(.dbd.O)--NH-phenyl which phenyl is optionally substituted with
one or more independently selected halogens or C.sub.1-C.sub.4
alkyl groups; a five to six-membered heterocycle; a C.sub.6-C.sub.8
aryl which is optionally substituted with one or more groups
independently selected from a hydroxy, a halogen, a C.sub.1-C.sub.4
alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a C.sub.1-C.sub.4
alkoxy group or an amino group which is optionally substituted with
one or more C.sub.1-C.sub.4 alkyl groups; an aryloxy which is
optionally substituted with one or more of the following
independently selected groups: a hydroxy, a halogen, a
C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a
C.sub.1-C.sub.4 alkoxy group or an amino group which is optionally
substituted with one or more C.sub.1-C.sub.4 alkyl groups; (c) a
C.sub.2 to C.sub.8 alkenyl; (d) a C.sub.3-C.sub.8 cycloalkyl
optionally substituted with a C.sub.1-C.sub.6 alkyl; (e) a
C.sub.6-C.sub.8 aryl which is optionally substituted with one or
more of the following independently selected groups: a hydroxy; a
halogen; a C.sub.1-C.sub.4 straight chain or branched chain alkyl
which is optionally substituted with one or more independently
selected halogen or hydroxy groups; a C.sub.1-C.sub.4 alkoxy which
is optionally substituted with one or more independently selected
halogen or phenyl groups; a C.sub.3-C.sub.8 cycloalkyl which is
optionally substituted with one or more independently selected
C.sub.1-C.sub.4 alkyl groups; a C.sub.6-C.sub.8 aryl which is
optionally substituted with one or more independently selected
C.sub.1-C.sub.4 alkyl groups; an aryloxy which is optionally
substituted with one or more of the following independently
selected groups: a hydroxy, a halogen, a C.sub.1-C.sub.4 alkyl
group, a C.sub.1-C.sub.4 haloalkyl group, a C.sub.1-C.sub.4 alkoxy
group, or an amino group which is optionally substituted with one
or more independently selected C.sub.1-C.sub.4 alkyl groups; a five
to six-membered heterocycle which is optionally substituted with
one or more independently selected C.sub.1-C.sub.4 alkyl, oxo, or
C.sub.6-C.sub.8 aryl which is optionally substituted with one or
more of the following independently selected groups: a hydroxy, a
halogen, a C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl
group, a C.sub.1-C.sub.4 alkoxy group, or an amino group which is
optionally substituted with one or more independently selected
C.sub.1-C.sub.4 alkyl groups; a naphthyl group which is optionally
substituted with an amino or aminoalkyl or alkoxy group; a
--C(O)--NR.sub.xR.sub.y group; a --C(O)--R.sub.x group; a
isoindole-1,3-dione group; a nitro group; a cyano group; a
--SO.sub.3H group; alkylthio group; alkyl sulfonyl group; a
--NR.sub.x--C(O)--R.sub.z group; a --NR.sub.xR.sub.y group; a
--NR.sub.x--SO.sub.2--R.sub.z group; a
--NR.sub.x--C(O)--NR.sub.xR.sub.y group; a
--NR.sub.x--C(O)O--R.sub.z group; (f) a C.sub.10-C.sub.14 aryl
group optionally substituted with one or more independently
selected halogens, amino groups or aminoalkyl groups, or alkoxy
groups; (g) a --C(O)--NR.sub.xR.sub.y group; (h) a five or six
membered heterocycle which is optionally substituted with one or
more independently selected oxo groups; halogens; C.sub.1-C.sub.4
alkyl groups; C.sub.1-C.sub.4 alkoxy groups; C.sub.1-C.sub.4
haloalkyl groups; C.sub.1-C.sub.4 haloalkoxy groups; aryloxy
groups; --NR.sub.xR.sub.y groups; alkylthio groups; --C(O)--R.sub.x
groups; or C.sub.6 to C.sub.8 aryl groups which are optionally
substituted with one or more independently selected halogens,
C.sub.1-C.sub.4 alkyl groups, C.sub.1-C.sub.4 alkoxy groups; (i) a
heterocycle group having two to three ring structures that is
optionally substituted with one or more independently selected
halogens, oxo groups, C.sub.1-C.sub.4 alkyl groups, C.sub.1-C.sub.4
haloalkyl groups, or C.sub.1-C.sub.4 alkoxy groups; wherein R.sub.x
is hydrogen, a C.sub.1-C.sub.6 alkyl group, or R.sub.x and R.sub.y
together with the atoms to which they are attached form a four to
seven membered carbocycle or heterocycle; R.sub.y is hydrogen, a
C.sub.1-C.sub.6 alkyl group; an aryl group optionally substituted
with one or more independently selected C.sub.1-C.sub.4 alkyl
groups, or R.sub.x and R.sub.y together with the atoms to which
they are attached form a four to seven membered carbocycle or
heterocycle; and R.sub.z is an C.sub.1-C.sub.6 alkyl optionally
substituted with an aryl or a halogen; or an aryl optionally
substituted with a halogen, a C.sub.1-C.sub.6 alkyl, or a
C.sub.1-C.sub.6 alkoxy; or a pharmaceutically acceptable salt,
hydrate, solvate, clathrate, racemate, stereoisomer, or polymorph
thereof.
33. The compound of claim 32, wherein: R.sub.1 is hydrogen,
C.sub.1-C.sub.6 alkyl, or Na.sup.+, or Mg.sup.2+; R.sub.3 is absent
or independently selected from halogen, hydroxy, C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.4 alkoxy, or nitro; W is selected from: (a) a
C.sub.1-C.sub.8 straight chain or branched chain alkyl which is
optionally substituted with one or more of the following: a
C.sub.1-C.sub.6 alkyl, a halogen, a five to six-membered
heterocycle, a C.sub.6-C.sub.8 aryl which is optionally substituted
with one or more of the following: a hydroxy, a halogen, a
C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a
C.sub.1-C.sub.4 alkoxy group or an amino group which is optionally
substituted with one or more C.sub.1-C.sub.4 alkyl groups; an
aryloxy which is optionally substituted with one or more of the
following: a hydroxy, a halogen, a C.sub.1-C.sub.4 alkyl group, a
C.sub.1-C.sub.4 haloalkyl group, a C.sub.1-C.sub.4 alkoxy group or
an amino group which is optionally substituted with one or more
C.sub.1-C.sub.4 alkyl groups; (b) a C.sub.2 to C.sub.8 alkenyl; (c)
a C.sub.3-C.sub.8 cycloalkyl optionally substituted with a C.sub.1
to C.sub.6 alkyl; (d) a C.sub.6-C.sub.8 aryl which is optionally
substituted with one or more of the following: a hydroxy, a
halogen; a C.sub.1-C.sub.4 straight chain or branched chain alkyl
which is optionally substituted with one or more independently
selected halogen or hydroxy groups; a C.sub.1-C.sub.4 alkoxy which
is optionally substituted with one or more independently selected
halogen or phenyl groups; a C.sub.3-C.sub.8 cycloalkyl which is
optionally substituted with one or more independently selected
C.sub.1-C.sub.4 alkyl groups; a C.sub.6-C.sub.8 aryl which is
optionally substituted with one or more independently selected
C.sub.1-C.sub.4 alkyl groups; an aryloxy which is optionally
substituted with one or more of the following: a hydroxy, a
halogen, a C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl
group, a C.sub.1-C.sub.4 alkoxy group, or an amino group which is
optionally substituted with one or more C.sub.1-C.sub.4 alkyl
groups; a five to six-membered heterocycle which is optionally
substituted with one or more independently selected C.sub.1-C.sub.4
alkyl, oxo, or C.sub.6-C.sub.8 aryl which is optionally substituted
with one or more of the following: a hydroxy, a halogen, a
C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a
C.sub.1-C.sub.4 alkoxy group, or an amino group which is optionally
substituted with one or more C.sub.1-C.sub.4 alkyl groups; a
naphthyl group which is optionally substituted with an amino or
aminoalkyl group; a --C(O)--NR.sub.xR.sub.y group; a
--C(O)--R.sub.x group; a isoindole-1,3-dione group; a nitro group;
a cyano group; a --SO.sub.3H group; alkylthio group; alkyl sulfonyl
group; a --NR.sub.x--C(O)--R.sub.z group; a --NR.sub.xR.sub.y
group; a --NR.sub.x--SO.sub.2--R.sub.z group; a
--NR.sub.x--C(O)--NR.sub.xR.sub.y group; a
--NR.sub.x--C(O)O--R.sub.z group; (e) a --C(O)--NR.sub.xR.sub.y
group; (f) a five or six membered heterocycle which is optionally
substituted with one or more oxo groups, halogens, C.sub.1 to
C.sub.4 alkyl groups, C.sub.1 to C.sub.4 haloalkyl groups,
--C(O)--R.sub.x groups, and/or C.sub.6 to C.sub.8 aryl groups which
are optionally substituted with one or more independently selected
halogens, C.sub.1 to C.sub.4 alkyl groups, C.sub.1 to C.sub.4
alkoxy groups, aryloxy groups, --NR.sub.xR.sub.y groups, and/or
alkylthio groups; (g) a heterocycle group having two to three ring
structures that is optionally substituted with one or more
halogens, C.sub.1 to C.sub.4 alkyl groups, C.sub.1 to C.sub.4
haloalkyl groups, and/or C.sub.1 to C.sub.4 alkoxy groups; wherein
R.sub.x is hydrogen, a C.sub.1 to C.sub.6 alkyl group, or R.sub.x
and R.sub.y together with the atoms to which they are attached form
a four to seven membered carbocycle or heterocycle; R.sub.y is
hydrogen, a C.sub.1 to C.sub.6 alkyl group; an optionally
substituted aryl, or R.sub.x and R.sub.y together with the atoms to
which they are attached form a four to seven membered carbocycle or
heterocycle; and R.sub.z is an C.sub.1 to C.sub.6 alkyl optionally
substituted with an aryl or a halogen; or an aryl optionally
substituted with a halogen, a C.sub.1 to C.sub.6 alkyl, or a
C.sub.1 to C.sub.6 alkoxy; or a pharmaceutically acceptable salt,
hydrate, solvate, clathrate, racemate, stereoisomer, or polymorph
thereof.
34. The compound of claim 32, wherein the compound of Formula (1-D)
is selected from a compound of Formula (1-D1): ##STR01310## wherein
all other variables are as previously defined.
35. The compound of claim 32, wherein W is selected from the group
consisting of: ##STR01311## ##STR01312## ##STR01313## ##STR01314##
##STR01315## ##STR01316## ##STR01317## ##STR01318## ##STR01319##
##STR01320## ##STR01321## ##STR01322## ##STR01323##
##STR01324##
36. The compound of claim 32, wherein the compound of Formula (1-C)
is selected from the group consisting of: ##STR01325## ##STR01326##
##STR01327## ##STR01328## ##STR01329## ##STR01330## ##STR01331##
and a pharmaceutically acceptable salt, hydrate, solvate,
clathrate, racemate, stereoisomer or polymorph thereof.
37. The compound of claim 32, wherein the compound of Formula (1-D)
is selected from the group consisting of: ##STR01332## ##STR01333##
##STR01334## ##STR01335## ##STR01336## ##STR01337## ##STR01338##
##STR01339## and a pharmaceutically acceptable salt, hydrate,
solvate, clathrate, racemate, stereoisomer or polymorph
thereof.
38. A method for preventing or treating a disease associated with a
gene having a nonsense mutation encoding a premature stop codon in
mRNA in a patient in need thereof, comprising administering to said
patient a compound of Formula (1-C) or Formula (1-D) ##STR01340##
wherein: R.sub.1 is hydrogen, C.sub.1-C.sub.6 alkyl, or Na.sup.+,
or Mg.sup.2+; R.sub.3 is absent or independently selected from
halogen, hydroxy, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 alkoxy, or
nitro; W is selected from: (a) a C.sub.2-C.sub.6 alkynyl,
optionally substituted with a phenyl; (b) a C.sub.1-C.sub.8
straight chain or branched chain alkyl which is optionally
substituted with one or more of the following independently
selected groups: a C.sub.1-C.sub.6 alkyl; a halogen; a
--C(.dbd.O)--NH-phenyl which phenyl is optionally substituted with
one or more independently selected halogens or C.sub.1-C.sub.4
alkyl groups; a five to six-membered heterocycle; a C.sub.6-C.sub.8
aryl which is optionally substituted with one or more groups
independently selected from a hydroxy, a halogen, a C.sub.1-C.sub.4
alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a C.sub.1-C.sub.4
alkoxy group or an amino group which is optionally substituted with
one or more C.sub.1-C.sub.4 alkyl groups; an aryloxy which is
optionally substituted with one or more of the following
independently selected groups: a hydroxy, a halogen, a
C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a
C.sub.1-C.sub.4 alkoxy group or an amino group which is optionally
substituted with one or more C.sub.1-C.sub.4 alkyl groups; (c) a
C.sub.2 to C.sub.8 alkenyl; (d) a C.sub.3-C.sub.8 cycloalkyl
optionally substituted with a C.sub.1-C.sub.6 alkyl; (e) a
C.sub.6-C.sub.8 aryl which is optionally substituted with one or
more of the following independently selected groups: a hydroxy; a
halogen; a C.sub.1-C.sub.4 straight chain or branched chain alkyl
which is optionally substituted with one or more independently
selected halogen or hydroxy groups; a C.sub.1-C.sub.4 alkoxy which
is optionally substituted with one or more independently selected
halogen or phenyl groups; a C.sub.3-C.sub.8 cycloalkyl which is
optionally substituted with one or more independently selected
C.sub.1-C.sub.4 alkyl groups; a C.sub.6-C.sub.8 aryl which is
optionally substituted with one or more independently selected
C.sub.1-C.sub.4 alkyl groups; an aryloxy which is optionally
substituted with one or more of the following independently
selected groups: a hydroxy, a halogen, a C.sub.1-C.sub.4 alkyl
group, a C.sub.1-C.sub.4 haloalkyl group, a C.sub.1-C.sub.4 alkoxy
group, or an amino group which is optionally substituted with one
or more independently selected C.sub.1-C.sub.4 alkyl groups; a five
to six-membered heterocycle which is optionally substituted with
one or more independently selected C.sub.1-C.sub.4 alkyl, oxo, or
C.sub.6-C.sub.8 aryl which is optionally substituted with one or
more of the following independently selected groups: a hydroxy, a
halogen, a C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl
group, a C.sub.1-C.sub.4 alkoxy group, or an amino group which is
optionally substituted with one or more independently selected
C.sub.1-C.sub.4 alkyl groups; a naphthyl group which is optionally
substituted with an amino or aminoalkyl or alkoxy group; a
--C(O)--NR.sub.xR.sub.y group; a --C(O)--R.sub.x group; a
isoindole-1,3-dione group; a nitro group; a cyano group; a
--SO.sub.3H group; alkylthio group; alkyl sulfonyl group; a
--NR.sub.x--C(O)--R.sub.z group; a --NR.sub.xR.sub.y group; a
--NR.sub.x--SO.sub.2--R.sub.z group; a
--NR.sub.x--C(O)--NR.sub.xR.sub.y group; a
--NR.sub.x--C(O)O--R.sub.z group; (f) a C.sub.10-C.sub.14 aryl
group optionally substituted with one or more independently
selected halogens, amino groups or aminoalkyl groups, or alkoxy
groups; (g) a --C(O)--NR.sub.xR.sub.y group; (h) a five or six
membered heterocycle which is optionally substituted with one or
more independently selected oxo groups; halogens; C.sub.1-C.sub.4
alkyl groups; C.sub.1-C.sub.4 alkoxy groups; C.sub.1-C.sub.4
haloalkyl groups; C.sub.1-C.sub.4 haloalkoxy groups; aryloxy
groups; --NR.sub.xR.sub.y groups; alkylthio groups; --C(O)--R.sub.x
groups; or C.sub.6 to C.sub.8 aryl groups which are optionally
substituted with one or more independently selected halogens,
C.sub.1-C.sub.4 alkyl groups, C.sub.1-C.sub.4 alkoxy groups; (i) a
heterocycle group having two to three ring structures that is
optionally substituted with one or more independently selected
halogens, oxo groups, C.sub.1-C.sub.4 alkyl groups, C.sub.1-C.sub.4
haloalkyl groups, or C.sub.1-C.sub.4 alkoxy groups; wherein R.sub.x
is hydrogen, a C.sub.1-C.sub.6 alkyl group, or R.sub.x and R.sub.y
together with the atoms to which they are attached form a four to
seven membered carbocycle or heterocycle; R.sub.y is hydrogen, a
C.sub.1-C.sub.6 alkyl group; an aryl group optionally substituted
with one or more independently selected C.sub.1-C.sub.4 alkyl
groups, or R.sub.x and R.sub.y together with the atoms to which
they are attached form a four to seven membered carbocycle or
heterocycle; and R.sub.z is an C.sub.1-C.sub.6 alkyl optionally
substituted with an aryl or a halogen; or an aryl optionally
substituted with a halogen, a C.sub.1-C.sub.6 alkyl, or a
C.sub.1-C.sub.6 alkoxy; or a pharmaceutically acceptable salt,
hydrate, solvate, clathrate, racemate, stereoisomer, or polymorph
thereof, wherein the premature stop codon results in either or both
premature mRNA translation termination or nonsense-mediated mRNA
decay, and wherein the disease is selected from cancer, a lysosomal
storage disorder, an autoimmune disease, a blood disease, a
collagen disease, diabetes, a cardiovascular disease, a pulmonary
disease, an inflammatory disease, a central nervous system disease,
heart disease, kidney disease, a muscular dystrophy, macular
degeneration, retinitis pigmentosa, amyloidosis, giantism,
dwarfism, hypothyroidism, hyperthyroidism, aging or obesity.
39. The method of claim 38, wherein: R.sub.1 is hydrogen,
C.sub.1-C.sub.6 alkyl, or Na.sup.+, or Mg.sup.2+; R.sub.3 is absent
or independently selected from halogen, hydroxy, C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.4 alkoxy, or nitro; W is selected from: (a) a
C.sub.1-C.sub.8 straight chain or branched chain alkyl which is
optionally substituted with one or more of the following: a
C.sub.1-C.sub.6 alkyl, a halogen, a five to six-membered
heterocycle, a C.sub.6-C.sub.8 aryl which is optionally substituted
with one or more of the following: a hydroxy, a halogen, a
C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a
C.sub.1-C.sub.4 alkoxy group or an amino group which is optionally
substituted with one or more C.sub.1-C.sub.4 alkyl groups; an
aryloxy which is optionally substituted with one or more of the
following: a hydroxy, a halogen, a C.sub.1-C.sub.4 alkyl group, a
C.sub.1-C.sub.4 haloalkyl group, a C.sub.1-C.sub.4 alkoxy group or
an amino group which is optionally substituted with one or more
C.sub.1-C.sub.4 alkyl groups; (b) a C.sub.2 to C.sub.8 alkenyl; (c)
a C.sub.3-C.sub.8 cycloalkyl optionally substituted with a C.sub.1
to C.sub.6 alkyl; (d) a C.sub.6-C.sub.8 aryl which is optionally
substituted with one or more of the following: a hydroxy, a
halogen; a C.sub.1-C.sub.4 straight chain or branched chain alkyl
which is optionally substituted with one or more independently
selected halogen or hydroxy groups; a C.sub.1-C.sub.4 alkoxy which
is optionally substituted with one or more independently selected
halogen or phenyl groups; a C.sub.3-C.sub.8 cycloalkyl which is
optionally substituted with one or more independently selected
C.sub.1-C.sub.4 alkyl groups; a C.sub.6-C.sub.8 aryl which is
optionally substituted with one or more independently selected
C.sub.1-C.sub.4 alkyl groups; an aryloxy which is optionally
substituted with one or more of the following: a hydroxy, a
halogen, a C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl
group, a C.sub.1-C.sub.4 alkoxy group, or an amino group which is
optionally substituted with one or more C.sub.1-C.sub.4 alkyl
groups; a five to six-membered heterocycle which is optionally
substituted with one or more independently selected C.sub.1-C.sub.4
alkyl, oxo, or C.sub.6-C.sub.8 aryl which is optionally substituted
with one or more of the following: a hydroxy, a halogen, a
C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a
C.sub.1-C.sub.4 alkoxy group, or an amino group which is optionally
substituted with one or more C.sub.1-C.sub.4 alkyl groups; a
naphthyl group which is optionally substituted with an amino or
aminoalkyl group; a --C(O)--NR.sub.xR.sub.y group; a
--C(O)--R.sub.x group; a isoindole-1,3-dione group; a nitro group;
a cyano group; a --SO.sub.3H group; alkylthio group; alkyl sulfonyl
group; a --NR.sub.x--C(O)--R.sub.z group; a --NR.sub.xR.sub.y
group; a --NR.sub.x--SO.sub.2--R.sub.z group; a
--NR.sub.x--C(O)--NR.sub.xR.sub.y group; a
--NR.sub.x--C(O)O--R.sub.z group; (e) a --C(O)--NR.sub.xR.sub.y
group; (f) a five or six membered heterocycle which is optionally
substituted with one or more oxo groups, halogens, C.sub.1 to
C.sub.4 alkyl groups, C.sub.1 to C.sub.4 haloalkyl groups,
--C(O)--R.sub.x groups, and/or C.sub.6 to C.sub.8 aryl groups which
are optionally substituted with one or more independently selected
halogens, C.sub.1 to C.sub.4 alkyl groups, C.sub.1 to C.sub.4
alkoxy groups, aryloxy groups, --NR.sub.xR.sub.y groups, and/or
alkylthio groups; (g) a heterocycle group having two to three ring
structures that is optionally substituted with one or more
halogens, C.sub.1 to C.sub.4 alkyl groups, C.sub.1 to C.sub.4
haloalkyl groups, and/or C.sub.1 to C.sub.4 alkoxy groups; wherein
R.sub.x is hydrogen, a C.sub.1 to C.sub.6 alkyl group, or R.sub.x
and R.sub.y together with the atoms to which they are attached form
a four to seven membered carbocycle or heterocycle; R.sub.y is
hydrogen, a C.sub.1 to C.sub.6 alkyl group; an optionally
substituted aryl, or R.sub.x and R.sub.y together with the atoms to
which they are attached form a four to seven membered carbocycle or
heterocycle; and R.sub.z is an C.sub.1 to C.sub.6 alkyl optionally
substituted with an aryl or a halogen; or an aryl optionally
substituted with a halogen, a C.sub.1 to C.sub.6 alkyl, or a
C.sub.1 to C.sub.6 alkoxy; or a pharmaceutically acceptable salt,
hydrate, solvate, clathrate, racemate, stereoisomer, or polymorph
thereof.
40. The method of claim 38, wherein the compound of Formula (1-D)
is selected from a compound of Formula (1-D1): ##STR01341## wherein
all other variables are as previously defined.
41. The method of claim 38, wherein W for the compound of Formula
(1-C) or (1-D) is selected from the group consisting of:
##STR01342## ##STR01343## ##STR01344## ##STR01345## ##STR01346##
##STR01347## ##STR01348## ##STR01349## ##STR01350## ##STR01351##
##STR01352## ##STR01353## ##STR01354##
42. The method of claim 38, wherein the autoimmune disease is
immunodeficiency, severe combined immunodeficiency, rheumatoid
arthritis or graft versus host disease; wherein the blood disease
is familial polycythemia, hemophilia, Von Willebrand disease,
ataxia-telangiectasia or .beta.-thalassemia; wherein the collagen
disease is epidermolysis bullosa, Marfan syndrome, osteogenesis
imperfecta or cirrhosis; wherein the inflammatory disease is
arthritis, rheumatoid arthritis or osteoarthritis; wherein the
central nervous system disease is multiple sclerosis, classical
late infantile neuronal ceroid lipofuscinosis, Alzheimer's disease
or Tay Sachs disease; wherein the lysosomal storage disorder is
tuberous sclerosis, Niemann Pick disease, mucopolysaccharidosis
type VII, metachromatic leukodystrophy, Sandhoff disease,
mucopolysaccharidosis type IIIA or mucopolysaccharidosis type VI;
wherein the kidney disease is kidney stones; wherein the
cardiovascular disease is familial hypercholesterolemia or
atherosclerosis; wherein the pulmonary disease is cystic fibrosis;
and, wherein the muscular dystrophy is Duchenne muscular
dystrophy.
43. The method of claim 38, wherein the disease is cancer
associated with a genetic or a somatic nonsense mutation in a tumor
suppressor gene in a human subject in need thereof.
44. The method of claim 43, wherein the tumor suppressor gene is
selected from the group consisting of PTEN, BRCA1, BRCA2, Rb, and
p53.
45. The method of claim 43, wherein the cancer is of the head and
neck, eye, skin, mouth, throat, esophagus, chest, bone, blood,
lung, colon, sigmoid, rectum, stomach, prostate, breast, ovaries,
kidney, liver, pancreas, brain, intestine, heart or adrenals.
46. The method of claim 43, wherein the cancer is a solid tumor
cancer selected from sarcoma, carcinoma, fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic
neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma or
retinoblastoma.
47. The method of claim 43, wherein the cancer is a blood-born
tumor selected from acute lymphoblastic leukemia, acute
lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,
acute myeloblastic leukemia, acute promyelocytic leukemia, acute
monoblastic leukemia, acute erythroleukemic leukemia, acute
megakaryoblastic leukemia, acute myelomonocytic leukemia, acute
nonlymphocytic leukemia, acute undifferentiated leukemia, chronic
myelocytic leukemia, chronic lymphocytic leukemia, hairy cell
leukemia, or multiple myeloma.
48. The method of claim 38, for inhibiting the growth of a cancer
cell or for selectively producing a protein in a mammal by the
suppression of a genetic or somatic nonsense mutation.
49. The method of claim 38, wherein the compound of Formula 1-C is
selected from the group consisting of: ##STR01355## ##STR01356##
##STR01357## ##STR01358## ##STR01359## ##STR01360## ##STR01361##
and a pharmaceutically acceptable salt, hydrate, solvate,
clathrate, racemate, stereoisomer or polymorph thereof.
50. The method of claim 38, wherein the compound of Formula 1-D is
selected from the group consisting of: ##STR01362## ##STR01363##
##STR01364## ##STR01365## ##STR01366## ##STR01367## ##STR01368##
##STR01369## and a pharmaceutically acceptable salt, hydrate,
solvate, clathrate, racemate, stereoisomer or polymorph thereof.
Description
RELATED APPLICATIONS
[0001] The present application claims priority to and the benefit
under 35 U.S.C. .sctn.119 of U.S. Application Nos. 60/617,653,
filed Oct. 13, 2004, and 60/624,170, filed Nov. 3, 2004. U.S.
Application No. 60/624,170, filed Nov. 3, 2004, is herein
incorporated by reference in its entirety. The present application
claims priority to and the benefit under 35 U.S.C. .sctn.119 of
U.S. Application Nos. 60/617,655, filed Oct. 13, 2004, 60/617,634,
filed Oct. 13, 2004; 60/617,633, filed Oct. 13, 2004, 60/617,670,
filed Oct. 13, 2004, all of which applications are herein
incorporated by reference in their entireties. The present
application also incorporates by reference herein in their
entireties International Patent Applications entitled "Compounds
for Nonsense Suppression, and Methods for Their Use," filed on Oct.
13, 2005 and identified as Attorney Docket Numbers 19025.040,
19025.041, 19025.043, and 19025.044.
FIELD OF THE INVENTION
[0002] The present invention relates to methods and compounds or
compositions for treating or preventing diseases associated with
nonsense mutations in an mRNA by administering the compounds or
compositions of the present invention. More particularly, the
present invention relates to methods and compounds or compositions
for suppressing premature translation termination associated with a
nonsense mutation in an mRNA.
BACKGROUND OF THE INVENTION
[0003] Gene expression in cells depends upon the sequential
processes of transcription and translation. Together, these
processes produce a protein from the nucleotide sequence of its
corresponding gene.
[0004] Transcription involves the synthesis of mRNA from DNA by RNA
polymerase. Transcription begins at a promoter region of the gene
and continues until termination is induced, such as by the
formation of a stem-loop structure in the nascent RNA or the
binding of the rho gene product.
[0005] Protein is then produced from mRNA by the process of
translation, occurring on the ribosome with the aid of tRNA, tRNA
synthetases and various other protein and RNA species. Translation
comprises the three phases of initiation, elongation and
termination. Translation is initiated by the formation of an
initiation complex consisting of protein factors, mRNA, tRNA,
cofactors and the ribosomal subunits that recognize signals on the
mRNA that direct the translation machinery to begin translation on
the mRNA. Once the initiation complex is formed, growth of the
polypeptide chain occurs by the repetitive addition of amino acids
by the peptidyl transferase activity of the ribosome as well as
tRNA and tRNA synthetases. The presence of one of the three
termination codons (UAA, UAG, UGA) in the A site of the ribosome
signals the polypeptide chain release factors (RFs) to bind and
recognize the termination signal. Subsequently, the ester bond
between the 3' nucleotide of the tRNA located in the ribosome's P
site and the nascent polypeptide chain is hydrolyzed, the completed
polypeptide chain is released, and the ribosome subunits are
recycled for another round of translation.
[0006] Mutations of the DNA sequence in which the number of bases
is altered are categorized as insertion or deletion mutations
(e.g., frameshift mutations) and can result in major disruptions of
the genome. Mutations of the DNA that change one base into another
and result in an amino acid substitution are labeled missense
mutations. Base substitutions are subdivided into the classes of
transitions (one purine to another purine, or one pyrimidine to
another pyrimidine) and transversions (a purine to a pyrimidine, or
a pyrimidine to a purine).
[0007] Transition and transversion mutations can result in a
nonsense mutation changing an amino acid codon into one of the
three stop codons. These premature stop codons can produce aberrant
proteins in cells as a result of premature translation termination.
A nonsense mutation in an essential gene can be lethal and can also
result in a number of human diseases, such as, cancers, lysosomal
storage disorders, the muscular dystrophies, cystic fibrosis and
hemophilia, to name a few.
[0008] The human p53 gene is the most commonly mutated gene in
human cancer (Zambetti, G. P. and Levine, A., FASEB 7:855-865
(1993)). Found in both genetic and spontaneous cancers, over 50
different types of human cancers contain p53 mutations and
mutations of this gene occur in 50-55% of all human cancers
(Hollstein, M., et al., Nucleic Acids Res. 22:3551-55 (1994);
International Agency for Research on Cancer (IARC) database).
Approximately 70% of colorectal cancer, 50% of lung cancer and 40%
of breast cancers contain mutant p53 (Koshland, D., Science
262:1953 (1993)). Aberrant forms of p53 are associated with poor
prognosis, more aggressive tumors, metastasis, and lower 5 year
survival rates (Id). p53's role in the induction of cell growth
arrest and/or apoptosis upon DNA damage is believed to be essential
for the destruction of mutated cells that would have otherwise
gained a growth advantage. In addition, p53 sensitizes rapidly
dividing cells to apoptotic signals. Of greater than 15,000
reported mutations in the p53 gene, approximately 7% are nonsense
mutations. Accordingly, there is a need for a safe and effective
treatment directed to p53 nonsense mutations.
[0009] In bacterial and eukaryotic strains with nonsense mutations,
suppression of the nonsense mutation can arise as a result of a
mutation in one of the tRNA molecules so that the mutant tRNA can
recognize the nonsense codon, as a result of mutations in proteins
that are involved in the translation process, as a result of
mutations in the ribosome (either the ribosomal RNA or ribosomal
proteins), or by the addition of compounds known to alter the
translation process (for example, cycloheximide or the
aminoglycoside antibiotics). The result is that an amino acid will
be incorporated into the polypeptide chain, at the site of the
nonsense mutation, and translation will not prematurely terminate
at the nonsense codon. The inserted amino acid will not necessarily
be identical to the original amino acid of the wild-type protein,
however, many amino acid substitutions do not have a gross effect
on protein structure or function. Thus, a protein produced by the
suppression of a nonsense mutation would be likely to possess
activity close to that of the wild-type protein. This scenario
provides an opportunity to treat diseases associated with nonsense
mutations by avoiding premature termination of translation through
suppression of the nonsense mutation.
[0010] The ability of aminoglycoside antibiotics to promote
read-through of eukaryotic stop codons has attracted interest in
these drugs as potential therapeutic agents in human diseases
caused by nonsense mutations. One disease for which such a
therapeutic strategy may be viable is classical late infantile
neuronal ceroid lipofuscinosis (LINCL), a fatal childhood
neurodegenerative disease with currently no effective treatment.
Premature stop codon mutations in the gene CLN2 encoding the
lysosomal tripeptidyl-peptidase 1 (TPP-I) are associated with
disease in approximately half of children diagnosed with LINCL. The
ability of the aminoglycoside gentamicin to restore TPP-I activity
in LINCL cell lines has been examined. In one patient-derived cell
line that is compound heterozygous for a commonly seen nonsense
mutation (Arg208Stop) and a different rare nonsense mutation,
approximately 7% of normal levels of TPP-I were maximally restored
with gentamicin treatment. These results suggest that
pharmacological suppression of nonsense mutations by
aminoglycosides or functionally similar pharmaceuticals may have
therapeutic potential in LINCL (Sleat et. al., Eur. J. Ped. Neurol.
5:Suppl A 57-62 (2001)).
[0011] In cultured cells having premature stop codons in the Cystic
Fibrosis Transmembrane Conductance Regulator (CFTR) gene, treatment
with aminoglycosides led to the production of full-length CFTR
(Bedwell et. al., Nat. Med. 3:1280-1284 (1997); Howard et. al. Nat.
Med. 2: 467-469 (1996)). In mouse models for Duchenne muscular
dystrophy, gentamicin sulfate was observed to suppress
translational termination at premature stop codons resulting in
full-length dystrophin (Barton-Davis et. al., J. Clin. Invest.
104:375-381 (1999)). A small increase in the amount of full-length
dystrophin provided protection against contraction-induced damage
in the mdx mice. The amino acid inserted at the site of the
nonsense codon was not determined in these studies.
[0012] Accordingly, small molecule therapeutics or prophylactics
that suppress premature translation termination by mediating the
misreading of the nonsense codon would be useful for the treatment
of a number of diseases. The discovery of small molecule drugs,
particularly orally bioavailable drugs, can lead to the
introduction of a broad spectrum of selective therapeutics or
prophylactics to the public which can be used against disease
caused by nonsense mutations is just beginning.
[0013] Clitocine
(6-Amino-5-nitro-4-(.beta.-D-ribo-furanosylamino)pyrimidine) is a
naturally occurring exocyclic amino nucleoside that was first
isolated from the mushroom Clitocybe inversa (Kubo et al., Tet.
Lett. 27: 4277 (1986)). The total synthesis of clitocine has also
been reported. (Moss et al., J. Med. Chem. 31:786-790 (1988) and
Kamikawa et al., J. Chem. Soc. Chem. Commun. 195 (1988)). Clitocine
has been reported to possess insecticidal activity and cytostatic
activity against leukemia cell lines (Kubo et al., Tet. Lett. 27:
4277 (1986) and Moss et al., J. Med Chem. 31:786-790 (1988)).
However, the use of clitocine as a therapeutic for diseases
associated with a nonsense mutation has not been disclosed until
now. Nor has anyone reported the development of an analogue or
derivative of clitocine that has utility as a therapeutic for
cancer or a disease associated with a nonsense mutation.
[0014] Thus, there remains a need to develop characterize and
optimize lead molecules for the development of novel drugs for
treating or preventing diseases associated with nonsense mutations
of mRNA. Accordingly, it is an object of the present invention to
provide such compounds.
[0015] All documents referred to herein are incorporated by
reference into the present application as though fully set forth
herein.
SUMMARY OF THE INVENTION
[0016] In accordance with the present invention, compounds that
suppress premature translation termination associated with a
nonsense mutation in mRNA have been identified, and methods for
their use provided.
[0017] In one aspect of the invention, compounds of Formula (1) are
provided which are useful for suppressing premature translation
termination associated with a nonsense mutation in mRNA, and for
treating diseases associated with nonsense mutations in mRNA:
##STR00001##
[0018] wherein:
[0019] X, Y, and Z are independently selected from N, S, O, and C
wherein at least one of X, Y or Z is a heteroatom;
[0020] R.sub.1 is hydrogen, a C.sub.1-C.sub.6 alkyl, or Na+, or
Mg.sup.2+;
[0021] R.sub.2 is independently absent; a hydrogen; a
--CH.dbd.N--OH group; a cyano group; a C.sub.1-C.sub.6 alkyl which
is optionally substituted with a hydroxy group; or a carbonyl group
which is optionally substituted with a hydrogen, a hydroxyl, or a
C.sub.1-C.sub.4 alkoxy group;
[0022] R.sub.3 is independently absent, a halogen, a hydroxy, a
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 alkoxy, or a nitro
group;
[0023] R.sub.4 is independently absent, a hydrogen, a
C.sub.1-C.sub.6 alkyl, or when taken together with W, R.sub.4 may
be a bond, and W and the heterocycle to which R.sub.4 and W are
attached form an eleven to thirteen membered hetero-tricycle ring
structure;
[0024] W is selected from: [0025] (a) a C.sub.2-C.sub.6 alkynyl,
optionally substituted with a phenyl; [0026] (b) a C.sub.1-C.sub.8
straight chain or branched chain alkyl which is optionally
substituted with one or more of the following independently
selected groups: a C.sub.1-C.sub.6 alkyl; a halogen; a
--C(.dbd.O)--NH-phenyl which phenyl is optionally substituted with
one or more independently selected halogens or C.sub.1-C.sub.4
alkyl groups; a five to six-membered heterocycle; a C.sub.6-C.sub.8
aryl which is optionally substituted with one or more groups
independently selected from a hydroxy, a halogen, a C.sub.1-C.sub.4
alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a C.sub.1-C.sub.4
alkoxy group or an amino group which is optionally substituted with
one or more C.sub.1-C.sub.4 alkyl groups; an aryloxy which is
optionally substituted with one or more of the following
independently selected groups: a hydroxy, a halogen, a
C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a
C.sub.1-C.sub.4 alkoxy group or an amino group which is optionally
substituted with one or more C.sub.1-C.sub.4 alkyl groups; [0027]
(C) C.sub.2 to C.sub.8 alkenyl; [0028] (d) a C.sub.3-C.sub.6
cycloalkyl optionally substituted with a C.sub.1-C.sub.6 alkyl;
[0029] (c) a C.sub.6-C.sub.8 aryl which is optionally substituted
with one or more of the following independently selected groups: a
hydroxy; a halogen; a C.sub.1-C.sub.4 straight chain or branched
chain alkyl which is optionally substituted with one or more
independently selected halogen or hydroxy groups; a C.sub.1-C.sub.4
alkoxy which is optionally substituted with one or more
independently selected halogen or phenyl groups; a C.sub.3-C.sub.8
cycloalkyl which is optionally substituted with one or more
independently selected C.sub.1-C.sub.4 alkyl groups; a
C.sub.6-C.sub.8 aryl which is optionally substituted with one or
more independently selected C.sub.1-C.sub.4 alkyl groups; an
aryloxy which is optionally substituted with one or more of the
following independently selected groups: a hydroxy, a halogen, a
C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a
C.sub.1-C.sub.4 alkoxy group, or an amino group which is optionally
substituted with one or more independently selected C.sub.1-C.sub.4
alkyl groups; a five to six-membered heterocycle which is
optionally substituted with one or more independently selected
C.sub.1-C.sub.4 alkyl, oxo, or C.sub.6-C.sub.8 aryl which is
optionally substituted with one or more of the following
independently selected groups: a hydroxy, a halogen, a
C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a
C.sub.1-C.sub.4 alkoxy group, or an amino group which is optionally
substituted with one or more independently selected C.sub.1-C.sub.4
alkyl groups, a naphthyl group which is optionally substituted with
an amino or aminoalkyl or alkoxy group; a --C(O)--NR.sub.xR.sub.y
group; a --C(O)--R.sub.x group; a isoindole-1,3-dione group; a
nitro group; a cyano group; a --SO.sub.3H group; alkylthio group;
alkyl sulfonyl group; a --NR.sub.x--C(O)--R.sub.z group; a
--NR.sub.xR.sub.y group; a --NR.sub.x--SO.sub.2--R.sub.2 group; a
--NR.sub.x--C(O)--NR.sub.xR.sub.y group; a
--NR.sub.x--C(O)O--R.sub.z group; [0030] (f) a C.sub.10-C.sub.14
aryl group optionally substituted with one or more independently
selected halogens, amino groups or aminoalkyl groups, or alkoxy
groups; [0031] (g) a --C(O)--NR.sub.xR.sub.y group; [0032] (h) a
five or six membered heterocycle which is optionally substituted
with one or more independently selected oxo groups; halogens;
C.sub.1-C.sub.4 alkyl groups; C.sub.1-C.sub.4 alkoxy groups;
C.sub.1-C.sub.4 haloalkyl groups; C.sub.1-C.sub.4 haloalkoxy
groups; aryloxy groups; --NR.sub.xR.sub.y groups; alkylthio groups;
--C(O)--R.sub.x groups; or C.sub.6 to C.sub.8 aryl groups which are
optionally substituted with one or more independently selected
halogens, C.sub.1-C.sub.4 alkyl groups C.sub.1-C.sub.4 alkoxy
groups; [0033] (i) a heterocycle group having two to three ring
structures that is optionally substituted with one or more
independently selected halogens, oxo groups, C.sub.1-C.sub.4 alkyl
groups, C.sub.1-C.sub.4 haloalkyl groups, or C.sub.1-C.sub.4 alkoxy
groups; [0034] (j) or W together with R.sub.4, including where
R.sub.4 is a bond, and the heterocycle to which R.sub.4 and W are
attached form an eleven to thirteen membered hetero-tricycle ring
structure; [0035] wherein R.sub.x is hydrogen, a C.sub.1-C.sub.6
alkyl group, or R.sub.x and R.sub.y together with the atoms to
which they are attached form a four to seven membered carbocycle or
heterocycle; [0036] R.sub.y is hydrogen, a C.sub.1-C.sub.6 alkyl
group; an aryl group optionally substituted with one or more
independently selected C.sub.1-C.sub.4 alkyl groups, or R.sub.x and
R.sub.y together with the atoms to which they are attached form a
four to seven membered carbocycle or heterocycle; and [0037]
R.sub.x is an C.sub.1-C.sub.6 alkyl optionally substituted with an
aryl or a halogen; or an aryl optionally substituted with a
halogen, a C.sub.1-C.sub.6 alkyl, or a C.sub.1-C.sub.6 alkoxy;
[0038] or a pharmaceutically acceptable salt, hydrate, solvate,
clathrate, racemate, stereoisomer, or polymorph of said compound of
Formula 1.
[0039] In another aspect of the invention, methods are provided for
the suppression of premature translation termination associated
with a nonsense mutation, and for the prevention or treatment of
diseases associated with nonsense mutations of mRNA. Such diseases
include, but are not limited to, genetic diseases caused by
premature translation termination associated with a nonsense
mutation, such as a CNS disease, an inflammatory disease, a
neurodegenerative disease, an autoimmune disease, a cardiovascular
disease, or a pulmonary disease; more preferably the disease is
cancer (or other proliferative diseases), amyloidosis, Alzheimer's
disease, atherosclerosis, giantism, dwarfism, hypothyroidism,
hyperthyroidism, cystic fibrosis, aging, obesity, Parkinson's
disease, Niemann Pick's disease, familial hypercholesterolemia,
retinitis pigmentosa, Marfan syndrome, lysosomal storage disorders,
the muscular dystrophies, cystic fibrosis, hemophilia, or classical
late infantile neuronal ceroid lipofuscinosis (LINCL).
