U.S. patent application number 15/443180 was filed with the patent office on 2017-08-24 for bifluorodioxalane-amino-benzimidazole kinase inhibitors for the treatment of cancer, autoimmuneinflammation and cns disorders.
This patent application is currently assigned to 4SC DISCOVERY GMBH. The applicant listed for this patent is 4SC DISCOVERY GMBH. Invention is credited to Johann LEBAN, Mirko ZAJA.
Application Number | 20170240556 15/443180 |
Document ID | / |
Family ID | 49782305 |
Filed Date | 2017-08-24 |
United States Patent
Application |
20170240556 |
Kind Code |
A1 |
LEBAN; Johann ; et
al. |
August 24, 2017 |
BIFLUORODIOXALANE-AMINO-BENZIMIDAZOLE KINASE INHIBITORS FOR THE
TREATMENT OF CANCER, AUTOIMMUNEINFLAMMATION AND CNS DISORDERS
Abstract
The invention relates to a compound of the general formula (I)
or a physiologically functional derivative, solvate or salt
thereof, ##STR00001## wherein A is a bond, alkyl or alkoxy
optionally substituted with one or more R'' as defined herein,
*--N(R''')CO--, *--CON(R''')--, *--N(R''')CON(R''')--, --S--,
--SO--, *--N(R''')--, *--N(R''')CO--, *--CON(R''')--, --CO--,
*--COO--, *--OOC--, *--SO.sub.2N(R''')--, --SO.sub.2, or
*--N(R''')--SO.sub.2--, wherein R''' is as defined herein and *
specifies the point of attachment to X; X is aryl, cycloalkyl,
aralkyl, heterocyclyl or heteroaryl, which may be substituted with
one or more R.sup.X further described herein; L is a bond or
*--N(R.sup.N)CO--, *--CON(R.sup.N)--, *--N(R.sup.N)--,
*--C.dbd.N(R.sup.N)--, *--N(R.sup.N)-alkyl-, *-alkyl-N(R.sup.N)--,
*--N(R.sup.N)CON(R.sup.N)--, *--CO--, *--CO.sub.2--, alkyl,
*-alkyl-O-alkyl-, *--NCO--CH.dbd.CH--, *--CH.dbd.CH--CONH--,
*--SO.sub.2N(R.sup.N)--, *--N(R.sup.N)SO.sub.2--, or heterocyclyl,
wherein * specifies the point of attachment to X; Y is H, alkyl,
aryl, aralkyl, cycloalkyl, heterocyclyl or heteroaryl, which may be
substituted with one or more R.sup.Y further described herein; and
R and R.sup.N are further described herein; as well as their use as
a medicament, a pharmaceutical composition comprising them, a
method of treatment or prevention of a medical condition entailing
the administration thereof, and the use thereof in the manufacture
of a medicament for the treatment or prevention of a medical
condition, particularly autoimmune inflammatory disorders, CNS
disorders, sleeping disorders, or proliferative diseases including
cancer. The invention further relates to a specific process for the
preparation of said compounds.
Inventors: |
LEBAN; Johann; (Wien,
AT) ; ZAJA; Mirko; (Muenchen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
4SC DISCOVERY GMBH |
Planegg-Martinsried |
|
DE |
|
|
Assignee: |
4SC DISCOVERY GMBH
Planegg-Martinsried
DE
|
Family ID: |
49782305 |
Appl. No.: |
15/443180 |
Filed: |
February 27, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14411795 |
Dec 29, 2014 |
9580438 |
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PCT/EP2013/063537 |
Jun 27, 2013 |
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15443180 |
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61664936 |
Jun 27, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 491/02 20130101;
C07D 491/056 20130101; A61P 35/00 20180101; A61P 29/00 20180101;
C07D 519/00 20130101; A61P 25/20 20180101; A61P 25/00 20180101;
A61P 43/00 20180101; A61P 37/02 20180101 |
International
Class: |
C07D 491/056 20060101
C07D491/056; C07D 519/00 20060101 C07D519/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 2, 2012 |
EP |
12174669.7 |
Claims
1. A compound of the general formula (I) or a physiologically
functional derivative, solvate or salt thereof, ##STR00225##
wherein R is independently selected from the group comprising H,
halogen, C.sub.1-6-alkyl, C.sub.1-6-haloalkyl,
C.sub.1-6-haloalkoxy, OH, C.sub.1-6-alkoxy, --S--R''', --SO--R''',
nitro, --N(R''').sub.2, --NH(R'''), --NHCO(R'''), --CONH.sub.2,
--CONH(R'''), --CO(R'''), --COH, --COO(R'''), --COOH,
--SO.sub.2NH.sub.2, --SO.sub.2NH(R'''), --SO.sub.2(R'''), and
--NH--SO.sub.2(R'''), wherein in the cases where said group R is
C.sub.1-6-alkyl, C.sub.1-6-haloalkyl, C.sub.1-6-haloalkoxy, or
C.sub.1-6-alkoxy, said group R may be substituted with one or more
substituents R'' independently selected from the group comprising
H, halogen, OH, nitro, --NH.sub.2, --N(C.sub.1-6-alkyl).sub.2,
--NH(C.sub.1-6-alkyl), --NHCO(C.sub.1-6-alkyl), --CONH.sub.2,
--CONH(C.sub.1-6-alkyl), --CO(C.sub.1-6-alkyl), --COH,
--COO(C.sub.1-6-alkyl), --COOH, and --CN, and wherein R''' is
independently selected from the group comprising H,
C.sub.1-6-alkyl, C.sub.1-6-haloalkyl, aryl, heteroaryl, cycloalkyl
and heterocyclyl; R.sup.N is independently selected from the group
comprising H, alkyl, haloalkyl, OH, aryl, heteroaryl, cycloalkyl,
heterocyclyl, --SO--R''', --NH.sub.2, --N(R''').sub.2, --NH(R'''),
--NHCO(R'''), --CONH.sub.2, --CONH(R'''), --CO(R'''), --COH,
--COO(R'''), --COOH, --SO.sub.2NH.sub.2, --SO.sub.2NH(R'''),
--SO.sub.2(R'''), and --NH--SO.sub.2(R'''), wherein R''' is as
defined above, wherein in the cases where said group R.sup.N is
alkyl, haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl,
said group R.sup.N may be substituted with one or more substituents
R''' as defined above; A is independently selected from
*--N(R.sup.a)CO--, *--CON(R.sup.a)--, *--SO.sub.2N(R.sup.a)--, and
*--N(R.sup.a)--SO.sub.2--, wherein R.sup.a is selected from H and
C.sub.1-4-alkyl, and wherein * specifies the point of attachment to
X; X is independently selected from the group comprising aryl,
cycloalkyl, aralkyl, heterocyclyl and heteroaryl, wherein said
group X may be substituted with one or more R.sup.X independently
selected from the group comprising halogen, C.sub.1-6-alkyl,
C.sub.1-6-haloalkyl, C.sub.1-6-haloalkoxy, OH, C.sub.1-6-alkoxy,
aryl, heteroaryl, cycloalkyl, heterocyclyl, --S--C.sub.1-6-alkyl,
--S--C.sub.1-6-haloalkyl, nitro, --NH.sub.2,
--N(C.sub.1-6-alkyl).sub.2, --NH(C.sub.1-6-alkyl),
--NHCO(C.sub.1-6-alkyl), --CONH.sub.2, --CONH(C.sub.1-6-alkyl),
--CO(C.sub.1-6-alkyl), --COH, --COO(C.sub.1-6-alkyl), --COOH,
--SO.sub.2NH.sub.2, --SO.sub.2NH(C.sub.1-6-alkyl),
--SO.sub.2(C.sub.1-6-alkyl), --NH--SO.sub.2(C.sub.1-6-alkyl),
C.sub.3-6-cycloalkyl, and --CN; L is independently a bond or a
linker group selected from the group comprising *--NHCO--,
*--CONH--, *--NH--, *--N(C.sub.1-4-alkyl)-,
*--C.dbd.N(C.sub.1-4-alkyl)-, *--NH--C.sub.1-4-alkyl-,
*--C.sub.1-4-alkyl-NH--, *--NHCONH--, *--CO--, *-SO.sub.2--,
C.sub.1-4-alkyl, *--C.sub.1-2-alkyl-O--C.sub.1-2-alkyl-,
*--NHCO--CH.dbd.CH--, *--CH.dbd.CH--CONH--, *--SO.sub.2NH--,
*--NHSO.sub.2--, and pyridinyl, wherein * specifies the point of
attachment to X; and Y is independently selected from the group
comprising H, alkyl, aryl, aralkyl, cycloalkyl, heterocyclyl and
heteroaryl, wherein said group Y may optionally be substituted with
one or more R.sup.Y independently selected from the group
comprising halogen, C.sub.1-6-alkyl, C.sub.1-6-haloalkyl,
C.sub.1-6-haloalkoxy, OH, C.sub.1-6-alkoxy, aryl, heteroaryl,
cycloalkyl, heterocyclyl, --S--C.sub.1-6-alkyl,
--S--C.sub.1-6-haloalkyl, nitro, --NH.sub.2,
--N(C.sub.1-6-alkyl).sub.2, --NH(C.sub.1-6-alkyl),
--NHCO(C.sub.1-6-alkyl), --CONH.sub.2, --CONH(C.sub.1-6-alkyl),
--CO(C.sub.1-6-alkyl), --COH, --COO(C.sub.1-6-alkyl), --COOH,
--SO.sub.2NH.sub.2, --SO.sub.2NH(C.sub.1-6-alkyl),
--SO.sub.2(C.sub.1-6-alkyl), --NH--SO.sub.2(C.sub.1-6-alkyl),
C.sub.1-6-alkyl-heterocyclyl, cycloalkyl and --CN.
2. A compound according to claim 1, which is a compound of the
general formula (Ia) or a physiologically functional derivative,
solvate or salt thereof, ##STR00226## wherein X is independently
selected from the group comprising aryl, cycloalkyl, aralkyl,
heterocyclyl and heteroaryl, wherein said group X may be
substituted with one or more R.sup.X independently selected from
the group comprising halogen, C.sub.1-6-alkyl, C.sub.1-6-haloalkyl,
C.sub.1-6-haloalkoxy, OH, C.sub.1-6-alkoxy, aryl, heteroaryl,
cycloalkyl, heterocyclyl, --S--C.sub.1-6-alkyl,
--S--C.sub.1-6-haloalkyl, nitro, --NH.sub.2,
--N(C.sub.1-6-alkyl).sub.2, --NH(C.sub.1-6-alkyl),
--NHCO(C.sub.1-6-alkyl), --CONH.sub.2, --CONH(C.sub.1-6-alkyl),
--CO(C.sub.1-6-alkyl), --COH, --COO(C.sub.1-6-alkyl), --COOH,
--SO.sub.2NH.sub.2, --SO.sub.2NH(C.sub.1-6-alkyl),
--SO.sub.2(C.sub.1-6-alkyl), --NH--SO.sub.2(C.sub.1-6-alkyl),
C.sub.3-6-cycloalkyl, and --CN; L is independently a bond or a
linker group selected from the group comprising *--NHCO--,
*--CONH--, *--NH--, *--N(C.sub.1-4-alkyl)-,
*--C.dbd.N(C.sub.1-4-alkyl)-, *--NH--C.sub.1-4-alkyl-,
*--C.sub.1-4-alkyl-NH--, *--NHCONH--, *--CO--, *--SO.sub.2--,
C.sub.1-4-alkyl, *--C.sub.1-2-alkyl-O--C.sub.1-2-alkyl-,
*--NHCO--CH.dbd.CH--, *--CH.dbd.CH--CONH--, *--SO.sub.2NH--,
*--NHSO.sub.2--, and pyridinyl, wherein * specifies the point of
attachment to X; and Y is independently selected from the group
comprising H, alkyl, aryl, aralkyl, cycloalkyl, heterocyclyl and
heteroaryl, wherein said group Y may optionally be substituted with
one or more R.sup.Y independently selected from the group
comprising halogen, C.sub.1-6-alkyl, C.sub.1-6-haloalkyl,
C.sub.1-6-haloalkoxy, OH, C.sub.1-6-alkoxy, aryl, heteroaryl,
cycloalkyl, heterocyclyl, --S--C.sub.1-6-alkyl,
--S--C.sub.1-6-haloalkyl, nitro, --NH.sub.2,
--N(C.sub.1-6-alkyl).sub.2, --NH(C.sub.1-6-alkyl),
--NHCO(C.sub.1-6-alkyl), --CONH.sub.2, --CONH(C.sub.1-6-alkyl),
--CO(C.sub.1-6-alkyl), --COH, --COO(C.sub.1-6-alkyl), --COOH,
--SO.sub.2NH.sub.2, --SO.sub.2NH(C.sub.1-6-alkyl),
--SO.sub.2(C.sub.1-6-alkyl), --NH--SO.sub.2(C.sub.1-6-alkyl),
C.sub.1-6-alkyl-heterocyclyl, cycloalkyl and --CN.
3. The compound according to claim 1 or a physiologically
functional derivative, solvate or salt thereof, wherein X is
independently selected from the group comprising aryl, aralkyl,
cycloalkyl, heterocyclyl and heteroaryl, wherein said group X may
be substituted with one or more R.sup.X independently selected from
the group comprising F, Cl, Br, I, C.sub.1-6-alkyl,
C.sub.1-6-haloalkyl, C.sub.1-6-haloalkoxy, OH, C.sub.1-6-alkoxy,
nitro, --NH.sub.2, --N(C.sub.1-6-alkyl).sub.2,
--NH(C.sub.1-6-alkyl), --NHCO(C.sub.1-6-alkyl), --CONH.sub.2,
--CONH(C.sub.1-6-alkyl), --SO.sub.2NH.sub.2,
--SO.sub.2NH(C.sub.1-6-alkyl), --SO.sub.2(C.sub.1-6-alkyl),
C.sub.3-6-cycloalkyl, --NH--SO.sub.2(C.sub.1-6-alkyl), --COOH,
--COO--C.sub.1-6-alkyl, and --CN; L is independently a bond or a
linker group selected from the group comprising *--NHCO--, *--NH--,
*--NHCH.sub.2--, *--NHCONH--, *--NHCO--CH.dbd.CH--,
*--NHSO.sub.2--, *--SO.sub.2--, and pyridinyl, wherein * specifies
the point of attachment to X; and Y is independently selected from
the group comprising H, aryl, cycloalkyl, heterocyclyl and
heteroaryl, wherein said group Y may optionally be substituted with
one or more R.sup.Y independently selected from the group
comprising F, Cl, Br, C.sub.1-6-alkyl, C.sub.1-6-haloalkyl,
C.sub.1-6-haloalkoxy, C.sub.1-6-alkoxy,
C.sub.1-6-alkyl-morpholinyl, and nitro.
4. The compound according to claim 1 or a physiologically
functional derivative, solvate or salt thereof, wherein X is
independently selected from the group comprising
1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1H-1,2,3-triazolyl,
1H-1,2,4-triazolyl, 1H-pyrazolyl, 1H-pyrrolyl, phenyl,
benzo[b]thiophenyl, cyclohexyl, furyl, isoxazolyl, oxazolyl,
imidazolyl, 1H-pyrazolyl, pyrazinyl, pyridyl, quinolinyl,
1-(naphthalen-2-yl)ethyl, thiazolyl, benzyl and thiophenyl, wherein
said group X may be substituted with one or more R.sup.X
independently selected from the group comprising F, Cl, Br, methyl,
tert-butyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy, OH,
acetyl, methylcarbamoyl, methoxy, nitro, --NH.sub.2, --NEt.sub.2,
--NMe.sub.2, --NHEt, --NHCOCH.sub.3, --CONH.sub.2,
--SO.sub.2NH.sub.2, --SO.sub.2Me, --NH--SO.sub.2Me, --COOH, and
--CN; L is independently a bond or a linker group selected from the
group comprising comprising *--NHCO--, *--NH--, *--NHCH.sub.2--,
*--NHCONH--, *--NHCO--CH.dbd.CH--, *-pyridinyl-, --SO.sub.2--, and
*--NHSO.sub.2--, wherein * specifies the point of attachment to X;
and Y is independently selected from the group comprising H,
phenyl, furyl, thiophenyl, pyridyl, pyrimidyl,
2,3-dihydrobenzo[b][1,4]dioxinyl, 2,3-dihydrobenzofuranyl,
benzo[d][1,3]dioxolyl, thieno[3,2-d]pyrimidinyl,
2-oxo-2,3-dihydrobenzoimidazolyl, pyrrolidinyl, tetrazolyl,
piperidinyl, pyrazolyl, 1,2,4-oxadiazolyl, 1,2,3-thiadiazolyl,
pyrrolyl, imidazolyl, isoxazolyl, thiazolyl, thiomorpholinyl, and
morpholinyl, wherein said group Y may be substituted with one or
two R.sup.Y independently selected from the group comprising F, Cl,
methyl, isopropyl, tert-butyl, trifluoromethyl, trifluoromethoxy,
methoxy, methylcarbamoyl, cyclopropyl, 2-morpholinoethyl, and
nitro.
5. The compound according to claim 1 or a physiologically
functional derivative, solvate or salt thereof, wherein X is
independently selected from the group comprising ##STR00227##
wherein * specifies the point of attachment to the central moiety,
# specifies the point of attachment to L and wherein the group X
may be substituted with one or more R.sup.X; L is independently a
bond or a linker group selected from the group comprising
*--NHCO--, *--NH--, *--NHCH.sub.2--, *--NHCONH--,
*--NHCO--CH.dbd.CH--, *-pyridinyl-, --SO.sub.2--, and
*--NHSO.sub.2--, wherein * specifies the point of attachment to X;
Y is independently H or selected from the group comprising
##STR00228## wherein * specifies the point of attachment to L and
wherein the group Y may be substituted with one or more R.sup.Y; or
wherein X is selected from the group comprising ##STR00229##
wherein * specifies the point of attachment to the central moiety,
wherein L is a bond, Y is H, and wherein the group X may be
substituted with one or more R.sup.X; wherein each R.sup.Y is
independently selected from the group comprising F, Cl, methyl,
isopropyl, tert-butyl, trifluoromethyl, trifluoromethoxy, methoxy,
methylcarbamoyl, cyclopropyl, 2-morpholinoethyl, and nitro; and
wherein each R.sup.X is independently selected from the group
comprising F, Cl, Br, methyl, tert-butyl, trifluoromethyl,
trifluoromethoxy, difluoromethoxy, OH, acetyl, methylcarbamoyl,
methoxy, nitro, --NH.sub.2, --NEt.sub.2, --NMe.sub.2, --NHEt,
--NHCOCH.sub.3, --CONH.sub.2, --SO.sub.2NH.sub.2, --SO.sub.2Me,
--NH--SO.sub.2Me, --COOH and --CN.
6. The compound according to claim 1, wherein said compound is
selected from one of the following: ##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## or a
physiologically functional derivative, solvate or salt thereof.
7. The compound according to claim 1, wherein said compound is
selected from one of the following: TABLE-US-00004 No. Structure 1
##STR00261##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-2-(2-(trifluoromethoxy)benzamido)thiazole-4-carboxamide 2
##STR00262## N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-1-(4-fluorophenyl)-5-methyl-1H-
pyrazole-4-carboxamide 3 ##STR00263##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(2,3-dihydrobenzofuran-5-
yl)thiazole-4-carboxamide 11 ##STR00264## N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-2-
(furan-2-carboxamido)thiazole-4-carboxamide 17 ##STR00265##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-5-(2-(trifluoromethoxy)benzamido)-
1,2,4-thiadiazole-3-carboxamide 18 ##STR00266##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)isonicotinamide 19 ##STR00267##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(2,5-
dimethoxyphenylsulfonamido)thiazole-4-carboxamide 20 ##STR00268##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)thiophene-3-carboxamide 21 ##STR00269##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(isonicotinamido)thiazole-4- carboxamide 26
##STR00270## N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(3-(2-
(trifluoromethoxy)phenyl)ureido)thiazole-4-carboxamide 29
##STR00271## 5-bromo-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)thiophene-3-carboxamide 30 ##STR00272##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-5-nitrothiophene-3-carboxamide 31 ##STR00273##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)benzo[b]thiophene-3-carboxamide 33 ##STR00274##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-2-methylfuran- 3-carboxamide 37
##STR00275## N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-morpholinothiazole-4-carboxamide 38 ##STR00276##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)-5-(2,3-dihydrobenzofuran-5-
yl)thiophene-3-carboxamide 41 ##STR00277##
3-chloro-N-(2,2-difluoro-5H-[1,3]dioxolo
[4',5':4,5]benzo[1,2-d]imidazol-6-yl)benzamide 48 ##STR00278##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(2-
(trifluoromethoxy)benzamido)oxazole-5-carboxamide 49 ##STR00279##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)benzamide 51 ##STR00280##
3-(2,4-dichlorobenzamido)-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-1H-
1,2,4-triazole-5-carboxamide 52 ##STR00281##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-(2-(trifluoromethoxy)benzamido)-
1H-1,2,4-triazole-5-carboxamide 56 ##STR00282##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-methoxybenzamide 57 ##STR00283##
5-(3-chlorobenzamido)-N-(2,2-difluoro-5H-[1,3]dioxolo
[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-1,2,4-thiadiazole-3-
carboxamide 60 ##STR00284## 4-amino-N-(2,2-difluoro-5H-[1,3]dioxolo
[4',5':4,5]benzo[1,2-d]imidazol-6-yl)benzamide 62 ##STR00285##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-4-sulfamoylbenzamide 65 ##STR00286##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-3-nitrobenzamide 66 ##STR00287##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-(trifluoromethyl)benzamide 69 ##STR00288##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-2-(4-methoxybenzamido)thiazole-4-carboxamide 70 ##STR00289##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-fluorobenzamide 75 ##STR00290##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-4-(dimethylamino)benzamide 76 ##STR00291##
4-(benzylamino)-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6- yl)benzamide 77
##STR00292## N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-4-(ethylamino)benzamide 78 ##STR00293##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)nicotinamide 80 ##STR00294##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)cyclohexanecarboxamide 81 ##STR00295##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)-2-(thiophen-3-yl)thiazole-4- carboxamide 82
##STR00296## N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(2,3-dihydrobenzo[b][1,4]dioxin-6-
yl)thiazole-4-carboxamide 83 ##STR00297##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)-5-sulfamoylthiophene-3- carboxamide 87
##STR00298## 88 ##STR00299##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-4-nitro-1H- pyrrole-2-carboxamide 91
##STR00300## 5-(4-chlorobenzamido)-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-
1,2,4-thiadiazole-3-carboxamide 92 ##STR00301##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-5-(4-fluorobenzamido)-1,2,4-
thiadiazole-3-carboxamide 94 ##STR00302##
N.sup.1-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-fluoroterephthalamide 100 ##STR00303##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-3- sulfamoylbenzamide 101 ##STR00304##
N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-4-sulfamoyl-1H-pyrrole-2-carboxamide 102 ##STR00305##
N-(2,2-difluoro-5H-[1,3]dioxolo
[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-3-
(dimethylamino)benzamide
104 ##STR00306## 4-acetyl-N-(2,2-difluoro-5H-[1,3]dioxolo
[4',5':4,5]benzo[1,2-d]imidazol-6-yl)- 1H-pyrrole-2-carboxamide 105
##STR00307## methyl 3-((2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)carbamoyl)benzoate 108 ##STR00308##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-3- (methylsulfonamido)benzamide 111
##STR00309## N.sup.1-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)terephthalamide 112 ##STR00310##
N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-
6-sulfamoylnicotinamide 113 ##STR00311##
N-(2,2-difluoro-5H-[1,3]dioxolo [4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-3-hydroxybenzamide 114 ##STR00312##
3-acetyl-N-(2,2-difluoro-5H-[1,3]dioxolo
[4',5':4,5]benzo[1,2-d]imidazol-6-yl) benzamide 116 ##STR00313##
3-cyano-N-(2,2-difluoro-5H-[1,3]dioxolo
[4',5':4,5]benzo[1,2-d]imidazol-6-yl) benzamide 117 ##STR00314##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-4-(morpholinosulfonyl)benzamide 118 ##STR00315##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-(1H-tetrazol-5-yl)benzamide 121 ##STR00316##
N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-3-(difluoromethoxy)benzamide 122 ##STR00317##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-3-(pyrrolidin-1- yl)benzamide 123
##STR00318## N-(2,2-difluoro-5H-[1,3]dioxolo
[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-5- methoxynicotinamide 124
##STR00319## N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-2- methoxyisonicotinamide 125
##STR00320## N-(2,2-difluoro-5H-[1,3]dioxolo
[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-2-
(dimethylamino)isonicotinamide 126 ##STR00321##
4-amino-N-(2,2-difluoro-5H-[1,3]dioxolo
[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-3- (trifluoromethyl)benzamide
128 ##STR00322## N-(2,2-difluoro-5H-[1,3]dioxolo
[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-3- (piperidin-1-yl)benzamide
130 ##STR00323## N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-3-(3,5-
dimethyl-1H-pyrazol-1-yl)benzamide 131 ##STR00324##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)-3-(5-isopropyl-1,2,4-
oxadiazol-3-yl)benzamide 132 ##STR00325##
3-(5-(tert-butyl)-1,2,4-oxadiazol-3-yl)-N-(2,2-
difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)benzamide 133 ##STR00326##
(S)-N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(6-methoxynaphthalen-2- yl)propanamide 134
##STR00327## N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)-2-(4-methyl-1,2,3-
thiadiazol-5-yl)thiazole-4-carboxamide 135 ##STR00328##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-[2,4'-bithiazole]- 4-carboxamide 136
##STR00329## N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-2-(thiophen-2- yl)oxazole-4-carboxamide
137 ##STR00330## N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)-3-(5-methyl-1,2,4- oxadiazol-3-yl)benzamide
138 ##STR00331## N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-5-(thiophen-2-
yl)isoxazole-3-carboxamide 139 ##STR00332## ethyl
1-(3-((2,2-difluoro-5H-[1,3]dioxolo
[4',5':4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)
phenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylate 140 ##STR00333##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-3-(1H- imidazol-2-yl)benzamide 141
##STR00334## N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-2-(5-
methylisoxazol-3-yl)thiazole-4-carboxamide 142 ##STR00335##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-4-(1H-pyrazol-1-yl)benzamide 144 ##STR00336##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-5-(1,3-dimethyl-1H-pyrazol-4-
yl)isoxazole-3-carboxamide 147 ##STR00337##
3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-N-(2,2-
difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)benzamide 148 ##STR00338##
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-3-(5-methyl-1H- tetrazol-1-yl)benzamide
149 ##STR00339## N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-3-(1H-tetrazol-1- yl)benzamide 1B
##STR00340## N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-3,5-dimethoxybenzamide 2B ##STR00341## N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-3-(thiazol-2-yl)benzamide 3B ##STR00342## N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-2-(3-methoxyphenyl)acetamide 4B ##STR00343##
N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-5-(furan-2-yl)nicotinamide 5B ##STR00344##
N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-3-(1H-pyrazol-3-yl)benzamide 6B ##STR00345##
N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-2-(furan-2-yl)thiazole-4-carboxamide 7B ##STR00346##
N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-5-(dimethylamino)nicotinamide 8B ##STR00347##
2-(3-chlorophenyl)-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2- d]imidazol-6-yl)acetamide 9B
##STR00348## 4-amino-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-methoxybenzamide 10B ##STR00349##
N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-5-
(thiophene-3-carboxamido)-1,2,4-thiadiazole-3-carboxamide 11B
##STR00350## N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-2-(thiophen-2-yl)thiazole-4-carboxamide 12B ##STR00351##
N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-2-(3-fluorophenyl)thiazole-4-carboxamide 13B ##STR00352##
N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-1-methyl-1H-pyrazole-4-carboxamide 14B ##STR00353##
N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-2-thiomorpholinoisonicotinamide 15B ##STR00354##
4-amino-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-1H-pyrrole-2-carboxamide 16B ##STR00355## N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-2-(furan-3-yl)thiazole-4-carboxamide 17B ##STR00356##
2-(3-chlorophenyl)-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)thiazole-4-carboxamide 22B ##STR00357##
N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-4-(1H-tetrazol-1-yl)benzamide 23B ##STR00358##
N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-5-(thiophen-2-yl)nicotinamide 25B ##STR00359##
N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-5-morpholinonicotinamide
or a physiologically functional derivative, solvate or salt
thereof.
