U.S. patent application number 16/301729 was filed with the patent office on 2019-09-19 for treatment of neurological disorders.
The applicant listed for this patent is PIQUR THERAPEUTICS AG, UNIVERSITAT BASEL. Invention is credited to Florent BEAUFILS, Claudia BRANDT, Doriano FABBRO, Paul HEBEISEN, Petra HILLMANN-WULLNER, Wolfgang LOSCHER, Hoa Huu Phuc NGUYEN, Denise RAGEOT, Alexander Markus SELE.
Application Number | 20190284178 16/301729 |
Document ID | / |
Family ID | 56024138 |
Filed Date | 2019-09-19 |
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United States Patent
Application |
20190284178 |
Kind Code |
A1 |
RAGEOT; Denise ; et
al. |
September 19, 2019 |
TREATMENT OF NEUROLOGICAL DISORDERS
Abstract
The present invention is relates to a compound of formula (I),
##STR00001## wherein X.sup.1, X.sup.2 and X.sup.3 are,
independently of each other, N or CH; with the proviso that at
least two of X.sup.1, X.sup.2 and X.sup.3 are N; Y is N or CH;
R.sup.1 and R.sup.2 are independently of each other (iii) a
morpholinyl of formula (II) ##STR00002## wherein the arrow denotes
the bond in formula (I); and wherein R.sup.3 and R.sup.4 are
independently of each other H, C.sub.1-C.sub.3alkyl optionally
substituted with one or two OH, C.sub.1-C.sub.2fluoroalkyl,
C.sub.1-C.sub.2alkoxy, C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl,
CN, or C(O)O--C.sub.1-C.sub.2alkyl; or R.sup.3 and R.sup.4 form
together a bivalent residue --R.sup.5R.sup.6-- selected from
C.sub.1-C.sub.3alkylene optionally substituted with 1 to 4 F,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--, or any of
the structures ##STR00003## wherein the arrows denote the bonds in
formula (II); or (iv) a saturated 6-membered heterocyclic ring Z
selected from thiomorpholinyl and piperazinyl, optionally
substituted by 1 to 3 R.sup.7; wherein R.sup.7 is independently at
each occurrence C.sub.1-C.sub.3alkyl optionally substituted with
one or two OH, C.sub.1-C.sub.2fluoroalkyl,
C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl,
C.sub.3-C.sub.6cycloalkyl; or two R.sup.7 substituents form
together a bivalent residue --R.sup.8R.sup.9-- selected from
C.sub.1-C.sub.3alkylene optionally substituted with 1 to 4 F,
--CH.sub.2--O--CH.sub.2-- or --O--CH.sub.2CH.sub.2--O--; with the
proviso that at least one of R.sup.1 and R.sup.2 is a morpholinyl
of formula II; and prodrugs, metabolites, tautomers, solvates and
pharmaceutically acceptable salts thereof, for use in the
prevention or treatment of a neurological disorder in a
subject.
Inventors: |
RAGEOT; Denise;
(Saint-Louis, FR) ; HEBEISEN; Paul; (Basel,
CH) ; BEAUFILS; Florent; (Bartenheim, FR) ;
FABBRO; Doriano; (Arlesheim, CH) ; HILLMANN-WULLNER;
Petra; (Oberengstringen, CH) ; NGUYEN; Hoa Huu
Phuc; (Tubingen, DE) ; LOSCHER; Wolfgang;
(Hannover, DE) ; BRANDT; Claudia; (Langenhagen,
DE) ; SELE; Alexander Markus; (Basel, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PIQUR THERAPEUTICS AG
UNIVERSITAT BASEL |
Basel
Basel |
|
CH
CH |
|
|
Family ID: |
56024138 |
Appl. No.: |
16/301729 |
Filed: |
May 17, 2017 |
PCT Filed: |
May 17, 2017 |
PCT NO: |
PCT/EP2017/025136 |
371 Date: |
November 14, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 498/08 20130101;
C07D 417/14 20130101; A61P 43/00 20180101; A61K 31/53 20130101;
C07D 413/14 20130101; A61K 31/5386 20130101; A61P 25/08 20180101;
A61P 25/16 20180101; A61P 25/00 20180101; C07D 519/00 20130101;
C07D 451/02 20130101; A61P 25/14 20180101; C07D 451/14 20130101;
A61K 31/541 20130101; A61K 31/5377 20130101; A61P 25/28
20180101 |
International
Class: |
C07D 413/14 20060101
C07D413/14; C07D 451/02 20060101 C07D451/02; C07D 417/14 20060101
C07D417/14; C07D 451/14 20060101 C07D451/14; C07D 498/08 20060101
C07D498/08; C07D 519/00 20060101 C07D519/00; A61P 25/08 20060101
A61P025/08; A61P 25/28 20060101 A61P025/28 |
Foreign Application Data
Date |
Code |
Application Number |
May 18, 2016 |
EP |
16170107.3 |
Claims
1. A compound of formula (I), ##STR00284## wherein X.sup.1, X.sup.2
and X.sup.3 are, independently of each other, N or CH; with the
proviso that at least two of X.sup.1, X.sup.2 and X.sup.3 are N; Y
is N or CH; R.sup.1 and R.sup.2 are independently of each other (i)
a morpholinyl of formula (II) ##STR00285## wherein the arrow
denotes the bond in formula (I); and wherein R.sup.3 and R.sup.4
are independently of each other H, C.sub.1-C.sub.3alkyl optionally
substituted with one or two OH, C.sub.1-C.sub.2fluoroalkyl,
C.sub.1-C.sub.2alkoxy, C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl,
CN, or C(O)O--C.sub.1-C.sub.2alkyl; or R.sup.3 and R.sup.4 form
together a bivalent residue --R.sup.5R.sup.6-- selected from
C.sub.1-C.sub.3alkylene optionally substituted with 1 to 4 F,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--, or any of
the structures ##STR00286## wherein the arrows denote the bonds in
formula (II); or (ii) a saturated 6-membered heterocyclic ring Z
selected from thiomorpholinyl and piperazinyl, optionally
substituted by 1 to 3 R.sup.7; wherein R.sup.7 is independently at
each occurrence C.sub.1-C.sub.3alkyl optionally substituted with
one or two OH, C.sub.1-C.sub.2fluoroalkyl,
C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl,
C.sub.3-C.sub.6cycloalkyl; or two R.sup.7 substituents form
together a bivalent residue --R.sup.8R.sup.9-- selected from
C.sub.1-C.sub.3alkylene optionally substituted with 1 to 4 F,
--CH.sub.2--O--CH.sub.2-- or --O--CH.sub.2CH.sub.2--O--; with the
proviso that at least one of R.sup.1 and R.sup.2 is a morpholinyl
of formula II; and prodrugs, metabolites, tautomers, solvates and
pharmaceutically acceptable salts thereof, for use in the
prevention or treatment of a neurological disorder in a
subject.
2. The compound of formula (I) for use according to claim 1,
wherein said R.sup.1 and said R.sup.2 are independently of each
other selected from ##STR00287## ##STR00288## ##STR00289##
3. The compound of formula (I) for use according to any one of the
claims 1 to 2, wherein R.sup.1 and R.sup.2 are independently of
each other selected from ##STR00290##
4. The compound of formula (I) for use according to claim 1,
wherein said compound is selected from
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyridin-2-amine-
;
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyrimidin-2-am-
ine;
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.-
1]octan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1,3,5-triazin-2-y-
l)-4-(difluoromethyl)pyridin-2-amine;
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1,3,5-triazin-2-y-
l)-4-(difluoromethyl)pyrimidin-2-amine;
5-(4,6-bis((S)-3-methylmorpholino)-1,3,5-triazin-2-yl)-4-(difluoromethyl)-
pyridin-2-amine;
5-(4,6-bis((S)-3-methylmorpholino)-1,3,5-triazin-2-yl)-4-(difluoromethyl)-
pyrimidin-2-amine;
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine;
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyrimidin-2-amine;
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-((S)-3-methylmorpholino)-1,3-
,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-((S)-3-methylmorpholino)-1,3-
,5-triazin-2-yl)-4-(difluoromethyl)pyrimidin-2-amine;
4-(difluoromethyl)-5-(4-morpholino-6-(piperazin-1-yl)-1,3,5-triazin-2-yl)-
pyridin-2-amine;
4-(difluoromethyl)-5-(4-morpholino-6-(piperazin-1-yl)-1,3,5-triazin-2-yl)-
pyrimidin-2-amine;
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-(piperazin-1-yl)-1,3,5-
-triazin-2-yl)pyridin-2-amine;
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-(piperazin-1-yl)-1,3,5-
-triazin-2-yl)pyrimidin-2-amine;
4-(difluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridin-2-amine;
4'-(difluoromethyl)-2,6-dimorpholino-[4,5'-bipyrimidin]-2'-amine;
4-(difluoromethyl)-5-(4,6-dimorpholinopyrimidin-2-yl)pyridin-2-amine;
4'-(difluoromethyl)-4,6-dimorpholino-[2,5'-bipyrimidin]-2'-amine;
4-(difluoromethyl)-5-(4-morpholino-6-thiomorpholino-1,3,5-triazin-2-yl)py-
ridin-2-amine;
4-(difluoromethyl)-5-(4-morpholino-6-thiomorpholino-1,3,5-triazin-2-yl)py-
rimidin-2-amine;
5-(6-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-2-(3-oxa-8-azabicyclo[3.2.1]oc-
tan-8-yl)pyrimidin-4-yl)-4-(difluoromethyl)pyridin-2-amine;
5-(2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholinopyrimidin-4-yl)-4--
(difluoromethyl)pyridin-2-amine;
2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-4'-(difluoromethyl)-6-morpholino--
[4,5'-bipyrimidin]-2'-amine;
5-(2,6-bis((S)-3-methylmorpholino)pyrimidin-4-yl)-4-(difluoromethyl)pyrid-
in-2-amine;
4'-(difluoromethyl)-2,6-bis((S)-3-methylmorpholino)-[4,5'-bipyrimidin]-2'-
-amine;
(S)-4-(difluoromethyl)-5-(6-(3-methylmorpholino)-2-morpholinopyrim-
idin-4-yl)pyridin-2-amine;
(S)-4'-(difluoromethyl)-6-(3-methylmorpholino)-2-morpholino-[4,5'-bipyrim-
idin]-2'-amine;
5-(4-(8-Oxa-3-azabicyclo[3.2.1]octan-3-yl)-6-(8-oxa-3-azabicyclo[3.2.1]oc-
tan-3-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
5-[4,6-bis(2,2-dimethylmorpholin-4-yl)-1,3,5-triazin-2-yl]-4-(difluoromet-
hyl)pyridin-2-amine;
(S)-4-(difluoromethyl)-5-(2-(3-methylmorpholino)-6-morpholinopyrimidin-4--
yl)pyridin-2-amine;
(S)-4'-(difluoromethyl)-2-(3-methylmorpholino)-6-morpholino-[4,5'-bipyrim-
idin]-2'-amine;
4-(difluoromethyl)-5-[4-[(2S,6R)-2,6-dimethylmorpholin-4-yl]-6-[(3R)-3-me-
thylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
5-[4,6-bis[(2R,6S)-2,6-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(dif-
luoromethyl)pyridin-2-amine;
5-[4,6-bis(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-1,3,5-triazin-2-yl]-4-
-(difluoromethyl)pyridin-2-amine;
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-(3-ox-
a-8-azabicyclo[3.2.1]octan-8-yl)-1,3,5-triazin-2-yl]pyridin-2-amine;
5-[4,6-bis(3,3-dimethylmorpholin-4-yl)-,3,5-triazin-2-yl]-4-(difluorometh-
yl)pyridin-2-amine;
5-[4,6-bis[(3R,5S)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(dif-
luoromethyl)pyridin-2-amine;
5-[4,6-bis[(3R)-3-methylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(difluorome-
thyl)pyridin-2-amine;
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-morpholino-1,3,5-t-
riazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-[(3R,5S)-3,5-dimethylmorpholin-4-yl]-6-[(3R)-3-me-
thylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-[(3R)-3-methylmorp-
holin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-[(3R)-3-(methoxymethyl)morpholin-4-yl]-6-[(3R)-3--
methylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-[(3R)-
-3-methylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-(3-oxa-6-azabicyc-
lo[3.1.1]heptan-6-yl)-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-(6-oxa-3-azabicyc-
lo[3.1.1]heptan-3-yl)-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-[(1R,4R)-2-oxa-5--
azabicyclo[2.2.1]heptan-5-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-[(1S,4S)-2-oxa-5--
azabicyclo[2.2.1]heptan-5-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
5-[4,6-bis[(3R)-3-ethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(difluoromet-
hyl)pyridin-2-amine;
5-[4,6-bis(8-oxa-5-azaspiro[3.5]nonan-5-yl)-1,3,5-triazin-2-yl]-4-(difluo-
romethyl)pyridin-2-amine;
5-[4,6-bis[(3R)-3-isopropylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(difluor-
omethyl)pyridin-2-amine
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-[(3R,5S)-3,5-dimet-
hylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-[(3R)-3-(methoxyme-
thyl)morpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
[(3R)-4-[4-[6-amino-4-(difluoromethyl)-3-pyridyl]-6-(3,3-dimethylmorpholi-
n-4-yl)-1,3,5-triazin-2-yl]morpholin-3-yl]methanol;
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-(3,7-dioxa-9-azabi-
cyclo[3.3.1]nonan-9-yl)-1,3,5-triazin-2-yl]pyridin-2-amine;
5-[4-(4-cyclopropylpiperazin-1-yl)-6-(3,3-dimethylmorpholin-4-yl)-1,3,5-t-
riazin-2-yl]-4-(difluoromethyl)pyridin-2-amine;
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-[4-(2-methoxyethyl-
)piperazin-1-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
[(3R)-4-[4-[6-amino-4-(difluoromethyl)-3-pyridyl]-6-[(3R)-3-methylmorphol-
in-4-yl]-1,3,5-triazin-2-yl]morpholin-3-yl]methanol;
4-(difluoromethyl)-5-[4-[(3R,5R)-3,5-dimethylmorpholin-4-yl]-6-[(3R)-3-me-
thylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-[(3S,5S)-3,5-dimethylmorpholin-4-yl]-6-[(3R)-3-me-
thylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-morpholino-6-(3-oxa-9-azabicyclo[3.3.1]nonan-9-yl-
)-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-(3-ox-
a-9-azabicyclo[3.3.1]nonan-9-yl)-1,3,5-triazin-2-yl]pyridin-2-amine;
5-[4,6-bis[(3S,5S)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(dif-
luoromethyl)pyridin-2-amine;
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-morph-
olino-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-[(3
S)-3-ethylmorpholin-4-yl]-6-[(3R)-3-methylmorpholin-4-yl]-1,3,5-triazin-2-
-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-[(3R)-3-ethylmorpholin-4-yl]-6-[(3R)-3-methylmorp-
holin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-(8-oxa-5-azaspiro-
[3.5]nonan-5-yl)-1,3,5-triazin-2-yl]pyridin-2-amine; and tautomers,
solvates and pharmaceutically acceptable salts thereof.