[0040] In one embodiment, the invention is directed to methods for
suppressing premature translation termination associated with a
nonsense mutation in mRNA comprising administering a
nonsense-suppressing amount of at least one compound of the
invention to a subject in need thereof.
[0041] In yet another embodiment, methods for treating cancers,
lysosomal storage disorders, a muscular dystrophy, cystic fibrosis,
hemophilia, or classical late infantile neuronal ceroid
lipofuscinosis are provided comprising administering a
therapeutically effective amount of at least one compound of the
invention to a subject in need thereof.
[0042] These and other aspects of the invention will be more
clearly understood with reference to the following preferred
embodiments and detailed description.
CERTAIN EMBODIMENTS
[0043] 1. A method of treating or preventing a disease resulting
from a somatic mutation comprising administering to a patient in
need thereof an effective amount of a compound of Formula 1, or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
racemate, stereoisomer, or polymorph of said compound of Formula 1.
2. The method of embodiment 1, wherein the compound, or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate
polymorph, racemate, stereoisomer, or polymorph thereof, is
administered as a composition comprising the compound and a
pharmaceutically acceptable carrier or diluent. 3. The method of
embodiment 1, wherein the administration is intravenous. 4. A
method of treating or preventing an autoimmune disease, a blood
disease, a collagen disease, diabetes, a neurodegenerative disease,
a cardiovascular disease, a pulmonary disease, or an inflammatory
disease or central nervous system disease comprising administering
to a patient in need thereof an effective amount of a compound of
Formula 1, or a pharmaceutically acceptable salt, hydrate, solvate,
clathrate, racemate, stereoisomer, or polymorph thereof. 5. The
method of embodiment 4, wherein the administration is intravenous.
6. The method of embodiment 4, wherein the autoimmune disease is
rheumatoid arthritis or graft versus host disease. 7. The method of
embodiment 4, wherein the inflammatory disease is arthritis. 8. The
method of embodiment 4, wherein the central nervous system disease
is multiple sclerosis, muscular dystrophy, Duchenne muscular
dystrophy, Alzheimer's disease, a neurodegenerative disease or
Parkinson's disease. 9. The method of embodiment 4, wherein the
blood disorder is hemophilia, Von Willebrand disease,
ataxia-telangiectasia, .beta.-thalassemia or kidney stones. 10. The
method of embodiment 4, wherein the collagen disease is
osteogenesis imperfecta or cirrhosis. 11. A method of treating or
preventing familial polycythemia, immunodeficiency, kidney disease,
cystic fibrosis, familial hypercholesterolemia, retinitis
pigmentosa, amyloidosis, hemophilia, Alzheimer's disease, Tay Sachs
disease, Niemann Pick disease, Parkinson's disease,
atherosclerosis, giantism, dwarfism, hyperthyroidism, aging,
obesity. Duchenne muscular dystrophy or Marfan syndrome comprising
administering to a patient in need thereof an effective amount of a
compound of Formula 1, or a pharmaceutically acceptable salt,
hydrate, solvate, clathrate, racemate, stereoisomer, polymorph
thereof. 12. The method of embodiment 11, wherein the
administration is intravenous. 13. A method of treating or
preventing cancer in a human comprising administering to a human in
need thereof an effective amount of a compound of Formula 1, or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
racemate, stereoisomer, polymorph thereof. 14. The method of
embodiment 13, wherein the administration is intravenous. 15. The
method of embodiment 13, wherein the cancer is of the head and
neck, eye, skin, mouth, throat, esophagus, chest, bone, blood,
lung, colon, sigmoid, rectum, stomach, prostate, breast, ovaries,
kidney, liver, pancreas, brain, intestine, heart or adrenals. 16.
The method of embodiment 13, wherein the compound, or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
racemate, stereoisomer, or polymorph thereof, comprises a
pharmaceutically acceptable carrier or diluent. 17. The method of
embodiment 13, wherein the cancer is a solid tumor. 18. The method
of embodiment 13, wherein the cancer is sarcoma, carcinoma,
fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic
neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,
retinoblastoma, a blood-born tumor or multiple myeloma. 19. The
method of embodiment 13, wherein the cancer is acute lymphoblastic
leukemia, acute lymphoblastic B-cell leukemia, acute lymphoblastic
T-cell leukemia, acute myeloblastic leukemia, acute promyelocytic
leukemia, acute monoblastic leukemia, acute erythroleukemic
leukemia, acute megakaryoblastic leukemia, acute myelomonocytic
leukemia, acute nonlymphocytic leukemia, acute undifferentiated
leukemia, chronic myelocytic leukemia, chronic lymphocytic
leukemia, hairy cell leukemia, or multiple myeloma. 20. A method of
treating or preventing a disease associated with a mutation of the
p53 gene comprising administering to a patient in need thereof an
effective amount of a compound of Formula 1, or a pharmaceutically
acceptable salt, hydrate, solvate, clathrate, racemate,
stereoisomer, or polymorph thereof. 21. The method of embodiment
20, wherein the administration is intravenous. 22. The method of
embodiment 20, wherein the disease is sarcoma, carcinomas,
fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic
neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma or
retinoblastoma. 23. A method of inhibiting the growth of a cancer
cell comprising contacting the cancer cell with an effective amount
of a compound of Formula 1, or a pharmaceutically acceptable salt,
hydrate, solvate, clathrate, racemate, stereoisomer, or polymorph
thereof. 24. A method for selectively producing a protein in a
mammal comprising,
[0044] transcribing a gene containing a nonsense mutation in the
mammal; and
[0045] providing an effective amount of a compound of the present
invention to said mammal, wherein said protein is produced by said
mammal.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 provides schematic representations of constructs for
luciferase: based assays to evaluate the suppression of a nonsense
mutation.
[0047] FIG. 2 provides schematic representations of the luciferase
constructs engineered to harbor one or more epitope tags in the
N-terminus of the luciferase protein.
[0048] FIG. 3 provides schematic representations of constructs for
luciferase based assays to evaluate readthrough efficiency.
[0049] FIG. 4 provides results from mdx mouse cells and muscle.
DETAILED DESCRIPTION OF THE INVENTION
[0050] Premature translation termination can produce aberrant
proteins which can be lethal or can cause a number of diseases,
including as non-limiting examples, cancers, lysosomal storage
disorders, the muscular dystrophies, cystic fibrosis and
hemophilia. In accordance with the present invention, compounds
that suppress nonsense mutations have been identified, and methods
for their use provided.
[0051] A. Compounds of the Invention
[0052] In one aspect of the invention, compounds of the invention
are provided which are useful in suppression of a nonsense
mutation. In certain embodiments, the compounds of the invention
specifically suppresses a nonsense mutation, while in other
embodiments, the compounds of the invention suppress a nonsense
mutation as well as treat a disease, including as non-limiting
examples, cancers, lysosomal storage disorders, the muscular
dystrophies, cystic fibrosis and hemophilia.
[0053] Preferred compounds of the present invention useful in the
suppression of a nonsense mutation include those of Formula (1) as
shown below.
##STR00002##
wherein:
[0054] X, Y, and Z are independently selected from N, S, O, and C
wherein at least one of X, Y or Z is a heteroatom;
[0055] R.sub.1 is hydrogen, a C.sub.1-C.sub.6 alkyl, or Na+, or
Mg.sup.2+;
[0056] R.sub.1 is hydrogen, a C.sub.1-C.sub.6 alkyl, or Na+, or
Mg.sup.2+;
[0057] R.sub.2 is independently absent; a hydrogen; a
--CH.dbd.N--OH group; a cyano group; a C.sub.1-C.sub.6 alkyl which
is optionally substituted with a hydroxy group; or a carbonyl group
which is optionally substituted with a hydrogen, a hydroxyl, or a
C.sub.1-C.sub.4 alkoxy group;
[0058] R.sub.3 is independently absent, a halogen, a hydroxy, a
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 alkoxy, or a nitro
group;
[0059] R.sub.4 is independently absent, a hydrogen, a
C.sub.1-C.sub.6 alkyl, or when taken together with W, R.sub.4 may
be a bond, and W and the heterocycle to which R.sub.4 and W are
attached form an eleven to thirteen membered hetero-tricycle ring
structure;
[0060] W is selected from: [0061] (a) a C.sub.2-C.sub.6 alkynyl,
optionally substituted with a phenyl; [0062] (b) a C.sub.1-C.sub.8
straight chain or branched chain alkyl which is optionally
substituted with one or more of the following independently
selected groups; a C.sub.1-C.sub.6 alkyl; a halogen; a
--C(.dbd.O)NH-phenyl which phenyl is optionally substituted with
one or more independently selected halogens or C.sub.1-C.sub.4
alkyl groups; a five to six-membered heterocycle; a C.sub.6-C.sub.8
aryl which is optionally substituted with one or more groups
independently selected from a hydroxy, a halogen, a C.sub.1-C.sub.4
alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a C.sub.1-C.sub.4
alkoxy group or an amino group which is optionally substituted with
one or more C.sub.1-C.sub.4 alkyl groups; an aryloxy which is
optionally substituted with one or more of the following
independently selected groups: a hydroxy, a halogen, a
C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a
C.sub.1-C.sub.4 alkoxy group or an amino group which is optionally
substituted with one or more C.sub.1-C.sub.4 alkyl groups; [0063]
(c) C.sub.2 to C.sub.8 alkenyl; [0064] (d) a C.sub.3-C.sub.8
cycloalkyl optionally substituted with a C.sub.1-C.sub.6 alkyl;
[0065] (e) a C.sub.6-C.sub.8 aryl which is optionally substituted
with one or more of the following independently selected groups: a
hydroxy; a halogen; a C.sub.1-C.sub.4 straight chain or branched
chain alkyl which is optionally substituted with one or more
independently selected halogen or hydroxy groups; a C.sub.1-C.sub.4
alkoxy which is optionally substituted with one or more
independently selected halogen or phenyl groups; a C.sub.3-C.sub.8
cycloalkyl which is optionally substituted with one or more
independently selected C.sub.1-C.sub.4 alkyl groups; a
C.sub.6-C.sub.8 aryl which is optionally substituted with one or
more independently selected C.sub.1-C.sub.4 alkyl groups; an
aryloxy which is optionally substituted with one or more of the
following independently selected groups: a hydroxy, a halogen, a
C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a
C.sub.1-C.sub.4 alkoxy group, or an amino group which is optionally
substituted with one or more independently selected C.sub.1-C.sub.4
alkyl groups; a five to six-membered heterocycle which is
optionally substituted with one or more independently selected
C.sub.1-C.sub.4 alkyl, oxo, or C.sub.6-C.sub.8 aryl which is
optionally substituted with one or more of the following
independently selected groups: a hydroxy, a halogen, a
C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a
C.sub.1-C.sub.4 alkoxy group, or an amino group which is optionally
substituted with one or more independently selected C.sub.1-C.sub.4
alkyl groups; a naphthyl group which is optionally substituted with
an amino or aminoalkyl or alkoxy group; a --C(O)--NR.sub.xR.sub.y
group; a --C(O)--R.sub.x group; a isoindole-1,3-dione group; a
nitro group; a cyano group; a --SO.sub.3H group; alkylthio group;
alkyl sulfonyl group; a --NR.sub.x--C(O)--R.sub.z group; a
--NR.sub.xR.sub.y group; a --NR.sub.x--SO.sub.2--R.sub.z group; a
--NR.sub.x--C(O)--NR.sub.xR.sub.y group; a
--NR.sub.x--C(O)O--R.sub.x group; [0066] (f) a C.sub.10-C.sub.14
aryl group optionally substituted with one or more independently
selected halogens, amino groups or aminoalkyl groups, or alkoxy
groups; [0067] (g) a --C(O)--NR.sub.xR.sub.y group; [0068] (h) a
five or six membered heterocycle which is optionally substituted
with one or more independently selected oxo groups; halogens;
C.sub.1-C.sub.4 alkyl groups; C.sub.1-C.sub.4 alkoxy groups;
C.sub.1-C.sub.4 haloalkyl groups; C.sub.1-C.sub.4 haloalkoxy
groups; aryloxy groups; --NR.sub.xR.sub.y groups; alkylthio groups;
--C(O)--R.sub.x groups; or C.sub.6 to C.sub.8 aryl groups which are
optionally substituted with one or more independently selected
halogens, C.sub.1-C.sub.4 alkyl groups, C.sub.1-C.sub.4 alkoxy
groups; [0069] (i) a heterocycle group having two to three ring
structures that is optionally substituted with one or more
independently selected halogens, oxo groups, C.sub.1-C.sub.4 alkyl
groups, C.sub.1-C.sub.4 haloalkyl groups, or C.sub.1-C.sub.4 alkoxy
groups; [0070] (j) or W together with R.sub.4, including where
R.sub.4 is a bond, and the heterocycle to which R.sub.4 and W are
attached form an eleven to thirteen membered hetero-tricycle ting
structure; [0071] wherein R.sub.x is hydrogen, a C.sub.1-C.sub.6
alkyl group, or R.sub.x and R.sub.y together with the atoms to
which they are attached form a four to seven membered carbocycle or
heterocycle; [0072] R.sub.y is hydrogen, a C.sub.1-C.sub.6 alkyl
group; an aryl group optionally substituted with one or more
independently selected C.sub.1-C.sub.4 alkyl groups, or R.sub.x and
R.sub.y together with the atoms to which they are attached form a
four to seven membered carbocycle or heterocycle; and [0073]
R.sub.z is an C.sub.1-C.sub.6 alkyl optionally substituted with an
aryl or a halogen; or an aryl optionally substituted with a
halogen, a C.sub.1-C.sub.6 alkyl, or a C.sub.1-C.sub.6 alkoxy;
[0074] or a pharmaceutically acceptable salt, hydrate, solvate,
clathrate, racemate, stereoisomer, or polymorph of said compound of
Formula 1.
[0075] In another embodiment, compounds of the present invention
useful in the suppression of a nonsense mutation include compounds
of Formula (1) wherein:
[0076] X, Y, and Z are independently selected from N, S, O, and C
wherein at least one of X, Y or Z is a heteroatom;
[0077] R.sub.1 is hydrogen or a C.sub.1-C.sub.6 alkyl; or Na.sup.+
or Mg.sup.2+
[0078] R.sub.2 is independently absent; hydrogen; a C.sub.1-C.sub.6
alkyl which is optionally substituted with a hydroxy group; a
carbonyl group which is optionally substituted with a hydroxyl, a
C.sub.1-C.sub.4 alkoxy group; a --CH.dbd.N--OH group; or a cyano
group;
[0079] R.sub.3 is absent, a halogen, a hydroxy, a C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.4 alkoxy, or a nitro group;
[0080] R.sub.4 is absent; a C.sub.1 to C.sub.6 alkyl; or together
with W and the heterocycle to which R.sub.4 and W are attached form
an eleven to thirteen membered hetero-tricycle ring structure;
[0081] W is selected from: [0082] a C.sub.1-C.sub.8 straight chain
or branched chain alkyl which is optionally substituted with one or
more of the following: a C.sub.1-C.sub.6 alkyl, a halogen, a five
to six-membered heterocycle, a C.sub.6-C.sub.8 aryl which is
optionally substituted with one or more of the following: a
hydroxy, a halogen, a C.sub.1-C.sub.4 alkyl group, a
C.sub.1-C.sub.4 haloalkyl group, a C.sub.1-C.sub.4 alkoxy group or
an amino group which is optionally substituted with one or more
C.sub.1-C.sub.4 alkyl groups; an aryloxy which is optionally
substituted with one or more of the following: a hydroxy, a
halogen, a C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl
group, a C.sub.1-C.sub.4 alkoxy group or an amino group which is
optionally substituted with one or more C.sub.1-C.sub.4 alkyl
groups; [0083] C.sub.2 to C.sub.8 alkenyl; [0084] a C.sub.3-C.sub.8
cycloalkyl optionally substituted with a C.sub.1 to C.sub.6 alkyl;
[0085] a C.sub.6-C.sub.8 aryl which is optionally substituted with
one or more of the following: a hydroxy, a halogen; a
C.sub.1-C.sub.4 straight chain or branched chain alkyl which is
optionally substituted with one or more independently selected
halogen or hydroxy groups; a C.sub.1-C.sub.4 alkoxy which is
optionally substituted with one or more independently selected
halogen or phenyl groups; a C.sub.3-C.sub.8 cycloalkyl which is
optionally substituted with one or more independently selected
C.sub.1-C.sub.4 alkyl groups; a C.sub.6-C.sub.8 aryl which is
optionally substituted with one or more independently selected
C.sub.1-C.sub.4 alkyl groups; an aryloxy which is optionally
substituted with one or more of the following: a hydroxy, a
halogen, a C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl
group, a C.sub.1-C.sub.4 alkoxy group, or an amino group which is
optionally substituted with one or more C.sub.1-C.sub.4 alkyl
groups; a five to six-membered heterocycle which is optionally
substituted with one or more independently selected C.sub.1-C.sub.4
alkyl, oxo, or C.sub.6-C.sub.8 aryl which is optionally substituted
with one or more of the following: a hydroxy, a halogen, a
C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a
C.sub.1-C.sub.4 alkoxy group, or an amino group which is optionally
substituted with one or more C.sub.1-C.sub.4 alkyl groups; a
naphthyl group which is optionally substituted with an amino or
aminoalkyl group; a --C(O)--NR.sub.xR.sub.y group; a
--C(O)--R.sub.x group; a isoindole-1,3-dione group; a nitro group;
a cyano group; a --SO.sub.3H group; alkylthio group; alkyl sulfonyl
group; a --NR.sub.x--C(O)--R.sub.z group; a --NR.sub.xR.sub.y
group; a --NR.sub.x--SO.sub.2--R.sub.z group; a
--NR.sub.x--C(O)--NR.sub.xR.sub.y group; a
--NR.sub.x--C(O)O--R.sub.z group; [0086] a --C(O)--NR.sub.xR.sub.y
group; [0087] a five or six membered heterocycle which is
optionally substituted with one or more oxo groups, halogens,
C.sub.1 to C.sub.4 alkyl groups, C.sub.1 to C.sub.4 haloalkyl
groups, --C(O)--R.sub.x groups, and/or C.sub.6 to C.sub.8 aryl
groups which are optionally substituted with one or more
independently selected halogens, C.sub.1 to C.sub.4 alkyl groups,
C.sub.1 to C.sub.4 alkoxy groups, aryloxy groups, --NR.sub.xR.sub.y
groups, and/or alkylthio groups; [0088] a heterocycle group having
two to three ring structures that is optionally substituted with
one or more halogens, C.sub.1 to C.sub.4 alkyl groups, C.sub.1 to
C.sub.4 haloalkyl groups, and/or C.sub.1 to C.sub.4 alkoxy groups;
[0089] or W together with R.sub.4 and the heterocycle to which
R.sub.4 and W are attached form an eleven to thirteen membered
hetero-tricycle ring structure; [0090] wherein R.sub.x is hydrogen,
a C.sub.1 to C.sub.6 alkyl group, or R.sub.x and R.sub.y together
with the atoms to which they are attached form a four to seven
membered carbocycle or heterocycle; [0091] R.sub.y is hydrogen, a
C.sub.1 to C.sub.6 alkyl group; an optionally substituted aryl, or
R.sub.x and R.sub.y together with the atoms to which they are
attached form a four to seven membered carbocycle or heterocycle;
and [0092] R.sub.z is an C.sub.1 to C.sub.6 alkyl optionally
substituted with an aryl or a halogen; or an aryl optionally
substituted with a halogen, a C.sub.1 to C.sub.6 alkyl, or a
C.sub.1 to C.sub.6 alkoxy;
[0093] or a pharmaceutically acceptable salt, hydrate, solvate,
clathrate, racemate, stereoisomer, or polymorph of said compound of
Formula 1.
[0094] In a preferred embodiment of Formula 1, when Y and Z are
both N, and X is O, the --C(O)--O--R.sub.1 group of the phenyl ring
is not in the meta position. In an alternative embodiment, when Y
and Z are both N, and X is O, the --C(O)--O--R.sub.1 group of the
phenyl ring is in the ortho or para position.
[0095] In a preferred embodiment of Formula 1, when W is a five or
six membered optionally substituted heterocycle, the heterocycle
may be selected from the group consisting of: a thienyl group, a
furyl group, a pyrazinyl group, a pyrimidinyl group, a pyridazinyl
group, a piperidyl group and a pyridyl group; and the heterocycle
may be optionally substituted with one or more independently
selected oxo groups; halogens; C.sub.1 to C.sub.4 alkyl groups;
C.sub.1 to C.sub.4 haloalkyl groups; --C(O)--R.sub.x groups; and/or
C.sub.6 to C.sub.8 aryl groups which are optionally substituted
with one or more independently selected halogens, C.sub.1 to
C.sub.4 alkyl groups, C.sub.1 to C.sub.4 alkoxy groups, aryloxy
groups, --NR.sub.xR.sub.y groups, and/or alkylthio groups
[0096] In another preferred embodiment of Formula 1, when W is a
five or six membered optionally substituted heterocycle, the
optionally substituted heterocycle may be selected from the group
consisting of: a thienyl group; a furyl group; a pyrazinyl group
which is optionally substituted with a C.sub.1-C.sub.4 alkyl group;
a pyrimidinyl group optionally substituted with one or two oxo
groups; a pyridazinyl group which is optionally substituted with
one or two oxo groups; a piperidyl group which is optionally
substituted with a --C(O)--R.sub.x group; and a pyridyl group which
is optionally substituted with one or more of the following: a
halogen; a C.sub.1-C.sub.4 alkyl group; C.sub.1-C.sub.4 haloalkyl
group; a C.sub.6-C.sub.8 aryl group which is optionally substituted
with one more independently selected C.sub.1-C.sub.4 alkyl groups;
a C.sub.1-C.sub.4 alkoxy group; an aryloxy group; --NR.sub.xR.sub.y
group; and an alkylthio group.
[0097] In yet another preferred embodiment of Formula 1, when W is
an optionally substituted heterocycle having two to three ring
structures, the heterocycle may be selected from the group
consisting of: a benzodioxolyl group; a benzo[1,3]dioxinyl group
which is optionally substituted with one or more independently
selected halogens; a benzimidazolyl group which is optionally
substituted with one or more independently selected C.sub.1-C.sub.4
alkyl or C.sub.1-C.sub.4 haloalkyl groups; a benzothiazolyl group;
a benzotriazolyl group which is optionally substituted with one or
more independently selected C.sub.1-C.sub.4 alkyl groups; a
benzothienyl group which is optionally substituted with one or more
independently selected C.sub.1-C.sub.4 alkyl groups; a
benzo[1,2,5]oxadiazolyl group; a 2,3-dihydrobenzo[1,4]dioxinyl
group; a benzofuryl group; a quinoxalinyl group; an indolyl group;
a quinolinyl group; and a substituent selected from the group
consisting of: (* indicating bond of attachment):
##STR00003##
[0098] As recognized by one of skill in the art, certain compounds
of the invention may include at least one chiral center, and as
such may exist as racemic mixtures or as enantiomerically pure
compositions. As used herein, "enantiomerically pure" refers to
compositions consisting substantially of a single isomer,
preferably consisting of 90%, 92%, 95%, 98%, 99%, or 100% of a
single isomer.
[0099] As used herein, the term "alkyl" generally refers to
saturated hydrocarbyl radicals of straight, branched or cyclic
configuration including methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl,
cyclohexyl, n-heptyl, octyl, n-octyl, and the like. In some
embodiments, alkyl substituents may be C.sub.1 to C.sub.8, C.sub.3
to C.sub.8, C.sub.1 to C.sub.6, or C.sub.1 to C.sub.4 alkyl groups.
In certain embodiments, the alkyl group may be optionally
substituted with one or more halogen or alkoxy groups. For
instance, the alkyl group may include one or more halogen
substituents to form a haloalkyl, including monohaloalkyl,
dihaloalkyl, and trihaloalkyl.
[0100] As used herein, "alkenyl" generally refers to linear,
branched or cyclic alkene radicals having one or more carbon-carbon
double bonds, such as C.sub.2 to C.sub.6 alkylene groups including
3-propenyl.
[0101] As used herein, "aryl" refers to a carbocyclic aromatic ring
structure. Included in the scope of aryl groups are aromatic rings
having from five to twenty carbon atoms. Aryl ring structures
include compounds having one or more ring structures, such as
mono-, bi-, or tricyclic compounds. Examples of aryl groups that
include phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl,
phenanthrenyl (i.e., phenanthrene), and naphthyl (i.e.,
naphthalene) ring structures. In certain embodiments, the aryl
group may be optionally substituted.
[0102] As used herein, "heterocycle" refers to cyclic ring
structures in which one or more atoms in the ring, the
heteroatom(s), is an element other than carbon. Heteroatoms are
typically O, S or N atoms. Included within the scope of
heterocycle, and independently selectable, are O, N, and S
heterocycle ring structures. The heterocyclic ring structure may
include compounds having one or more ring structures, such as
mono-, bi-, or tricyclic compounds, and may be aromatic, i.e., the
ring structure may be a heteroaryl. Heterocycle may include a
benzofused heterocyclic ring structure. Non-limiting exemplary
heterocyclo groups include morpholinyl, pyrrolidinonyl,
pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl,
valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,
tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl,
tetrahydrothiophenyl or tetrahydrothiopyranyl, benzodioxolyl,
benzothiazolyl, dihydrobenzodioxine, dihydroisoindolyl,
dihydrobenzoimidazolyl and the like. In certain embodiments, the
heterocycle may optionally be substituted. As used herein,
"heteroaryl" refers to cyclic aromatic ring structures in which one
or more atoms in the ring, the heteroatom(s), is an element other
than carbon. Heteroatoms are typically O, S or N atoms. Included
within the scope of heteroaryl, and independently selectable, are
O, N, and S heteroaryl ring structures. The ring structure may
include compounds having one or more ring structures, such as
mono-, bi-, or tricyclic compounds. In some embodiments, the
heteroaryl groups may be selected from heteroaryl groups that
contain two or more heteroatoms, three or more heteroatoms, or four
or more heteroatoms. Heteroaryl ring structures may be selected
from those that contain five or more atoms, six or more atoms, or
eight or more atoms. In a preferred embodiment, the heteroaryl
including five to ten atoms. Examples of heteroaryl ring structures
include: acridine, benzimidazole, benzoxazole, benzodioxole,
benzofuran, 1,3-diazine, 1,2-diazine, 1,2-diazole,
1,4-diazanaphthalene, furan, furazan, imidazole, indole, isoxazole,
isoquinoline, isothiazole, oxazole, purine, pyridazine, pyrazole,
pyridine, pyrazine, pyrimidine, pyrrole, quinoline, quinoxaline,
thiazole, thiophene, 1,3,5-triazine, 1,2,4-triazine,
1,2,3-triazine, tetrazole and quinazoline.
[0103] As used herein, "alkoxy" generally refers to a group with
the structure --O--R. In certain embodiments, R may be an alkyl
group, such as a C.sub.1 to C.sub.8, C.sub.1 to C.sub.6 alkyl
group, or C.sub.1 to C.sub.4 alkyl group. In certain embodiments,
the R group of the alkoxy may optionally be substituted with at
least one halogen. For example, the R group of the alkoxy may be a
haloalkyl, i.e., haloalkoxy.
[0104] Halogen substituents may be independently selected from the
halogens such as fluorine, chlorine, bromine, iodine, and
astatine.
[0105] For the purposes of this invention, where one or more
functionalities or substituents are incorporated into a compound of
the invention, including preferred embodiments, each functionality
or substituent appearing at any location within the disclosed
compounds may be independently selected, and as appropriate,
independently substituted. Further, where a more generic
substituent is set forth for any position in the molecules of the
present invention, it is understood that the generic substituent
may be replaced with more specific substituents, and the resulting
molecules are within the scope of the molecules of the present
invention.
[0106] With reference is Formula 1, preferred W groups include
those shown in the table below (* indicates the bond of
attachment).
TABLE-US-00001 ##STR00004## ##STR00005## ##STR00006## ##STR00007##
##STR00008## ##STR00009## ##STR00010## ##STR00011## ##STR00012##
##STR00013## ##STR00014## ##STR00015## ##STR00016## ##STR00017##
##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022##
##STR00023## ##STR00024## ##STR00025## ##STR00026## ##STR00027##
##STR00028## ##STR00029## ##STR00030## ##STR00031## ##STR00032##
##STR00033## ##STR00034## ##STR00035## ##STR00036## ##STR00037##
##STR00038## ##STR00039## ##STR00040## ##STR00041## ##STR00042##
##STR00043## ##STR00044## ##STR00045## ##STR00046## ##STR00047##
##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052##
##STR00053## ##STR00054## ##STR00055## ##STR00056## ##STR00057##
##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062##
##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067##
##STR00068## ##STR00069## ##STR00070## ##STR00071## ##STR00072##
##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077##
##STR00078## ##STR00079## ##STR00080## ##STR00081## ##STR00082##
##STR00083## ##STR00084## ##STR00085## ##STR00086## ##STR00087##
##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092##
##STR00093## ##STR00094## ##STR00095## ##STR00096## ##STR00097##
##STR00098## ##STR00099## ##STR00100## ##STR00101## ##STR00102##
##STR00103## ##STR00104## ##STR00105## ##STR00106## ##STR00107##
##STR00108## ##STR00109## ##STR00110## ##STR00111## ##STR00112##
##STR00113## ##STR00114## ##STR00115## ##STR00116## ##STR00117##
##STR00118## ##STR00119## ##STR00120## ##STR00121## ##STR00122##
##STR00123## ##STR00124## ##STR00125## ##STR00126## ##STR00127##
##STR00128##
##STR00129## ##STR00130## ##STR00131## ##STR00132## ##STR00133##
##STR00134## ##STR00135## ##STR00136## ##STR00137## ##STR00138##
##STR00139## ##STR00140## ##STR00141## ##STR00142## ##STR00143##
##STR00144## ##STR00145## ##STR00146## ##STR00147## ##STR00148##
##STR00149## ##STR00150## ##STR00151## ##STR00152## ##STR00153##
##STR00154## ##STR00155## ##STR00156## ##STR00157## ##STR00158##
##STR00159## ##STR00160## ##STR00161## ##STR00162## ##STR00163##
##STR00164## ##STR00165## ##STR00166## ##STR00167## ##STR00168##
##STR00169## ##STR00170## ##STR00171## ##STR00172## ##STR00173##
##STR00174## ##STR00175## ##STR00176## ##STR00177## ##STR00178##
##STR00179## ##STR00180## ##STR00181## ##STR00182## ##STR00183##
##STR00184## ##STR00185## ##STR00186## ##STR00187## ##STR00188##
##STR00189## ##STR00190## ##STR00191## ##STR00192## ##STR00193##
##STR00194## ##STR00195## ##STR00196## ##STR00197## ##STR00198##
##STR00199## ##STR00200## ##STR00201## ##STR00202## ##STR00203##
##STR00204## ##STR00205## ##STR00206## ##STR00207## ##STR00208##
##STR00209## ##STR00210## ##STR00211## ##STR00212## ##STR00213##
##STR00214## ##STR00215## ##STR00216## ##STR00217## ##STR00218##
##STR00219## ##STR00220## ##STR00221##
[0107] In a preferred embodiment, compounds of Formula 1 include
the compounds of Formula 1-A:
##STR00222##
[0108] With reference to Formula 1-A, in a preferred embodiment,
the carboxy group is preferably in the meta or para position. In
another preferred embodiment, the carboxy group is preferably in
the para position. Further, R.sub.3 is preferably absent, a
halogen, a C.sub.1-C.sub.4 alkoxy, or a nitro group. In one
preferred embodiment of the compounds of Formula 1-A, W is a
C.sub.6-C.sub.8 aryl, optionally substituted as in Formula 1. In
another embodiment of Formula 1-A, preferred W groups are shown in
the table below,
TABLE-US-00002 ##STR00223## ##STR00224## ##STR00225## ##STR00226##
##STR00227## ##STR00228## ##STR00229## ##STR00230## ##STR00231##
##STR00232## ##STR00233## ##STR00234## ##STR00235## ##STR00236##
##STR00237## ##STR00238## ##STR00239## ##STR00240## ##STR00241##
##STR00242## ##STR00243## ##STR00244## ##STR00245## ##STR00246##
##STR00247## ##STR00248## ##STR00249## ##STR00250## ##STR00251##
##STR00252## ##STR00253## ##STR00254## ##STR00255## ##STR00256##
##STR00257## ##STR00258## ##STR00259## ##STR00260## ##STR00261##
##STR00262## ##STR00263## ##STR00264## ##STR00265## ##STR00266##
##STR00267## ##STR00268## ##STR00269## ##STR00270## ##STR00271##
##STR00272## ##STR00273## ##STR00274## ##STR00275## ##STR00276##
##STR00277## ##STR00278## ##STR00279## ##STR00280## ##STR00281##
##STR00282## ##STR00283## ##STR00284## ##STR00285## ##STR00286##
##STR00287## ##STR00288## ##STR00289## ##STR00290## ##STR00291##
##STR00292## ##STR00293## ##STR00294## ##STR00295## ##STR00296##
##STR00297## ##STR00298## ##STR00299## ##STR00300## ##STR00301##
##STR00302## ##STR00303## ##STR00304## ##STR00305## ##STR00306##
##STR00307## ##STR00308## ##STR00309## ##STR00310## ##STR00311##
##STR00312## ##STR00313## ##STR00314## ##STR00315## ##STR00316##
##STR00317## ##STR00318## ##STR00319## ##STR00320## ##STR00321##
##STR00322## ##STR00323## ##STR00324## ##STR00325## ##STR00326##
##STR00327## ##STR00328## ##STR00329## ##STR00330## ##STR00331##
##STR00332## ##STR00333## ##STR00334## ##STR00335## ##STR00336##
##STR00337## ##STR00338## ##STR00339## ##STR00340## ##STR00341##
##STR00342## ##STR00343## ##STR00344## ##STR00345## ##STR00346##
##STR00347##
##STR00348## ##STR00349## ##STR00350## ##STR00351## ##STR00352##
##STR00353## ##STR00354## ##STR00355## ##STR00356## ##STR00357##
##STR00358## ##STR00359## ##STR00360## ##STR00361## ##STR00362##
##STR00363## ##STR00364## ##STR00365## ##STR00366## ##STR00367##
##STR00368## ##STR00369## ##STR00370## ##STR00371## ##STR00372##
##STR00373## ##STR00374## ##STR00375## ##STR00376## ##STR00377##
##STR00378## ##STR00379## ##STR00380## ##STR00381## ##STR00382##
##STR00383## ##STR00384##
[0109] In another preferred embodiment, compounds of Formula 1
include the compounds of Formula 1-B:
##STR00385##
1-B
[0110] With reference to Formula 1-B, in an embodiment, the carboxy
group is preferably in the para position. In another embodiment, W
is preferably a C.sub.6-C.sub.8 aryl, optionally substituted as in
Formula 1; and more preferably a phenyl optionally substituted with
a C.sub.1-C.sub.4 alkyl. A preferred compound of Formula 1-B is
shown below.
##STR00386##
[0111] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-C:
##STR00387##
[0112] With reference to Formula 1-C, in an embodiment, the carboxy
group is in the para position. In another embodiment, W is
preferably a C.sub.6-C.sub.8 aryl, optionally substituted as in
Formula 1.
[0113] In another embodiment, preferred W groups include those
shown in the table below:
TABLE-US-00003 ##STR00388## ##STR00389## ##STR00390## ##STR00391##
##STR00392## ##STR00393## ##STR00394## ##STR00395## ##STR00396##
##STR00397## ##STR00398## ##STR00399## ##STR00400## ##STR00401##
##STR00402## ##STR00403## ##STR00404## ##STR00405## ##STR00406##
##STR00407## ##STR00408## ##STR00409## ##STR00410## ##STR00411##
##STR00412## ##STR00413## ##STR00414## ##STR00415## ##STR00416##
##STR00417## ##STR00418## ##STR00419## ##STR00420## ##STR00421##
##STR00422## ##STR00423## ##STR00424## ##STR00425## ##STR00426##
##STR00427## ##STR00428## ##STR00429##
[0114] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-D:
##STR00430##
[0115] With reference to Formula 1-D, in an embodiment, the carboxy
group is preferably in the meta or para position. Further, R.sub.1
is preferably hydrogen or methyl. R.sub.3 is preferably in the meta
position. In one embodiment, W is preferably a C.sub.6-C.sub.8
aryl, optionally substituted as in Formula 1. In another
embodiment, preferred W groups include those shown in the table
below.
TABLE-US-00004 ##STR00431## ##STR00432## ##STR00433## ##STR00434##
##STR00435## ##STR00436## ##STR00437## ##STR00438## ##STR00439##
##STR00440## ##STR00441## ##STR00442## ##STR00443## ##STR00444##
##STR00445## ##STR00446## ##STR00447##
[0116] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-E:
##STR00448##
[0117] With reference to Formula 1-E, in an embodiment, the carboxy
group is preferably in the mete, or para position. Further, in an
embodiment, R.sub.4 is preferably hydrogen. In one embodiment, W is
preferably a C.sub.6-C.sub.8 aryl, optionally substituted as in
Formula 1. In another embodiment, preferred W groups include those
shown in the table below.
TABLE-US-00005 ##STR00449## ##STR00450## ##STR00451## ##STR00452##
##STR00453## ##STR00454## ##STR00455## ##STR00456## ##STR00457##
##STR00458## ##STR00459## ##STR00460## ##STR00461## ##STR00462##
##STR00463## ##STR00464## ##STR00465## ##STR00466## ##STR00467##
##STR00468## ##STR00469## ##STR00470## ##STR00471## ##STR00472##
##STR00473## ##STR00474## ##STR00475##
[0118] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-F:
##STR00476##
[0119] With reference to Formula 1-F, in an embodiment, the carboxy
group is preferably in the meta or para position, more preferably
the meta position. In a further embodiment, R.sub.4 is preferably
hydrogen. In one embodiment. W is preferably a C.sub.6-C.sub.8
aryl, optionally substituted as in Formula 1. In another
embodiment, preferred W groups include those shown in the table
below.
TABLE-US-00006 ##STR00477## ##STR00478## ##STR00479## ##STR00480##
##STR00481## ##STR00482## ##STR00483## ##STR00484## ##STR00485##
##STR00486## ##STR00487## ##STR00488## ##STR00489## ##STR00490##
##STR00491## ##STR00492## ##STR00493## ##STR00494## ##STR00495##
##STR00496## ##STR00497## ##STR00498## ##STR00499## ##STR00500##
##STR00501## ##STR00502## ##STR00503## ##STR00504## ##STR00505##
##STR00506## ##STR00507## ##STR00508##
[0120] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-G:
##STR00509##
[0121] With reference to Formula 1-G, in an embodiment, the carboxy
group is preferably in the meta position. In one embodiment, W is
preferably a C.sub.6-C.sub.8 aryl, optionally substituted as in
Formula 1. In another embodiment, preferred W groups include those
shown in the table below.
TABLE-US-00007 ##STR00510## ##STR00511## ##STR00512##
##STR00513##
[0122] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-H:
##STR00514##
[0123] With reference to Formula 1-H, in an embodiment, the carboxy
group is preferably in the meta position. In a further embodiment,
R.sub.2 is preferably hydrogen or a C.sub.1 to C.sub.4 alkyl.
R.sub.3, if present, is preferably in the ortho position, and is
preferably a hydroxy group. In one embodiment, W is preferably a
C.sub.6-C.sub.8 aryl, optionally substituted as in Formula 1, and
more preferably a phenyl group. In another embodiment, a preferred
compound of Formula 1-H is shown below.
##STR00515##
[0124] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-I:
##STR00516##
[0125] With reference to Formula 1-I, in an embodiment, the carboxy
group is preferably in the meta position. In a further embodiment,
R.sub.4 is preferably hydrogen. In one embodiment of Formula 1-I, W
is preferably a C.sub.6-C.sub.8 aryl, optionally substituted as in
Formula 1. In another embodiment of Formula 1-I, W is preferably a
naphthyl group; a pyridyl group; or W together with R.sub.4 and the
heterocycle to which R.sub.4 and W are attached form an eleven to
thirteen membered hetero-tricycle ring structure. In a preferred
embodiment of Formula 1-I, W together with R.sub.4 and the
heterocycle to which R.sub.4 and W are attached form a
hetero-tricycle ring structure as follows, wherein the * indicates
the bond of attachment to the phenyl ring of Formula 1-I.
##STR00517##
[0126] In yet another embodiment, preferred W groups of compounds
of Formula 1-I include those shown in the table below.
TABLE-US-00008 ##STR00518## ##STR00519## ##STR00520## ##STR00521##
##STR00522## ##STR00523## ##STR00524## ##STR00525## ##STR00526##
##STR00527## ##STR00528## ##STR00529## ##STR00530## ##STR00531##
##STR00532## ##STR00533## ##STR00534## ##STR00535## ##STR00536##
##STR00537## ##STR00538## ##STR00539## ##STR00540## ##STR00541##
##STR00542## ##STR00543## ##STR00544## ##STR00545## ##STR00546##
##STR00547## ##STR00548## .psi. W taken together with R.sub.4 as in
Formula 1
[0127] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-J:
##STR00549##
[0128] With reference to Formula 1-J, in an embodiment, the carboxy
group is preferably in the meta position. In a further embodiment,
R.sub.4 is preferably hydrogen. In one embodiment, W is preferably
a C.sub.6-C.sub.8 aryl, optionally substituted as in Formula 1, and
more preferably a phenyl optionally substituted with a C.sub.1 to
C.sub.4 alkyl group. In another embodiment, a preferred compound of
Formula 1-J is shown below.
##STR00550##
[0129] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-K:
##STR00551##
[0130] With reference to Formula 1-K, in an embodiment, the carboxy
group is preferably in the meta or para position. In a further
embodiment, R.sub.1 is preferably hydrogen or a methyl. R.sub.2, if
present, is preferably hydrogen; a C.sub.1-C.sub.6 alkyl which is
optionally substituted with a hydroxy group; a carbonyl group which
is optionally substituted with a hydroxyl or a C.sub.1-C.sub.4
alkoxy group; a --CH.dbd.N--OH group; or a cyano group. In one
embodiment, W is preferably a C.sub.6-C.sub.8 aryl, optionally
substituted as in Formula 1, or a naphthyl group optionally
substituted as in Formula 1. In another embodiment, preferred W
groups include those shown in the table below.
TABLE-US-00009 ##STR00552## ##STR00553## ##STR00554## ##STR00555##
##STR00556## ##STR00557## ##STR00558## ##STR00559## ##STR00560##
##STR00561## ##STR00562## ##STR00563## ##STR00564## ##STR00565##
##STR00566## ##STR00567##
[0131] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-L:
##STR00568##
[0132] With reference to Formula 1-L, in an embodiment, the carboxy
group is preferably in the meta or para position. In a further
embodiment, R.sub.1 is preferably hydrogen or a C.sub.1 to C.sub.4
alkyl group, R.sub.2 is preferably hydrogen. In one embodiment, W
is preferably a C.sub.6-C.sub.8 aryl, optionally substituted as in
Formula 1, and more preferably a phenyl optionally substituted with
a C.sub.1 to C.sub.4 alkyl group. In another embodiment, preferred
W groups include those shown in the table below.