8. The compound according to claim 1 or a physiologically
functional derivative, solvate or salt thereof for use as a
medicament.
9. The compound according to claim 1 or a physiologically
functional derivative, solvate or salt thereof for use in the
treatment or prevention of a medical condition selected from the
group comprising autoimmune inflammatory disorders, CNS disorders,
sleeping disorders, and proliferative diseases including
cancer.
10. A pharmaceutical composition comprising a compound according to
claim 1 or a physiologically functional derivative, solvate or salt
thereof and one or more pharmaceutically acceptable excipients.
11. A method of treatment or prevention of a medical condition
selected from the group comprising autoimmune inflammatory
disorders, CNS disorders, sleeping disorders, and proliferative
diseases including cancer, which comprises the administration of an
effective amount of a compound according to claim 1, or a
physiologically functional derivative, solvate or salt thereof to a
subject in need thereof.
12. Use of a compound according to claim 1, or a physiologically
functional derivative, solvate or salt thereof in the manufacture
of a medicament for the treatment or prevention of a medical
condition selected from the group comprising autoimmune
inflammatory disorders, CNS disorders, sleeping disorders, and
proliferative diseases including cancer.
13. Process for the preparation of a compound according to claim 1,
wherein A is an --CONH-- or --NHCO--, the process comprising the
step of coupling a compound of below Formula IV with a compound of
below formula II; ##STR00360## wherein Y, L and X are as defined
above, and wherein either R.sup.1 is NH.sub.2 and R.sup.2 is COOH
or COOCl, or wherein R.sup.2 is NH.sub.2 and R.sup.1 is COOH or
COOCl.
Description
BACKGROUND OF THE INVENTION
[0001] Casein kinase 1 (CK1) is a family of highly related,
constitutively active serine/threonine protein kinases (Christenson
E, De Maggio A J and Hockstra M F. (1997). Recent Results Cancer
Res. 143, 263-274.; Gross S D and Anderson R A. (1998). Cell Signal
10, 699-711).
[0002] CK1 is ubiquitously expressed in eukaryotes. Mammalian
family members comprise at least seven mammalian CK1 isoforms
(.alpha., .beta., .gamma.1, .gamma.2, .gamma.3, .delta. and
.delta.) and their various splice variants. (Fish K J, Cegielska A,
Getman M E, Landes G M and Virshup D M. (1995). J. Biol. Chem. 270,
14875-14883.; Graves P R, Haas D W, Hagedorn C H, De Paoli-Roach A
A and Roach P J. (1993). J. Biol. Chem. 268, 6394-6401.; Rowles J,
Slaughter C, Moomaw C, Hsu J and Cobb M H. (1991). Proc. Natl.
Acad. Sci. USA 88, 9548-9552.; Zhai L, Graves P R, Robinson L C,
Italiano M, Culbertson M R, Rowles J, Cobb M H, De Paoli-Roach A A
and Roach P J. (1995). J. Biol. Chem. 270, 12717-12724).
[0003] These isoforms share a high degree of similarity within
their protein kinase domains. For example CK1.delta. and
CK1.epsilon. are 98% identical in this region (Fish K J, Cegielska
A, Getman M E, Landes G M and Virshup D M. (1995). J. Biol. Chem.
270, 14875-14883.; Graves P R, Haas D W, Hagedorn C H, De
Paoli-Roach A A and Roach P J. (1993). J. Biol. Chem. 268,
6394-6401), but show considerable variation in the presence, length
and primary structure of the C-terminal non-catalytic domain
(Christenson E, De Maggio A J and Hockstra M F. (1997). Recent
Results Cancer Res. 143, 263-274). These variable C-terminal
domains are responsible for substrate specificity of the different
isoforms (Cegielska A, Gietzen K F, Rivers A and Virshup D M.
(1998). J. Biol. Chem. 273, 1357-1364.; Graves P R and Roach P J.
(1995). J. Bio. Chem. 270, 21689-21694.) and are involved in the
regulation of the interaction with other proteins, and/or
subcellular structures.
[0004] Autophosphorylation, (Cegielska A, Gietzen K F, Rivers A and
Virshup D M. (1998). J. Biol. Chem. 273, 1357-1364; and perhaps
dimerization in the case of CK1.delta. Longenecker K L, Roach P J
and Hurley T D. (1998). Acta Crystallogr. D. Biol. Crystallogr. 54,
473-475) are additional mechanisms that regulate CK1 activity,
specificity, and sub-cellular localization. The list of known
substrates of the CK1 family is still increasing, and so far
includes cytoskeleton proteins such as spectrin, troponin, myosin
and tau (Simkowski K W and Tao M. (1980) J. Biol. Chem. 255,
6456-6461; Singh T J, Akatsuka A, Blake K R and Huang K P. (1983).
Arch. Biochem. Biophys. 220, 615-622.; Singh T J, Grunke-Iqbal I
and Iqbal K. (1995). J. Neurochem. 64, 1420-1423; and
transcriptional components such as RNA polymerases I and II, SV40 T
antigen and CREM Dahmus M E. (1981). J. Biol. Chem. 256,
11239-11243.; de Groot R P, den Hertog J, Vandenhee de J R, Grois J
and Sassone-Corsi P. (1993). EMBO J, 12, 3903-3911.; Grasser F A,
Scheidtmann K H, Tuazon P T, Traugh J A and Walter G. (1988).
Virology 165, 13-22).
[0005] CK1 isoforms can influence the development of tumors in many
ways. Their ability to modulate p53 and Mdm2 functions through
site-directed phosphorylation, their function in centrosome and
spindle regulation, the opposite roles of CK1 isoforms in Wnt
signaling and their involvement in impeding apoptosis demonstrate
the potential role of CK1 family members in proliferative diseases
on multiple levels. Different isoforms seem to play an important
role in the development and progression of certain tumor types.
Therefore, the interest in targeting CK1 for drug development has
increased within the last 5 years. The role of the casein kinase 1
(CK1) family in different signaling pathways is linked to cancer
development (Uwe Knippschild et. al., Onkologie 2005; 28:508-514).
Further information on specific disorders connected with the casein
kinase 1 (CK1) family has been published in: Uwe Knippschild et
al., Cellular Signalling 17 (2005) 675-689.
[0006] The Wnt and Hedgehog pathways are involved in the regulation
of stem cell identity and the initiation and maintenance of many
tumor types. Dysregulation of these pathways play an important role
in cancer stem cell maintainence. Casein kinases 1.epsilon. and
1.delta. are mainly positive modulators of the Wnt and the Hh
pathways. Thus, selective inhibition of Casein kinases 1.epsilon.
and 1.delta. inhibit the proliferation and self renewal of cancer
stem cells.
[0007] Multiple members of the casein kinase I family of
serine/threonine protein kinases can have positive or negative
effects on individual regulatory elements of the Wnt and Hedgehog
pathway, which in summary leads to inhibition. These roles,
including recent results on Casein kinase 1 (CK1) phosphorylation
and activation of LRP6, CKI phosphorylation of Ci and mediation of
Ci-Slimb/.beta.-TrCP binding were revieved ("CKI, There's more than
one: casein kinase I family members in Wnt and Hedgehog signaling"
Price M A, Genes & Dev. 2006. 20: 399-410). Both the Wnt and Hh
signaling pathways are important in many developmental patterning
events.
[0008] Alzheimer's desease is an age-related disorder characterized
in part by the appearance of intracellular lesions composed of
filamentous aggregates of the microtubule-assiciated protein tau.
Abnormal tau phosphorylation accompanies tau aggregation and is
considered to be an upstream pathological event in this desease.
Enzymes implicated in tau hyperphosphorylation in Alzheimer's
desease include members of the casein kinase 1 family.
(Kannanayakal, T J et al. Nuerosci Lett. 2008; 432(2): 141-5.)
[0009] The circadian clock links our daily cycles of sleep and
activity to the external environment. Deregulation of the clock is
implicated in a number of human disorders, including depression,
seasonal affective disorder and metabolic disorders. Casein kinase
1 epsilon (CK1.epsilon.) and casein kinase 1 delta (CK1.delta.) are
Ser-Thr protein kinases, which are closely related to each other
and serve as key clock regulators. This was demonstrated by
mutations in CK1.delta. and CK1.epsilon. that dramatically alter
the circadian period. Therefore, inhibitors of CK1 have utility in
treating circadian disorders. (Walten, K. M. et al. JPET
330:430-439, 2009.)
[0010] Several publications describe casein kinase inhibitors:
Wager Travis et. al. Abstracts of Papers, 238.sup.th ACS National
Meeting, Washington, D.C., United States, Aug. 16-20, 2009; Oumata
Nassima et al. Journal of Medicinal Chemistry Volume 51 Issue 17
Pages 5229-5242 Journal 2008; Mashhoon N. et al. J Biol Chem 200;
275(26): 20052-60; Pfeifer C. et al. J Med Chem. 2009 Dec. 10;
52(23):7618-30.
[0011] In addition several patent applications regarding CK1.delta.
and/or .epsilon. inhibitors were published: US 2010/0179154 A1; US
2004/0110808 A1; US 2008/0027124 A1; US 2009/0099237 A1.
[0012] Several patents and patent applications described certain
specific acyl-amino-benzimidazoles with pharmacological activity:
EP 1 388 341 A1 and US 2004/0110808 A1; U.S. Pat. No. 7,132,438 B2;
WO 2007/064932 A2; DE 27 54 930 Al; US 2003/0144286.
BRIEF SUMMARY OF THE INVENTION
[0013] In one aspect, the present invention provides compounds of
Formula (I), and in further embodiments compounds of Formula (Ia),
and their respective physiologically functional derivatives or
salts, where the groups A, X, L, and Y are detailed further herein
below.
##STR00002##
[0014] In another aspect, the present invention provides methods
for preparation of compounds of Formulae (I) or (Ia),
physiologically functional derivatives or salts thereof, as
detailed further herein below.
[0015] In another aspect, the present invention provides methods
for the treatment or prevention of certain medical conditions, the
method comprising the administration of compounds of Formulae (I)
or (Ia), physiologically functional derivatives or salts thereof,
to a subject in need thereof, as detailed further herein below.
[0016] In another aspect, the present invention provides the use of
compounds of Formulae (I) or (Ia), physiologically functional
derivatives or salts thereof, in the manufacture of a medicament
for the treatment or prevention of certain medical conditions, as
detailed further herein below.
[0017] In another aspect, the present invention provides compounds
of Formulae (I) or (Ia), physiologically functional derivatives or
salts thereof, for use in the treatment or prevention of certain
medical conditions, as detailed further herein below.
[0018] In another aspect, the present invention provides
pharmaceutical compositions comprising compounds of Formulae (I) or
(Ia), physiologically functional derivatives or salts thereof and
one or more pharmaceutically acceptable excipients.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 shows the inhibition of anchorage independent growth
of colonies of the pancreatic cancer cell line PANC1 by the
compound of Example 1 (columns marked as"A"), compared with DMSO as
control. a) The compound of Example 1 reduces anchorage independent
growth of PANC1 colonies. b) The compound of Example 1 is
particularly potent in the inhibition of PANC1 macrocolonies, which
represent a subpopulation the colonies of a).
DETAILED DESCRIPTION OF THE INVENTION
[0020] Described herein is a compound of the general formula (I) or
a physiologically functional derivative, solvate or salt
thereof,
##STR00003##
wherein
[0021] R is independently selected from the group comprising H,
halogen, alkyl, haloalkyl, haloalkoxy, OH, alkoxy, aryl,
heteroaryl, cycloalkyl, heterocyclyl, --S--R''', --SO--R''', nitro,
--NH.sub.2, --N(R''').sub.2, --NH(R'''), --NHCO(R'''),
--CONH.sub.2, --CONH(R'''), --CO(R'''), --COH, --COO(R'''), --COOH,
--SO.sub.2NH.sub.2, --SO.sub.2NH(R'''), --SO.sub.2(R'''),
--NH--SO.sub.2(R'''), and --CNCOOR''', wherein in the cases where
said group R is alkyl, haloalkyl, haloalkoxy, alkoxy, aryl,
heteroaryl, cycloalkyl, or heterocyclyl, said group R may be
substituted with one or more substituents R'' independently
selected from the group comprising H, halogen, alkyl, haloalkyl,
haloalkoxy, OH, alkoxy, aryl, heteroaryl, cycloalkyl, heterocyclyl,
--S-alkyl, --S-haloalkyl, nitro, --NH.sub.2, --N(alkyl).sub.2,
--NH(alkyl), --NHCO(alkyl), --CONH.sub.2, --CONH(alkyl),
--CO(alkyl), --COH, --COO(alkyl), --COOH, --SO.sub.2NH.sub.2,
--SO.sub.2NH(alkyl), --SO.sub.2(alkyl), --NH--SO.sub.2(alkyl), and
--CN, and wherein R''' is independently selected from the group
comprising H, alkyl, haloalkyl, aryl, heteroaryl, cycloalkyl and
heterocyclyl;
[0022] R.sup.N is independently selected from the group comprising
H, alkyl, haloalkyl, OH, aryl, heteroaryl, cycloalkyl,
heterocyclyl, --SO--R''', --NH.sub.2, --N(R''').sub.2, --NH(R'''),
--NHCO(R'''), --CONH.sub.2, --CONH(R'''), --CO(R'''), --COH,
--COO(R'''), --COOH, --SO.sub.2NH.sub.2, --SO.sub.2NH(R'''),
--SO.sub.2(R'''), and --NH--SO.sub.2(R'''), wherein in the cases
where said group R.sup.N is alkyl, haloalkyl, aryl, heteroaryl,
cycloalkyl, or heterocyclyl, said group R.sup.N may be substituted
with one or more substituents R''' as defined above, and wherein
R''' is as defined above;
[0023] A is independently selected from the group comprising a
bond, alkyl optionally substituted with one or more substituents
R'' as defined above, alkoxy optionally substituted with one or
more substituents R'' as defined above, *--N(R''')CO--,
*--CON(R''')--, *--N(R''')CON(R''')--, --S--, --SO--, *--N(R''')--,
*--N(R''')CO--, *--CON(R''')--, --CO--, *--COO--, *--OOC--,
*--SO.sub.2N(R''')--, --SO.sub.2, and *--N(R''')--SO.sub.2--,
wherein R''' is as defined above and wherein * specifies the point
of attachment to X;
[0024] X is independently selected from the group comprising aryl,
cycloalkyl, aralkyl, heterocyclyl and heteroaryl, wherein said
group X may be substituted with one or more R.sup.X independently
selected from the group comprising halogen, alkyl, haloalkyl,
haloalkoxy, OH, alkoxy, aryl, heteroaryl, cycloalkyl, heterocyclyl,
--S--(R'''), nitro, --NH.sub.2, --N(R''').sub.2, --NH(R'''),
--NHCO(R'''), --CONH.sub.2, --CONH(R'''), --CO(R'''), --COH,
--COO(R'''), --COOH, --SO.sub.2NH.sub.2, --SO.sub.2NH(R'''),
--SO.sub.2(R'''), --NH--SO.sub.2(R'''), cycloalkyl, and --CN, and
wherein R''' is as defined above;
[0025] L is independently a bond or a linker group selected from
the group comprising *--N(R.sup.N)CO--, *--CON(R.sup.N)--,
*--N(R.sup.N)--, *--C.dbd.N(R.sup.N)--, *--N(R.sup.N)-alkyl-,
*-alkyl-N(R.sup.N)--, *--N(R.sup.N)CON(R.sup.N)--, *--CO--,
*--SO.sub.2--, alkyl, *-alkyl-O-alkyl-, *--NCO--CH.dbd.CH--,
*--CH.dbd.CH--CONH--, *--SO.sub.2N(R.sup.N)--,
*--N(R.sup.N)SO.sub.2--, and heterocyclyl, wherein * specifies the
point of attachment to X; and wherein R.sup.N is as defined
above;
[0026] and wherein
[0027] Y is independently selected from the group comprising H,
alkyl, aryl, aralkyl, cycloalkyl, heterocyclyl and heteroaryl,
wherein said group Y may optionally be substituted with one or more
R.sup.Y independently selected from the group comprising halogen,
alkyl, haloalkyl, haloalkoxy, OH, alkoxy, aryl, heteroaryl,
cycloalkyl, heterocyclyl, --S--(R'''), nitro, --NH.sub.2,
--N(R''').sub.2, --NH(R'''), --NHCO(R'''), --CONH.sub.2,
--CONH(R'''), --CO(R'''), --COH, --COO(R'''), --COOH,
--SO.sub.2NH.sub.2, --SO.sub.2NH(R'''), --SO.sub.2(R'''),
--NH--SO.sub.2(R'''), cycloalkyl, and --CN, and wherein R''' is as
defined above.
[0028] Embodiments of the present invention are enumerated in the
following:
[0029] 1. A compound of the general formula (I) or a
physiologically functional derivative, solvate or salt thereof,
##STR00004##
wherein
[0030] R is independently selected from the group comprising H,
halogen, C.sub.1-6-alkyl, C.sub.1-6-haloalkyl,
C.sub.1-6-haloalkoxy, OH, C.sub.1-6-alkoxy, --S--R''', --SO--R',
nitro, --N(R''').sub.2, --NH(R'''), --NHCO(R'''), --CONH.sub.2,
--CONH(R'''), --CO(R'''), --COH, --COO(R'''), --COOH,
--SO.sub.2NH.sub.2, --SO.sub.2NH(R'''), --SO.sub.2(R'''), and
--NH--SO.sub.2(R'''),
[0031] wherein in the cases where said group R is C.sub.1-6-alkyl,
C.sub.1-6-haloalkyl, C.sub.1-6-haloalkoxy, or C.sub.1-6-alkoxy,
said group R may be substituted with one or more substituents R''
independently selected from the group comprising H, halogen, OH,
nitro, --NH.sub.2, --N(C.sub.1-6-alkyl).sub.2,
--NH(C.sub.1-6-alkyl), --NHCO(C.sub.1-6-alkyl), --CONH.sub.2,
--CONH(C.sub.1-6-alkyl), --CO(C.sub.1-6-alkyl), --COH,
--COO(C.sub.1-6-alkyl), --COOH, and --CN,
[0032] and wherein R''' is independently selected from the group
comprising H, C.sub.1-6-alkyl, C.sub.1-6-haloalkyl, aryl,
heteroaryl, cycloalkyl and heterocyclyl;
[0033] R.sup.N is independently selected from the group comprising
H, alkyl, haloalkyl, OH, aryl, heteroaryl, cycloalkyl,
heterocyclyl, --SO--R''', --NH.sub.2, --N(R''').sub.2, --NH(R'''),
--NHCO(R'''), --CONH.sub.2, --CONH(R'''), --CO(R'''), --COH,
--COO(R'''), --COOH, --SO.sub.2NH.sub.2, --SO.sub.2NH(R'''),
--SO.sub.2(R'''), and --NH--SO.sub.2(R'''), wherein R''' is as
defined above,
[0034] wherein in the cases where said group R.sup.N is alkyl,
haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl, said
group R.sup.N may be substituted with one or more substituents R'''
as defined above;
[0035] A is independently selected from *--N(R.sup.a)CO--,
*--CON(R.sup.a)--, *--SO.sub.2N(R.sup.a)--, and
*--N(R.sup.a)--SO.sub.2--,
[0036] wherein R.sup.1 is selected from H and C.sub.1-4-alkyl,
[0037] and wherein * specifies the point of attachment to X; X is
independently selected from the group comprising aryl, cycloalkyl,
aralkyl, heterocyclyl and heteroaryl, wherein said group X may be
substituted with one or more R.sup.X independently selected from
the group comprising halogen, C.sub.1-6-alkyl, C.sub.1-6-haloalkyl,
C.sub.1-6-haloalkoxy, OH, C.sub.1-6-alkoxy, aryl, heteroaryl,
cycloalkyl, heterocyclyl, --S--C.sub.1-6-haloalkyl,
--S--C.sub.1-6-haloalkyl, nitro, --NH.sub.2,
--N(C.sub.1-6-alkyl).sub.2, --NH(C.sub.1-6-alkyl),
--NHCO(C.sub.1-6-alkyl), --CONH.sub.2, --CONH(C.sub.1-6-alkyl),
--CO(C.sub.1-6-alkyl), --COH, --COO(C.sub.1-6-alkyl), --COOH,
--SO.sub.2NH.sub.2, --SO.sub.2NH(C.sub.1-6-alkyl),
--SO.sub.2(C.sub.1-6-alkyl), --NH--SO.sub.2(C.sub.1-6-alkyl),
C.sub.3-6-cycloalkyl, and --CN;
[0038] L is independently a bond or a linker group selected from
the group comprising *--NHCO--, *--CONH--, *--NH--,
*--N(C.sub.1-4-alkyl)-, *--C.dbd.N(C.sub.1-4-alkyl)-,
*--NH--C.sub.1-4-alkyl-, *--C.sub.1-4-alkyl-NH--, *--NHCONH--,
*--CO--, *--SO.sub.2--, C.sub.1-2-alkyl-O--C.sub.1-2-alkyl-,
*--NHCO--CH.dbd.CH--, *--CH.dbd.CH--CONH--, *--SO.sub.2NH--,
*--NHSO.sub.2--, and pyridinyl, wherein * specifies the point of
attachment to X; and
[0039] Y is independently selected from the group comprising H,
alkyl, aryl, aralkyl, cycloalkyl, heterocyclyl and heteroaryl,
wherein said group Y may optionally be substituted with one or more
R.sup.Y independently selected from the group comprising halogen,
C.sub.1-6-alkyl, C.sub.1-6-haloalkyl, C.sub.1-6-haloalkoxy, OH,
C.sub.1-6-alkoxy, aryl, heteroaryl, cycloalkyl, heterocyclyl,
--S--C.sub.1-6-alkyl, --S--C.sub.1-6-haloalkyl, nitro, --NH.sub.2,
--N(C.sub.1-6-alkyl).sub.2, --NH(C.sub.1-6-alkyl),
--NHCO(C.sub.1-6-alkyl), --CONH.sub.2, --CONH(C.sub.1-6-alkyl),
--CO(C.sub.1-6-alkyl), --COH, --COO(C.sub.1-6-alkyl), --COOH,
--SO.sub.2NH.sub.2, --SO.sub.2NH(C.sub.1-6-alkyl),
--SO.sub.2(C.sub.1-6-alkyl), --NH--SO.sub.2(C.sub.1-6-alkyl),
C.sub.1-6-alkyl-heterocyclyl, cycloalkyl and --CN.
[0040] 2. A compound according to the present invention, in
particular according to above item 1, which is a compound of the
general formula (Ia) or a physiologically functional derivative,
solvate or salt thereof,
##STR00005##
wherein
[0041] X is independently selected from the group comprising aryl,
cycloalkyl, aralkyl, heterocyclyl and heteroaryl, wherein said
group X may be substituted with one or more R.sup.X independently
selected from the group comprising halogen, C.sub.1-6-alkyl,
C.sub.1-6-haloalkyl, C.sub.1-6-haloalkoxy, OH, C.sub.1-6-alkoxy,
aryl, heteroaryl, cycloalkyl, heterocyclyl, --S--C.sub.1-6-alkyl,
--S--C.sub.1-6-haloalkyl, nitro, --NH.sub.2,
--N(C.sub.1-6-alkyl).sub.2, --NH(C.sub.1-6-alkyl),
--NHCO(C.sub.1-6-alkyl), --CONH.sub.2, --CONH(C.sub.1-6-alkyl),
--CO(C.sub.1-6-alkyl), --COH, --COO(C.sub.1-6-alkyl), --COOH,
--SO.sub.2NH.sub.2, --SO.sub.2NH(C.sub.1-6-alkyl),
--SO.sub.2(C.sub.1-6-alkyl), --NH--SO.sub.2(C.sub.1-6-alkyl),
C.sub.3-6-cycloalkyl, and --CN;
[0042] L is independently a bond or a linker group selected from
the group comprising *--NHCO--, *--CONH--, *--NH--,
*--N(C.sub.1-4-alkyl)-, *--C.dbd.N(C.sub.1-4-alkyl)-,
*--NH--C.sub.1-4-alkyl-, *--C.sub.1-4-alkyl-NH--, *--NHCONH--,
*--CO--, *--SO.sub.2--, C.sub.1-4-alkyl,
*--C.sub.1-2-alkyl-O--C.sub.1-2-alkyl-, *--NHCO--CH.dbd.CH--,
*--CH.dbd.CH--CONH--, *--SO.sub.2NH--, *--NHSO.sub.2--, and
pyridinyl, wherein * specifies the point of attachment to X;
and
[0043] Y is independently selected from the group comprising H,
alkyl, aryl, aralkyl, cycloalkyl, heterocyclyl and heteroaryl,
wherein said group Y may optionally be substituted with one or more
R.sup.Y independently selected from the group comprising halogen,
C.sub.1-6-alkyl, C.sub.1-6-haloalkyl, C.sub.1-6-haloalkoxy, OH,
C.sub.1-6-alkoxy, aryl, heteroaryl, cycloalkyl, heterocyclyl,
--S--C.sub.1-6-alkyl, --S--C.sub.1-6-haloalkyl, nitro, --NH.sub.2,
--N(C.sub.1-6-alkyl).sub.2, --NH(C.sub.1-6-alkyl),
--NHCO(C.sub.1-6-alkyl), --CONH.sub.2, --CONH(C.sub.1-6-alkyl),
--CO(C.sub.1-6-alkyl), --COH, --COO(C.sub.1-6-alkyl), --COOH,
--SO.sub.2NH.sub.2, --SO.sub.2NH(C.sub.1-6-alkyl),
--SO.sub.2(C.sub.1-6-alkyl), --NH--SO.sub.2(C.sub.1-6-alkyl),
C.sub.1-6-alkyl-heterocyclyl, cycloalkyl and -CN.
[0044] 3. The compound according to the present invention, in
particular according to above item 1 or 2 or a physiologically
functional derivative, solvate or salt thereof, wherein
[0045] X is independently selected from the group comprising aryl,
cycloalkyl, heterocyclyl and heteroaryl, wherein said group X may
be substituted with one or more R.sup.X independently selected from
the group comprising F, Cl, Br, I, C.sub.1-6-alkyl,
C.sub.1-6-haloalkyl, C.sub.1-6-haloalkoxy, OH, C.sub.1-6-alkoxy,
nitro, --NH.sub.2, --N(C.sub.1-6-alkyl).sub.2,
--NH(C.sub.1-6-alkyl), --NHCO(C.sub.1-6-alkyl), --CONH.sub.2,
--CONH(C.sub.1-6-alkyl), --SO.sub.2NH.sub.2,
--SO.sub.2NH(C.sub.1-6-alkyl), --SO.sub.2(C.sub.1-6-alkyl),
C.sub.3-6-cycloalkyl, --NH--SO.sub.2(C.sub.1-6-alkyl),
--COO--C.sub.1-6-alkyl, and --CN;
[0046] L is independently a bond or a linker group selected from
the group comprising *--NHCO--, *--NH--, *--NHCH.sub.2--,
*--NHCONH--, *--NHCO--CH.dbd.CH--, *--NHSO.sub.2--, *--SO.sub.2--,
and pyridinyl, wherein * specifies the point of attachment to X;
and
[0047] Y is independently selected from the group comprising H,
aryl, cycloalkyl, heterocyclyl and heteroaryl, wherein said group Y
may optionally be substituted with one or more R.sup.Y
independently selected from the group comprising F, Cl, Br,
C.sub.1-6-alkyl, C.sub.1-6-haloalkyl, C.sub.1-6-haloalkoxy,
C.sub.1-6-alkoxy, C.sub.1-6-alkyl-morpholinyl, and nitro.