5. The compound of formula (I) according to claim 1, wherein said
compound is selected from the group consisting of
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyrimidin-2-ami-
ne;
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.1-
]octan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1,3,5-triazin-2-y-
l)-4-(difluoromethyl)pyridin-2-amine;
5-(4,6-bis((S)-3-methylmorpholino)-1,3,5-triazin-2-yl)-4-(difluoromethyl)-
pyrimidin-2-amine;
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine;
4-(difluoromethyl)-5-(4-morpholino-6-(piperazin-1-yl)-1,3,5-triazin-2-yl)-
pyrimidin-2-amine;
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyridin-2-amine-
; and
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5--
triazin-2-yl)pyrimidin-2-amine;
5-[4,6-bis(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-1,3,5-triazin-2-yl]-4-
-(difluoromethyl)pyridin-2-amine;
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-(3-ox-
a-8-azabicyclo[3.2.1]octan-8-yl)-1,3,5-triazin-2-yl]pyridin-2-amine;
5-[4,6-bis(3,3-dimethylmorpholin-4-yl)-1,3,5-triazin-2-yl]-4-(difluoromet-
hyl)pyridin-2-amine;
5-[4,6-bis[(3R,5S)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(dif-
luoromethyl)pyridin-2-amine;
5-[4,6-bis[(3R)-3-methylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(difluorome-
thyl)pyridin-2-amine;
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-morpholino-1,3,5-t-
riazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-[(3R,5S)-3,5-dimethylmorpholin-4-yl]-6-[(3R)-3-me-
thylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-[(3R)-3-methylmorp-
holin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-[(3R)-3-(methoxymethyl)morpholin-4-yl]-6-[(3R)-3--
methylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-[(3R)-
-3-methylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-(3-ox-
a-9-azabicyclo[3.3.1]nonan-9-yl)-1,3,5-triazin-2-yl]pyridin-2-amine;
5-[4,6-bis[(3S,5S)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(dif-
luoromethyl)pyridin-2-amine;
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-morph-
olino-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-[(3
S)-3-ethylmorpholin-4-yl]-6-[(3R)-3-methylmorpholin-4-yl]-1,3,5-triazin-2-
-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-[(3R)-3-ethylmorpholin-4-yl]-6-[(3R)-3-methylmorp-
holin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-(8-oxa-5-azaspiro-
[3.5]nonan-5-yl)-1,3,5-triazin-2-yl]pyridin-2-amine and tautomers,
solvates and pharmaceutically acceptable salts thereof.
6. The compound of formula (I) according to claim 1, wherein said
compound is selected from
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.1]oc-
tan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
and
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine; and tautomers, solvates and
pharmaceutically acceptable salts thereof.
7. The compound of formula (I) for use according to any one of the
claims 1 to 6, wherein R.sup.1 and R.sup.2 are independently of
each other a morpholinyl of formula (II).
8. The compound of formula (I) for use according to claim 7,
wherein R.sup.1 is equal to R.sup.2.
9. The compound of formula (I) for use according to claim 7,
wherein R.sup.1 is not equal to R.sup.2.
10. A compound for use according to any one of the claims 1 to 9,
wherein the neurological disorder is epilepsy or a
neurodegenerative disease.
11. A compound for use according to any one of the claims 1 to 10,
wherein the neurological disorder is a neurodegenerative disease,
and wherein the neurodegenerative disease is selected from the
group consisting of Huntington's disease, spinocerebellar ataxias,
Parkinson's disease, morbus Alzheimer, amyotrophic lateral
sclerosis (ALS), cystic fibrosis, familial amyloidotic
polyneuropathy, spongiform encephalopathies, dementia with Lewy
bodies, frontotemporal dementia with Parkinsonism, spinocerebellar
ataxias, spinal and bulbar muscular atrophy, hereditary
dentatorubral-pallidoluysian atrophy, familial British dementia,
familial Danish dementia and prion disease.
12. A compound for use according to claim 11, wherein the
neurodegenerative disease is Huntington's disease.
13. A compound for use according to any one of the claims 1 to 10,
wherein the neurological disorder is epilepsy.
14. A compound for use according to claim 13, wherein the epilepsy
is symptomatic epilepsy, and wherein said symptomatic epilepsy is
caused by brain injury, brain tumor, brain infection,
adrenoleukodystrophy, Rasmussen's syndrome, Sturge-Weber syndrome,
megalencephaly, polyhydramnios, tuberous sclerosis complex (TSC),
symptomatic epilepsy syndrome, PMSE, PTEN mutations or focal
cortical dysplasia (FCD).
15. A compound for use according to any one of claims 13 to 14,
wherein the epilepsy is symptomatic epilepsy, wherein said
symptomatic epilepsy is due to a disease characterized by
upregulation of mTOR ("TORopathy").
Description
[0001] The present invention relates to compositions for use in the
prevention or treatment of a neurological disorder in a
subject.
RELATED ART
[0002] The protein kinase mTOR (mammalian target of rapamycin) is
an integrating factor in energy metabolism, differentiation, growth
and survival of the cell. Additionally, mTOR has critical functions
in brain-specific mechanisms such as synaptic plasticity, learning
and cortical development (Wong, M., Biomed J, 2013. 36(2): p.
40-50). The mTOR signaling pathway is activated in many diseases.
In particular, various neurological disorders such as Huntington's
disease and epileptic seizures have been linked to the
phosphatidylinositol-3-kinase (PI3K)/mTOR pathway.
[0003] So far available mTOR inhibitors such as rapamycin, a
macrolide antibiotic with immunosuppressive and anti-inflammatory
potency, and the closely related rapalogs bind allosterically to
the FKBP binding pocket of mTORC1 (Laplante, M. and D. M. Sabatini,
Cell, 2012. 149(2): p. 274-93.). Prominent downstream effectors of
mTOR are S6 kinase (S6K), S6 ribosomal protein (S6rP) and
4E-binding protein (4E-BP). Inhibitory effects on mTORC2 appear
negligible resulting in a partial inhibition of mTOR effects in
neurological disorders. Furthermore, these compounds show
undesirable physicochemical properties. A first generation of ATP
site directed mTOR inhibitors like INK128 has been developed. These
inhibitors inhibit mTORC1 and mTORC2 and block all the functions of
mTOR but on the other hand, lack high target specificity which
might result in lower tolerability. Additionally, a non-favorable
PK profile limits penetration over the blood brain barrier and,
therefore, target inhibition in the CNS, which is needed for
treatment of CNS disorders.
[0004] Aggregation of proteins can lead to various diseases.
Certain proteins are toxic in the central nervous system (CNS)
despite the fact that they are ubiquitously expressed. These
neurodegenerative diseases include disorders in which the
pathological proteins may accumulate within the nucleus, as is the
case with polyglutamine expansion diseases (such as Huntington's
disease and spinocerebellar ataxias), disorders characterized by
cytoplasmic inclusions (such as .alpha.-synuclein in Parkinson's
disease), as well as disorders in which pathological proteins
accumulate extracellularly (for example in prion diseases) or both
intracellularly and extracellularly (for example, tau and
amyloid-.beta. (A.beta.) in Alzheimer's disease) (Aguzzi, A. and T.
O'Connor, Nat Rev Drug Discov, 2010. 9(3): p. 237-248).
[0005] Huntington's disease, an autosomal dominant disorder,
involves relatively selective neurodegeneration in the basal
ganglia and cortex, related to trinucleotide repeat expansion of
polyglutamine on the huntingtin protein (HTT). Mutant huntingtin
protein (mHTT) is not effectively cleared from neurons, leading to
accumulation of toxic intracellular aggregates and associated
neuronal death. Lowering levels of mHTT or transforming mHTT into
less toxic species are the most promising strategies for
development of effective therapies. Besides HTT gene silencing
approaches, attempts to enhance clearance of the mHTT protein,
particularly by autophagy, have been initiated (Nopoulos, P. C.,
Dialogues Clin Neurosci, 2016. 18(1): p. 91-98).
[0006] Autophagy is a cellular pathway through which damaged or
pathological proteins and organelles are engulfed by a
double-membrane autophagosome vesicle, which fuses with the
lysosome, leading to cargo degradation (Nyfeler, B., et al.,
Methods Mol Biol, 2012. 821: p. 239-250.). Autophagy may play a key
role in neurodegenerative proteinopathies, such as Huntington's
disease, which are characterized by the accumulation of misfolded
proteins. Impaired autophagy and protein aggregation have also been
shown to be connected to other neurological disorders, like
Alzheimer's and Parkinson's disease.
[0007] Activation of the mammalian target of rapamycin (mTOR)
signaling pathway is known to reduce macroautophagy (Jung, C. H.,
et al., FEBS Lett, 2010. 584(7): p. 1287-95), and mHTT promotes
mTOR signaling (Pryor, W. M., et al., Sci Signal, 2014. 7(349): p.
ra103). In mouse models and human brains of Huntington's disease,
mTOR has been shown to be sequestered in polyglutamine aggregates.
Furthermore, mTOR inhibitors have been shown to enhance autophagy
and consequently reduce mHTT accumulation and associated neuronal
death in cellular and animal models of Huntington's disease
(Ravikumar B. et al., Nat Genet, 2004. 36: p. 585-595.; Floto R A
et al., Autophagy, 2007. 3: p. 620-622). Allosteric mTOR inhibitors
(e.g. rapamycin/rapalogs) mediate clearance of mHTT fragments and
protect against mHTT-induced toxicity in non-neuronal cells by
stimulating autophagy (Ravikumar, B. et al., Hum Mol Genet, 2002.
11(9): p. 1107-1117). However, recent studies showed that rapalogs
are inefficient in preventing neurodegeneration in the R6/2 mouse
HD model (Fox, J. H., et al., Mol Neurodegener, 2010. 5: p.
26).
[0008] Compared to allosteric mTOR inhibitors, catalytic ATP-site
directed mTOR inhibitors were recently shown to be more potent at
inducing autophagic flux and reducing mHTT accumulation in
non-neuronal cells (Roscic, A., et al., J Neurochem, 2011. 119(2):
p. 398-407) as well as to reduce mHTT aggregate accumulation and
toxicity, and prevented medium spiny neuron degeneration in
corticostriatal brain slices of R6/2 mice (Proenca, C. C., et al.,
PLoS One, 2013. 8(7): p. e68357.). Unfortunately, currently
available catalytic mTOR inhibitors lack sufficient brain
penetration and might be too toxic for long-term, in vivo proof-of
concept (PoC) studies.
[0009] The most advanced therapeutic approaches to date are based
on HTT gene silencing using antisense oligonucleotides or siRNAs by
central administration. In HD animal model these approaches have
convincingly demonstrated a strong reduction in disease
progression.
[0010] Current key challenges of the antisense approach is its
invasive central administration as well as whether these antisense
molecule achieve sufficient spread in human brain.
[0011] Among the 50 million people with activated epilepsy
worldwide, 30-40% are therapy resistant (Loscher, W. et al., Nat
Rev Drug Discov, 2013. 12(10): p. 757-776.). Examples of current
standard treatment are phenobarbital and levetiracetam.
Phenobarbital is a first generation anti-seizure drug with a
pronounced anticonvulsant efficacy but also with severe side
effects like strong sedation in human patients and animal models.
Levetiracetam belongs to the second generation anti-seizure drugs
and is one of the most widely used drugs in the treatment of
epilepsy.
[0012] Several genetic defects associated with elevated mTOR
signaling (TORopathies) lead to epileptic seizures. TSC seizures
are a form of epilepsy caused by an autosomal dominant inactivation
mutation of TSC1 or TSC2 that induces constitutive mTOR activation
resulting in the formation of benign tumors, mental retardation and
seizures. In clinical studies, everolimus treatment decreased
seizure frequency in patients with TSC and in patients with
TSC-associated SEGA. Other "TORopathies" causing epilepsy are
polyhydramnios, megalencephaly, symptomatic epilepsy syndrome,
PMSE, focal cortical dysplasia (FCD) and "TORopathies" associated
with PTEN mutations (Cardamone, M., et al., J Pediatr, 2014.
164(5): p. 1195-200; Sadowski, K., et al., Pharmacol Rep, 2015.
67(3): p. 636-46).
[0013] Epileptogenesis is the period between the occurrence of a
pro-epileptic insult (e.g. brain injury or genetic defect) and the
first spontaneous seizure. Many factors that are involved in
different types of epileptogenesis have been described, and mTOR
activation is one factor that has been observed in many types of
epilepsies (Sadowski, K., K. Kotulska-Jozwiak, and S. Jozwiak,
Pharmacol Rep, 2015. 67(3): p. 636-46.). All current seizure
suppressing agents used act symptomatically and do not alter
epileptogenesis. mTOR activation is involved in different processes
during epileptogenesis: neuronal growth and formation of
hypertrophic neural cells, inhibition of autophagy and
neuroinflammation. Furthermore, there is data that indicates that
the mTOR inhibitor rapamycin influences epileptogenesis and the
number/strength of seizures in different animal models of
epilepsy.
[0014] A major drawback of the current therapies is the limited
ability or even inability of the reported mTOR or dual mTOR/PI3K
inhibitors to cross the blood brain barrier (BBB), let alone the
cytotoxic potential of the compounds of the state of the art, which
makes the same of limited medical benefit for the treatment of
neurological disorders.
SUMMARY OF THE INVENTION
[0015] It has now surprisingly been found that the compounds of the
invention are useful for the treatment of neurological disorders.
In particular, it has been found that the compounds of the present
invention are specific inhibitors of mTOR or dual inhibitors of
PI3K/mTOR and that they are able to penetrate the blood brain
barrier, i.e. they have been found to inhibit the PI3K/mTOR pathway
in the brain. In addition, the compounds of the invention have
surprisingly been found to be non-cytotoxic and orally
bioavailable. Notably, the compounds of the invention have
surprisingly been found to induce autophagy in vitro and in vivo.
Moreover, the compounds of the invention have been found to reduce
formation of neurotoxic mutant huntingtin aggregates in vitro and
to reduce electroshock induced seizures in mice.
[0016] Due to the proven efficacy in preclinical models, the well
tolerated and blood brain barrier penetrating mTOR inhibitors of
the present invention have the potential to be developed into
clinical candidates for the treatment or prevention of neurological
disorders, such as HD and epilepsy, which are so far devastating
and often deadly diseases without any treatment options.
[0017] Thus, in a first aspect of the invention, there is provided
a compound of formula (I),
##STR00004##
wherein X.sup.1, X.sup.2 and X.sup.3 are, independently of each
other, N or CH; with the proviso that at least two of X.sup.1,
X.sup.2 and X.sup.3 are N;
Y is N or CH;
[0018] R.sup.1 and R.sup.2 are independently of each other
[0019] (i) a morpholinyl of formula (II)
##STR00005##
wherein the arrow denotes the bond in formula (I); and wherein
R.sup.3 and R.sup.4 are independently of each other H,
C.sub.1-C.sub.3alkyl optionally substituted with one or two OH,
C.sub.1-C.sub.2fluoroalkyl, C.sub.1-C.sub.2alkoxy,
C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl, CN, or
C(O)O--C.sub.1-C.sub.2alkyl; or R.sup.3 and R.sup.4 form together a
bivalent residue --R.sup.5R6-- selected from
C.sub.1-C.sub.3alkylene optionally substituted with 1 to 4 F,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--, or any of
the structures
##STR00006##
wherein the arrows denote the bonds in formula (II); or
[0020] (ii) a saturated 6-membered heterocyclic ring Z selected
from thiomorpholinyl and piperazinyl, optionally substituted by 1
to 3 R.sup.7; wherein R.sup.7 is independently at each occurrence
C.sub.1-C.sub.3alkyl optionally substituted with one or two OH,
C.sub.1-C.sub.2fluoroalkyl,
C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl,
C.sub.3-C.sub.6cycloalkyl; or two R.sup.7 substituents form
together a bivalent residue --R.sup.8R9-- selected from
C.sub.1-C.sub.3alkylene optionally substituted with 1 to 4 F,
--CH.sub.2--O--CH.sub.2-- or --O--CH.sub.2CH.sub.2--O--;
with the proviso that at least one of R.sup.1 and R.sup.2 is a
morpholinyl of formula II; and prodrugs, metabolites, tautomers,
solvates and pharmaceutically acceptable salts thereof, for use in
the prevention or treatment of a neurological disorder in a
subject.