TABLE-US-00010 ##STR00569## ##STR00570## ##STR00571## ##STR00572##
##STR00573## ##STR00574## ##STR00575## ##STR00576##
[0133] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-M:
##STR00577##
[0134] With reference to Formula 1-M, in an embodiment, the carboxy
group is preferably in the meta position. In a further embodiment,
R.sub.2 is preferably hydrogen. In one embodiment, W is preferably
a C.sub.6-C.sub.8 aryl, optionally substituted as in Formula 1, and
more preferably a phenyl optionally substituted with a C.sub.1 to
C.sub.4 alkyl group. In another embodiment, a preferred compound of
Formula 1-M is shown below.
##STR00578##
[0135] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-N:
##STR00579##
[0136] With reference to Formula 1-N, in an embodiment, the carboxy
group is preferably in the meta position. In a further embodiment,
R.sub.4 is preferably hydrogen. In one embodiment, W is preferably
a C.sub.6-C.sub.8 aryl, optionally substituted as in Formula 1, and
more preferably a phenyl optionally substituted with a C.sub.1 to
C.sub.4 alkyl group. In another embodiment, a preferred compound of
Formula 1-N is shown below.
##STR00580##
[0137] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-O:
##STR00581##
[0138] With reference to Formula 1-O, in an embodiment, the carboxy
group is preferably in the meta position. In a further embodiment.
R.sub.2 is preferably hydrogen. In one embodiment, W is preferably
a C.sub.6-C.sub.8 aryl, optionally substituted as in Formula 1, and
more preferably a phenyl optionally substituted with a C.sub.1 to
C.sub.4 alkyl group. In another embodiment, a preferred compound of
Formula 1-J is shown below.
##STR00582##
[0139] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-P:
##STR00583##
[0140] With reference to Formula 1-P, in an embodiment, the carboxy
group is preferably in the meta position. In a further embodiment,
R.sub.2 is preferably hydrogen. In one embodiment, W is preferably
a C.sub.6-C.sub.8 aryl, optionally substituted as in Formula 1, and
more preferably a phenyl optionally substituted with a C.sub.1 to
C.sub.4 alkyl group. In another embodiment, a preferred compound of
Formula 1-P is shown below.
##STR00584##
[0141] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-Q:
##STR00585##
[0142] With reference to Formula 1-Q, in an embodiment, the carboxy
group is preferably in the meta position. In a further embodiment,
R.sub.2 is preferably hydrogen. In one embodiment, W is preferably
a C.sub.6-C.sub.8 aryl, optionally substituted as in Formula 1, and
more preferably a phenyl optionally substituted with a C.sub.1 to
C.sub.4 alkyl group. In another embodiment, a preferred compound of
Formula 1-Q is shown below.
##STR00586##
[0143] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-R:
##STR00587##
[0144] With reference to Formula 1-R, in an embodiment, the carboxy
group is preferably in the meta position. In a further embodiment.
R.sub.2 is preferably hydrogen. In one embodiment, W is preferably
a C.sub.6-C.sub.8 aryl, optionally substituted as in Formula 1, and
more preferably a phenyl optionally substituted with a C.sub.1 to
C.sub.4 alkyl group. In another embodiment, a preferred compound of
Formula 1-R is shown below.
##STR00588##
[0145] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-S;
##STR00589##
[0146] With reference to Formula 1-S, in an embodiment, the carboxy
group is preferably in the meta position. In a further embodiment,
R.sub.2 is preferably hydrogen or a carbonyl group optionally
substituted as in Formula 1. R.sub.4 is preferably hydrogen. In one
embodiment, W is preferably a C.sub.6-C.sub.8 aryl, optionally
substituted as in Formula 1, and more preferably a phenyl
optionally substituted with a C.sub.1 to C.sub.4 alkyl group. In
another embodiment, a preferred compound of Formula 1-S is shown
below.
##STR00590##
[0147] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-T:
##STR00591##
[0148] With reference to Formula 1-T in an embodiment, the carboxy
group is preferably in the meta position. In a further embodiment,
R.sub.2 is preferably carbonyl optionally substituted as in Formula
1 or hydrogen. R.sub.4 is preferably hydrogen. In one embodiment, W
is preferably a C.sub.6-C.sub.8 aryl, optionally substituted as in
Formula 1, and more preferably a phenyl optionally substituted with
a C.sub.1 to C.sub.4 alkyl group. In another embodiment, a
preferred compound of Formula 1-T is shown below.
##STR00592##
[0149] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-U:
##STR00593##
[0150] With reference to Formula 1-U, in an embodiment, the carboxy
group is preferably in the meta position. In one embodiment, W is
preferably a C.sub.6-C.sub.8 aryl, optionally substituted as in
Formula 1, and more preferably a phenyl optionally substituted with
a C.sub.1 to C.sub.4 alkyl group. In another embodiment, a
preferred compound of Formula 1-U is shown below.
##STR00594##
[0151] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-V:
##STR00595##
[0152] With reference to Formula 1-V, in an embodiment, the carboxy
group is preferably in the meta position. In one embodiment, W is
preferably a C.sub.6-C.sub.8 aryl, optionally substituted as in
Formula 1, and more preferably a phenyl optionally substituted with
a C.sub.1 to C.sub.4 alkyl group. In another embodiment, a
preferred compound of Formula 1-V is shown below.
##STR00596##
[0153] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-W:
##STR00597##
[0154] With reference to Formula 1-W, in an embodiment, the carboxy
group is preferably in the meta position. In a further embodiment,
R.sub.2 is preferably hydrogen. In one embodiment, W is preferably
a C.sub.6-C.sub.8 aryl, optionally substituted as in Formula 1, and
more preferably a phenyl optionally substituted with a C.sub.1 to
C.sub.4 alkyl group. In another embodiment, a preferred compound of
Formula 1-W is shown below.
##STR00598##
[0155] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-X:
##STR00599##
[0156] With reference to Formula 1-X, in an embodiment, the carboxy
group is preferably in the meta position. In a further embodiment,
R.sub.2 is preferably hydrogen. In one embodiment, W is preferably
a C.sub.6-C.sub.8 aryl, optionally substituted as in Formula 1, and
more preferably a phenyl optionally substituted with a C.sub.1 to
C.sub.4 alkyl group. In another embodiment, a preferred compound of
Formula 1-X is shown below.
##STR00600##
[0157] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-Y:
##STR00601##
[0158] With reference to Formula 1-Y, in an embodiment, the carboxy
group is preferably in the meta position. In a further embodiment,
R.sub.4 is preferably hydrogen. In one embodiment, W is preferably
a C.sub.6-C.sub.8 aryl, optionally substituted as in Formula 1, and
more preferably a phenyl optionally substituted with a C.sub.1 to
C.sub.4 alkyl group. In another embodiment, a preferred compound of
Formula 1-Y is shown below.
##STR00602##
[0159] In yet another embodiment, preferred compounds of Formula 1
include the compounds of Formula 1-Z:
##STR00603##
[0160] With reference to Formula 1-Z, in an embodiment, the carboxy
group is preferably in the meta position. In one embodiment, W is
preferably a C.sub.6-C.sub.8 aryl, optionally substituted as in
Formula 1; a pyridyl group; or a thienyl group. In another
embodiment, preferred W groups include those shown in the table
below.
TABLE-US-00011 ##STR00604## ##STR00605## ##STR00606## ##STR00607##
##STR00608## ##STR00609## ##STR00610## ##STR00611## ##STR00612##
##STR00613## ##STR00614## ##STR00615## ##STR00616## ##STR00617##
##STR00618##
[0161] In another aspect of the invention, compounds of Formula (2)
are provided which are useful for suppressing premature translation
termination associated with a nonsense mutation in mRNA, and for
treating diseases associated with nonsense mutations in mRNA;
##STR00619##
[0162] wherein:
[0163] X, Y, and Z are independently selected from N, S, O, and C
wherein at least one of X, Y or Z is a heteroatom;
[0164] R.sub.1 is hydrogen, a C.sub.1-C.sub.6 alkyl, or Na+, or
Mg.sup.2+;
[0165] R.sub.2 is independently absent; a hydrogen; a
--CH.dbd.N--OH group; a cyano group; a C.sub.1-C.sub.6 alkyl which
is optionally substituted with a hydroxy group; or a carbonyl group
which is optionally substituted with a hydrogen, a hydroxyl, or a
C.sub.1-C.sub.4 alkoxy group;
[0166] R.sub.3 is independently absent, a halogen, a hydroxy, a
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.4 alkoxy, or a nitro
group;
[0167] R.sub.4 is independently absent, a hydrogen, a
C.sub.1-C.sub.6 alkyl, or when taken together with W, R.sub.4 may
be a bond, and W and the heterocycle to which R.sub.4 and W are
attached form an eleven to thirteen membered hetero-tricycle ring
structure;
[0168] q is 0, 1, or 2;
[0169] W is selected from: [0170] (a) a C.sub.2-C.sub.6 alkynyl,
optionally substituted with a phenyl; [0171] (b) a C.sub.1-C.sub.8
straight chain or branched chain alkyl which is optionally
substituted with one or more of the following independently
selected groups: a C.sub.1-C.sub.6 alkyl; a halogen; a
--C(.dbd.O)NH-phenyl which phenyl is optionally substituted with
one or more independently selected halogens or C.sub.1-C.sub.4
alkyl groups; a five to six-membered heterocycle; a C.sub.6-C.sub.8
aryl which is optionally substituted with one or more groups
independently selected from a hydroxy, a halogen, a C.sub.1-C.sub.4
alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a C.sub.1-C.sub.4
alkoxy group or an amino group which is optionally substituted with
one or more C.sub.1-C.sub.4 alkyl groups; an aryloxy which is
optionally substituted with one or more of the following
independently selected groups: a hydroxy, a halogen, a
C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a
C.sub.1-C.sub.4 alkoxy group or an amino group which is optionally
substituted with one or more C.sub.1-C.sub.4 alkyl groups; [0172]
(c) C.sub.2 to C.sub.8 alkenyl; [0173] (d) a C.sub.3-C.sub.8
cycloalkyl optionally substituted with a C.sub.1-C.sub.6 alkyl;
[0174] (e) a C.sub.6-C.sub.8 aryl which is optionally substituted
with one or more of the following independently selected groups: a
hydroxy; a halogen; a C.sub.1-C.sub.4 straight chain or branched
chain alkyl which is optionally substituted with one or more
independently selected halogen or hydroxy groups; a C.sub.1-C.sub.4
alkoxy which is optionally substituted with one or more
independently selected halogen or phenyl groups; a C.sub.3-C.sub.8
cycloalkyl which is optionally substituted with one or more
independently selected C.sub.1-C.sub.4 alkyl groups: a
C.sub.6-C.sub.8 aryl which is optionally substituted with one or
more independently selected C.sub.1-C.sub.4 alkyl groups; an
aryloxy which is optionally substituted with one or more of the
following independently selected groups: a hydroxy, a halogen, a
C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a
C.sub.1-C.sub.4 alkoxy group, or an amino group which is optionally
substituted with one or more independently selected C.sub.1-C.sub.4
alkyl groups; a five to six-membered heterocycle which is
optionally substituted with one or more independently selected
C.sub.1-C.sub.4 alkyl, oxo, or C.sub.6-C.sub.8 aryl which is
optionally substituted with one or more of the following
independently selected groups: a hydroxy, a halogen, a
C.sub.1-C.sub.4 alkyl group, a C.sub.1-C.sub.4 haloalkyl group, a
C.sub.1-C.sub.4 alkoxy group, or an amino group which is optionally
substituted with one or more independently selected C.sub.1-C.sub.4
alkyl groups; a naphthyl group which is optionally substituted with
an amino or aminoalkyl or alkoxy group; a --C(O)--NR.sub.xR.sub.y
group; a --C(O)--R.sub.x group; a isoindole-1,3-dione group; a
nitro group; a cyano group; a --SO.sub.3H group; alkylthio group;
alkyl sulfonyl group; a --NR.sub.x--C(O)--R.sub.z group; a
--NR.sub.xR.sub.y, group; a --NR.sub.x--SO.sub.2--R.sub.z group; a
--NR.sub.x--C(O)--NR.sub.xR.sub.y group; a
--NR.sub.x--C(O)O--R.sub.z group; [0175] (f) a C.sub.10-C.sub.14
aryl group optionally substituted with one or more independently
selected halogens, amino groups or aminoalkyl groups, or alkoxy
groups; [0176] (g) a --C(O)--NR.sub.xR.sub.y group; [0177] (h) a
five or six membered heterocycle which is optionally substituted
with one or more independently selected oxo groups; halogens;
C.sub.1-C.sub.4 alkyl groups; C.sub.1-C.sub.4 alkoxy groups;
C.sub.1-C.sub.4 haloalkyl groups; C.sub.1-C.sub.4 haloalkoxy
groups; aryloxy groups; --NR.sub.xR.sub.y groups; alkylthio groups;
--C(O)--R.sub.x groups; or C.sub.6 to C.sub.8 aryl groups which are
optionally substituted with one or more independently selected
halogens, C.sub.1-C.sub.4 alkyl groups, C.sub.1-C.sub.4 alkoxy
groups; [0178] (i) a heterocycle group having two to three ring
structures that is optionally substituted with one or more
independently selected halogens, oxo groups, C.sub.1-C.sub.4 alkyl
groups, C.sub.1-C.sub.4 haloalkyl groups, or C.sub.1-C.sub.4 alkoxy
groups; [0179] (j) or W together with R.sub.4, including where
R.sub.4 is a bond, and the heterocycle to which R.sub.4 and W are
attached form an eleven to thirteen membered hetero-tricycle ring
structure; [0180] wherein R.sub.x is hydrogen, a C.sub.1-C.sub.6
alkyl group, or R.sub.x and R.sub.y together with the atoms to
which they are attached form a four to seven membered carbocycle or
heterocycle; [0181] R.sub.y is hydrogen, a C.sub.1-C.sub.6 alkyl
group; an aryl group optionally substituted with one or more
independently selected C.sub.1-C.sub.4 alkyl groups, or R.sub.x and
R.sub.y together with the atoms to which they are attached form a
four to seven membered carbocycle or heterocycle; and [0182]
R.sub.2 is an C.sub.1-C.sub.6 alkyl optionally substituted with an
aryl or a halogen; or an aryl optionally substituted with a
halogen, a C.sub.1-C.sub.6 alkyl, or a C.sub.1-C.sub.6 alkoxy;
[0183] or a pharmaceutically acceptable salt, hydrate, solvate,
clathrate, racemate, stereoisomer, or polymorph of said compound of
Formula 2.
[0184] In an embodiment of Formula 2, preferred substituents for
Formula 2 may be chosen as for Formula 1. In a preferred embodiment
of Formula 2, substituents for Formula 2 may be chosen as described
for Formula 1-E.
[0185] In a preferred embodiment of Formula 2, q is 0. In another
preferred embodiment of Formula 2, q is 1 or 2. In a preferred
embodiment of Formula 2, q is 1. In another embodiment of Formula
2, q is 2.
[0186] In a preferred embodiment of Formula 2, R.sub.3 is hydrogen,
q is 1 and the --CH2-COOR1 group is in the para position relative
to the 5-membered ring containing the X, Y, and Z substituents.
[0187] In other embodiments of compounds of Formula 2, Z is oxygen.
Y is nitrogen, and both R.sub.2 groups are absent. In a related
more preferred embodiment, X is carbon and R.sub.4 is hydrogen. In
either of the previous two embodiments q is preferably 1.
[0188] In even more preferred compounds of Formula 2 embodiments, W
is a phenyl ring substituted with one or more independently
selected halogens. In another more preferred embodiments, Z is
oxygen, Y is nitrogen, both R.sub.2 groups are absent, X is carbon,
R.sub.4 is hydrogen, q is 1, and W is a phenyl ring substituted
with two independently selected halogens. In an even more preferred
embodiment, the compound of Formula 2 is:
##STR00620##
[0189] Compounds of Formula 2 are useful in methods of treatment,
and the preparation of pharmaceutical compositions as recited for
compounds of Formula 1.
[0190] Preferred compounds of the invention include the following
compounds in Table X:
TABLE-US-00012 TABLE X ##STR00621## 1 ##STR00622## 2 ##STR00623## 3
##STR00624## 4 ##STR00625## 5 ##STR00626## 6 ##STR00627## 7
##STR00628## 8 ##STR00629## 12 ##STR00630## 13 ##STR00631## 14
##STR00632## 15 ##STR00633## 16 ##STR00634## 17 ##STR00635## 18
##STR00636## 19 ##STR00637## 21 ##STR00638## 22 ##STR00639## 23
##STR00640## 24 ##STR00641## 25 ##STR00642## 26 ##STR00643## 27
##STR00644## 28 ##STR00645## 29 ##STR00646## 30 ##STR00647## 31
##STR00648## 32 ##STR00649## 33 ##STR00650## 34 ##STR00651## 35
##STR00652## 36 ##STR00653## 37 ##STR00654## 38 ##STR00655## 39
##STR00656## 40 ##STR00657## 41 ##STR00658## 42 ##STR00659## 43
##STR00660## 44 ##STR00661## 45 ##STR00662## 46 ##STR00663## 47
##STR00664## 48 ##STR00665## 49 ##STR00666## 50 ##STR00667## 51
##STR00668## 53 ##STR00669## 54 ##STR00670## 55 ##STR00671## 60
##STR00672## 62 ##STR00673## 63 ##STR00674## 64 ##STR00675## 65
##STR00676## 66 ##STR00677## 67 ##STR00678## 68 ##STR00679## 69
##STR00680## 70 ##STR00681## 71 ##STR00682## 72 ##STR00683## 73
##STR00684## 82 ##STR00685## 83 ##STR00686## 84 ##STR00687## 85
##STR00688## 86 ##STR00689## 87 ##STR00690## 88 ##STR00691## 89
##STR00692## 90 ##STR00693## 91 ##STR00694## 92 ##STR00695## 93
##STR00696## 94 ##STR00697## 95 ##STR00698## 96 ##STR00699## 97
##STR00700## 98 ##STR00701## 99 ##STR00702## 100 ##STR00703## 101
##STR00704## 102 ##STR00705## 103 ##STR00706## 104 ##STR00707## 106
##STR00708## 107 ##STR00709## 108 ##STR00710## 109 ##STR00711## 110
##STR00712## 111 ##STR00713## 112 ##STR00714## 113 ##STR00715## 114
##STR00716## 115 ##STR00717## 116 ##STR00718## 117 ##STR00719## 118
##STR00720## 119 ##STR00721## 120 ##STR00722## 121 ##STR00723## 122
##STR00724## 123 ##STR00725## 124 ##STR00726## 125 ##STR00727## 126
##STR00728## 127 ##STR00729## 128 ##STR00730## 129 ##STR00731## 130
##STR00732## 131 ##STR00733## 132 ##STR00734## 133 ##STR00735## 134
##STR00736## 135 ##STR00737## 136 ##STR00738## 137 ##STR00739## 138
##STR00740## 139 ##STR00741## 142 ##STR00742## 143 ##STR00743## 144
##STR00744## 145
##STR00745## 146 ##STR00746## 147 ##STR00747## 148 ##STR00748## 149
##STR00749## 150 ##STR00750## 151 ##STR00751## 152 ##STR00752## 153
##STR00753## 154 ##STR00754## 155 ##STR00755## 156 ##STR00756## 157
##STR00757## 158 ##STR00758## 159 ##STR00759## 160 ##STR00760## 161
##STR00761## 162 ##STR00762## 163 ##STR00763## 164 ##STR00764## 165
##STR00765## 166 ##STR00766## 167 ##STR00767## 168 ##STR00768## 169
##STR00769## 170 ##STR00770## 171 ##STR00771## 172 ##STR00772## 173
##STR00773## 174 ##STR00774## 175 ##STR00775## 176 ##STR00776## 177
##STR00777## 178 ##STR00778## 179 ##STR00779## 180 ##STR00780## 181
##STR00781## 182 ##STR00782## 183 ##STR00783## 184 ##STR00784## 185
##STR00785## 186 ##STR00786## 187 ##STR00787## 188 ##STR00788## 189
##STR00789## 190 ##STR00790## 191 ##STR00791## 192 ##STR00792## 193
##STR00793## 194 ##STR00794## 195 ##STR00795## 196 ##STR00796## 197
##STR00797## 198 ##STR00798## 199 ##STR00799## 200 ##STR00800## 201
##STR00801## 202 ##STR00802## 203 ##STR00803## 204 ##STR00804## 205
##STR00805## 206 ##STR00806## 207 ##STR00807## 208 ##STR00808## 209
##STR00809## 210 ##STR00810## 211 ##STR00811## 212 ##STR00812## 213
##STR00813## 214 ##STR00814## 215 ##STR00815## 216 ##STR00816## 217
##STR00817## 218 ##STR00818## 219 ##STR00819## 220 ##STR00820## 221
##STR00821## 222 ##STR00822## 223 ##STR00823## 224 ##STR00824## 225
##STR00825## 226 ##STR00826## 227 ##STR00827## 228 ##STR00828## 229
##STR00829## 230 ##STR00830## 231 ##STR00831## 232 ##STR00832## 233
##STR00833## 234 ##STR00834## 235 ##STR00835## 236 ##STR00836## 237
##STR00837## 238 ##STR00838## 239 ##STR00839## 240 ##STR00840## 241
##STR00841## 242 ##STR00842## 243 ##STR00843## 244 ##STR00844## 245
##STR00845## 246 ##STR00846## 247 ##STR00847## 248 ##STR00848## 249
##STR00849## 250 ##STR00850## 251 ##STR00851## 252 ##STR00852## 253
##STR00853## 254 ##STR00854## 255 ##STR00855## 258 ##STR00856## 259
##STR00857## 260 ##STR00858## 261 ##STR00859## 262 ##STR00860## 263
##STR00861## 264 ##STR00862## 265 ##STR00863## 266 ##STR00864## 267
##STR00865## 268 ##STR00866## 269 ##STR00867## 270 ##STR00868## 271
##STR00869## 272 ##STR00870## 273
##STR00871## 274 ##STR00872## 278 ##STR00873## 279 ##STR00874## 280
##STR00875## 281 ##STR00876## 282 ##STR00877## 283 ##STR00878## 284
##STR00879## 285 ##STR00880## 286 ##STR00881## 292 ##STR00882## 293
##STR00883## 294 ##STR00884## 295 ##STR00885## 296 ##STR00886## 297
##STR00887## 298 ##STR00888## 299 ##STR00889## 300 ##STR00890## 301
##STR00891## 302 ##STR00892## 303 ##STR00893## 304 ##STR00894## 305
##STR00895## 306 ##STR00896## 307 ##STR00897## 308 ##STR00898## 309
##STR00899## 315 ##STR00900## 316 ##STR00901## 317 ##STR00902## 318
##STR00903## 319 ##STR00904## 401 ##STR00905## 402 ##STR00906## 596
##STR00907## 601 ##STR00908## 606 ##STR00909## 609 ##STR00910## 610
##STR00911## 615 ##STR00912## 620 ##STR00913## 621 ##STR00914## 622
##STR00915## 624 ##STR00916## 626 ##STR00917## 628 ##STR00918## 324
##STR00919## 141 ##STR00920## 275 ##STR00921## 407 ##STR00922## 408
##STR00923## 409 ##STR00924## 410 ##STR00925## 411 ##STR00926## 412
##STR00927## 413 ##STR00928## 414 ##STR00929## 415 ##STR00930## 416
##STR00931## 417 ##STR00932## 418 ##STR00933## 419 ##STR00934## 420
##STR00935## 421 ##STR00936## 422 ##STR00937## 430 ##STR00938## 431
##STR00939## 432 ##STR00940## 433 ##STR00941## 434 ##STR00942## 435
##STR00943## 436 ##STR00944## 437 ##STR00945## 438 ##STR00946## 439
##STR00947## 440 ##STR00948## 441 ##STR00949## 442 ##STR00950## 443
##STR00951## 444 ##STR00952## 445 ##STR00953## 446 ##STR00954## 447
##STR00955## 448 ##STR00956## 449 ##STR00957## 450 ##STR00958## 451
##STR00959## 452 ##STR00960## 453 ##STR00961## 140 ##STR00962## 349
##STR00963## 364 ##STR00964## 394 ##STR00965## 395 ##STR00966## 396
##STR00967## 397 ##STR00968## 398 ##STR00969## 399 ##STR00970## 403
##STR00971## 404 ##STR00972## 405 ##STR00973## 406 ##STR00974## 506
##STR00975## 507 ##STR00976## 508 ##STR00977## 509 ##STR00978## 510
##STR00979## 511 ##STR00980## 512 ##STR00981## 513 ##STR00982## 559
##STR00983## 560 ##STR00984## 561 ##STR00985## 562 ##STR00986## 563
##STR00987## 564 ##STR00988## 565 ##STR00989## 569 ##STR00990## 570
##STR00991## 571 ##STR00992## 572 ##STR00993## 576 ##STR00994## 577
##STR00995## 578
##STR00996## 288 ##STR00997## 527 ##STR00998## 528 ##STR00999## 542
##STR01000## 543 ##STR01001## 544 ##STR01002## 545 ##STR01003## 546
##STR01004## 547 ##STR01005## 548 ##STR01006## 549 ##STR01007## 550
##STR01008## 553 ##STR01009## 554 ##STR01010## 555 ##STR01011## 556
##STR01012## 557 ##STR01013## 558 ##STR01014## 579 ##STR01015## 580
##STR01016## 581 ##STR01017## 582 ##STR01018## 583 ##STR01019## 584
##STR01020## 585 ##STR01021## 586 ##STR01022## 587 ##STR01023## 588
##STR01024## 589 ##STR01025## 590 ##STR01026## 591 ##STR01027## 592
##STR01028## 593 ##STR01029## 594 ##STR01030## 595 ##STR01031## 629
##STR01032## 630 ##STR01033## 631 ##STR01034## 632 ##STR01035## 633
##STR01036## 634 ##STR01037## 635 ##STR01038## 636 ##STR01039## 637
##STR01040## 638 ##STR01041## 639 ##STR01042## 640 ##STR01043## 641
##STR01044## 642 ##STR01045## 643 ##STR01046## 644 ##STR01047## 645
##STR01048## 646 ##STR01049## 647 ##STR01050## 648 ##STR01051## 649
##STR01052## 650 ##STR01053## 651 ##STR01054## 276 ##STR01055## 325
##STR01056## 329 ##STR01057## 330 ##STR01058## 331 ##STR01059## 332
##STR01060## 333 ##STR01061## 334 ##STR01062## 335 ##STR01063## 336
##STR01064## 337 ##STR01065## 338 ##STR01066## 339 ##STR01067## 340
##STR01068## 341 ##STR01069## 342 ##STR01070## 343 ##STR01071## 344
##STR01072## 345 ##STR01073## 346 ##STR01074## 351 ##STR01075## 352
##STR01076## 353 ##STR01077## 354 ##STR01078## 355 ##STR01079## 356
##STR01080## 357 ##STR01081## 358 ##STR01082## 359 ##STR01083## 360
##STR01084## 361 ##STR01085## 362 ##STR01086## 363 ##STR01087## 287
##STR01088## 551 ##STR01089## 552 ##STR01090## 75 ##STR01091## 289
##STR01092## 350 ##STR01093## 365 ##STR01094## 366 ##STR01095## 367
##STR01096## 368 ##STR01097## 369 ##STR01098## 370 ##STR01099## 371
##STR01100## 372 ##STR01101## 373 ##STR01102## 374 ##STR01103## 375
##STR01104## 376 ##STR01105## 377 ##STR01106## 378 ##STR01107## 379
##STR01108## 380 ##STR01109## 381 ##STR01110## 382 ##STR01111## 383
##STR01112## 384 ##STR01113## 385 ##STR01114## 386 ##STR01115## 387
##STR01116## 388 ##STR01117## 389 ##STR01118## 390 ##STR01119## 391
##STR01120## 392 ##STR01121## 393
##STR01122## 310 ##STR01123## 290 ##STR01124## 463 ##STR01125## 464
##STR01126## 465 ##STR01127## 466 ##STR01128## 467 ##STR01129## 468
##STR01130## 469 ##STR01131## 470 ##STR01132## 471 ##STR01133## 472
##STR01134## 473 ##STR01135## 474 ##STR01136## 475 ##STR01137## 652
##STR01138## 653 ##STR01139## 654 ##STR01140## 655 ##STR01141## 656
##STR01142## 657 ##STR01143## 658 ##STR01144## 659 ##STR01145## 660
##STR01146## 661 ##STR01147## 662 ##STR01148## 663 ##STR01149## 664
##STR01150## 476 ##STR01151## 477 ##STR01152## 478 ##STR01153## 479
##STR01154## 480 ##STR01155## 481 ##STR01156## 482 ##STR01157## 483
##STR01158## 484 ##STR01159## 485 ##STR01160## 486 ##STR01161## 487
##STR01162## 488 ##STR01163## 489 ##STR01164## 490 ##STR01165## 491
##STR01166## 521 ##STR01167## 522 ##STR01168## 523 ##STR01169## 524
##STR01170## 525 ##STR01171## 526 ##STR01172## 529 ##STR01173## 530
##STR01174## 531 ##STR01175## 532 ##STR01176## 533 ##STR01177## 534
##STR01178## 566 ##STR01179## 567 ##STR01180## 568 ##STR01181## 573
##STR01182## 574 ##STR01183## 575 ##STR01184## 291 ##STR01185## 492
##STR01186## 493 ##STR01187## 494 ##STR01188## 495 ##STR01189## 496
##STR01190## 497 ##STR01191## 498 ##STR01192## 499 ##STR01193## 500
##STR01194## 501 ##STR01195## 502 ##STR01196## 503 ##STR01197## 504
##STR01198## 505 ##STR01199## 514 ##STR01200## 515 ##STR01201## 516
##STR01202## 517 ##STR01203## 518 ##STR01204## 519 ##STR01205## 520
##STR01206## 535 ##STR01207## 536 ##STR01208## 537 ##STR01209## 538
##STR01210## 539 ##STR01211## 540 ##STR01212## 541 ##STR01213## 311
##STR01214## 277 ##STR01215## 312 ##STR01216## 321 ##STR01217## 313
##STR01218## 320 ##STR01219## 314 ##STR01220## 322 ##STR01221## 323
##STR01222## 326 ##STR01223## 327 ##STR01224## 348 ##STR01225## 400
##STR01226## 423 ##STR01227## 424 ##STR01228## 425 ##STR01229## 426
##STR01230## 427 ##STR01231## 428 ##STR01232## 429 ##STR01233## 454
##STR01234## 455 ##STR01235## 456 ##STR01236## 457 ##STR01237## 458
##STR01238## 459 ##STR01239## 460 ##STR01240## 461 ##STR01241## 462
##STR01242## 605
[0191] The above compounds are listed only to provide examples that
may be used in the methods of the invention. Based upon the instant
disclosure, the skilled artisan would recognize other compounds
intended to be included within the scope of the presently claimed
invention that would be useful in the methods recited herein.
[0192] B. Preparation of Compounds of the Invention
[0193] Compounds of the invention may be produced in any manner
known in the art. By way of example, compounds of the invention may
be prepared according to the general methodologies described below.
For instance, certain 1,3,4-oxadiazoles of Formula 1-A may be
prepared by the methodology depicted in Scheme A1 below:
##STR01243##
[0194] In accordance with Scheme A1, benzonitriles of structure A1
can be converted to tetrazoles of structure A2 by treatment with,
e.g., sodium azide. Treatment of the tetrazoles A2 with an
activated carboxylic acid, e.g., an acid chloride or an acid
activated with a dehydrating agent, e.g., dicyclohexyl carbodiimide
in a suitable solvent, affords the 1,3,4-oxadiazole compounds of
Formula 1-A. Suitable solvents include, but are not limited to,
e.g., toluene or dichloroethane. The reaction can usually carried
out within a temperature range of 60-150.degree. C.
[0195] In another embodiment, certain 1,3,4-oxadiazoles of Formula
1-A may be prepared by the methodology described in Scheme A2
below.
##STR01244##
[0196] In accordance with Scheme A2, activated benzoic acids of
structure A3 can be reacted with substituted hydrazides to give
substituted benzoyl hydrazides of structure A4. The activating
group may be a halide (e.g., an acid chloride or bromide) or
derived from treatment of the benzoic acid with a dehydrating
agent, e.g., dicyclohexyl carbodiimide). Optionally, a base, e.g.,
triethylamine, may be employed. Compounds of type A4 can then be
dehydrated to form compounds of Formula 1-A. Typical dehydrating
agents include, but are not limited to, e.g., dicyclohexyl
carbodiimide, or phosphorous oxychloride. The reaction is usually
carried out within a range of 20-120.degree. C.
[0197] In yet another embodiment, certain 1,3,4-oxadiazoles of
Formula 1-A may be prepared by the methodology depicted in Scheme
A3 below:
##STR01245##
[0198] In accordance with Scheme A3, commercially available,
acid-labile resin such as trityl resin, 2-chlorotrityl chloride
resin, phenylacetamidomethyl (PAM) resin, and p-alkoxybenzyl
alcohol resin can be used in this invention. The coupling of
carboxylic acid compounds A6 and trityl resin A5 (here
X=2-chlorotrityl chloride) can be performed in a suitable solvent
such as dichloromethane, dimethylformamide or tetrahydrofuran in
the presence of a tertiary amine reagent such as
diisopropylethylamine or triethylamine. The resin-bound ester A7
can be treated with hydrazides in the presence of
hexafluorophosphate (PyBOP) or equivalents such as
diisopropylcarbodiimide,
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (PyBOP), bromotrispyrrolidinophosphonium
hexafluorophosphate (PyBrOP) or
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)
to give acyl hydrazides A8. Alternatively, the hydrazide resin A10
can be conveniently prepared from A7 under usual amide linkage
formation reactions using diisopropyl carbodiimide or equivalents
such as benzotriazole-1-yl-oxytrispyrrolidinophosphonium
hexafluorosposphate (PyBOP), bromotrispyrrolidinophosphonium
hexafluorophosphate (PyBrOP),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)
with or without diisopropylethylamine in dimethylformamide.
Alternatively, the resin-bound hydrazide resin A10 can be reacted
with a carboxylic acid using diisopropylcarbodiimide or equivalents
such as benzotriazole-1-yloxytrispyrrolidinophosphonium
hexafluorophosphate (PyBOP), bromotrispyrrolidinophosphonium
hexafluorophosphate (PyBrOP) or
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)
to form A8. A ring-closure reaction on resin-bound A8 can be
effected by the treatment of 2-chloro-1,3-dimethylimidazolidinium
chloride in an inert solvent such as dichloromethane,
tetrahydrofuran, dioxane or dimethylformamide with bases such as
diisopropylethylamine or triethylamine to afford the
1,3,4-oxdiazole compound A9. The resin-bound oxadiazole compound A9
is cleaved under acidic conditions such as 2N trifluoroacetic acid
in dichloromethane, or 3N acetic acid in dichloromethane, to afford
the desired compound of Formula 1-A.
[0199] Certain 1,3,4-thiadiazoles of Formula 1-B can be prepared by
the methodology described in Scheme B below:
##STR01246##
[0200] In accordance with Scheme B, treatment of benzoyl hydrazides
B1 with a thionating reagent, e.g., Lawesson's reagent or
phosphorous pentasulfide in a suitably nonreactive organic solvent,
e.g., toluene or dioxane, at a temperature range from
50-120.degree. C. can furnish 1,3,4-thiadiazole compounds of
Formula 1-B.
[0201] Certain 1,2,4-oxadiazoles of Formula 1-C can be prepared by
the methodology depicted in Scheme C1 below:
##STR01247##
[0202] In accordance with Scheme C1, the benzonitrile compound C1
can be converted to the hydroxyamidine C2 by treatment with
hydroxylamine or hydroxylamine-HCl. The reaction with
hydroxylamine-HCl is usually performed in the presence of a base,
such as triethylamine, potassium carbonate or
diisopropylethylamine. The reaction can be carried out in a solvent
such as methanol, ethanol, tert-butanol, tetrahydrofuran or
dimethylformamide, and at temperatures ranging from ambient to the
reflux temperature of the chosen solvent. The hydroxyamidine
compound C2 is acylated with acyl derivative C3 to give compound
C4, wherein the group L represents some leaving group, such as
halo, imidazoyl or p-nitrophenol, etc. The reaction is usually
carried out with a base, such as triethylamine or
diisopropylethylamine, in a solvent such as dichloromethane,
tetrahydrofuran or dimethylformamide. In an alternative method, the
acylation is conveniently carried out under usual ester linkage
formation reactions, wherein the group L represents hydroxy, using
diisopropyl carbodiimide or equivalents such as
benzotriazole-1-yl-oxytrispyrrolidinophosphonium
hexafluorophosphate, bromotrispyrrolidinophosphonium
hexafluorophosphate or
1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride
without or with diisopropylethylamine. The ring-closure of the
acylated compound C4 can be accomplished with or without a base
such as triethylamine or diisopropylethylamine, in a solvent such
as dichloromethane, tetrahydrofuran, toluene or dimethylformamide,
and at temperatures ranging from ambient to the reflux temperature
of the chosen solvent.
[0203] Certain 1,2,4-oxadiazole compounds of Formula 1-C may also
be prepared by the method described above using solid phase
chemistry as described in Scheme C2, below:
##STR01248##
[0204] In accordance with Scheme C2, commercially available,
acid-labile resin C5 such as trityl resin, 2-chlorotrityl chloride
resin, phenylacetamidomethyl (PAM) resin, and p-alkoxybenzyl
alcohol resin can be used in this example. The coupling of benzoic
acid compounds C6 and trityl resin (here X=2-chlorotrityl chloride)
can be performed in a suitable solvent such as dichloromethane,
dimethylformamide or toluene in the presence of a tertiary amine
reagent such as diisopropylethylamine or triethylamine. The
resin-bound cyanobenzoic ester C7 can be treated with hydroxylamine
in an inert solvent such as ethanol, tetrahydrofuran, dioxane or
dimethylformamide or mixtures with or without diisopropylethylamine
to afford the hydroxyamidine compound C8. The resin-bound
hydroxyamidine compound C8 can be acylated with a reagent (WCOY),
wherein the group Y represents some leaving group, such as halo,
imidazoyl, p-nitrophenol, etc. The reaction is typically carried
out in the presence of a base, such as diisopropylethylamine or
triethylamine, in an inert solvent such as dichloromethane,
tetrahydrofuran or dimethylformamide or mixtures. Alternatively,
the acylation is conveniently carried out with a reagent (WCOY),
wherein the group Y represents hydroxy, using
diisopropylcarbodiimide or equivalents such as
benzotriazole-1-yloxytrispyrrolidinophosphonium
hexafluorophosphate, bromotrispyrrolidinophosphonium
hexafluorophosphate or
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride without
or with diisopropylethylamine in dimethylformamide. The resin-bound
acylated compound C9 is cleaved under acidic conditions such as 2N
trifluoroacetic acid in dichloromethane, or 3N acetic acid in
dichloromethane, to afford the desired intermediate compound C10. A
ring-closure reaction on free acid compound C10 can be effected by
heating in an inert solvent such as toluene, tetrahydrofuran,
dioxane or dimethylformamide or mixtures with or without a base
reagent such as diisopropylethylamine, triethylamine or
tetrabutylammonium fluoride to afford the 1,2,4-oxdiazole compounds
of Formula 1-C.
[0205] Certain 1,2,4-oxadiazoles of Formula 1-D can be prepared by
the methodology depicted in Scheme D1 below:
##STR01249##
[0206] In accordance with Scheme D1, acyl chlorides of structure D1
can be treated with a hydroxyamidine reagent D2 in the presence of
a base, such as N-methylmorpholine, N,N-diisopropylethylamine, or
triethylamine, in an inert solvent such as dichloromethane,
tetrahydrofuran or dimethylformamide or mixtures. Hydroxyamidine
compounds D2 can be conveniently prepared from treatment of
nitrites with hydroxylamine in an inert solvent such as, e.g.,
ethanol, dioxane, or tetrahydrofuran. Ring-closure of the compound
D3 can be effected by heating in an inert solvent such as toluene,
tetrahydrofuran, dioxane or dimethylformamide or mixtures with or
without a base reagent such as diisopropylethylamine, triethylamine
or tetrabutylammonium fluoride to afford the 1,2,4-oxadiazole
compounds of Formula 1-D.
[0207] Certain 1,2,4-oxadiazole compounds of Formula 1-D may also
be prepared by the method described above using solid phase
chemistry as described in Scheme D2, below:
##STR01250##
[0208] In accordance with Scheme D2, commercially available,
acid-labile resin D4, such as trityl resin, 2-chlorotrityl chloride
resin, phenylacetamidomethyl (PAM) resin, or p-alkoxybenzyl alcohol
resin, is used in this example. The coupling of benzoic acid
compound D5 and trityl resin (here X=2-chlorotrityl chloride) can
be performed in a suitable solvent such as dichloromethane,
dimethylformamide, or toluene in the presence of a tertiary amine
reagent such as diisopropylethylamine or triethylamine to give
acylated resin D6. In an alternative method, the acylated resin D6
is conveniently prepared by standard ester linkage formation
conditions using diisopropylcarbodiimide (for phenylacetamidomethyl
resin and p-alkoxybenzyl alcohol resin) or equivalents such as
benzotriazole-1-yloxytrispyrrolidinophosphonium hexafluorophosphate
(PyBOP), bromotrispyrrolidinophosphonium hexafluorophosphate
(PyBrOP) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC) without or with diisopropylethylamine in
dimethylformamide. The resin-bound carboxybenzoic ester D6 can be
treated with cyanuric fluoride and a tertiary amine base, such as
N-methyl morpholine, triethylamine, or N,N-diisopropylethylamine,
in an inert solvent such as dichloromethane, dioxane,
tetrahydrofuran, or dimethylformamide to afford the acyl fluoride
compound D7.
[0209] The combinatorial chemistry method may use multi-reaction
vessels, where a different combination of reagents used in each
vessel to provide library compounds of interest. The resin-bound
acyl fluoride compound D7 is treated with a reagents of structure
D8 in the presence of a base, such as N-methylmorpholine,
N,N-diisopropylethylamine, or triethylamine, in an inert solvent
such as dichloromethane, tetrahydrofuran or dimethylformamide or
mixtures to give compounds D9. Hydroxyamidines D8 can be
conveniently prepared from treatment of nitriles with hydroxylamine
in an inert solvent such as ethanol, dioxane, or tetrahydrofuran.
The resin-bound acylated compound D9 can be cleaved under acidic
conditions such as 2N trifluoroacetic acid in dichloromethane or 3N
acetic acid in dichloromethane, to afford the desired compound D10.
Ring-closure of free acid compound D10 can be effected by heating
in an inert solvent such as toluene, tetrahydrofuran, dioxane or
dimethylformamide or mixtures with or without a base reagent such
as diisopropylethylamine, triethylamine or tetrabutylammonium
fluoride to afford the 1,2,4-oxdiazole compounds of Formula
1-D.
[0210] Certain oxazoles of Formula 1-E can be prepared by the
methodology described in Scheme E1 below:
##STR01251##
[0211] In accordance with Scheme E1, .alpha.-Bromoketones of
structure E1 can be converted to .alpha.-aminoketones of structure
E2 with such reagents as e.g., urotropine. Reaction of the
.alpha.-aminoketones E2 with activated acids of type E3 in the
presence of base can give compounds of structure E4. The activated
acid E3 can be either an acid chloride or an acyl imidazolide.
Dehydration of the intermediate E4 with reagents such as
phosphorous pentoxide or phosphorous oxychloride within a
temperature range from ambient to 120.degree. C. gives the oxazoles
of Formula 1-E.