[0048] 4. The compound according to the present invention, in
particular according to any of the above items 1 to 3 or a
physiologically functional derivative, solvate or salt thereof,
wherein
[0049] X is independently selected from the group comprising aryl,
aralkyl, cycloalkyl, heterocyclyl and heteroaryl, wherein said
group X may be substituted with one or more R.sup.X independently
selected from the group comprising F, Cl, Br, I, C.sub.1-6-alkyl,
C.sub.1-6-haloalkyl, C.sub.1-6-haloalkoxy, OH, C.sub.1-6-alkoxy,
nitro, --NH.sub.2, --N(C.sub.1-6-alkyl).sub.2,
--NH(C.sub.1-6-alkyl), --NHCO(C.sub.1-6-alkyl), --CONH.sub.2,
--CONH(C.sub.1-6-alkyl), --SO.sub.2NH.sub.2,
--SO.sub.2NH(C.sub.1-6-alkyl), --SO.sub.2(C.sub.1-6-alkyl),
C.sub.3-6-cycloalkyl, --NH--SO.sub.2(C.sub.1-6-alkyl),
--COO--C.sub.1-6-alkyl, --COOH, and --CN;
[0050] L is independently a bond or a linker group selected from
the group comprising *--NHCO--, *--NH--, *--NHCH.sub.2--,
*--NHCONH--, *--NHCO--CH.dbd.CH--, *--NHSO.sub.2--, *--SO.sub.2--,
and pyridinyl, wherein * specifies the point of attachment to X;
and
[0051] Y is independently selected from the group comprising H,
aryl, cycloalkyl, heterocyclyl and heteroaryl, wherein said group Y
may optionally be substituted with one or more R.sup.Y
independently selected from the group comprising F, Cl, Br,
C.sub.1-6-alkyl, C.sub.1-6-haloalkyl, C.sub.1-6-haloalkoxy,
C.sub.1-6-alkoxy, C.sub.1-6-alkyl-morpholinyl, and nitro.
[0052] 5. The compound according to the present invention, in
particular according to any of the above items 1 to 4 or a
physiologically functional derivative, solvate or salt thereof,
wherein
[0053] X is independently selected from the group comprising
1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1H-1,2,3-triazolyl,
1H-1,2,4-triazolyl, 1H-pyrazolyl, 1H-pyrrolyl, phenyl,
benzo[b]thiophenyl, cyclohexyl, furyl, isoxazolyl, oxazolyl,
imidazolyl, 1H-pyrazolyl, pyrazinyl, pyridyl, quinolinyl,
1-(naphthalen-2-yl)ethyl, thiazolyl and thiophenyl, wherein said
group X may be substituted with one or more R.sup.X independently
selected from the group comprising F, Cl, Br, methyl, tert-butyl,
trifluoromethyl, trifluoromethoxy, difluoromethoxy, OH, acetyl,
methylcarbamoyl, methoxy, nitro, --NH.sub.2, --NEt.sub.2,
--NMe.sub.2, --NHEt, --NHCOCH.sub.3, --CONH.sub.2,
--SO.sub.2NH.sub.2, --SO.sub.2Me, --NH--SO.sub.2Me, and --CN;
[0054] L is independently a bond or a linker group selected from
the group comprising comprising *--NHCO--, *--NH--,
*--NHCH.sub.2--, *--NHCONH--, *--NHCO--CH.dbd.CH--, *-pyridinyl-,
--SO.sub.2--, and *--NHSO.sub.2--, wherein * specifies the point of
attachment to X; and
[0055] Y is independently selected from the group comprising H,
phenyl, furyl, thiophenyl, pyridyl, pyrimidyl,
2,3-dihydrobenzo[b][1,4]dioxinyl, 2,3-dihydrobenzofuranyl,
benzo[d][1,3]dioxolyl, thieno[3,2-d]pyrimidinyl,
2-oxo-2,3-dihydrobenzoimidazolyl, pyrrolidinyl, tetrazolyl,
piperidinyl, pyrazolyl, 1,2,4-oxadiazolyl, 1,2,3-thiadiazolyl,
pyrrolyl, imidazolyl, isoxazolyl, thiazolyl, and morpholinyl,
wherein said group Y may be substituted with one or two R.sup.Y
independently selected from the group comprising F, Cl, methyl,
isopropyl, tert-butyl, trifluoromethyl, trifluoromethoxy, methoxy,
methylcarbamoyl, cyclopropyl, 2-morpholinoethyl, and nitro.
[0056] 6. The compound according to the present invention, in
particular according to any of the above items 1 to 5 or a
physiologically functional derivative, solvate or salt thereof,
wherein
[0057] X is independently selected from the group comprising
1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1H-1,2,3-triazolyl,
1H-1,2,4-triazolyl, 1H-pyrazolyl, 1H-pyrrolyl, phenyl,
benzo[b]thiophenyl, cyclohexyl, furyl, isoxazolyl, oxazolyl,
imidazolyl, 1H-pyrazolyl, pyrazinyl, pyridyl, quinolinyl,
1-(naphthalen-2-yl)ethyl, thiazolyl, benzyl and thiophenyl, wherein
said group X may be substituted with one or more R.sup.X
independently selected from the group comprising F, Cl, Br, methyl,
tert-butyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy, OH,
acetyl, methylcarbamoyl, methoxy, nitro, --NH.sub.2, --NEt.sub.2,
--NMe.sub.2, --NHEt, --NHCOCH.sub.3, --CONH.sub.2,
--SO.sub.2NH.sub.2, --SO.sub.2Me, --NH--SO.sub.2Me, --COOH, and
--CN;
[0058] L is independently a bond or a linker group selected from
the group comprising comprising *--NHCO--, *--NH--,
*--NHCH.sub.2--, *--NHCONH--, *--NHCO--CH.dbd.CH--, *-pyridinyl-,
--SO.sub.2--, and *--NHSO.sub.2--, wherein * specifies the point of
attachment to X; and
[0059] Y is independently selected from the group comprising H,
phenyl, furyl, thiophenyl, pyridyl, pyrimidyl,
2,3-dihydrobenzo[b][1,4]dioxinyl, 2,3-dihydrobenzofuranyl,
benzo[d][1,3]dioxolyl, thieno[3,2-d]pyrimidinyl,
2-oxo-2,3-dihydrobenzoimidazolyl, pyrrolidinyl, tetrazolyl,
piperidinyl, pyrazolyl, 1,2,4-oxadiazolyl, 1,2,3-thiadiazolyl,
pyrrolyl, imidazolyl, isoxazolyl, thiazolyl, thiomorpholinyl, and
morpholinyl, wherein said group Y may be substituted with one or
two R.sup.Y independently selected from the group comprising F, Cl,
methyl, isopropyl, tert-butyl, trifluoromethyl, trifluoromethoxy,
methoxy, methylcarbamoyl, cyclopropyl, 2-morpholinoethyl, and
nitro.
[0060] 7. The compound according to the present invention, in
particular according to any of the above items 1 to 6 or a
physiologically functional derivative, solvate or salt thereof,
wherein
[0061] X is independently selected from the group comprising
##STR00006##
[0062] wherein * specifies the point of attachment to the central
moiety, # specifies the point of attachment to L and wherein the
group X may be substituted with one or more R.sup.X;
[0063] L is independently a bond or a linker group selected from
the group comprising *--NHCO--, *--NH--, *--NHCH.sub.2--,
*--NHCONH--, *--NHCO--CH.dbd.CH--, *-pyridinyl-, --SO.sub.2--, and
*--NHSO.sub.2--, wherein * specifies the point of attachment to
X;
[0064] Y is independently H or selected from the group
comprising
##STR00007##
[0065] wherein * specifies the point of attachment to L and wherein
the group Y may be substituted with one or more R.sup.Y; or
[0066] wherein X is selected from the group comprising
##STR00008##
[0067] wherein * specifies the point of attachment to the central
moiety, wherein L is a bond, Y is H, and wherein the group X may be
substituted with one or more R.sup.X;
[0068] wherein each R.sup.Y is independently selected from the
group comprising F, Cl, methyl, isopropyl, tert-butyl,
trifluoromethyl, trifluoromethoxy, methoxy, methylcarbamoyl,
cyclopropyl, 2-morpholinoethyl, and nitro; and
[0069] wherein each R.sup.X is independently selected from the
group comprising F, Cl, Br, methyl, tert-butyl, trifluoromethyl,
trifluoromethoxy, difluoromethoxy, OH, acetyl, methylcarbamoyl,
methoxy, nitro, --NH.sub.2, --NEt.sub.2, --NMe.sub.2, --NHEt,
--NHCOCH.sub.3, --CONH.sub.2, --SO.sub.2NH.sub.2, --SO.sub.2Me,
--NH--SO.sub.2Me, and --CN.
[0070] 8. The compound according to the present invention, in
particular according to any of the above items 1 to 7 or a
physiologically functional derivative, solvate or salt thereof,
wherein
[0071] X is independently selected from the group comprising
##STR00009##
[0072] wherein * specifies the point of attachment to the central
moiety, # specifies the point of attachment to L and wherein the
group X may be substituted with one or more R.sup.X;
[0073] L is independently a bond or a linker group selected from
the group comprising *--NHCO--, *--NH--, *--NHCH.sub.2--,
*--NHCONH--, *--NHCO--CH.dbd.CH--, *-pyridinyl-, --SO.sub.2--, and
*--NHSO.sub.2--, wherein * specifies the point of attachment to
X;
[0074] Y is independently H or selected from the group
comprising
##STR00010##
[0075] wherein * specifies the point of attachment to L and wherein
the group Y may be substituted with one or more R.sup.Y; or
[0076] wherein X is selected from the group comprising
##STR00011##
[0077] wherein * specifies the point of attachment to the central
moiety, wherein L is a bond, Y is H, and wherein the group X may be
substituted with one or more R.sup.X;
[0078] wherein each R.sup.Y is independently selected from the
group comprising F, Cl, methyl, isopropyl, tert-butyl,
trifluoromethyl, trifluoromethoxy, methoxy, methylcarbamoyl,
cyclopropyl, 2-morpholinoethyl, and nitro; and
[0079] wherein each R.sup.X is independently selected from the
group comprising F, Cl, Br, methyl, tert-butyl, trifluoromethyl,
trifluoromethoxy, difluoromethoxy, OH, acetyl, methylcarbamoyl,
methoxy, nitro, --NH.sub.2, --NEt.sub.2, --NMe.sub.2, --NHEt,
--NHCOCH.sub.3, --CONH.sub.2, --SO.sub.2NH.sub.2, --SO.sub.2Me,
--NH--SO.sub.2Me, --COOH and --CN.
[0080] In certain embodiments of the present invention, Y is a
phenyl group optionally substituted with one or more R.sup.Y as
defined herein, more particularly a phenyl group substituted with
one or more R.sup.Y as defined herein, wherein at least one of the
substituents R.sup.Y is located in the meta of the phenyl group
position, wherein the ortho positions of the phenyl group are H,
and wherein said ortho and meta positions are in relation to the
point of attachment to L, or in the case where L is a bond, the
point of attachment to X, respectively.
[0081] In certain embodiments of the present invention, R.sup.X is
in each occurrence independently selected from the group comprising
alkyl, alkylsulfonyl, halogen, haloalkyl, hydroxy, amino,
alkylamino, dialkylamino, benzylamino, nitro, alkoxy, haloalkoxy
and cyano, more particularly selected from the group comprising
methyl, methylsulfonyl, fluorine, chlorine, bromine,
trifluoromethyl, hydroxyl, amino, dimethylamino, ethylamino,
diethylamino, benzylamino, nitro, methoxy, trifluoromethoxy and
cyano.
[0082] In certain embodiments of the present invention, L
independently is a bond or selected from the group comprising
*--NHCO--, *--NH SO.sub.2--, *--SO.sub.2--, *--CONH--, *--NH--,
--NHCONH--, and *--SO.sub.2NH--, particularly *--NHCO--, *--NH,
*--NHCONH--, and *--NHSO.sub.2--, wherein * specifies the point of
attachment to X. More particularly, L independently is *--NHCO,
wherein * specifies the point of attachment to X.
[0083] In certain embodiments of the present invention, R.sup.Y is
in each occurrence independently selected from the group comprising
alkyl, halogen, haloalkyl, alkoxy, haloalkoxy and nitro, more
particularly selected from the group comprising methyl, chlorine,
fluorine, methoxy, trifluoromethoxy and nitro.
[0084] Particular compounds of the present invention are those
having an IC.sub.50 on cancer cell growth inhibition of 1 .mu.M or
lower, more particularly 100 nM or lower. Said IC.sub.50 is
particularly determined as described herein below in the exemplary
section (under "9. Determination of proliferation inhibition on a
panel of cancer cell lines; Proliferation Assay"), and particular
cancer types in this context are the ones described therein.
[0085] Furthermore, particular compounds of the present invention
are those having an IC.sub.50 on CK1 delta and/or epsilon
inhibition of 1 .mu.M or lower, even more particularly 200 nM or
lower. Said IC.sub.50 is particularly determined as described
herein below in the exemplary section (under "8. Determination of
the inhibitory capacity; Kinase Assay"--Data shown in tables 1 and
2).
[0086] 9. The compound according to the present invention, in
particular according to any of the above items 1 to 8, wherein said
compound is selected from one of the compounds 1 to 149 enumerated
in the example section, or a physiologically functional derivative,
solvate or salt thereof.
[0087] 10. The compound according to the present invention, in
particular according to any of the above items 1 to 9, wherein said
compound is selected from one of the compounds 1 to 149 or 1B to
26B enumerated in the example section, or a physiologically
functional derivative, solvate or salt thereof.
[0088] 11. The compound according to the present invention, in
particular according to any of the above items 1 to 10, wherein
said compound is selected from one of the following compounds as
enumerated in the example section: 1, 2, 3, 11, 17, 18, 19, 20, 21,
26, 29, 30, 31, 33, 37, 38, 41, 48, 49, 51, 52, 56, 57, 60, 62, 65,
66, 69, 70, 75, 76, 77, 78, 80, 81, 82, 83, 87, 88, 91, 92, 94,
100, 101, 102, 104, 105, 108, 111, 112, 113, 114, 116, 117, 118,
121, 122, 123, 124, 125, 126, 128, 130, 131, 132, 133, 134, 135,
136, 137, 138, 139, 140, 141, 142, 144, 147, 148, and 149,
[0089] or a physiologically functional derivative, solvate or salt
thereof.
[0090] 12. The compound according to the present invention, in
particular according to any of the above items 1 to 11, wherein
said compound is selected from one of the following compounds as
enumerated in the example section: 1, 2, 3, 11, 17, 18, 19, 20, 21,
26, 29, 30, 31, 33, 37, 38, 41, 48, 49, 51, 52, 56, 57, 60, 62, 65,
66, 69, 70, 75, 76, 77, 78, 80, 81, 82, 83, 87, 88, 91, 92, 94,
100, 101, 102, 104, 105, 108, 111, 112, 113, 114, 116, 117, 118,
121, 122, 123, 124, 125, 126, 128, 130, 131, 132, 133, 134, 135,
136, 137, 138, 139, 140, 141, 142, 144, 147, 148, and 149, 1B, 2B,
3B, 4B, 5B, 6B, 7B, 8B, 9B, 10B, 11B, 12B, 13B, 14B, 15B, 16B, 17B,
22B, 23B, and 25B.
[0091] or a physiologically functional derivative, solvate or salt
thereof.
[0092] 13. The compound according to the present invention, in
particular according to any of the above items 1 to 12 or a
physiologically functional derivative, solvate or salt thereof for
use as a medicament.
[0093] 14. The compound according to the present invention, in
particular according to any of the above items 1 to 12 or a
physiologically functional derivative, solvate or salt thereof for
use in the treatment or prevention of a medical condition selected
from the group comprising autoimmuneinflammatory disorders, more
particularly selected from the group comprising inflammatory Bowel
disease, multiple sclerosis, rheumathoid arthritis, autoimmune
uveitis, CNS disorders, particularly selected from the group
comprising Alzheimer's desease, Parkinson's desease, Down's
syndrom, sleeping disorders, particularly sleeping disorders in
connection with the circadian clock mechanism, and proliferative
diseases including cancer.
[0094] 15. A pharmaceutical composition comprising a compound
according to the present invention, in particular according to any
of the above items 1 to 12 or a physiologically functional
derivative, solvate or salt thereof and one or more
pharmaceutically acceptable excipients.
[0095] Furthermore, the present invention relates to a method of
treatment or prevention of the medical conditions specified herein,
which comprises the administration of an effective amount of a
compound according to the present invention, or a physiologically
functional derivative, solvate or salt thereof to a subject in need
thereof.
[0096] Furthermore, the present invention relates to the use of a
compound according to the present invention, or a physiologically
functional derivative, solvate or salt thereof in the treatment or
prevention of the medical conditions specified herein.
[0097] 16. A method of treatment or prevention of a medical
condition selected from the group comprising autoimmune
inflammatory disorders, CNS disorders, sleeping disorders, and
proliferative diseases including cancer, which comprises the
administration of an effective amount of a compound according to
the present invention, in particular according to any of the above
items 1 to 12, or a physiologically functional derivative, solvate
or salt thereof to a subject in need thereof.
[0098] 17. Use of a compound according to the present invention, in
particular according to any of the above items 1 to 12, or a
physiologically functional derivative thereof, solvate or salt in
the manufacture of a medicament for the treatment or prevention of
a medical condition selected from the group comprising autoimmune
inflammatory disorders, CNS disorders, sleeping disorders, and
proliferative diseases including cancer.
[0099] 18. Process for the preparation of a compound according to
the present invention, in particular according to any of the above
items 1 to 12, wherein A is an --CONH-- or --NHCO--, the process
comprising the step of coupling a compound of below Formula IV with
a compound of below formula II;
##STR00012##
[0100] wherein Y, L and X are as defined above, and
[0101] wherein either R.sup.1 is NH.sub.2 and R.sup.2 is COOH or
COOCl, or wherein R.sup.2 is NH.sub.2 and R.sup.1 is COOH or COOCl,
particularly R.sup.1 is NH.sub.2 and R.sup.2 is COOH or COOCl.
[0102] The compounds of the present invention interact with Casein
kinase delta and/or epsilon, suggesting their applicability in
prevention and/or therapy of medical conditions wherein the
function of Casein kinase delta and/or epsilon plays a role.
[0103] Particularly, the difluoromethylenedioxy group of the
compounds according to the present invention unexpectedly shows two
molecular interactions with the side chain residues of glutamyl 52
and tyrosin 56 of Casein kinase delta. Such additional binding
interaction in addition to the hydrogen binding network of the
benzimidazol heterocycle of the central moiety was not known from
the prior art, and contributes to the favourable inhibitory
activity of the compounds according to the present invention.
[0104] This unexpected finding shows, that an acyl
amino-benzimidazol derivative containing a difloromethylendioxo
residue on the benzole ring has specific interactions to kasein
kinase delta.
[0105] The following definitions are meant to further define
certain terms used in the context of the present invention. If a
particular term used herein is not specifically defined, the term
shoud not be considered to be indefinite. Rather, such terms are to
be construed in accordance with their meaning as regularly
understood by the skilled artisan in the field of art to which the
invention is directed, particularly in the field of organic
chemistry, pharmaceutical sciences and medicine.
[0106] As used herein, an alkyl group is to be understood to
encompass alkanyl, alkenyl, alkynyl, wherein alkanyl means a
completely saturated hydrocarbon chain, alkenyl means a hydrocarbon
chain comprising at least one carbon-carbon double bond, alkynyl
means a hydrocarbon chain comprising at least one carbon-carbon
triple bond (including a hydrocarbon chain comprising one or more
carbon-carbon double bonds and at least one carbon-carbon triple
bond). In the context of the present invention, an alkanyl group,
if not stated otherwise, particularly denotes a linear or branched
C.sub.1-C.sub.6-alkanyl, particularly a linear or branched
C.sub.1-C.sub.5-alkanyl; an alkenyl group, if not stated otherwise,
particularly denotes a linear or branched C.sub.2-C.sub.6-alkenyl;
and an alkynyl group, if not stated otherwise, particularly denotes
a linear or branched C.sub.2-C.sub.6-alkynyl group. In particular
embodiments the alkyl group is selected from the group comprising
--CH.sub.3, --C.sub.2H.sub.5, --CH.dbd.CH.sub.2, --C.ident.CH,
--C.sub.3H.sub.7, --CH(CH.sub.3).sub.2,
--CH.sub.2--CH.dbd.CH.sub.2, --C(CH.sub.3).dbd.CH.sub.2,
--CH.dbd.CH--CH.sub.3, --C.ident.C--CH.sub.3,
--CH.sub.2--C.ident.CH, --C.sub.4H.sub.9,
--CH.sub.2--CH(CH.sub.3).sub.2, --CH(CH.sub.3)--C.sub.2H.sub.5,
--C(CH.sub.3).sub.3, --C.sub.5H.sub.11, --C.sub.6H.sub.13,
--C(R').sub.3, --C.sub.2(R').sub.5, --CH.sub.2--C(R').sub.3,
--C.sub.3(R').sub.7, --C.sub.2H.sub.4--C(R').sub.3,
--C.sub.2H.sub.4--CH.dbd.CH.sub.2, --CH.dbd.CH--C.sub.2H.sub.5,
--CH.dbd.C(CH.sub.3).sub.2, --CH.sub.2--CH.dbd.CH--CH.sub.3,
--CH.dbd.CH--CH.dbd.CH.sub.2, --C.sub.2H.sub.4--C.ident.CH,
--C.ident.C--C.sub.2H.sub.5, --CH.sub.2--C.ident.C--CH.sub.3,
--C.ident.C--CH.dbd.CH.sub.2, --CH.dbd.CH--C.ident.CH,
--C.ident.C--C.ident.CH, --C.sub.2H.sub.4--CH(CH.sub.3).sub.2,
--CH(CH.sub.3)--C.sub.3H.sub.7,
--CH.sub.2--CH(CH.sub.3)--C.sub.2H.sub.5,
--CH(CH.sub.3)--CH(CH.sub.3).sub.2,
--C(CH.sub.3).sub.2--C.sub.2H.sub.5, --CH.sub.2--C(CH.sub.3).sub.3,
--C.sub.3H.sub.6--CH.dbd.CH.sub.2, --CH.dbd.CH--C.sub.3H.sub.7,
--C.sub.2H.sub.4--CH.dbd.CH--CH.sub.3,
--CH.sub.2--CH.dbd.CH--C.sub.2H.sub.5,
--CH.sub.2--CH.dbd.CH--CH.dbd.CH.sub.2,
--CH.dbd.CH--CH.dbd.CH--CH.sub.3,
--CH.dbd.CH--CH.sub.2--CH.dbd.CH.sub.2,
--C(CH.sub.3).dbd.CH--CH.dbd.CH.sub.2,
--CH.dbd.C(CH.sub.3)--CH.dbd.CH.sub.2,
--CH.dbd.CH--C(CH.sub.3).dbd.CH.sub.2,
--CH.sub.2--CH.dbd.C(CH.sub.3).sub.2,
C(CH.sub.3).dbd.C(CH.sub.3).sub.2, --C.sub.3H.sub.6--C.ident.CH,
--C.ident.C--C.sub.3H.sub.7, --C.sub.2H.sub.4--C.ident.C--CH.sub.3,
--CH.sub.2--C.ident.C--C.sub.2H.sub.5,
--CH.sub.2--C.ident.C--CH.sub.3, CH.dbd.CH.sub.2,
--CH.sub.2--CH.dbd.CH--C.ident.CH,
--CH.sub.2--C.ident.C--C.ident.CH,
--C.ident.C--CH.dbd.CH--CH.sub.3, --CH.dbd.CH--C.ident.C--CH.sub.3,
--C.ident.C--C--CH.sub.3, --C.ident.C--CH.sub.2--CH.dbd.CH.sub.2,
--CH.dbd.CH--CH.sub.2--C.ident.CH,
--C.ident.C--CH.sub.2--C.ident.CH,
--C(CH.sub.3).dbd.CH--CH.dbd.CH.sub.2,
--CH.dbd.C(CH.sub.3)--CH.dbd.CH.sub.2,
--CH.dbd.CH--C(CH.sub.3).dbd.CH.sub.2,
--C(CH.sub.3).dbd.CH--C.ident.CH, --CH.dbd.C(CH.sub.3)--C.ident.CH,
--C.ident.C--C(CH.sub.3).dbd.CH.sub.2,
--C.sub.3H.sub.6--CH(CH.sub.3).sub.2,
--C.sub.2H.sub.4--CH(CH.sub.3)--C.sub.2H.sub.5,
--CH(CH.sub.3)--C.sub.4H.sub.9,
--CH.sub.2--CH(CH.sub.3)--C.sub.3H.sub.7,
--CH(CH.sub.3)--CH.sub.2--CH(CH.sub.3).sub.2,
--CH(CH.sub.3)--CH(CH.sub.3)--C.sub.2H.sub.5,
--CH.sub.2--CH(CH.sub.3)--CH(CH.sub.3).sub.2,
--CH.sub.2--C(CH.sub.3).sub.2--C.sub.2H.sub.5,
--C(CH.sub.3).sub.2--C.sub.3H.sub.7,
--C(CH.sub.3).sub.2--CH(CH.sub.3).sub.2,
--C.sub.2H.sub.4--C(CH.sub.3).sub.3,
--CH(CH.sub.3)--C(CH.sub.3).sub.3,
--C.sub.4H.sub.8--CH.dbd.CH.sub.2, --CH.dbd.CH--C.sub.4H.sub.9,
--C.sub.3H.sub.6--CH.dbd.CH--CH.sub.3,
--CH.sub.2--CH.dbd.CH--C.sub.3H.sub.7,
--C.sub.2H.sub.4--CH.dbd.CH--C.sub.2H.sub.5,
--CH.sub.2--C(CH.sub.3).dbd.C(CH.sub.3).sub.2,
--C.sub.2H.sub.4--CH.dbd.C(CH.sub.3).sub.2,
--C.sub.4H.sub.8--C.ident.CH, --C.ident.C--C.sub.4H.sub.9,
--C.sub.3H.sub.6--C.ident.C--CH.sub.3,
--CH.sub.2--C.ident.C--C.sub.3H.sub.7, and
--C.sub.2H.sub.4--C.ident.C--C.sub.2H.sub.5. The alkyl, alkanyl,
alkenyl, and alkynyl groups as defined above, including the groups
enumerated as examples and particular embodiments thereof, are
optionally substituted by one or more sub stituents R'.