[0021] Further aspects and embodiments of the present invention
will be become apparent as this description continues.
DESCRIPTION OF FIGURES
[0022] FIG. 1A: Pharmacokinetic analysis of Compound 3 ("Cpd. 3")
levels in Sprague Dawley (SD) rats. After a single oral
administration of Cpd. 3 (10 mg/kg), tissue samples were analyzed
for compound levels at different time points by LC-MS. n=3
[0023] FIG. 1B: Pharmacokinetic analysis of Cpd. 3 and Compound 8
(Cpd. 8) levels in balb 6 mice. After a single oral administration
of Cpd. 3 or Cpd. 8 (50 mg/kg), tissue samples were analyzed for
compound levels at different time points by LC-MS. n=3
[0024] FIG. 2 A: Pharmacodynamic analysis of Cpd. 3 administered to
balb 6 mice in a single oral application of 50 mg/kg. Brain lysates
were analyzed by western blot. mTOR signaling is inhibited by Cpd.3
as indicated by reduction of S6 phosphorylation at time points
between 30 minutes and 8 hours. Quantification of western blot
bands shows significance of reduction of mTOR signaling pathway.
n=3, analysis of variance (ANOVA), *** p<0.0005, ** p<0.005,
* p<0.05
[0025] FIG. 2B: Pharmacodynamic analysis of Cpd. 8 administered to
balb 6 mice in a single oral application of 50 mg/kg. Brain lysates
were analyzed by western blot. mTOR signaling is inhibited by Cpd.
8 as indicated by reduction of S6 phosphorylation at time points
between 30 minutes and 8 hours. Quantification of western blot
bands shows significance of reduction of mTOR signaling pathway.
n=3, ANOVA, *** p<0.0005, ** p<0.005, * p<0.05
[0026] FIG. 2C: Pharmacodynamic analysis of Cpd. 3 administered to
balb 6 mice in a single oral application of 50 mg/kg. Thigh muscle
lysates were analyzed by western blot. mTOR signaling is inhibited
by Cpd. 3 as indicated by reduction of S6 phosphorylation at time
points between 30 minutes and 8 hours. Quantification of western
blot bands shows significance of reduction of mTOR signaling
pathway. n=3, ANOVA, *** p<0.0005, ** p<0.005, *
p<0.05
[0027] FIG. 2D: Pharmacodynamic analysis of Cpd. 8 administered to
balb 6 mice in a single oral application of 50 mg/kg. Thigh muscle
lysates were analyzed by western blot. mTOR signaling is inhibited
by Cpd. 8 as indicated by reduction of S6 phosphorylation at time
points between 30 minutes and 8 hours. Quantification of western
blot bands shows significance of reduction of mTOR signaling
pathway. n=3, ANOVA, *** p<0.0005, ** p<0.005, *
p<0.05
[0028] FIG. 3A: Western blot analysis of lysed mouse brains from
pilocarpine-pretreated, epileptic mice and their untreated
counterparts. mTOR signaling is elevated in the pilocarpine status
epilepticus (SE) model. n=9 (naive mice), n=8 (epileptic mice),
ANOVA * p<0.05
[0029] FIG. 3B: Western Blot analysis of lysed mouse brain from
epileptic/pilocarpine pre-treated and naive mice that were treated
with a single oral dose of Compound 3 and 8 significantly inhibited
S6 phosphorylation in brain. Stronger effects were observed in
naive mice. Everolimus did not show significant mTOR signaling
inhibition in brain. MEAN.+-.SEM, ANOVA, * p<0.05, # p<0.05,
n=5
[0030] FIG. 4: After treatment of epileptic and nonepileptic mice
with mTOR inhibitors, Phenobarbital or Levetiracetam maximal
electroshock seizure threshold test (MEST) was performed.
Antiepileptic effect of inhibitors was observed.
[0031] FIG. 4 A, B: MEST of naive mice.
[0032] FIG. 4 C, D: MEST of pilocarpine treated, epileptic
mice.
[0033] Pheno=Phenobarbital, LEV=Levetiracetam, Evero=Everolimus,
Rapa=Rapamycin, Brackets: dose provided in mg/kg, pretreatment time
in h, route of administration per oral (po) or intra peritoneal
(ip). A, C: CC50 in mA.+-.standard error of the mean (SEM); B, D:
Change of CC50 in % to vehicle CC50, ANOVA and post hoc Dunnett's
test, * p<0.05
[0034] FIG. 5: Cell viability and protein synthesis of immortalized
striatal cells derived from a knock-in mouse model expressing
full-length HTT with 7 (STHdh.sup.Q7/Q7) or 111 CAG-repeats
(STHdh.sup.Q111/Q111) after incubation with different
concentrations of Cpd. 3 and Cpd. 8 and the mTORC1/2 inhibitor INK
128 (Sapanisertib) (100 nM) and rapamycin (400 nM).
[0035] FIG. 5 A+B Lactate dehydrogenase (LDH) assay in A:
STHdh.sup.Q7/Q7. B: STHdh.sup.Q111/Q111. Both, Cpd. 3 and Cpd. 8
reduced LDH production at time points between 24 h and 48 h;
n=3-6
[0036] FIG. 5 C+D PrestoBlue assay detecting the mitochondrial
activity of STHdh.sup.Q7/7 and STHdh.sup.Q111/Q111 at different
time points after incubation with Cpd. 3, Cpd. 8 and reference
compounds INK128 (100 nM) and rapamycin (400 nM). Compounds were
well tolerated only leading to slight inhibition after 72 hours of
incubation with 1230 nM of Cpd. 3. n=6-9
[0037] ANOVA *** p<0.0005, ** p<0.005, * p<0.05
[0038] FIG. 6: Inhibition of the mTOR signaling pathway and
induction of autophagy in STHdh.sup.Q7/Q7 and STHdh.sup.Q111/Q111
cells after treatment with Cpd. 3 (200 nM, 400 nM, 1230 nM), Cpd. 8
(130 nM, 1230 nM), INK 128 (100 nM), rapamycin (400 nM) or DMSO
control for 4 hours. Western blot analysis of cell lysates.
[0039] FIG. 6 A+B: Decrease in phosphorylation of mTOR signaling
molecules mTOR, S6rp and 4E-PB is concentration dependent. n=3
[0040] FIG. 6 C+D: Induction of autophagy indicated by increase in
LC3-II. n=3 [0041] ANOVA *** p<0.0005, ** p<0.005, *
p<0.05
[0042] FIG. 7: Reduction of mHTT aggregate formation on human
embryonic kidney cells (HEK293) cells transfected with exon 1 of
mHTT with a Q51 or Q19 extension, respectively.
[0043] FIG. 7 A+B: Filter trap assay of lysed HEK293 cell
transfected with exon 1 of mHTT with a Q51 pre-treated with Cpd. 8
(130 nM, 1230 nM), cpd. 3 (400 nM), 1230 nM), INK128 (100 nM),
rapamycin (400 nM) or DMSO control and Q19 extension treated with
DMSO control. Treatment with mTOR inhibitors significantly reduced
aggregate formation. n=3, ANOVA *** p<0.0005, ** p<0.005, *
p<0.05
[0044] FIG. 7 C+D: Immuno staining of HEK293 cell transfected with
exon 1 of mHTT with a Q51 pre-treated with Cpd. 8 (130 nM, 1230
nM), cpd. 3 (400 nM), 1230 nM), INK128 (100 nM), rapamycin (400 nM)
or DMSO control. Aggregates were manually counted in 10000 cells
per sample. ANOVA **** p<0.00005, *** p<0.0005, **
p<0.005, * p<0.05
[0045] FIG. 8A: Induction of autophagy in brains of BALB/C mice 0.5
h, 4 h, 8 h, respectively, after a single, oral administration of
Cpd. 3, 50 mg/kg. Brain lysates were analyzed by western blot.
Autophagic markers LC3-II and p62 were altered at time points
between 30 minutes and 8 hours. Quantification of western blot
bands shows induction of autophagy. n=3, ANOVA, *** p<0.0005, **
p<0.005, * p<0.05
[0046] FIG. 8B: Induction of autophagy in brains of BALB/C mice
after a single, oral administration of Cpd. 8, 50 mg/kg. Brain
lysates were analyzed by western blot. Autophagic markers LC3-II
and p62 were altered at time points between 30 minutes and 8 hours.
Quantification of western blot bands shows induction of autophagy.
n=3, ANOVA, *** p<0.0005, ** p<0.005, * p<0.05.
[0047] FIG. 9A: Body weight of TSC1 GFAP mice treated with vehicle,
Cpd. R, Cpd. 3 and Cpd. 8 at different postnatal days before (P21)
and during (P22-P50) treatment. Mean.+-.SEM. As some animals died
during observation period n varied between 6 and 14.
[0048] FIG. 9B: Effects of specific treatment regimens on
spontaneous electrographic seizures in Tsc1 GFAP mice. Cpd. 3 and
Cpd. 8 strongly reduce spontaneous seizures while the reference
molecule Cpd. R had a weaker effect. Data are presented as
mean.+-.SEM (N=27-48 recordings/group) for each week of testing.
Statistical significance is marked by a multiplicity-adjusted
p<0.05 with vehicle as a comparator (Wilcoxon non-parametrical
test).
[0049] FIG. 10: IHC analysis of striata from R6/2 mice and one wt
animal. Mice were treated with vehicle, Cpd. 3 and Cpd.8 for 11.5
weeks and striata were sectioned. After staining for mutHTT
aggregates, number of aggregates was counted and area of aggregates
was analyzed in 4 samples. While the number of mutHTT aggregates
was unchanged, the area covered by these aggregates was
significantly reduced after Cpd. 3 treatment indicating that mutHTT
aggregates were smaller after treatment. (ANOVA, p<0.05)
DETAILED DESCRIPTION OF THE INVENTION
[0050] Reference will now be made in detail to the presented and
further aspects and the presented and further embodiments of the
invention, examples of which are illustrated in the accompanying
structures and formulas. While the invention will be described in
conjunction with the enumerated embodiments, it will be understood
that they are not intended to limit the invention to those
embodiments. One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. The present
invention is in no way limited to the methods and materials herein
described.
[0051] Features, integers and characteristics, described in
conjunction with a particular aspect, embodiment or example of the
invention are to be understood to be applicable to any other
aspect, embodiment or example described herein unless incompatible
therewith. All of the features disclosed in this specification
(including any accompanying claims, abstract and drawings), and/or
all of the steps of any method or process so disclosed, may be
combined in any combination, except combinations where at least
some of such features and/or steps are mutually exclusive. The
invention is not restricted to the details of any foregoing
embodiments. The invention extends to any novel one, or any novel
combination, of the features disclosed in this specification
(including any accompanying claims, abstract and drawings), or to
any novel one, or any novel combination, of the steps of any method
or process so disclosed.
[0052] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0053] For the purposes of interpreting this specification, the
following definitions will apply and whenever appropriate, terms
used in the singular will also include the plural and vice versa.
It is to be understood that the terminology used herein is for the
purpose of describing particular embodiments only and is not
intended to be limiting.
Definitions
[0054] The terms "comprising", "having", and "including" are to be
construed as open-ended terms (i.e., meaning "including, but not
limited to,") unless otherwise noted.
[0055] The terms "individual," "subject" or "patient" are used
herein interchangeably. In a preferred embodiment, the subject is a
human.
[0056] The terms "treatment"/"treating" as used herein include: (1)
preventing or delaying the appearance of clinical symptoms of the
state, disorder or condition developing in a subject that may be
afflicted with or predisposed to the state, disorder or condition
but does not yet experience or display clinical or subclinical
symptoms of the state, disorder or condition; (2) inhibiting the
state, disorder or condition (e.g. arresting, reducing or delaying
the development of the disease, or a relapse thereof in case of
maintenance treatment, of at least one clinical or subclinical
symptom thereof); and/or (3) relieving the condition (i.e. causing
regression of the state, disorder or condition or at least one of
its clinical or subclinical symptoms). The benefit to a patient to
be treated is either statistically significant or at least
perceptible to the patient or to the physician. However, it will be
appreciated that when a medicament is administered to a patient to
treat a disease, the outcome may not always be effective treatment.
In one embodiment, the terms "treatment"/"treating" as used herein,
refer to a therapeutic treatment.
[0057] In another embodiment, the terms "treatment"/"treating" as
used herein, refer to a prophylactic treatment.
[0058] The term "chiral" refers to molecules, which have the
property of non-superimposability of the mirror image partner,
while the term "achiral" refers to molecules, which are
superimposable on their mirror image partner.
[0059] The term "stereoisomers" refers to compounds, which have
identical chemical constitution, but differ with regard to the
arrangement of the atoms or groups in space.
[0060] "Diastereomer" refers to a stereoisomer with two or more
centers of chirality in which the compounds are not mirror images
of one another. Diastereomers have different physical properties,
e.g. melting points, boiling points, spectral properties, and
chemical and biological reactivities. Mixtures of diastereomers may
be separated under high resolution analytical procedures such as
electrophoresis and chromatography.
[0061] "Enantiomers" refer to two stereoisomers of a compound which
are non-superimposable mirror images of one another. Stereochemical
definitions and conventions used herein generally follow S. P.
Parker, Ed., McRaw-Hiff Dictionary of Chemical Terms (1984),
McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,
"Stereochemistry of Organic Compounds", John Wiley & Sons,
Inc., New York, 1994. The compounds of the invention may contain
asymmetric or chiral centers, and therefore exist in different
stereoisomeric forms. It is intended that all stereoisomeric forms
of the compounds of the invention, including but not limited to,
diastereomers, enantiomers and atropisomers, as well as mixtures
thereof such as racemic mixtures, form part of the present
invention. Many organic compounds exist in optically active forms,
i.e., they have the ability to rotate the plane of plane-polarized
light. In describing an optically active compound, the prefixes D
and L, or R and S, are used to denote the absolute configuration of
the molecule about its chiral center(s). The prefixes d and 1 or
(+) and (-) are employed to designate the sign of rotation of
plane-polarized light by the compound, with (-) or l meaning that
the compound is levorotatory. A compound prefixed with (+) or d is
dextrorotatory. For a given chemical structure, these stereoisomers
are identical except that they are mirror images of one another. A
specific stereoisomer may also be referred to as an enantiomer, and
a mixture of such isomers is often called an enantiomeric or a
scalemic mixture. A 50:50 mixture of enantiomers is referred to as
a racemic mixture or a racemate. The term "tautomer" or "tautomeric
form" refers to structural isomers of different energies, which are
interconvertible via a low energy barrier. For example, proton
tautomers include interconversions via migration of a proton, such
as keto-enol and imine-enamine isomerizations.
[0062] The phrase "pharmaceutically acceptable salt" as used
herein, refers to pharmaceutically acceptable organic or inorganic
salts of a compound of the invention, in particular acid addition
salts. Exemplary salts include, but are not limited to, sulfate,
citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,
bisulfate, phosphate, acid phosphate, isonicotinate, lactate,
salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,
bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,
gluconate, glucuronate, saccharate, formate, benzoate, glutamate,
methanesulfonate (mesylate), ethanesulfonate, benzenesulfonate,
p-toluenesulfonate, and pamoate salts. A pharmaceutically
acceptable salt may involve the inclusion of another molecule such
as an acetate ion, a succinate ion or other counter ion. The
counter ion may be any organic or inorganic moiety that stabilizes
the charge on the parent compound. Furthermore, a pharmaceutically
acceptable salt may have more than one charged atom in its
structure. Instances where multiple charged atoms are part of the
pharmaceutically acceptable salt can have multiple counter ions.