[0212] Certain oxazoles of Formula 1-E can also be prepared by the
methodology depicted in Scheme E2 below;
##STR01252##
[0213] In accordance with Scheme E2, carboxylic acids E5 in which
one carboxyl group is activated as the acid chloride or similar
activating group can react with ethylamines of the type E6 (in
which L.sub.2 is a leaving group) in the presence of a base, such
as triethylamine, to give the dihydrooxazole E7. Reaction of E7
with N-bromosuccinimide in refluxing carbon tetrachloride with a
catalytic amount of a radical initiator such as
azobisisobutyronitrile gives the bromooxazole E8. The bromooxazole
E8 can react with arylboronic acids E9 in the presence of a Pd
catalyst such as, but not limited to, tetrakistriphenylphosphine
palladium(0) or dichlorobis(triphenylphosphine)palladium(II) and a
base such as cesium fluoride or potassium carbonate and a solvent
such as toluene, dimethylformamide or dimethoxyethane to give the
oxazole compounds Formula 1-E.
[0214] Certain oxazoles of Formula 1-F can be prepared by the
methodology described in Scheme F below:
##STR01253##
[0215] In accordance with Scheme F, amide formation of esters of
structure F1 and ammonium hydroxide can be performed in a suitable
solvent such as water, tetrahydrofuran, dioxane or
dimethylformamide or a mixture with heating to give compounds of
structure F2. Heating compounds of structure F2 with
.alpha.-bromoketones in inert solvents such as toluene,
tetrahydrofuran, dioxane or dimethylformamide or mixtures, at
temperatures of 60-150.degree. C. can afford the desired oxazole
compounds of Formula 1-F.
[0216] Certain pyrazoles of Formula 1-G can be prepared by the
methodology depicted in Scheme G1 shown below or by those skilled
in the art.
##STR01254##
[0217] In accordance with Scheme G1, substituted diketones G3 can
be prepared by the treatment of substituted acetophenones G2 with
sodium hydride in a suitable solvent such as tetrahydrofuran and
subsequent reaction with cyanobenzoic esters of type G1. In a 1-pot
microwave sequence, the 1,3-diketones of structure G3 can be
reacted with 1.1 equivalents of anhydrous hydrazine in a protic
solvent such as ethanol at a power of 300 W and a temperature not
exceeding 100.degree. C. to afford, pyrazole benzonitriles of type
G4 which is then subsequently reacted with six equivalents of
aqueous 1N sodium hydroxide under identical microwave conditions to
afford pyrazole acids of Formula 1-G.
[0218] Certain pyrazoles of Formula 1-G can also be prepared by the
methodology described in Scheme G2 below:
##STR01255##
[0219] In accordance with Scheme G2, reaction of esters of type G5
with substituted acetophenones of type G6 in the presence of a
base, e.g., sodium hydride, in a suitable solvent such as
tetrahydrofuran, can give 1,3-diketones of structure G7. Hydrolysis
of the ester affords carboxylic acids of structure G8. The acid can
then be reacted with hydrazine in a protic solvent such as ethanol
at reflux to afford the pyrazoles of Formula 1-G.
[0220] Certain thiazoles of Formula 1-I can be prepared by the
methodology depicted in Scheme I shown below.
##STR01256##
[0221] In accordance with Scheme I, benzonitriles of structure I1
can be converted to thioamide compounds of structure I2 by
treatment with dithiophosphoric acid diethyl ester in inert
solvents such as water, tetrahydrofuran, dioxane or
dimethylformamide or mixtures at reflux temperature. Alternatively,
hydrogen sulfide gas can be used for the conversion of the nitrile
to the thioamide. Reaction of the thioamides I2 with
.alpha.-bromoketones I3 with heating in inert solvents such as
toluene, tetrahydrofuran, dioxane or dimethylformamide or mixtures,
afford the desired thiazole compounds of Formula 1-I.
[0222] Certain thiazoles of Formula 1-J can be prepared by the
methodology depicted in Scheme J below:
##STR01257##
[0223] In accordance with Scheme J, .alpha.-Aminoketones of
structure J1 can be reacted with activated carboxylic acid
derivatives of type J2, e.g., acid chlorides or acyl imidazolides,
in a suitable non-reactive organic solvent, optionally in the
presence of a base, e.g., triethylamine, to give compounds of
structure J3. Heating compounds of type J3 with phosphorous
pentasulfide in the presence of a solvent. e.g., pyridine, can give
the thiazoles of Formula 1-J.
[0224] Certain isoxazoles of Formula 1-K can be prepared by the
methodology depicted in Scheme K below:
##STR01258##
[0225] In accordance with Scheme K, oximes of structure K2 can be
derived from commercial benzaldehydes of structure K1 using
hydroxylamine hydrochloride and a base such as pyridine in a protic
solvent, such as ethanol. Reaction of oxime K2 with
N-chlorosuccinimide in dimethylformamide in the presence of gaseous
hydrochloric acid catalyst can afford .alpha.-chlorooximes of
structure K3. Treatment of K3 in a suitable organic solvent such as
dichloromethane with a base such as triethylamine at 0.degree. C.
to room temperature and a substituted acetylene, available
commercially or prepared by those skilled in the art, can afford an
isoxazole ester of Formula 1-K.
[0226] Certain isoxazoles of Formula 1-L can be prepared by the
methodology depicted in Scheme L.
##STR01259##
[0227] In accordance with Scheme L, oximes of structure L2 can be
derived from commercial benzaldehydes of structure L1 using
hydroxylamine hydrochloride and a base such as pyridine in a protic
solvent, preferably ethanol. Reaction of oxime L2 with
N-chlorosuccinimide in dimethylformamide in the presence of gaseous
hydrochloric acid catalyst can afford .alpha.-chlorooxime of
structure L3. Treatment of L3 in a suitable organic solvent such as
dichloromethane with a base such as triethylamine at 0.degree. C.
or room temperature and a substituted acetylene L4, prepared by
those skilled in the art using a two step sequence from the
corresponding iodides L5 can afford isoxazoles of Formula 1-L.
Alternatively, other halides of formula L5, such as bromides and
chlorides in place of the iodide can also be used to effect the two
step transformation to acetylene L4 by those skilled in the
art.
[0228] Certain imidazoles of Formula 1-M can be prepared by the
methodology depicted in Scheme M shown below:
##STR01260##
[0229] In accordance with Scheme M, heating amidines of structure
M1 with .alpha.-bromoketones of structure M2 in the presence of a
non-reactive solvent affords the imidazoles of Formula 1-M. The
amidines may be obtained commercially or prepared by methods known
by those skilled in the art for example by treatment of the
appropriate nitrite precursors with, e.g., sodium amide or sodium
hexamethyldisilazide. The reaction between M1 and M2 can be carried
out at a temperature range from ambient to 150.degree. C.
[0230] Certain imidazoles of Formula 1-N can be prepared by the
methodology depicted in Scheme N.
##STR01261##
[0231] In accordance with Scheme N, heating .alpha.-bromoketones of
structure N1 with amidines of structure N2 in the presence of a
non-reactive solvent affords the imidazoles of Formula 1-N. The
amidines may be prepared by methods known by those skilled in the
art, for example by treatment of the appropriate nitrile precursors
with, e.g., lithium or sodium hexamethyldisilazide. The reaction
between N1 and N2 can be carried out at a temperature range from
ambient to 150.degree. C.
[0232] Certain thiazoles of Formula 1-O can be prepared by the
methodology depicted in Scheme O shown below:
##STR01262##
[0233] In accordance with Scheme O, reaction of the thioamides O1
with .alpha.-bromoketones O2 with heating in inert solvents such as
toluene, tetrahydrofuran, dioxane or dimethylformamide or mixtures,
afford the desired thiazole compounds of Formula 1-O. The
thioamides may be purchased commercially, prepared from amides with
reagents such as Lawesson's reagent or phosphorous pentasulfide or
prepared from nitriles with such reagents as hydrogen sulfide or
dithiophosphoric acid diethyl ester.
[0234] Certain thiazoles of Formula 1-P can be prepared by the
methodology depicted in Scheme P1 shown below:
##STR01263##
[0235] In accordance with Scheme P1, .alpha.-Bromoketones of
structure P1 can be convened to .alpha.-aminoketones of structure
P2 with, e.g., urotropine. Reaction of the .alpha.-aminoketones P2
with carboxylic acid derivatives of type P3 in the presence of base
gives compounds of structure P4. Thio-dehydration and concomitant
cyclization of the intermediate P4 with reagents such as
phosphorous pentasulfide within a temperature range from ambient to
120.degree. C. gives the thiazoles of Formula 1-P.
[0236] Certain thiazoles of Formula 1-P can also be prepared by the
methodology depicted in Scheme P2 shown below:
##STR01264##
[0237] In accordance with Scheme P2, .alpha.-Aminoketones P5
(prepared as described in Scheme R) can be reacted with activated
carboxylic acid derivatives (P6) e.g., acid chlorides or acyl
imidazolides, in a suitable solvent, optionally in the presence of
a base, e.g., triethylamine, to give compounds of structure P7.
Thiodehydration of P7 with reagents such as phosphorous
pentasulfide or Lawesson's reagent within a temperature range from
ambient to 120.degree. C. gives thiazole compounds Formula 1-P.
[0238] Certain oxazoles of Formula 1-Q can be prepared by the
methodology depicted in Scheme Q below:
##STR01265##
[0239] In accordance with Scheme Q, commercially available
carboxamides of structure Q1 or carboxamides prepared from
commercially available acid chlorides or carboxylic acids can be
reacted with .alpha.-bromoketones of structure Q2 to give oxazole
compounds of Formula 1-Q. The reaction can be carried out in inert
solvents such as toluene, tetrahydrofuran, dioxane or
dimethylformamide or mixtures, at temperatures of 60-150.degree.
C.
[0240] Certain oxazoles of the Formula 1-R can be prepared by the
methodology described in Scheme R below:
##STR01266##
[0241] In accordance with Scheme R, .alpha.-Bromoketones of
structure R1 can be converted to .alpha.-aminoketones R3 by initial
displacement with sodium azide to give the .alpha.-azidoketones R2.
Conversion to the .alpha.-aminoketones R3 can be carried out by
reduction of the .alpha.-azidoketones via catalytic hydrogenation
in the presence of acid, such as hydrochloric acid. The reduction
can be carried out from 1-4 atmospheres of pressure in the presence
of either protic or non-protic solvents. The active catalyst can be
e.g., platinum or palladium metal on charcoal. The
.alpha.-aminoketones R3 can then be reacted with activated
carboxylic acid derivatives (R4) e.g., acid chlorides or acyl
imidazolides, in a suitable solvent, optionally in the presence of
a base, e.g., triethylamine, to give compounds of structure R5.
Dehydration of the intermediate R5 with reagents such as
phosphorous pentoxide or phosphorous oxychloride within a
temperature range from ambient to 120.degree. C. gives the oxazoles
of Formula 1-R.
[0242] Certain furans of Formula 1-T can be prepared by the
methodology depicted in Scheme T.
##STR01267##
[0243] In accordance with Scheme T, reaction between ketoesters of
structure T1 and .alpha.-bromoketones of structure T2 afford
intermediate compounds of structure T3. Ketoesters T1 can be
obtained by a number of methods known by those skilled in the art.
Heating intermediate compounds T3 under conditions that facilitate
dehydration can give the furan compounds of Formula 1-T, wherein
R.sub.2 is an ester group. The reaction can be carried out in the
presence of an acid, e.g., HCl or p-toluenesulfonic acid, or heated
in the presence of a reagent such as phosphorous oxychloride or
phosphorous pentoxide to induce dehydration and cyclization.
[0244] Decarboxylation of the intermediate T3 gives compounds of
type T4. Conditions for the decarboxylation reaction can include
heating with a nucleophilic reagent in a nonreactive solvent, e.g.,
sodium chloride in H.sub.2O-DMSO or LiI in pyridine or selective
hydrolysis, trifluoroacetic acid if the ester to be decarboxylated
is t-butyl or catalytic reduction if the ester to be decarboxylated
is a benzyl ester. Heating the resultant intermediate compounds T4
under conditions that facilitate dehydration can give the furan
compounds of Formula 1-T. The reaction can be carried out in the
presence of an acid, such as HCl or p-toluenesulfonic acid, or
heated in the presence of a reagent such as phosphorous oxychloride
or phosphorous pentoxide to induce dehydration and cyclization.
[0245] Certain 1,2,4-thiadiazoles of Formula 1-U can be prepared by
the methodology depicted in Scheme U.
##STR01268##
[0246] In accordance with Scheme U, heating amide compounds of
structure U1 with a thionating agent, e.g., trichloromethyl
sulfenyl chloride (U2) can give the oxathiazole intermediate
compounds U3. The reaction is typically carried out in a
non-reactive solvent, e.g., toluene or xylenes and heated at
80-150.degree. C. Reaction of the oxathiazole compounds thus formed
with nitrites of structure U4 at high temperature can give the
1,2,4-thiadiazoles compounds of Formula 1-U.
[0247] Certain 1,2,4-thiadiazoles of Formula 1-V can be prepared by
the methodology depicted in Scheme V.
##STR01269##
[0248] In accordance with Scheme V, heating primary amide compounds
of structure V1 with a thionating agent, such as trichloromethyl
sulfenyl chloride (V2), can give intermediate oxathiazole compounds
V3. This reaction is typically carried out in a non-reactive
solvent, such as toluene or xylenes, and heated at 80-150.degree.
C. Reaction of the oxathiazole compounds thus formed with nitriles
of structure V4 at high temperature can give the 1,2,4-thiadiazoles
compounds of Formula 1-V.
[0249] Certain thiophenes of Formula 1-W can be prepared by the
methodology depicted in Scheme W.
##STR01270##
[0250] 2,4-dibromo or diiodothiophenes W1 can be reacted with
boronic acid compounds W2 with an appropriate catalyst, such as
tetrakis (triphenylphosphine) palladium (0), bis
(triphenylphosphine) palladium (II) dichloride or palladium acetate
with added phosphine ligand, to give compounds of structure W3.
These reactions are carried out in a suitable solvent, such as DMF,
toluene, dimethoxyethane or dioxane at a temperature range of
ambient to 150.degree. C. in the presence of added base. The
coupling reaction typically takes place at the more reactive
halogen, typically at the 2-position of the thiophene. A second
coupling reaction is then carried out with boronic acid W4, which
reacts with the remaining bromide or iodide under similar
conditions to give compounds of Formula 1-W.
[0251] Certain thiophenes of Formula 1-X may be prepared by similar
methodology as described above. This is depicted in Scheme X.
##STR01271##
[0252] In accordance, with Scheme X, 2,4-dibromo or
diiodothiophenes X1 can be reacted with boronic acid compounds X2
with an appropriate catalyst, e.g., tetrakis (triphenylphosphine)
palladium (0), his (triphenylphosphine) palladium (II) dichloride
or palladium acetate with added phosphine ligand to give compounds
of structure X3. The reactions are carried out in a suitable
solvent, such as DMF, toluene, dimethoxyethane or dioxane at a
temperature range of ambient to 150.degree. C. in the presence of
added base. The coupling reaction typically takes place at the more
reactive halogen, typically at the 2-position of the thiophene. A
second coupling reaction is then carried out with boronic acid X4
which reacts with the remaining bromide or iodide under similar
conditions to give compounds of Formula 1-X.
[0253] Certain thiophenes of Formula 1-Y can be prepared by the
methodology depicted in Scheme Y.
##STR01272##
[0254] In accordance with. Scheme Y, 2,4-dibromo or
diiodothiophenes Y1 can be reacted with boronic acid compounds Y2
with an appropriate catalyst, such as tetrakis (triphenylphosphine)
palladium (0), bis (triphenylphosphine) palladium (II) dichloride
or palladium acetate with added phosphine ligand, to give compounds
of structure Y3 as described in the previous examples. A second
coupling reaction is then carried out with boronic acid Y4, which
reacts with the remaining bromide or iodide under similar
conditions to give compounds of Formula 1-Y.
[0255] Certain 1,2,4-triazoles of Formula 1-Z can be prepared by
the methodology depicted in Scheme Z.
##STR01273##
[0256] In accordance with Scheme Z, cyanobenzoic acids of structure
Z1 can be converted to methoxyimidates Z2 by treatment of Z1 with
HCl in methanol in the cold, such as 0.degree. C. Reaction of Z2
with substituted hydrazides (Z3) in the presence of a base and a
suitable nonreactive solvent gives an intermediate, which is then
heated in the presence of an appropriate solvent (e.g., dioxane) or
a mixture of solvents at a temperature range of 60-150.degree. C.
to give the desired cyclized compounds of Formula 1-Z.
[0257] In certain preferred embodiments, compounds of the invention
may be resolved to enantiomerically pure compositions or
synthesized as enantiomerically pure compositions using any method
known in art. By way of example, compounds of the invention may be
resolved by direct crystallization of enantiomer mixtures, by
diastereomer salt formation of enantiomers, by the formation and
separation of diasteriomers or by enzymatic resolution of a racemic
mixture.
[0258] These and other reaction methodologies may be useful in
preparing the compounds of the invention, as recognized by one of
skill in the art. Various modifications to the above schemes and
procedures will be apparent to one of skill in the art, and the
invention is not limited specifically by the method of preparing
the compounds of the invention.
[0259] C. Methods of the Invention
[0260] In another aspect of the invention, methods are provided for
the suppression of premature translation termination, which may be
associated with a nonsense mutation, and for the prevention or
treatment of diseases. In a preferred embodiment, such diseases are
associated with mutations of mRNA, especially nonsense mutations.
Exemplary diseases include, but are not limited to, cancer,
lysosomal storage disorders, the muscular dystrophies, cystic
fibrosis, hemophilia, epidermolysis bullosa and classical late
infantile neuronal ceroid lipofuscinosis. In this embodiment,
methods for treating cancer, lysosomal storage disorders, a
muscular dystrophy, cystic fibrosis, hemophilia, or classical late
infantile neuronal ceroid lipofuscinosis are provided comprising
administering a therapeutically effective amount of at least one
compound of the invention to a subject in need thereof.
[0261] In one embodiment, the present invention is directed to
methods for increasing the expression of one or more specific,
functional proteins. Any compound of the invention can be used to
specifically increase expression of functional protein. In another
embodiment, a specific increase in expression of functional protein
occurs when premature translation termination is suppressed by
administering a therapeutically effective amount of at least one
compound of the invention to a subject in need thereof. In a
preferred embodiment premature translation termination is
associated with a nonsense mutation in mRNA. In another embodiment,
a specific increase in expression of functional protein occurs when
mRNA decay is reduced in a patient. In a preferred embodiment, the
abnormality in a patient is caused by mutation-mediated mRNA decay.
In a particularly preferred embodiment, mutation-mediated mRNA
decay is the result of a nonsense mutation. The methods of the
present invention are not limited by any particular theory.
[0262] The invention encompasses methods of treating and preventing
diseases or disorders ameliorated by the suppression of premature
translation termination, nonsense-mediated mRNA decay, or premature
translation termination and nonsense-mediated mRNA decay in a
patient which comprise administering to a patient in need of such
treatment or prevention a therapeutically effective amount of a
compound of the invention.
[0263] In one embodiment, the present invention encompasses the
treatment or prevention of any disease that is associated with a
gene exhibiting premature translation termination,
nonsense-mediated mRNA decay, or premature translation termination
and nonsense-mediated mRNA decay. In one embodiment, the disease is
due, in part, to the lack of or reduced expression of the gene
resulting from a premature stop codon. Specific examples of genes
which may exhibit premature translation termination and/or
nonsense-mediated mRNA decay and diseases associated with premature
translation termination and/or nonsense-mediated mRNA decay are
found in U.S. Provisional Patent Application No. 60/390,747,
titled: Methods For Identifying Small Molecules That Modulate
Premature Translation Termination And Nonsense Mediated mRNA Decay,
filed Jun. 21, 2002, and International Application PCT/US03/19760,
filed Jun. 23, 2003, both of which are incorporated herein by
reference in their entirety.
[0264] Diseases ameliorated by the suppression of premature
translation termination, nonsense-mediated mRNA decay, or premature
translation termination and nonsense-mediated mRNA decay include,
but are not limited to: genetic diseases, somatic diseases,
cancers, autoimmune diseases, blood diseases, collagen diseases,
diabetes, neurodegenerative diseases, proliferative diseases,
cardiovascular diseases, pulmonary diseases, inflammatory diseases
or central nervous system diseases.
[0265] In one embodiment, diseases to be treated or prevented by
administering to a patient in need thereof an effective amount of a
compound of the invention include, but are not limited to,
amyloidosis, hemophilia, Alzheimer's disease, Tay Sachs disease,
Niemann Pick disease, atherosclerosis, giantism, dwarfism,
hypothyroidism, hyperthyroidism, aging, obesity, Parkinson's
disease, cystic fibrosis, muscular dystrophy, heart disease, kidney
stones, ataxia-telangiectasia, familial hypercholesterolemia,
retinitis pigmentosa, Duchenne muscular dystrophy, epidermolysis
bullosa and Marfan syndrome. In one embodiment, the diseases are
associated with a nonsense mutation.
[0266] In one embodiment, the compounds of the invention are useful
for treating or preventing an autoimmune disease. In one
embodiment, the autoimmune disease is associated with a nonsense
mutation. In a preferred embodiment, the autoimmune disease is
rheumatoid arthritis or graft versus host disease.
[0267] In another embodiment, the compounds of the invention are
useful for treating or preventing a blood disease. In one
embodiment, the blood disease is associated with a nonsense
mutation. In a preferred embodiment, the blood disease is
hemophilia, Von Willebrand disease, .beta.-thalassemia
[0268] In another embodiment, the compounds of the invention are
useful for treating or preventing a collagen disease. In one
embodiment, the collagen disease is associated with a nonsense
mutation. In a preferred embodiment, the collagen disease is
osteogenesis imperfecta or cirrhosis.
[0269] In another embodiment, the compounds of the invention are
useful for treating or preventing diabetes. In one embodiment, the
diabetes is associated with a nonsense mutation.
[0270] In another embodiment, the compounds of the invention are
useful for treating or preventing an inflammatory disease. In one
embodiment, the inflammatory disease is associated with a nonsense
mutation. In a preferred embodiment, the inflammatory disease is
arthritis, rheumatoid arthritis or osteoarthritis.
[0271] In another embodiment, the compounds of the invention are
useful for treating or preventing a central nervous system disease.
In one embodiment, the central nervous system disease is associated
with a nonsense mutation. In one embodiment, the central nervous
system disease is a neurodegenerative disease. In a preferred
embodiment, the central nervous system disease is multiple
sclerosis, muscular dystrophy, Duchenne muscular dystrophy,
Alzheimer's disease, Tay Sachs disease, Niemann Pick disease, late
infantile neuronal ceroid lipofuscinosis (LINCL) or Parkinson's
disease.
[0272] In another preferred embodiment, the compounds of the
invention are useful for treating or preventing cancer,
particularly in humans. In a preferred embodiment, the cancer is of
the head and neck, eye, skin, mouth, throat, esophagus, chest,
bone, blood, lung, colon, sigmoid, rectum, stomach, prostate,
breast, ovaries, kidney, liver, pancreas, brain, intestine, heart
or adrenals. In one embodiment, the cancer is a solid tumor. In one
embodiment, the cancer is associated with a nonsense mutation. In
another embodiment, the cancer is associated with a genetic
nonsense mutation. In another embodiment, the cancer is associated
with a somatic mutation. Without being limited by any theory, the
use of the compounds of the invention against cancer may relate to
its action against mutations of the p53 gene.
[0273] In one embodiment, the cancer is not a blood cancer. In
another embodiment, the cancer is not leukemia. In another
embodiment, the cancer is not multiple myeloma. In another
embodiment, the cancer is not prostate cancer.
[0274] In another preferred embodiment, the compounds of the
invention are useful for treating or preventing cancer associated
with a mutation of tumor suppressor gene. Such genes include, but
are not limited to PTEN, BRCA1, BRCA2, Rb, and the p53 gene. In one
embodiment, the mutation is a genetic mutation. In another
embodiment, the mutation is a somatic mutation. The methods of the
invention are particularly useful for treating or preventing a
cancer associated with a nonsense mutation in the in a tumor
suppressor gene. In a preferred embodiment, the methods of the
invention are particularly useful for treating or preventing a
cancer associated with a p53 gene due to the role of p53 in
apoptosis. Without being limited by theory, it is thought that
apoptosis can be induced by contacting a cell with an effective
amount of a compound of the invention resulting in suppression of
the nonsense mutation, which, in turn, allows the production of
full-length p53 to occur. Nonsense mutations have been identified
in the p53 gene and have been implicated in cancer. Several
nonsense mutations in the p53 gene have been identified (see, e.g.,
Masuda et al., 2000, Tokai J Exp Clin Med. 25(2):69-77; Oh et al.,
2000, Mol Cells 10(3):275-80; Li et al., 2000, Lab Invest.
80(4):493-9; Yang et al., 1999, Zhonghua Zhong Liu Za Zhi
21(2):114-8; Finkelstein et al., 1998, Mol Diagn. 3(1):37-41;
Kajiyama et al., 1998, Dis Esophagus. 11(4):279-83; Kawamura et
al., 1999, Leuk Res. 23(2):115-26; Radig et al., 1998, Hum Pathol,
29(11):1310-6; Schuyer et al., 1998, Int J Cancer 76(3):299-303;
Wang-Gohrke et al., 1998, Oncol Rep. 5(1):65-8; Fulop at al., 1998,
J Reprod Med. 43(2):119-27; Ninomiya et al., 1997, J Dermatol Sci.
14(3):173-8; Hsieh et al., 1996, Cancer Let. 100(1-2):107-13; Rall
et al., 1996, Pancreas. 12(1):10-7; Fukutomi et al., 1995, Nippon
Rinsho. 53(11):2764-8; Frebourg et al., 1995, Am J Hum Genet.
56(3):608-15; Dove et al., 1995, Cancer Surv. 25:335-55; Adamson et
al., 1995, Br J Haematol. 89(1):61-6; Grayson et al., 1994, Am J
Pediatr Hematol Oncol. 16(4):341-7; Lepelley et al., 1994,
Leukemia. 8(8):1342-9; McIntyre et al., 1994, J Clin Oncol.
12(5):925-30; Horio et al., 1994, Oncogene. 9(4):1231-5; Nakamura
et al., 1992, Jpn J Cancer Res. 83(12):1293-8; Davidoff et al.,
1992, Oncogene. 7(1):127-33: and Ishioka et al., 1991, Biochem
Biophys Res Commun. 177(3):901-6; the disclosures of which are
hereby incorporated by reference herein in their entireties). Any
disease associated with a p53 gene encoding a premature translation
codon including, but not limited to, the nonsense mutations
described in the references cited above, can be treated or
prevented by compounds of the invention.
[0275] In other embodiments, diseases to be treated or prevented by
administering to a patient in need thereof an effective amount of a
compound of the invention include, but are not limited to, solid
tumors such as sarcoma, carcinomas, fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic
neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,
retinoblastoma, a blood-born tumor or multiple myeloma.
[0276] In another embodiment, diseases to be treated or prevented
by administering to a patient in need thereof an effective amount
of a compound of the invention include, but are not limited to, a
blood-born tumor such as acute lymphoblastic leukemia, acute
lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,
acute myeloblastic leukemia, acute promyelocytic leukemia, acute
monoblastic leukemia, acute erythroleukemic leukemia, acute
megakaryoblastic leukemia, acute myelomonocytic leukemia, acute
nonlymphocytic leukemia, acute undifferentiated leukemia, chronic
myelocytic leukemia, chronic lymphocytic leukemia, hairy cell
leukemia, or multiple myeloma. See e.g., Harrison's Principles of
Internal Medicine, Eugene Braunwald et al., eds., pp. 491-762 (15th
ed. 2001).
[0277] In yet another embodiment, the invention encompasses the
treatment of a human afflicted with a solid tumor or a blood
tumor.
[0278] In a preferred embodiment, the invention encompasses a
method of treating or preventing a disease ameliorated by
modulation of premature translation termination, nonsense-mediated
mRNA decay, or premature translation termination and
nonsense-mediated mRNA decay, or ameliorating one or more symptoms
associated therewith comprising contacting a cell with a
therapeutically effective amount of a compound of the invention.
Cells encompassed by the present methods include animal cells,
mammalian cells, bacterial cells, and virally infected cells. In
one embodiment, the nonsense mutation is a genetic mutation (i.e.,
the nonsense codon was present in the progenitor DNA). In another
embodiment, the nonsense mutation is a somatic mutation (i.e., the
nonsense codon arose spontaneously or from mutagenesis).
[0279] In certain embodiments, a compound of the invention is
administered to a subject, including but not limited to a plant,
reptile, avian, amphibian or preferably a mammal, more preferably a
human, as a preventative measure against a disease associated with
premature translation termination, nonsense-mediated mRNA decay, or
premature translation termination and nonsense-mediated mRNA
decay.
[0280] In a preferred embodiment, it is first determined that the
patient is suffering from a disease associated with premature
translation termination and/or nonsense-mediated mRNA decay. In
another embodiment, the patient has undergone a screening process
to determine the presence of a nonsense mutation comprising the
steps of screening a subject, or cells extracted therefrom, by an
acceptable nonsense mutation screening assay. In a preferred
embodiment, the DNA of the patient can be sequenced or subjected to
Southern Blot, polymerase chain reaction (PCR), use of the Short
Tandem Repeat (STR), or polymorphic length restriction fragments
(RFLP) analysis to determine if a nonsense mutation is present in
the DNA of the patient. In one embodiment, it is determined whether
the nonsense mutation is a genetic mutation or a somatic mutation
by comparison of progenitor DNA. Alternatively, it can be
determined if altered levels of the protein with the nonsense
mutation are expressed in the patient by western blot or other
immunoassays. In another embodiment, the patient is an unborn child
who has undergone screening in utero for the presence of a nonsense
mutation. Administration of a compound of the invention can occur
either before or after birth. In a related embodiment, the therapy
is personalized in that the patient is screened for a nonsense
mutation screening assay and treated by the administration of one
or more compounds of the invention; particularly, the patient may
be treated with a compound particularly suited for the mutations in
question; e.g., depending upon the disease type, cell type, and the
gene in question. Such methods are well known to one of skill in
the art.
[0281] In another embodiment, the cells (e.g., animal cells,
mammalian cells, bacterial cells, plant cells and virtually
infected cells) are screened for premature translation termination
and/or nonsense-mediated mRNA decay with a method such as that
described above (i.e., the DNA of the cell can be sequenced or
subjected to Southern Blot, polymerase chain reaction (PCR), use of
the Short Tandem Repeat (STR), or polymorphic length restriction
fragments (RFLP) analysis to determine if a nonsense mutation is
present in the DNA of the cell; the RNA of the cell can be
subjected to quantitative real time PCR to determine transcript
abundance).
[0282] Specific methods of the invention further comprise the
administration of an additional therapeutic agent (i.e., a
therapeutic agent other than a compound of the invention). In
certain embodiments of the present invention, the compounds of the
invention can be used in combination with at least one other
therapeutic agent. Therapeutic agents include, but are not limited
to non-opioid analgesics; non-steroid anti-inflammatory agents;
steroids, antiemetics; .beta.-adrenergic blockers; anticonvulsants;
antidepressants; Ca.sup.2+-channel blockers; anticancer agent(s)
and antibiotics and mixtures thereof.
[0283] In certain embodiments, the compounds of the invention can
be administered or formulated in combination with anticancer
agents. Suitable anticancer agents include, but are not limited to:
alkylating agents; nitrogen mustards; folate antagonists; purine
antagonists; pyrimidine antagonists; spindle poisons; topoisomerase
inhibitors; apoptosis inducing agents; angiogenesis inhibitors;
podophyllotoxins; nitrosoureas; cisplatin; carboplatin; interferon;
asparaginase; tamoxifen; leuprolide; flutamide; megestrol;
mitomycin; bleomycin; doxorubicin; irinotecan and taxol.
[0284] In certain embodiments, the compounds of the invention can
be administered or formulated in combination with antibiotics. In
certain embodiments, the antibiotic is an aminoglycoside (e.g.,
tobramycin), a cephalosporin (e.g., cephalexin, cephradine,
cefuroxime, cefprozil, cefaclor, cefixime or cefadroxil), a
clarithromycin (e.g., clarithromycin), a macrolide (e.g.,
erythromycin), a penicillin (e.g., penicillin V) or a quinolone
(e.g., ofloxacin, ciprofloxacin or norfloxacin). In a preferred
embodiment, the antibiotic is active against Pseudomonas
aeruginosa.
[0285] Without intending to be limited by theory, it is believed
that the methods of the present invention act through a combination
of mechanisms that suppress nonsense mutations. In preferred
embodiments, the methods of the invention comprise administering a
therapeutically effective amount of at least one compound of the
invention, e.g., a compound of Formula 1. Relative activity of the
compounds of the invention may be determined by any method known in
the art, including the assay described in Example 2 herein.
[0286] Compounds of the invention can be characterized with an in
vitro luciferase nonsense suppression assay. Luciferase assays are
included in the methods of the present invention. Luciferase can be
used as a functional reporter gene assay (light is only produced if
the protein is functional), and luciferase is extremely sensitive
(Light intensity is proportional to luciferase concentration in the
nM range). In one embodiment, an assay of the present invention is
a cell-based luciferase reporter assay. In a preferred cell-based
luciferase reporter assay, a luciferase reporter construct
containing a premature termination codon (UGA, UAA, or UAG) is
stably transfected in 293 Human Embryonic Kidney cells.
[0287] In another assay of the present invention, a preferred assay
is a biochemical assay consisting of rabbit reticulocyte lysate and
a nonsense-containing luciferase reporter mRNA. In another assay of
the present invention, the assay is a biochemical assay consisting
of prepared and optimized cell extract (Lie & Macdonald, 1999,
Development 126(22):4989-4996 and Lie & Macdonald, 2000,
Biochem. Biophys. Res. Commun. 270(2):473-481. In the biochemical
assay, mRNA containing a premature termination codon (UGA, UAA, or
UAG) is used as a reporter in an in vitro translation reaction
using rabbit reticulocyte lysate supplemented with tRNA, hemin,
creatine kinase, amino acids, KOAc, Mg(OAc)2, and creatine
phosphate. Translation of the mRNA is initiated within a virus
derived leader sequence, which significantly reduces the cost of
the assay because capped RNA is not required. Synthetic mRNA is
prepared in vitro using the T7 promoter and the MegaScript in vitro
transcription kit (Ambion, Inc.; Austin, Tex.). In assays of the
present invention, addition of gentamicin, an aminoglycoside known
to allow readthrough of premature termination codons, results in
increased luciferase activity and can be used as an internal
standard. Assays of the present invention can be used in
high-throughput screens. Hundreds of thousands of compounds can be
screened in cell-based and biochemical assays of the present
invention. In a preferred aspect, a functional cell-based assay
similar to the one described.
[0288] Compounds of the present invention include compounds capable
of increasing specific, functional protein expression from mRNA
molecules comprising premature termination codons. In one
embodiment, compounds of the present invention can preferentially
suppress premature translation termination. For example, a compound
of the present invention can be capable of suppressing a nonsense
mutation if the mutation results in UAA, but not capable of
suppressing a nonsense mutation if the mutation results in UAG.
Another non-limiting example can occur when a compound of the
present invention can be capable of suppressing a nonsense mutation
if the mutation results in UAA and is followed, in-frame by a
cytosine at the +1 position, but not capable of suppressing a
nonsense mutation if the mutation results in UAA and is followed,
in-frame by an adenine at the +1 position.
[0289] A stable cell line harboring the UGA nonsense-containing
luciferase gene can be treated with a test compound. In this
aspect, cells can be grown in standard medium supplemented with 1%
penicillin-streptomycin (P/S) and 10% fetal bovine serum (FBS) to
70% confluency and split 1:1 the day before treatment. The next
day, cells are trypsinized and 40,000 cells are added to each well
of a 96-well tissue culture dish. Serial dilutions of each compound
are prepared to generate a six-point dose response curve spanning 2
logs (30 .mu.M to 0.3 .mu.M). The final concentration of the DMSO
solvent remains constant at 1% in each well. Cells treated with 1%
DMSO serve as the background standard, and cells treated with
gentamicin serve as a positive control.
[0290] To address the effects of the nonsense-suppressing compounds
on mRNAs altered in specific inherited diseases, a bronchial
epithelial cell line harboring a nonsense codon at amino acid 1282
(W1282X) can be treated with a compound of the invention and CFTR
function is monitored as a cAMP-activated chloride channel using
the SPQ assay (Yang et al, Hum. Mol. Genet. 2(8):1253-1261 (1993)
and Howard et al, Nat. Med. 2(4):467-469 (1996)). The increase in
SPQ fluorescence in cells treated with a compound of the invention
is compared to those treated with cAMP and untreated cells. An
increase in SPQ fluorescence in cells is consistent with
stimulation of CFTR-mediated halide efflux and an increase in
readthrough of the nonsense codon. Full-length CFTR expression from
this nonsense-containing allele following treatment with a compound
of the invention demonstrates that cystic fibrosis cell lines
increase chloride channel activity when treated with a compound of
the invention.
[0291] D. Metabolites of the Compounds of the Invention
[0292] Also falling within the scope of the present invention are
the in vivo metabolic products of the compounds described herein.
Such products may result for example from the oxidation, reduction,
hydrolysis, amidation, esterification and the like of the
administered compound, primarily due to enzymatic processes.
Accordingly, the invention includes compounds produced by a process
comprising contacting a compound of this invention with a mammalian
tissue or a mammal for a period of time sufficient to yield a
metabolic product thereof. Such products typically are identified
by preparing a radio-labeled (e.g. C.sup.14 or H.sup.3) compound of
the invention, administering it in a detectable dose (e.g., greater
than about 0.5 mg/kg) to a mammal such as rat, mouse, guinea pig,
monkey, or to man, allowing sufficient time for metabolism to occur
(typically about 30 seconds to 30 hours), and isolating its
conversion products from urine, blood or other biological samples.
These products are easily isolated since they are labeled (others
are isolated by the use of antibodies capable of binding epitopes
surviving in the metabolite). The metabolite structures are
determined in conventional fashion, e.g., by MS or NMR analysis. In
general, analysis of metabolites may be done in the same way as
conventional drug metabolism studies well-known to those skilled in
the art. The conversion products, so long as they are not otherwise
found in vivo, are useful in diagnostic assays for therapeutic
dosing of the compounds of the invention even if they possess no
biological activity of their own.
[0293] E. Pharmaceutical Compositions of the Invention
[0294] While it is possible for the compounds of the present
invention to be administered neat, it may be preferable to
formulate the compounds as pharmaceutical compositions.
[0295] As such, in yet another aspect of the invention,
pharmaceutical compositions useful in the methods of the invention
are provided. The pharmaceutical compositions of the invention may
be formulated with pharmaceutically acceptable excipients such as
carriers, solvents, stabilizers, adjuvants, diluents, etc.,
depending upon the particular mode of administration and dosage
form. The pharmaceutical compositions should generally be
formulated to achieve a physiologically compatible pH, and may
range from a pH of about 3 to a pH of about 11, preferably about pH
3 to about pH 7, depending on the formulation and route of
administration. In another embodiment, pharmaceutical compositions
of the invention may be formulated so that the pH is adjusted to
about pH 4 to about pH 7. In alternative embodiments, it may be
preferred that the pH is adjusted to a range from about pH 5 to
about pH 8.
[0296] More particularly, the pharmaceutical compositions of the
invention comprise a therapeutically or prophylactically effective
amount of at least one compound of the present invention, together
with one or more pharmaceutically acceptable excipients.
Optionally, the pharmaceutical compositions of the invention may
comprise a combination of compounds of the present invention, or
may include a second active ingredient useful in the treatment of
cancer, diabetic retinopathy, or exudative macular
degeneration.
[0297] Formulations of the present invention, e.g., for parenteral
or oral administration, are most typically solids, liquid
solutions, emulsions or suspensions, while inhalable formulations
for pulmonary administration are generally liquids or powders, with
powder formulations being generally preferred. A preferred
pharmaceutical composition of the invention may also be formulated
as a lyophilized solid that is reconstituted with a physiologically
compatible solvent prior to administration. Alternative
pharmaceutical compositions of the invention may be formulated as
syrups, creams, ointments, tablets, and the like.
[0298] The pharmaceutical compositions of the invention can be
administered to the subject via any drug delivery route known in
the art. Specific exemplary administration routes include oral,
ocular, rectal, buccal, topical, nasal, ophthalmic, subcutaneous,
intramuscular, intravenous (bolus and infusion), intracerebral,
transdermal, and pulmonary.
[0299] The term "pharmaceutically acceptable excipient" refers to
an excipient for administration of a pharmaceutical agent, such as
the compounds of the present invention. The term refers to any
pharmaceutical excipient that may be administered without undue
toxicity. Pharmaceutically acceptable excipients are determined in
part by the particular composition being administered, as well as
by the particular method used to administer the composition.
Accordingly, there exists a wide variety of suitable formulations
of pharmaceutical compositions of the present invention (see, e.g.,
Remington's Pharmaceutical Sciences, 18.sup.th Ed., Mack Publishing
Co., 1990).
[0300] Suitable excipients may be carrier molecules that include
large, slowly metabolized macromolecules such as proteins,
polysaccharides, polylactic acids, polyglycolic acids, polymeric
amino acids, amino acid copolymers, and inactive virus particles;
Other exemplary excipients include antioxidants such as ascorbic
acid; chelating agents such as EDTA; carbohydrates such as dextrin,
hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid;
liquids such as oils, water, saline, glycerol and ethanol; wetting
or emulsifying agents; pH buffering substances; and the like.
Liposomes are also included within the definition of
pharmaceutically acceptable excipients.
[0301] The pharmaceutical compositions of the invention may be
formulated in any form suitable for the intended method of
administration. When intended for oral use for example, tablets,
troches, lozenges, aqueous or oil suspensions, non-aqueous
solutions, dispersible powders or granules (including micronized
particles or nanoparticles), emulsions, hard or soft capsules,
syrups or elixirs may be prepared. Compositions intended for oral
use may be prepared according to any method known to the art for
the manufacture of pharmaceutical compositions, and such
compositions may contain one or more agents including sweetening
agents, flavoring agents, coloring agents and preserving agents, in
order to provide a palatable preparation.
[0302] Pharmaceutically acceptable excipients particularly suitable
for use in conjunction with tablets include, for example, inert
diluents, such as celluloses, calcium or sodium carbonate, lactose,
calcium or sodium phosphate; disintegrating agents, such as
croscarmellose sodium, cross-linked povidone, maize starch, or
alginic acid; binding agents, such as povidone, starch, gelatin or
acacia; and lubricating agents, such as magnesium stearate, stearic
acid or talc. Tablets may be uncoated or may be coated by known
techniques including microencapsulation to delay disintegration and
adsorption in the gastrointestinal tract and thereby provide a
sustained action over a longer period. For example, a time delay
material such as glyceryl monostearate or glyceryl distearate alone
or with a wax may be employed.
[0303] Formulations for oral use may be also presented as hard
gelatin capsules where the active ingredient is mixed with an inert
solid diluent, for example celluloses, lactose, calcium phosphate
or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with non-aqueous or oil medium, such as
glycerin, propylene glycol, polyethylene glycol, peanut oil, liquid
paraffin or olive oil.