[0107] As used herein, an aryl group denotes an aromatic mono- or
polycyclic hydrocarbon ring system, which may optionally be fused
to one or more cycloalkyl or heterocyclyl rings, and wherein the
total number of ring atoms in the aryl group is six to fourteen,
particularly six to ten. The point of attachment of of said aryl
group to the central moiety may be located on the mono- or
polycyclic hydrocarbon ring system or on the optionally fused
cycloalkyl or heterocyclyl ring. Examples of the aryl group are
phenyl, naphthyl, indenyl, azulenyl, fluorenyl,
1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl,
2,3-dihydroindenyl, 1,5-dihydro-s-indacenyl,
1,6-dihydro-as-indacenyl, 1H-cyclopenta[a]naphthyl and
1H-cyclopenta[b]naphthyl, phenalenyl, phenanthrenyl, anthracenyl,
1,6-dihydropentalenyl, 1,6a-dihydropentalenyl,
1,2,3,4-tetrahydroanthracenyl, 1,2,3,4-tetrahydrophenanthrenyl,
2,3-dihydro-1H-cyclopenta[a]naphthalenyl,
2,3-dihydro-1H-cyclopenta[b]naphthalenyl,
2,3-dihydro-1H-phenalenyl,
2,3-dihydrobenzo[b]thiophenyl-1,1-dioxide,
1,2,3,4-tetrahydroisoquinolinyl, 1,2,3,4-tetrahydroquinolinyl,
2,3-dihydrobenzo[b][1,4]dioxinyl, 2,3-dihydrobenzo[b]thiophenyl,
2,3-dihydrobenzofuranyl, 2-oxo-2,3-dihydrobenzoimidazolyl,
benzo[d][1,3]dioxolyl, chromanyl, indazolinyl and indolinyl. In
particular embodiments, the aryl group is phenyl,
2,3-dihydrobenzo[b][1,4]dioxinyl, 2,3-dihydrobenzofuranyl,
2-oxo-2,3-dihydrobenzoimidazolyl or benzo[d][1,3]dioxolyl, more
particularly phenyl. The aryl groups as defined above, including
the groups enumerated as examples and particular embodiments
thereof, are optionally substituted by one or more sub stituents
R'.
[0108] As used herein, a heteroaryl group denotes an aromatic mono-
or polycyclic hydrocarbon ring system wherein one or more carbon
atoms are replaced by heteroatoms independently selected from the
group comprising O, N and S, wherein the aromatic mono- or
polycyclic hydrocarbon ring system may optionally be fused to one
or more cycloalkyl or heterocyclyl rings, and wherein the total
number of ring atoms in the heteroaryl group is five to fourteen,
particularly five to ten, more particularly five or six. The point
of attachment of of said heteroaryl group to the central moiety may
be located on the mono- or polycyclic hydrocarbon ring system or on
the optionally fused cycloalkyl or heterocyclyl ring. Examples of
the heteroaryl group are thiadiazole, thiazol-2-yl, thiazol-4-yl,
thiazol-5-yl, isothiazol-3-yl, isothiazol-4-yl, isothiazol-5-yl,
oxazol-2-yl, oxazol-4-yl, oxazol-5-yl, isooxazol-3-yl,
isooxazol-4-yl, isooxazol-5-yl, 1,2,4-oxadiazol-3-yl,
1,2,4-oxadiazol-5-yl, 1,2,5-oxadiazol-3-yl, benzooxazol-2-yl,
benzooxazol-4-yl, benzooxazol-5-yl, benzoisooxazol-3-yl,
benzoisooxazol-4-yl, benzoisooxazol-5-yl, 1,2,5-oxadiazol-4-yl,
1,3,4-oxadiazol-2-yl, 1,2,4-thiadiazol-3-yl, 1,2,4-thiadiazol-5-yl,
1,3,4-thiadiazol-2-yl, isothiazol-3-yl, isothiazol-4-yl,
isothiazol-5-yl, benzoisothiazol-3-yl, benzoisothiazol-4-yl,
benzoisothiazol-5-yl, 1,2,5-thiadiazol-3-yl, 1-imidazolyl,
2-imidazolyl, 1,2,5-thiadiazol-4-yl, 4-imidazolyl,
benzoimidazol-4-yl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-furanyl,
3-furanyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
2-pyranyl, 3-pyranyl, 4-pyranyl, 2-pyrimidinyl, 4-pyrimidinyl,
5-pyrimidinyl, pyrid-2-yl, pyrid-3-yl, pyrid-4-yl, pyrid-5-yl,
pyrid-6-yl, 3-pyridazinyl, 4-pyridazinyl, 2-pyrazinyl, 1-pyrazolyl,
3-pyrazolyl, 4-pyrazolyl, 1,2,3-triazol-4-yl, 1,2,3-triazol-5-yl,
1,2,4-triazol-3-yl, 1,2,4-triazol-5-yl, 1H-tetrazol-2-yl,
1H-tetrazol-3-yl, tetrazolyl, acridyl, phenazinyl, carbazolyl,
phenoxazinyl, indolizine, 2-indolyl, 3-indolyl, 4-indolyl,
5-indolyl, 6-indolyl, 7-indolyl, 1-isoindolyl, 3-isoindolyl,
4-isoindolyl, 5-isoindolyl, 6-isoindolyl, 7-isoindolyl,
2-indolinyl, 3-indolinyl, 4-indolinyl, 5-indolinyl, 6-indolinyl,
7-indolinyl, benzo[b]furanyl, benzofurazane, benzothiofurazane,
benzotriazol-1-yl, benzotriazol-4-yl, benzotriazol-5-yl,
benzotriazol-6-yl, benzotriazol-7-yl, benzotriazine,
benzo[b]thiophenyl, benzimidazolyl, benzothiazolyl, quinazolinyl,
quinoxazolinyl, cinnoline, quinolinyl, tetrahydroquinolinyl,
isoquinolinyl, or tetrahydroisoquinolinyl, purine, phthalazine,
pteridine, thiatetraazaindene, thiatriazaindene,
isothiazolopyrazine, 6-pyrimidinyl, 2,4-dimethoxy-6-pyrimidinyl,
benzimidazol-2-yl, 1H-benzimidazolyl, benzimidazol-4-yl,
benz-imidazol-5-yl, benzimidazol-6-yl, benzimidazol-7-yl,
tetrazole, tetrahydro-thieno[3,4-d]imidazol-2-one,
pyrazolo[5,1-c][1,2,4]triazine, isothiazolopyrimidine,
pyrazolotriazine, pyrazolopyrimidine, imidazopyridazine,
imidazopyrimidine, imidazopyridine, imidazolotriazine,
triazolotriazine, triazolopyridine, triazolopyrazine,
triazolopyrimidine, or triazolopyridazine. The heteroaryl groups as
defined above, including the groups enumerated as examples and
particular embodiments thereof, are optionally substituted by one
or more substituents R'.
[0109] As used herein, a cycloalkyl group denotes a non-aromatic,
mono- or polycyclic completely saturated or partially unsaturated
hydrocarbon ring system. Said cycloalkyl is particularly mono- or
bicyclic, more particularly monocyclic. Said cycloalkyl is
particularly completely saturated. Said cycloalkyl particularly
comprises 3 to 10 carbon atoms, more particularly 5 to 7 carbon
atoms. Even more particularly, said cycloalkyl is selected from the
group comprising cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, 1-norbornyl, 2-norbonryl, 7-norbornyl, 1-adamantyl,
and 2-adamantyl, yet even more particularly said cycloalkyl is
cyclohexyl or cyclopropyl. The cycloalkyl groups as defined above,
including the groups enumerated as examples and particular
embodiments thereof, are optionally substituted by one or more
substituents R'.
[0110] As used herein, a heterocyclyl group denotes a non-aromatic
mono- or polycyclic completely saturated or partially unsaturated
hydrocarbon ring system, wherein one or more of the carbon atoms
are replaced by a heteroatom independently selected from N, O, or
S. Said heterocyclyl is particularly mono- or bicyclic, more
particularly monocyclic. Said heterocyclyl is particularly
completely saturated. Said heterocyclyl particularly comprises a
sum of 5 to 10 carbon and heteroatoms, more particularly a sum of 5
to 7 carbon and heteroatoms, even more particularly a sum of 6
carbon and heteroatoms. Even more particularly said heterocyclyl is
selected from the group comprising morpholinyl, piperidinyl,
dioxanyl, piperazinyl, thiomorpholinyl, piperidinyl, pyrrolidinyl,
tetrahydrofuranyl, isoxazolidinyl, thiomorpholinyl,
tetrahydrothiofuranyl and tetrahydropyranyl, more particularly
selected from the group comprising morpholinyl, piperidinyl,
dioxanyl, piperazinyl, thiomorpholinyl, piperidinyl, and
pyrrolidinyl. The heterocyclyl groups as defined above, including
the groups enumerated as examples and particular embodiments
thereof, are optionally substituted by one or more sub stituents
R'.
[0111] As used herein, a halo or halogen group denotes fluorine,
chlorine, bromine or iodine; particularly chlorine or fluorine.
[0112] As used herein, a haloalkyl group denotes an alkyl group
wherein one or more, particularly at least half, more particularly
all of the hydrogen atoms on the hydrocarbon chain are replaced by
halogen atoms. The haloalkyl group is particularly selected from
the group comprising --C(R.sup.10).sub.3,
--CH.sub.2--C(R.sup.10).sub.3, --C(R.sup.10).sub.2--CH.sub.3,
--C(R.sup.10).sub.2--C(R.sup.10).sub.3,
--C(R.sup.10).sub.2--CH(R.sup.10).sub.2,
--CH.sub.2--CH(R.sup.10).sub.2, --CH(R.sup.10)--C(R.sup.10).sub.3,
--CH(R.sup.10)--CH.sub.3, and --C.sub.2H.sub.4--C(R.sup.10).sub.3,
more particularly --C(R.sup.10).sub.3, wherein R.sup.10, R.sup.10
represents halogen, particularly F. More particularly, haloalkyl is
--CF.sub.3, --CHF.sub.2, --CH.sub.2CF.sub.3, or CF.sub.2Cl.
[0113] As used herein, an alkoxy group denotes an O-alkyl group,
the alkyl group as defined above. The alkoxy group is particularly
selected from the group comprising methoxy, ethoxy and propoxy,
more particularly methoxy.
[0114] As used herein, alkylthio group denotes an --S-alkyl group,
the alkyl group as defined above, particularly methylthio.
[0115] As used herein, a haloalkoxy group denotes an O-haloalkyl
group, haloalkyl group being defined as defined above. The
haloalkoxy group is particularly selected from the group comprising
--OC(R.sup.10).sup.3, --OCR.sup.10(R.sup.10').sub.2,
--OCH.sub.2--C(R.sup.10).sub.3, and
--OC.sub.2H.sub.4--C(R.sup.10).sub.3, wherein R.sup.10, R.sup.10'
represent F, Cl, Br or I, particularly F.
[0116] An alkylamino group is an NH-alkyl or N-dialkyl group, the
alkyl group as defined above, particularly mono- or dimethylamino,
mono- or diethylamino or isopropylamino, more particularly
dimethylamino.
[0117] An arylalkyl or aralkyl group denotes a linear or branched
C1-C6-alkyl as defined herein substituted with at least one aryl
group as defined herein. Exemplary arylalkyl groups include styryl,
benzyl, phenylethyl, 1-(naphthalen-2-yl)ethyl, particularly the
arylalkyl group is styryl or 1-(naphthalen-2-yl)ethyl, wherein
styryl is particularly optionally substituted at its phenyl part as
defined above for the aryl group. In other particular embodiments,
the arylalkyl group is benzyl, styryl or
1-(naphthalen-2-yl)ethyl.
[0118] R' is independently selected from the group comprising
halogen, C.sub.1-6-alkyl, C.sub.1-6-haloalkyl,
C.sub.1-6-haloalkoxy, OH, C.sub.1-6-alkoxy, aryl, heteroaryl,
cycloalkyl, heterocyclyl, --S--C.sub.1-6-alkyl,
--S--C.sub.1-6-haloalkyl, nitro, --NH.sub.2,
--N(C.sub.1-6-alkyl).sub.2, --NH(C.sub.1-6-alkyl),
--NHCO(C.sub.1-6-alkyl), --CONH.sub.2, --CONH(C.sub.1-6-alkyl),
--CO(C.sub.1-6-alkyl), --COH, --COO(C.sub.1-6-alkyl), --COOH,
--SO.sub.2NH.sub.2, --SO.sub.2NH(C.sub.1-6-alkyl),
--SO.sub.2(C.sub.1-6-alkyl), and --CN, particularly selected from
the group comprising F, Cl, C.sub.1-3-alkyl, C.sub.1-3-haloalkyl,
C.sub.1-3-haloalkoxy, OH, C.sub.1-3-alkoxy, phenyl, pyridyl,
pyrrolyl, thiophenyl, furanyl, cyclopentyl, cyclohexyl,
piperidinyl, piperazinyl, tetrahydrofuranyl, morpholinyl,
--S--C.sub.1-3-alkyl, --S--C.sub.1-3-haloalkyl, nitro, --NH.sub.2,
--N(C.sub.1-3-alkyl).sub.2, --NH(C.sub.1-3-alkyl),
--NHCO(C.sub.1-3-alkyl), --CONH.sub.2, --CONH(C.sub.1-3-alkyl),
--CO(C.sub.1-3-alkyl), --COH, --COO(C.sub.1-3-alkyl), --COOH,
--SO.sub.2NH.sub.2, --SO.sub.2NH(C.sub.1-3-alkyl),
--SO.sub.2(C.sub.1-3-alkyl), and --CN, more particularly selected
from the group comprising F, Cl, methyl, ethyl, isopropyl,
trifluoromethyl, trifluoromethoxy, OH, methoxy, methylsulfanyl,
trifluoromethylsulfanyl, nitro, --NH.sub.2, dimethylamino,
diethylamino, diisopropylamino, methylamino, ethylamino,
isopropylamino, --NHCO-methyl, --CONH.sub.2, --CONH-methyl,
--CONH-ethyl, --CO-methyl, --CO-ethyl, --COO-methyl, --COO-ethyl,
--COOH, --SO.sub.2NH.sub.2, --SO.sub.2NH-methyl, --SO.sub.2-methyl,
--SO.sub.2NH-ethyl, --SO.sub.2-ethyl, and --CN, even more
particularly selected from the group comprising F, Cl, methyl,
ethyl, isopropyl, trifluoromethyl, trifluoromethoxy, OH, methoxy,
nitro, --NH.sub.2, dimethylamino, diethylamino, methylamino,
ethylamino, --CONH.sub.2, --COO-methyl, --COO-ethyl, --COOH, and
--CN, yet even more particularly selected from the group comprising
F, Cl, methyl, trifluoromethyl, trifluoromethoxy, OH, methoxy,
nitro, --NH.sub.2, and --CN, most particularly selected from the
group comprising F, Cl, methyl, trifluoromethyl, trifluoromethoxy,
OH, and methoxy. Particularly, R' is not substituted by further
groups.
[0119] Where chemically feasible from the viewpoint of molecule
stability and/or chemical valence rules, a nitrogen heteroatom as
defined herein, e.g. in the context of "heteroaryl" and
"heterocycle", may include the N-oxide.
[0120] Where chemically feasible from the viewpoint of molecule
stability under physiological conditions and/or chemical valence
rules, the definition of a sulfur heteroatom as defined herein,
e.g. in the context of "heteroaryl" and "heterocycle", may include
the sulfur oxide and/or the sulfur dioxide, respectively.
[0121] As used herein the term "substituted with" or "substituted
by" means that one or more hydrogen atoms connected to a carbon
atom or heteroatom of a chemical group or entity are exchanged with
a substituent group, respectively; e.g. substituted aryl comprises
4-hydroxyphenyl, wherein the H-atom in the 4-position of the phenyl
group is exchanged with a hydroxyl group. Said hydrogen atom(s) to
be replaced may be attached to a carbon atom or heteroatom, and may
be expressedly shown in a specific formula, such as for example in
an --NH-- group, or may not expressedly be shown but intrinsically
be present, such as for example in the typical "chain" notation
which is commonly used to symbolize e.g. hydrocarbons. The skilled
person will readily understand that particularly such substituents
or substituent patterns are excluded, which lead to compounds which
are not stable and/or not accessible via the synthesis methods
known in the art.
[0122] As used herein, the term "central moiety" means the
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)
part of the molecule which is shown in the below graphic
representation.
##STR00013##
[0123] Unless specified otherwise, references to the compounds
according to the present invention include the pharmaceutically
acceptable derivatives, solvates or salts thereof as described
herein, as well as to salts of said pharmaceutically acceptable
derivatives, solvates of salts and pharmaceutically acceptable
derivatives, and optionally solvates of salts of pharmaceutically
acceptable derivatives.
[0124] As used herein, the term "pharmaceutically acceptable
derivative" of a compound according to the present invention is for
instance a prodrug of said compound, wherein at least one of the
following groups are derivatized as specified in the following: A
carboxylic acid group is derivatized into an ester, a hydroxyl
group is derivatized into an ester, a carboxylic acid is
derivatized into an amide, an amine is derivatized into an amide, a
hydroxyl group is derivatized into a phosphate ester.
[0125] As used herein, the term "tautomer" used in reference to the
compouns according to the present invention, in particular includes
tautomers that typically form with respect to substituted
benzimidazol groups. As an illustration two tautomeric forms of an
exemplary substituted benzimidazol moiety, as is present in the
compounds according to the present invention, are shown:
##STR00014##
[0126] The compounds according to the present invention are to be
understood to comprise all tautomeric forms thereof, even if not
expressedly shown in the formulae described herein, including
Formulae (I) and (Ia). Throughout this specification, whenever a
chemical formula, generic or otherwise, discloses a compound having
a 1H-benzimidazole moiety that is unsubstituted at the 1 position,
as shown on the left-hand side of the above exemplary illustration,
said chemical formula it is to be understood to implicitly also
relate to compounds wherein the benzimidazole moiety is
tautomerized to form the structure as shown on the right-hand side
of the above exemplary illustration.
[0127] The compounds of Formulae (I) and (Ia) as defined herein are
to be understood to encompass, where applicable, all stereoismers
of said compounds, unless specified otherwise. The term
"stereoisomer" as used herein refers to a compound with at least
one stereogenic centre, which may be R- or S-configured, as defined
by the according IUPAC rules, and encompasses enantiomers and
diastereomers as commonly understood by the skilled person. It has
to be understood, that in compounds with more than one stereogenic
centre, each of the individual stereogenic centres may
independently from each other be R- or S-configured. The term
"stereoisomer" as used herein also refers to salts of the compounds
herein described with optically active acids or bases.
[0128] In the present invention, the salts of the compounds
according to the present invention are particularly
pharmaceutically acceptable salts of the compounds according to the
present invention. Pharmaceutically acceptable salts are such salts
which are usually considered by the skilled person to be suitable
for medical applications, e.g. because they are not harmful to
subjects which may be treated with said salts, or which give rise
to side effects which are tolerable within the respective
treatment. Usually, said pharmaceutically acceptable salts are such
salts which are considered as acceptable by the regulatory
authorities, such as the US Food and Drug Administration (FDA), the
European Medicines Agency (EMA), or the Japanese Ministry of
Health, Labor and Welfare Pharmaceuticals and Medical Devices
Agency (PMDA). However, the present invention in principle also
encompasses salts of the compounds according to the present
invention which are as such not pharmaceutically acceptable, e.g.
as intermediates in the production of the compounds according to
the present invention or physiologically functional derivatives
thereof, or as intermediates in the production pharmacologically
acceptable salts of the compounds according to the present
invention or physiologically functional derivatives thereof.
[0129] In each case, the skilled person can readily determine
whether a certain compound according to the present invention or
pharmaceutically acceptable derivative thereof can form a salt,
i.e. whether said compound according to the present invention or
pharmaceutically acceptable derivative thereof has a group which
may carry a charge, such as e.g. an amino group, a carboxylic acid
group, etc.
[0130] Exemplary salts of the compounds of the present invention
are acid addition salts or salts with bases, particularly
pharmacologically tolerable inorganic and organic acids and bases
customarily used in pharmacy, which are either water insoluble or,
particularly, water-soluble acid addition salts. Salts with bases
may--depending on the substituents of the compounds of the present
invention--also be suitable.
[0131] Pharmacologically intolerable salts, which can be obtained,
for example, as process products during the preparation of the
compounds according to the invention on an industrial scale, are
also encompassed by the present invention and, if desired, may be
converted into pharmacologically tolerable salts by processes known
to the person skilled in the art.
[0132] According to expert's knowledge the compounds of the
invention as well as their salts may contain, e.g. when isolated in
crystalline form, varying amounts of solvents. Included within the
scope of the invention are therefore solvates and in particular
hydrates of the compounds of the present invention as well as
solvates and in particular hydrates of the salts and/or
physiologically functional derivatives of the compounds of the
present invention. More particularly the invention encompasses
hydrates of the compounds, salts and/or physiologically functional
derivatives according to the present invention, comprising one, two
or one half water molecule, with respect to their
stoichiometry.
[0133] As used herein, the term "room temperature", "rt" or "r.t."
relates to a temperature of about 25.degree. C., unless specified
otherwise.
[0134] As used herein, the term "stable" specifies a compound in
which the chemical structure is not altered when the compound is
stored at a temperature from about -80.degree. C. to about
+40.degree. C., particularly from about -80.degree. C. to
+25.degree. C. in the absence of light, moisture or other
chemically reactive conditions for at least one week, particularly
at least one month, more particularly at least six months, even
more particularly, at least one year, and/or a compound which under
IUPAC standard conditions and in the absence of light, moisture or
other chemically reactive conditions maintains its structural
integrity long enough to be useful for therapeutic or prophylactic
administration to a patient, i.e. at least one week. Compounds
which are not stable as described above are particularly not
encompassed by the present invention. In particular, such compounds
which at IUPAC standard conditions spontaneously decompose within a
period of less then one day are regarded as not being stable
compounds. The skilled person will readily recognize, based on his
general knowledge in his field of expertise, which compounds and
which substitution patterns result in stable compounds.
[0135] As used herein, the term "treatment" includes complete or
partial healing of a disease, prevention of a disease, alleviation
of a disease or stop of progression of a given disease.
[0136] As used herein, the term "medicament" includes the compounds
of formula (I) as described herein, pharmacologically acceptable
salts or physiologically functional derivatives thereof, which are
to be administered to a subject in pure form, as well as
compositions comprising at least one compound according to the
present invention, a pharmacologically acceptable salt or
physiologically functional derivative thereof, which is suitable
for administration to a subject.
[0137] The compounds according to the present invention and their
pharmacologically acceptable salts and physiologically functional
derivatives can be administered to animals, particularly to
mammals, and in particular to humans as therapeutics per se, as
mixtures with one another or particularly in the form of
pharmaceutical preparations or compositions which allow enteral
(e.g. oral) or parenteral administration and which comprise as
active constituent a therapeutically effective amount of at least
one compound according to the present invention, or a salt or
physiologically functional derivative thereof, in addition to e.g.
one or more components selected from the group comprising customary
adjuvants, pharmaceutically innocuous excipients, carriers,
buffers, diluents, and/or other customary pharmaceutical
auxiliaries.
[0138] The pharmaceutical compositions, medical uses and methods of
treatment according to the present invention may comprise the more
than one compound according to the present invention.
[0139] Pharmaceutical compositions comprising a compound according
to the present invention, or a pharmaceutically acceptable salt or
physiologically functional derivative may optionally comprise one
or more further therapeutically active substances which are not
compounds of formula (I) according to the present invention. As
used herein, the term "therapeutically active substance" specifies
a substance which upon administration can induce a medical effect
in a subject. Said medical effect may include the medical effect
described herein for the compounds of formula (I) of the present
invention, but may also, in the case of therapeutically active
substances which are to be co-administered with the compounds
according to the present invention, include other
medicalsubstances, such as for example but not exclusively
irinotecan, oxaliplatin, capecitabine, 5-fluorouracil, cetuximab
(Erbitux), panitumumab (Vectibix), bevacizumab (Avastin),
vincristine, vinblastine, vinorelbine, vindesine, taxol, amsacrine,
etoposide, etoposide phospahte, Teniposide, actinomycin,
anthracyclines, doxorubicin, valrubicin, valrubicin, idarubicin,
epirubicin, bleomycin, plicamycin, mitomycin, mechlorethamine,
cyclophosphamide, chlorambucil, ifosfamide and other kinase
inhibitors.
[0140] The term "pharmaceutically acceptable" is well known to the
skilled person and particularly means that the respective entity is
not harmful to the subject to which the entity or the composition
comprising the entity is administered, that said entity is stable
and that said entity is chemically compatible (i.e. non-reactive)
with other ingredients of the respective pharmaceutical
composition.
[0141] Medicaments and pharmaceutical compositions according to the
present invention, comprising at least one compound according to
the present invention or a pharmacologically acceptable salt or a
physiologically functional derivative therof include those suitable
for oral, rectal, bronchial, nasal, topical, buccal, sub-lingual,
vaginal or parenteral (including transdermal, subcutaneous,
intramuscular, intrapulmonary, intravascular, intracranial,
intraperitoneal, intravenous, intraarterial, intracerebral,
intraocular injection or infusion) administration, or those in a
form suitable for administration by inhalation or insufflation,
including powders and liquid aerosol administration, or by
controlled release (e.g. sustained release, pH-controlled release,
delayed, release, repeat action release, prolonged release,
extended release) systems. Suitable examples of controlled release
systems include semipermeable matrices of solid hydrophobic
polymers containing the compound of the invention, which matrices
may be in form of shaped articles, e.g. films or microcapsules or
colloidal drug carriers e.g. polymeric nanoparticles, or controlled
release solid dosage forms, e.g. core tablets or multi-layer
tablets.
[0142] The production of medicaments or pharmaceutical compositions
comprising the compounds according to the present invention and
their application can be performed according to methods which are
well-known to the medical practitioner.
[0143] Pharmaceutically acceptable carriers used in the preparation
of a pharmaceutical composition or medicament comprising a compound
according to the present invention, a pharmacologically acceptable
salt or physiologically functional derivative thereof, can be
either solid or liquid. Solid form pharmaceutical compositions
comprising a compound according to the present invention, a
pharmacologically acceptable salt or physiologically functional
derivative thereof, include powders, tablets, pills, capsules,
sachets, suppositories, and dispersible granules. A solid carrier
may comprise one or more components, which may also act as
diluents, flavouring agents, solubilizers, lubricants, suspending
agents, binders, preservatives, tablet disintegrating agents, or an
encapsulating material.
[0144] In powders, the carrier is a finely divided solid, which is
in a mixture with the finely divided active component. In tablets,
the active component is mixed with the carrier having the necessary
binding capacity in suitable proportions and compacted in the shape
and size desired. The tabletting mixture can be granulated, sieved
and compressed or direct compressed. Suitable carriers are
magnesium carbonate, magnesium stearate, talc, sugar, lactose,
pectin, dextrin, starch, gelatine, tragacanth, methylcellulose,
sodium carboxymethylcellulose, a low melting wax, cocoa butter, and
the like. The term "preparation" is intended to include the
formulation of the active compound with encapsulating material as
carrier providing a capsule in which the active component, with or
without carriers, is surrounded by a carrier, which is thus in
association with it. Similarly, sachets and lozenges are included.
Tablets, powders, capsules, pills, sachets, and lozenges can be
used as solid forms suitable for oral administration.
[0145] For preparing suppositories, a low melting wax, such as a
mixture of fatty acid glyceride or cocoa butter, is first melted
and the active component is dispersed homogeneously therein, as by
stirring. The molten homogenous mixture is then poured into
conveniently sized moulds, allowed to cool, and thereby to
solidify. Compositions suitable for vaginal administration may be
presented as peccaries, tampons, creams, gels, pastes, foams or
sprays containing in addition to the active ingredient such
carriers as are known in the art to be appropriate. Liquid
preparations include solutions, suspensions, and emulsions, for
example, water or water-propylene glycol solutions. For example,
parenteral injection liquid preparations can be formulated as
solutions in aqueous polyethylene glycol solution.