Hence, a pharmaceutically acceptable salt can have one or more
charged atoms and/or one or more counter ion.
[0063] If the compound of the invention is a base, the desired
pharmaceutically acceptable salt may be prepared by any suitable
method available in the art, for example, treatment of the free
base with an inorganic acid, such as hydrochloric acid, hydrobromic
acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric
acid and the like, or with an organic acid, such as acetic acid,
trifluoroacetic acid, maleic acid, succinic acid, mandelic acid,
fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic
acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid
or galacturonic acid, an alpha hydroxy acid, such as citric acid or
tartaric acid, an amino acid, such as aspartic acid or glutamic
acid, an aromatic acid, such as benzoic acid or cinnamic acid, a
sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic
acid, or the like.
[0064] The phrase "pharmaceutically acceptable" indicates that the
substance or composition must be compatible chemically and/or
toxicologically, with the other ingredients comprising a
formulation, and/or the mammal being treated therewith.
[0065] A "solvate" refers to an association or complex of one or
more solvent molecules and a compound of the invention. Examples of
solvents that form solvates include, but are not limited to, water,
isopropanol, ethanol, methanol, dimethyl sulfoxide (DMSO), ethyl
acetate, acetic acid, and ethanolamine. The term "hydrate" refers
to the complex where the solvent molecule is water.
[0066] The term "protecting group" refers to a substituent that is
commonly employed to block or protect a particular functionality
during the reaction of other functional groups on the compound. For
example, an "amino-protecting group" is a substituent attached to
an amino group that blocks or protects the amino functionality in
the compound. Suitable amino-protecting groups include acetyl,
trifluoroacetyl, tert-butoxycarbonyl (BOC), benzyloxycarbonyl and
9-fluorenylmethylenoxycarbonyl (Fmoc). For a general description of
protecting groups and their use, see T. W. Greene, Protective
Groups in Organic Synthesis, John Wiley & Sons, New York,
1991.
[0067] The terms "compound of this invention" and "compounds of the
present invention" and "compounds of formula (I)" include
stereoisomers, geometric isomers, tautomers, solvates,
pharmaceutically acceptable salts, and solvates of the salts
thereof.
[0068] The term "mammal" includes, but is not limited to, humans,
mice, rats, guinea pigs, monkeys, dogs, cats, horses, cows, pigs,
and sheep. The term "mammal", as used herein, preferably refers to
humans.
[0069] The terms "Alzheimer's disease" and "morbus Alzheimer" are
used herein interchangeably.
[0070] The term "neurological disorder" generally refers to a
disorder affecting the nervous system, including the central
nervous system and the peripheral nervous system. Neurological
disorders, in particular central nervous system (CNS) disorders,
encompass numerous afflictions, including inter alia
neurodegenerative diseases, such as Huntington's disease and a
large number of central nervous system dysfunctions, such as
epilepsy.
[0071] The term "neurodegenerative disease" refers to a disease
which is caused by damage to the central nervous system and can be
identified by progressive dysfunction, degeneration and death of
specific populations of neurons which are often synaptically
interconnected. Neurodegeneration causes dysfunction of movement
and/or mental function. Further, the term "neurodegenerative
disease" as used herein describes neurodegenerative diseases which
are associated with or caused by protein misfolding and/or
aggregation. Exemplary neurodegenerative diseases include
Huntington's disease, spinocerebellar ataxias, Parkinson's disease,
morbus Alzheimer, amyotrophic lateral sclerosis (ALS), cystic
fibrosis, familial amyloidotic polyneuropathy, spongiform
encephalopathies, dementia with Lewy bodies, frontotemporal
dementia with Parkinsonism, spinocerebellar ataxias, spinal and
bulbar muscular atrophy, hereditary dentatorubral-pallidoluysian
atrophy, familial British dementia, familial Danish dementia and
prion disease.
[0072] The terms "epilepsy" as used herein refer to any chronic
neurological disorder characterized by recurrent seizures. Each
seizure may appear to be unprovoked or may be triggered or provoked
by stress, anxiety, sleep deprivation, illness, chemical exposure
(e.g., drug abuse or alcohol consumption), photic stimulation
(e.g., a flashing/flickering light), and/or the like. The disorder
may have a cause that is unknown ("idiopathic epilepsy") or may be
caused, for example, by head trauma, a brain tumor, a genetic
predisposition, an infection, a developmental defect, or any
combination thereof, among others ("symptomatic epilepsy").
Exemplary types of epileptic seizures include partial or focal
onset seizures, which are localized (at least initially) within the
brain, and generalized seizures, which are distributed widely
within the brain. Partial seizures may be further categorized as
simple partial seizures, which do not affect consciousness, and
complex partial seizures, which do affect consciousness.
Generalized seizures, which produce a loss of consciousness, may
include absence, atonic, clonic, myoclonic, tonic, and tonic-clonic
seizures, among others. Epilepsy and/or an epileptic seizure may be
diagnosed by any suitable technique or combination of techniques
including electroencephalography (EEG), magnetoencephalography,
magnetic resonance imaging (MRI), positron emission tomography
(PET), single photon emission computed tomography (SPECT), or
video-EEG, among others.
[0073] The term "seizure," as used herein, means a neurological
event characterized by abnormal electrical activity in the brain
that results in at least one clinical symptom. The electrical
activity may be characterized by hypersynchrony, hyperactivity,
and/or hyperexcitability of neurons in a portion or all of the
brain. Exemplary symptoms produced by seizures may include sudden
and involuntary muscle contraction (e.g., convulsions), numbness of
a part or all of the body, memory loss, loss of consciousness,
inability to concentrate, hallucinations, and/or the like. Seizures
thus may affect motor, autonomic, cognitive, sensory (visual,
auditory, olfactory, taste, feel), and/or emotional function, among
others. Each seizure may be characterized either as an epileptic
seizure, produced by epilepsy, or a non-epileptic seizure with any
other cause.
[0074] As indicated above, classification of epilepsy as
"symptomatic" indicates that a probable cause exists and a specific
course of therapy to eliminate that cause may be tried, whereas
classification as "idiopathic" indicates that no obvious cause can
be found.
[0075] The term "TSC" relates to a form of epilepsy caused by an
autosomal dominant inactivation mutation of TSC1 or TSC2 that
induces constitutive mTOR activation resulting in the formation of
benign tumors, mental retardation and seizures. Therefore, TSC is a
model form of epilepsy to investigate effects of mTOR inhibitors on
epileptogenesis and epileptic seizures. Subependymal giant cell
astrocytomas (SEGAs) develop in 90% of patients with TSC. In the
brain, disturbances of normal cellular development and function can
lead to epilepsy and neurocognitive, behavioral, and psychiatric
deficits. Epilepsy occurs in 80-90% of patients with TSC, and drug
resistance is common (Curatolo, P., R. Moavero, and P. J. de Vries,
The Lancet Neurology. 14(7): p. 733-745). Inhibition of mTOR with
rapamycin has positive effects on behavior, reduces tumor formation
and suppresses seizures in TSC mouse models. Early treatment with
rapamycin prevents the development of epilepsy and premature death
(Meikle, L., et al., J Neurosci, 2008. 28(21): p. 5422-5432). A
brief treatment with rapamycin in adult mice rescued not only the
synaptic plasticity, but also the behavioral deficits a
heterozygous model of TSC (Ehninger, D., et al., Nat Med, 2008.
14(8): p. 843-848.). Furthermore, rapamycin treatment could reverse
cellular abnormalities of neurons with TSC mutation (Goto, J., et
al., Proceedings of the National Academy of Sciences, 2011.
108(45): p. E1070-E1079.).
[0076] Other "TORopathies", i.e. primary diseases that are due to
upregulation of mTOR, causing epilepsy are polyhydramnios,
megalencephaly, symptomatic epilepsy syndrome, PMSE, focal cortical
dysplasia (FCD) and "TORopathies" associated with PTEN
mutations.
[0077] The term "PMSE" refers to polyhydramnios, megalencephaly,
and symptomatic epilepsy, a rare syndrome found in some Amish
children characterized by an abnormally large brain, cognitive
disability, and severe, treatment-resistant epilepsy.
[0078] The term "PTEN" refers to phosphatase and tensin homolog, a
phosphatase that dephosphorylates the 3' phosphate of the inositol
ring in PIP3. PTEN is a tumor suppressor that is commonly mutated
in cancer, leading to increased signaling of the mTOR pathway. PTEN
deletion in the CNS can be associated with seizures.
[0079] The term "epileptogenesis" refers to the period between the
occurrence of a pro-epileptic insult (e.g. brain injury or genetic
defect) and the first spontaneous seizure. "Epileptogenesis" is
divided into primary epileptogenesis (until first seizure) and
secondary epileptogenesis (progression of epilepsy after occurrence
of a first seizure). The compounds of the invention may be used
both during primary epileptogenesis and during secondary
epileptogenesis (partial seizures) as well as in fully developed
epileptic disorders (generalized seizures).
[0080] Treating epilepsy means, for example to suppress one or more
seizures and/or to suppress seizure activity, i.e. to reduce the
frequency of seizures; to reduce the severity, physical extent,
and/or duration of at least one seizure; to substantially prevent
at least one seizure; or to slow down/reduce/prohibit
epileptogenesis; or any combination thereof. Seizure suppression
for a particular subject may be measurable directly from the
subject (e.g., if a seizure is in progress during treatment)
and/or, more typically, may be a statistically predicted outcome
based on results from controlled tests or clinical trials with a
group of subjects.
[0081] The term "effective amount" means an amount of a compound of
the present invention that (i) treats or prevents the particular
disease, condition, or disorder, (ii) attenuates, ameliorates, or
eliminates one or more symptoms of the particular disease,
condition, or disorder, or (iii) prevents or delays the onset of
one or more symptoms of the particular disease, condition, or
disorder described herein.
[0082] In case of epilepsy, the effective amount of the drug may
reduce strength or number of seizures as well as events during
epileptogenesis leading to development of epilepsy.
[0083] In the case of neurodegenerative diseases, the effective
amount of the drug may reduce the amount of protein aggregates in
neurons or other cells and in the CNS in general as well as
associated cognitive, psychological and motor symptoms. For therapy
of neurodegenerative diseases, efficacy can be measured, for
example, by assessing the number of protein aggregates, cognitive
and motor function.
[0084] The term "maximum tolerated dose" (MTD) refers to the
highest dose of a drug or treatment that does not cause
unacceptable side effects. Typically, the maximum tolerated dose is
determined in the species that needs to be treated, e.g. in rodents
or in humans during a clinical trial by testing increasing doses on
different groups of a given species until the highest dose with
acceptable side effects is found. The compounds of the present
invention have been found to have a MTD within their therapeutic
window (see Examples 3 and 4).
[0085] The term "prodrug" as used in this application refers to a
precursor or derivative form of a compound of the invention that
may have improved properties such as better solubility, reduced
cytotoxicity or increased bioavailability compared to the parent
compound or drug and is capable of being activated or converted
into the more active parent form. The prodrugs of this invention
include, but are not limited to, derivatives of the amino group
connected to the pyridine or pyrimidine nucleus in which one or two
hydrogens are replaced by a suitable substituent, or derivatives of
the ring amino function if R.sup.2 is piperazin-1-yl. Examples of
such prodrugs are compounds acylated by an amino acid selected from
the 20 most often occurring natural L-alpha-amino acids, acylated
by a dipeptide such as L-Ala-L-Ala, by carbonic acid, sulfuric acid
or phosphoric acid, as well as pharmaceutically acceptable salts
thereof.
[0086] A "metabolite" is a product produced through metabolism in
the body of a specified compound or salt thereof. Metabolites of a
compound may be identified using routine techniques known in the
art and their activities determined using tests such as those
described herein. Such products may result for example from the
oxidation, reduction, hydrolysis, amidation, deamidation,
esterification, deesterification, enzymatic cleavage, and the like,
of the administered compound. In particular, compounds of formula
(I) as defined hereinbefore, which are oxygenated or hydroxylated
at any one position in the morpholine, piperazine or thiomorpholine
ring R.sup.1 and/or R.sup.2 are considered metabolites. Further
metabolites considered are thiomorpholine S-oxides and
thiomorpholine S,S-dioxides. Accordingly, the invention is also
directed to metabolites of compounds of the invention, including
compounds produced by a process comprising contacting a compound of
this invention with a mammal for a period of time sufficient to
yield a metabolic product thereof.
[0087] In a first aspect, the present invention provides for a
compound of formula (I),
##STR00007##
wherein X.sup.1, X.sup.2 and X.sup.3 are, independently of each
other, N or CH; with the proviso that at least two of X.sup.1,
X.sup.2 and X.sup.3 are N;
Y is N or CH;
[0088] R.sup.1 and R.sup.2 are independently of each other
[0089] (i) a morpholinyl of formula (II)
##STR00008##
wherein the arrow denotes the bond in formula (I); and wherein
R.sup.3 and R.sup.4 are independently of each other H,
C.sub.1-C.sub.3alkyl optionally substituted with one or two OH,
C.sub.1-C.sub.2fluoroalkyl, C.sub.1-C.sub.2alkoxy,
C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl, CN, or
C(O)O--C.sub.1-C.sub.2alkyl; or R.sup.3 and R.sup.4 form together a
bivalent residue --R.sup.5R6-- selected from
C.sub.1-C.sub.3alkylene optionally substituted with 1 to 4 F,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--, or any of
the structures
##STR00009##
wherein the arrows denote the bonds in formula (II); or
[0090] (ii) a saturated 6-membered heterocyclic ring Z selected
from thiomorpholinyl and piperazinyl, optionally substituted by 1
to 3 R.sup.7; wherein R.sup.7 is independently at each occurrence
C.sub.1-C.sub.3alkyl optionally substituted with one or two OH,
C.sub.1-C.sub.2fluoroalkyl,
C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl,
C.sub.3-C.sub.6cycloalkyl; or two R.sup.7 substituents form
together a bivalent residue --R.sup.8R.sup.9-- selected from
C.sub.1-C.sub.3alkylene optionally substituted with 1 to 4 F,
--CH.sub.2--O--CH.sub.2-- or --O--CH.sub.2CH.sub.2--O--;
with the proviso that at least one of R.sup.1 and R.sup.2 is a
morpholinyl of formula II; and prodrugs, metabolites, tautomers,
solvates and pharmaceutically acceptable salts thereof, for use in
the prevention or treatment of a neurological disorder in a
subject.
[0091] In another aspect, the invention provides for a compound of
formula (I),
##STR00010##
[0092] wherein
X.sup.1, X.sup.2 and X.sup.3 are, independently of each other, N or
CH; with the proviso that at least two of X.sup.1, X.sup.2 and
X.sup.3 are N; Y is N or CH; R.sup.1 is 4-morpholinyl,
2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl or
3-aza-8-oxabicyclo[3.2.1]oct-3-yl; and R.sup.2 is 4-morpholinyl,
2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl,
3-aza-8-oxabicyclo[3.2.1]oct-3-yl, piperazin-1-yl,
4-methylpiperazin-1-yl, or 4-thiomorpholinyl.
[0093] Each alkyl moiety either alone or as part of a larger group
such as alkoxy is a straight or branched chain and is preferably
C.sub.1-C.sub.3alkyl, more preferably C.sub.1-C.sub.2alkyl.