[0304] In another embodiment, pharmaceutical compositions of the
invention may be formulated as suspensions comprising a compound of
the present invention in admixture with at least one
pharmaceutically acceptable excipient suitable for the manufacture
of a suspension. In yet another embodiment, pharmaceutical
compositions of the invention may be formulated as dispersible
powders and granules suitable for preparation of a suspension by
the addition of suitable excipients.
[0305] Excipients suitable for use in connection with suspensions
include suspending agents, such as sodium carboxymethylcellulose,
methylcellulose, hydroxypropyl methylcelluose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth, gum acacia, dispersing or
wetting agents such as a naturally occurring phosphatide (e.g.,
lecithin), a condensation product of an alkylene oxide with a fatty
acid (e.g., polyoxyethylene stearate), a condensation product of
ethylene oxide with a long chain aliphatic alcohol (e.g.,
heptadecaethyleneoxycetanol), a condensation product of ethylene
oxide with a partial ester derived from a fatty acid and a hexitol
anhydride (e.g., polyoxyethylene sorbitan monooleate); and
thickening agents, such as carbomer, beeswax, hard paraffin or
cetyl alcohol. The suspensions may also contain one or more
preservatives such as acetic acid, methyl and/or n-propyl
p-hydroxy-benzoate; one or more coloring agents; one or more
flavoring agents; and one or more sweetening agents such as sucrose
or saccharin.
[0306] The pharmaceutical compositions of the invention may also be
in the form of oil, in-water emulsions. The oily phase may be a
vegetable oil, such as olive oil or arachis oil, a mineral oil,
such as liquid paraffin, or a mixture of these. Suitable
emulsifying agents include naturally-occurring gums, such as gum
acacia and gum tragacanth; naturally occurring phosphatides, such
as soybean lecithin, esters or partial esters derived from fatty
acids; hexitol anhydrides, such as sorbitan monooleate; and
condensation products of these partial esters with ethylene oxide,
such as polyoxyethylene sorbitan monooleate. The emulsion may also
contain sweetening and flavoring agents. Syrups and elixirs may be
formulated with sweetening agents, such as glycerol, sorbitol or
sucrose. Such formulations may also contain a demulcent, a
preservative, a flavoring or a coloring agent.
[0307] Additionally, the pharmaceutical compositions of the
invention may be in the form of a sterile injectable preparation,
such as a sterile injectable aqueous emulsion or oleaginous
suspension. This emulsion or suspension may be formulated according
to the known art using those suitable dispersing or wetting agents
and suspending agents which have been mentioned above. The sterile
injectable preparation may also be a sterile injectable solution or
suspension in a non-toxic parenterally acceptable diluent or
solvent, such as a solution in 1,2-propane-diol. The sterile
injectable preparation may also be prepared as a lyophilized
powder. Among the acceptable vehicles and solvents that may be
employed are water, Ringer's solution, and isotonic sodium chloride
solution. In addition, sterile fixed oils may be employed as a
solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid may likewise be used in
the preparation of injectables.
[0308] Generally, the compounds of the present invention useful in
the methods of the present invention are substantially insoluble in
water and are sparingly soluble in most pharmaceutically acceptable
protic solvents and in vegetable oils. However, the compounds are
generally soluble in medium chain fatty acids (e.g., caprylic and
capric acids) or triglycerides and have high solubility in
propylene glycol esters of medium chain fatty acids. Also
contemplated in the invention are compounds which have been
modified by substitutions or additions of chemical or biochemical
moieties which make them more suitable for delivery (e.g., increase
solubility, bioactivity, palatability, decrease adverse reactions,
etc.), for example by esterification, glycosylation, PEGylation,
etc.
[0309] In a preferred embodiment, the compounds of the present
invention may be formulated for oral administration in a
lipid-based formulation suitable for low solubility compounds.
Lipid-based formulations can generally enhance the oral
bioavailability of such compounds. As such, a preferred
pharmaceutical composition of the invention comprises a
therapeutically or prophylactically effective amount of a compound
of the present invention, together with at least one
pharmaceutically acceptable excipient selected from the group
consisting of: medium chain fatty acids or propylene glycol esters
thereof (e.g., propylene glycol esters of edible fatty acids such
as caprylic and capric fatty acids) and pharmaceutically acceptable
surfactants such as polyoxyl 40 hydrogenated castor oil.
[0310] In an alternative preferred embodiment, cyclodextrins may be
added as aqueous solubility enhancers. Preferred cyclodextrins
include hydroxypropyl, hydroxyethyl, glucosyl, maltosyl and
maltotriosyl derivatives of .alpha.-, .beta.-, and
.gamma.-cyclodextrin. A particularly preferred cyclodextrin
solubility enhancer is hydroxypropyl-.beta.-cyclodextrin (HPBC),
which may be added to any of the above-described compositions to
further improve the aqueous solubility characteristics of the
compounds of the present invention. In one embodiment, the
composition comprises 0.1% to 20%
hydroxypropyl-.beta.-cyclodextrin, more preferably 1% to 15%
hydroxypropyl-.beta.-cyclodextrin, and even more preferably from
2.5% to 10% hydroxypropyl-.beta.-cyclodextrin. The amount of
solubility enhancer employed will depend on the amount of the
compound of the present invention in the composition.
[0311] The therapeutically effective amount, as used herein, refers
to an amount of a pharmaceutical composition of the invention to
treat, ameliorate, or modulate an identified disease or condition,
or to exhibit a detectable therapeutic or inhibitory effect. The
effect can be detected by, for example, assays of the present
invention. The effect can also be the prevention of a disease or
condition where the disease or condition is predicted for an
individual or a high percentage of a population.
[0312] The precise effective amount for a subject will depend upon
the subject's body weight, size, and health; the nature and extent
of the condition; the therapeutic or combination of therapeutics
selected for administration, the protein half-life, the mRNA
half-life and the protein localization. Therapeutically effective
amounts for a given situation can be determined by routine
experimentation that is within the skill and judgment of the
clinician.
[0313] For any compound, the therapeutically effective amount can
be estimated initially either in cell culture assays, e.g., of
neoplastic cells, or in animal models, usually rats, mice, rabbits,
dogs, or pigs. The animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans. Therapeutic/prophylactic efficacy and
toxicity may be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, e.g., ED.sub.50 (the dose
therapeutically effective in 50% of the population) and LD.sub.50
(the dose lethal to 0.50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index, and
it can be expressed as the ratio, LD.sub.50/ED.sub.50.
Pharmaceutical compositions that exhibit large therapeutic indices
are preferred. The data obtained from cell culture assays and
animal studies may be used in formulating a range of dosage for
human use. The dosage contained in such compositions is preferably
within a range of circulating concentrations that include an
ED.sub.50 with little or no toxicity. The dosage may vary within
this range depending upon the dosage form employed, sensitivity of
the patient, and the route of administration.
[0314] More specifically, the concentration-biological effect
relationships observed with regard to the compound(s) of the
present invention indicate an initial target plasma concentration
ranging from approximately 5 .mu.g/mL to approximately 100
.mu.g/mL, preferably from approximately 10 .mu.g/mL to
approximately 50 .mu.g/mL, more preferably from approximately 10
.mu.g/mL to approximately 25 .mu.g/mL. To achieve such plasma
concentrations, the compounds of the invention may be administered
at doses that vary from 0.1 .mu.g to 100,000 mg, depending upon the
route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and is generally
available to practitioners in the art. In general the dose will be
in the range of about 1 mg/day to about 10 g/day, or about 0.1 g to
about 3 g/day, or about 0.3 g to about 3 g/day, or about 0.5 g to
about 2 g/day, in single, divided, or continuous doses for a
patient weighing between about 40 to about 100 kg (which dose may
be adjusted for patients above or below this weight range,
particularly children under 40 kg).
[0315] The magnitude of a prophylactic or therapeutic dose of a
particular active ingredient of the invention in the acute or
chronic management of a disease or condition will vary, however,
with the nature and severity of the disease or condition, and the
route by which the active ingredient is administered. The dose, and
perhaps the dose frequency, will also vary according to the age,
body weight, and response of the individual patient. Suitable
dosing regimens can be readily selected by those skilled in the art
with due consideration of such factors. In general, the recommended
daily dose range for the conditions described herein lie within the
range of from about 1 mg/kg to about 150 mg/kg per day. In one
embodiment, the compound of the invention is given as a single
once-a-day dose. In another embodiment, the compound of the
invention is given as divided doses throughout a day. More
specifically, the daily dose is administered in a single dose or in
equally divided doses. Preferably, a daily dose range should be
from about 5 mg/kg to about 100 mg/kg per day, more preferably,
between about 10 mg/kg and about 90 mg/kg per day, even more
preferably 20 mg/kg to 60 mg/kg per day. In managing the patient,
the therapy should be initiated at a lower dose, perhaps about 200
mg to about 300 mg, and increased if necessary up to about 600 mg
to about 4000 mg per day as either a single dose or divided doses,
depending on the patient's global response. It may be necessary to
use dosages of the active ingredient outside the ranges disclosed
herein in some cases, as will be apparent to those of ordinary
skill in the art. Furthermore, it is noted that the clinician or
treating physician will know how and when to interrupt, adjust, or
terminate therapy in conjunction with individual patient
response.
[0316] The phrases "therapeutically effective amount",
"prophylactically effective amount" and "therapeutically or
prophylactically effective amount," as used herein encompass the
above described dosage amounts and dose frequency schedules.
Different therapeutically effective amounts may be applicable for
different diseases and conditions, as will be readily known by
those of ordinary skill in the art. Similarly, amounts sufficient
to treat or prevent such diseases, but insufficient to cause, or
sufficient to reduce, adverse effects associated with conventional
therapies are also encompassed by the above described dosage
amounts and dose frequency schedules.
[0317] The exact dosage will be determined by the practitioner, in
light of factors related to the subject that requires treatment.
Dosage and administration are adjusted to provide sufficient levels
of the active agent(s) or to maintain the desired effect. Factors
which may be taken into account include the severity of the disease
state, general health of the subject, age, weight, and gender of
the subject, diet, time, protein of interest half-life, RNA of
interest half-life, frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
[0318] F. Combination Therapy
[0319] It is also possible to combine any compound of the present
invention with one or more other active ingredients useful in the
treatment of diseases associated with nonsense mutations of mRNA as
described herein, including compounds in a unitary dosage form, or
in separate dosage forms intended for simultaneous or sequential
administration to a patient in need of treatment. When administered
sequentially, the combination may be administered in two or more
administrations. In an alternative embodiment, it is possible to
administer one or more compounds of the present invention and one
or more additional active ingredients by different routes.
[0320] The skilled artisan will recognize that a variety of active
ingredients may be administered in combination with the compounds
of the present invention that may act to augment or synergistically
enhance the nonsense mutation-suppressing activity of the compounds
of the invention.
[0321] According to the methods of the invention, the combination
of active ingredients may be: (1) co-formulated and administered or
delivered simultaneously in a combined formulation; (2) delivered
by alternation or in parallel as separate formulations; or (3) by
any other combination therapy regimen known in the art. When
delivered in alternation therapy, the methods of the invention may
comprise administering or delivering the active ingredients
sequentially, e.g., in separate solution, emulsion, suspension,
tablets, pills or capsules, or by different injections in separate
syringes. In general, during alternation therapy, an effective
dosage of each active ingredient is administered sequentially,
i.e., serially, whereas in simultaneous therapy, effective dosages
of two or more active ingredients are administered together.
Various sequences of intermittent combination therapy may also be
used.
[0322] G. Gene Therapy
[0323] The compounds of the present invention or other nonsense
compounds can be utilized in combination with gene therapy. In this
embodiment, a gene can be introduced or provided to a mammal,
preferably a human that contains a specified nonsense mutation in
the desired gene. In a preferred aspect, the desired gene is
selected from the group consisting of IGF1, EPO, p53, p19ARF, p21,
PTEN, EI 24 and ApoAI. In order to obtain expression of the
full-length polypeptide in a patient or mammal, the patient or
mammal would be provided with an effective amount of a compound of
the present invention or other nonsense compound when such
polypeptide is desired.
[0324] There are two major approaches to getting nucleic acid that
contain a nonsense mutation (optionally contained in a vector) into
the patient's cells: in vivo and ex vivo. For in vivo delivery the
nucleic acid is injected directly into the patient, usually at the
sites where the polypeptide is required, i.e., the site of
synthesis of the polypeptide, if known, and the site (e.g. solid
tumor) where biological activity of the polypeptide is needed. For
ex vivo treatment, the patient's cells are removed, the nucleic
acid is introduced into these isolated cells, and the modified
cells are administered to the patient either directly or, for
example, encapsulated within porous membranes that are implanted
into the patient (see e.g., U.S. Pat. Nos. 4,892,538 and
5,283,187). There are a variety of techniques available for
introducing nucleic acids into viable cells. The techniques vary
depending upon whether the nucleic acid is transferred into
cultured cells in vitro, or transferred in vivo in the cells of the
intended host. Techniques suitable for the transfer of nucleic acid
into mammalian cells in vitro include the use of liposomes,
electroporation, microinjection, transduction, cell fusion.
DEAE-dextran, the calcium phosphate precipitation method, etc.
Transduction involves the association of a replication-defective,
recombinant viral (preferably retroviral) particle with a cellular
receptor, followed by introduction of the nucleic acids contained
by the particle into the cell. A commonly used vector for ex vivo
delivery of the gene is a retrovirus.
[0325] The currently preferred in vivo nucleic and transfer
techniques include transfection with viral or non-viral vectors
(such as adenovirus, lentivirus, Herpes simplex 1 virus, or
adeno-associated virus (AAV)) and lipid-based systems (useful
lipids for lipid-mediated transfer of the gene are, for example,
DOTMA, DOPE, and DC-Chol; see, e.g., Tonkinson et al. Cancer
Investigation, 14 (1): 54-65 (1996)). The most preferred vectors
for use in gene therapy are viruses, most preferably adenoviruses,
AAV, lentiviruses, or retroviruses. A viral vector such as a
retroviral vector includes at least one transcriptional
promoter/enhancer or locus-defining element(s), or other elements
that control gene expression by other means such as alternate
splicing, nuclear RNA export, or post-translational modification of
messenger. In addition, a viral vector such as a retroviral vector
includes a nucleic acid sequence that, when transcribed with a gene
encoding a polypeptide, is operably linked to the coding sequence
and acts as a translation initiation sequence. Such vector
constructs also include a packaging signal, long terminal repeats
(LTRs) or portions thereof, and positive and negative strand primer
binding sites appropriate to the virus used (if these are not
already present in the viral vector). In addition, such vector
typically includes a signal sequence for secretion of the
polypeptide from a host cell in which it is placed. Preferably the
signal sequence for this purpose is a mammalian signal sequence,
most preferably the native signal sequence for the polypeptide.
Optionally, the vector construct may also include a signal that
directs polyadenylation, as well as one or more restriction sites
and a translation termination sequences. By way of example, such
vectors will typically include a 5' LTR, a tRNA binding site, a
packaging signal, a origin of second-strand DNA synthesis, and a 3'
LTR or a portion thereof. Other vectors can be used that are
non-viral, such as cationic lipids, polylysine, and dendrimers.
[0326] In some situations, it is desirable to provide the nucleic
acid source with an agent that targets the target cells, such as an
antibody specific for a cell-surface membrane protein or the target
cell, a ligand for a receptor on the target cell, etc. Where
liposomes are employed, proteins that bind to a cell-surface
membrane protein associated with endocytosis may be used for
targeting and/or to facilitate uptake, e.g., capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins that undergo internalization in cycling, and proteins that
target intracellular localization and enhance intracellular
half-life. The technique of receptor-mediated endocytosis is
described, for example, by Wu et al., J. Biol. Chem. 262: 4429-4432
(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA, 87:
3410-3414 (1990). For a review of the currently known gene marking
and gene therapy protocols, see, Anderson et al., Science 256:
808-813 (1992). See also WO 93/25673 and the references cited
therein.
[0327] Suitable gene therapy and methods for making retroviral
particles and structural proteins can be found in, e.g. U.S. Pat.
Nos. 5,681,746; 6,800,604 and 6,800,731.
[0328] To assist in understanding the present invention, the
following Examples are included. The experiments relating to this
invention should not, of course, be construed as specifically
limiting the invention and such variations of the invention, now
known or later developed, which would be within the purview of one
skilled in the art are considered to fall within the scope of the
invention as described herein and hereinafter claimed.
EXAMPLES
[0329] The present invention is described in more detail with
reference to the following non-limiting examples, which are offered
to more fully illustrate the invention, but are not to be construed
as limiting the scope thereof. The examples illustrate the
preparation of certain compounds of the invention, and the testing
of these compounds in vitro and/or in vivo. Those of skill in the
art will understand that the techniques described in these examples
represent techniques described by the inventors to function well in
the practice of the invention, and as such constitute preferred
modes for the practice thereof. However, it should be appreciated
that those of skill in the art should in light of the present
disclosure, appreciate that many changes can be made in the
specific methods that are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1: Preparation of Compounds of the Invention
Example A: Preparation of
3-[5-(4-isopropylphenyl)-[1,3,4]oxadiazol-2-yl]benzoic acid.
(Compound No. 6)
##STR01274##
[0331] Step A: A suspension of methyl 3-cyanobenzoate (5.05 g, 31.4
mmol), sodium azide (3.06 g, 47.0 mmol) and triethylamine
hydrochloride (6.47 g, 47 mmol) in 60 mL of toluene is heated at
reflux for 12 h and then cooled to rt. The heterogeneous mixture is
diluted with H.sub.2O and the phases are separated. The organic
layer is extracted with saturated NaHCO.sub.3, and the aqueous
phases are combined and washed with EtOAc. After discarding the
organic layer, the combined aqueous phases are acidified with 6N
HCl to approximately pH 2 and the resultant thick paste is
extracted with EtOAc (2.times.). The combined organic layers are
washed with saturated NaCl and then are dried and are concentrated
to give 5.30 g (83%) of methyl 3-(1H-tetrazol-5-yl)benzoate as a
white solid: mp 180-181.degree. C.; MS m/z 205.1 [MH.sup.+].
[0332] Step B: A suspension of methyl 3-(1H-tetrazol-5-yl)benzoate
(0.41 g, 2.0 mmol), 4-isopropyl benzoic acid (0.33 g, 2.0 mmol) and
dicyclohexyl carbodiimide (0.41 g, 2.0 mmol) in dichloroethane (10
mL) is heated at reflux for 20 h. After cooling to t, the mixture
is filtered and the solids are rinsed with methylene chloride. The
filtrate is washed with saturated NaHCO.sub.3 and then dried and
concentrated to a solid. Flash chromatography over silica gel
(EtOAc/CH.sub.2Cl.sub.2, 2-5%) gave 0.54 g (84%) of methyl
3-[5-(4-isopropylphenyl)-[1,3,4]oxadiazol-2-yl]benzoate as a tan
solid: mp 74-77.degree. C., .sup.1H NMR: (CDCl.sub.3) .delta. 8.74
(t, J=1.5, 1H), 8.33 (dt, J=1.5, 7.8, 1H), 8.20 (1=1.5, 7.8, 1H),
8.06 (dt, J=1.5, 8.4, 2H), 7.61 (t, J=7.9, 1H), 7.39 (dd, J=1.8,
8.4, 2H), 3.99 (s, 3H), 3.00 (septet, J=6.9, 1 H), 1.31 (d, J=6.9,
6); MS m/z 323.2 [MH.sup.+].
[0333] Step C: A solution of methyl
3-[5-(4-isopropylphenyl)-[1,3,4]oxadiazol-2-yl]benzoate (0.48 g,
1.49 mmol) in THF (10 mL) is treated with 1N NaOH (2.25 mL, 2.25
mmol) and is heated at reflux for 5 h. After cooling to rt, and
basifying with saturated NaHCO.sub.3, the aqueous phase is
extracted with EtOAc. The organic layer is then extracted with
NaHCO.sub.3 (2.times.). The aqueous phases are combined, acidified
to pH 2 and extracted with EtOAc (3.times.) and then are dried and
are concentrated to give a white solid. Recrystallization
(EtOAc/hexanes) gives 324 mg (71%) of
3-[5-(4-isopropylphenyl)-[1,3,4]-oxadiazol-2-yl]benzoic acid as
white needles: mp 202-204.degree. C., .sup.1H NMR: (DMSO-d.sub.6)
.quadrature. 8.54 (br s, 1H), 8.28 (d, J=7.8, 1H), 8.12 (d, J=7.8,
1H), 7.97 (d, J=8.1, 2H), 7.71 (t, J=7.7, 1H), 7.43 (d, =7.7, 1H),
2.95 (septet, J=6.9, 1H), 1.20 (d, J=6.9, 6H); MS m/z 309.2
[MH.sup.+], 307.2 [MH.sup.-].
[0334] In similar fashion, utilizing the above steps, the following
compounds are prepared from the appropriate cyanobenzoates and
carboxylic acid starting materials: Compound Nos: 1, 2, 3, 4, 5, 7,
8, 85, 86, 175, 222, 223, 224, 225, 278, 279, 283, 284, 285, 286,
292, 293, 315, 316, 317, 318, 319, 401, 402, 596, 601, 605, 606,
610, 615, 620, 621, 622, 624, 626, 628.
Example B: Preparation of
3-[5-(4-tertbutylphenyl)-[1,3,4]oxadiazol-2-yl]benzoic acid
(Compound No. 29
##STR01275##
[0336] Step A: 20 g of 2-chlorotrityl chloride resin (Rapp
polymere, Germany) is agitated in dry dimethylformamide (100 mL)
for 10 min and the solvent is then drained. To the resin is added a
solution of isophthalic acid (8.0 g, 48.2 mmol) in 1%
disopropylethylamine in dimethylformamide (150 mL) and then is
agitated for 4 h at room temperature. The solvents are drained and
the resin is washed sequentially with dichloromethane (3.times.200
mL.times.1 min), dimethylformamide (3.times.200 mL.times.1 min),
methanol (3.times.200 mL.times.1 min), and dichloromethane
(3.times.200 mL.times.1 min). The resin is vacuum dried for 4 h at
room temperature. The desired product is analyzed by cleavage of a
small amount of the reacted resin with
triethylsilane/trifluoroacetic acid/dichloromethane.
[0337] Step B: To a suspension of isophthalic resin that is
prepared in step A above (200 mg, 0.2 mmole) in DMF (3 mL) is added
PyBOP (520 mg, 1.0 mmole). After agitation for 5 min at room
temperature, 4-t-butylbenzohydrazide (1 mmol) is then added to the
reaction mixture. The reaction mixture is agitated overnight at
room temperature. The solvents are drained and the resin is washed
with dichloromethane (3.times.20 mL.times.10 min), DMF (3.times.20
mL.times.10 min), MeOH (3.times.20 mL.times.10 min), and
dichloromethane (3.times.20 mL.times.10 min). The resin is vacuum
dried for 4 h. The desired product is analyzed by cleavage of a
small amount of the reacted resin with
triethylsilane/trifluoroacetic acid/dichloromethane.
[0338] Step C: To a suspension of hydrazide resin from step B,
above (200 mg, 0.1 mmol) in dichloromethane is added
2-chloro-1,3-dimethylimidazolidinium chloride (CDC, 33.6 mg, 0.2
mmol) and triethylamine (56 .mu.L, 0.4 mmole) followed by agitation
at room temperature overnight. The solvents are drained and the
resin is washed with dichloromethane (3.times.20 mL.times.10 min),
DMF (3.times.20 mL.times.10 min), MeOH (3.times.20 mL.times.10
min), and dichloromethane (3.times.20 mL.times.10 min). The resin
is treated with 20% TFA in dichloromethane (4 mL) for 1 h at room
temperature. The resin is removed and the filtrate is concentrated
under reduced pressure to afford
3-[5-(4-tert-butylphenyl)-[1,3,4]oxadiazol-2-yl]benzoic acid. The
desired product is purified by preparative LC/MS. MS m/z 323.1
[M+H].sup.+ (95% purity).
[0339] The following compounds are prepared using the procedures
described above starting from either isophthalic acid or
terephthalic acid in step A and are reacted with the appropriate
hydrazine derivatives: Compound Nos: 22, 23, 24, 25, 26, 27, 28,
30, 31, 32, 33, 34, 35, 36, 37, 38, 62, 63, 64, 65, 66, 67, 123,
124, 125, 126, 27, 128, 129, 130, 131, 132, 133, 134, 135, 136,
137, 138, 139, 226, 228, 230, 232, 234, 236, 239, 241, 243, 245,
247, 249, 250, 252, 254, 103, 104, 105, 106, 107, 108, 109, 110,
111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 176,
177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,
190, 191, 192, 258, 259, 260, 261, 262, 263, 264, 272, 161, 162,
163, 170, 169, 166, 173, 167, 172, 168, 174, 171, 164, 165, 172,
265, 15, 16, 17, 18, 19, 21, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 53, 54, 55, 68, 69, 70, 71, 72, 73, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97 98, 99, 100, 101, 102, 142, 143, 144,
145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,
158, 159, 209, 210, 211, 212, 213 214, 215, 216, 217, 218, 219,
220, 221, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,
204, 205, 206, 207, 208, 294, 295, 296, 297, 298, 299, 300, 301,
302, 303, 304, 227, 229, 231, 233, 235, 237, 238, 240, 242, 244,
246, 248, 251, 253, 255, 266, 267, 268, 269, 270, 271, 273, 274,
305, 306, 307, 308, 309, 160.
Example C:
4-{5-[3-(toluene-4-sulfonylamino)phenyl]-[1,3,4]oxadiazol-2-yl}-
benzoic acid (Compound No. 13)
##STR01276##
[0341] Step A: A Parr bottle is charged with methyl
4-[5-(3-nitrophenyl)-[1,3,4]oxadiazol-2-yl]benzoate (4.04 g, 12.43
mmol), 0.80 g of 10% Pd--C, THF (200 mL) and EtOAc (50 mL) and the
mixture is hydrogenated at 50 psi for 5 h. The reaction mixture is
then diluted with saturated NaHCO.sub.3 and EtOAc and then is
filtered. The filtrate layers are separated and the aqueous layers
are extracted with additional EtOAc. The combined organic phases
are washed with H.sub.2O and saturated NaCl and then is dried and
concentrated to give 2.34 g (64%) of methyl
4-[5-(3-aminophenyl)-[1,3,4]oxadiazol-2-yl]benzoate a yellow solid;
.sup.1H NMR (DMSO-d.sub.6) .delta. 8.20-8.14 (m, 4H), 7.31 (t.
J=1.4, 1H), 7.22 (d. J=3.3, 2), 6.78 (m, 1H), 5.53 (br s, 2H), 3.88
(s, 3H).
[0342] Step B: A suspension of methyl
4-[5-(3-aminophenyl)-[1,3,4]oxadiazol-2-yl]benzoate (0.30 g, 1.02
mmol) pyridine (0.12 mL, 1.53 mmol) and p-toluenesulfonyl chloride
(0.23 g, 1.22 mmol) in CH.sub.2Cl.sub.2 (10 mL) is stirred at room
temperature overnight. The resultant mixture is diluted with
H.sub.2O and CH.sub.2Cl.sub.2 and is filtered to give 0.25 g (55%)
of methyl
4-{5-[3-(toluene-4-sulfonylamino)-phenyl]-[1,3,4]oxadiazol-2-yl}benzoate
as a white solid: mp 227-228.degree. C.; .sup.1H NMR (DMSO-d.sub.6)
.delta. 10.67 (s, 1H), 8.20-8.13 (m, 4H), 7.85 (br s, 1H),
7.76-7.69 (m, 3H), 7.46 (dt, J=2.7, 10.2, 1H), 7.36-7.33 (m, 3H),
3.88 (s, 3H), 2.30 (s, 3H); MS m/z 450.0 [MH.sup.+], 448.0
[MH.sup.-].
[0343] Step C: A suspension of methyl
4-{5-[3-(toluene-4-sulfonylamino)-phenyl]-[1,3,4]oxadiazol-2-yl}benzoate
(225 mg, 0.50 mmol) in THF (10 mL) and 1N NaOH (0.55 m L, 0.55
mmol) is heated at reflux overnight. After cooling to room
temperature, the reaction mixture is partitioned in EtOAC and
saturated NaHCO.sub.3. The phases are separated and the organic
layer is extracted with saturated NaHCO.sub.3 (3.times.). The
aqueous phases are combined and acidified to pH 2 with 6N HCl. The
resultant heterogeneous mixture is filtered and dried to obtain 129
mg (5.9%) of
4-{5-[3-(toluene-4-sulfonylamino)-phenyl]-[1,3,4]oxadiazol-2-yl}benzoic
acid as a tan powder: mp>275.degree. C.; .sup.1H NMR:
(DMSO-d.sub.6) .delta. 10.62 (br s, 1H), 8.18-8.11 (m, 4H), 7.84
(t, J=0.8, 1H), 7.76-7.67 (m, 3H), 7.47 (t, J=7.9, 1H), 7.35 (7.32
(m, 3H), 2.30 (s, 3H); MS m/z 436.0 [MH.sup.+], 434.0
[MH.sup.-].
[0344] Utilizing steps B-C above and substituting other sulfonyl
chlorides or acid chlorides the following compounds are prepared:
Compound Nos: 12 and 14.
Example D: Preparation of
4-(5-{3-[3-(4-isopropylphenyl)ureido]phenyl}-[1,3,4]oxadiazol-2-yl)benzoi-
c acid (Compound No. 60)
##STR01277##
[0346] Step A: A suspension of methyl
4-[5-(3-aminophenyl)-[1,3,4]oxadiazol-2-yl]benzoate from Example C
step A (0.30 g, 1.02 mmol) and 4-isopropylphenyl isocyanate (0.20
mL, 1.22 mmol) in dichloroethane (10 mL) is stirred for 3 days at
room temperature. The reaction mixture is filtered and the solid is
washed with CH.sub.2Cl.sub.2, to afford 0.28 g (60%) of methyl
4-(5-{3-[3-(4-isopropyl-phenyl)-ureido]-phenyl}-[1,3,4]oxadiazol-2-yl)ben-
zoate as a white solid: mp>270.degree. C., .sup.1H NMR:
(DMSO-d.sub.6) .quadrature. 8.97 (br s, 1H), 8.63 (br s, 1H), 8.33
(t, J=1.8, 1H), 8.22-8.13 (m, 4H), 7.70 (dt, J=1.7, 7.5, 1 H),
7.60-7.47 (m, 2H), 7.36 (d, J=8.4, 2H), 7.14 (d, J=8.4, 2H); 2.82
(septet, J=6.8, 1H), 1.18 (d, J=6.8, 6H); MS m/z 457.2 [MH.sup.+],
455.3 [MH.sup.-].
[0347] Step B: A suspension of methyl
4-(5-{3-[3-(4-isopropylphenyl)ureido]phenyl}-[1,3,4]oxadiazol-2-yl)benzoa-
te (0.23 g, 0.50 mmol) in THF (10 mL) and 1N NaOH (0.56 mL, 0.56
mmol) is heated at reflux for 2.5 h. After cooling to rt, the
reaction mixture is diluted with H.sub.2O, acidified to pH 2 with
6N HCl and is extracted with EtOAC (3.times.) and is dried and
concentrated to give 170 mg (77%) of methyl
4-(5-{3-[3-(4-isopropylphenyl)ureido]phenyl}-[1,3,4]oxadiazol-2-
-yl)benzoate as an off-white solid: mp>270.degree. C., .sup.1H
NMR: .quadrature. (DMSO-d.sub.6) 8.97 (br s, 1H), 8.63 (br s, 1H),
8.33 (br s, 1H), 8.20-8.10 (m, 4H), 7.70-7.45 (m, 3H), 7.35 (d,
J=7.2, 2H), 7.12 (d, J=7.2, 2H), 2.81 (m, 1H), 1.16 (d, J=5.4, 6H);
MS m/z 443.2 [MH.sup.+], 441.2 [MH.sup.-].
Example E: Preparation of
3-[5-(4-morpholin-4-yl-phenyl)-[1,3,4]oxadiazol-2-yl]benzoic acid
(Compound No. 82)
##STR01278##
[0349] Step A: A flame-dried tube is charged with Cs.sub.2CO.sub.3
(0.38 g, 1.17 mmol), (tris)-dibenzylidineacetone dipalladium (16
mg, 0.017 mmol), racemic-BINAP (21 mg, 0.033 mmol) and methyl
3-[5-(4-bromophenyl)-[1,3,4]oxadiazol-2-yl]benzoate (preparation by
the method of Example A, step B) (0.30 g, 0.83 mmol). After
evacuating and flushing with N.sub.2, morpholine (0.09 mL, 1.00
mmol) and toluene (3.6 mL) are added and the reaction is heated at
reflux for 24 h and then is cooled to room temperature. The
heterogeneous mixture is filtered, washed with EtOAc and is
concentrated. The residue is purified by flash chromatography over
silica gel (EtOAc/CH.sub.2Cl.sub.2, 5-10%) to afford 0.19 g (63%)
of methyl
3-[5-(4-morpholin-4-yl-phenyl)-[1,3,4]oxadiazol-2-yl]-benzoate as a
yellow solid: mp 150-151.degree. C., .sup.1H NMR: (CDCl.sub.3)
.delta. 8.72 (t, J=1.8, 1H), 8.34 (d, J=7.8, 1H), 8.19 (d, J=7.8,
1H), 8.03 (d, J=8.7, 2H), 7.61 (t, J=7.8, 1H) 6.99 (d, J=8.7, 2H),
3.99 (s, 3H), 3.89-3.87 (m, 4H), 3.39-3.30 (m, 4H).
[0350] Step B: A solution of methyl
3-[5-(4-morpholin-4-yl-phenyl)-[1,3,4]oxadiazol-2-yl]benzoate (0.14
g, 0.38 mmol) in THF (10 mL) and 1N NaOH (0.46 mL, 0.46 mmol) is
heated at reflux for 15 h. After cooling to room temperature, the
reaction mixture is diluted with H.sub.2O and the aqueous phase is
extracted with EtOAc. The organic layer is back-extracted with
saturated NaHCO.sub.3. The combined aqueous phases are acidified to
pH 4.5 with 0.5 N NaH.sub.2PO.sub.4 and extracted with EtOAC
(3.times.) to give, after drying and concentrating, 0.11 g (82%) of
3-[5-(4-morpholin-4-yl-phenyl)-[1,3,4]oxadiazol-2-yl]benzoic acid
as a yellow solid: mp 235-237.degree. C.; .sup.1H NMR:
(DMSO-d.sub.6) 8.56 (br s, 1H), 8.32 (d, J=7.5, 1H), 8.13 (d,
J=7.5, 1H), 7.95 (d, J=8.4, 2H), 7.73 (t, J=7.8, 1H), 7.11 (d,
J=8.4, 2H), 3.76-3.72 (m, 4H), 3.32-327 (m, 4H); MS m/z 352.3
[MH.sup.+], 350.3 [MH.sup.-].
[0351] In similar fashion, the following compounds are prepared by
reaction of methyl
3-[5-(4-bromophenyl)-[1,3,4]oxadiazol-2-yl]benzoate with the
appropriate amines following steps A-B above: Compound Nos: 83, 84,
and 280.
Example F: Preparation of
3-[5-(3'-methylbiphenyl-4-yl)-[1,3,4]oxadiazol-2-yl]benzoic acid
(Compound No. 281)
##STR01279##
[0353] Step A: A flame-dried tube is charged with 0.40 g, 1.11
mmol) of methyl 3-[5-(4-bromophenyl)-[1,3,4]oxadiazol-2-yl]benzoate
(from Example A, step B) m-tolyl boronic acid (0.21 g, 1.55 mmol),
(tris)-dibenzylidineacetone dipalladium (10 mg, 0.011 mmol) and KF
(0.19 g, 3.33 mmol). The tube is flushed with N.sub.2 followed by
the addition of THF (4 mL) and a solution of 0.7 M tri-tert-butyl
phosphine in hexane (0.08 m, 0.027 mmol). The reaction is stirred
at room temperature for 15 h and then is heated at reflux for 2 h.
After cooling to room temperature, the reaction is filtered, washed
with EtOAc, and the filtrate is washed with saturated NaHCO.sub.3
and then is dried and is concentrated. Flash chromatography
(EtOAc/CH.sub.2Cl.sub.2, 0-2%) over silica gel gives 0.18 g (44%)
of methyl
3-[5-(3'-methyl-biphenyl-4-yl)-[1,3,4]oxadiazol-2-yl]benzoate as a
white solid: mp 147-148.degree. C., .sup.1H NMR: (CDCl.sub.3)
.delta. 8.77 (t, J=3.1, 1H), 8.37 (dd, J=1.0, 7.5, 1H), 8.24-8.20
(m, 3H), 7.76 (d, J=8.4, 2H), 7.64 (t, J=7.8, 1H), 7.47-7.44 (m,
2H), 3.34 (t, J=7.8, 1H), 7.22 (d, J=7.2, 1H), 4.00 (s, 3H), 2.46
(s, 3H); MS m/z 371.2 [MH.sup.+].
[0354] Step B: A solution of methyl
3-[5-(3'-methy)-biphenyl-4-yl)-[1,3,4]oxadiazol-2-yl]benzoate (0.15
g, 0.41 mmol) in THF (5 mL) and 1N NaOH (0.51 mL, 0.51 mmol) and
H.sub.2O (1 mL) is heated at reflux overnight. After cooling to rt,
the reaction mixture is diluted with H.sub.2O and the pH is
adjusted to 4.5-5 by addition of NaH.sub.2PO.sub.4 and 1N HCl. The
mixture is extracted with EtOAc (3.times.) and then is dried and
concentrated to give
3-[5-(3'-methylbiphenyl-4-yl)-[1,3,4]oxadiazol-2-yl]benzoic acid as
a white solid: mp 240-242.degree. C.; .sup.1H NMR: .delta.
(DMSO-d.sub.6) 8.53 (s, 1H), 8.27 (dt, J=1.35, 8.1, 1 H), 8.14-8.08
(m, 3H), 7.80 (d, J=8.1, 2H), 7.70 (t, J=7.8, 1H), 7.49-7.45 (m,
2H), 7.32 (t, J=7.8, 1H), 7.17 (d, J=7.8, 1H), 2.36 (s, 3H); MS m/z
357.2 [MH.sup.+], 355.3 [MH.sup.-].
[0355] The following compound is made by the above procedure by
substituting methyl
3-[5-(6-bromopyridin-3-yl)-[1,3,4]oxadiazol-2-yl]benzoate, as
prepared as in Example A step B: Compound No. 282.
Example G: Preparation of
4-[5-(4-isopropylphenyl)-[1,3,4]thiadiazol-2-yl]benzoic acid
(Compound No. 324)
##STR01280##
[0357] Step A: A 0.degree. C. solution of 4-isopropylbenzohydrazide
(0.73 g, 4.10 mmol) in THF (20 mL) is treated with Et.sub.3N (0.62
mL) and methyl 4-chlorocarbonylbenzoate (0.90 g, 4.51 mmol). The
reaction is then warmed to room temperature and is stirred
overnight. The reaction mixture is then washed with H.sub.2O and is
extracted with EtOAc (3.times.). The combined organic phases are
washed with H.sub.2O and saturated NaCl and then is dried and
concentrated in vacuo to a solid. Purification by flash
chromatography over silica gel using EtOAc/CH.sub.2Cl.sub.2 (0-15%)
as eluent gives 0.86 g (62%) of
4-[N'-(4-isopropylbenzoyl)-hydrazinocarbonyl]benzoic acid as a
white solid: mp 235-237.degree. C.; MS m/z 341.2 [MH.sup.+], 339.2
[MH.sup.-].
[0358] Step B: A suspension of
4-[N'-(4-isopropylbenzoyl)-hydrazinocarbonyl]benzoic acid (0.25 g,
0.74 mmol) from step A above and Lawesson's reagent (0.59 g, 1.47
mmol) in CH.sub.2Cl.sub.2 (10 mL) is heated at reflux for 18 h and
then is cooled to room temperature. The crude reaction mixture is
concentrated in vacuo and is purified by flash chromatography
(EtOAC/CH.sub.2Cl.sub.2, 0-1%) to give 022 g (88%) of methyl
4-[5-(4-isopropylphenyl)-[1,3,4]thiadiazol-2-yl]benzoate as a white
solid: mp 147-151.degree. C.; .sup.1H NMR: (DMSO-d.sub.6) .delta.
8.16-8.09 (m, 4H), 7.93 (d, J=8.1, 2H), 7.45 (d, J=8.1, 2H), 3.89
(s, 3H), 2.98 (septet, J=6.8, 1H), 1.24 (d, J=6.9, 6H); MS m/z
339.2 [MH.sup.+].
[0359] Step C: A solution of methyl
4-[5-(4-isopropylphenyl)-[1,3,4]thiadiazol-2-yl]-benzoate (96 mg,
0.28 mmol) in THF is treated with 1N NaOH (0.36 mL, 0.36 mmol) and
H.sub.2O (0.65 mL) and the biphasic reaction mixture is heated at
reflux for 3 h and then is cooled to room temperature. After
diluting with additional H.sub.2O, sufficient 6N HCl is added until
the pH is adjusted to 2, resulting in the formation of a white
solid precipitate. The solid is filtered, washed with H.sub.2O and
is dried to give 60 mg (65%) of
4-[5-(4-isopropylphenyl)-[1,3,4]thiadiazol-2-yl]-benzoic acid:
m/p>300.degree. C.; .sup.1H NMR: (DMSO-d.sub.6) .delta.
7.99-7.88 (m, 6H), 7.44 (d, J8.1, 2H), 2.97 (septet, J=6.9, 1H),
1.24 (d, J=6.9, 6H); MS m/z 325.1 [MH.sup.+], 323.2 [MH.sup.-].
Example H. Preparation of
4-[5-(4-isopropylphenyl)-[1,2,4]oxadiazol-3-yl]benzoic acid
(Compound No. 275)
##STR01281##
[0361] Step A: To a solution of hydroxylamine, that is prepared
from 2.19 g (31.5 mmol) of NH.sub.2OH.HCl and 1.26 g (31.5 mmol) of
NaOH, in H.sub.2O/EtOH (1/1, 50 mL) is added methyl 4-cyanobenzoate
(4.83 g, 30.0 mmol). The reaction mixture is stirred at 90.degree.
C. overnight. The solvent is then replaced by EtOH/Hexanes (9/1, 50
mL) and stirred for 0.5 h at room temperature. The solid is removed
by filtration and the filtrate is evaporated to dryness to give a
white powder, which is further recrystallized from EtOH/Hexane to
give white needles (4.53 g, 77.8/%): MS m/z 195 [MH.sup.+]
[0362] Step B: To a 0.degree. C. solution of the above
hydroxyamidine (0.39 g, 2.05 mmol), 4-isopropylbenzoic acid (0.34
g, 2.05 mmol) and dichloromethane (10 mL) is added HOBt (0.28 g,
2.05 mmol) followed by DCC (0.42 g, 2.05 mmol). The mixture is
stirred at room temperature overnight. The precipitate is removed
by filtration and the filtrate is concentrated, followed by
chromatography over silica gel to give methyl
4-((Z)-amino{[(4-isopropylbenzoyl)oxy]imino}methyl)benzoate (0.60
g, 77%): MS m/z 341 [MH.sup.+].
[0363] Step C: The intermediate that is prepared above (0.48 g, 1.4
mmol) is heated in toluene (5.0 mL) at 130.degree. C. overnight,
cooled and chromatographed (silica gel, EtOAc/Hexanes, 2/8) to
provide methyl
4-[5-(4-isopropylphenyl)-1,2,4-oxadiazol-3-yl]benzoate as a white
powder (0.41 g, 91%): MS m/z 323 [MH.sup.+].