[0146] The compounds according to the present invention may be
formulated for parenteral administration (e.g. by injection, for
example bolus injection or continuous infusion) and may be
presented in unit dose form in ampoules, pre-filled syringes, small
volume infusion or in multi-dose containers with an added
preservative. The compositions may take such forms as suspensions,
solutions, or emulsions in oily or aqueous vehicles, and may
contain formulation agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, the active ingredient may be in
powder form, obtained by aseptic isolation of sterile solid or by
lyophilization from solution, for re-constitution with a suitable
vehicle, e.g. sterile, pyrogen-free water, before use.
[0147] Aqueous solutions suitable for oral administration can be
prepared by dissolving the active component in water and adding for
example suitable colorants, flavours, stabilizing and thickening
agents, as desired. Aqueous suspensions suitable for oral use can
be made by dispersing the finely divided active component in water
with viscous material, such as natural or synthetic gums, resins,
methylcellulose, sodium carboxymethylcellulose, or other well known
suspending agents.
[0148] Also included are solid form preparations, which are
intended to be converted, shortly before administration, to liquid
form preparations for oral administration. Such liquid forms
include solutions, suspensions, and emulsions. These preparations
may contain, in addition to the active component, for example
colorants, flavours, stabilizers, buffers, artificial and natural
sweeteners, dispersants, thickeners, solubilizing agents, and the
like.
[0149] In an embodiment of the present invention the medicament is
applied topically, e.g. in the form of transdermal therapeutic
systems (e.g. patches) or topical formulations (e.g. liposomes,
cremes, ointment, lotion, gels, dispersion, suspension, spray,
solution, foam, powder). This may be suitable to reduce possible
side effects and, where appropriate, limit the necessary treatment
to those areas affected.
[0150] Particularly the medicament may comprise carrier materials
or excipients, including but not limited to a lipophilic phase (as
for example Vaseline, paraffines, triglycerides, waxes,
polyalcylsiloxanes), oils (olive oil, peanut oil, castor oil,
triglyceride oil), emulsifier (as for example lecithin,
phosphatidylglyceroles, alkyl alcohols, sodium lauryl sulfate,
polysorbats, Cholesterol, sorbitan fatty acid ester,
polyoxyethylene fatty acid glycerol and -ester, poloxamers),
preservatives (for instance benzalkonium chloride, chlorobutanol,
parabene or thiomersal), flavouring agents, buffer substances (for
example salts of acetic acid, citric acid, boric acid, phosphoric
acid, tatric acid, trometamole or trolamine), solvents (for
instance polyethylenglycols, glycerol, ethanol, isopropanol or
propyleneglycol) or solubilizers, agents for achieving a depot
effect, salts for modifying the osmotic pressure, carrier materials
for patches (for instance polypropylene,
ethylene-vinylacetat-copolymer, polyacrylates, silicon) or
antioxidants (for example ascorbate, tocopherol,
butylhydroxyanisole, gallic acid esters or butylhydroxytoluol).
[0151] Ointments and creams may, for example, be formulated with an
aqueous or oily base with the addition of suitable thickening
and/or gelling agents. Lotions may be formulated with an aqueous or
oily base and will in general also contain one or more emulsifying
agents, stabilizing agents, dispersing agents, suspending agents,
thickening agents, or colouring agents.
[0152] Compositions suitable for topical administration in the
mouth include lozenges comprising the active agent in a flavoured
base, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert base such as gelatine
and glycerine or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
[0153] Solutions or suspensions may be applied directly to the
nasal cavity by conventional means, for example with a dropper,
pipette or spray. The compositions may be provided in single or
multi-dose form. In the latter case of a dropper or pipette, this
may be achieved by the patient administering an appropriate,
predetermined volume of the solution or suspension. In the case of
a spray, this may be achieved for example by means of a metering
atomizing spray pump.
[0154] Administration to the respiratory tract may also be achieved
by means of an aerosol formulation in which the active ingredient
is provided in a pressurized pack with a suitable propellant such
as a chlorofluorocarbon (CFC), for example dichlorodifluoromethane,
trichlorofluoromethane, or dichlorotetrafluoroethane, carbon
dioxide, or other suitable gas. The aerosol may conveniently also
contain a surfactant such as lecithin. The dose of drug may be
controlled by provision of a metered valve.
[0155] Alternatively the medicament may be provided in the form of
a dry powder, for example a powder mix of the compound in a
suitable powder base such as lactose, starch, starch derivatives
such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone
(PVP). Conveniently the powder carrier will form a gel in the nasal
cavity. The powder composition may be presented in unit dose form,
for example in capsules or cartridges of, e.g., gelatine, or
blister packs from which the powder may be administered by means of
an inhaler.
[0156] In compositions for administration to the respiratory tract,
including intranasal compositions, the compound will generally have
a small particle size for example of the order of 5 microns or
less. Such a particle size may be obtained by means known in the
art, for example by micronization.
[0157] When desired, compositions adapted to give sustained release
of the active ingredient may be employed.
[0158] The pharmaceutical preparations are particularly in unit
dosage forms. In such form, the preparation is subdivided into unit
doses containing appropriate quantities of the active component.
The unit dosage form can be a packaged preparation, the package
containing discrete quantities of preparation, such as packaged
tablets, capsules, and powders in vials or ampoules. Also, the unit
dosage form can be a capsule, tablet, sachet, or lozenge itself, or
it can be the appropriate number of any of these in packaged form.
Tablets or capsules for oral administration and liquids for
intravenous administration and continuous infusion are particular
compositions.
[0159] Further details on techniques for formulation and
administration may be found in the 21.sup.st edition of Remington's
Pharmaceutical Sciences (Maack Publishing Co. Easton, Pa.).
[0160] The compounds of the present invention may be used in
combination with radiation therapy, or in combination with
radiation therapy and other active compounds, already known for the
treatment of the medical conditions disclosed herein, whereby a
favourable additive or amplifying effect is noticed.
[0161] To prepare the pharmaceutical preparations, pharmaceutically
inert inorganic or organic excipients can be used. To prepare
pills, tablets, coated tablets and hard gelatine capsules, for
example, lactose, cornstarch or derivatives thereof, talc, stearic
acid or its salts, etc. can be used. Excipients for soft gelatine
capsules and suppositories are, for example, fats, waxes,
semi-solid and liquid polyols, natural or hardened oils etc.
Suitable excipients for the production of solutions and syrups are,
for example, water, sucrose, invert sugar, glucose, polyols etc.
Suitable excipients for the production of injection solutions are,
for example, water, alcohols, glycerol, polyols or vegetable
oils.
[0162] The dose can vary within wide limits and is to be suited to
the individual conditions in each individual case. For the above
uses the appropriate dosage will vary depending on the mode of
administration, the particular condition to be treated and the
effect desired. In general, however, satisfactory results are
achieved at dosage rates of about 1 to 100 mg/kg animal body weight
particularly 1 to 50 mg/kg. Suitable dosage rates for larger
mammals, for example humans, are of the order of from about 10 mg
to 3 g/day, conveniently administered once, in divided doses 2 to 4
times a day, or in sustained release form.
[0163] The compounds of the present invention are suitable for the
treatment of inflammatory diorders and hyperproliferative diseases,
such as benign and malignant forms of neoplasia, including
cancer.
[0164] Exemplary types of cancer in the comtext of the present
invention are hepatocarcinoma, adrenocortical carcinoma,
AIDS-related cancers including AIDS-related lymphoma, anal cancer,
basal cell carcinoma, bile duct cancer, bone cancer, brain tumors
including brain stem glioma, cerebellar astrocytoma, cerebral
astrocytoma, malignant glioma, ependymoma, medulloblastoma,
supratentorial primitive neuroectodermal tumors, visual pathway and
hypothalamic glioma, breast cancer, bronchial adenomas/carcinoids,
Burkitt's lymphoma, gastrointestinal, carcinoma of unknown primary
site, central nervous system lymphoma, cervical cancer, chronic
myeloproliferative disorders, colon cancer, colorectal cancer,
cutaneous T-cell lymphoma, endometrial cancer, ependymoma,
esophageal cancer, extracranial germ cell tumor, extragonadal germ
cell tumor, ovarian germ cell tumor, eye cancer including
intraocular melanoma and retinoblastoma, gallbladder cancer,
gastrointestinal carcinoid tumor, gestational trophoblastic tumor,
glioma, childhood brain stem glioma, head and neck cancer,
hematologic cancer, adult and childhood (primary) hepatocellular
cancer, hypopharyngeal cancer, islet cell or pancreatic cancer,
renal cancer, laryngeal cancer, acute lymphoblastic leukemia, adult
and childhood acute myeloid leukemia, chronic lymphocytic leukemia,
chronic myelogenous leukemia, hairy cell leukemia, lip and oral
cavity cancer, liver cancer, lung cancer, including non-small cell
lung cancer and small cell lung cancer, Hodgkin's lymphoma,
non-Hodgkin's lymphoma, primary central nervous system lymphoma,
Waldenstrom's macroglobulinemia, merkel cell carcinoma,
mesothelioma, metastatic squamous neck cancer with occult primary
site, multiple endocrine neoplasia syndrome, multiple
myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic
syndromes, myelodysplastic myeloproliferative diseases, multiple
myeloma, chronic myeloproliferative disorders, nasal cavity and
paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral
cancer, oropharyngeal cancer, osteosarcoma/malignant fibrous
histiocytoma of bone, ovarian cancer, ovarian epithelial cancer,
ovarian low malignant potential tumor, pancreatic cancer,
parathyroid cancer, penile cancer, pheochromocytoma, pineoblastoma
and supratentorial primitive neuroectodermal tumors, pituitary
tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary
blastoma, prostate cancer, rectal cancer, renal pelvis and ureter
cancer, transitional cell cancer, rhabdomyosarcoma, salivary gland
cancer, Ewing's sarcoma, Kaposi's sarcoma, soft tissue sarcoma,
uterine sarcoma, sezary syndrome, skin cancer, including melanoma
and non-melanoma skin cancer, small intestine cancer, squamous cell
carcinoma, gastric cancer, supratentorial primitive neuroectodermal
tumors, testicular cancer, thymoma, thymoma and thymic carcinoma,
thyroid cancer, trophoblastic tumor, gestational, endometrial
uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer,
Waldenstrom's macroglobulinemia, Wilms' tumor.
[0165] In a more particular embodiment of the present invention,
the compounds of the present invention may be used in the treatment
of the following cancer types: Prostate, bladder, kidney (i.e.
renal), muscle, ovary, skin, lung, pancreas, breast, cervix, colon,
liver, connective tissue, placenta, bone, brain, uterus, salivary
gland, or testes.
[0166] In particular embodiments of the present invention, in said
cancer, the hedgehog signaling pathway is activated.
[0167] In particular embodiments of the present invention, in cells
of said cancer, the hedgehog signaling pathway is activated.
[0168] In the present invention patients wherein in said cancer, or
in cells of said cancer, the hedgehog signaling pathway is
activated are in short referred to as "Hedgehog dependent
patients", and patients wherein in said cancer, or in the cells of
said cancer, the hedgehog signaling pathway is not activated are in
short referred to as "Hedgehog independent patients". In the
present invention, patients e.g. can be classified as Hedgehog
dependent patients if the cancer said patient is suffering from is
described in the known scientific literature as being associated
with aberrant Hedgehog signaling pathway activity. In the present
invention, patients can also be stratified into Wnt dependent
patients and Wnt independent patients by a procedure comprising the
steps of [0169] 1) providing a sample from said patient, wherein
said sample comprises cancer cells from said patient, [0170] 2)
optionally subjecting said sample to a work-up step, [0171] 3)
adding a labeled antibody which specifically binds to at least one
protein playing a role in the hedgehog signaling pathway, or [0172]
adding a first antibody which specifically binds to at least one
protein playing a role in the hedgehog signaling pathway, and
subsequently adding a second antibody which specifically binds to
said first antibody, and wherein said second antibody is a labeled
antibody, [0173] 4) washing said sample after step 3, [0174] 5)
determining whether said labeled antibody is detectable in said
sample after step 4), [0175] 6) if in step 5) said marker moiety is
detectable, classifying said patient as Hedgehog dependent patient,
and if in step 5) said marker moiety is not detectable, classifying
said patient as Hedgehog independent patient.
[0176] Antibodies used in the present invention are typically
monoclonal antibodies.
[0177] The label in said labeled antibody can be selected from any
label typically used as antibody label in the field of
biochemistry, cellular biology, immunochemistry, etc., for a label
selected from the group comprising a fluorescence label, a dye, a
FRET label, a radioactive label. moiety, or an enzymatically active
moiety. Said enzymatically active moiety can process a reaction
which in turn results in the release of a detectable substance,
e.g. a dye.
[0178] In the above method of stratifying patients into Hedgehog
dependent patients and Hedgehog independent patients, the work-up
step is e.g. in particular embodiments selected from the group
comprising preservation, embedding, slicing and staining.
Preservation can be performed by cryopreservation or fixation by
e.g. formaldehyde or ethanol. Embedding the tumor material prepares
it for slicing. Staining can be performed with direct or indirect
methods. For further information and examples see DOI:
10.1354/vp.42-4-405 J. A. Ramos-Vara, Technical Aspects of
Immunohistochemistry, (2005) 42: 405 Vet Pathol.
[0179] In the context of the present invention, the expression
"said labeled antibody is detectable" means that by the state of
the art measurement methods used for detecting said label, no
signal relating to said label is detectable, and/or said signal is
not significant in relation to the background noise generated by
said measurement method.
[0180] In the above method to stratify patients into Hedgehog
dependent patients and Hedgehog independent patients, washing step
4 is to remove unbound and/or unspecifically bound antibodies from
step 3. In particular embodiments, said washing step comprises
washing with a buffer, e.g. a PBS buffer, and optionally a serum
protein, e.g. BSA. Washing step 4 can be repeated as necessary to
obtain a suitable signal/noise ratio, e.g. 2 or more, 3 or more, 4
or more times.
[0181] In certain embodiments of the above method to stratify
patients into Hedgehog dependent cancer patients and Hedgehog
independent cancer patients, background signal by unspecific
binding of antibodies is excluded by an isotype control. This
control can be utilized when working with monoclonal primary
antibodies. A comparative sample treated as above is incubated with
antibody diluent, supplemented with a non-immune immunoglobulin of
the same isotype (for example, IgG.sub.1, IgG2.sub.A, IgG2.sub.B,
IgM) and concentration as the aforementioned antibody. The sample
is then incubated with the labeled antibody and detection reagents.
These steps will help ensure that what appears to be specific
staining was not caused by non-specific interactions of
immunoglobulin molecules with the sample. Examples and a further
description of this method can be found in "Tissue
Microarrays--Methods in Molecular Biology Volume 664, 2010, pp
113-126 , Immunohistochemical Analysis of Tissue Microarrays;
Ronald Simon, Martina Mirlacher, and Guido Sauter".
[0182] In the context of the present invention the G
protein-coupled receptor Smoothened is interchangeably abbreviated
as "Smoothened" and "Smo".
[0183] In the context of the present invention the expression "the
activation of the hedgehog signaling pathway" in particular refers
to the activation of expression of primary target genes of the
Hedgehog signaling pathway, including GLI, HHIP, Ptch, more
particularly of GLI expression via the hedgehog pathway. Typically,
GLI expression is triggered via binding of hedgehog to the Smo/Ptch
(Smoothened/Patched) complex and thereupon GLI expression via
signalling by Smo.
[0184] In the context of the present invention said at least one
protein playing a role in the hedgehog signaling pathway can e.g.
be selected from the group comprising Patched, GLI, Smoothened,
HHIP, Hedgehog and SUFU.
[0185] In the context of the present invention, the term "GLI"
refers to members of the GLI protein family, such as GLI1, GLI2,
GLI3 in particular embodiments and, unless specified otherwise
particularly to GLI1.
[0186] In general, and unless specified otherwise, the proteins,
genes and/or gene expression products as defined herein in certain
embodiments also include variants of said proteins, genes and gene
expression products, such as isoforms, homologs and mutants
thereof, which share at least 95% sequence homology, more
particularly at least 97% sequence homology, even more particularly
at least 99% sequence homology with , the proteins, genes and/or
gene expression products as defined herein, and in the case of
proteins and/or gene expression products in certain embodiments
have essentially the same enzymatic activity as , the proteins
and/or gene expression products as defined herein, wherein however
the enzymatic activity of said variants may differ (i.e. be higher
or lower than) from the proteins, and/or gene expression products
as defined herein by up to two orders of magnitude, particularly up
to one order of magnitude, more particularly up to a factor of
2.
[0187] As used herein, the term "hedgehog signaling pathway" means
a cellular signaling pathway comprising an interaction with a
protein of the family known as hedgehog proteins, such as e.g. the
proteins commonly known as "sonic hedgehog" (UniProtKB/Swiss-Prot:
Q15465), "indian hedgehog" (UniProtKB/Swiss-Prot: Q14623) and
"desert hedgehog" (UniProtKB/Swiss-Prot: 043323) (Ingham and
McMahon, 2001).
[0188] As used herein, the term "sample" in principle comprises
samples from natural sources, such as a sample obtainable from a
mammal, and artificial samples, which are obtainable by admixing
several ingredients, wherein said ingredients may or may not be
derived from natural sources, and may e.g. comprise ingredients
selected from the group comprising synthetic and/or natural
proteins, peptides, oligo- or polynucleic acids, etc. In certain
embodiments, samples are from natural sources, which include bodily
fluids and/or tissue samples, such as bodily fluid and/or a tissue
sample obtainable from mammals. Said samples from natural sources
can be used in the present invention with or without further
processing after being obtained from their source, e.g. a mammal.
Such processing can for instance comprise separation,
fractionation, dilution, dispersion, mechanical treatment such as
sonification, or grinding, concentration, removal of certain
components of said sample, or addition of compounds, such as salts,
buffers, detergents, etc.
[0189] As used herein, the term "bodily fluid" or "body fluid"
specifies a fluid or part of a fluid originating from the body of a
patient, including fluids that are excreted or secreted from the
body of the patient, including but not limited to blood, including
peripheral blood, serum, plasma, urine, interstitial fluid, liquor,
aqueous humour and vitreous humour, bile, breast milk,
cerebrospinal fluid, endolymph, perilymph, ejaculate, gastric
juice, mucus, peritoneal fluid, pleural fluid, saliva, sweat, tears
and vaginal secretion, particularly peripheral blood, serum, plasma
and urine. Said bodily fluid itself may or may not comprise
diseased and/or non-diseased cells.
[0190] As used herein, the term "tissue sample" specifies a
non-fluid material or solid originating from the body of a patient.
Tissue samples include, but are not limited to samples of bone
material, bone marrow, skin, hair follicle, mucosa, brain,
cartilage, muscles, lung, kidney, stomach, intestines, bladder and
liver. Said tissue sample itself may or may not comprise diseased
cells, and may for instance be a sample taken from a diseased
region of a patient's body, such as a biopsy of a tumor. In certain
embodiments the tissue sample is selected from skin, hair follicle
or oral mucosa.
[0191] In the embodiments of the present invention, the sample is
obtained from the patient by any method and/or means commonly known
to the skilled person in the field of medicine, e.g. in certain
embodiments blood sample taking by venipuncture.
[0192] As used herein, the term "peripheral blood" specifies blood
obtained from the circulation remote from the heart, i.e. the blood
in the systemic circulation, as for example blood from acral
areas.
[0193] As used herein, the term "whole blood" specifies unmodified
blood comprising cells and fluid, as obtained from the donor of
said blood, such as a patient.
[0194] As used herein, the term "small molecule" is to be
understood as commonly used in the field of pharmacology and means
a low molecular weight organic compound which is not a polymer, and
which usually has a molecular weight of about 800 Daltons or lower,
particularly, 700 Daltons or lower, more particularly 600 Daltons
or lower, even more particularly 500 Daltons or lower. From a
functional point of view, a small molecule has to molecular weight
which allows the molecule to rapidly diffuse across cell membranes
and/or reach the interior of a mammalian cell.
[0195] In particular embodiments of the present invention, in said
cancer, the Wnt signaling pathway is activated.
[0196] In particular embodiments of the present invention, in cells
of said cancer, the Wnt signaling pathway is activated.
[0197] Multiple cancer types are associated with aberrant Wnt
signaling pathway activity. As reviewed by Anastas and Moon (Nature
Reviews Cancer, 2013, January, p. 11-26) hematological and solid
tumors including AML, ALL, Breast cancer , Adrenocortical cancer,
Colorectal Cancer, Oesophageal carcinoma, Gastric Cancer,
Glioblastoma, Lung Cancer, Prostate Cancer, Melanoma, HCC, Sarcoma,
Ovarian carcinoma, Pancreatic Cancer exhibit an aberrant Wnt
signaling pathway.
[0198] In the present invention patients wherein in said cancer, or
in cells of said cancer, the Wnt signaling pathway is activated are
in short referred to as "Wnt dependent patients", and patients
wherein in said cancer, or in the cells of said cancer, the Wnt
signaling pathway is not activated are in short referred to as "Wnt
independent patients". In the present invention, patients e.g. can
be classified as Wnt dependent patients if the cancer said patient
is suffering from is described in the known scientific literature
as being associated with aberrant Wnt signaling pathway activity,
such as e.g. the cancer types described above by Anastas and Moon.
In the present invention, patients can also be stratified into Wnt
dependent patients and Wnt independent patients by by a procedure
comprising the steps of [0199] 1) providing a sample from said
patient, wherein said sample comprises cancer cells from said
patient, [0200] 2) optionally subjecting said sample to a work-up
step, [0201] 3) adding a labeled antibody which specifically binds
to at least one protein playing a role in the Wnt signaling
pathway, or [0202] adding a first antibody which specifically binds
to at least one protein playing a role in the Wnt signaling
pathway, and subsequently adding a second antibody which
specifically binds to said first antibody, and wherein said second
antibody is a labeled antibody, [0203] 4) washing said sample after
step 3, [0204] 5) determining whether said labeled antibody is
detectable in said sample after step 4), [0205] 6) if in step 5)
said marker moiety is detectable, classifying said patient as Wnt
dependent patient, and if in step 5) said marker moiety is not
detectable, classifying said patient as Wnt independent
patient.
[0206] Said patients can further be stratified into Wnt dependent
patients and Wnt independent patients by determining by providing a
sample comprising cancer cells of said patient, culturing said
cancer cells in growth medium and comparing whether growth of said
cancer cells can be inhibited by a known Wnt inhibitor, as compared
to a control group of said cancer cells which is not treated said
Wnt inhibitor.
[0207] The label in said labeled antibody can be selected from any
label typically used as antibody label in the field of
biochemistry, cellular biology, immunochemistry, etc., for a label
selected from the group comprising a fluorescence label, a dye, a
FRET label, a radioactive label. moiety, or an enzymatically active
moiety. Said enzymatically active moiety can process a reaction
which in turn results in the release of a detectable substance,
e.g. a dye.
[0208] In the above method of stratifying patients into Wnt
dependent patients and Wnt independent patients, the work-up step
is e.g. in particular embodiments selected from the group
comprising preservation, embedding, slicing and staining.
Preservation can be performed by cryopreservation or fixation by
e.g. formaldehyde or ethanol. Embedding the tumor material prepares
it for slicing. Staining can be performed with direct or indirect
methods. For further information and examples see DOI:
10.1354/vp.42-4-405 J. A. Ramos-Vara, Technical Aspects of
Immunohistochemistry, (2005) 42: 405 Vet Pathol.
[0209] In the above method to stratify patients into Wnt dependent
patients and Wnt independent patients, washing step 4 is to remove
unbound and/or unspecifically bound antibodies from step 3. In
particular embodiments, said washing step comprises washing with a
buffer, e.g. a PBS buffer, and optionally a serum protein, e.g.
BSA. Washing step 4 can be repeated as necessary to obtain a
suitable signal/noise ratio, e.g. 2 or more, 3 or more, 4 or more
times.
[0210] In certain embodiments of the above method to stratify
patients into Wnt dependent cancer patients and Wnt independent
cancer patients, background signal by unspecific binding of
antibodies is excluded by an isotype control. This control can be
utilized when working with monoclonal primary antibodies. A
comparative sample treated as above is incubated with antibody
diluent, supplemented with a non-immune immunoglobulin of the same
isotype (for example, IgG.sub.1, IgG2.sub.A, IgG2.sub.B, IgM) and
concentration as the aforementioned antibody. The sample is then
incubated with the labeled antibody and detection reagents. These
steps will help ensure that what appears to be specific staining
was not caused by non-specific interactions of immunoglobulin
molecules with the sample. Examples and a further description of
this method can be found in "Tissue Microarrays--Methods in
Molecular Biology Volume 664, 2010, pp 113-126, Immunohistochemical
Analysis of Tissue Microarrays; Ronald Simon, Martina Mirlacher,
and Guido Sauter".
[0211] In the context of the present invention the expression "the
activation of the Wnt signaling pathway" in particular refers to
the activation of expression of primary target genes of the Wnt
signaling pathway, e.g. selected from the group comprising c-myc,
Cyclin D, TCF1, LEF1, PPARdelta, c-jun, fra-1, MMP7, Axin2, ITF-2,
CD44, BMP4, Survivin, VEGF, FGF18, FGF9, FGF20, Jagged, DKK1, LGR5,
SOX2, SOX9, and OCT4.
[0212] In the context of the present invention said at least one
protein playing a role in the Wnt signaling pathway can e.g. be
selected from the group comprising Frizzled receptors (particularly
FZD4, 5, 6, 7, ROR1, ROR2), Wnt ligands (particularly WNT1, 2, 3A,
5A, 7A), Wnt inhibitory factors (particularly WIF1), secreted
frizzled-related proteins (particularly SFRP3, 4), nuclear
B-Catenin, and TCF/LEF family members (particularly LEF1,
TCF7L2).
[0213] As used herein, the term "Wnt signaling pathway" means a
cellular signaling pathway comprising an interaction with a protein
of the family known as Wnt proteins (UniProtKB/Swiss-Prot: e.g.
P56704, O00755, Q9GZT5, O00744, Q93098, P41221, Q93097, O14905,
O14904, Q9UBV4, O96014, Q9H1J7, P56706, Q9Y6F9, Q9H1J5, P56705,
P56703, P09544, or P04628).
[0214] General Synthesis Procedure
[0215] In the following description of the synthesis procedures for
compounds of the present invention, which are exemplified for the
amides of Formula (Ia), the residues X, L and Y, where present,
have the meaning as defined above.
##STR00015## ##STR00016##
EXAMPLES
1. Synthesis of Precursors
1.1 Synthesis of
N-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)acetamide
##STR00017##
[0217] A solution of 5-Amino-2,2-difluoro-1,3-benzodioxole (26.0 g,
150.186 mmol) in dry toluene (410 ml) and acetic anhydride (16.2
ml, 1.15 eq.) was stirred at 100.degree. C. for 2 h.
[0218] Subsequently, the solvent was removed under reduced
pressure. The crude product was dissolved in 100 ml methanol to
remove traces of acetic anhydride. The solvent was subsequently
evaporated. The obtained crude product was recrystallized from
toluene. The obtained product was filtered off and dried under high
vacuo to obtain greyish-beige crystals (30.5 g, 92.5% yield, 98%
purity).
[0219] .sup.1H NMR (DMSO-d.sub.6+CCl.sub.4): 2.04 (3H, s,
CH.sub.3), 7.20-7.23 (1H, dd, CH-arom.), 7.30-7.33 (1H, s,
CH-arom.), 7.74-7.75 (1H, d, CH-arom.), 10.12 (1H, s, NH).