Examples include in particular methyl, ethyl, n-propyl and
prop-2-yl (iso-propyl). Examples of an alkoxy include in particular
methoxy, ethoxy, n-propoxy and iso-propoxy. As described herein,
alkoxy may include further substituents such as halogen atoms
leading to haloalkoxy moieties.
[0094] The term "alkoxyalkyl" refers to an R--O--R' moiety in which
the R and R' groups are alkyl groups as defined herein. Examples
include methoxymethyl, methoxyethyl, ethoxyethyl and
methoxypropyl.
[0095] Each alkylene moiety is a straight or branched chain and is,
particularly for example, --CH.sub.2--, --CH.sub.2--CH.sub.2--,
--CH(CH.sub.3)--, --CH.sub.2--CH.sub.2--CH.sub.2--,
--CH(CH.sub.3)--CH.sub.2--, or --CH(CH.sub.2CH.sub.3)--, preferably
--CH.sub.2--, --CH.sub.2--CH.sub.2-- or --CH(CH.sub.3)--.
[0096] Each haloalkyl moiety either alone or as part of a larger
group such as haloalkoxy is an alkyl group substituted by one or
more of the same or different halogen atoms. Haloalkyl moieties
include for example 1 to 5 halo substituents, or 1 to 3 halo
substituents. Examples include in particular fluoromethyl,
difluoromethyl, trifluoromethyl, chlorodifluoromethyl and
2,2,2-trifluoro-ethyl.
[0097] Each haloalkenyl moiety either alone or as part of a larger
group such as haloalkenyloxy is an alkenyl group substituted by one
or more of the same or different halogen atoms. Examples include
2-difluoro-vinyl and 1,2-dichloro-2-fluoro-vinyl. Haloalkenyl
moieties include for example 1 to 5 halo substituents, or 1 to 3
halo substituents.
[0098] Each cycloalkyl moiety can be in mono- or bi-cyclic form,
typically and preferably in mono-cyclic form, and preferably
contains 3 to 6 carbon atoms. Preferred examples of monocyclic
cycloalkyl groups include in particular cyclopropyl, cyclobutyl,
cyclopentyl and cyclohexyl.
[0099] The term "heterocyclic ring" refers to a saturated or
partially unsaturated carbocyclic ring containing one to three
heteroatoms selected from nitrogen, oxygen and sulfur as ring
members. Such rings do not contain adjacent oxygen atoms, adjacent
sulfur atoms, or adjacent oxygen and sulfur atoms within the ring.
Preferred examples include in particular tetrahydrofuranyl,
pyrrolidinyl, pyrazolidinyl, imidazolidinyl, piperidinyl,
piperazinyl, dioxanyl, morpholinyl, oxazolidinyl and
isooxazolidinyl.
[0100] Where a group is said to be optionally substituted,
preferably there are optionally 1-3 substituents, more preferably
optionally 1-2 substituents.
[0101] Certain compounds of formula (I) may contain one or two or
more centers of chirality and such compounds may be provided as
pure enantiomers or pure diastereoisomers as well as mixtures
thereof in any ratio. The compounds of the invention also include
all tautomeric forms of the compounds of formula (I).
[0102] In a preferred embodiment, the present invention provides
for the compound of formula (I) as defined herein and tautomers,
solvates and pharmaceutically acceptable salts thereof.
[0103] In another preferred embodiment, the present invention
provides for the compound of formula (I), wherein X.sup.1, X.sup.2
and X.sup.3 are N.
[0104] In another preferred embodiment, (i) said X.sup.1 and said
X.sup.2 are N, and said X.sup.3 is CH; (ii) said X.sup.1 and said
X.sup.3 are N, and said X.sup.2 is CH; or (iii) said X.sup.2 and
said X.sup.3 are N, and said X.sup.1 is CH, and preferably
tautomers, solvates and pharmaceutically acceptable salts thereof.
In another embodiment, (i) said X.sup.1 and said X.sup.2 are N, and
said X.sup.3 is CH; or (ii) said X.sup.2 and said X.sup.3 are N,
and said X.sup.1 is CH, and preferably tautomers, solvates and
pharmaceutically acceptable salts thereof. In another preferred
embodiment, said X.sup.1 and said X.sup.3 are N, and said X.sup.2
is CH; and preferably tautomers, solvates and pharmaceutically
acceptable salts thereof.
[0105] In another preferred embodiment, said Y is N, and preferably
tautomers, solvates and pharmaceutically acceptable salts thereof.
In another preferred embodiment, said Y is CH, and preferably
tautomers, solvates and pharmaceutically acceptable salts
thereof.
[0106] In another preferred embodiment, said R.sup.1 and said
R.sup.2 are independently of each other selected from
##STR00011## ##STR00012## ##STR00013##
[0107] In another preferred embodiment, said R.sup.1 and said
R.sup.2 are independently of each other selected from
##STR00014##
[0108] In another preferred embodiment, said R.sup.1 and said
R.sup.2 are independently of each other selected from
##STR00015##
[0109] In another preferred embodiment, said compound is selected
from [0110]
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyridin--
2-amine; [0111]
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyrimidin-2-ami-
ne; [0112]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicycl-
o[3.2.1]octan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
[0113]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1,3,5-tria-
zin-2-yl)-4-(difluoromethyl)pyridin-2-amine; [0114]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1,3,5-triazin-2-y-
l)-4-(difluoromethyl)pyrimidin-2-amine; [0115]
5-(4,6-bis((S)-3-methylmorpholino)-1,3,5-triazin-2-yl)-4-(difluoromethyl)-
pyridin-2-amine; [0116]
5-(4,6-bis((S)-3-methylmorpholino)-1,3,5-triazin-2-yl)-4-(difluoromethyl)-
pyrimidin-2-amine; [0117]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine; [0118]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyrimidin-2-amine; [0119]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-((S)-3-methylmorpholino)-1,3-
,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine; [0120]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-((S)-3-methylmorpholino)-1,3-
,5-triazin-2-yl)-4-(difluoromethyl)pyrimidin-2-amine; [0121]
4-(difluoromethyl)-5-(4-morpholino-6-(piperazin-1-yl)-1,3,5-triazin-2-yl)-
pyridin-2-amine; [0122]
4-(difluoromethyl)-5-(4-morpholino-6-(piperazin-1-yl)-1,3,5-triazin-2-yl)-
pyrimidin-2-amine; [0123]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-(piperazin-1-yl)-1,3,5-
-triazin-2-yl)pyridin-2-amine; [0124]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-(piperazin-1-yl)-1,3,5-
-triazin-2-yl)pyrimidin-2-amine; [0125]
4-(difluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridin-2-amine;
[0126]
4'-(difluoromethyl)-2,6-dimorpholino-[4,5'-bipyrimidin]-2'-amine;
[0127]
4-(difluoromethyl)-5-(4,6-dimorpholinopyrimidin-2-yl)pyridin-2-ami-
ne; [0128]
4'-(difluoromethyl)-4,6-dimorpholino-[2,5'-bipyrimidin]-2'-amin- e;
[0129]
4-(difluoromethyl)-5-(4-morpholino-6-thiomorpholino-1,3,5-triazi-
n-2-yl)pyridin-2-amine; [0130]
4-(difluoromethyl)-5-(4-morpholino-6-thiomorpholino-1,3,5-triazin-2-yl)py-
rimidin-2-amine; [0131]
5-(6-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-2-(3-oxa-8-azabicyclo[3.2.1]oc-
tan-8-yl)pyrimidin-4-yl)-4-(difluoromethyl)pyridin-2-amine; [0132]
5-(2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholinopyrimidin-4-yl)-4--
(difluoromethyl)pyridin-2-amine; [0133]
2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-4'-(difluoromethyl)-6-morpholino--
[4,5'-bipyrimidin]-2'-amine; [0134]
5-(2,6-bis((S)-3-methylmorpholino)pyrimidin-4-yl)-4-(difluoromethyl)pyrid-
in-2-amine; [0135]
4'-(difluoromethyl)-2,6-bis((S)-3-methylmorpholino)-[4,5'-bipyrimidin]-2'-
-amine; [0136]
(S)-4-(difluoromethyl)-5-(6-(3-methylmorpholino)-2-morpholinopyrimidin-4--
yl)pyridin-2-amine; [0137]
(S)-4'-(difluoromethyl)-6-(3-methylmorpholino)-2-morpholino-[4,5'-bipyrim-
idin]-2'-amine; [0138]
5-(4-(8-Oxa-3-azabicyclo[3.2.1]octan-3-yl)-6-(8-oxa-3-azabicyclo[3.2.1]oc-
tan-3-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
[0139]
5-[4,6-bis(2,2-dimethylmorpholin-4-yl)-1,3,5-triazin-2-yl]-4-(difluoromet-
hyl)pyridin-2-amine; [0140]
(S)-4-(difluoromethyl)-5-(2-(3-methylmorpholino)-6-morpholinopyrimidin-4--
yl)pyridin-2-amine; [0141]
(S)-4'-(difluoromethyl)-2-(3-methylmorpholino)-6-morpholino-[4,5'-bipyrim-
idin]-2'-amine; [0142]
4-(difluoromethyl)-5-[4-[(2S,6R)-2,6-dimethylmorpholin-4-yl]-6-[(3R)-3-me-
thylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0143]
5-[4,6-bis[(2R,6S)-2,6-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(dif-
luoromethyl)pyridin-2-amine; [0144]
5-[4,6-bis(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-1,3,5-triazin-2-yl]-4-
-(difluoromethyl)pyridin-2-amine; [0145]
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-(3-ox-
a-8-azabicyclo[3.2.1]octan-8-yl)-1,3,5-triazin-2-yl]pyridin-2-amine;
[0146]
5-[4,6-bis(3,3-dimethylmorpholin-4-yl)-1,3,5-triazin-2-yl]-4-(difl-
uoromethyl)pyridin-2-amine; [0147]
5-[4,6-bis[(3R,5S)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(dif-
luoromethyl)pyridin-2-amine; [0148]
5-[4,6-bis[(3R)-3-methylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(difluorome-
thyl)pyridin-2-amine; [0149]
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-morpholino-1,3,5-t-
riazin-2-yl]pyridin-2-amine; [0150]
4-(difluoromethyl)-5-[4-[(3R,5S)-3,5-dimethylmorpholin-4-yl]-6-[(3R)-3-me-
thylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0151]
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-[(3R)-3-methylmorp-
holin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0152]
4-(difluoromethyl)-5-[4-[(3R)-3-(methoxymethyl)morpholin-4-yl]-6-[(3R)-3--
methylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0153]
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-[(3R)-
-3-methylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0154]
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-(3-oxa-6-azabicyc-
lo[3.1.1]heptan-6-yl)-1,3,5-triazin-2-yl]pyridin-2-amine; [0155]
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-(6-oxa-3-azabicyc-
lo[3.1.1]heptan-3-yl)-1,3,5-triazin-2-yl]pyridin-2-amine; [0156]
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-[(1R,4R)-2-oxa-5--
azabicyclo[2.2.1]heptan-5-yl]-1,3,5-triazin-2-yl]pyridin-2-amine;
[0157] 4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-[(1
S,4S)-2-oxa-5-azabicyclo[2.2.1]heptan-5-yl]-1,3,5-triazin-2-yl]pyridin-2--
amine; [0158]
5-[4,6-bis[(3R)-3-ethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(difluoromet-
hyl)pyridin-2-amine; [0159]
5-[4,6-bis(8-oxa-5-azaspiro[3.5]nonan-5-yl)-1,3,5-triazin-2-yl]-4-(difluo-
romethyl)pyridin-2-amine; [0160]
5-[4,6-bis[(3R)-3-isopropylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(difluor-
omethyl)pyridin-2-amine [0161]
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-[(3R,5S)-3,5-dimet-
hylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0162]
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-[(3R)-3-(methoxyme-
thyl)morpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0163]
[(3R)-4-[4-[6-amino-4-(difluoromethyl)-3-pyridyl]-6-(3,3-dimethylmorpholi-
n-4-yl)-1,3,5-triazin-2-yl]morpholin-3-yl]methanol; [0164]
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-(3,7-dioxa-9-azabi-
cyclo[3.3.1]nonan-9-yl)-1,3,5-triazin-2-yl]pyridin-2-amine; [0165]
5-[4-(4-cyclopropylpiperazin-1-yl)-6-(3,3-dimethylmorpholin-4-yl)-1,3,5-t-
riazin-2-yl]-4-(difluoromethyl)pyridin-2-amine; [0166]
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-[4-(2-methoxyethyl-
)piperazin-1-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0167]
[(3R)-4-[4-[6-amino-4-(difluoromethyl)-3-pyridyl]-6-[(3R)-3-methylmorphol-
in-4-yl]-1,3,5-triazin-2-yl]morpholin-3-yl]methanol; [0168]
4-(difluoromethyl)-5-[4-[(3R,5R)-3,5-dimethylmorpholin-4-yl]-6-[(3R)-3-me-
thylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0169]
4-(difluoromethyl)-5-[4-[(3S,5S)-3,5-dimethylmorpholin-4-yl]-6-[(3R)-3-me-
thylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0170]
4-(difluoromethyl)-5-[4-morpholino-6-(3-oxa-9-azabicyclo[3.3.1]nonan-9-yl-
)-1,3,5-triazin-2-yl]pyridin-2-amine; [0171]
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-(3-ox-
a-9-azabicyclo[3.3.1]nonan-9-yl)-1,3,5-triazin-2-yl]pyridin-2-amine;
[0172]
5-[4,6-bis[(3S,5S)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-
-4-(difluoromethyl)pyridin-2-amine; [0173]
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-morph-
olino-1,3,5-triazin-2-yl]pyridin-2-amine; [0174]
4-(difluoromethyl)-5-[4-[(3
S)-3-ethylmorpholin-4-yl]-6-[(3R)-3-methylmorpholin-4-yl]-1,3,5-triazin-2-
-yl]pyridin-2-amine; [0175]
4-(difluoromethyl)-5-[4-[(3R)-3-ethylmorpholin-4-yl]-6-[(3R)-3-methylmorp-
holin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0176]
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-(8-oxa-5-azaspiro-
[3.5]nonan-5-yl)-1,3,5-triazin-2-yl]pyridin-2-amine.