[0364] Step D: The methyl ester prepared as above (0.37 g, 1.15
mmol) is treated with BBr.sub.3 (1M in dichloromethane, 2.3 mL, 2.3
mmol) in dichloromethane (10 mL) at room temperature overnight. The
volatiles are removed in vacuo and the residue is treated with
water and the crude product is recrystallized from chloroform to
furnish the desired product,
4-[5-(4-isopropyl-phenyl)-[1,2,4]oxadiazol-3-yl]benzoic acid (0.23
g, 66%): mp. 210-213.degree. C.; .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta. 1.23 (d, 6H), 2.89-2.99 (m, 1H), 7.33 (d, 2H), 8.03-8.17
(m, 6H); MS m/z 307 [MH.sup.-].
[0365] The following compounds are prepared essentially following
the steps above with substitution of the appropriate carboxylic
acid derivative in step B: Compound Nos: 141 and 407.
Example I: Preparation of
4-[5-(4-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzoic acid (Compound
No. 412)
##STR01282##
[0367] Step A: 40 g of 2-chlorotrityl chloride resin (Rapp
polymere, Germany), is agitated in dimethylformamide (200 mL) for
10 min and the solvent is drained. To the resin is added a solution
of 4-cyanobenzoic acid (12.71 g, 96.4 mmol) in 300 mL of 1%
diisopropylethyl amine/dimethylformamide and is agitated 4 h at
room temperature. The solvents are drained and the resin is washed
with dichloromethane (3.times.200 mL.times.1 min),
dimethylformamide (3.times.200 mL.times.1 min), methanol
(3.times.200 mL.times.1 min), and dichloromethane (3.times.200
mL.times.1 min). The resin is vacuum dried for 4 h. The desired
product is analyzed by cleavage of a small amount of the reacted
resin with triethylsilane/trifluoroacetic acid/dichloromethane
(10/50/40): MS m/z 148 [MH.sup.+] (97% purity).
[0368] Step B: The 4-cyanobenzoic resin in ethanol (300 mL) is
agitated for 10 min at room temperature, and then the solvent is
drained. To a solution of hydroxylamine hydrochloride (35.81 g, 516
mmol) in ethanol (200 mL) is added diisopropylethylamine (89.3 mL,
516 mmol) and the mixture is stirred for 5 min at room temperature.
To the resin is added the above reaction mixture and agitated for
24 h at 40.degree. C. The solvents are drained, and the resin is
washed with dichloromethane (3.times.200 mL.times.10 min),
dimethylformamide (3.times.200 mL.times.10 min), methanol
(3.times.200 mL.times.10 min), and dichloromethane (3.times.200
mL.times.10 min). The resin is vacuum dried for 4 h. The desired
product is analyzed by cleavage of a small amount of the reacted
resin with triethylsilane/trifluoroacetic acid/dichloromethane
(10/50/40): MS m/z 181 [MH.sup.+] (92% purity).
[0369] Step C: To a suspension of hydroxyamidine resin (500 mg, 0.4
mmol) in anhydrous dichloromethane (3 mL) is added 4-fluorobenzoyl
chloride (95 .delta.L, 0.8 mmol) and diisopropylethylamine (138
.delta.L, 0.8 mmol). The reaction mixture is agitated overnight at
room temperature. The solvents are drained, and the resin is washed
with dichloromethane (3.times.10 mL.times.10 min),
dimethylformamide (3.times.10 mL.times.10 min), methanol
(3.times.10 mL.times.10 min), and dichloromethane (3.times.10
mL.times.10 min). The resin is vacuum dried for 4 h. The desired
product is analyzed by cleavage of a small amount of the reacted
resin with triethylsilane/trifluoroacetic acid/dichloromethane
(10/50/40): MS m/z 303 [MH.sup.+].
[0370] Step D: To a suspension of acylated resin in anhydrous
dichloromethane (1.5 mL) is added 50% trifluoroacetic acid in
dichloromethane (1.5 mL). The reaction mixture is agitated for 2 h
at room temperature. The resin is removed and the filtrate is
concentrated under reduced pressure. The residue is dissolved in
10% dimethylformamide in toluene (4 mL) and then is stirred for 2 h
at 130.degree. C. The solvents are removed and the desired product,
4-[5-(4-fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzoic acid, is
purified by preparative LC/MS: MS m/z 285 [MH.sup.+].
[0371] The following compounds are prepared using the procedures
described above: Compound Nos: 408, 409, 410, 411, 412, 413, 414,
415, 416, 417, 418, 419, 420, 421, 422, 430, 431, 432, 433, 434,
435, 436, 437, 438, 439, 440, 441, 442, 443, 445, 446, 447, 448,
449, 450, 451,452, 453, 444.
Example J: Preparation of
4-[3-(4-isopropylphenyl)-1,2,4-oxadiazol-5-yl]benzoic acid
(Compound No. 140)
##STR01283##
[0373] Step A: To a solution of hydroxylamine, prepared from 3.13 g
(45.0 mmol) of NH.sub.2OH.HCl and 1.89 g (45 mmol) of NaOH, in
H.sub.2O/EtOH (1/1, 50 mL) is added 4-isopropylbenzonitrile (4.35
g, 30.0 mmol). The reaction mixture is stirred at 90.degree. C.
overnight. The solvent is then replaced by EtOH/Hexanes (9/1, 50
mL) and is stirred for 0.5 h at room temperature. The solid is
removed by filtration and the filtrate is evaporated to dryness to
give a colorless oil, N-hydroxy-4-isopropylbenzenecarboximidamide
in quantitative yield: MS m/z 195 [MH.sup.+].
[0374] Step B: To a 0.degree. C. solution of the intermediate
prepared above, (0.27 g, 1.50 mmol), triethylamine (0.18 g, 0.25 m,
1.8 mmol) in dichloromethane (10 mL) is added methyl
4-(chlorocarbonyl)benzoate (0.32 g, 1.58 mmol). The mixture is then
stirred at room temperature for 4 h. The mixture is then washed
with water and brine, and is dried over anhydrous sodium sulfate
and is filtered. The solvent is replaced with toluene and is
stirred at 130.degree. C. in a sealed tube overnight. The crude
product obtained after the removal of the solvent is
chromatographed to provide methyl
4-[3-(4-isopropylphenyl)-1,2,4-oxadiazol-5-yl]benzoate (0.38 g,
79%): MS m/s 323 [MH.sup.+].
[0375] Step C: The methyl ester prepared above (0.37 g, 1.15 mmol)
is treated with BBr.sub.3 (1M in dichloromethane, 2.3 mL, 2.3 mmol)
in dichloromethane (10 mL) at room temperature overnight. The
volatiles are removed in vacuo and the residue is treated with
water and the crude product is recrystallized from chloroform to
furnish the desired product,
4-[3-(4-isopropylphenyl)-1,2,4-oxadiazol-5-yl]benzoic acid (0.34 g,
97%): mp 253-255.degree. C. .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta. 1.25 (d, 6H), 2.90-3.00 (m, 1H), 7.31 (d, 2H), 8.01-8.24
(m, 6H); MS m/z 307 [MH.sup.-].
[0376] In similar fashion, utilizing the above steps, the following
compounds are prepared by substitution of the appropriate
benzonitriles in step A above and reaction with methyl
3-(chlorocarbonyl)benzoate starting materials in step B above:
Compound Nos: 349, 364, 394, 396, 397, 398, 399, 403, 404, 405, and
406.
Example K: Preparation 3-[3-(2-fluorophenyl)-[1,2,4]
oxadiazole-5-yl] benzoic acid (Compound No. 506)
##STR01284##
[0378] Steps A-C (1-pot): To a solution of 2-fluorobenzonitrile
(484 mg, 4.00 mmol, Aldrich) in 3 mL of t-BuOH is added 274 .mu.L
(4.48 mmol, 1.12 equiv.) of a solution of 50% NH.sub.2OH/H.sub.2O.
The solution is heated to 73.degree. C. for 20 h, an additional
portion of 50% NH.sub.2OH/H.sub.2O is added (100 .mu.L, 1.60 mmol,
0.38 equiv.), and the mixture is heated for 2 days at 73.degree. C.
The resulting mixture of crude 2-fluoro-N-hydroxybenzamidine is
then cooled to 10.degree. C., diluted with 3 mL of t-BuOH, and
treated with Et.sub.3N (836 .mu.L, 6 mmol), followed by
3-chlorocarbonylbenzoic acid methyl ester (1.19 g, 6 mmol) to form
the intermediate O-acylated hydroxybenzamidine by slow warming of
the mixture to room temperature over a 1-2 h period. This
suspension is then heated to 90.degree. C., stirred for 3 days,
cooled to room temperature, diluted with 200 mL of 20%
THF/Et.sub.2O and filtered. The organic solution is washed with 1N
aqueous NaOH (2.times.50 mL), water (2.times.50 mL), dried
(MgSO.sub.4) and concentrated in vacuo to afford
3-[3-(2-fluorophenyl)-[1,2,4]oxadiazol-5-yl]benzoic acid methyl
ester which is taken directly into the next reaction without
further purification: MS m/z 299.33, calcd for
C.sub.16H.sub.12FN.sub.3O.sub.3 (MH.sup.+) 299.
[0379] Step D: The crude solid from step 3 (>93% pure by LC/MS)
is diluted with 40 mL of 50% THF/H.sub.2O, is heated to 65.degree.
C. for 5 h and cooled to room temperature. The solution is adjusted
to pH 4 by the slow addition of 6N aqueous HCl solution and
filtered. The resulting solid is washed with 30% Et.sub.2O/hexanes
and dried overnight at 70.degree. C. (10 torr) to afford 1.07 g
(94% over 4-steps) of 3-[3-(2-fluorophenyl)-[1,2,4]
oxadiazole-5-yl] benzoic acid as a white fluffy powder: mp
233-234.degree. C.; .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta.
7.45 (m, 2H), 7.66 (m, 1H), 7.79 (t, J=7.7 Hz, 1H), 8.13 (m, 1H),
8.24 (dt, J=8.0 Hz, 1.4 Hz, 1H), 8.39 (dt, J=8.0, 1.6 Hz, 1H), 8.65
(t, J=1.6 Hz, 1H); MS m/z 285.26, calcd for
C.sub.15H.sub.10FN.sub.3O.sub.2 (MH.sup.+) 285.
[0380] The following compounds are made essentially by the
procedures shown above starting from the appropriate substituted
nitriles: Compound Nos: 507, 508, 509, 510, 511, 512, 513, 559,
560, 561,562, 563, 564, 565, 569, 571, 572, 576, 577, 578, and
570.
Example L: Preparation of
3-[5-(4-isopropylphenyl)-oxazol-2-yl]benzoic acid (Compound No.
288)
##STR01285##
[0382] Step A: To a solution of hexamethylenetetraamine (7.0 g, 50
mmol) in 70 mL of dry toluene is added a solution of
2-bromo-1-(p-isopropylphenyl)ethanone (12 g, 50 mmol) in 40 mL dry
toluene at 0.degree. C. The reaction mixture is stirred overnight.
The solid formed is removed by filtration, washed with 20 mL of
toluene and then the solid (hexamethylenetetraammonium salt) is
added to a solution of concentrated hydrochloric acid (8.5 mL) in
80 mL of ethanol. The mixture is stirred for 24 h in the dark at
room temperature. The white solid (ammonium chloride) is removed by
filtration and the filtrate is evaporated. The residue is
recrystallized from ethanol/ether to give
2-amino-1-(p-isopropylphenyl)ethanone hydrochloride (7 g, 33 mmol)
as a yellow solid (70%).
[0383] Step B: A solution of isophthalic acid mono ethyl ester (5.2
g, 27 mmol) in 20 mL of thionyl chloride is refluxed for 3 h and
then is concentrated to remove excess thionyl chloride. The residue
is dissolved in dry THF (10 mL) and added dropwise to a solution of
2-amino-1-(p-isopropylphenyl)ethanone hydrochloride (4.7 g, 22
mmol) and pyridine (5 mL, 61 mmol) in dry THF (30 mL) at 0.degree.
C. After stirring for 24 h, the solvent is evaporated. The residue
is dissolved in 10 mL of water, is extracted with CH.sub.2Cl.sub.2,
is washed with brine and dried over Na.sub.2SO.sub.4. After
concentration in vacuo, the residue is purified by column
chromatography to give
N-[2-(4-isopropylphenyl)-2-oxo-ethyl]-isophthalamic acid ethyl
ester as a brown solid (5.5 g, 71%).
[0384] Step C: A solution of the above ester (500 mg, 1.42 mmol) in
5 mL of phosphorus oxychloride is refluxed for 2.5 h. After
evaporation of the solvent, the residue is dissolved in 20 mL of
conc. ammonia solution, is extracted with EtOAC, is washed with
brine and dried over Na.sub.2SO.sub.4. Concentration of the solvent
gives crude ethyl 3-[5-(4-isopropylphenyl)-oxazol-2-yl]benzoate as
a brown oil (340 mg, 72%).
[0385] Step D: A mixture of ethyl
3-[5-(4-isopropylphenyl)-oxazol-2-yl]benzoate (150 mg, 0.45 mg) and
lithium hydroxide (94 mg, 2.24 mmol) in methanol/water (9 mL/3 mL)
is stirred for 2 h. After evaporation of the solvent, the residue
is dissolved in 10 mL of water, treated with 1 g of citric acid,
extracted with EtOAc, washed with brine and dried over
Na.sub.2SO.sub.4. The solvent is concentrated in vacuo and the
product is recrystallized from CH.sub.2Cl.sub.2/hexane to give 71
mg (52%) of 3-[5-(4-isopropylphenyl)-oxazol-2-yl]benzoic acid a
pale yellow solid: mp 150-153.degree. C.; .sup.1H NMR (CDCl.sub.3)
.delta. 8.97 (br s, 1H), 8.33 (d, J=7.6, 1H), 8.21 (d, J=7.6, 1H),
7.68 (d, J=7.6, 2H), 7.62 (t, J=7.8, 1H), 7.50 (br s, 1H), 7.33 (d,
J=7.6, 2H), 2.97 (septet, J=6.8, 1H), 1.25 (d, J=6.9, 6H); MS m/z
308.2 [MH.sup.+].
Example M: Preparation of 4-[5-(2,
4-difluorophenyl)oxazol-2-yl]benzoic acid (Compound No. 548)
##STR01286##
[0387] Step A: Methyl 4-(4,5-dihydro-oxazol-2-yl)-benzoate: To a
solution of methyl 4-chlorocarbonylbenzoate (10.92 g, 54.98 mmol)
in toluene (200 mL) at room temperature is added 2-bromoethylamine
hydrobromide (10.25 g, 50.0 mmol) with stirring. The reaction
mixture is stirred at room temperature as triethylamine (35.0 ml,
251 mmol) is added. The reaction mixture is heated at reflux for 15
h and then cooled to room temperature. Water (200 mL) is added and
the mixture is extracted with CH.sub.2Cl.sub.2 (4.times.50 mL). The
extract is washed with water (2.times.50 mL), saturated aqueous
NaCl (2.times.50 mL) and dried over MgSO.sub.4, filtered and
concentrated on a rotary evaporator to give 6.86 g of methyl
4-(4,5-dihydro-oxazol-2-yl)benzoate as a tan solid in 67% %
yield.
[0388] Step B: Methyl 4-(5-bromo-oxazol-2-yl)benzoate: Methyl
4-(4,5-dihydrooxazol-2-yl)benzoate (6.86 g, 33.43 mmol) is
suspended in CCl.sub.4 (335 mL). N-bromosuccinimide (18.45 g, 103.7
mmol) is added followed by addition of azobisisobutyronitrile (50
mg). The reaction mixture is purged with nitrogen (5
vacuum/nitrogen cycles) and is heated to reflux for 17 h. The solid
is filtered, washed with CCl.sub.4, and discarded. The filtrate is
washed with a solution of saturated aqueous Na.sub.2S.sub.2O.sub.3
(40 mL), dried over MgSO.sub.4, filtered and concentrated on a
rotary evaporator to give the crude product. The product is further
purified by silica gel chromatography eluting with 1-6% ethyl
acetate/hexanes to give 4.42 g (47%) of methyl
4-(5-bromooxazol-2-yl) benzoate as a white solid.
[0389] Step C: Methyl
4-[5-(2,4-difluorophenyl)oxazol-2-yl]benzoate: Methyl
4-(5-bromooxazol-2-yl)benzoate (2.23 g, 7.91 mmol) is dissolved in
anhydrous dimethoxyethane (26 mL) and stirred at 25.degree. C.
2,4-Difluorophenyl boronic acid (1.39 g, 8.80 mmol), cesium
fluoride (2.89 g, 19.0 mmol) and
Dichlorobis(triphenylphosphine)palladium(II) (0.281 g, 0.40 mmol)
are then added. The reaction mixture is heated to reflux under
nitrogen for 16 h. The reaction mixture is cooled to room
temperature, the solid is filtered, is washed with dimethoxyethane
and is discarded. The filtrate is diluted with water to precipitate
the product, which is filtered, washed with water and dried to give
the crude product as a tan solid. The product is purified by silica
gel chromatography (10-20% ethyl acetate/hexanes) to give 1.16 g
(47%) of methyl 4-[5-(2,4-difluorophenyl)oxazol-2-yl]benzoate as a
light yellow solid.
[0390] Step D: 4-[5-(2,4-Difluoro-phenyl)oxazol-2-yl]benzoic acid:
Methyl 4-[5-(2,4-difluorophenyl)-oxazol-2-yl]benzoate is suspended
in a mixture of t-butanol (6 mL) and water (2 mL). Sodium hydroxide
(0.24 g, 6.0 mmol) is added and the reaction mixture is heated to
reflux for 15 h. The reaction mixture is cooled to room temperature
and acidified to pH 3 by addition of 10% aqueous hydrochloric acid
to precipitate the product. The solid is filtered, washed with
water (3.times.10 mL), and dried to give 1.04 g (94%) of
4-[5-(2,4-difluorophenyl)oxazol-2-yl]benzoic acid as a white solid:
mp 301-302.degree. C., .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.
7.30 (1H, dt, J=2.4, 8.1), 7.52 (1H, m), 7.71 (1H, d, J=3.6),
8.03-8.11 (m, 4H), 8.22 (2H, d, J36.9); MS m/z 302.32
[MH.sup.+].
[0391] The following compounds are made by the method described
above utilizing the appropriate boronic acids: Compounds Nos: 542,
543, 544, 545, 546, 547, 549, 550, 553, 554, 555, 556, 557, 558,
579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591,
592, 593, 594, 595, 527, 528, 629, 630, 631, 632, 633, 634, 635,
636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648,
649, 649 and 650.
Example N: Preparation of
3-[4-(4-pyrrolidin-1-yl-phenyl)-oxazol-2-yl]benzoic acid (Compound
No. 335)
##STR01287##
[0393] Step A: A solution of isophthalic acid methyl ester (20 g)
in ammonium hydroxide (100 mL) is stirred for 18 h at 120.degree.
C. The solvent is removed under reduced pressure and the desired
product is obtained as white solid.
[0394] Step B: To a solution of isophthalamic acid above, (160 mg,
0.96 mmol) in DMF (2 mL) is added
2-bromo-1-(4-pyrrolidin-1-yl-phenyl)-ethanone (158 mg, 0.96 mmol)
at room temperature. The reaction mixture is stirred for 18 h at
150.degree. C. and then cooled to ambient temperature. The solvent
is removed under reduced pressure and the desired product,
3-[4-(4-pyrrolidin-1-yl-phenyl)oxazol-2-yl]benzoic acid,
(MH.sup.+=355.0) is purified by prep. LC-MS.
[0395] The following compounds are prepared using the procedure
described above by substitution of the appropriate bromo or
chloroketones: Compound Nos: 351, 352, 353, 354, 355, 356, 357,
358, 359, 360, 361, 362, 363, 329, 330, 331, 332, 333, 334, 335,
336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 325, and
276.
Example O: Preparation of
3-[2-(4-isopropylphenyl)-oxazol-4-yl]benzoic acid (Compound No.
313)
##STR01288##
[0397] Step A: A solution of 4-isopropylbenzamide (301 mg, 1.85
mmol) and methyl 3-(2-bromoacetyl)benzoate (500 mg, 1.85 mmol) in 5
ml m-xylene is heated at 140-150.degree. C. for 7 h. After cooling,
the reaction is poured into water, extracted with EtOAc, dried over
MgSO.sub.4 and the product is purified by flash chromatography to
give 161 mg (27%) of methyl
3-[2-(4-isopropylphenyl)-oxazol-4-yl]-benzoate.
[0398] Step B: A solution of methyl
3-[2-(4-isopropylphenyl)oxazol-4-yl]benzoate (100 mg, 0.311 mmol)
and LiOH (64 mg, 1.56 mmol) in methanol/H.sub.2O (5 m/1.7 mL) is
stirred at room temperature for 0.5 h. The reaction mixture is then
heated to 45.degree. C. and stirred for 3 h. Upon completion of the
reaction, the solvent is removed under reduced pressure. The
residue is dissolved in 10 mL of water, neutralized, extracted with
EtOAc, and then washed with brine, dried over Na.sub.2SO.sub.4, and
concentrated to afford 90 mg (94%) of
3-[2-(4-isopropylphenyl)oxazol-4-yl]benzoic acid: mp
187-189.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta. 8.53 (br s,
1H), 8.12-8.04 (m, 5H), 7.56 (t, J=7.6, 1H), 7.34 (d, J=7.6, 2H),
2.98 (septet, J=36.8, 1H), 1.30 (d, J=6.8, 6H); MS m/z 308.3
[MH.sup.+].
Example P: Preparation of
3-[2-(4-isopropylphenyl)-oxazol-3-yl]-benzoic acid (Compound No.
320)
##STR01289##
[0400] Step A; A solution of 3-acetylbenzoic acid (0.67 g, 4.1
mmol) and a catalytic amount of TsOH in 50 mL of methanol is
refluxed for 20 h. The solvent is removed by evaporation and the
residue is dissolved in 50 mL of ether, washed with 20 mL of 5%
NaHCO.sub.3 and 20 mL of brine, dried (Na.sub.2SO.sub.4),
evaporated to give methyl 3-acetylbenzoate (0.71 g, 97%) as a pale
yellow oil.
[0401] Step B: A solution of methyl 3-acetylbenzoate (6.6 g, 37
mmol) in a mixture of ethyl ether and 1,4-dioxane (V:V=10:1, total
57.5 mL) is treated dropwise with bromine (1.91 mL, 37 mmol) over
30 min at room temperature. After the addition, the mixture is
stirred for an additional 40 min. The mixture is then treated with
an aqueous solution of NaHCO.sub.3 (4 g, 47 mmol in 40 mL) under
ice cooling and extracted with EtOAc (2.times.100 mL). The organic
layer is washed in turn with 50 mL of saturated NaHCO.sub.3, 50 mL
of water and 50 mL of brine, dried over anhydrous MgSO.sub.4, and
evaporated. The residue is purified by silica gel column
chromatography (petroleum ether-EtOAc, 15:1) to give methyl
3-(bromoacetyl)benzoate (6.5 g, 68%) as a white solid.
[0402] Step C: To a solution of methyl 3-(bromoacetyl)benzoate (3.8
g, 14.8 mmol) in 20 mL of DMF is added NaN.sub.3 at room
temperature and the mixture is stirred for 35 min. The reaction
mixture is diluted with 100 mL of ice water and extracted with
ether (3.times.50 mL). The combined organic layer is washed in turn
with water (2.times.40 mL), brine (40 mL) and dried over MgSO.sub.4
and concentrated to give methyl 3-(2-azidoacetyl)benzoate (2.1 g,
65%) as a gray solid.
[0403] Step D: A mixture of methyl 3-(2-azidoacetyl)benzoate (1.89
g, 8.6 mmol), 0.4 g 10% Pd--C in 40 mL of MeOH and 2.5 mL of conc.
HCl is hydrogenated at 1 atm overnight at room temperature. After
filtering the catalyst, the filtrate is evaporated and dried to
give the amine hydrochloride salt (1.25 g, 63.2%) as a white
solid.
[0404] Step E: To a solution of the hydrochloride salt of methyl
3-(2-aminoacetyl)benzoate (1.2 g, 5.2 mmol) in 10 mL of dry THF
cooled to 0C is added 5 mL of absolute pyridine. The mixture is
stirred for 30 min and to it is added dropwise a solution of
4-isopropylbenzoyl chloride in THE (10 mmol in 5 mL of solvent)
over 15 min. After the addition, the reaction mixture is stirred
for 2 h and evaporated. The residue is dissolved in 100 mL of EtOAc
and washed with water (3.times.0.30 mL), brine (30 mL), and dried
over Na.sub.2SO.sub.4. The residue is purified by silica gel column
chromatography (petroleum ether/EtOAc, 3/1) to give methyl
3-[2-(4-isopropylbenzylamino)acetyl]benzoate (1.2 g, 67.7%) as a
pale yellow solid.
[0405] Step F: A solution of methyl
3-[2-(4-isopropylbenzylamino)acetyl]benzoate (0.5 g, 1.5 mmol) in
10 mL of POCl.sub.3 is refluxed for 6 hr and cooled to room
temperature. The reaction mixture is added to 100 mL of ice water
and adjusted to pH 10 with 2N NaOH. Then the mixture is extracted
with EtOAc (2.times.50 mL) and the organic layer is washed with
water (2.times.50 mL), brine (50 mL), dried over Na.sub.2SO.sub.4
and evaporated. The residue is purified by silica gel column
chromatography (petroleum ether/EtOAc, 3/1) to give methyl
3-[2-(4-isopropylphenyl)oxazol-5-yl]benzoate (0.4 g, 84.5%) as a
pale yellow solid.
[0406] Step G: To a solution of methyl
3-[2-(4-isopropylphenyl)oxazol-S-yl]benzoate (0.4 g, 1.25 mmol) in
5 mL of MeOH is added LiOH (0.1 g) in 10 mL of water and the
reaction is heated to reflux for 2 h. The MeOH is removed by
evaporation and acidified to pH 3 with 6N HCl and stirred for 15
min. The mixture is filtered and washed with water (3.times.10 mL),
petroleum ether (10 mL) and dried to give
3-[2-(4-isopropylphenyl)oxazol-5-yl]benzoic acid (0.35 g, 91%) as a
gray solid: mp 193-195.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta.
8.46 (br s, 1H), 8.10-8.05 (m, 3H), 7.95 (d, J=7.6, 2H), 7.58 (t,
J=7.6, 1H), 7.56 (s, 1H), 7.37 (d, J=8.4, 2H), 2.99 (septet, J=6.9,
1H), 1.29 (d, J=6.9, 6H); mass spectrum (m/z) 308.2 [MH.sup.+].
Example Q: Preparation of
3-[5-(4-fluorophenyl)-1H-pyrazol-3-yl]benzoic acid (Compound No.
552)
##STR01290##
[0408] Step A: Preparation of
3-[3-(4-fluorophenyl)-3-oxopropionyl]benzonitrile. To a mixture of
methyl 3-cyanobenzoate (1.05 g, 6.52 mmol) and sodium hydride (0.69
g, 60% in hexanes, 17.25 mmol) in THF (15 mL) is added a solution
of 4-fluoroacetylphenone (0.86 g, 6.22 mmol) in THF (5 mL). The
resulting mixture is heated at reflux under stirring for 1 h until
the starting material is consumed as judged by TLC. After cooling
to room temperature, the mixture is added to 15 mL of 1N HCl and
the solution is extracted with ethyl acetate (2.times.20 mL). The
combined organic layers are washed with saturated NaHCO.sub.3, and
then brine, dried over MgSO.sub.4, and removed under reduced
pressure. The residue is further purified by flash column, eluting
with hexane and 50% methyl enechloride in hexane in sequence. The
solid isolated is suspended in ethyl ether, and filtered to afford
1.28 g (74%) of 3-[3-(4-fluorophenyl)-3-oxopropionyl]benzonitrile
as white powder. The obtained compound is >95% pure as
determined by .sup.1HNMR and LC-MS: .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta. 8.25 (m, 1H), 8.20 (m, 1H), 8.03 (m, 2H), 7.83
(m, 1H), 7.64 (t, J=7.8, 1H), 7.20 (m, 2H), 6.79 (s, 1H); MS (ES-)
266.25.
[0409] Step B: Preparation of
3-[5-(4-fluorophenyl)-1H-pyrazol-3-yl]benzonitrile. To a solution
of 3-[3-(4-fluorophenyl)-3-oxo-propionyl]benzonitrile (250 mg,
0.895 mmol) in 3 mL of anhydrous EtOH is added anhydrous hydrazine
(30 .delta.L, 0.985 mmol) and the sealed reaction mixture is heated
to 100.degree. C. over 21 min in the microwave (Power 300 W, 1 min
ramp to 100.degree. C., 20 min hold) to afford a crude solution of
3-[5-(4-fluorophenyl)-1H-pyrazol-3-yl]benzonitrile: MS m/z 264.29,
calcd for C.sub.16H.sub.11FN.sub.3 (MH.sup.+) 264.
[0410] Step C: Preparation of
3-[5-(4-fluorophenyl)-1H-pyrazol-3-yl]benzoic acid. To the mixture
of 3-[5-(4-fluorophenyl)-1H-pyrazol-3-yl]-benzonitrile is added 5N
aqueous sodium hydroxide (1 mL, 5 mmol) and the mixture is resealed
and is heated to 10.degree. C. over 31 min in the microwave (Power
300 W, 1 min ramp to 100.degree. C., 30 min hold) to afford a crude
solution containing 3-[5-(4-fluorophenyl)-1H-pyrazol-3-yl]benzoic
acid which is cooled to room temperature. The solution is adjusted
to pH 4 by the slow addition of 2N aqueous HCl solution and
filtered. The resulting solid is washed with water (2.times.5 mL),
50% Et.sub.2O/hexanes (2.times.5 mL), and dried (50.degree. C., 1
torr) overnight to afford 211.5 mg (86%-2 steps) of
3-[5-(4-fluorophenyl)-1H-pyrazol-3-yl]benzoic acid as a white
powder; mp 270.5-272.degree. C.; .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 7.27 (m, 3H), 7.56 (t, J=7.7 Hz, 1H), 7.89
(m, 3H), 8.05 (d, J=7.7 Hz, 1H), 8.39 (m, 1H); MS m/z 283.32, calcd
for C.sub.16H.sub.12FN.sub.2O.sub.2 (MH.sup.+) 283.
[0411] In essentially the same manner the following compound is
made: Compound No. 551.
Example R: Preparation of
3-[5-(4-isopropylphenyl)-2H-pyrazol-3-yl]benzoic acid (Compound No.
287)
##STR01291##
[0413] Step A: To a suspension of sodium hydride (1.56 g, 39 mmol,
60% dispersion in mineral oil) in anhydrous THF (50 mL) is added a
20 mL of a THF solution of 1-(4-isopropyl-phenyl)ethanone (4.86 g,
30 mmol) and isophthalic acid dimethyl ester (5.83 g, 30 mmol) and
stirred for 30 min at room temperature. The mixture is heated at
reflux for 5 h, cooled on ice and quenched with the addition of 3.5
mL of concentrated HCl, and then concentrated. The crude methyl
3-[3-(4-isopropylphenyl)-3-oxo-propionyl]-benzoate is dissolved in
dichloromethane, purified by flash chromatography using
dichloromethane/petroleum ether, 1/1 as eluent to give 6.4 g of
intermediate as an oil (66%).
[0414] Step B: To 600 mg of methyl
3-[3-(4-isopropylphenyl)-3-oxo-propionyl]benzoate in 25 mL of 4/1
MeOH/H.sub.2O is added 518 mg of LiOH.H.sub.2O in one portion, and
the reaction is heated at reflux for 2 hrs, cooled to room
temperature and neutralized with aq. HCl. The precipitate is
filtered, washed with water, dried and recrystallized from EtOH to
give 350 mg of 3-[3-(4-isopropylphenyl)-3-oxo-propionyl]benzoic
acid as a white solid.
[0415] Step C: To 310 mg of
3-[3-(4-isopropylphenyl)-3-oxo-propionyl]-benzoic acid in 5 mL of
EtOH is added 0.05 mL hydrazine monohydrate. The reaction mixture
is refluxed for 24 h, and cooled to room temperature. The
precipitate is collected, washed with EtOH, and recrystallized from
toluene to give 190 mg of
3-[5-(4-isopropylphenyl)-2H-pyrazol-3-yl]benzoic acid as a white
solid: .sup.1H NMR (DMSO-d.sub.6) .delta. 8.40 (br s, 1H), 8.04 (d,
J=7.2, 1H), 7.87 (d, J=7.6, 1H), 7.74 (d, J=7.6, 2H), 7.54 (t,
J=7.6, 1H), 7.30 (d, J=7.6, 2H), 7.19 (s, 1H) 2.90 (septet, J=6.8,
1H), 1.21 (d, J36.8, 6H); MS m/z 308.2 [MH.sup.+].
Example S: Preparation of 3-(4-p-tolylthiazol-2-yl)benzoic acid
(Compound No. 350)
##STR01292##
[0417] Step A: To a solution of 3-cyanobenzoic acid (1:2 g, 8.2
mmol) in THF (50 mL) is added dithiophosphoric acid diethyl ester
and water (5 mL) and the mixture is then stirred for 18 h at
80.degree. C. The solvents are removed under reduced pressure and
the desired 3-thiocarbamoyl benzoic acid is obtained as white
solid.
[0418] Step B: To a solution of the 3-thiocarbamoyl benzoic acid in
anhydrous DMF is added 2-bromo-1-p-tolyl-ethanone and the reaction
is stirred for 18 h at 150.degree. C. The solvent is removed under
a nitrogen stream and the desired product,
3-(4-p-tolylthiazol-2-yl)benzoic acid, is purified by preparative
LC-MS.
[0419] The following compounds are prepared using the procedures
described above: Compound Nos: 365, 366, 367, 368, 369, 370, 371,
372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,
385, 386, 387, 388, 389, 390, 391, 392, and 393.
Example T: Preparation of
3-[4-(4-isopropylphenyl)triazol-2-yl]-benzoic acid (Compound No.
289)
##STR01293##
[0421] Step A: Ethyl 3-cyanobenzoate (3.36 g, 19.2 mmol) is
dissolved in 10 mL of anhydrous DMF and the solution is heated to
70-75.degree. C. H.sub.2S is bubbled through the solution and 0.5
ML hexahydropyridine is added. After 2 h, the reaction mixture is
poured into 50 mL of water and the precipitate that formed is
collected and dried in vacuo to give 3 g of ethyl
3-thiocarbamoylbenzoate.
[0422] Step B: A solution of ethyl 3-thiocarbamoylbenzoate (0.5 g,
2.39 mmol) and 2-bromo-1-(4-isopropylphenyl)-ethanone (576 mg, 2.39
mmol) in 2 mL of anhydrous DMF is heated at 60-65.degree. C. for 2
h. After the reaction is complete, the reaction is poured into
water, extracted with EtOAc, dried over MgSO.sub.4 and purified by
flash chromatography affording 0.7 g (84%) of ethyl
3-[4-(4-isopropylphenyl)thiazol-2-yl]benzoate.
[0423] Step C: A solution of ethyl
3-[4-(4-isopropylphenyl)thiazol-2-yl]-benzoate (92 mg, 0.26 mmol)
and LiOH (55 mg, 1.3 mmol) in methanol/H.sub.2O (5 mL/1.7 mL) is
stirred at room temperature for 0.5 h. The reaction mixture is then
heated to 50.degree. C. and stirred for 3 h. Upon completion, the
solvent is removed in vacuo and the residue is dissolved in 10 mL
of water, neutralized, extracted with EtOAC, washed with brine and
then dried over Na.sub.2SO.sub.4, and concentrated to afford 82 mg
(97.6%) of 3-[4-(4-isopropylphenyl)thiazol-2-yl]benzoic acid: mp
206-208.degree. C.; .sup.1H NMR: (CDCl.sub.3) .delta.8.74 (br s,
1H), 8.33 (d, J=7.6, 1H), 8.17 (d, J=7.6, 1H), 7.93 (d, J=8.0, 2H),
7.59 (t, J=7.6, 1H), 7.48 (s, 1H), 7.32 (d, J=8.0, 1H), 2.97
(septet, J=6.9, 1H), 1.30 (d, J=6.8, 61); MS m/z 324.2 [MH.sup.+].
The following compounds are prepared using the procedures described
above: Compound Nos: 350, 365, 366, 367, 368, 369, 370, 371, 372,
373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385,
386, 387, 388, 389, 390, 391, 392, 393.
Example U: Preparation of
3-[5-(4-Isopropyl-phenyl)thiazol-2-yl]benzoic acid (Compound No.
310)
##STR01294##
[0425] Step A: A mixture of
N-[2-(4-isopropylphenyl)-2-oxoethyl]isophthalamic acid ethyl ester
(from Example L step B above, 500 mg 1.42 mmol) and phosphorus
pentasulphide (1.0 g, 4.5 mmol) in 10 mL of dry pyridine is
refluxed for 2 h, and after cooling, the mixture is poured into ice
water (20 mL) and saturated ammonia solution (10 mL). The reaction
is extracted with EtOAc, washed with brine and dried over
Na.sub.2SO.sub.4. After concentration, the residue is purified by
column chromatography to give ethyl
3-[5-(4-isopropylphenyl)-thiazol-2-yl]-benzoate as a brown oil (150
mg, 30%).
[0426] Step B: A mixture of ethyl
3-[5-(4-isopropylphenyl)thiazol-2-yl]benzoate (120 mg, 0.34 mmol)
and lithium hydroxide (94 mg 2.24 mmol) in methanol/water (9 mL/3
mL) is stirred for 2 h. After the evaporation of the solvent, the
residue is dissolved in 10 mL of water, treated with 1 g of citric
acid, and then extracted with EtOAc. The organic phase is washed
with brine, dried over Na.sub.2SO.sub.4, concentrated and the crude
product is recrystallized from CH.sub.2Cl.sub.2/hexane to give
3-[5-(4-isopropylphenyl)thiazol-2-yl]benzoic acid as a brown solid
(64 mg, 59%): mp 218-220.degree. C.; .sup.1H NMR (DMSO-d.sub.6)
8.78 (br s, 1H), 8.22-8.16 (m, 2H), 8.07 (s, 1H), 7.61-7.55 (m,
3H), 7.30 (d, J=8.0, 2H), 2.96 (septet, J=6.4, 1H), 1.32 (d, J=6.4,
6H); MS m/z 324.3 [MH.sup.+].
Example V: Preparation of
3-[2-(4-isopropylphenyl)thiazol-4-yl]-benzoic acid (Compound No.
312)
##STR01295##
[0428] Step A: 4-isopropylbenzonitrile (2.0 g, 14 mmol) is
dissolved in 10 ml of anhydrous DMF and the solution is heated to
70-75.degree. C. H.sub.2S is slowly bubbled through the solution
and 0.5 mL of hexahydropyridine is added. After 1.5 h. the reaction
mixture is poured into 50 mL of water and the precipitate that
formed is collected and dried in vacuo to give 1.5 g of
4-isopropylthiobenzamide.
[0429] Step B: A solution of 4-isopropyl-thiobenzamide (331 mg,
1.85 mmol) and ethyl 3-(2-bromoacetyl)benzoate (500 mg, 1.85 mmol)
in 5 mL of anhydrous DMF is heated at 60-65.degree. C. for 2 h. The
product mixture is poured into water, extracted with EtOAc, dried
over MgSO.sub.4 and concentrated in vacuo. Purification by flash
chromatography gives 468 mg (72%) of methyl
3-[2-(4-isopropylphenyl)thiazol-4-yl]benzoate.
[0430] Step C: A solution of methyl
3-[2-(4-isopropylphenyl)thiazol-4-yl]benzoate (92 mg, 0.262 mmol)
and LiOH (55 mg, 1.3 mmol) in methanol/H.sub.2O (5 mL/1.7 mL) is
stirred at mom temperature for 0.5 h. The reaction mixture is then
heated to 55.degree. C. and stirred for 3 h. The solvent is then
removed under reduced pressure and the residue is dissolved in 10
mL of water, neutralized with acid, extracted with EtOAc, and then
is washed with brine, and dried over Na.sub.2SO.sub.4, and
concentrated to give 92 mg of
3-[2-(4-isopropylphenyl)thiazol-4-yl]benzoic acid: mp
193-195.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta. 8.68 (br s,
1H), 8.29 (d, J=8.0, 2H), 8.08, (d, J=7.6, 1H), 7.98 (d, J=8.0,
2H), 7.59-7.55 (m, 2H), 7.33 (d, J=8.0, 2H), 2.98 (septet, J=6.8,
1H), 1.31 (d, J=6.8, 6H); MS m/z 324.3 [MH.sup.+].
Example W: Preparation of
3-[2-(4-isopropylphenyl)thiazol-5-yl]benzoic acid (Compound No.
321)
##STR01296##
[0432] Step A: A solution of methyl
3-[2-(4-isopropylbenzylamino)acetyl]benzoate (Example P, Step E)
(0.6 g, 1.8 mmol) and 1 g P.sub.2S.sub.5 in 5 mL of pyridine is
refluxed for 6 h and cooled to room temperature. The reaction
mixture is added to 100 mL of ice water and adjusted to pH 10 with
2N NaOH. The mixture is extracted with EtOAc (2.times.50 mL) and
the organic layer is washed with water (2.times.50 mL), brine (50
mL), dried over Na.sub.2SO.sub.4 and evaporated. The residue is
purified by silica gel column chromatography (petroleum
ether/EtOAc, 3/1) to give methyl
3-[2-(4-isopropylphenyl)thiazol-5-yl]benzoate (0.31 g, 52.0%) as a
pale yellow solid.
[0433] Step B: To a solution of methyl
3-[2-(4-isopropylphenyl)thiazol-5-yl]benzoate (0.31 g, 0.92 mmol)
in 5 mL of MeOH is added LiOH (0.1 g) dissolved in 10 mL of water
and the reaction is heated to reflux for 1 h. The MeOH is removed
by evaporation and the reaction is acidified to pH 3 with 6N HCl
and then stirred for 15 min. The mixture is filtered and washed
with water (3.times.10 mL), petroleum ether (10 mL) and dried to
give 3-[2-(4-isopropylphenyl)thiazol-5-yl]benzoic acid (0.28 g,
94%) as a pale yellow solid: mp 237-239.degree. C.; .sup.1H NMR
(DMSO-d.sub.6) .delta. 8.37 (s, 1H), 8.17 (br s, 1H), 7.99-7.88 (m,
4H), 7.59 (t, J=7.8, 1H), 7.39 (d, J=8.0, 2H), 2.95 (septet, J=7.2,
1H), 1.22 (d, J=7.2, 6 H); MS m/z 322.0 [MH.sup.+].
Example X: Preparation of
3-[5-(4-chlorophenyl)isoxazol-3-yl]benzoic acid (Compound No.