1.2 Synthesis of
N-(2,2-difluoro-6-nitrobenzo[d][1,3]dioxol-5-yl)acetamide
##STR00018##
[0221] N-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)acetamide (11.39 g,
52.938 mmol) was dissolved in glacial acetic acid (50.4 ml). To the
resulting mixture was added dropwise a mixture of fuming nitric
acid (6.1 ml, 3.35 eq.) in glacial acetic acid (20.2 ml, 0.28 eq.).
After addition, the reaction mixture was stirred for 22 h at room
temperature. The reaction mixture was stirred at 60.degree. C.
overnight.
[0222] Subsequently, the reaction mixture was poured into a of ice
and water mixture. The resulting precipitate was filtered off by
suction filtration. The crude product was then purified by column
chromatography on a silica gel flash column (eluent DCM:MeOH 95:5).
The product was isolated and concentrated in vacuo to obtain a
yellow solid (6.7 g, 49% yield, 100% purity).
[0223] .sup.1H NMR (DMSO-d.sub.6+CCl.sub.4): 2.09 (3H, s,
CH.sub.3), 7.76 (1H, s, CH-arom.), 8.15 (1H, s, CH-arom.), 10.33
(1H, s, NH).
1.3 Synthesis of
2,2-difluoro-6-nitrobenzo[d][1,3]dioxol-5-amine
##STR00019##
[0225] N-(2,2-difluoro-6-nitrobenzo[d][1,3]dioxol-5-yl)acetamide
(6.76 g, 25.985 mmol) was dissolved in methanol (676 ml). The
reaction mixture was cooled to 0.degree. C. Then sodium methylate
(ca. 25% in methanol) (30.3 ml, 5 eq.) was added and the resulting
mixture was stirred for 20 min at 0.degree. C. and subsequently for
25 min at 5.degree. C. The reaction was then interrupted by adding
glacial acetic acid (37.2 ml, 25 eq.).
[0226] The solvent was removed under reduced pressure, whereupon a
liquid-oily residue formed. The last traces of solvent, together
with the remaining glacial acetic acid, were removed by a two-fold
co-evaporation with toluene. Upon addition of toluene, a white
solid precipitated, which was filtered off by suction filtration.
The filtrate was concentrated in vacuo and dried under reduced
pressure. The crude product was purified by column chromatography
on a silica gel flash column (eluent DCM:MeOH 95:5). The first spot
as observed by thin layer chromatography under the same eluent
conditions was isolated and the fractions containing the product
were collected, concentrated in vacuo and dried to obtain an orange
powder.
[0227] .sup.1H NMR (DMSO-d6; CCl.sub.4): 6.95 (1H, s, CH-arom.),
7.79 (2H, s, NH.sub.2), 7.95 (1H, s, CH-arom.).
1.4 Synthesis of 2,2-difluorobenzo[d][1,3]dioxole-5,6-diamine
##STR00020##
[0229] 2,2-difluoro-6-nitrobenzo[d][1,3]dioxol-5-amine (770 mg,
3.53 mmol) was dissolved in dry methanol (100 ml) under an argon
atmosphere and hydrogenated using Raney Nickel as catalyst for 1 h
at room temperature.
[0230] The reaction mixture was filtered over CELLITE.RTM. and
washed with methanol. The solvent was evaporated and concentrated
in vacuo. A purple solid precipitated and was dried under reduced
pressure. The crude greyish product (530 mg, 53% yield) was
purified by column chromatography on a silica gel flash column
(eluent DCM:MeOH 95:5). The selected fractions were combined and
concentrated in vacuo. The crude product was precipitated as a
hydrochloride using a 1.25 M hydrogen chloride solution in ethanol
and an excess of ethyl acetate. The resulting suspension was
stirred overnight. Subsequently, the solid was filtered off
Afterwards the obtained
2,2-difluorobenzo[d][1,3]dioxole-5,6-diamine hydrochloride was
dissolved in water and extracted with ethylacetate. The aqueous
layer was alkalized to pH 8-10 and extracted with ethyl acetate.
The organic layer was then dried over magnesium sulfate,
concentrated in vacuo and dried to obtain a brown solid (98%
purity).
[0231] .sup.1H NMR (DMSO-d6+CCl.sub.4): 4.52 (4H, s,
2.times.NH.sub.2), 6.52 (2H, s, CH-arom.).
1.5 Synthesis of
2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-amine
hydrobromide
##STR00021##
[0233] To a solution of 5,6-Diamino-2,2-difluoro-1,3-benzodioxole
(4.91 g, 0.0261 mol) in dry methanol (98 ml) was added cyanogen
bromide (3.23 g, 1.3 eq.). The reaction mixture was stirred
overnight at room temperature.
[0234] The reaction mixture was subsequently concentrated in vacuo.
The residue was then washed with dichloromethane and the
precipitate was filtered off and dried to obtain a brown solid
(6.72 g, 88% yield, 98% purity).
[0235] .sup.1H NMR (DMSO-d6+CCl.sub.4): 7.47 (2H, s,
2.times.CH-arom.), 8.46 (2H, s, NH.sub.2), 12.58 (1H, s, NH).
2. Synthesis of Final Compounds (I)
[0236] 2.1 General Procedure 1
##STR00022##
[0237] To a solution of II (1-2 eq.) in dry DMF, dioxane or
dichloromethane, particularly dry DMF (dimethylformamide) (3-10 ml)
were added IV (1 mmol). HBTU
(2-(1H-Benzotriazole-1-yl)-1,1,3,3-Tetramethyluronium
hexafluorophosphate) (1-1.4 eq.), DIPEA (N,N-Diisopropylethylamine)
(1-5 eq.) or triethylamine (5 eq.), particularly DIPEA, and
optionally DMAP (4-Dimethylaminopyridine) (0.1 eq.) were also added
to the reaction mixture. The reaction temperature was usually in
the range of from rt to 85.degree. C., particularly rt. The
reaction was usually allowed to process overnight (which as used
herein specifies a duration of approximately between 12 and 24 h,
depending on the reaction velocity).
[0238] After completion of the reactions, the reaction solution was
subjected to one or more after-treatments including:
[0239] A) Extraction with organic solvents: The residue obtained
from the reaction was dissolved in an organic solvent (such as
ethyl acetate or dichloromethane) and was washed at least once with
an aqueous 5% NaHCO.sub.3, aqueous 5% citric acid and water. The
organic layer was dried over magnesium sulfate and concentrated in
vacuo.
[0240] B) Chromatography: The crude product obtained from the
reaction was purified by column chromatography on a silica gel
flash column, by preparative TLC (thin layer chromatography) or
preparative HPLC (high pressure liquid chromatography) with a
defined eluent proportion. After completion of the reaction, the
crude product was purified by siliga gel flash column
chromatography (CHCl.sub.3/EtOH=80:1+1 drop of HOAc).
[0241] C) Recrystallization: The crude product was crystallized
from ethanol (with activated carbon).
[0242] D) Precipitation: After completion of the reaction, the
reaction mixture was diluted with water, hexane or an aqueous
Na.sub.2CO.sub.3-solution and/or was poured into ice water and the
formed precipitate was filtered off.
[0243] E) Washing: The obtained solid (e.g. obtained by filtration)
was washed with water, aqueous HCl or Na.sub.2CO.sub.3-solution
and/or organic solvents.
[0244] F) Suspending, followed by filtration: The crude product was
suspended in Et.sub.2O (diethyether), filtered off and dried.
[0245] G) Neutralization and recovery: After completion of the
reaction, the solvent was evaporated in vacuo, water was added and
the precipitate was formed. An aqueous 3% ammonia solution, or
alternatively a sodium hydrogen carbonate solution was added to the
suspension till pH=8. After 30 min of stirring the precipitate was
filtered off or the dissolved product was extracted.
2.2 Synthesis of
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-2-(2-
-(trifluoromethoxy)benzamido)thiazole-4-carboxamide (1)
##STR00023##
[0247]
2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-amine
(3.28 mmol) and
2-(2-(trifluoromethoxy)benzamido)thiazole-4-carboxylic acid (3.28
mmol) were dissolved in dry dimethylformamide,
2-(1H-Benzotriazole-1-yl)-1,1,3,3-Tetramethyluronium
hexafluorophosphate (HBTU) (3.28 mmol) and
N,N-Diisopropylethylamine (DIPEA) (6.55 mmol) were added and the
reaction mixture was stirred for 16 h at 85.degree. C. The reaction
mixture was then concentrated in vacuo. The residue was dissolved
in EtOAc and was washed twice with an aqueous 5% sodium hydrogen
carbonate solution, aqueous 5% citric acid solution and water. The
organic layer was dried over magnesium sulfate and was concentrated
in vacuo. The product was purified by silica gel flash
chromatography with petrol ether/ethyl acetate. .sup.1H NMR
(DMSO+d.sub.6; CCl.sub.4): 7.48 (2H, s, CH-arom.), 7.54-7.59 (2H,
m, CH-arom.), 7.70-7.76 (1H, m, CH-arom.), 7.80-7.83 (1H, dd,
CH-arom.), 8.35 (1H, s, CH-thiazol), 11.14 (1H, s, NH), 12.49 (1H,
s, NH), 13.12 (1H, s, NH).
2.3 General Procedure 2
##STR00024##
[0249] II (1-1.5 eq.) was added to the suspension of IV (1 eq.) in
dry pyridine (1-3 ml). The resulting mixture was stirred at rt
overnight.
[0250] After completion of the reaction, the reaction solution,
where necessary, was subjected to after-treatments such as the ones
defined above.
2.4 Synthesis of
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)benza-
mide (49)
##STR00025##
[0252] Benzoylchloride (0.179 g, 1.5 eq.) was added to the
suspension of IV (0.25 g, 0.85 mmol) in dry pyridine (3 ml). The
resulting mixture was stirred at rt overnight. The reaction mixture
was diluted with water (20 ml) and the formed precipitate was
filtered off, washed with water and recrystallized from ethanol to
obtain a pure product (0.1 g, 37% yield). .sup.1H NMR (400 MHz,
DMSO-d.sub.6+CCl.sub.4): 7.32 (2H, s, CH-arom.), 7.44-7.54 (2H, m,
CH-arom.), 7.54-7.64 (1H, m, CH-arom.), 8.13 (2H, d, CH-arom.),
12.29 (2H, s, NH).
2.5 General Procedure 3
[0253] I (1-1.5 eq.) was refluxed in SOCl.sub.2 (3-5 ml) for 2 h.
An excess of SOCl.sub.2 was evaporated in vacuo and pyridine (3 ml)
was added to the resulting residue. The mixture was then stirred
for 10 min., IV (1 mmol) was added and the resulting mixture was
stirred overnight at rt. After completion of the reaction, the
reaction solution was subjected, where necessary, to
after-treatments such as the ones defined above.
2.6 Synthesis of
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-4-me-
thyl-1,2,3-thiadiazole-5-carboxamide (42)
##STR00026##
[0255] 4-Methyl-1,2,3-thiadiazole-5-carboxylic acid (0.105 g, 1.43
eq.) was refluxed in SOCl.sub.2 (4 ml) for 2 h. Excess SOCl.sub.2
was evaporated in vacuo and pyridine (3 ml) was added to the
resulting residue. The obtained mixture was stirred for 10 min,
2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]-benzo[1,2-d]imidazol-6-amine
hydrobromide (0.15 g, 0.51 mmol) was added and stirring was
continued overnight. Water (25 ml) was then added and the resulting
suspension was stirred for 1 h. The obtained precipitate was
filtered off, washed with water, dried and recrystallized from
ethanol (with activated carbon) to obtain a pure solid (90 mg, 52%
yield). .sup.1H NMR (400 MHz, DMSO+CCl.sub.4): 2.96 (3H, s,
CH.sub.3), 7.33 (2H, s, CH-benzimidazole), 12.78 (2H, s, NH).
2.7 General Procedure 4
##STR00027##
[0257] V (1-1.5 eq.) was added to a suspension of compound 65 (1
mmol) in DCE (1,2-dichloroethene) (15 ml) and the mixture was
stirred for 1 h. Then sodiumtriacetoxyborohydride (2 eq.), and
acetic acid (0.5-2 ml) were added and the reaction mixture was
stirred for 2 days. After completion of the reaction, the reaction
solution was subjected, where necessary, to after-treatments such
as the ones defined above
2.8 Synthesis of
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-4-(e-
thylamino)benzamide (77)
##STR00028##
[0259] Acetaldehyde (0.013 ml, 1.1 eq.) was added to a suspension
of compound 65 (0.07 g, 0.21 mmol) in DCE (15 ml) and the mixture
was stirred for 1 h. Then sodiumtriacetoxyborohydride (0.089 g, 2
eq.) and acetic acid (0.3 ml) were added and the reaction mixture
was stirred for 2 days. The mixture was then neutralized with an
aqueous 10% sodium hydrogen carbonate solution and extracted with
dichloromethane. The combined organic extracts were dried over
MgSO.sub.4, filtered and concentrated in vacuo. The residue was
purified by siliga gel flash column chromatography
(CHCl.sub.3/EtOH=80:1+1 drop of HOAc) to obtain the pure product
(20.3 mg, 39% yield). .sup.1H NMR (400 MHz, DMSO-d.sub.6): 1.24
(3H, t, CH.sub.3), 3.16 (2H, q, CH.sub.2), 6.29 (1H, s, NH), 6.57
(2H, d, CH-arom.), 7.32 (2H, s, CH-benzimidazole), 7.93 (2H, d,
CH-arom.), 11.48 (1H, s, NH), 12.40 (1H, s, NH)
3. Alternative Procedures for the Synthesis of Final Compounds
3.1 Synthesis of
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-5-(2-
,3-dihydrobenzofuran-5-yl)thiophene-3-carboxamide (38)
##STR00029##
[0261] Compound 29 (0.1 g, 0.2486 mmol) and
(2,3-dihydrobenzofuran-5-yl)boronic acid (0.612 g, 1.5 eq) were
suspended in DME (1,2-dimethoxyethane).
Tetrakis(triphenylphosphine)-palladium(0) (0.03 g, 0.2 eq) and an
aqueous 2M Na.sub.2CO.sub.3 solution (0.5 ml, 4 eq.) were added.
The reaction mixture was stirred 20 h at 100.degree. C.,
subsequently dissolved in ethyl acetate and washed twice with
water. The organic layer was concentrated in vacuo, the residue
dissolved in methanol and purified by preparative HPLC to obtain
the pure product (4 mg, 4% yield).
3.2 Synthesis of
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)cyclo-
hexanecarboxamide (80)
##STR00030##
[0263] IV (0.06 g, 0.2 mmol) was refluxed in chloroform with
cyclohexanecarbonyl chloride (32 .mu.l, 1.1 eq.) in the presence of
DIPEA (0.1 ml, 0.57 eq.) during 2 days. Subsequently, the solvent
was evaporated and the residue was purified by silica gel flash
column chromatography (eluent: ethyl acetate) to obtain the pure
product (0.04 g, 61% yield). .sup.1H-NMR (400 MHz, DMSO-d6):
1.26-1.35 (5H, m, CH2), 1.38-1.42 (1H, m, CH2), 1.51-1.86 (4H, m,
CH2), 3.07-3.45 (1H, m, CH), 7.18 (1H, s, CH-arom.), 7.31 (1H, s,
Ch-arom.), 11.36 (1H, s, NH), 12.11 (1H, s, NH).
3.3 Synthesis of
4-amino-N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6--
yl)benzamide (60)
##STR00031##
[0265]
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl-
)-4-nitrobenzamide (0.32 g, 0.88 mmol) was suspended in ethanol (20
ml), hydrazine hydrate (0.3 ml) and Pd/C (0.16 g), were added to
the suspension and the mixture was refluxed during 1.5 h. The
catalyst was then filtered off; the solution was evaporated in
vacuum till dryness. The residue was washed with water to obtain
the pure product (0.23 g, 79% yield). NMR .sup.1H (400 MHz,
DMSO-d.sub.6): 5.68 (2H, s, CH-arom.), 6.68 (2H, d, CH-arom.), 7.24
(2H, s, NH.sub.2), 7.86 (2H, d, CH-arom.), 11.37 (1H, s, NH), 12.31
(1H, s, NH).
4. Synthesis of Intermediates
4.1 Synthesis of ethyl
2-(2-(trifluoromethoxy)benzamido)thiazole-4-carboxylate
##STR00032##
[0267] To a solution of Ethyl 2-aminothiazole-4-carboxylate (12 g,
70 mmol, 1 eq.) in dry THF (300 ml) was added DIPEA (250 ml, 209
mmol, 3 eq.). A solution of 2-(Trifluoromethoxy)-benzoyl chloride
(19 g, 84 mmol, 1.2 eq.) in THF (50 ml) was then added dropwise at
0.degree. C. The reaction mixture was stirred for 24 h at rt. Water
(50 ml) was then added and THF was removed under reduced pressure.
The obtained residue was extracted with DCM (dichloromethane). The
organic layer was dried over MgSO.sub.4, filtered and concentrated
under reduced pressure. The residue was purified by flash column
chromatography on silica gel (PE/EtOAc 80:20). The product was
obtained as a white solid (12 g, 33 mmol, 48% yield). .sup.1H NMR
(DMSO-d.sub.6): 1.28-0.133 (3H, t, CH.sub.3), 4.26-4.33 (2H, q,
CH.sub.2), 7.51-7.57 (2H, m, CH-arom.), 7.68-7.74 (1H, m,
CH-arom.), 7.78-7.81 (1H, dd, CH-arom.), 8.14 (1H, s, CH-thiazole),
13.11 (1H, s, NH).
4.2 Synthesis of
2-(2-(trifluoromethoxy)benzamido)thiazole-4-carboxylic acid
##STR00033##
[0269] Ethyl
2-(2-(trifluoromethoxy)benzamido)thiazole-4-carboxylate (10 g, 28
mmol, 1 eq.) was dissolved in THF (20 ml) and an aqueous 2M NaOH
solution (110 ml) was added at rt. The reaction mixture was stirred
for 24 h at rt. THF was then removed under reduced pressure. The
residual aqueous phase was acidified to pH=1-2 using 15% aqueous
HCl. The precipitate was collected by filtration, washed with water
and dried. The product was obtained as a white solid (10.4 g, 31
mmol, yield >90%). .sup.1H NMR (DMSO-d.sub.6): 7.51-7.57 (2H, m,
CH-arom.), 7.68-7.74 (1H, m, CH-arom.), 7.78-7.81 (1H, dd,
CH-arom.), 8.06 (1H, s, CH-thiazole), 13.01 (1H, s, NH).
4.3 Synthesis of ethyl
2-((4-methyl-6-(trifluoromethyl)pyrimidin-2-yl)amino)thiazole-4-carboxyla-
te
##STR00034##
[0271] Ethyl 2-(diaminomethyleneamino)thiazole-4-carboxylate (2 g,
9,3 mmol, 1 eq.) was dissolved in EtOH (200 ml) and
1,1,1-trifluoropentane-2,4-dione (1.2 ml, 9.3 mmol, 1 eq.) was
added. The reaction mixture was stirred under reflux for 3.5 h.
EtOH was then partly removed under reduced pressure until a
precipitate was formed. The precipitate was collected by
filtration, washed with EtOH and dried. The product was obtained as
a light yellow solid (2.2 g, 6.6 mmol, 71% yield). .sup.1H NMR
(DMSO-d.sub.6): 1.28-1.33 (3H, t, CH.sub.3), 2.58 (3H, s,
CH.sub.3), 4.25-4.32 (2H, q, CH.sub.2), 7.45 (1H, s,
CH-pyrimidine), 8.04 (1H, s, CH-thiazole), 12.39 (1H, s, NH).
4.4 Synthesis of
2-((4-methyl-6-(trifluoromethyl)pyrimidin-2-yl)amino)thiazole-4-carboxyli-
c acid
##STR00035##
[0273] Ethyl
2-(4-methyl-6-(trifluoromethyl)pyrimidin-2-ylamino)thiazole-4-carboxylate
(2.18 g, 6.56 mmol, 1 eq.) and LiOH (550 mg, 13.1 mmol, 2 eq.) were
dissolved in a mixture of MeOH and water (52 ml:18 ml). The
reaction mixture was stirred at room temprature for 2 h. The
reaction mixture was then acidified to pH=2 using an aqueous 10%
HCl solution. MeOH was then removed under reduced pressure. The
precipitate formed was collected by filtration to afford the
product as a light yellow solid (2 g, 6.5 mmol, 99% yield). .sup.1H
NMR (DMSO-d6): 2.64 (3H, s, CH.sub.3), 7.50 (1H, s, CH-pyrimidine),
8.03 (1H, s, CH-thiazole), 12.39 (1H, s, NH).
4.5 Synthesis of ethyl
2-((2,6-dimethoxypyrimidin-4-yl)amino)thiazole-5-carboxylate
##STR00036##
[0275] 1-(2,6-dimethoxypyrimidin-4-yl)thiourea (6.133 g, 28.626
mmol) was suspended in dry DMF. The bromo pyruvate (6.699 g, 34.351
mmol) was dissolved in dry DMF and added dropwise to the mixture.
The suspension cleared off and was stirred for 2 h at rt. The
mixture was concentrated in vacuo to obtain the product as a
yellowish solid (12.9 g, yield: >100%). LC/MS [M+H].sup.-:
310.96
4.6 Synthesis of
2-((2,6-dimethoxypyrimidin-4-yl)amino)thiazole-5-carboxylic
acid
##STR00037##
[0277] ethyl
2-((2,6-dimethoxypyrimidin-4-yl)amino)thiazole-5-carboxylate (9.0
g, 29 mmol) was suspended in EtOH (100 ml) and 2N NaOH (50 ml) was
added and the mixture was stirred for 4 h at it A precipitate
formed. Further 2N NaOH (40 ml) was added and the reaction mixture
was stirred for 16 h at rt. The mixture was concentrated in vacuo.
Upon addition of HCl (2 N) a precipitate was formed, which was
filtered off and washed with water. The precipitate was dried in
vacuo to obtain 11.58 g (89% yield) of the pure product. LC/MS
[M+H].sup.+: 284,36
4.7 Synthesis of ethyl
5-(2-(trifluoromethoxy)benzamido)-1,2,4-thiadiazole-3-carboxylate
##STR00038##
[0279] 2-(Trifluoromethoxy)benzoyl chloride (1.1 g, 6.5 mmol, 1
eq.) was dissolved in THF (130 ml) and 2-(trifluoromethoxy)benzoyl
chloride (1.5 g, 6.5 mmol, 1 eq.) and DIPEA (1.1 ml, 6.5 mmol, 1
eq.) were added. The reaction mixture was stirred at room
temperature for 18 h. Subsequently, all volatiles were removed
under reduced pressure. The obtained residue was triturated with
ice water. The precipitate formed was collected by filtration and
dried. The crude product was purified by flash column
chromatography on silica gel (DCM/MeOH 95:5). The product was
obtained as a light yellow solid (1.05 g, 2.9 mmol, 45% yield) and
was used as such further. LC/MS [M+H].sup.+: 361.86; .sup.1H NMR
(DMSO-d.sub.6): 1.31-1.36 (3H, t, CH.sub.3), 4.34-4.41 (2H, q,
CH.sub.2), 7.56-7.58 (2H, m, CH-aromat), 7.75-7.81 (1H, m,
CH-aromat), 7.88-7.92 (1H, dd, CH-aromat), 13.97 (1H, s, NH).
4.8 Synthesis of
5-(2-(trifluoromethoxy)benzamido)-1,2,4-thiadiazole-3-carboxylic
acid
##STR00039##
[0281] Ethyl
5-(2-(trifluoromethoxy)benzamido)-1,2,4-thiadiazole-3-carboxylate
(1 g, 2.9 mmol, 1 eq.) and LiOH (244 mg, 5.8 mmol, 2 eq.) were
dissolved in a mixture of EtOH and H.sub.2O (3:1) (40 ml) and the
reaction mixture was stirred at room temperature for 2 h. The
mixture was then acidified to pH=5 using an aqueous 10% HCl
solution. The reaction mixture was then concentrated in vacuo.
After cooling with an ice-bath, a precipitate formed, which was
filtered off, washed with water and dried in vacuo. The product was
obtained as a white solid (638 mg, 1.9 mmol, 66% yield). LC/MS
[M+H].sup.+: 333.87
4.9 Synthesis of ethyl
2-(2,3-dihydrobenzofuran-5-yl)oxazole-4-carboxylate
##STR00040##
[0283] 2-Chloro-oxazole-4-carboxylic acid ethyl ester (1.5 g, 8.5
mmol, 1 eq.) and 2,3-Dihydro-1-benzofuran-5-ylboronic acid (2.1 g,
12.8 mmol, 1.5 eq.) were suspended in DME (170 ml).
Tetrakis(triphenylphosphine)palladium(0) (987 mg, 0.85 mmol, 0.1
eq.) and an aqueous 2M sodium carbonate solution (17.1 ml, 34.2
mmol, 4 eq.) were added. The reaction mixture was stirred at
90.degree. C. for 6 h. DME was then removed under reduced pressure.
The obtained residue was dissolved inEtOAc and was washed three
times with water. The organic layer was dried over MgSO.sub.4,
filtered and concentrated in vacuo. The obtained crude product was
purified by flash column chromatography on silica gel (PE/EtOAc 9:1
to 8:2). The fractions containing the product were collected and
concentrated in vacuo. The product was obtained as a yellow oil
(472 mg, Purity: ca. 50%). LC/MS [M+H].sup.-: 259.10
4.10 Synthesis of
2-(2,3-dihydrobenzofuran-5-yl)oxazole-4-carboxylic acid
##STR00041##
[0285] To a solution of Ethyl
2-(2,3-dihydrobenzofuran-5-yl)oxazole-4-carboxylate (472 mg, 1.8
mmol, 1 eq.) in a mixture of THF and H.sub.2O (2:1; 15 ml) was
added LiOH (402 mg, 9.6 mmol, 5 eq.) at rt. The reaction mixture
was stirred at rt for 18 h. The reaction mixture was then
concentrated in vacuo and partitioned between water (50 ml) and
Et.sub.2O (50 ml). The aqueous layer was acidified to pH=2-3 with
an aqueous 1 M HCl solution. The resulting aqueous layer was
extracted twice with EtOAc. The organic layer obtained from the
extraction with EtOAc was dried over MgSO.sub.4, filtered and
concentrated in vacuo. The product was obtained as a brown
crystalline solid (168 mg, 0.7 mmol, 40% yield). LC/MS [M+H].sup.+:
231.87
4.11 Synthesis of ethyl
2-(3-(2-(trifluoromethoxy)phenyl)ureido)thiazole-4-carboxylate
##STR00042##
[0287] To a solution of ethyl 2-aminothiazole-4-carboxylate (1 g,
5.8 mmol, 1 eq.) in dry DCM (20 ml) was added a mixture of
1-isocyanato-2-(trifluoromethoxy)benzene (1.2 g, 5.8 mmol, 1 eq.)
in dry DCM (10 ml). The reaction mixture was stirred at room
temperature for 3 h. The precipitate formed was collected by
filtration, washed with DCM and dried. The product was obtained as
a white solid (1.9 g, 5 mmol, 86% yield). LC/MS [M+H].sup.+:
376.12
4.12 Synthesis of
2-(3-(2-(trifluoromethoxy)phenyl)ureido)thiazole-4-carboxylic
acid
##STR00043##
[0289] To a solution of ethyl
2-(3-(2-(trifluoromethoxy)phenyl)ureido)thiazole-4-carboxylate (1.5
g, 4.11 mmol, 1 eq.) in dry dioxane (10 ml) was added dropwise an
aqueous 2M NaOH solution (2.3 ml, 4.5 mmol, 1.1 eq.) at 0.degree.