[0177] In another preferred embodiment, said compound is selected
from [0178]
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyridin--
2-amine; [0179]
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyrimidin-2-ami-
ne; [0180]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicycl-
o[3.2.1]octan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
[0181]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1,3,5-tria-
zin-2-yl)-4-(difluoromethyl)pyridin-2-amine; [0182]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1,3,5-triazin-2-y-
l)-4-(difluoromethyl)pyrimidin-2-amine; [0183]
5-(4,6-bis((S)-3-methylmorpholino)-1,3,5-triazin-2-yl)-4-(difluoromethyl)-
pyridin-2-amine; [0184]
5-(4,6-bis((S)-3-methylmorpholino)-1,3,5-triazin-2-yl)-4-(difluoromethyl)-
pyrimidin-2-amine; [0185]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine; [0186]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyrimidin-2-amine; [0187]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-((S)-3-methylmorpholino)-1,3-
,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine; [0188]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-((S)-3-methylmorpholino)-1,3-
,5-triazin-2-yl)-4-(difluoromethyl)pyrimidin-2-amine; [0189]
4-(difluoromethyl)-5-(4-morpholino-6-(piperazin-1l-yl)-1,3,5-triazin-2-yl-
)pyridin-2-amine; [0190]
4-(difluoromethyl)-5-(4-morpholino-6-(piperazin-1l-yl)-1,3,5-triazin-2-yl-
)pyrimidin-2-amine; [0191]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-(piperazin-1-yl)-1,3,5-
-triazin-2-yl)pyridin-2-amine; [0192]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-(piperazin-1-yl)-1,3,5-
-triazin-2-yl)pyrimidin-2-amine; [0193]
4-(difluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridin-2-amine;
[0194]
4'-(difluoromethyl)-2,6-dimorpholino-[4,5'-bipyrimidin]-2'-amine;
[0195]
4-(difluoromethyl)-5-(4,6-dimorpholinopyrimidin-2-yl)pyridin-2-ami-
ne; [0196]
4'-(difluoromethyl)-4,6-dimorpholino-[2,5'-bipyrimidin]-2'-amin- e;
[0197]
4-(difluoromethyl)-5-(4-morpholino-6-thiomorpholino-1,3,5-triazi-
n-2-yl)pyridin-2-amine; [0198]
4-(difluoromethyl)-5-(4-morpholino-6-thiomorpholino-1,3,5-triazin-2-yl)py-
rimidin-2-amine; [0199]
5-(6-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-2-(3-oxa-8-azabicyclo[3.2.1]oc-
tan-8-yl)pyrimidin-4-yl)-4-(difluoromethyl)pyridin-2-amine; [0200]
5-(2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholinopyrimidin-4-yl)-4--
(difluoromethyl)pyridin-2-amine; [0201]
2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-4'-(difluoromethyl)-6-morpholino--
[4,5'-bipyrimidin]-2'-amine; [0202]
5-(2,6-bis((S)-3-methylmorpholino)pyrimidin-4-yl)-4-(difluoromethyl)pyrid-
in-2-amine; [0203]
4'-(difluoromethyl)-2,6-bis((S)-3-methylmorpholino)-[4,5'-bipyrimidin]-2'-
-amine; [0204]
(S)-4-(difluoromethyl)-5-(6-(3-methylmorpholino)-2-morpholinopyrimidin-4--
yl)pyridin-2-amine; [0205]
(S)-4'-(difluoromethyl)-6-(3-methylmorpholino)-2-morpholino-[4,5'-bipyrim-
idin]-2'-amine; [0206]
5-(4-(8-Oxa-3-azabicyclo[3.2.1]octan-3-yl)-6-(8-oxa-3-azabicyclo[3.2.1]oc-
tan-3-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
[0207]
5-[4,6-bis(2,2-dimethylmorpholin-4-yl)-1,3,5-triazin-2-yl]-4-(difluoromet-
hyl)pyridin-2-amine; [0208]
(S)-4-(difluoromethyl)-5-(2-(3-methylmorpholino)-6-morpholinopyrimidin-4--
yl)pyridin-2-amine; [0209]
(S)-4'-(difluoromethyl)-2-(3-methylmorpholino)-6-morpholino-[4,5'-bipyrim-
idin]-2'-amine; [0210]
4-(difluoromethyl)-5-[4-[(2S,6R)-2,6-dimethylmorpholin-4-yl]-6-[(3R)-3-me-
thylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0211]
5-[4,6-bis[(2R,6S)-2,6-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(dif-
luoromethyl)pyridin-2-amine; [0212]
5-[4,6-bis(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-1,3,5-triazin-2-yl]-4-
-(difluoromethyl)pyridin-2-amine; [0213]
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-(3-ox-
a-8-azabicyclo[3.2.1]octan-8-yl)-1,3,5-triazin-2-yl]pyridin-2-amine;
[0214]
5-[4,6-bis(3,3-dimethylmorpholin-4-yl)-1,3,5-triazin-2-yl]-4-(difl-
uoromethyl)pyridin-2-amine; [0215]
5-[4,6-bis[(3R,5S)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(dif-
luoromethyl)pyridin-2-amine; [0216]
5-[4,6-bis[(3R)-3-methylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(difluorome-
thyl)pyridin-2-amine; [0217]
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-morpholino-1,3,5-t-
riazin-2-yl]pyridin-2-amine; [0218]
4-(difluoromethyl)-5-[4-[(3R,5S)-3,5-dimethylmorpholin-4-yl]-6-[(3R)-3-me-
thylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0219]
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-[(3R)-3-methylmorp-
holin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0220]
4-(difluoromethyl)-5-[4-[(3R)-3-(methoxymethyl)morpholin-4-yl]-6-[(3R)-3--
methylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0221]
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-[(3R)-
-3-methylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0222]
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-(3-ox-
a-9-azabicyclo[3.3.1]nonan-9-yl)-1,3,5-triazin-2-yl]pyridin-2-amine;
[0223] 5-[4,6-bis[(3 S,5
S)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(difluoromethyl)pyri-
din-2-amine; [0224]
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-morph-
olino-1,3,5-triazin-2-yl]pyridin-2-amine; [0225]
4-(difluoromethyl)-5-[4-[(3
S)-3-ethylmorpholin-4-yl]-6-[(3R)-3-methylmorpholin-4-yl]-1,3,5-triazin-2-
-yl]pyridin-2-amine; [0226]
4-(difluoromethyl)-5-[4-[(3R)-3-ethylmorpholin-4-yl]-6-[(3R)-3-methylmorp-
holin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0227]
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-(8-oxa-5-azaspiro-
[3.5]nonan-5-yl)-1,3,5-triazin-2-yl]pyridin-2-amine;
[0228] In another preferred embodiment, said compound is selected
from [0229]
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyrimidi-
n-2-amine; [0230]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.1]oc-
tan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
[0231]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1,3,5-triazin-2-y-
l)-4-(difluoromethyl)pyridin-2-amine; [0232]
5-(4,6-bis((S)-3-methylmorpholino)-1,3,5-triazin-2-yl)-4-(difluoromethyl)-
pyrimidin-2-amine; [0233]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine; [0234]
4-(difluoromethyl)-5-(4-morpholino-6-(piperazin-1-yl)-1,3,5-triazin-2-yl)-
pyrimidin-2-amine; [0235]
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyridin-2-amine-
; and [0236]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyrimidin-2-amine; [0237]
5-[4,6-bis(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-1,3,5-triazin-2-yl]-4-
-(difluoromethyl)pyridin-2-amine; [0238]
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-(3-ox-
a-8-azabicyclo[3.2.1]octan-8-yl)-1,3,5-triazin-2-yl]pyridin-2-amine;
[0239]
5-[4,6-bis(3,3-dimethylmorpholin-4-yl)-1,3,5-triazin-2-yl]-4-(difl-
uoromethyl)pyridin-2-amine; [0240]
5-[4,6-bis[(3R,5S)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(dif-
luoromethyl)pyridin-2-amine; [0241]
5-[4,6-bis[(3R)-3-methylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(difluorome-
thyl)pyridin-2-amine; [0242]
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-morpholino-1,3,5-t-
riazin-2-yl]pyridin-2-amine; [0243]
4-(difluoromethyl)-5-[4-[(3R,5S)-3,5-dimethylmorpholin-4-yl]-6-[(3R)-3-me-
thylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0244]
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-[(3R)-3-methylmorp-
holin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0245]
4-(difluoromethyl)-5-[4-[(3R)-3-(methoxymethyl)morpholin-4-yl]-6-[(3R)-3--
methylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0246]
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-[(3R)-
-3-methylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0247]
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-(3-ox-
a-9-azabicyclo[3.3.1]nonan-9-yl)-1,3,5-triazin-2-yl]pyridin-2-amine;
[0248] 5-[4,6-bis[(3 S,5
S)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(difluoromethyl)pyri-
din-2-amine; [0249]
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-morph-
olino-1,3,5-triazin-2-yl]pyridin-2-amine; [0250]
4-(difluoromethyl)-5-[4-[(3
S)-3-ethylmorpholin-4-yl]-6-[(3R)-3-methylmorpholin-4-yl]-1,3,5-triazin-2-
-yl]pyridin-2-amine; [0251]
4-(difluoromethyl)-5-[4-[(3R)-3-ethylmorpholin-4-yl]-6-[(3R)-3-methylmorp-
holin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine; [0252]
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-(8-oxa-5-azaspiro-
[3.5]nonan-5-yl)-1,3,5-triazin-2-yl]pyridin-2-amine
[0253] In another preferred embodiment, said compound is selected
from [0254]
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyrimidi-
n-2-amine; [0255]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.1]oc-
tan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
[0256]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1,3,5-triazin-2-y-
l)-4-(difluoromethyl)pyridin-2-amine; [0257]
5-(4,6-bis((S)-3-methylmorpholino)-1,3,5-triazin-2-yl)-4-(difluoromethyl)-
pyrimidin-2-amine; [0258]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine; [0259]
4-(difluoromethyl)-5-(4-morpholino-6-(piperazin-1-yl)-1,3,5-triazin-2-yl)-
pyrimidin-2-amine; [0260]
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyridin-2-amine-
; and [0261]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyrimidin-2-amine;
[0262] In another preferred embodiment, said compound is selected
from [0263]
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyrimidi-
n-2-amine; [0264]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.1]oc-
tan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
[0265]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine; and tautomers, solvates and
pharmaceutically acceptable salts thereof.
[0266] In another very preferred embodiment, said compound is
selected from
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2-
.1]octan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
and [0267]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,-
5-triazin-2-yl)pyridin-2-amine; and tautomers, solvates and
pharmaceutically acceptable salts thereof.
[0268] In another very preferred embodiment, said compound is
selected from
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2-
.1]octan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
and [0269]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,-
5-triazin-2-yl)pyridin-2-amine.
[0270] In another very preferred embodiment, said compound of
formula (I) is
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyrimidin-2--
amine.
[0271] In another very preferred embodiment, said compound of
formula (I) is
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyrimidin-2--
amine; and tautomers, solvates and pharmaceutically acceptable
salts thereof.
[0272] In another very preferred embodiment, said compound of
formula (I) is
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.1-
]octan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine.
[0273] In another very preferred embodiment, said compound of
formula (I) is
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.1-
]octan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
and tautomers, solvates and pharmaceutically acceptable salts
thereof.
[0274] In another very preferred embodiment, said compound of
formula (I) is
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-tr-
iazin-2-yl)pyridin-2-amine.
[0275] In another very preferred embodiment, said compound of
formula (I) is
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-tr-
iazin-2-yl)pyridin-2-amine; and tautomers, solvates and
pharmaceutically acceptable salts thereof.
[0276] In another preferred embodiment, said R.sup.1 and R.sup.2
are independently of each other a morpholinyl of formula (II). In
one preferred embodiment, said R.sup.1 is equal to R.sup.2. In
another preferred embodiment, said R.sup.1 is not equal to
R.sup.2.
[0277] In another preferred embodiment, said R.sup.1 and R.sup.2
are independently of each other a morpholinyl of formula (II) and
said saturated 6-membered heterocyclic ring Z.
[0278] In another preferred embodiment, within said morpholinyl of
formula (II)
##STR00016##
R.sup.3 and R.sup.4 are independently of each other H,
C.sub.1-C.sub.3alkyl optionally substituted with one or two OH,
C.sub.1-C.sub.2fluoroalkyl, C.sub.1-C.sub.2alkoxy,
C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl, CN, or
C(O)O--C.sub.1-C.sub.2alkyl; or R.sup.3 and R.sup.4 form together a
bivalent residue --R.sup.5R.sup.6-- selected from
C.sub.1-C.sub.3alkylene optionally substituted with 1 to 4 F,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--, or any of
the structures
##STR00017##
wherein the arrows denote the bonds in formula (II).
[0279] In the instance that R3 and R4 together form a bivalent
residue and are bound to vicinal carbon atoms annulated morpholinyl
substituents are formed. In the instance that R3 and R4 together
form a bivalent residue and are spanning across the morpholine ring
bridged morpholinyl substituents are formed. In the instance that
R3 and R4 together form a bivalent residue and are bound to the
same carbon atom of the morpholine, spiro morpholinyl substituents
are formed.
[0280] In a preferred embodiment, R.sup.3 and R.sup.4 form together
a bivalent residue --R.sup.5R.sup.6-- selected from
C.sub.1-C.sub.3alkylene optionally substituted with 1 to 4 F,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--, or any of
the structures
##STR00018##
[0281] and forming a bridged morpholinyl substituent.
In another preferred embodiment, said R.sup.1 and R.sup.2 are
independently of each other a morpholinyl of formula (II), wherein
R.sup.3 and R.sup.4 form together a bivalent residue leading to a
bridged morpholinyl, wherein R.sup.3 and R.sup.4 form together a
bivalent residue --R.sup.5R.sup.6-- selected from
C.sub.1-C.sub.3alkylene, preferably C.sub.1-C.sub.2alkylene,
--CH.sub.2CF.sub.2--, --CHFCHF--, --CH.sub.2CF.sub.2CH.sub.2--,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--, or any of
the structures
##STR00019##
wherein the arrows denote the bonds in formula (II).
[0282] In a further preferred embodiment, said morpholinyl of
formula (II)
##STR00020##
is independently of each other a morpholinyl of said formula (II),
wherein R.sup.3 and R.sup.4 are independently of each other H,
C.sub.1-C.sub.3alkyl, CH.sub.2OH, CH.sub.2CH.sub.2OH, CH.sub.2F,
CHF.sub.2, CF.sub.3, CH.sub.2CF.sub.3, C.sub.1-C.sub.2alkoxy,
C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl, CN, or
C(O)O--C.sub.1-C.sub.2alkyl; or R.sup.3 and R.sup.4 form together a
bivalent residue --R.sup.5R.sup.6-- selected from
C.sub.1-C.sub.3alkylene, preferably C.sub.1-C.sub.2alkylene,
--CH.sub.2CF.sub.2--, --CHFCHF--, --CH.sub.2CF.sub.2CH.sub.2--,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--, or any of
the structures
##STR00021##
wherein the arrows denote the bonds in formula (II).
[0283] In a further preferred embodiment, said morpholinyl of
formula (II) is independently of each other a morpholinyl of said
formula (II), wherein R.sup.3 and R.sup.4 are independently of each
other H or CH.sub.3.