479)
##STR01297##
[0435] Step A: Preparation of 3-(hydroxyiminomethyl)benzoic acid
methyl ester: To a solution of methyl 3-formylbenzoate (5 g, 30.5
mmol, Acros) in 50 mL of anhydrous EtOH is added hydroxylamine
hydrochloride (2.40 g, 33.60 mmol) and pyridine (4.0 mL, 49.5
mmol). The mixture is heated to reflux for 2 h, cooled to room
temperature and concentrated in vacuo. The residue is dissolved in
500 mL of Et.sub.2O, partitioned with 1N aqueous HCl solution
(2.times.50 mL), water (2.times.50 mL), dried (MgSO.sub.4) and
concentrated in vacuo to afford 5.5 g (100%) of methyl
3-(hydroxyiminomethyl)-benzoate as a white powder; mp
107-108.degree. C.; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 3.94
(s, 3H), 7.46 (t, J=7.5 Hz, 1H), 7.80 (d, J=7.8 Hz, 1H), 8.05 (d.
J=7.5 Hz, 1H), 8.20 (m, 2H), 8.41 (s, 1H); MS m/z 178.23
[MH.sup.+].
[0436] Step B: Preparation of 3-carbomethoxyphenyl hydroximoyl
chloride: To a solution of methyl 3-(hydroxyiminomethyl)benzoate
(5.5 g, 30.5 mmol) in 7 mL of DMF cooled to 0.degree. C. is added
NCS (4.97 g, 36.8 mmol), followed by 1-2 mL of gaseous HCl added by
pipette from the headgas of a bottle of concentrated hydrochloric
acid. The mixture over 15 min produced a strongly exothermic
reaction which is controlled through the use of an ice bath. The
mixture is stirred for 90 min. dissolved in 200 mL of 90%
Et.sub.2O/THF, washed with water (4.times.50 mL portions), brine
(50 mL), and dried (MgSO.sub.4). The solution is concentrated in
vacuo until about 5 mL of solvent remained, 150 mL of hexane is
added to precipitate the product, and the slurry filtered after 1-2
h to afford 4.5 g (61%) of 3-carbomethoxyphenyl hydroximoyl
chloride as a white powder. This material is kept in the freezer in
a dessicator to maintain stability: MS m/z 214.20 [MH.sup.+].
[0437] Step C: Preparation of methyl
3-[5-(4-chlorophenyl)isoxazol-3-yl]benzoate: To a solution of
3-carbomethoxyphenyl hydroximoyl chloride (2.0 g, 9.4 mmol) and
1-chloro-4-ethynylbenzene (2.6 g, 19.9 mmol, Aldrich) in 50 mL of
CH.sub.2Cl.sub.2 cooled to 0.degree. C. is added Et.sub.3N (1.8 mL,
12.9 mmol). The reaction mixture is allowed to warm to room
temperature over 1-2 h and is stirred for 24 h. The solution is
diluted with 200 mL of CH.sub.2Cl.sub.2, washed with 1N aqueous
NaOH solution (75 mL), water (75 mL), dried (MgSO.sub.4) and
concentrated in vacuo until .about.15 mL volume remained. The
solution is diluted with 8 mL of MeOH, and 110 mL of hexanes is
added and the solvent is slowly concentrated at ambient temperature
until significant precipitation occurred. The slurry is filtered
and the solid product dried (70.degree. C. at 10 torr) to afford
2.25 g (77%) of methyl 3-[5-(4-chlorophenyl)isoxazol-3-yl]benzoate
as a white powder. The precipitation procedure is repeated on the
concentrated mother liquor to afford an additional 310 mg (11%):
.sup.1H NMR (300 MHz, Acetone-d.sub.6) .delta. 3.95 (s, 3H), 7.55
(s, 1H), 7.62 (d. J=8.5 Hz, 1.4 Hz, 2H), 7.70 (t, J=7.8 Hz, 2H),
7.98 (d, J=8.4 Hz, 2H), 8.13 (dm, J=7.8 Hz, 1H), 8.20 (dm, J=7.8
Hz, 1H), 8.54 (s, 1H); MS m/z 314.21, calcd for
C.sub.17H.sub.13ClNO.sub.3 (MH.sup.+) 314.
[0438] Step D: Preparation of
3-[5-(4-chlorophenyl)isoxazol-3-yl]benzoic acid: A solution of 2.56
g (8.2 mmol) of methyl 3-[5-(4-chlorophenyl)isoxazol-3-yl]benzoate
in 56 mL of 50% THF/H.sub.2O is heated to 65.degree. C. for 3 h and
cooled to room temperature. The solution is adjusted to pH 4 by the
slow addition of 6N aqueous HCl solution and filtered. The
resulting solid is washed with water, 30% Et.sub.2O/hexanes and
then dried overnight at 70.degree. C. (10 torr) to afford 1.81 g
(74%) of 3-[5-(4-chlorophenyl)isoxazol-3-yl]benzoic acid as a white
fluffy powder. An additional 376 mg (15%) is obtained from
precipitation of the mother liquor: mp 293-295.degree. C.; .sup.1H
NMR (300 MHz. DMSO-d.sub.4) .delta. 7.65 (m, 3H), 7.80 (s, 1H),
7.94 (d, J=8.8 Hz, 2H), 8.06 (dm, J=8.0 Hz, 1H), 8.14 (dm, J=7.9
Hz, 1H), 8.44 (m, 1H); MS m/z 300.19, calcd for
C.sub.16H.sub.11ClNO.sub.3 (MH.sup.+) 300.
[0439] Utilizing essentially the same procedures described above
and substituting other acetylene derivatives in step 4 gave the
following compounds: Compound Nos; 463, 464, 465.466, 467, 468,
469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 480, 481, 482,
483, 484, 485, 486, 487, 488, 489, 490, 491, 521, 522, 523, 524,
525, 526, 529, 530, 531, 532, 533, 534, 566, 567, 568, 573, 574,
and 575.
Example Y: Preparation of
3-[3-(4-chlorophenyl)isoxazol-5-yl]benzoic acid (Compound No.
503)
##STR01298##
[0441] Step A: Preparation of 4-chlorobenzaldehyde oxime: To a
solution of 4-chlorobenzaldehyde (2.83 g, 20.15 mmol, Aldrich) in
17 mL of anhydrous EtOH is added hydroxylamine hydrochloride (1.61
g, 22.5 mmol) and pyridine (2.5 mL, 30.9 mmol). The mixture is
heated to reflux for 3 h, cooled to room temperature and
concentrated in vacuo. The residue is dissolved in 125 mL of
Et.sub.2O, partitioned with 1N aqueous HCl solution (2.times.30 L),
water (2.times.30 mL), dried (MgSO.sub.4) and concentrated in vacuo
to afford 2.77 g (88.4%) of 4-chlorobenzaldehyde oxime as a white
powder: MS m/z 156.00, calcd for C.sub.7H.sub.7ClNO (MH.sup.+) 156.
For reference regarding preparation, see Luca, L. D.; Giacomelli,
G.; Riu, A.; J. Org. Chem. 2001, 66(20), 6823-6825.
[0442] Step B: Preparation of 4-chlorophenyl hydroximinoyl
chloride: To a solution of 4-chlorobenzaldehyde oxime (1.22 g, 7.9
mmol) in 2 mL of DMF cooled to 0.degree. C. is added NCS (1.20 g,
8.30 mmol), followed 1-2 mL of gaseous HCl added by pipette from
the headgas of a bottle of concentrated hydrochloric acid. The
mixture over 15 min is produced a strongly exothermic reaction
which is controlled through the use of an ice bath. The mixture is
stirred 120 min, dissolved in 125 mL of Et.sub.2O, washed with
water (3.times.35 mL portions), brine (35 mL), and dried
(MgSO.sub.4). The solution is concentrated in vacuo to afford 1.46
g (98%) of 4-chlorophenyl hydroximinoyl chloride as a white powder.
This material is kept in the freezer in a dessicator to maintain
stability: MS m/z 190.02, calcd for C.sub.7H.sub.6Cl.sub.2NO
(MH.sup.+) 190.
[0443] Two Step Preparation of ethyl 3-ethylynylbenzoate from ethyl
3-iodobenzoate: To a solution of ethyl 3-iodobenzoate (25 g, 90.6
mmol) in 43 mL of DMF is added trimethylsilylacetylene (17 mL,
119.5 mmol) and Et.sub.3N (25 mL, 181.1 mmol). This mixture is
degassed under argon several times, CuI (175 mg, 0.92 mmol) is
added, followed by 1.04 g of Pd(PPh.sub.3).sub.4 catalyst. The
reaction mixture is heated to 50.degree. C. for 24 h, cooled to
room temperature and diluted with 400 mL of 50% Et.sub.2O/hexanes.
This mixture is partitioned with water (4.times.75 mL portions),
dried (MgSO.sub.4) and concentrated to afford 23.29 g of a brown
oil. This residue is chromatographed over 200 g of SiO.sub.2
(eluted with 30% CH.sub.2Cl.sub.2/hexanes) to afford 22.2 g (99%)
of 3-trimethylsilanylethynylbenzoic acid ethyl ester as a pale
yellow oil which is taken directly into the next reaction: MS m/z
247.12, calcd for C.sub.14H.sub.19SiO.sub.2 (MH.sup.+) 247.
[0444] This material is dissolved in 250 mL of EtOH, 1.25 g (9.0
mmol) of K.sub.2CO.sub.3 catalyst is added, the mixture stirred at
room temperature for 5 h, and concentrated in vacuo. The residue is
chromatographed over 200 g of SiO.sub.2 (eluted with 40%
CH.sub.2Cl.sub.2/hexanes) to afford 15.7 g (100%) of ethyl
3-ethynylbenzoate (90% pure by LC/MS) as an orange solid. This
material is recrystallized from the minimum amount of hexanes to
afford 12.2 g (78% overall-two steps) as a pale yellow solid: mp
36-38.degree. C.; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.38
(t, J=7.2 Hz, 3H), 3.12 (s, 1H), 4.36 (q, J=7.2 Hz, 2H), 7.40 (t,
J=7.8 Hz, 2H), 7.63 (dt, J=7.8 Hz, 1.5 Hz, 1H), 8.14 (t, J=1.5 Hz,
1H); MS m/z 174.98, calcd for C.sub.11H.sub.11O.sub.2(MH.sup.+)
175.
[0445] Step C: Preparation of ethyl
3-[3-(4-chlorophenyl)isoxazol-5-yl]benzoate: To a solution of
4-chlorophenyl hydroxyiminoyl chloride (0.60 g, 3.15 mmol) and
ethyl 3-ethynylbenzoate in 25 mL of CH.sub.2Cl.sub.2 is added
Et.sub.3N (0.66 mL, 4.73 mmol) and the mixture is stirred 48 h. The
solution is diluted with 60 mL of CH.sub.2Cl.sub.2, washed with 1N
aqueous NaOH solution (30 mL), water (30 mL), dried (MgSO.sub.4)
and concentrated in vacuo. The solid residue is recrystallized from
the minimum amount of Et.sub.2O/hexanes to afford 700 mg (68%) of
ethyl 3-[3-(4-chlorophenyl)isoxazol-5-yl]benzoate as a white
powder: .sup.1H NMR (300 MHz, Acetone-d.sub.6) .delta. 1.41 (t,
J=7.2 Hz, 3H) 4.41 (q, J=7.2 Hz, 2H), 7.57 (m, 3H), 7.72 (t, J=7.8
Hz, 1H), 7.70 (dt, J=8.4, 1.8 Hz, 2H), 8.15 (tt, J=8.5, 1.8 Hz,
2H), 8.55 (t, J=1.5 Hz, 1H); MS m/z 314.21, calcd for
C.sub.18H.sub.15ClNO.sub.3 (MH.sup.+) 328.
[0446] Step D: Preparation of
3-[3-(4-chlorphenyl)isoxazol-5-yl]benzoic acid: A solution of 678
mg (2.1 mmol) of ethyl 3-[3-(4-chlorophenyl)isoxazol-5-yl]benzoate
in 14 mL of 50% THF/H.sub.2O is heated to 60.degree. C. for 5 h and
cooled to room temperature. The solution is adjusted to pH 4 by the
slow addition of 6N aqueous HCl solution and filtered. The
resulting solid is washed with water, hexanes and dried overnight
at 70.degree. C. (10 torr) to afford 594 mg (96%) of
3-[3-(4-chlorophenyl)isoxazol-5-yl]benzoic acid as a white fluffy
powder: mp 265-266.degree. C.; .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 7.61 (dm, J=8.7 Hz, 2H), 7.70 (t, J=7.8 Hz, 1H), 7.82 (s,
1H), 7.95 (dm, J=8.7 Hz, 2H), 8.06 (dm, J=8.0 Hz, 1H), 8.13 (dm,
J=8.0 Hz, 1H), 8.41 (m, 1H); MS m/z 300.16, calcd for
C.sub.16H.sub.11ClNO.sub.3 (MH.sup.+) 300.
[0447] Utilizing essentially the same procedures described above
and substituting other benzaldehyde derivatives in Step A gave the
following compounds: Compound Nos: 492, 493, 494, 495, 496, 497,
498, 499, 500, 501, 502, 503, 504, 505, 514, 515, 516, 517, 518,
0.519, 520, 535, 536, 537, 538, 539, 540, and 541.
Example Z: Preparation of
3-[2-(4-isopropylphenyl)-3H-imidazol-4-yl]benzoic acid (Compound
No. 311)
##STR01299##
[0449] Step A: 4-Isopropylbenzamidine (356 mg, 2.20 mmol) and 514
mg (2.00 mmol) of methyl 3-(2-bromo-acetyl)-benzoate in 20 mL of
CHCl.sub.3 is heated at reflux for 8 h, cooled to room temperature
and evaporated. The residue is partitioned between aqueous
K.sub.2CO.sub.3 and EtOAc, separated and the organic layer is
washed with brine, dried over Na.sub.2SO.sub.4, filtered and
evaporated. The residue is purified by column chromatography to
give 210 mg (33%) of methyl
3-[2-(4-isopropylphenyl)-3H-imidazol-4-yl]-benzoate as a yellow
solid.
[0450] Step B; To a suspension of 190 mg (0.60 mmol) of methyl
3-[2-(4-isopropylphenyl)-3H-imidazol-4-yl]-benzoate in 6 mL of
aqueous MeOH (5/1) is added 120 mg LiOH.H.sub.2O. The reaction is
heated at reflux for 1 h, cooled to room temperature, and
neutralized with acetic acid. The precipitate is filtered and
washed with water and air-dried. The resulting white solid is
recrystallized from acetone to afford 140 mg (76%) of
3-[2-(4-isopropylphenyl)-3H-imidazol-4-yl]-benzoic acid as a white
solid: .sup.1H NMR (DMSO-d.sub.6) .delta. 8.42 (br s, 1H), 8.06 (d,
J=7.6, 1H), 7.91 (d, J=7.6, 2H), 7.83 (br s, 1H), 7.75 (d, J=7.2,
1H), 7.47 (t, J=7.2, 1H), 7.32 (d, J=8.0, 2H), 2.91 (septet, J=6.6,
1 H), 1.22 (d, J=6.6, 6H); MS m/z 307.2 [MH.sup.+].
Example AA. Preparation of
3-[5-(4-isopropylphenyl)-1H-imidazol-2-yl]benzoic acid (Compound
No. 277)
##STR01300##
[0452] Step A: To 1.20 g (4.97 mmol) of
2-bromo-1-(4-isopropylphenyl)ethanone in 50 mL of CHCl.sub.3 is
added 1.05 g (5.47 mmol) of ethyl 3-carbamimidoylbenzoate and the
reaction is heated at refluxed for 3 h, then cooled to room
temperature, and basified with aqueous K.sub.2CO.sub.3. The organic
layer is separated and dried over K.sub.2CO.sub.3, filtered and
evaporated. The residue is purified by flash column chromatography
on silica gel to give 0.85 g of ($1%) of methyl
3-[5-(4-isopropylphenyl)-H-imidazo-2-yl]benzoate as a white
solid.
[0453] Step B: To a suspension of 480 mg (1.47 mmol) of methyl
3-[5-(4-isopropylphenyl)-1H-imidazol-2-yl]benzoate in 14 mL of
aqueous MeOH/H.sub.2O (5/1) is added 103 mg LIOH.H.sub.2O, and the
reaction is heated at reflux for 1 h, cooled to room temperature,
and neutralized with acetic acid. The precipitate is filtered and
washed with water and air-dried. The resulting
3-[5-(4-isopropylphenyl)-1H-imidazol-2-yl]benzoic acid is
recrystallized from acetone to afford 300 mg (63%) of a white
solid: mp 296-298.degree. C.; .sup.1H NMR (DMSO-d.sub.6) .delta.
8.56 (br s, 1H), 8.23 (d, J=8.0, 1H), 7.87 (d, J=8.0, 2H), 7.56 (d,
J=8.0, 2H), 7.31 (t, J=8.0, 1H), 7.23 (br s, 1H), 7.10 (d, J=7.6,
2H), 2.93 (septet, J=6.8, 1H), 1.11 (d, J=6.8, 6H).
Example BB: Preparation of
2-(3-carboxyphenyl)-4-(4-isopropylphenyl)furan-3-carboxylic acid
(Compound No. 314)
##STR01301##
[0455] Step A: A mixture of 680 mg (2.90 mmol) of methyl
3-(2-methoxycarbonylacetyl)benzoate, 30 mL of acetone, 4.08 g of
K.sub.2CO.sub.3 and 780 mg (3.24 mmol) of
2-bromo-1-(4-isopropylphenyl)-ethanone is heated at reflux for 30
min. The solvent is then removed under reduced pressure, the
residue is partitioned between aqueous HCl and EtOAc. The organic
layer is washed with brine, dried over Na.sub.2SO.sub.4, filtered
and evaporated. The residue is purified by column chromatography to
give 660 mg (57%) of methyl
3-[4-(4-isopropylphenyl)-2-methoxycarbonyl-4-oxo-butyryl]benzoate
as a yellow oil.
[0456] Step B: To 480 mg (1.21 mmol) of methyl
3-[4-(4-isopropylphenyl)-2-methoxycarbonyl-4-oxo-butyryl]-benzoate
in 10 mL of MeOH is added 15 mL of 6N HCl and the reaction is
heated at reflux for 5 h. The reaction mixture is cooled to room
temperature and extracted with EtOAc. The combined organic phases
are washed with water, brine, dried over Na.sub.2SO.sub.4, filtered
and then evaporated. The residue is purified by column
chromatography to give 260 mg (57%) of methyl
5-(4-isopropylphenyl)-2-(3-methoxycarbonylphenylfuran-3-carboxylate
as a yellow oil.
[0457] Step C: To 260 mg (0.69 mmol) of methyl
5-(4-isopropylphenyl)-2-(3-methoxycarbonylphenyl)-furan-3-carboxylate
in 12 mL of 5:1 MeOH/H.sub.2O is added 160 mg of LiOH.H.sub.2O and
the reaction is heated at reflux for 1 h, cooled to room
temperature, and neutralized with acetic acid. The precipitate is
collected, washed with water and dried. The crude product is
recrystallized from acetone to afford 140 mg (58%) of
2-(3-carboxyphenyl)-5-(4-isopropylphenyl)furan-3-carboxylic acid as
a yellow solid: mp 236-239.degree. C.; .sup.1H NMR (DMSO-d.sub.6)
.delta. 13.0 (br s, 1H), 8.60 (br s, 1H), 8.28 (d, J=7.6, 1 H),
7.98 (d, J=7.6, 114), 7.74 (d, J=7.6, 2H), 7.74 (t, J=8.0, 1H),
7.33 (d, J=8.0, 2H), 7.25 (s, 1H), 2.91 (septet, J=6.6, 1H), 1.21
(d, J=6.6, 6H); MS m/z 349.0 [MH.sup.-].
Example CC: Preparation of
3-[5-(4-isopropylphenyl)furan-2-yl]benzoic acid (Compound No.
322)
##STR01302##
[0459] Step A: A suspension of
3-(3-methoxycarbonylphenyl)-3-oxo-propyl acid ethyl ester (1.8 g,
7.2 mmol) and powdered K.sub.2CO.sub.3 (4 g, 29 mmol) and
4-(bromoacetyl) isopropylbenzene (1.8 g, 7.5 mmol) in dry acetone
is refluxed for 2 h. The solvent is removed by evaporation and the
residue is added into 50 mL of ice water, acidified to pH 4 with 6N
HCl and extracted with ethyl acetate (3.times.30 mL). The organic
phase is washed with 30 mL of water and 30 mL of brine, dried over
anhydrous Na.sub.2SO.sub.4, evaporated and purified by silica gel
column chromatography (petroleum ether/ethyl acetate, 6/1) to give
methyl
3-[2-ethoxycarbonyl-4-(4-isopropylphenyl)-4-oxobutyryl]benzoate
(1.5 g, 51%) as pale yellow oil.
[0460] Step 8: A mixture of methyl
3-[2-ethoxycarbonyl-4-(4-isopropylphenyl)-4-oxobutyryl]benzoate
(1.5 g, 3.7 mmol), 0.29 g of NaCl and 0.15 mL of water in 20 mL of
DMSO is heated to 140-150.degree. C. and stirred for 3.5 hr. The
mixture is cooled to room temperature and added into 50 mL of
ice-water. Then the mixture is extracted with ether (3.times.50 mL)
and the combined organic layer is washed with water (2.times.50
mL), brine (50 mL) and then dried over Na.sub.2SO.sub.4 and
evaporated. The residue is purified by silica gel column
chromatography to obtain methyl
1-[4-(4-isopropylphenyl)-4-oxo-butyryl]benzoate (0.7 g) as a pale
yellow solid.
[0461] Step C: A solution of methyl
3-[4-(4-isopropylphenyl)-4-oxo-butyryl]benzoate (0.7 g) and a
catalytic amount of TsOH in 10 mL of absolute toluene is refluxed
overnight. The reaction mixture is diluted with 100 mL of EtOAc and
washed with water (2.times.50 mL), brine (50 mL) and then dried
over Na.sub.2SO.sub.4 and evaporated. The residue is purified by
silica gel column chromatography to give methyl
3-[5-(4-isopropyl-phenyl)furan-2-yl]benzoate (0.3 g) as yellow
oil.
[0462] Step D: To a solution of methyl
3-[5-(4-isopropyl-phenyl)furan-2-yl]benzoate (0.3 g) in 5 mL of THE
is added LiOH (0.2 g) dissolved in 15 mL water, and the reaction is
stirred for 2 h. The reaction mixture is cooled to room temperature
and extracted with ether (2.times.30 mL). The organic layer is
washed with water (2.times.30 mL), brine (30 mL), dried over
Na.sub.2SO.sub.4 and evaporated. The residue is purified by
preparative HPLC to give 3-[5-(4-isopropylphenyl)furan-2-yl]benzoic
acid (7 mg, 0.62%, over 3 steps) as a white solid: mp
172-176.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta. 8.46 (s, 1H),
7.99 (t, J=7.8, 2H), 7.70 (d, J=8, 2H), 7.53 (t, J=7.8, 1H), 7.29,
J=8.0, 2H), 6.84 (d, J=3.6, 1H), 6.71 (d, J=3.6, 1H), 2.95 (septet,
6.8, 1H), 1.29 (d, J=6.8, 6H).
Example DD: Preparation of
3-[5-(4-isopropylphenyl)-[1,2,4]thiadiazol-3-yl]-benzoic acid
(Compound No. 323)
##STR01303##
[0464] Step A: A solution of isophthalamic acid methyl ester (1.0
g, 5.6 mmol) and trichloromethyl sulfenyl chloride (1.039 g, 5.6
mmol, 0.6 mL) in 10 mL of anhydrous toluene is heated to reflux
overnight under nitrogen. The mixture is cooled to room temperature
and water is added to quench the reaction. The residue is
partitioned between water and EtOAc and then the organic layer is
washed with brine, dried over Na.sub.2SO.sub.4, filtered and
evaporated. The residue is purified by flash chromatography and 274
mg (21%) of methyl 3-(2-oxo-[1,3,4]oxathiazol-5-yl)benzoate is
obtained.
[0465] Step B: To 4-isopropylbenzonitrile (795 mg, 5.5 mmol) at
190.degree. C., methyl 3-(2-oxo-[1,3,4]oxathiazol-S-yl)-benzoate
(260 mg, 1.1 mmol) is added in three equal portions at 5-minutes
interval. The reaction is stirred for another 30 min. The mixture
is cooled to room temperature and the residue is partitioned
between water and EtOAc. The organic layer is washed with brine,
dried over Na.sub.2SO.sub.4, filtered and evaporated. The residue
is purified by flash chromatography to give 11 mg (3%) of methyl
3-[5-(4-isopropylphenyl)-[1,2,4]thiadiazol-3-yl]benzoate.
[0466] Step C: To a solution of 11 mg of the above ester in 4 mL of
3/1 MeOH/HO.sub.2O is added 7 mg of LiOH.H.sub.2O. The mixture is
stirred at 40-50.degree. C. overnight, cooled to room temperature
and neutralized with 3N hydrochloric acid. The mixture is extracted
with EtOAc, washed with brine and dried over Na.sub.2SO.sub.4.
Removal of the solvent gave 8 mg (79%) of
3-[5-(4-isopropylphenyl)-[1,2,4]thiadiazol-3-yl]benzoic acid: mp
165-167.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta. 8.75 (br s,
1H), 8.33-8.27 (m, 4H), 7.67 (t, J=7.8, 1H), 7.37 (d, J=7.6, 2H),
3.00 (septet, J=6.8, 1H), 1.31 (d, J=6.8, 6H); MS m/z 325.1
[MH.sup.+].
Example EE: Preparation of
3-[3-(4-isopropylphenyl)-[1,2,4]thiadiazol-5-yl]benzoic acid
(Compound No. 326)
##STR01304##
[0468] Step A: A solution of 4-isopropylbenzamide (1.0 g, 6.1 mmol)
and trichloromethyl sulfenyl chloride (1.14 g, 6.1 mmol) in 10 mL
of anhydrous toluene is heated to reflux overnight. The mixture is
cooled to room temperature and water is added to quench the
reaction. The residue is partitioned between water and EtOAc and
the organic layer is washed with brine, dried over
Na.sub.2SO.sub.4, filtered and evaporated. The residue is purified
by flash chromatography to give 250 mg (18%) of
5-(4-isopropylphenyl)-[1,3,4]oxathiazol-2-one.
[0469] Step B: To ethyl 3-cyanobenzoate (2.77 g, 15.8 mmol) at
190.degree. C., 5-(4-isopropylphenyl)-[1,3,4]oxathiazol-2-one (250
mg, 1.1 mmol) is added in three equal portions at 5-minutes
interval. The reaction is stirred for another 30 min. The mixture
is cooled to room temperature, and the residue is partitioned
between water and EtOAc. The organic layer is washed with brine,
dried over Na.sub.2SO.sub.4, filtered and evaporated. The residue
is purified by flash chromatography to give 12 mg (3%) of ethyl
3-[3-(4-isopropylphenyl)-[1,2,4]thiadiazol-5-yl]benzoate.
[0470] Step C: To a solution of 12 mg of the above ester in 4 mL of
3/1 MeOH/H.sub.2O is added 7 mg of LiOH.H.sub.2O. The mixture is
stirred at 40-50.degree. C. overnight, cooled to room temperature
and neutralized with 3N hydrochloric acid. The mixture is extracted
with EtOAc, washed with brine and dried over Na.sub.2SO.sub.4.
Removal of the solvent gave 6 mg (54%) of
3-[3-(4-isopropylphenyl)-[1,2,4]thiadiazol-5-yl]benzoic acid: mp
166-168.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta. 8.74 (br s,
1H), 8.33-8.25 (m, 5H), 7.67 (t, J=8.0, 1H), 7.37 (d, J=8.0, 2H),
3.00 (septet, J=6.8, 1H), 1.31 (d, J=6.8, 6H); MS m/z 325.1
[MH.sup.+].
Example FF: Preparation of
3-[4-(4-isopropylphenyl)-thiophen-2-yl]-benzoic acid (Compound No.
327)
##STR01305##
[0472] Step A: To a solution of 2,4-dibromothiophene (433 mg, 1.8
mmol) and 3-(ethoxycarbonyl)phenyl boronic acid (347 mg, 1.8 mmol)
in ethanol/toluene/water (10 mL/5 mL/3 mL), 568 mg of
Na.sub.2CO.sub.3 is added. After degasification twice, a catalytic
amount of Pd(PPh.sub.3).sub.4 is added under a nitrogen atmosphere.
The reaction mixture is stirred at 80.degree. C. overnight. The
mixture is cooled to room temperature, filtered and evaporated. The
residue is partitioned between water and EtOAc. The organic layer
is then washed with brine, dried over Na.sub.2SO.sub.4, filtered
and evaporated. The residue is purified by flash chromatography to
give 350 mg (63%) of ethyl 3-(4-bromothiophen-2-yl)-benzoate.
[0473] Step B: To a solution of ethyl
3-(4-bromothiophen-2-yl)benzoate (350 mg, 1.1 mmol) and
4-isopropylphenyl boronic acid (187 mg, 1.1 mmol) in
ethanol/toluene/water (10 mL/5 mL/3 mL), 358 mg Na.sub.2CO.sub.3 is
added. After degasification twice, a catalytic amount of
Pd(PPh.sub.3).sub.4 is added under a nitrogen atmosphere. The
reaction mixture is stirred at 80.degree. C. until TLC analysis
indicated the reaction is complete. The mixture is cooled to room
temperature, filtered and evaporated. The residue is partitioned
between water and EtOAc. The organic layer is then washed with
brine, dried over Na.sub.2SO.sub.4, filtered and evaporated. The
residue is purified by flash chromatography to give 150 mg (38%) of
ethyl 3-[4-(4-isopropylphenyl)thiophen-2-yl]benzoate.
[0474] Step C: To a solution of 50 mg of ethyl
3-[4-(4-isopropylphenyl)thiophen-2-yl]benzoate in 4 mL of 3/1
MeOH/H.sub.2O is added 30 mg of LiOH.H.sub.2O and the mixture is
stirred at 40-50.degree. C. until TLC analysis indicated the
reaction is complete. The mixture is cooled to room temperature and
neutralized with 3N hydrochloric acid. The mixture is extracted
with EtOAc, washed with brine and dried over Na.sub.2SO.sub.4.
Removal of the solvent gave 30 mg (65%) of
3-[4-(4-isopropylphenyl)thiophen-2-yl]benzoic acid: mp
220-222.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta. 8.35 (br s,
1H), 8.02 (d, J=8.0, 1H), 7.83 (d, J=8.0, 1H), 7.60-7.57 (m, 3H),
7.52 (t, J=8.0, 1H), 7.45 (s, 1H), 7.29 (s, 1H), 2.95 (septet,
J=6.8, 1H), 1.28 (d, J=6.8, 6H); MS m/z 323.1 [MH.sup.+].
Example GG: Preparation of
3-[5-(4-isopropylphenyl)thiophen-3-yl]benzoic acid (Compound No.
348)
##STR01306##
[0476] Step A: To a solution of 2,4-dibromothiophene (500 mg, 2.1
mmol) and 4-isopropylphenyl boronic acid (339 mg, 2.1 mmol) in
ethanol/toluene/water (10 mL/5 mL/3 mL), 657 mg Na.sub.2CO.sub.3 is
added. After degasification twice, a catalytic amount of
Pd(PPh.sub.3).sub.4 is added under a nitrogen atmosphere. The
reaction mixture is stirred at 80.degree. C. overnight. The mixture
is cooled to room temperature, filtered and evaporated. The residue
is partitioned between water and EtOAc. The organic layer is washed
with brine, dried over Na.sub.2SO.sub.4, filtered and evaporated.
The residue is purified by flash chromatography to give 207 mg
(36%) of 4-bromo-2-(4-isopropylphenyl)thiophene.
[0477] Step B: To a solution of
4-bromo-2-(4-isopropylphenyl)thiophene (207 mg, 0.7 mmol) and
3-(ethoxycarbonyl)phenyl boronic acid (143 mg, 0.7 mmol) in
ethanol/toluene/water (10 mL/5 mL/3 mL), 234 mg Na.sub.2CO.sub.3 is
added. After degasification twice, a catalytic amount of
Pd(PPh.sub.3).sub.4 is added under a nitrogen atmosphere. The
reaction mixture is stirred at 80.degree. C. until TLC analysis
indicated the reaction is complete. The mixture is cooled to room
temperature, filtered and evaporated. The residue is partitioned
between water and EtOAc. The organic layer is washed with brine,
dried over Na.sub.2SO.sub.4, filtered and evaporated. The residue
is purified by flash chromatography to give 180 mg (70%) of ethyl
3-[5-(4-isopropylphenyl)thiophen-3-yl]benzoate.
[0478] Step C: To a solution of 100 mg of ethyl
3-[5-(4-isopropylphenyl)thiophen-3-yl]benzoate in 4 mL of 3/1
MeOH/H.sub.2O is added 65 mg LiOH.H.sub.2O. The mixture is stirred
at 40-50.degree. C. overnight, cooled to room temperature and
neutralized with 3 N hydrochloric acid. The mixture is extracted
with EtOAc, washed with brine and dried over Na.sub.2SO.sub.4.
After removal of the solvent, 80 mg (87%) of
3-[5-(4-isopropylphenyl)thiophen-3-yl]benzoic acid is obtained: mp
208-209.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta. 8.37 (br s,
1H), 8.01 (d, J=7.2, 1H), 7.87 (d, J=7.6, 1H), 7.66 (d, J=1.6, 1H),
7.56 (d, J=8.0, 2H), 7.51 (t, J=8.0, 1H), 7.40 (d, J=1.6, 18), 7.29
(d, J=8.0, 2H), 2.95 (septet, J=7.2, 1H), 1.28 (d, J=7.2, 6H); MS
m/z (m/z) 323.2 [MH.sup.+].
Example HH: Preparation of
3-[3-(4-isopropylphenyl)thiophen-2-yl]benzoic acid (Compound No.
400)
##STR01307##
[0480] Step A: To a stirred solution of thiophene (5.94 g, 71 mmol)
in an equal volume of toluene at 0.degree. C. bromine (23 g, 142
mmol) in 50 mL of toluene is added as rapidly as possible and
stirred for another 0.5 h. Then 5 g sodium hydroxide is added. The
mixture is partitioned between water and EtOAc, dried over sodium
sulfate and evaporated. The residue, 2,5-dibromothiophene, is
purified by distillation.
[0481] Step B: To a solution of 2,5-dibromothiophene (1.0 g, 4.0
mmol) and 3-(ethoxycarbonyl)phenyl boronic acid (793 mg, 4.0 mmol)
in ethanol/toluene/water (10 mL/5 mL/3 mL), 1.32 g of
Na.sub.2CO.sub.3 is added. After degasification twice, a catalytic
amount of Pd(PPh.sub.3).sub.4 is added under a nitrogen atmosphere.
The reaction mixture is stirred at 80.degree. C. overnight. The
mixture is cooled to room temperature, filtered and evaporated. The
residue is partitioned between water and EtOAc. The organic layer
is washed with brine, dried over Na.sub.2SO.sub.4, filtered and
evaporated. The residue is purified by flash chromatography to give
456 mg (36%) of ethyl 3-(5-bromothiophen-2-yl)-benzoate.
[0482] Step C: To a solution of ethyl
3-(5-bromothiophen-2-yl)-benzoate (200 mg, 0.6 mmol) and
4-isopropylphenyl boronic acid (105 mg, 0.6 mmol) in
ethanol/toluene/water (10 mL/5 mL/3 mL), 204 mg Na.sub.2CO.sub.3 is
added. After degasification twice, a catalytic amount of
Pd(PPh.sub.3).sub.4 is added under a nitrogen atmosphere. The
reaction mixture is stirred at 80.degree. C. until TLC analysis
indicated the reaction is complete. The mixture is cooled to room
temperature, filtered and evaporated. The residue is partitioned
between water and EtOAc; the organic layer is washed with brine,
dried over Na.sub.2SO.sub.4, filtered and evaporated. The residue
is purified by flash chromatography to give 154 mg (69%) of ethyl
3-[5-(4-isopropylphenyl)thiophen-2-yl]benzoate.
[0483] Step D: To a solution of 100 mg of ethyl
3-[5-(4-isopropylphenyl)thiophen-2-yl]benzoate in 4 mL of 3/1
MeOH/H.sub.2O is added 46 mg of LiOH.H.sub.2O. The mixture is
stirred at 40-50.degree. C. overnight, cooled to room temperature
and neutralized with 3N hydrochloric acid. The mixture is extracted
with EtOAc, washed with brine and dried over Na.sub.2SO.sub.4.
After removal of the solvent, 78 mg (85%) of
3-[5-(4-isopropylphenyl)thiophen-2-yl]benzoic acid is obtained: mp
233-235.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta. 8.16 (s, 1H),
7.80 (d, J=7.8, 1H), 7.65 (d, J=8.1, 1H), 7.42 (d, J=8.1, 2H), 732
(t, J=7.8, 1H), 7.22 (d, J=3.6, 1H), 7.13 (d, J=3.6, 1H), 7.11 (d,
J=8.4, 2H), 2.80 (septet, 6.8, 1H), 1.14 (d, 3=6.9, 6H); MS m/z
321.5 [MH.sup.-].
Example II: Preparation of
3-[5-(4-methoxy-phenyl)-2H-[1,2,4]triazol-3-yl]-benzoic acid
(Compound No. 424)
##STR01308##
[0485] Step A: m-Methoxycarbonyimidoyl benzoic acid methyl ester
hydrochloride: To a solution of methyl 3-cyanobenzoate (0.65 g,
4.03 mmol) in methanol (15 mL) is added acetyl chloride (12 mL)
dropwise at 0.degree. C. After the addition, the reaction mixture
is stirred for 6 h at 0.degree. C. to room temperature. Solvent
removal gives a white solid that is purified by washing with
diethyl ether and is used immediately in the next step.
[0486] Step B: Methyl
3-(5-(4-methoxyphenyl-2H-[1,2,4-]triazol-3-yl)benzoate: A solution
of sodium methoxide (0.5 N in methanol) (8.5 mL, 4.25 mmol) in
anhydrous ethanol (30 ml) is added to a room temperature solution
of m-methoxycarbonyimidoyl benzoic acid methyl ester hydrochloride
in ethanol (10 mL). The milky slurry is stirred at room temperature
for 30 min and then filtered. The filtrate is condensed to of the
volume, to which is added 4-methoxybenzohydrazide (0.55 g, 3.31
mmol) and dioxane (10 mL). The resulting mixture is heated to
reflux for 15 h. Addition of 1N HCl to afford a white solid (0.66
g, 71.0% yield), which is collected by filtration and washed with
water, then water/ethanol (1/5). The obtained compound is >90%
pure as determined by LC-MS; MS m/z 310 [MH.sup.+].
[0487] Step C: 3-(5-4-methoxy-phenyl-2H-[1,2,4]triazol-3-yl)benzoic
acid: A mixture of methyl
3-[5-(4-methoxyphenyl)-2H-[1,2,4]triazol-3-yl]benzoate (0.32 g,
1.04 mmol) in 1N NaOH (3.0 mL, 3.00 mmol)/THF (1:1) is stirred at
reflux for 6 h until complete consumption of the starting material
is determined by TLC. The THF is stripped off in vacuo. A white
solid is precipitated after addition of 1N HCl. The desired product
(0.26 g, 85.2% yield) is collected by filtration and washed with
water, then diethyl ether in sequence: mp 287-289.degree. C.;
.sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta. 13.20 (1H, s), 8.64
(1H, s), 8.27 (1H, dd, J17.7, 0.8 Hz), 8.01 (3H, m), 7.60 (1H, t,
J=1.2 Hz), 7.61 (2H, m), 2.49 (3H, s); MS m/z 296 [MH.sup.+].
[0488] Melting point and mass spec data for certain preferred
compounds of the Invention is shown in the table 1 below.
TABLE-US-00013 Compound Melting Point (.degree. C.) MS (ES+) 1
>260. 310.1 2 187-189 310.1 3 247-248 310.1 4 268-270 310.2 5
262-265 309.2 6 202-204 309.2 7 227-229 309.3 8 >270 312.2 12
>270 358 13 >275 436 14 >275 324.1 15 281.3 16 301.1 17
273.2 18 268.2 19 257.2 21 283.3 22 297.2 23 297.2 24 297.2 25
281.2 26 281.2 27 301.1 28 301.1 29 323.3 30 283.2 31 283.2 32
344.1 33 344.1 34 312.2 35 312.2 36 312.2 37 327.3 38 311.3 39
297.2 40 297.2 41 297.2 42 281.2 43 281.2 44 301.1 45 301.1 46
323.3 47 283.3 48 283.2 49 344.1 50 344.1 51 312.2 53 312.2 54
327.3 55 311.3 60 >270 443.2 62 335.2 63 317.3 64 317.3 65 343.3
66 315.2 67 232.2 68 335.2 69 317.3 70 317.3 71 343.3 72 315.2 73
233.2 75 82 235-237 352.3 83 275-277 350.3 84 279-282 427.3 85
220-222 324.3 86 243-245 324.3 87 355.3 88 311.2 89 359.3 90 338.3
91 339.3 92 343.3 93 358.3 94 335.3 95 318.2 96 359.1 97 360.3 98
337.3 99 359.3 100 412.3 101 309.2 102 359.3 103 295.2 104 309.3
106 393.1 107 285.2 108 309.3 109 295.3 110 336.1 111 326.3 112
336.1 113 311.3 114 335.2 115 360.1 116 285.2 117 335.2 118 295.3
119 294.3 120 326.2 121 359.2 122 327.3 123 433.4 124 313.3 125
332.3 126 345.3 127 309.2 128 342.2 129 325.2 130 351.2 131 348.3
132 359.3 133 371.2 134 411.3 135 401.4 136 331.2 137 318.2 138
318.2 139 318.2 140 253-255 .sup. 307 (ES-) 141 237-9 323 142 295.2
143 309.3 144 393.1 145 285.2 146 309.3 147 295.3 148 336.1 149
327.3 150 336.1 151 311.3 152 335.2 153 360.1 154 285.2 155 335.2
156 295.3 157 295.3 158 327.2 159 360.2 160 327.1 161 355.3 162
311.2 163 359.3 164 338.3 165 339.3 166 358.3 167 335.2 168 360.1
169 373.3 170 359.3 171 412.3 172 318.2 173 309.2 174 360.3 175
236-238 354.1 176 336.1 177 360.1 178 311.3 179 331.1 180 325.3 181
319.4 182 319.4 183 341.3 184 295.3 185 336.1 186 295.3 187 285.2
188 309.2 189 336.1 190 360.1 191 311.3 192 331.1 193 325.3 194
319.4 195 319.4 196 341.3 197 325.3 198 332.3 199 319.4 200 319.4
201 295.3 202 336.1 203 295.3 204 285.2 205 295.3 206 379.3 207
407.2 208 311.2 209 321.3 210 313.3 211 345.3 212 309.2 213 342.2
214 325.3 215 351.2 216 349.3 217 360.3 218 372.1 219 412.3 220
318.2 221 318.2 222 278-282 302.2 223 272-274 311.2 224 240-250
311.2 225 >285 302.1 226 324.2 227 324.2 228 325.2 229 325.2 230
334.3 231 334.3 232 319.2 233 319.2 234 336.3 235 336.3 236 337.3
237 337.3 238 301.2 239 283.2 240 283.2 241 285.2 242 285.2 243
350.3 244 350.3 245 379.1 246 379.1 247 417.3 248 417.3 249 351.3
250 309.2 251 309.2 252 343.2 253 343.2 254 334.3 255 334.3 258
314.3 259 302.1 260 337.1 261 282.2 262 328.2 263 324.3 264 336.2
265 336.2 266 439.1
267 376.2 268 316.8 269 363.3 270 331.7 271 292.3 272 205-208 337.1
273 347.1 274 302.7 275 210-213 .sup. 307 (ES-) 276 235-237 308.4
277 296-298 307.4 278 >310 338.2 279 228-235 338.2 280 274-276
336.2 281 240-242 357.2 282 274-275 358.2 283 220-226 352.2 284
282-291 352.2 285 253-256 378.3 286 >310 378.3 287 307.2 288
150-153 308.2 289 206-208 324.2 290 222-225 308.2 291 200-212 292
275-278 346.1-348.1 293 274-275 346.1-348.1 294 314.3 295 >310
306.2 296 302.7 297 337.1 298 282.2 299 347.1 300 328.2 301 324.3
302 336.2 303 337.1 304 302.7 305 292.2 306 331.7 307 363.3 308
315.7 309 376.1 310 218-220 324.3 311 283-285 307.2 312 193-195
324.3 313 236-239 308.3 314 236-239 349.0 (ES-) 315 >310 306.2
316 >270 306.2 317 >290 (decomp) 306.2 318 >310 306.2 319
304-320 306.1 320 193-195 308.2 321 237-239 322 322 172-176 323
165-167 325.1 324 >300 325.1 325 280.1 326 166-168 325.2 327
220-222 323.1 329 311.2 330 334.2 331 316.3 332 296.2 333 337.3 334
291.2 335 335.3 336 300.1 337 342.2 338 335.1 339 326.3 340 302.2
341 326.3 342 402.2 343 322.3 344 296.2 345 345.1 346 293.3 348
208-209 323.2 349 281.2 350 296.1 351 345.1 352 322.3 353 335.1 354
302.2 355 332.2 356 345.1 357 338.3 358 324.2 359 267.2 360 310.2
361 336.3 362 284.2 363 300.1 364 315.2 365 351.4 366 312.3 367
307.3 368 353.4 369 338.4 370 312.3 371 350.3 372 316.1 373 310.3
374 318.3 375 418.3 376 300.3 377 318.3 378 348.3 379 316.4 380
326.3 381 340.3 382 352.4 383 338.4 384 294.3 385 342.3 386 351.2
387 283.3 388 332.3 389 327.3 390 330.4 391 383.3 392 322.2 394
267.1 395 285.1 396 303.1 397 309.2 398 311.2 399 325.2 400 233-235
321.5 (ES-) 401 152-155 343.1 402 174-177 338.2 403 365 404 330.1
405 341.2 406 315.1 407 230-235 285.3 408 303.2 409 335.2 410 267.3
411 312.2 412 301.3 413 285.2 414 247.3 415 357.3 416 281.3 417 253
418 297.2 419 303.2 420 257.2 421 285.2 422 359.3 423 309-311
346.09 424 287-289 296.18 425 >310 284.2 426 >310 284.2 427
>300 266.38 428 >310 267.19 429 >300 342.2 430 267.3 431
312.2 432 301.3 433 285.2 434 247.3 435 357.3 436 281.3 437 253 438
331 439 302 440 315 441 314.1 442 324 443 316.1 444 351.1 445 397.2
446 311.2 447 231.2 448 310.2 449 311 450 268.2 451 292.2 452 268.1
453 302.1 454 309.19 455 >300 282.17 456 235-238 334.17 457
>300 280.19 458 247-250 296.45 459 287-290 296.18 460 275-278
326.2 461 295-298 310.21 462 282-285 272.16 463 221-222 284.12 464
.sup. 112-113.5 298.25 465 298.25 466 298.25 467 280.25 468 316.24
469 314.21 470 310.23 471 352.23 472 378.10, 380 473 308.26 474
310.28 475 242-243 284.22 476 266.5-268.sup. 284.23 477 245-247
266.25 478 260-262 302.24 479 293-295 300.19 480 249.5-251.sup.