C. The reaction mixture was stirred at rt for 8 h. After addition
of an aqueous 2M HCl solution (2.3 ml) a solid precipitated. The
solid was collected by filtration, washed with water and dried. The
product was obtained as a white solid (1.4 g, 3.9 mmol, 95% yield).
LC/MS [M+H].sup.+: 348.06
4.13 Synthesis of methyl
3-(2-(trifluoromethoxy)benzamido)-1H-1,2,4-triazole-5-carboxylate
##STR00044##
[0291] 2-(trifluoromethoxy)benzoyl chloride (0.26 ml, 1.2 eq.) was
added dropwise to a suspension of methyl
3-amino-1H-1,2,4-triazole-5-carboxylate (0.22 g, 1.4 mmol) in
pyridine (3 ml). The reaction mixture was stirred at rt overnight.
Then the mixture was diluted with water (20 ml) and stirred for 30
min. A colorless oil was formed. The Water/pyridine solution was
decanted and the residual oil was treated with hexane to obtain
0.167 g of a colorless precipitate, which was used in the
subsequent synthesis steps without further characterization.
4.14 Synthesis of
3-(2-(trifluoromethoxy)benzamido)-1H-1,2,4-triazole-5-carboxylic
acid
##STR00045##
[0293] To a solution of methyl
3-(2-(trifluoromethoxy)benzamido)-1H-1,2,4-triazole-5-carboxylate
(0.16 g) in the mixture of ethanol, water and potassium hydroxide
(15 ml/15 ml/0.15 g) was refluxed for 15 min. Ethanol was
subsequently removed. The resulting solution was diluted with water
and neutralized with HCl until pH=3. The colorless precipitate was
filtered off and washed with water to obtain 0.117 g of the pure
product. NMR .sup.1H (400 MHz, DMSO-d.sub.6): 7.41 (1H, d,
CH-arom.), 7.49 (1H, t, CH-arom.), 7.65 (1H, t, CH-arom.), 7.73
(1H, d, CH-arom.), 12.29 (1H.s, NH), 12.98 (1H, s, OH), 14.03 (1H,
s, NH).
4.15 Synthesis of ethyl
5-acetamido-1H-1,2,4-triazole-3-carboxylate
##STR00046##
[0295] A suspension of ethyl
5-amino-1H-1,2,4-triazole-3-carboxylate in acetic anhydride was
refluxed for 30 min. Excess acetic anhydride was evaporated. Water
was added to the residue and the mixture was stirred overnight. The
colourless product was filtered off, washed with water and dried to
obtain 0.134 g (53% yield) of the product. .sup.1H-NMR (40 MHz,
DMSO-d.sub.6): 1.35 (3H. t. OCH.sub.2CH.sub.3), 2.12 (3H, s,
COCH.sub.3), 4.3 (2H, q, OCH.sub.2CH.sub.3), 11.71 (1H, s, NHCO),
13.74 (1H, s, NH-triazole).
4.16 Synthesis of 5-acetamido-1H-1,2,4-triazole-3-carboxylic
acid
##STR00047##
[0297] A solution of ethyl
5-acetamido-1H-1,2,4-triazole-3-carboxylate (0.13 g, 0.66 mmol) in
NaOH/H.sub.2O (0.079 g/5 ml) was stirred for 6 h. The solution was
then acidified with conc. HCl to pH=2 and the colorless precipitate
was filtered off and dried to give the pure product (0.076 g, 69%
yield). .sup.1H-NMR (40 MHz, DMSO-d.sub.6): 2.12 (3H,s,
COCH.sub.3), 11.65 (1H, s, NHCO), 13.67 (1H, s, NH-triazole).
4.17 Synthesis of
(6-Amino-2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-5-yl-
)(1-methyl-1H-pyrrol-2-yl)methanone
##STR00048##
[0299] The compound was synthesized by the aforementioned general
synthesis protocols 1, D and C as described above.
[0300] Other carboxylic acid derivatives were synthezised
analogously to the aforemnetioned synthesis protocols, which are to
be understood as exemplary synthesis protocols.
5. Alternative Procedures for the Synthesis of Final Compounds
5.1 Synthesis of
4-Amino-N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6--
yl)-1H-pyrrole-2-carboxamide (15B)
##STR00049##
[0302]
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl-
)-4-nitro-1H-pyrrole-2-carboxamide (XX) (110.0 mg, 0.313 mmol) was
dissolved in 10 ml methanol and palladium on carbon (10%/C, 53.33
mg, 0.501 mmol) was added. The reaction flask was then purged with
hydrogen and the reaction mixture was stirred for 2 h under an
hydrogen atmosphere. The reaction mixture was filtered over celite
and the filtrate was concentrated in vacuo. The crude solid was
washed with water and dried. The product was obtained as a brown
solid (28 mg, 0.09 mmol, 28% yield).
5.2 Synthesis of
3-((2,2-Difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)carb-
amoyl)benzoic acid (18B)
##STR00050##
[0304] To a solution of ethyl
2-(2,3-dihydrobenzofuran-5-yl)oxazole-4-carboxylate (32 mg, 0.085
mmol) in 6 ml THF/H.sub.2O (1:1), Lithiumhydroxid monohydrate (56%
LiOH, 25 mg, 0.597 mmol) was added at room temperature The reaction
mixture was stirred overnight at room temperature. The reaction
mixture was concentrated in vacuo and the aqueous layer was
acidified to pH 2-3 with an aqueous 1 mol/1 hydrochlorid acid
solution. The resulting solid was filtered off and dried. The
product was obtained as a bright brown solid (14 mg, 0.04 mmol, 46%
yield).
5.3 Synthesis of
N-(2,2-Difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-1-me-
thyl-1H-pyrrole-2-carboxamide (20B)
##STR00051##
[0306] A suspension of
(6-amino-2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-5-yl-
)(1-methyl-1H-pyrrol-2-yl)methanone (37.82 mg, 0.118 mmol) in 3 ml
xylene and 1 ml DMF was refluxed for 4 h. The reaction mixture was
lyophilized. The crude product was purified by preparative TLC
(DCM:MeOH 9:1) (PLC silica gel 60 F.sub.254, 1 mm). The highest
spot was isolated and purified by a second preparative TLC with the
same conditions as described above. The product was obtained as a
beige solid (6 mg, 0.02 mmol, 16% yield).
6. Analysis of Synthesis Products
[0307] Analytical LC/ESI-MS parameters: Waters 2700 Autosampler.
1.times. Waters 1525 Multisolvent Delivery System 5 .mu.L sample
loop. Column, Phenomenex Onyx.TM. Monolythic C18 50.2 mm, with
stainless steel 2 .mu.m prefilter. Eluent A, H.sub.2O+0.1% HCOOH;
eluent B, MeCN. Gradient, 5% B to 100% B within 3.80 min, then
isocratic for 0.20 min, then back to 5% B within 0.07 min, then
isocratic for 0.23 min; flow, 0.6 mL/min and 1.2 mL/min. Waters
Micromass ZQ single quadrupol mass spectrometer with electrospray
source. MS method, MS4_15minPM-80-800-35V; positive/negative ion
mode scanning, m/z 80-800 or 80-900 in 1 s; capillary voltage, 3.50
kV; cone voltage, 35 V; multiplier voltage, 650 V; probe and
desolvation gas temperature, 120.degree. C. and 300.degree. C.,
respectively. Waters 2487 Dual .lamda. Absorbance Detector set to
254 nm. Software: Waters Masslynx V 4.0.
7. Exemplary Compounds of the Present Invention
TABLE-US-00001 [0308] TABLE 1 Exemplary compounds. The symbols in
the columns "CK1 delta Assay" and "CK1 epsilon Assay" have the
following meanings: +++: IC.sub.50 < 200 nM, ++: IC.sub.50
200-1000 nM, +: IC.sub.50 > 1 .mu.M, each determined according
to the kinase assays as described herein below. The symbols in the
column "HH Assay" have the following meanings: +++: IC.sub.50
.ltoreq. 500 nM, ++: IC.sub.50 > 500-1000 nM, +: IC.sub.50 1-15
.mu.M, each determined according to the Hedgehog reporter assay as
described herein below. The symbols in the column "Wnt Assay" have
the following meanings: +++: IC.sub.50 < 5 .mu.M, ++: IC.sub.50
5-20 .mu.M, each determined according to the Wnt reporter assay as
described herein below. The indications in the column "General
procedure/aftertreatment" refers to the protocols as described
above. CK1 CK1 General delta epsiplon HH Wnt procedure/ No.
Structure Assay Assay Assay Assay [M + H].sup.+ aftertreatment 1
##STR00052## +++ +++ + +++ 527.85 1 A, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(2-
(trifluoromethoxy)benzamido)thiazole-4-carboxamide 2 ##STR00053##
+++ ++ + 415.89 1 A, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-1-(4-fluorophenyl)-5-methyl-1H-
pyrazole-4-carboxamide 3 ##STR00054## +++ ++ +++ 442.88 1 A, F
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(2,3-dihydrobenzofuran-5-
yl)thiazole-4-carboxamide 4 ##STR00055## ++ ++ 401.05 1 E
N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-2-phenylthiazole-4-carboxamide 5 ##STR00056## + + 469.00 1 A, F
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(4-(trifluoromethyl)phenyl)thiazole-4-
carboxamide 6 ##STR00057## + + 468.76 1 A, F
2-(2,3-dichlorophenyl)-N-(2,2-difluoro-
5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)thiazole-4-carboxamide 7 ##STR00058## + + + 334.74
1 A, F 3-amino-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)pyrazine-2-carboxamide 8 ##STR00059## ++ ++ 477.89
1 A 2-(4-chlorobenzamido)-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)thiazole-4-carboxamide 9 ##STR00060## + + 454.02 1 A, F
2-cinnamamido-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)oxazole-
4-carboxamide 10 ##STR00061## ++ + 417.88 1 F
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(furan-2-carboxamido)oxazole-4- carboxamide 11
##STR00062## +++ +++ 434.03 1 A, F N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-2-
(furan-2-carboxamido)thiazole-4-carboxamide 12 ##STR00063## ++ +
457.94 1 A, F N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(2-methoxybenzamido)oxazole-4- carboxamide 14
##STR00064## + + 479.80 1 A, B
1-(3,4-dichlorophenyl)-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-3,5-dimethyl-1H-pyrazole-4-carboxamide 15 ##STR00065## + +
411.8 1 A, F N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-3,5-dimethyl-1-phenyl-1H-pyrazole-4- carboxamide 16
##STR00066## ++ ++ 451.88 1 A, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-1-phenyl-3-(trifluoromethyl)-1H-
pyrazole-4-carboxamide 17 ##STR00067## +++ +++ 528.91 1 A, B
N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-
5-(2-(trifluoromethoxy)benzamido)-1,2,4- thiadiazole-3-carboxamide
18 ##STR00068## +++ ++ 320.11 1 A, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)isonicotinamide 19 ##STR00069## +++ +++
539.8 1 A, B N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(2,5-
dimethoxyphenylsulfonamido)thiazole-4-carboxamide 20 ##STR00070##
+++ ++ 323.95 2 D, E N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)thiophene-3- carboxamide 21 ##STR00071##
+++ +++ 444.83 1 D, A, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(isonicotinamido)thiazole-4- carboxamide 22
##STR00072## ++ + 414.73 1 D, B 2-amino-6-bromo-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)pyrazine-2-carboxamide 23 ##STR00073## + + 499.86 1 D, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-((4-
(trifluoromethoxy)phenyl)amino)thiazole-4-carboxamide 24
##STR00074## ++ ++ 393.70 1 A, D
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-morpholinooxazole-4-carboxamide 25 ##STR00075##
+ + 499.84 1 F N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-((4-methyl-6-(trifluoromethyl)pyrimidin-
2-yl)amino)thiazole-4-carboxamide 26 ##STR00076## +++ +++ 542.97 1
A, B N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(3-(2-
(trifluoromethoxy)phenyl)ureido)thiazole-4-carboxamide 27
##STR00077## + + + 319.71 1 A
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)pyrazine-2- carboxamide 28 ##STR00078##
++ ++ 351.81 1 A, B N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazole-6-yl)-2,5-dimethylthiophene-3- carboxamide 29
##STR00079## +++ +++ 403.64 1 A, E 5-bromo-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazole-6-
yl)-thiophene-3-carboxamide 30 ##STR00080## +++ +++ 368.74 1 A, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazole-6-yl)-5-nitrothiophene-3-carboxamide 31 ##STR00081##
+++ ++ 373.90 1 A, F
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)benzo[b]thiophene-3- carboxamide 32
##STR00082## ++ +++ 307.95 1 D, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)furan-3- carboxamide 33 ##STR00083## +++
++ 321.85 1 A, B N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-2-methylfuran- 3-carboxamide 34
##STR00084## + + 335.86 1 A, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)-2,5-dimethylfuran-3- carboxamide 35
##STR00085## ++ + 308.90 1 A, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-oxazole-4- carboxamide 36 ##STR00086##
++ + 470.78 1 A, F
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-1-(3-fluorophenyl)-5-(trifluoromethyl)-
1H-1,2,3-triazole-4-carboxamide 37 ##STR00087## +++ ++ 410.40 1 A,
E N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-morpholinothiazole-4-carboxamide 38 ##STR00088##
+++ ++ 441.77 see description of alternative procedures
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-5-(2,3-dihydrobenzofuran-5-
yl)thiophene-3-carboxamide 39 ##STR00089## + ++ 444.80 1 A, E
2-(benzo[d][1,3]dioxol-5-yl)-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)thiazole-4-carboxamide 40 ##STR00090## + + 426.84 1 A, F
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(2,3-dihydrobenzofuan-5-yl)oxazole-4-
carboxamide 41 ##STR00091## +++ +++ 351.8 2 D, E, C
3-chloro-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6- yl)benzamide 42
##STR00092## + + 339.81 3 D, E, C N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide 43 ##STR00093## ++ ++
385.7 2 D, E, C 2,4-dichloro-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol- 6-yl)benzamide 44
##STR00094## ++ ++ 324.8 3 D, E, C
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)thiazole-4- carboxamide 45 ##STR00095##
++ ++ 323.8 2 D, E, C N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)thiophene-2- carboxamide 46 ##STR00096##
++ ++ 347.8 2 D, E, C
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-4-methoxybenzamide 47 ##STR00097## ++ + 401.7 2 D,
E, C N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-2- (trifluoromethoxy)benzamide 48
##STR00098## +++ ++ 511.7 1 D.E, B, C
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(2-
(trifluoromethoxy)benzamido)oxazole-5-carboxamide 49 ##STR00099##
+++ +++ 317.9 2 D, E, C N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)benzamide 50 ##STR00100## + 398.8 3 D, G,
B N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-5-methyl-3-
phenylisoxazole-4-carboxamide 51 ##STR00101## +++ ++ 496.02 1 D, E,
C 3-(2,4-dichlorobenzamido)-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-1H-
1,2,4-triazole-5-carboxamide 52 ##STR00102## +++ ++ 512.31 1 D, E,
c N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-(2-(trifluoromethoxy)benzamido)-
1H-1,2,4-triazole-5-carboxamide 54 ##STR00103## ++ + 353.8 2 D, E,
C N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]
benzo[1,2-d]imidazol-6-yl)-2,6- difluorobenzamide 55 ##STR00104##
++ ++ 402.40 1 2-bromo-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)thiazole-4-carboxamide 56 ##STR00105## +++ +++ +++ +++ 347.8 2 D
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)-3-methoxybenzamide 57 ##STR00106## +++ +++
478.7 1 D, E 5-(3-chlorobenzamido-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-
1,2,4-thiazole-3-carboxamide 58 ##STR00107## ++ 363.21 1 D, E, C
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-4-nitrobenzamide 59 ##STR00108## + 369.24 1 D, E,
C N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)quinoline-2-carboxamide 60 ##STR00109## +++
332.9 see description of alternative procedures
4-amino-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol- 6-yl)benzamide 61
##STR00110## + + 487.14 1 D, E
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-(4-nitrobenzamido)-1H-1,2,4-triazole-5-
carboxamide 62 ##STR00111## +++ 396.61 1 D, F, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-4-sulfamoylbenzamide 63 ##STR00112## ++ 368.73 1
A, B N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-5-nitrothiophene-2-carboxamide 64 ##STR00113## +
357.72 1 A, B 3-chloro-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)thiophene-2-carboxamide 65 ##STR00114## +++ 362.78 1 A, E
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-nitrobenzamide 66 ##STR00115## +++ 386.45 2 D,
B, F N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-(trifluoromethyl)benzamide 67 ##STR00116## +
318.43 2 D N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6- yl)picolinamide 68
##STR00117## ++ + 331.85 1 D, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)-2-methylbenzamide 69 ##STR00118## +++ ++
473.76 1 D, B N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(4-methoxybenzamido)thiazole-4- carboxamide 70
##STR00119## +++ 336.22 2 D, C
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)-3-fluorobenzamide 71 ##STR00120## ++ 308.93 1
D, E N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)oxazole-5-carboxamide 72 ##STR00121## ++
308.82 1 D, B N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)isoxazole-5-carboxamide 73 ##STR00122## +
307.82 1 D, C N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)-1H-pyrazole-3-carboxamide 74 ##STR00123## ++
388.9 4 G, C 4-(diethylamino)-N-(2,2-difluoro-5H-[1,3]dioxolo
[4',5':4,5]benzo[1,2-d]imidazol-6-yl)benzamide 75 ##STR00124## +++
360.9 4 G, C N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-4-(dimethylamino)benzamide 76 ##STR00125## +++
422.8 4 G, C 4-(benzylamino)-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6- yl)benzamide 77
##STR00126## +++ 360.9 4 G, C
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-4-(ethylamino)benzamide 78 ##STR00127## +++ 318.8
1 D, E N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)nicotinamide 79 ##STR00128## ++ ++ 338.81 see
description of alternative procedures
2-amino-N-(2,2-difluoro-5H-[1,3]
dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)
thiophene-3-carboxamide 80 ##STR00129## +++ 323.9 see description
of alternative procedures
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)cyclohexamecarboxamide 81 ##STR00130## +++
406.7 1 E N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-2-
(thiophen-3-yl)thiazole-4-carboxamide 82 ##STR00131## +++ 459.2 1
D, E, B N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(2,3-dihydrobenzo[b][1,4]dioxin-6-
yl)thiazole-4-carboxamide 83 ##STR00132## +++ +++ 402.77 1 D, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-5-sulfamoylthiophene-3-carboxamide 84 ##STR00133##
+ + 308.89 1 A, E
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-1H-1,2,4-triazole-3-carboxamide 85 ##STR00134## ++
++ 401.76 1 D, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-5-(methylsulfonyl)thiophene-2- carboxamide 86
##STR00135## + + 367.74 1 A, B 5-chloro-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-2-hydroxybenzamide 87 ##STR00136## +++ ++ 332.88 1 A, D
3-amino-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6- yl)benzamide 88
##STR00137## +++ +++ 351.78 1 D, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-4-nitro-1H-pyrrole-2-carboxamide 89 ##STR00138##
++ ++ 458.8 1 D, E
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-5-(4-methylbenzamido)-1,2,4-
thiadiazole-3-carboxamide 90 ##STR00139## + + 474.7 1 D, E
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-5-(4-methoxybenzamido)-1,2,4-
thiadiazole-3-carboxamide 91 ##STR00140## +++ +++ 478.7 1 D, E
5-(4-chlorobenzamido-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-
1,2,4-thiazole-3-carboxamide 92 ##STR00141## +++ +++ 462.8 1 D, E
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-5-(4-fluorobenzamido)-1,2,4-
thiadiazole-3-carboxamide 93 ##STR00142## + 360.8 1 D, G, E
4-cyano-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-2-
fluorobenzamide 94 ##STR00143## +++ 378.8 1 D, G, E
N.sup.1-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-fluoroterephthalamide 95 ##STR00144## + + 365.8
1 E 3-acetamido-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-1H-1,2,4-triazole-5-carboxamide 96 ##STR00145## + + 308.9 1 E
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)-1H-1,2,4-triazole-5- carboxamide 97
##STR00146## ++ ++ 541.92 1 A, F N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-2-
(1-(thieno[3,2-d]pyrimidin-4-yl)piperidin-4-
yl)thiazole-4-carboxamide 98 ##STR00147## ++ + 570.27 1 A, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-2-(3-(2-morpholinoethyl)-2-oxo-2,3-dihydro-1H-
benzo[d]imidazol-1-yl)thiazole-4-carboxamide 99 ##STR00148## + +
477.84 1 A, B N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-((2,6-dimethoxypyrimidin-4-
yl)amino)thiazole-5-carboxamide 100 ##STR00149## +++ 397.13 1 A, B,
F N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-sulfamoylbenzamide 101 ##STR00150## +++ 385.75 1
A, B N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-4-sulfamoyl-1H-pyrrole-2-carboxamide 102 ##STR00151## +++ +++
++ 360.91 1 D, B N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-
3-(dimethylamino)benzamide 103 ##STR00152## ++ 307.87 1 A, B
N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-1H-imidazole-4-carboxamide 104 ##STR00153## +++ +++ 348.87 1
D, B 4-acetyl-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-
1H-pyrrole-2-carboxamide 105 ##STR00154## +++ +++ 375.73 1 D, B
methyl 3-((2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)carbamoyl)benzoate 106 ##STR00155## + 351.78 1 D, E
4-chloro-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol- 6-yl)benzamide 107
##STR00156## + 331.84 2 A, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-4-methylbenzamide 108 ##STR00157## +++ 410.74 1 D,
B N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-(methylsulfonamido)benzamide 109 ##STR00158## ++
385.72 2 A, B 3,4-dichloro-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol- 6-yl)benzamide 110
##STR00159## ++ 333.85 1 A, B 6-amino-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6- yl)nicotinamide 111
##STR00160## +++ +++ 360.85 1 A, F N.sup.1-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6- yl)terephthalamide
112 ##STR00161## +++ 397.77 1 A, F N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-
6-sulfamoylnicotinamide 113 ##STR00162## +++ 333.85 1 A, B
N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-3-hydroxybenzamide 114 ##STR00163## +++ 359.82 1 D, F
3-acetyl-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6- yl)benzamide 115
##STR00164## ++ 373.84 1 D, F 4-(tert-butyl)-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6- yl)benzamide 116
##STR00165## +++ 342.85 1 D, E 3-cyano-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6- yl)benzamide 117
##STR00166## +++ +++ 467.03 1 D, E
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-4-(morpholinosulfonyl)benzamide 118 ##STR00167##
+++ +++ 385.79 1 D, A, E
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-(1H-tetrazol-5-yl)benzamide 119 ##STR00168## ++
335.86 1 D, F N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-4-fluorobenzamide 120 ##STR00169## ++ 401.73 2 A,
B N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-(trifluoromethoxy)benzamide 121 ##STR00170## +++
384.98 1 D, B N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-3-(difluoromethoxy)benzamide 122 ##STR00171## +++ +++ 386.89 1
D, B N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-(pyrrolidin-1-yl)benzamide 123 ##STR00172## +++
+++ 348.76 1 D, E N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-5-
methoxynicotinamide 124 ##STR00173## +++ +++ 348.85 1 D, E
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-methoxyisonicotinamide 125 ##STR00174## +++ +
361.89 1 D N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-
2-(dimethylamino)isonicotinamide 126 ##STR00175## +++ 400.79 1 A, B
4-amino-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-3-
(trifluoromethyl)benzamide 127 ##STR00176## ++ 350.78 1 A, B
4-amino-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-3-
fluorobenzamide 128 ##STR00177## +++ 400.81 1 D, E
N-(2,2-difluoro-5H- [1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-3-(piperidin-1-yl)benzamide 129 ##STR00178## ++ + 462.74 1 D, E
3-(chlorobenzamido)-N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-1H-
1,2,4-triazole-5-carboxamide
130 ##STR00179## +++ +++ +++ 412.36 1 D, B N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-
3-(3,5-dimethyl-1H-pyrazol-1-yl)benzamide 131 ##STR00180## +++
428.35 1 D, B N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-3-
(5-isopropyl-1,2,4-oxadiazol-3-yl)benzamide 132 ##STR00181## +++
442.39 1 D, B 3-(5-(tert-butyl)-1,2,4-oxadiazol-3-yl)-N-(2,2-
difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)benzamide 133 ##STR00182## +++ +++ 426.12 2 D, C
(S)-N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(6-methoxymaphthalen-2- yl)propanamide 134
##STR00183## +++ +++ 423.01 1 E
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-2-(4-methyl-1,2,3-thiadiazol-5-
yl)thiazole-4-carboxamide 135 ##STR00184## +++ +++ 408.00 1 D, E, C
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-[2,4-bithiazole]-4-carboxamide 136 ##STR00185##
+++ ++ 391.02 1 D, E, C N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-
2-(thiophen-2-yl)oxazole-4-carboxamide 137 ##STR00186## +++ +++ ++
+++ 400.1 1 D, E, C
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-(5-methyl-1,2,4-oxadiazol-3- yl)benzamide 138
##STR00187## +++ +++ ++ 391.02 1 D, E, C N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-5-
(thiophen-2-yl)isoxazole-3-carboxamide 139 ##STR00188## +++ 483.14
1 D, E, B ethyl 1-(3-((2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)carbamoyl)phenyl)-2,5-dimethyl-1H-pyrrole-3- carboxylate 140
##STR00189## +++ +++ ++ +++ 384.10 1 D, E, C
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-3-(1H-imidazol-2-yl)benzamide 141 ##STR00190## +++
+++ 406.03 1 D, E, C N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-
yl)-2-(5-methylisoxazol-3-yl)thiazole-4- carboxamide 142
##STR00191## +++ + 384.1 1 D, E, C
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-4-(1H-pyrazol-1-yl)benzamide 143 ##STR00192## ++
++ 375.05 1 D, E, C N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-
6-yl)-5-(furan-2-yl)isoxazole-3-carboxamide 144 ##STR00193## +++ +
403.09 1 D, E, C
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-5-(1,3-dimethyl-1H-pyrazol-4-
yl)isoxazole-3-carboxamide 145 ##STR00194## + 323.06 1 D, E, C
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)-5-methylisoxazole-3- carboxamide 146
##STR00195## + 336.09 1 D, E, B
N-(2,2-difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo
[1,2-d]imidazol-6-yl)-1,5-dimethyl-1H-pyrazole- 3-carboxamide 147
##STR00196## +++ +++ ++ 426.10 1 D, C
3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-N-(2,2-
difluoro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2-
d]imidazol-6-yl)-benzamide 148 ##STR00197## +++ +++ +++ 400.09 1 D,
C N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-3-
(5-methyl-1H-tetrazol-1-yl)benzamide 149 ##STR00198## +++ +++ + ++
386.08 1 D, C N-(2,2-difluoro-5H-
[1,3]dioxolo[4',5':4,5]benzo[1,2-d]imidazol-6-yl)-
3-(1H-tetrazol-1-yl)benzamide
8. Further Exemplary Compounds of the Present Invention
TABLE-US-00002 [0309] TABLE 2 Further Exemplary compounds. The
symbols in the columns have the same meaning as in above Table 1.