[0284] In a further preferred embodiment, said morpholinyl of
formula (II) is independently of each other a morpholinyl of said
formula (II), wherein R.sup.3 and R.sup.4 are independently of each
other C.sub.2-C.sub.3alkyl, CH.sub.2OH, CH.sub.2CH.sub.2OH,
CH.sub.2F, CHF.sub.2, CF.sub.3, CH.sub.2CF.sub.3,
C.sub.1-C.sub.2alkoxy, C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl,
CN, or C(O)O--C.sub.1-C.sub.2alkyl; or R.sup.3 and R.sup.4 form
together a bivalent residue --R.sup.5R.sup.6-- selected from
--CH.sub.2-- or C.sub.3alkylene, preferably --CH.sub.2--,
--CH.sub.2CF.sub.2--, --CHFCHF--, --CH.sub.2CF.sub.2CH.sub.2--,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--, or any of
the structures
##STR00022##
wherein the arrows denote the bonds in formula (II). In a further
preferred embodiment, said morpholinyl of formula (II) is
independently of each other selected from
##STR00023## ##STR00024##
[0285] In a further preferred embodiment, said morpholinyl of
formula (II) is independently of each other selected from
##STR00025##
[0286] In a further preferred embodiment, said heterocyclic ring Z
is a saturated 6-membered heterocyclic ring Z selected from
thiomorpholinyl and piperazinyl, optionally substituted by 1 to 3
R.sup.7; wherein R.sup.7 is independently at each occurrence
C.sub.1-C.sub.3alkyl, CH.sub.2OH, CH.sub.2CH.sub.2OH, CH.sub.2F,
CHF.sub.2, CF.sub.3, CH.sub.2CF.sub.3,
C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl,
C.sub.3-C.sub.6cycloalkyl; or two R.sup.7 substituents form
together a bivalent residue --R.sup.8R.sup.9-- selected from
C.sub.1-C.sub.3alkylene optionally substituted with 1 to 4 F,
--CH.sub.2--O--CH.sub.2-- or --O--CH.sub.2CH.sub.2--O--;
[0287] In a further preferred embodiment, said heterocyclic ring Z
is selected from
##STR00026##
[0288] In another preferred embodiment of the present invention,
said R.sup.1 and said R.sup.2 are independently of each other a
morpholinyl of formula (II)
##STR00027##
[0289] wherein the arrow denotes the bond in formula (I); and
[0290] wherein R.sup.3 and R.sup.4 are independently of each other
H, C.sub.1-C.sub.3alkyl optionally substituted with one or two OH,
C.sub.1-C.sub.2fluoroalkyl, C.sub.1-C.sub.2alkoxy,
C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl, CN, or
C(O)O--C.sub.1-C.sub.2alkyl; or R.sup.3 and R.sup.4 form together a
bivalent residue --R.sup.5R.sup.6-- selected from
C.sub.1-C.sub.3alkylene optionally substituted with 1 to 4 F,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--, or any of
the structures
##STR00028##
[0291] wherein the arrows denote the bonds in formula (II).
[0292] In a further preferred embodiment, said R.sup.1 is equal to
said R.sup.2, and said R.sup.1 and said R.sup.2 are independently
of each other a morpholinyl of formula (II)
##STR00029##
[0293] wherein the arrow denotes the bond in formula (I); and
[0294] wherein R.sup.3 and R.sup.4 are independently of each other
H, C.sub.1-C.sub.3alkyl optionally substituted with one or two OH,
C.sub.1-C.sub.2fluoroalkyl, C.sub.1-C.sub.2alkoxy,
C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl, CN, or
C(O)O--C.sub.1-C.sub.2alkyl; or R.sup.3 and R.sup.4 form together a
bivalent residue --R.sup.5R.sup.6-- selected from
C.sub.1-C.sub.3alkylene optionally substituted with 1 to 4 F,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--, or any of
the structures
##STR00030## [0295] wherein the arrows denote the bonds in formula
(II).
[0296] In a further preferred embodiment of the present invention,
said R.sup.1 and said R.sup.2 are independently of each other a
morpholinyl of formula (II)
##STR00031##
[0297] wherein the arrow denotes the bond in formula (I); and
[0298] wherein R.sup.3 and R.sup.4 are independently of each other
H, C.sub.1-C.sub.3alkyl, CH.sub.2OH, CH.sub.2CH.sub.2OH, CH.sub.2F,
CHF.sub.2, CF.sub.3, CH.sub.2CF.sub.3, C.sub.1-C.sub.2alkoxy,
C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl, CN, or
C(O)O--C.sub.1-C.sub.2alkyl; or R.sup.3 and R.sup.4 form together a
bivalent residue --R.sup.5R.sup.6-- selected from
C.sub.1-C.sub.3alkylene, preferably C.sub.1-C.sub.2alkylene,
--CH.sub.2CF.sub.2--, --CHFCHF--, --CH.sub.2CF.sub.2CH.sub.2--,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--, or any of
the structures
##STR00032## [0299] wherein the arrows denote the bonds in formula
(II).
[0300] In a further preferred embodiment of the present invention,
R.sup.1 is equal to R.sup.2, and said R.sup.1 and said R.sup.2 are
a morpholinyl of formula (II)
##STR00033##
[0301] wherein the arrow denotes the bond in formula (I); and
[0302] wherein R.sup.3 and R.sup.4 are independently of each other
H, C.sub.1-C.sub.3alkyl, CH.sub.2OH, CH.sub.2CH.sub.2OH, CH.sub.2F,
CHF.sub.2, CF.sub.3, CH.sub.2CF.sub.3, C.sub.1-C.sub.2alkoxy,
C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl, CN, or
C(O)O--C.sub.1-C.sub.2alkyl; or R.sup.3 and R.sup.4 form together a
bivalent residue --R.sup.5R.sup.6-- selected from
C.sub.1-C.sub.3alkylene, preferably C.sub.1-C.sub.2alkylene,
--CH.sub.2CF.sub.2--, --CHFCHF--, --CH.sub.2CF.sub.2CH.sub.2--,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--, or any of
the structures
##STR00034## [0303] wherein the arrows denote the bonds in formula
(II). [0304] In another aspect and preferred embodiment, the
present invention provides for a compound of (I)
[0304] ##STR00035## [0305] wherein [0306] X.sup.1, X.sup.2 and
X.sup.3 are, independently of each other, N or CH; with the proviso
that at least two of X.sup.1, X.sup.2 and X.sup.3 are N; Y is N or
CH; and wherein [0307] R.sup.1 and R.sup.2 are independently of
each other a morpholinyl of formula (II)
[0307] ##STR00036## [0308] wherein the arrow denotes the bond in
formula (I); and R.sup.1 is not equal to R.sup.2, and at least one
of said R.sup.1 and said R.sup.2 are a morpholinyl of formula
(II),
##STR00037##
[0308] wherein R.sup.3 and R.sup.4 are independently of each other
C.sub.2-C.sub.3alkyl, CH.sub.2OH, CH.sub.2CH.sub.2OH, CH.sub.2F,
CHF.sub.2, CF.sub.3, CH.sub.2CF.sub.3, C.sub.1-C.sub.2alkoxy,
C.sub.1-C.sub.2alkoxyC.sub.1-C.sub.3alkyl, CN, or
C(O)O--C.sub.1-C.sub.2alkyl; or R.sup.3 and R.sup.4 form together a
bivalent residue --R.sup.5R.sup.6-- selected from --CH.sub.2-- or
C.sub.3alkylene, preferably --CH.sub.2--, --CH.sub.2CF.sub.2--,
--CHFCHF--, --CH.sub.2CF.sub.2CH.sub.2--,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--, or any of
the structures
##STR00038##
wherein the arrows denote the bonds in formula (II). [0309]
Preferably, said R.sup.3 and R.sup.4 form together a bivalent
residue --R.sup.5R.sup.6-- selected from --CH.sub.2-- or
C.sub.3alkylene, preferably --CH.sub.2--, --CH.sub.2CF.sub.2--,
--CHFCHF--, --CH.sub.2CF.sub.2CH.sub.2--,
--CH.sub.2--O--CH.sub.2--, --CH.sub.2--NH--CH.sub.2--, or any of
the structures
##STR00039##
[0310] In another preferred embodiment, R.sup.1 is 4-morpholinyl,
2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl or
3-aza-8-oxabicyclo[3.2.1]oct-3-yl; and R.sup.2 is 4-morpholinyl,
2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl,
3-aza-8-oxabicyclo[3.2.1]oct-3-yl, 4-piperazin-1-yl,
4-methylpiperazin-1-yl, or 4-thiomorpholinyl.
[0311] In another preferred embodiment, R.sup.1 is 4-morpholinyl,
2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl or
3-aza-8-oxabicyclo[3.2.1]oct-3-yl; and R.sup.2 is 4-morpholinyl,
2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl,
3-aza-8-oxabicyclo[3.2.1]oct-3-yl, 4-piperazin-1-yl,
4-methylpiperazin-1-yl, or 4-thiomorpholinyl, and X.sup.1, X.sup.2
and X.sup.3 are N; and tautomers, solvates and pharmaceutically
acceptable salts thereof. Preferably Y is N or CH; R.sup.1 is
4-morpholinyl, 2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl or
3-aza-8-oxabicyclo[3.2.1]oct-3-yl; and R.sup.2 is 4-morpholinyl,
2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl,
3-aza-8-oxabicyclo[3.2.1]oct-3-yl, 4-piperazin-1-yl,
4-methylpiperazin-1-yl, or 4-thiomorpholinyl; and tautomers,
solvates and pharmaceutically acceptable salts thereof.
[0312] In a further preferred embodiment, R.sup.1 is 4-morpholinyl,
2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl or
3-aza-8-oxabicyclo[3.2.1]oct-3-yl; and R.sup.2 is 4-morpholinyl,
2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl,
3-aza-8-oxabicyclo[3.2.1]oct-3-yl, 4-piperazin-1-yl,
4-methylpiperazin-1-yl, or 4-thiomorpholinyl, and X.sup.1 and
X.sup.3 are N, and X.sup.2 is CH; and tautomers, solvates and
pharmaceutically acceptable salts thereof. Preferably Y is N or CH;
R.sup.1 is 4-morpholinyl, 2-methyl-4-morpholinyl,
3-methyl-4-morpholinyl, octadeuterio-4-morpholinyl,
8-aza-3-oxabicyclo[3.2.1]oct-8-yl or
3-aza-8-oxabicyclo[3.2.1]oct-3-yl; and R.sup.2 is 4-morpholinyl,
2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl,
3-aza-8-oxabicyclo[3.2.1]oct-3-yl, 4-piperazin-1-yl,
4-methylpiperazin-1-yl, or 4-thiomorpholinyl; and tautomers,
solvates and pharmaceutically acceptable salts thereof.
[0313] In a preferred embodiment, R.sup.1 is 4-morpholinyl,
2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl or
3-aza-8-oxabicyclo[3.2.1]oct-3-yl; and R.sup.2 is 4-morpholinyl,
2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl,
3-aza-8-oxabicyclo[3.2.1]oct-3-yl, 4-piperazin-1-yl,
4-methylpiperazin-1-yl, or 4-thiomorpholinyl, and X.sup.1 and
X.sup.2 are N, and X.sup.3 is CH; and tautomers, solvates and
pharmaceutically acceptable salts thereof. Preferably, Y is N or
CH; R.sup.1 is 4-morpholinyl, 2-methyl-4-morpholinyl,
3-methyl-4-morpholinyl, octadeuterio-4-morpholinyl,
8-aza-3-oxabicyclo[3.2.1]oct-8-yl or
3-aza-8-oxabicyclo[3.2.1]oct-3-yl; and R.sup.2 is 4-morpholinyl,
2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl,
3-aza-8-oxabicyclo[3.2.1]oct-3-yl, 4-piperazin-1-yl,
4-methylpiperazin-1-yl, or 4-thiomorpholinyl; and tautomers,
solvates and pharmaceutically acceptable salts thereof.
[0314] In a preferred embodiment, R.sup.1 is 4-morpholinyl,
2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl or
3-aza-8-oxabicyclo[3.2.1]oct-3-yl; and R.sup.2 is 4-morpholinyl,
2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl,
3-aza-8-oxabicyclo[3.2.1]oct-3-yl, 4-piperazin-1-yl,
4-methylpiperazin-1-yl, or 4-thiomorpholinyl, and X.sup.2 and
X.sup.3 are N, and X.sup.1 is CH; and tautomers, solvates and
pharmaceutically acceptable salts thereof. Preferably, Y is N or
CH; R.sup.1 is 4-morpholinyl, 2-methyl-4-morpholinyl,
3-methyl-4-morpholinyl, octadeuterio-4-morpholinyl,
8-aza-3-oxabicyclo[3.2.1]oct-8-yl or
3-aza-8-oxabicyclo[3.2.1]oct-3-yl; and R.sup.2 is 4-morpholinyl,
2-methyl-4-morpholinyl, 3-methyl-4-morpholinyl,
octadeuterio-4-morpholinyl, 8-aza-3-oxabicyclo[3.2.1]oct-8-yl,
3-aza-8-oxabicyclo[3.2.1]oct-3-yl, 4-piperazin-1-yl,
4-methylpiperazin-1-yl, or 4-thiomorpholinyl; and tautomers,
solvates and pharmaceutically acceptable salts thereof.
[0315] In a further embodiment, there is provided a compound of
formula (I) according to the invention for use in the prevention or
treatment of a neurological disorder in a subject, wherein the
neurological disorder is epilepsy or a neurodegenerative
disease.
[0316] In a further embodiment, there is provided a compound of
formula (I) according to the invention for use in the prevention or
treatment of a neurodegenerative disease in a subject.
[0317] In a further embodiment, there is provided a compound of
formula (I) according to the invention for use in the prevention or
treatment of epilepsy in a subject.
[0318] In a further embodiment, there is provided a compound of
formula (I) according to the invention for use in the prevention or
treatment of epilepsy in a subject, wherein the compound of formula
(I) is administered to said subject during primary epileptogenesis
or during secondary epileptogenesis or in fully developed
epilepsy.
[0319] In a further embodiment, there is provided a compound of
formula (I) according to the invention for use in the prevention or
treatment of epilepsy in a subject, wherein the epilepsy is
symptomatic epilepsy or idiopathic epilepsy.
[0320] In a further embodiment, there is provided a compound of
formula (I) according to the invention for use in the prevention or
treatment of epilepsy in a subject, wherein the epilepsy is
symptomatic epilepsy.
[0321] In a further embodiment, there is provided a compound of
formula (I) according to the invention for use in the prevention or
treatment of epilepsy in a subject, wherein the epilepsy is
idiopathic epilepsy.
[0322] In a further embodiment, there is provided a compound of
formula (I) according to the invention for use in the prevention or
treatment of symptomatic epilepsy in a subject, wherein said
symptomatic epilepsy is caused by brain injury, brain tumor, brain
infection, adrenoleukodystrophy, Rasmussen's syndrome, Sturge-Weber
syndrome, megalencephaly, polyhydramnios, tuberous sclerosis
complex (TSC), symptomatic epilepsy syndrome, PMSE, PTEN mutations
or focal cortical dysplasia (FCD).
[0323] In one embodiment, there is provided a compound of formula
(I) according to the invention for use in the prevention or
treatment of symptomatic epilepsy in a subject, wherein said
symptomatic epilepsy is due to a disease characterized by
upregulation of mTOR ("TORopathy").
[0324] In one embodiment, there is provided a compound of formula
(I) for use in the prevention or treatment of symptomatic epilepsy
in a subject, wherein said symptomatic epilepsy is due to a disease
characterized by upregulation of mTOR ("TORopathy"), wherein said
"TORopathy" is selected from the group consisting of TSC,
polyhydramnios, megalencephaly, symptomatic epilepsy syndrome,
PMSE, focal cortical dysplasia (FCD) and a "TORopathy" associated
with PTEN mutations.
[0325] In a further embodiment, there is provided a compound of
formula (I) according to the invention for use in the prevention or
treatment of symptomatic epilepsy in a subject, wherein said
symptomatic epilepsy is caused by brain injury, brain infection,
adrenoleukodystrophy, Rasmussen's syndrome, Sturge-Weber syndrome,
megalencephaly, polyhydramnios, tuberous sclerosis complex (TSC),
symptomatic epilepsy syndrome, PMSE, PTEN mutations or focal
cortical dysplasia (FCD).
[0326] In yet a further embodiment, there is provided a compound of
formula (I) for use in the prevention or treatment of symptomatic
epilepsy in a subject, wherein said symptomatic epilepsy is caused
by brain injury, brain infection, adrenoleukodystrophy, Rasmussen's
syndrome or Sturge-Weber syndrome.
[0327] In a preferred embodiment, there is provided a compound of
formula (I) according to the invention for use in the prevention or
treatment of symptomatic epilepsy in a subject, wherein said
symptomatic epilepsy is caused by TSC.