296.22 481 201-203 296.22 482 283.5-285.sup. 364.09, 366 483
255-257 310.22 484 215-216 296.22 485 224-225 296.22 486 198-202
309.27 487 249-250 291.22 488 310.27 489 330.24 490 323.21 491
305.23 492 308.29 493 312.25 494 328.22 495 312.25 496 312.25 497
324.29 498 330.24 499 .sup. >278.5 302.21 500 246-247 296.27 501
231-232 284.24 502 264-265 284.22 503 265-266 300.16 504 .sup.
273-274.5 284.29 505 253-254 280.25 506 233-234 285.26 507
221.5-223.sup. 285.23 508 299-300 301.15, 299.19 509 281.5-283.sup.
297.26 510 245-246 311.25 511 217-218 351.23 512 243-244 281.28 513
249.5-251.sup. 335.27 514 298.25 515 298.25 516 298.25 517 316.24
518 310.29 519 314.17 520 294.26
521 298.27 522 298.27 523 316.27 524 310.29 525 314.27 526 294.31
527 243-245 346.1 528 233-235 296.2 529 292.5-293.5 284.18 530
318-319 284.19 531 317-319 302.18 532 298-300 296.22 533 273-275
300.05 534 297.5-299.sup. 280.23 535 302-303 284.24 536 319-321
284.22 537 322-323 284.23 538 324-326 302.23 539 297.5-299.5 296.24
540 320.5-322.5 300.19 541 307-308 280.25 542 284-285 350.2 543
286-287 334.24 544 240-242 296.22 545 239-240 334.24 546 222-224
350.2 547 241-243 326.25 548 298-299 302.19 549 295-296 302.22 550
272-273 284.18 551 238-239 295.34 552 270.5-272.sup. 283.32 553
264-265 284.31 554 245-246 284.31 555 276-277 300.28 556 272-274
300.28 557 269-270 310.3 558 246-247 272.27 559 281.22 560 315.28
561 365.22 562 350.29 563 386.33 564 347.22 565 299.4 566 294.38
567 348.33 568 308.3 569 283.5-285.sup. 281.22 570 292-293 315.28
571 282-283 365.22 572 298.5-301.sup. 350.29 573 257-259 280.27 574
281-282 334.31 575 248-250 296.3 576 233-235 347.22 577 304-305
299.4 578 234-236 386.33 579 228-229 284.31 580 .sup. 223-224.5
284.29 581 255-256 284.27 582 236-237 300.21 583 220-222 300.28 584
218-219 334.24 585 .sup. 250-251.5 334.33 586 223.5-225.sup. 334.21
587 210-211 350.32 588 207-208 280.32 589 194-195 280.3 590 174-175
294.32 591 213-215 308.33 592 224-225 322.34 593 244-246 296.29 594
207-208 296.29 595 223-224 310.29 596 >275 267.1 601 177-178
310.1 605 170-172 310.2 606 197-200 310.2 609 71-78 310.2 610
180-183 310.2 615 190-192 2 67.2 620 190-192 309.3 621 258-261
329.4 622 245-247 329.4 624 227-235 (decomp) 626 200-205 (decomp)
628 >300 629 302.35 630 301-302 302.32 631 308-309 324.35 632
248-249 280.36 633 256-257 280.36 634 233-234 294.37 635 233-234
308.37 636 258-259 322.44 637 249-251 296.34 638 245-246 310.36 639
260-263 300.26 640 291-292 302.25 641 273-274 302.25 642 259-261
344.20 643 284-286 280.24 644 236-237 308.30 645 305-306 296.27 646
210-211 373.27 647 220-222 358.27 648 291-292 282.20 649 295-297
318.18 650 191-193 316.26 651 251-253 290.29 652 338.27 653 364.35
654 348.2 655 394.32 656 360.31 657 324.27 658 363.33 659 336.4 660
294.32 661 340.23 662 324.26 663 322.38 664 323.32
Example 2: Nonsense Suppression Activity
[0489] A functional, cell-based translation assay based on
luciferase-mediated chemoluminescence (International Application
PCT/US2003/023185, filed on Jul. 23, 2003, hereby incorporated by
reference in its entirety) permits quantitative assessment of the
level of nonsense suppression. Human embryonic kidney cells (293
cells) are grown in medium containing fetal bovine serum (FBS).
These cells can be stably transfected with the luciferase gene
containing a premature termination codon at amino acid position
190. In place of the threonine codon (ACA) normally present in the
luciferase gene at this site, each of the 3 possible nonsense
codons (TAA, TAG, or TGA) and each of the 4 possible nucleotides
(adenine, thymine, cytosine, or guanine) at the contextually
important downstream +1 position following the nonsense codon are
introduced by site-directed mutagenesis. As such, amino acid 190 in
the luciferase gene containing a premature termination codon is
either TAA, TAG, or TGA. For each stop codon, the nucleotide
following amino acid 190 of luciferase gene containing a premature
termination codon can be replaced with an adenine, thymine,
cytosine, or guanine (A, T, C, G) such that these mutations do not
change the reading frame of the luciferase gene, Schematics of
these constructs are depicted in FIG. 1.
[0490] The nonsense suppression activity from a cell-based
luciferase reporter assay of the present invention as described
above shown in the table below (Table 2). Human Embryonic Kidney
293 cells are stably transfected with a luciferase reporter
construct comprising a UGA nonsense mutation at position 190, which
is followed, in-frame by an adenine nucleotide.
[0491] Activity measurements in Table 2 are determined in a
cell-based luciferase reporter assay of the present invention
construct containing a UGA premature termination codon. Gentamicin,
an aminoglycoside antibiotic known to allow readthrough of
premature termination codons, is used as an internal standard.
Activity measurements are based on the qualitative ratio between
the minimum concentration of compound required to produce a given
protein in a cell versus the amount of protein produced by the cell
at that concentration. Compounds which are found to have either or
both very high potency and very high efficacy of protein synthesis
are classified as "****". Compounds which are found to have
intermediate potency and/or efficacy of protein synthesis are
classified as "****"; "****" or "**". Similarly, compounds which
are found to have lower potency and/or efficacy of protein
synthesis are classified as "*".
[0492] Activity of the certain preferred compounds of the invention
is shown in the table below:
TABLE-US-00014 Compound No. Activity 1 **** 2 *** 3 *** 4 **** 5 **
6 **** 7 *** 8 * 12 * 13 * 14 * 15 ** 16 ** 17 ** 18 * 19 ** 21 *
22 * 23 ** 24 ** 25 ** 26 * 27 * 28 ** 29 ** 30 ** 31 * 32 * 33 **
34 * 35 * 36 * 37 * 38 * 39 * 40 * 41 ** 42 ** 43 ** 44 ** 45 ** 46
** 47 * 48 * 49 * 50 *** 51 * 53 * 54 * 55 ** 60 * 62 * 63 * 64 *
65 ** 66 * 67 * 68 ** 69 * 70 ** 71 * 72 * 73 * 75 * 82 * 83 ** 84
** 85 * 86 * 87 * 88 **** 89 * 90 ** 91 * 92 * 93 * 94 * 95 * 96 *
97 * 98 * 99 *** 100 * 101 * 102 *** 103 **** 104 *** 106 **** 107
** 108 * 109 *** 110 **** 111 * 112 * 113 * 114 ** 115 *** 116 **
117 *** 118 **** 119 *** 120 * 121 **** 122 * 123 * 124 ** 125 **
126 * 127 ** 128 * 129 *** 130 *** 131 ** 132 * 133 * 134 ** 135 *
136 *** 137 * 138 ** 139 ** 140 ** 141 *** 142 *** 143 *** 144 *
145 * 146 * 147 * 148 * 149 ** 150 * 151 * 152 * 153 * 154 ** 155 *
156 *** 157 * 158 * 159 ** 160 * 161 * 162 ** 163 * 164 * 165 * 166
* 167 * 168 ** 169 * 170 ** 171 * 172 ** 173 * 174 * 175 * 176 ****
177 * 178 * 179 * 180 ** 181 *** 182 * 183 * 184 ** 185 ** 186 *
187 ** 188 ** 189 *** 190 * 191 * 192 * 193 * 194 ** 195 ** 196 ***
197 * 198 ** 199 *** 200 * 201 **** 202 *** 203 * 204 ** 205 ***
206 ** 207 ** 208 ** 209 * 210 *** 211 ** 212 * 213 * 214 ** 215 **
216 * 217 * 218 ** 219 ** 220 ** 221 *** 222 * 223 *** 224 * 225 *
226 * 227 * 228 *** 229 *** 230 * 231 * 232 * 233 * 234 * 235 * 236
* 237 * 238 * 239 * 240 * 241 * 242 * 243 ** 244 * 245 *** 246 *
247 ** 248 * 249 * 250 * 251 ** 252 * 253 * 254 ** 255 * 258 * 259
* 260 ** 261 * 262 ** 263 * 264 * 265 * 266 *
267 * 268 ** 269 * 270 * 271 * 272 *** 273 ** 274 ** 275 *** 276
*** 277 ** 278 ** 279 ** 280 **** 281 ** 282 ** 283 ** 284 ** 285 *
286 ** 287 **** 288 **** 289 *** 290 **** 291 **** 292 ** 293 * 294
* 295 * 296 * 297 ** 298 * 299 ** 300 * 301 * 302 * 303 ** 304 *
305 * 306 * 307 * 308 ** 309 ** 310 *** 311 *** 312 *** 313 *** 314
* 315 ** 316 * 317 * 318 *** 319 * 320 *** 321 **** 322 *** 323 ***
324 * 325 *** 326 *** 327 **** 329 **** 330 ** 331 **** 332 ** 333
*** 334 *** 335 *** 336 **** 337 ** 338 *** 339 *** 340 *** 341 ***
342 *** 343 ** 344 *** 345 *** 346 *** 348 **** 349 **** 350 ****
351 *** 352 *** 353 *** 354 ** 355 *** 356 **** 357 *** 358 *** 359
*** 360 **** 361 ** 362 *** 363 *** 364 ** 365 *** 366 *** 367 ***
368 *** 369 *** 370 *** 371 *** 372 ** 373 *** 374 *** 375 *** 376
*** 377 *** 378 ** 379 *** 380 *** 381 *** 382 *** 383 ** 384 **
385 *** 386 *** 387 ** 388 *** 389 *** 390 ** 391 * 392 ** 393 *
394 *** 395 *** 396 * 397 *** 398 *** 399 **** 400 **** 401 * 402
** 403 * 404 ** 405 * 406 ** 407 **** 408 ** 409 **** 410 **** 411
** 412 **** 413 *** 414 *** 415 *** 416 * 417 * 418 * 419 **** 420
* 421 ** 422 * 423 **** 424 * 425 ** 426 * 427 * 428 * 429 *** 430
** 431 ** 432 * 433 ** 434 * 435 * 436 * 437 * 438 * 439 * 440 ***
441 * 442 *** 443 * 444 *** 445 **** 446 **** 447 * 448 *** 449 *
450 * 451 * 452 * 453 * 454 *** 455 ** 456 *** 457 **** 458 * 459
** 460 ** 461 * 462 * 463 **** 464 ** 465 *** 466 *** 467 *** 468
**** 469 *** 470 *** 471 *** 472 *** 473 *** 474 *** 475 **** 476
**** 477 **** 478 **** 479 **** 480 **** 481 **** 482 **** 483 *
484 ** 485 * 486 ** 487 ** 488 ** 489 *** 490 ** 491 ***** 492 **
493 *** 494 *** 495 **** 496 **** 497 ** 498 ** 499 ***** 500 *****
501 ***** 502 **** 503 **** 504 **** 505 ***** 506 **** 507 ****
508 **** 509 ***** 510 **** 511 **** 512 ***** 513 ***** 514 ** 515
** 516 ** 517 * 518 ** 519 **
520 *** 521 *** 522 **** 523 *** 524 *** 525 **** 526 *** 527 ****
528 *** 529 *** 530 ***** 531 ***** 532 ***** 533 *** 534 *** 535
*** 536 ** 537 ** 538 *** 539 **** 540 **** 541 **** 542 *** 543
*** 544 ***** 545 **** 546 **** 547 **** 548 **** 549 **** 550
***** 551 *** 552 *** 553 *** 554 **** 555 **** 556 *** 557 *****
558 **** 559 ** 560 ** 561 * 562 ** 563 * 564 * 565 ** 566 ** 567
** 568 ** 569 ** 570 ** 571 *** 572 * 573 **** 574 **** 575 *** 576
** 577 *** 578 * 579 *** 580 *** 581 *** 582 *** 583 *** 584 ***
585 *** 586 *** 587 *** 588 *** 589 **** 590 **** 591 *** 592 ****
593 *** 594 *** 595 **** 596 * 601 * 605 * 606 ** 609 * 610 * 615 *
620 * 621 *** 622 * 624 ***** 626 **** 628 *** 629 *** 630 *** 631
***** 632 ***** 633 ***** 634 ***** 635 ***** 636 ***** 637 *****
638 ***** 639 **** 640 **** 641 ** 642 **** 643 *** 644 **** 645
*** 646 *** 647 **** 648 **** 649 * 650 **** 651 **** 652 ** 653 **
654 ** 655 **** 656 * 657 ** 658 ** 659 ** 660 ** 661 ** 662 ** 663
** 664 **
[0493] The nonsense suppression activity in an assay as described
above is shown in the Table 3 below, for a construct with a UAG
nonsense mutation at position 190, followed by an adenine
nucleotide in-frame, (UAGA); and a construct with a UAA nonsense
mutation at position 190, followed by an adenine nucleotide
in-frame, (UAAA). "POS WB" indicates that a positive signal is
produced on a western blot when the compound of the invention is
used in an assay of the present invention. "ND" indicates that the
result is not determined.
TABLE-US-00015 Compound No. UAG UAA 4 ** ** (FA) (FA) ** ** (Na)
(Na) 5 ** 6 *** *** (FA) (FA) ** ** (Na) (Na) 7 *** *** (FA) (FA)
**** *** (Na) (Na) 499 *** 500 ** 501 * 502 * 503 * 504 * 505 ***
506 ** *** 507 * * 508 ** * 509 **** *** 510 *** ** 511 *** ** 512
**** *** 513 *** **
TABLE-US-00016 Compound No. UAGA UAAA 527 **** * 528 *** ** 548
POSWB 554 * * 557 * * 590 ** ** 592 *** *** 595 ** ** POSWB 478 ***
** 479 *** ** POSWB 480 ***** *** 481 *** *** 482 *** *** 525 *** *
573 ** * 574 ** *
Example 3: Readthrough Assay
[0494] A functional, cell-based translation assay based on
luciferase-mediated chemoluminescence (International Application
PCT/US2003/023185, filed on Jul. 23, 2003 and incorporated by
reference in its entirety) permits assessment of
translation-readthrough of the normal stop codon in a mRNA. Human
embryonic kidney cells (293 cells) are grown in medium containing
fetal bovine serum (FBS). These cells are stably transfected with
the luciferase gene containing a premature termination codon at
amino acid position 190. In place of the threonine codon (ACA)
normally present in the luciferase gene at this site, each of the 3
possible nonsense codons (TAA, TAG, or TGA) and each of the 4
possible nucleotides (adenine, thymine, cytosine, or guanine) at
the contextually important downstream +1 position following the
nonsense codon are introduced by site-directed mutagenesis. As
such, amino acid 190 in the luciferase gene containing a premature
termination codon is either TAA, TAG, or TGA. For each stop codon,
the nucleotide following amino acid 190 of luciferase gene
containing a premature termination codon are replaced with an
adenine, thymine, cytosine, or guanine (A, T, C, G) such that these
mutation do not change the reading frame of the luciferase gene.
Schematics of these constructs are depicted above in FIG. 1.
[0495] Another assay of the present invention can evaluate
compounds that promote nonsense mutation suppression. The
luciferase constructs described above in FIG. 1 are engineered to
harbor two epitope tags in the N-terminus of the luciferase
protein. Based on luciferase protein production, these constructs
qualitatively assess the level of translation-readthrough. The
presence of the full-length luciferase protein produced by
suppression of the premature termination codon is measured by
immunoprecipitation of the suppressed luciferase protein (using an
antibody against a His tag) followed by western blotting using an
antibody against the second epitope (the Xpress.TM. epitope;
Invitrogen.RTM.; Carlsbad, Calif.). These constructs are depicted
in FIG. 2.
[0496] Cells that harbor the constructs of FIG. 2 show increased
full-length protein production when treated with a compound of the
present invention. After treatment for 20 hours, cells containing
the constructs of FIG. 2 are collected and an antibody recognizing
the His epitope is used to immunoprecipitate the luciferase
protein. Following immunoprecipitation, western blotting is
performed using the antibody to the Xpress.TM. epitope
(Invitrogen.RTM.; Carlsbad, Calif.) to detect the truncated
luciferase (produced when no nonsense suppression occurs) and to
detect the full-length protein (produced by suppression of the
nonsense codon). Treatment of cells with a test compound produces
full-length protein and not a readthrough protein (See e.g., FIG.
3). The readthrough protein is produced if suppression of the
normal termination codon occurs. Compounds of the present invention
suppress the premature, i.e. nonsense mutation, but not the normal
termination codon in the luciferase mRNA.
[0497] Compounds of the present invention selectively act on
premature termination codons but not normal termination codons in
mammals.
[0498] Rats and dogs are administered high doses of compound (up to
1800 mg/kg) by gavage (oral) once daily for 14 days. After the
treatment, tissues are collected, lysates are prepared, and Western
blot analysis is performed. Selection of the proteins for
evaluation of normal termination codon readthrough is based
primarily on the corresponding mRNA having a second stop codon in
the 3'-UTR that is in-frame with the normal termination codon.
Between these 2 stop codons, each selected protein has an
intervening sequence of nucleotides that codes for an extension of
the protein in the event of ribosomal readthrough of the first
termination codon. If the compound has the capacity to induce
nonspecific, ribosomal readthrough, an elongated protein is
differentiated from the wild-type protein using Western blot.
Tissues are collected from rats and are analyzed for suppression of
the normal termination codon (UAA) in the vimentin mRNA. No
evidence of suppression is apparent. Tissues are collected from
dogs treated with compounds of the present invention. There is no
evidence of suppression of the normal termination codon of beta
actin, which harbors a UAG stop codon.
[0499] In healthy human volunteers, a single dose of a compound of
the present invention (200 mg/kg) is administered orally. Blood
samples are collected, plasma is prepared, and a Western blot is
conducted using plasma samples from female and male subjects.
C-reactive protein (CRP), which harbors a UGA termination codon, is
used to determine if treatment of subjects with compounds of the
present invention result in suppression of the normal termination
codon in the CRP mRNA. A luciferase assay in combination with a
premature termination assay demonstrates selective suppression of
premature termination codons but not normal termination codons.
Example 4: Animal Models
[0500] Animal model systems can also be used to demonstrate the
safety and efficacy of a compound of the present invention. The
compounds of the present invention can be tested for biological
activity using animal models for a disease, condition, or syndrome
of interest. These include animals engineered to contain the target
RNA element coupled to a functional readout system, such as a
transgenic mouse.
Cystic Fibrosis
[0501] Examples of animal models for cystic fibrosis include, but
are not limited to, cftr(-/-) mice (see, e.g., Freedman et al.,
2001, Gastroenterology 121(4); 950-7), cftr(tm1HGU/tm1HGU) mice
(see, e.g., Bernhard et al., 2001, Exp Lung Res 27(4):349-66),
CFTR-deficient mice with defective cAMP-mediated Cl(-) conductance
(see, e.g., Stotland et al., 2000, Pediatr Pulmonol 30(5):4.13-24),
and C57BL/6-Cftr(m1UNC)/Cftr(m1UNC) knockout mice (see, e.g.,
Stotland et al., 2000, Pediatr Pulmonol 30(5):413-24).
Muscular Dystrophy
[0502] Examples of animal models for muscular dystrophy include,
but are not limited to, mouse, hamster, cat, dog, and C. elegans.
Examples of mouse models for muscular dystrophy include, but are
not limited to, the dy-/- mouse (see, e.g., Connolly et al., 2002,
J. Neuroimmunol 127(1-2):80-7), a muscular dystrophy with myositis
(mdm) mouse mutation (see, e.g., Garvey et al., 2002, Genomics
79(2):146-9), the mdx mouse (see, e.g., Nakamura et al., 2001,
Neuromuscul Disord 11(3):251-9), the utrophin-dystrophin knockout
(dko) mouse (see, e.g., Nakamura et al., 2001, Neuromuscul Disord
11(3):251-9), the dy/dy mouse (see, e.g., Dubowitz et al., 2000,
Neuromuscul Disord 10(4-5):292-8), the mdx(Cv3) mouse model (see,
e.g., Pillers et al., 1999, Laryngoscope 109(8):1310-2), and the
myotonic ADR-MDX mutant mice (see, e.g., Kramer et al., 1998,
Neuromuscul Disord 8(8):542-50). Examples of hamster models for
muscular dystrophy include, but are not limited to,
sarcoglycan-deficient hamsters (see, e.g., Nakamura at al., 2001,
Am J Physiol Cell Physiol 281(2):C690-9) and the BIO 14.6
dystrophic hamster (see, e.g., Schlenker & Burbach, 1991, J
Appl Physiol 71(5):1655-62). An example of a feline model for
muscular dystrophy includes, but is not limited to, the
hypertrophic feline muscular dystrophy model (see, e.g., Gaschen
& Burgunder, 2001, Acta Neuropathol (Berl) 101(6):591-600).
Canine models for muscular dystrophy include, but are not limited
to, golden retriever muscular dystrophy (see, e.g., Fletcher et
al., 2001. Neuromuscul Disord 11(3):239-43) and canine X-linked
muscular dystrophy (see, e.g., Valentine et al., 1992, Am J Med
Genet 42(3):352-6). Examples of C. elegans models for muscular
dystrophy are described in Chamberlain & Benian, 2000, Curr
Biol 10(21):R795-7 and Culette & Sattelle, 2000, Hum Mol Genet
9(6):869-77.
Familial Hypercholesterolemia
[0503] Examples of animal models for familial hypercholesterolemia
include, but are not limited to, mice lacking functional LDL
receptor genes (see, e.g., Aji et al., 1997, Circulation
95(2):430-7), Yoshida rats (see, e.g., Fantappie et al., 1992, Life
Sci 50(24):1913-24), the JCR:LA-cp rat (see, e.g., Richardson et
al., 1998, Atherosclerosis 138(1):135-46), swine (see, e.g.,
Hasler-Rapacz et al., 1998, Am J Med Genet 76(5):379-86), and the
Watanabe heritable hyperlipidaemic rabbit (see, e.g., Tsutsumi et
al., 2000, Arzneimittelforschung 50(2):118-21; Harsch et al., 1998,
Br J Pharmacol 124(2):227-82; and Tanaka et al., 1995,
Atherosclerosis 114(1):73-82).
Human Cancer
[0504] An example of an animal model for human cancer, in general
includes, but is not limited to, spontaneously occurring tumors of
companion animals (see, e.g., Vail & MacEwen, 2000, Cancer
Invest 18(8):781-92). Examples of animal models for lung cancer
include, but are not limited to, lung cancer animal models
described by Zhang & Roth (1994, In Vivo 8(5):755-69) and a
transgenic mouse model with disrupted p53 function (see, e.g.,
Morris et al., 1998, J La State Med Soc 150(4):179-85). An example
of an animal model for breast cancer includes, but is not limited
to, a transgenic mouse that overexpresses cyclin D1 (see, e.g.,
Hosokawa et al., 2001, Transgenic Res 10(5):471-8). An example of
an animal model for colon cancer includes, but is not limited to, a
TCRbeta and p53 double knockout mouse (see, e.g., Kado et al.,
2001, Cancer Res 61(6):2395-8). Examples of animal models for
pancreatic cancer include, but are not limited to, a metastatic
model of Panc02 marine pancreatic adenocarcinoma (see, e.g., Wang
et al., 2001, Int J Pancreatol 29(1):37-46) and nu-nu mice
generated in subcutaneous pancreatic tumours (see, e.g., Ghaneh et
al, 2001, Gene Ther 8(3):199-208). Examples of animal models for
non-Hodgkin's lymphoma include, but are not limited to, a severe
combined immunodeficiency ("SCID") mouse (see, e.g., Bryant et al.,
2000, Lab Invest 80(4):553-73) and an IgHmu-HOX11 transgenic mouse
(see, e.g., Hough et al., 1998, Proc Natl Acad Sci USA
95(23):13853-8). An example of an animal model for esophageal
cancer includes, but is not limited to, a mouse transgenic for the
human papillomavirus type 16 E7 oncogene (see, e.g., Herber et al.,
1996, J Virol 70(3):1873-81). Examples of animal models for
colorectal carcinomas include, but are not limited to, Apc mouse
models (see, e.g., Fodde & Smits, 2001, Trends Mol Med
7(8):369-73 and Kuraguchi et al., 2000, Oncogene 19(50):5755-63).
An example of an animal model for neurofibromatosis includes, but
is not limited to, mutant NF1 mice (see, e.g., Cichowski et al.,
1996, Semin Cancer Biol 7(5):291-8). Examples of animal models for
retinoblastoma include, but are not limited to, transgenic mice
that expression the simian virus 40 T antigen in the retina (see,
e.g., Howes et al., 1994, Invest Ophthalmol Vis Sci 35(2):342-51
and Windle et al, 1990, Nature 343(6259):665-9) and inbred rats
(see, e.g., Nishida et al., 1981, Curr Eye Res 1(1):53-5 and
Kobayashi et al., 1982, Acta Neuropathol (Berl) 57(2-3):203-8).
Examples of animal models for Wilm's tumor include, but are not
limited to, a WT1 knockout mice (see, e.g., Scharnhorst et al.,
1997, Cell Growth Differ 8(2):133-43), a rat subline with a high
incidence of neuphroblastoma (see, e.g., Mesfin & Breech, 1996,
Lab Anim Sci 46(3):321-6), and a Wistar/Furth rat with Wilms' tumor
(see, e.g., Murphy et al., 1987, Anticancer Res 7(4B):717-9).
Retinitis Pigmentosa
[0505] Examples of animal models for retinitis pigmentosa include,
but are not limited to, the Royal College of Surgeons ("RCS") rat
(see, e.g., Vollrath et al., 2001, Proc Natl Acad Sci USA 98(22);
12584-9 and Hanitzsch et al., 1998, Aca Anal (Basel)
162(2-3):119-26), a rhodopsin knockout mouse (see, e.g., Jaissle et
al., 2001, Invest Ophthalmol Vis Sci 42(2):506-13), and Wag/Rij
rats (see, e.g., Lai et al., 1980, Am J Pathol 98(1):281-4).
Cirrhosis
[0506] Examples of animal models for cirrhosis include, but are not
limited to, CCl.sub.4-exposed rats (see, e.g., Kloehn et al., 2001,
Horm Metab Res 33(7):394-401) and rodent models instigated by
bacterial cell components or colitis (see, e.g., Vierling, 2001,
Best Pract Res Clin Gastroenterol 15(4):591-610).
Hemophilia
[0507] Examples of animal models for hemophilia include, but are
not limited to, rodent models for hemophilia A (see, e.g., Reipert
et al., 2000, Thromb Haemost 84(5):826-32; Jarvis et al., 1996,
Thromb Haemost 75(2):318-25; and Bi et al., 1995, Nat Genet
10(1):119-21), canine models for hemophilia A (see, e.g.,
Gallo-Penn et al., 1999, Hum Gene Ther 10(11):1791-802 and Connelly
et al, 1998, Blood 91(9); 3273-81), murine models for hemophilia B
(see, e.g., Snyder et al., 1999. Nat Med 5(1):64-70; Wang et al.,
1997, Proc Natl Acad Sci USA 94(21):11563-6; and Fang et al., 1996,
Gene Ther 3(3):217-22), canine models for hemophilia B (see, e.g.,
Mount et al., 2002, Blood 99(8):2670-6; Snyder et al., 1999, Natl
Med 5(1):64-70; Fang et al., 1996, Gene Ther 3(3):217-22); and Kay
et al., 1994, Proc Natl Acad Sci USA 91(6):2353-7), and a rhesus
macaque model for hemophilia B (see, e.g., Lozier et at, 1999,
Blood 93(6):1875-81).
Von Willebrand Disease
[0508] Examples of animal models for von Willebrand disease
include, but are not limited to, an inbred mouse strain RIIIS/J
(see, e.g., Nichols et al., 1994, 83(11):3225-31 and Sweeney et
al., 1990, 76(11):2258-65), rats injected with botrocetin (see,
e.g., Sanders et al., 1988, Lab Invest 59(4):443-52), and porcine
models for von Willebrand disease (see, e.g., Nichols et al., 1995,
Proc Natl Acad Sci USA 92(7):2455-9; Johnson & Bowie, 1992, J
Lab Clin Med 120(4):553-8); and Brinkhous et al., 1991, Mayo Clin
Proc 66(7):733-42).
.beta.-Thalassemia
[0509] Examples of animal models for .beta.-thalassemia include,
but are not limited to, murine models with mutations in globin
genes (see, e.g., Lewis et al., 1998, Blood 91(6):2152-6; Raja et
al., 1994, Br J Haematol 86(1):156-62; Popp et al., 1985,
445:432-44; and Skow et al., 1983, Cell 34(3): 1043-52).
Kidney Stones
[0510] Examples of animal models for kidney stones include, but are
not limited to, genetic hypercalciuric rats (see, e.g., Bushinsky
et al., 1999, Kidney Int 55(1):234-43 and Bushinsky et al., 1995,
Kidney Int 48(6):1705-13), chemically treated rats (see, e.g.,
Grases et al., 1998, Scand J Urol Nephrol 32(4):261-5; Burgess et
al., 1995, Urol Res 23(4):239-42; Kumar et al., 1991, J Urol
146(5):1384-9; Okada et al., 1985, Hinyokika Kiyo 31(4):565-77; and
Bluestone et al., 1975, Lab Invest 33(3):273-9), hyperoxaluria rats
(see, e.g., Jones et al., 1991, J Urol 145(4):868-74), pigs with
unilateral retrograde flexible nephroscopy (see, e.g., Seifmah et
al., 2001, 57(4):832-6), and rabbits with an obstructed upper
urinary tract (see, e.g., Itatani et at, 1979, Invest Urol
17(3):234-40).
Ataxia-Telangiectasia
[0511] Examples of animal models for ataxia-telangiectasia include,
but are not limited to, murine models of ataxia-telangiectasia
(see, e.g., Barlow et al., 1999, Proc Natl Acad Sci USA 96(17);
9915-9 and Inoue et al., 1986, Cancer Res 46(8):3979-82).
Lysosomal Storage Diseases
[0512] Examples of animal models for lysosomal storage diseases
include, but are not limited to, mouse models for
mucopolysaccharidosis type VII (see, e.g., Brooks et al., 2002,
Proc Natl Acad Sci USA. 99(9):6216-21; Monroy et al., 2002, Bone
30(2):352-9; Vogler et al., 2001, Pediatr Dev Pathol. 4(5):421-33;
Vogler et al., 2001, Pediatr Res. 49(3):342-8; and Wolfe et al.,
2000, Mol Ther. 2(6):552-6), a mouse model for metachromatic
leukodystrophy (see, e.g., Matzner et al., 2002, Gene Ther.
9(1):53-63), a mouse model of Sandhoff disease (see, e.g., Sango et
al., 2002, Neuropathol Appl Neurobiol. 28(1):23-34), mouse models
for mucopolysaccharidosis type III A (see, e.g., Bhattacharyya et
al., 2001, Glycobiology 11(1):99-10 and Bhaumik et al., 1999,
Glycobiology 9(12):1389-96.), arylsulfatase A (ASA)-deficient mice
(see, e.g., D'Hooge et al., 1999, Brain Res. 847(2):352-6 and
D'Hooge et at, 1999, Neurosci Lett. 273(2):93-6); mice with an
aspartylglucosaminuria mutation (see, e.g., Jalanko et al., 1998,
Hum Mol Genet. 7(2):265-72); feline models of mucopolysaccharidosis
type VI (see, e.g., Crawley et al., 1998, J Clin Invest.
101(1):109-19 and Norrdin et al., 1995, Bone 17(5):485-9); a feline
model of Niemann-Pick disease type C (see, e.g., March et al.,
1997, Acta Neuropathol (Berl). 94(2):164-72); acid
sphingomyelinase-deficient mice (see, e.g., Otterbach &
Stoffel, 1995, Cell 81(7):1053-6), and bovine mannosidosis (see,
e.g., Jolly et al., 1975, Birth Defects Orig Arctic Ser. 11
(6):273-8).
Tuberous Sclerosis
[0513] Examples of animal models for tuberous sclerosis ("TSC")
include, but are not limited to, a mouse model of TSC1 (see, e.g.,
Kwiatkowski et al, 2002, Hum Mol Genet. 11(5):525-34), a Tsc1 (TSC1
homologue) knockout mouse (see, e.g., Kobayashi et al., 2001, Proc
Natl Acad Sci USA. 2001 Jul. 17; 98(15); 8762-7), a TSC2 gene
mutant (Eker) rat model (see, e.g., Hino 2000, Nippon Rinsho
58(6):1255-61; Mizuguchi et al., 2000, J Neuropathol Exp Neurol.
59(3):188-9; and Hino et al., 1999, Prog Exp Tumor Res. 35:95-108);
and Tsc2(+/-) mice (see, e.g., Onda et al., 1999, J Clin Invest.
104(6):687-95).
Example 5: Mdx Mouse, an Animal Model Study
[0514] The mutation in the mdx mouse that causes premature
translation termination of the 427 kDa dystrophin polypeptide has
been shown to be a C to T transition at position 3185 in exon 23
(Sicinski et al., Science 244(4912):1578-1580 (1989)). Mouse
primary skeletal muscle cultures derived from 1-day old mdx mice
are prepared as described previously (Barton-Davis et al., J. Clin.
Invest. 104(4):375-381 (1999)). Cells are cultured for 10 days in
the presence of a compound of the invention. Culture medium is
replaced every four days and the presence of dystrophin in myoblast
cultures is detected by immunostaining as described previously
(Barton-Davis et al., J. Clin. Invest. 104(4):375-381 (1999)). A
primary monoclonal antibody to the C-terminus of the dystrophin
protein is used undiluted and rhodamine conjugated anti-mouse IgG
is used as the secondary antibody. The antibody detects the
full-length protein produced by suppression of the nonsense codon.
Staining is viewed using a Leica DMR microscope, digital camera,
and associated imaging software.
[0515] As previously described (Barton-Davis et al., J. Clin.
Invest. 104(4):375-381 (1999), compound is delivered by Alzet
osmotic pumps implanted under the skin of anesthetized mice. Two
doses of a compound of the invention are administered. Gentamicin
serves as a positive control and pumps filled with solvent only
serve as the negative control. Pumps are loaded with appropriate
compound such that the calculated doses to which tissue is exposed
are 10 mM and 20 mM. The gentamicin concentration is calculated to
achieve tissue exposure of approximately 200 mM. In the initial
experiment, mice are treated for 14 days, after which animals are
anesthetized with ketamine and exsanguinated. The tibialis anterior
(TA) muscle of the experimental animals is then excised, frozen,
and used for immunofluorescence analysis of dystrophin
incorporation into striated muscle. The presence of dystrophin in
TA muscles is detected by immunostaining, as described previously
(Barton-Davis et al., J. Clin. Invest. 104(4):375-381 (1999).
Western Blot Analysis
[0516] Quadricep muscles from an mdx mouse treated with a compound
of the present invention for 4 weeks are analyzed by western blot
using a commercially available antibody to dystrophin. Protein
extracted from the quadriceps of a wild-type mouse serve as a
positive control. Production of full-length dystrophin is observed
in the treated animal. The amount of full-length dystrophin
produced, as a result of nonsense suppression, but not limited by
this theory, is approximately 10% of wild-type levels of
expression.
Immunofluorescence
[0517] Male mdx mice (age 9-11 weeks) are treated with different
compounds of the present invention (n=2 at least for each
compound). These compounds are injected SQ once per day for two
weeks at 25 mg/kg. After 2 weeks of treatment, mice are sacrificed
for the removal of muscles to determine dystrophin readthrough
efficiency.
[0518] Immunofluorescence (IF) is performed on 10 .mu.m
cryosections using a dystrophin antibody. The antibody recognizes
an epitope C-terminal to the premature stop mutation found in mdx
mice. Image analysis is performed in an identical manner in all
sections. Images from treated and untreated mice are analyzed and a
signal greater than the signal on the untreated control is deemed
positive and indicates that suppression of the premature
termination codon in the dystrophin mRNA occurred.
Muscle Mechanics
[0519] Isolated whole muscle mechanics is performed on EDL muscles
from animals. Optimum muscle length (Lo) is defined as the length
that produced maximum twitch tension. Maximum tetanic force at to
is measured using a 120 Hz, 500 msec pulse at supramaximal voltage.
Protection against mechanical injury, induced by a series of 5
eccentric tetanic contractions, is monitored. These measurements
are performed using a 700 msec stimulation period during which the
muscle is held in an isometric contraction for the first 500 msec
followed by a stretch of 8 or 10% Lo at a rate of 0.5 Lo/sec.
Protection against mechanical injury is evaluated at 80 Hz
stimulation frequency. Damage is determined as the loss in force
between the first and last eccentric contraction. As shown in FIG.
4, treatment with compounds of the present invention result in
protection from damage induced by eccentric contractions of the EDL
muscle compared to the untreated control.
Example 6: Suppression of a Nonsense Mutation in the p53 Gene
[0520] For an animal model system, CAOV-3 cells (1.times.10.sup.7)
are injected into the flanks of nude/nude mice. After 12 days, mice
are randomized (10 mice per group) and treated subcutaneously (5
days per week) with 3 mg/kg of a compound of the present invention
or intraperitonealy (1 day per week) with 30 mg/kg of a compound of
the present invention. Tumor volumes are measured weekly.
Suppression of nonsense mutations in the p53 gene by a compound of
the present invention can inhibit cancer growth in vivo.
Example 7: Access to Specific Nucleotides of the 28S rRNA can be
Modified by Compounds of the Present Invention
[0521] Previous studies have demonstrated that gentamicin and other
members of the aminoglycoside family that decrease the fidelity of
translation bind to the A site of the 16S rRNA. By chemical
footprinting, UV cross-linking and NMR, gentamicin has been shown
to bind at the A site (comprised of nucleotides 1400-1410 and
1490-1500, E. coli numbering) of the rRNA at nucleotides 1406,
1407, 1494, and 1496 (Moazed & Noller, Nature 327(6121):389-394
(1978); Woodcock et al., EMBO J. 10(10):3099-3103 (1991); and
Schroeder et al., EMBO J. 19: 1-9 (2000).
[0522] Ribosomes prepared from HeLa cells are incubated with the
small molecules (at a concentration of 100 mM), followed by
treatment with chemical modifying agents (dimethyl sulfate [DMS]
and kethoxat [KE]). Following chemical modification, rRNA is
phenol-chloroform extracted, ethanol precipitated, analyzed in
primer extension reactions using end-labeled oligonucleotides
hybridizing to different regions of the three rRNAs and resolved on
6% polyacrylamide gels. Probes for primer extension cover the
entire 18S (7 oligonucleotide primers), 28S (24 oligonucleotide
primers), and 5S (one primer) rRNAs. Controls in these experiments
include DMSO (a control for changes in rRNA accessibility induced
by DMSO), paromomycin (a marker for 18S rRNA binding), and
anisomycin (a marker for 28S rRNA binding).
[0523] All publications and patent applications cited herein are
incorporated by reference to the same extent as if each individual
publication or patent application is specifically and individually
indicated to be incorporated by reference.
[0524] Although certain embodiments have been described in detail
above, those having ordinary skill in the art will clearly
understand that many modifications are possible in the embodiments
without departing from the teachings thereof. All such
modifications are intended to be encompassed within the claims of
the invention.
* * * * *