CK1 CK1 General delta epsilon HH WNT procedure/ No. Structure Assay
Assay Assay Assay [M + H].sup.+ after treatment 1B ##STR00199## +++
+++ +++ 378.0 1 D, E 2B ##STR00200## +++ +++ 401.0 1 D, E 3B
##STR00201## +++ +++ ++ 362.0 1 D, E 4B ##STR00202## +++ 385.1 1 D,
E 5B ##STR00203## +++ + 384.0 1 D, E, B, F 6B ##STR00204## +++ +++
++ 391.02 1 E 7B ##STR00205## +++ + 404.0 1 D, E 8B ##STR00206##
+++ +++ 366.0 1 D, E, B 9B ##STR00207## +++ +++ +++ 363.0 3 A, B
10B ##STR00208## +++ 451.00 1 D, E 11B ##STR00209## +++ + 407.00 1
E 12B ##STR00210## +++ 419.03 1 E 13B ##STR00211## +++ 322.07 1 D,
E 14B ##STR00212## +++ 420.1 1 D, E, B 15B ##STR00213## +++ ++
321.9 see Example 5.1 16B ##STR00214## +++ + 390.8 1 E, B 17B
##STR00215## +++ ++ 435.01 1 E, B 18B ##STR00216## ++ ++ 361.9 see
Example 5.2 19B ##STR00217## ++ 485.03 1 E 20B ##STR00218## ++ +
321.0 see Example 5.3 21B ##STR00219## ++ 394.0 1 D, E 22B
##STR00220## +++ 386.07 1 D, C 23B ##STR00221## +++ 401.0 1 D, E
24B ##STR00222## ++ 349.0 1 D, E or F 25B ##STR00223## +++ 404.0 1
D, E, F 26B ##STR00224## + 349.0 1 D, E, F
9. NMR Data for Exemplary Compounds of the Present Invention
[0310] 4: NMR .sup.1H (400 MHz, CDCl.sub.3): 7.12 (1H, s,
CH-benzimidazole), 7.24 (1H, s, CH-benzimidazole), 7.47 (3H, m,
CH-arom.), 7.90 (2H, m, CH-arom.), 8.29 (1H, s, CH-thiazole), 10.62
(1H, s, NH), 11.23 (1H, s, NH)
[0311] 17: .sup.1H NMR (400 MHz, DMSO-d6+CCl.sub.4): 7.50 (2H, s,
CH-benzimidaz.), 7.57-7.63 (2H, m CH-arom.), 7.74-7.80 (1H, m,
CH-arom.), 7.91-7.94 (1H, dd, CH-arom.), 12.36 (2H, s, NH).
[0312] 44: .sup.1H NMR (400 MHz, DMSO-d.sub.6+CCl.sub.4): 7.33 (2H,
s, CH-benzimidazole), 8.64 (1H, d, CH-thiazole), 9.20 (1H, d,
CH-thiazole), 11.08 (1H, s, NH), 12.40 (1H, s, NH).
[0313] 50: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 2.67 (3H, s,
CH.sub.3), 7.31 (2H, s, CH-benzimidazole), 7.39-7.54 (3H, m,
CH-arom.), 7.61-7.74 (2H, m, CH-arom.), 12.26 (2H, s, NH).
[0314] 51: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.39 (2H, s,
CH-arom.), 7.49 (1H, d, CH-arom.), 7.61 (1H, s, CH-arom.), 7.67
(1H, d, CH-arom.), 11.21 (1H, s, NH), 12.48 (2H, s, NH), 14.65 (1H,
s, NH).
[0315] 52: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.39 (2H, s,
CH-arom.), 7.42 (1H, d, CH-arom.), 7.51 (1H, t, CH-arom.), 7.67
(1H, t, CH-arom.), 7.77 (1H, d, CH-arom.), 11.15 (1H, s, NH), 12.43
(2H, s, NH), 14.64 (1H, s, NH).
[0316] 53: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.39 (2H, s,
CH-arom.), 7.54 (1H, t, CH-arom.), 7.63 (1H, d, CH-arom.), 8.07
(1H, d, CH-arom.), 8.15 (1H, s, CH-arom.), 11.30 (1H, s, NH), 12.48
(2H, s, 2.times.NH), 14.51 (1H, s, NH).
[0317] 54: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.13 (2H, t,
CH-arom.), 7.33 (2H, s, CH-arom.), 7.52-7.59 (1H, m, CH-arom.),
12.40 (2H, s, NH).
[0318] 55: .sup.1H NMR (400 MHz, DMSO-d.sub.6+CCl.sub.4): 7.32 (2H,
s, CH-benzimidazole), 8.61 (1H, s, CH-thiazole), 11.0-12.5 (2H, s,
2.times.NH).
[0319] 56: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 3.89 (3H, s,
CH.sub.3), 7.13 (2H, d, CH-arom.), 7.34 (2H, s, CH-arom.), 7.41
(1H, t, CH-arom.), 7.71 (2H, s, CH-arom.), 12.13 (1H, s, NH), 12.37
(1H, s NH).
[0320] 58: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.35 (2H, s,
CH-arom.), 8.32 (2H, d, CH-arom.), 8.37 (2H, d, CH-arom.), 12.51
(2H, s, 2.times.NH).
[0321] 59: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.39 (2H,s,
CH-arom.), 7.74 (1H, t, CH-arom.), 7.89 (1H, t, CH-arom.), 8.08
(1H, d, CH-arom.), 8.27 (1H, d, CH-arom.), 8.32 (1H, d, CH-arom.),
8.61 (2H, d, CH-arom.), 11.32 (1H, s, NH), 12.53 (1H, s, NH).
[0322] 61: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.42 (2H, s,
CH-arom.), 8.34 (2H, d, CH-arom.), 8.39 (2H, d, CH-arom.), 11.41
(1H, s, NH), 12.51 (2H, s, NH), 14.28 (1H, s, NH).
[0323] 74: .sup.1H NMR (400 MHz, CDCl.sub.3): 1.16 (6H, t,
2*CH.sub.3), 3.39 (4H, q, 2.times.CH.sub.2), 6.44 (1H, s,
CH-arom.), 6.57 (2H, d, CH-arom.), 7.07 (1H, s, CH-arom.), 7.92
(2H, d, CH-arom.), 11.78 (1H, s, NH), 12.72 (1H, s, NH).
[0324] 75: .sup.1H NMR (400 MHz, CDCl.sub.3): 3.03 (6H, s,
2.times.CH.sub.3), 6.50 (1H, s, CH-arom.), 6.60 (2H, d, CH-arom.),
7.09 (1H, s, CH-arom.), 7.91 (2H, d, CH-arom.), 11.71 (1H, s, NH),
12.32 (1H, s, NH)
[0325] 76: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 4.36 (2H, d,
CH.sub.2), 6.59 (2H, d, CH-arom.), 6.94 (1H, t, CH-arom.),
7.19-7.35 (6H, m, CH-arom.), 7.89 (2H, d, CH-arom.), 11.40 (1H, s,
NH), 12.31 (1H, s, NH).
[0326] 78: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.33 (2H, s,
CH-arom.), 7.47-7.51 (1H, m, CH-arom.), 8.45 (1H, dt, CH-arom.),
8.72 (1H, d, CH-arom.), 9.23 (1H, d, CH-arom.), 12.40 (1H, s,
NH).
[0327] 81: .sup.1H NMR (400 MHz, DMSO-d.sub.6+CCl.sub.4): 7.32 (2H,
s, CH-benzimidazole), 7.62 (1H, m, CH-thien.), 7.75 (1H, m,
CH-thien.), 8.27 (1H, m, CH-thien.), 8.49 (1H, s, CH-thiazole),
11.29 (1H, s, NH), 12.42 (1H, s, NH).
[0328] 89: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 2.44 (3H, s,
CH.sub.3), 7.39 (2H, dd, CH-arom), 7.42 (2H, s, CH-arom.), 8.10
(2H, dd, CH-arom.), 11.56-11.92 (1H, s, NH), 12.32-12.64 (1H, s,
NH), 13.70-14.94 (1H, s, NH).
[0329] 90: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 3.89 (3H, s, CH3),
7.06 (2H. dd, CH-arom.), 7.36 (2H, s, CH-arom.), 8.20 (2H, dd,
CH-arom.), 11.21-12.90 (2H, s, NH), 13.37-13.94 (1H, s, NH).
[0330] 91: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.36 (2H, s,
CH-benzimidazole), 7.58 (2H, dd, CH-arom.), 8.22 (2H, dd,
CH-arom.), 11.40-12.95 (3H, s, NH).
[0331] 92: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.32 (2H, d,
CH-arom.), 7.36 (2H, s, CH-arom.), 8.30 (2H, dd, CH-arom.),
11.22-12.80 (2H, s, NH), 13.17-14.49 (1H, s, NH).
[0332] 95: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 2.16 (3H, s,
CH.sub.3), 7.42 (2H, s, CH-arom.), 11.23 (1H, s, NH), 11.79 (1H, s,
NH), 12.49 (1H, s, NH), 14.05 (1H, s, NH).
[0333] 96: .sup.1H NMR (400 MHz, DMSO-d.sub.6): 7.41 (2H, s,
CH-arom.), 8.64 (1H, s, CH-triazole), 12.80-13.20 (3H, s,
3.times.NH).
10. Determination of the Inhibitory Capacity; Casein Kinase
Assays
[0334] The substrate CKltide (peptide HAAIGDDDDAYSITS-NH.sub.2) was
prepared in a concentration of 20 .mu.M in the freshly prepared
Base Reaction Buffer (20 mM Hepes (pH 7.5), 10 mM MgCl.sub.2, 1 mM
EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na.sub.3VO.sub.4, 2 mM
DTT, 1% DMSO). The recombinant protein Casein kinase 1 delta
(CSKN1D) was added to the substrate solution in a concentration of
5 nM and gently mixed. Dilution series in of compounds of the
present invention in DMSO were added to the reaction mixture,
followed after 20 min by addition of a mixture of ATP and
.sup.33P-ATP (specific activity 0.01 .mu.Ci/.mu.l final) to a final
concentration of 10 .mu.M. Reactions were carried out at 25.degree.
C. for 120 min, followed by spotting the reactions onto P81 ion
exchange filter paper. Unbound phosphate was removed by washing of
the filters in 0.75% phosphoric acid. After subtraction of the
background, which was derived from control reactions containing
inactive enzyme, kinase activity data were expressed as percent
remaining kinase activity in the test samples compared to vehicle
(DMSO) reactions. IC.sub.50 values and curve fits were obtained
using the program Prism.RTM. (Graph Pad Software).
[0335] The above assay was also used to determine the inhibitory
capacity of the compounds of the present invention on Casein Kinase
1 epsilon, wherein instead of CSKN1D, recombinant protein Casein
kinase 1 epsilon (CSKN1E) was added to the substrate solution in a
concentration of 30 nM and gently mixed.
[0336] The above tables land 2 provide an overview of the results
of the compounds in the CK1 delta and epsilon kinase Assays.
11. Determination of Proliferation Inhibition on a Panel of Cancer
Cell Lines; Proliferation Assay
[0337] For the determination of the inhibitory capacity of
compounds of the present invention (in the following: "test
compound(s)") on cell growth, cancer cells were seeded into
microtitre plates, treated with different concentrations of test
compound or left untreated, and after 72 h the protein content was
determined as an equivalent for the cell number.
[0338] Test compounds were dissolved in 100% DMSO and predilutions
in cell culture medium were prepared with a final concentration of
0.1% DMSO in medium. The cell culture medium for dilution of the
test compounds, consecutive culturing of cell lines and usage
during the assay was a cell line specific medium as recommended by
the cell line supplier and identical for all three applications
mentioned above. After a 24-hour pre-growth period of the seeded
cancer cells, the test compound containing media or medium
containing 0.1% DMSO as control was added to the cells. The cells
were allowed to grow at 37.degree. C. for 72 hours. In addition,
all experiments contained a few plates with cells that were
processed for measurement immediately after the 24 hours recovery
period. These plates contained information about the cell number
that existed before treatment, at time zero, and served to
calculate the cytotoxicity and/or growth inhibitory effects.
[0339] After treatment, cells were precipitated by addition of 10%
TCA (trichloracetic acid). Prior to fixation, the media was
aspirated. After an hour of incubation at 4.degree. C. the plates
were washed twice with 400 .mu.l of deionized water. Cells were
then stained with 100 .mu.l of a 0.08% wt/v SRB (sulforhodamine).
The plates were allowed to sit for at least 30 min and washed six
times with 1% acetic acid to remove unbound staining agent. The
plates were left to dry at room temperature and bound SRB was
solubilized with 100 .mu.l of 10 mM Tris base. Measurement of
optical density was performed at 560 nm on a Victor 2 plate reader
(Perkin Elmer, Germany).
[0340] One common way to express the effect of an anticancer agent
is to measure cell viability and survival in the presence of the
test agent as % Treated/Control.times.100. The relationship between
the viability and dose is called a dose response curve. Those dose
response curves were determined by using algorithms developed by
Oncolead GmbH & Co. KG, Munich, Germany, that can be compared
to commercial applications, e.g. XLfit.TM. (ID Business Solutions
Ltd., Guildford, UK) algorithm "205". The potency of a given test
compound to inhibit cell growth was specified as the IC.sub.50
value (drug concentration needed for 50% inhibition of cell
growth).
[0341] The IC.sub.50 of the compound of Example 56, determined as
described above, with the following cell lines was in all cases 1
.mu.M or lower: 22RV1 (prostate), 5637 (bladder), 786O (kidney),
A204 (muscle), A2780 (ovary), A375 (skin), A431 (skin), A549
(lung), A673 (muscle), ACHN (kidney), ASPC1 (pancreas), BT20
(breast), BXPC3 (pancreas), C33A (cervix), CAKI1 (kidney), CALU6
(lung), CASKI (cervix), CLS439 (bladder), COLO205 (colon), DLD1
(colon), DU145 (prostate), EFO21 (ovary), EJ28 (bladder), HCT116
(colon), HCT15 (colon), HEK293 (kidney), HELA (cervix), HEPG2
(liver), HS729 (muscle), HS578T (breast), HT1080 (connective
tissue), HT29 (colon), IMR90 (lung), IGROV1 (ovary), J82 (bladder),
JAR (placenta), JEG3 (placenta), JIMT1 (breast), LOVO (colon), MCF7
(breast), MDAMB435 (skin), MDAMB436 (breast), MDAMB468 (breast),
MG63 (bone), MHHES1 (bone), MIAPACA2 (pancreas), MT3 (breast),
NCIH292 (lung), NCIH358M (lung), NCIH460 (lung), NCIH82 (lung),
OVCAR3 (ovary), OVCAR4 (ovary), PANC1 (pancreas), PANC1005
(pancreas), PC3 (prostate), PLCPRFS (liver), RD (muscle), RDES
(bone), SAOS2 (bone), SF268 (brain), SF295 (brain), SKBR3 (breast),
SKHEP1 (liver), SKLMS1 (uterus), SKMEL28 (skin), SKMEL5 (skin),
SKNAS (brain), SKNSH (brain), SNB75 (brain), SW620 (colon), T24
(bladder), TE671 (muscle), U2OS (bone), U87MG (brain). UMUC3
(bladder), SKOV3 (ovary), in the following cases 100 nM or lower:
A2780 (ovary), A375 (skin), A673 (muscle), BXPC3 (pancreas), CALU6
(lung), EJ28 (bladder), HCT116 (colon), HCT15 (colon), JAR
(placenta), LOVO (colon), MCF7 (breast), MDAMB435 (skin), MG63
(bone), MHHES1 (bone), NCIH358M (lung), PC3 (prostate), SF295
(brain), SKMELS (skin), SKNAS (brain).
12. Inhibition of Anchorage Independent Growth, Soft Agar Colony
Formation
[0342] To analyze anchorage-independent growth, cells were seeded
in 12-well plates in 0.4% select agar.RTM. on top of 0.5% bottom
select agar.RTM. (Invitrogen) according to standard protocols. 8000
PANC1 cells were seeded in 400 .mu.l select agar and the compound
according to the present invention or DMSO as control was added in
a final volume of 400 .mu.l growth medium to the top agar. Cultures
were grown for 21 days at 37.degree. C. in a humidified atmosphere
of 5% CO.sub.2. Fresh growth medium was added against drying-out
twice per week. Colony growth in soft agar cultures was quantified
using Colony Counter software (Microtech Nition).
[0343] Depending on the addition of Culture plates exhibit growth
of cancer cell colonies, wherein some colonies develop into
macrocolonies having a diameter which is approximately .gtoreq.4
times larger than the median diameter over all cancer cell colonies
(in said well). The inhibition of both overall colony formation
(including macrocolony formation) and macrocolony formation alone
by compounds of the present invention was determined, results are
shown in FIG. 1. Macrocolonies of this cell line are rare but
highly clonogenic, tumor-initiating cells with high activity of the
Hedgehog signaling pathway. Inhibition of those macrocolonies in
vitro can be interpreted to relate to cancer stem cell inhibition
in tumor bearing patients (Eberl et al., EMBO Mol Med, 2011, 4,
218-233).
[0344] Growth medium Stock solutions (sterilized, stored at
4.degree. C.): 2.times. DMEM, GIBCO powder: 26.76 g DMEM powder
(GIBCO, with 4.5 g/l Glucose), 7.4 g NaHCO3, 220 mg Sodium Pyruvate
(Sigma, 11.0 mg/ml solution); dissolve in water, adjust pH with 2M
Hepes to 7.2; final volume 1 liter. 2.times.DMEM, PAA powder: 27 g
DMEM powder (PAA Art. No. G0006.3010, with 4.5 g/l Glucose, with
Sodium Pyruvate), 7.4 g NaHCO3; dissolve in water, adjust pH to
6.8-7.5 with 1N HCl; final volume 1 liter.
[0345] Before usage, add 50 ml FBS and 5 ml PenStrep (100.times.
stock) to 200 ml aliquot; store this 2.times. medium for maximum
6-8 weeks at 4.degree. C.
[0346] 2.times. stock solutions of 1% (bottom) and 0.8% (top)
Select Agar: 1 g or 0.8 g, respectively, Select Agar (Invitrogen,
Art. No. 30391-023) in 100 ml water (=1% or 0.8% final,
respectively). Store at 4.degree. C. until needed.
[0347] Bottom Agar (=0.5% agar final concentration): Melt "1%
Select Agar"-stock in microwave; cool down to 42.degree. C. in a
water bath; warm 2.times. DMEM to 37.degree. C.; mix Select Agar
and 2.times. DMEM solution 1:1 (avoid bubbles); plate 800 .mu.l
Agar/DMEM mix in each well; allow to cool and harden completely
(room temperature)can be stored at 4.degree. C. for up to 2 weeks
prior to use.
[0348] Top Agar (=0.4% agar final concentration, containing cells);
Melt "0.8% Select Agar"-stock in microwave; cool down to 40.degree.
C. in a water bath; warm 2.times. DMEM to 37.degree. C.; let bottom
agar plates warm to room temperature (or warm in 37.degree. C.
incubator); mix Select Agar and 2.times. DMEM solution 1:1 (avoid
bubbles); aliquot Agar/DMEM mix in 15 ml tubes and store at
40.degree. C.; Trypsinize cells and determine cell count; add 8000
cells per well to Agar/DMEM mix; again mix by gently inverting tube
and plate out; allow to cool and harden top agar in laminar flow
(.about.30 min; room temperature); pipette 400 .mu.l medium
(containing optional chemical substances) onto top agar to prevent
drying-out; Replace supernatant as appropriate (e.g. 2.times. per
week).
13. Wnt Reporter Assay
[0349] The Wnt reporter assay was performed in stably transfected
human HEK293 cells. Wnt signaling was induced by administration of
Wnt3a protein and read out by .beta.-Lactamase mediated cleavage of
CCF4-AM and subsequent fluorescent detection.
[0350] CellSensor LEF/TCF-bla FreeStyle 293F cells (invitrogen
#K1677) contain a beta-lactamase reporter gene under control of the
.beta.-catenin/LEF/TCF binding elements stably integrated into
FreeStyle 293F cells. This cell line can be used to detect
antagonists of the Wnt/.beta.-catenin signaling pathway when
stimulated by mouse Wnt3a or LiCl. The detection of beta-lactamase
is possible with LiveBLAzer FRET--B/G Loading Kit (invitrogen
#K1095) with CCF4-AM as subtrate.
[0351] Culture Medium: DMEM Medium +10% dialyzed FBS (PAN
2102-P290310), +1% NEAA (PAA M11-003), +1% P/S
(Penicillin/Streptomycin, PAA P11-010) +25 mM HEPES (PAA S11-001),
+5 .mu.g/ml Blasticidin (PAA P05-017).
[0352] Assay Medium: Opti-MEM (Gibco 11058-021) +0.5% dialyzed FBS
(PAN 2102-P290310), +1% NEAA (PAA M11-003), +1% P/S (PAA P11-010),
+10 mM HEPES (PAA S11-001), +1 mM Na-Pyruvate (PAA S11-003).
[0353] Cells (CellSensor LEF/TCF-bla FreeStyle 293F, invitrogen
K1677) were split with Culture Medium to reach a confluence at the
beginning of the assay of 80-90%.
[0354] At start of the assay, cells were harvested from culture and
resuspend in assay medium at density of 0,66*10 6 cells/ml.
Subsequently, 60.mu.1 cell suspension were pipetted in each well of
a Poly-D-Lysine 96 well Plate (BD #354640, vertical rows are marked
1 through 12, horizontal lanes are marked A through H),
TABLE-US-00003 1 2 3 4 5 6 7 8 9 10 11 12 A 120 .mu.l Medium B 60
.mu.l cells + 60 .mu.l 60 .mu.l 60 .mu.l 60 .mu.l 60 .mu.l 60 .mu.l
60 .mu.l 60 .mu.l 60 .mu.l 60 .mu.l 60 .mu.l C 60 .mu.l cells cells
cells cells cells cells cells cells cells cells medium D medium E
60 .mu.l cells + 60 .mu.l 60 .mu.l 60 .mu.l 60 .mu.l 60 .mu.l 60
.mu.l 60 .mu.l 60 .mu.l 60 .mu.l 60 .mu.l 60 .mu.l F 60 .mu.l cells
cells cells cells cells cells cells cells cells cells cells G
medium H 120 .mu.l Medium
[0355] Plates were subsequently incubated at 37.degree. C. for at
least 2 h.
[0356] To stimulate with Wnt3a, overnight prestimulation of Wnt
signalling pathway with LiCl is necessary. Subsequently, an 8M
aqueous LiCl solution was diluted 1:200 in assay medium, and 30
.mu.l of said diluted LiCl solution was plated into the appropriate
wells (see below pipetting scheme). Plates were then incubated at
37.degree. C. over night.
[0357] Subsequently, compounds and controls were prepared as
follows: mWnt3a (R&D 1324-WN, dissolved in PBS+0.1% BSA to 40
.mu.g/ml) was diluted 1:100 in assay medium (=400 ng/ml). Compounds
(Cpd) were diluted to a concentration of 10 mM in DMSO to prepare
dilution series in assay medium (final concentrations 30 .mu.M, 10
.mu.M, 3 .mu.M, 1 .mu.M, 0.3 .mu.M, 0.1 .mu.M, 0.03 .mu.M and 0.01
.mu.M), which were pipetted into the appropriate wells (see below
pipetting scheme). For each dilution of each compound, 3 replicates
were tested. Final DMSO concentration was 0.3%.
[0358] LiCl and mWnt3a stimulated cells were used as High control
(HC), unstimulated cells were used as low control, a blank was
prepared from cell free medium plus mWnt3a, and a functional
control was prepared with a known Wnt inhibitor (e.g. PKF118-310),
final concentrations were as above.
[0359] Pipetting scheme:
[0360] Subsequently, the well was incubated for 5 h at 37.degree.
C.
[0361] At t=23 hours, FRET reagent mix (Loading Kit with CCF4-AM,
Life Technologies #K1096) was prepared; per plate: 138 .mu.l
Solution B, 13.8 .mu.l FRET-reagent, and 2300 .mu.l Solution C. 24
.mu.l FRET reagent mix were plated to each well, subsequently
plates were incubated for at least 2 h in the dark (it is possible
to incubate over night). Subsequently, plates were measured in the
BMG FluorStar.RTM. plate reader at an emission waveltengths of 460
and 520 nm and with an excitation wavelength of 405 nm.
[0362] To determine potential cytotoxicity of the compounds, plates
were centrifuge and the supernatant was discarded by dumping the
plates. Subsequently, 100 .mu.l Opti-MEM and 50 .mu.l ViaLight
(Lonza #LT07-321) Lysis Buffer were added to each well, followed by
incubation for 10 min at room temperature (25.degree. C.).
Subsequently, 100 .mu.l ViaLight reagent were added to each well,
followed by incubation for 2 min at room temperature (25.degree.
C.) in the dark. Optionally, 200 .mu.l of the obtained suspension
were transferred to a white 96 well plate. The luminescence was
measured with a Tecan Ultra plate reader.
[0363] 13. Hedgehog Reporter Assay
[0364] In order to investigate the potency of test compounds to
inhibit the Hedgehog signaling pathway, a Gli-Reporter assay was
performed.
[0365] The "Gli Reporter--NIH3T3 Cell Line" contains the firefly
luciferase gene under the control of Gli responsive elements stably
integrated into murine NIH3T3 cells (cells purchased from Amsbio).
The luciferase expression correlates with activation of the
hedgehog signaling pathway. This cell line is validated for its
response to stimulation with murine Sonic Hedgehog and to treatment
with inhibitors of the hedgehog signaling pathway. A multiplexed
viability assay was used to discriminate inhibition on the pathway
activity from cell toxicity.
[0366] Growth Medium: DMEM, 10% Calf Serum, 1%
Penicillin/Streptomycin 500 .mu.g/ml Geneticin (G418 Stock 50
mg/ml).
[0367] Assay Medium: Opti-MEM.RTM. Reduced Serum Medium, 0.5% Calf
Serum, 1% non-essential amino acids, 1 mM Na-pyruvate, 10 mM HEPES,
1% Penicillin/Streptomycin.
[0368] 25.000 cells per well were seeded into a white 96 well plate
in 100 .mu.l growth medium and incubated over night at 37.degree.
C. and 5% CO.sub.2. After removing the supernatant the test
compounds and controls (known GLI inhibitor, e.g. GANT-61) were
added in different concentrations in a final volume of 45 .mu.l and
incubated for 1 h at 37.degree. C. and 5% CO.sub.2. For the
stimulation of the Hedgehog pathway 5 .mu.l of 10 .mu.g/ml
concentrated murine sonic hedgehog (SHH) was added to the cells. A
final concentration of 1 .mu.g/ml mSHH and 0.1% DMSO was reached
per well. After incubation for 24 h at 37.degree. C. the cells were
investigated for viability and reporter activity.
[0369] Viability: For the determination of the viability of the
treated cells the CellTiter-Fluor Kit from Promega was used.
Essentially, only proteases of viable cells are able to cleave the
cell-permeant substrate Gly-Phe-AFCoumarin (GF-AFC). By this
cleavage the fluorescent AFC is set free and can be detected in a
fluorescence reader. For this assay 10 .mu.l of GF-AFC substrate
(CellTiter-Fluor, Promega #G6082) was diluted in 2 ml assay buffer
from the CellTiter-Fluor Kit and 10 .mu.l of this dilution was
added per well to the cells and incubated for 30 min at 37.degree.
C. The fluorescence was measured with excitation at 380-400 nm and
emission at 505 nm.
[0370] Reporter activity: The firefly luciferase reporter activity
was detected with the ONE-Glo.TM. Luciferase Assay System from
Promega. For this assay 50 .mu.l ONE-Glo luciferase reagent
(Promega #E6120, contains cell lysis buffer and luciferin) was
added to each well and incubated at room temperature for 5 min.
Luminescence was detected in a plate reader and served as a degree
for reporter activity.
* * * * *