[0328] In a further embodiment, there is provided a compound of
formula (I) according to the invention for use in the prevention or
treatment of idiopathic epilepsy in a subject, wherein said
idiopathic epilepsy is selected from the group consisting of Doose
syndrome (myoclonic astatic epilepsy of childhood), West syndrome,
benign Rolandic epilepsy, Lennox-Gastaut syndrome, Landau-Kleffner
syndrome, symptomatic epilepsy syndrome, PMSE and juvenile
myoclonic epilepsy.
[0329] As indicated above, epilepsy can be partial or generalized.
Thus, in one embodiment, there is provided a compound of formula
(I) according to the invention for use in the prevention or
treatment of epilepsy in a subject, wherein the epilepsy is partial
epilepsy or generalized epilepsy.
[0330] In a further embodiment, there is provided a compound of
formula (I) for use in the prevention or treatment of epilepsy in a
subject, wherein the epilepsy is partial idiopathic epilepsy,
partial symptomatic epilepsy, generalized idiopathic epilepsy or
generalized symptomatic epilepsy.
[0331] In a preferred embodiment, there is provided a compound of
formula (I) for use in the prevention or treatment of partial
epilepsy in a subject, wherein said partial epilepsy is temporal
lobe epilepsy.
[0332] In a further embodiment, there is provided a compound of
formula (I) according to the invention for use in the prevention or
treatment of a neurodegenerative disease in a subject, wherein said
neurodegenerative disease is associated with or caused by protein
misfolding and/or protein aggregation.
[0333] In a further embodiment, there is provided a compound of
formula (I) according to the invention for use in the prevention or
treatment of a neurodegenerative disease in a subject, wherein the
neurodegenerative disease is selected from the group consisting of
Huntington's disease, spinocerebellar ataxias, Parkinson's disease,
morbus Alzheimer, amyotrophic lateral sclerosis (ALS), cystic
fibrosis, familial amyloidotic polyneuropathy, spongiform
encephalopathies, dementia with Lewy bodies, frontotemporal
dementia with Parkinsonism, spinocerebellar ataxias, spinal and
bulbar muscular atrophy, hereditary dentatorubral-pallidoluysian
atrophy, familial British dementia, familial Danish dementia and
prion disease.
[0334] In a preferred embodiment, there is provided a compound of
formula (I) according to the invention for use in the prevention or
treatment of a neurodegenerative disease in a subject, wherein the
neurodegenerative disease is selected from the group consisting of
Huntington's disease, morbus Alzheimer and prion disease.
[0335] In a particularly preferred embodiment, there is provided a
compound of formula (I) according to the invention for use in the
prevention or treatment of Huntington's disease in a subject.
[0336] In a preferred embodiment, there is provided a compound of
formula (I) according to the invention, wherein said compound is
selected from: [0337]
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyrimidi-
n-2-amine; [0338]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.1]oc-
tan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
[0339]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine; and tautomers, solvates and
pharmaceutically acceptable salts thereof, for use in the
prevention or treatment of a neurological disorder in a subject,
wherein the neurological disorder is epilepsy or Huntington's
disease.
[0340] In a further preferred embodiment, there is provided a
compound of formula (I) according to the invention, wherein said
compound is selected from: [0341]
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyrimidin-2-ami-
ne; [0342]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicycl-
o[3.2.1]octan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
[0343]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,-
5-triazin-2-yl)pyridin-2-amine; and tautomers, solvates and
pharmaceutically acceptable salts thereof, for use in the
prevention or treatment of epilepsy in a subject.
[0344] In a preferred embodiment, there is provided a compound of
formula (I) according to the invention, wherein said compound is
selected from: [0345]
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyrimidi-
n-2-amine; [0346]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.1]oc-
tan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
[0347]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine; and tautomers, solvates and
pharmaceutically acceptable salts thereof, for use in the
prevention or treatment of Huntington's disease in a subject.
[0348] In a preferred embodiment, there is provided a compound of
formula (I) according to the invention, wherein said compound is
selected from
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.1]oc-
tan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
and
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine; and tautomers, solvates and
pharmaceutically acceptable salts thereof, for use in the
prevention or treatment of a neurological disorder in a subject,
wherein the neurological disorder is epilepsy or Huntington's
disease.
[0349] In a preferred embodiment, there is provided a compound of
formula (I) according to the invention, wherein said compound is
selected from
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.1]oc-
tan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
and
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine; and tautomers, solvates and
pharmaceutically acceptable salts thereof, for use in the
prevention or treatment of epilepsy in a subject.
[0350] In a preferred embodiment, there is provided a compound of
formula (I) according to the invention, wherein said compound is
selected from
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.1]oc-
tan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
and
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine; and tautomers, solvates and
pharmaceutically acceptable salts thereof, for use in the
prevention or treatment of Huntington's disease in a subject.
[0351] In a preferred embodiment, there is provided the compound
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.1]oc-
tan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
and tautomers, solvates and pharmaceutically acceptable salts
thereof, for use in the prevention or treatment of a neurological
disorder in a subject, wherein the neurological disorder is
epilepsy or Huntington's disease.
[0352] In a particularly preferred embodiment, there is provided
the compound
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo-
[3.2.1]octan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
and tautomers, solvates and pharmaceutically acceptable salts
thereof, for use in the prevention or treatment of epilepsy in a
subject.
[0353] In a further particularly preferred embodiment, there is
provided the compound
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.1]oc-
tan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
and tautomers, solvates and pharmaceutically acceptable salts
thereof, for use in the prevention or treatment of Huntington's
disease in a subject.
[0354] In a preferred embodiment, there is provided the compound
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine; and tautomers, solvates and
pharmaceutically acceptable salts thereof, for use in the
prevention or treatment of a neurological disorder in a subject,
wherein the neurological disorder is epilepsy or Huntington's
disease.
[0355] In a particularly preferred embodiment, there is provided
the compound
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,-
3,5-triazin-2-yl)pyridin-2-amine; and tautomers, solvates and
pharmaceutically acceptable salts thereof, for use in the
prevention or treatment of epilepsy in a subject.
[0356] In a further particularly preferred embodiment, there is
provided the compound
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine; and tautomers, solvates and
pharmaceutically acceptable salts thereof, for use in the
prevention or treatment of Huntington's disease in a subject.
[0357] In a preferred embodiment, there is provided a compound of
formula (I) according to the invention, wherein R1 and R2 are
independently of each other a morpholinyl of formula (II); and
tautomers, solvates and pharmaceutically acceptable salts thereof,
for use in the prevention or treatment of a neurological disorder
in a subject, wherein the neurological disorder is epilepsy or
Huntington's disease.
[0358] In a preferred embodiment, there is provided a compound of
formula (I) according to the invention, wherein R1 and R2 are
independently of each other a morpholinyl of formula (II); and
tautomers, solvates and pharmaceutically acceptable salts thereof,
for use in the prevention or treatment of epilepsy in a
subject.
[0359] In a preferred embodiment, there is provided a compound of
formula (I) according to the invention, wherein R1 and R2 are
independently of each other a morpholinyl of formula (II); and
tautomers, solvates and pharmaceutically acceptable salts thereof,
for use in the prevention or treatment of Huntington's disease in a
subject.
[0360] In a preferred embodiment, there is provided a compound of
formula (I) according to the invention, wherein R1 is equal to R2;
and tautomers, solvates and pharmaceutically acceptable salts
thereof, for use in the prevention or treatment of a neurological
disorder in a subject, wherein the neurological disorder is
epilepsy or Huntington's disease.
[0361] In a preferred embodiment, there is provided a compound of
formula (I) according to the invention, wherein R1 is equal to R2;
and tautomers, solvates and pharmaceutically acceptable salts
thereof, for use in the prevention or treatment of epilepsy in a
subject.
[0362] In a preferred embodiment, there is provided a compound of
formula (I) according to the invention, wherein R1 is equal to R2;
and tautomers, solvates and pharmaceutically acceptable salts
thereof, for use in the prevention or treatment of Huntington's
disease in a subject.
[0363] In a preferred embodiment, there is provided a compound of
formula (I) according to the invention, wherein R1 is not equal to
R2; and tautomers, solvates and pharmaceutically acceptable salts
thereof, for use in the prevention or treatment of a neurological
disorder in a subject, wherein the neurological disorder is
epilepsy or Huntington's disease.
[0364] In a preferred embodiment, there is provided a compound of
formula (I) according to the invention, wherein R1 is not equal to
R2; and tautomers, solvates and pharmaceutically acceptable salts
thereof, for use in the prevention or treatment of epilepsy in a
subject.
[0365] In a preferred embodiment, there is provided a compound of
formula (I) according to the invention, wherein R1 is not equal to
R2; and tautomers, solvates and pharmaceutically acceptable salts
thereof, for use in the prevention or treatment of Huntington's
disease in a subject.
[0366] In a further aspect of the invention, there is provided a
method for treating or preventing a neurological disorder in a
subject, comprising administering an effective amount of a compound
of formula (I) according to the invention to said subject.
[0367] In one embodiment, there is provided a method for treating
or preventing a neurological disorder in a subject, comprising
administering an effective amount of a compound of formula (I)
according to the invention to said subject, wherein the
neurological disorder is epilepsy or Huntington's disease.
[0368] In a preferred embodiment, there is provided a method for
treating or preventing epilepsy in a subject, comprising
administering an effective amount of a compound of formula (I)
according to the invention to said subject.
[0369] In a further preferred embodiment, there is provided a
method for treating or preventing epilepsy in a subject, comprising
administering an effective amount of a compound of formula (I)
according to the invention to said subject, wherein said effective
amount of a compound of formula (I) according to the invention is
administered during primary epileptogenesis or during secondary
epileptogenesis or in fully developed epilepsy.
[0370] In a further preferred embodiment, there is provided a
method for treating or preventing Huntington's disease in a
subject, comprising administering an effective amount of a compound
of formula (I) according to the invention to said subject.
[0371] In a particularly preferred embodiment, there is provided a
method for treating or preventing a neurological disorder in a
subject, comprising administering an effective amount of a compound
of formula (I) according to the invention to said subject, wherein
said compound is selected from:
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyrimidin-2-ami-
ne;
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.1-
]octan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine; and tautomers, solvates and
pharmaceutically acceptable salts thereof; and wherein the
neurological disorder is epilepsy or Huntington's disease.
[0372] In a further aspect of the invention, there is provided the
use of a compound of formula (I) according to the invention for
treating or preventing a neurological disorder in a subject.
[0373] In one embodiment, there is provided the use of a compound
of formula (I) according to the invention for treating or
preventing a neurological disorder in a subject, wherein the
neurological disorder is epilepsy or Huntington's disease.
[0374] In a particularly preferred embodiment, there is provided
the use of a compound of formula (I) according to the invention for
treating or preventing a neurological disorder in a subject,
wherein said compound is selected from: [0375]
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyrimidin-2-ami-
ne; [0376]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicycl-
o[3.2.1]octan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
[0377]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,-
5-triazin-2-yl)pyridin-2-amine; and tautomers, solvates and
pharmaceutically acceptable salts thereof; and wherein the
neurological disorder is epilepsy or Huntington's disease.
[0378] In a further aspect of the invention, there is provided the
use of a compound of formula (I) according to the invention for the
manufacture of a medicament for treating or preventing a
neurological disorder in a subject.
[0379] In one embodiment, there is provided the use of a compound
of formula (I) according to the invention for the manufacture of a
medicament for treating or preventing a neurological disorder in a
subject, wherein the neurological disorder is epilepsy or
Huntington's disease.
[0380] In a particularly preferred embodiment, there is provided
the use of a compound of formula (I) according to the invention for
the manufacture of a medicament for treating or preventing a
neurological disorder in a subject, wherein said compound is
selected from: [0381]
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyrimidin-2-ami-
ne; [0382]
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicycl-
o[3.2.1]octan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine;
[0383]
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,-
5-triazin-2-yl)pyridin-2-amine; and tautomers, solvates and
pharmaceutically acceptable salts thereof; and wherein the
neurological disorder is epilepsy or Huntington's disease.
[0384] Most preferred for the present invention are the following
compounds shown by formula: (The names of the corresponding
structures were produced using ChemDraw Ultra, version 13.0.1 as
well as lower and upper software versions thereof, CambridgeSoft
Corp., Cambridge Mass.).
##STR00040## ##STR00041## ##STR00042## ##STR00043## ##STR00044##
##STR00045## ##STR00046## ##STR00047## ##STR00048## ##STR00049##
##STR00050## ##STR00051## ##STR00052## ##STR00053## ##STR00054##
##STR00055## ##STR00056##
Preparation of Compounds of the Invention
[0385] The compounds of the invention may be synthesized by
synthetic routes that include processes analogous to those well
known in the chemical arts, particularly in light of the
description contained herein. The starting materials are generally
available from commercial sources or are readily prepared using
methods well known to those skilled in the art.
[0386] In preparing compounds of the invention, protection of
remote functionality (e.g, primary or secondary amine) of
intermediates may be necessary. The need for such protection will
vary depending on the nature of the remote functionality and the
conditions of the preparation methods. Suitable amino-protecting
groups include tert-butyloxycarbonyl (BOC),
bis-tert-butyloxycarbonyl or dimethylaminomethylenyl. The need for
such protection is readily determined by one skilled in the art.
For a general description of protecting groups and their use, see
T. W. Greene, Protective Groups in Organic Synthesis, John Wiley
& Sons, New York, 1991.
[0387] Methods of Separation
[0388] In the methods of preparing the compounds of this invention,
it may be advantageous to separate reaction products from one
another and/or from starting materials. The desired products of
each step or series of steps are separated and/or purified to the
desired degree of homogeneity by the techniques common in the art.
Typically such separations involve multiphase extraction,
crystallization from a solvent or solvent mixture, distillation,
sublimation, or chromatography. Chromatography can involve any
number of methods including, for example: reverse-phase and normal
phase; high, medium and low pressure liquid chromatography methods
and apparatus; small scale analytical; and preparative thin or
thick layer chromatography, as well as techniques of small scale
thin layer and flash chromatography.
[0389] Selection of appropriate methods of separation depends on
the nature of the materials involved, for example, presence or
absence of polar functional groups in chromatography, stability of
materials in acidic and basic media in multiphase extraction, and
the like. One skilled in the art will apply techniques most likely
to achieve the desired separation.
EXAMPLES
[0390] The Examples are intended to illustrate the present
invention without restricting it.
[0391] The chemical reactions described in the Examples may be
readily adapted to prepare a number of other lipid kinase
inhibitors of the invention, and alternative methods for preparing
the compounds of this invention are deemed to be within the scope
of this invention. For example, the synthesis of non-exemplified
compounds according to the invention may be successfully performed
by modifications apparent to those skilled in the art, e.g., by
appropriately protecting interfering groups, by utilizing other
suitable reagents known in the art other than those described,
and/or by making routine modifications of reaction conditions.
Alternatively, other reactions disclosed herein or known in the art
will be recognized as having applicability for preparing other
compounds of the invention.
[0392] As a rule, .sup.1H NMR and mass spectra have been obtained
for the compounds prepared. In the Examples described below, unless
otherwise indicated, all temperatures are set forth in degrees
Celsius (.degree. C.). Reagents were purchased from commercial
suppliers such as Sigma Aldrich, Fluorochem, Acros, Lancaster, TCI
or Maybridge, and were used without further purification unless
otherwise indicated. The reactions set forth below were done
generally under a positive pressure of nitrogen or with a drying
tube (unless otherwise stated) in anhydrous solvents, and the
reaction flasks were typically fitted with rubber septa for the
introduction of substrates and reagents via syringe. Glassware was
oven dried. Column chromatography was performed using Merck silica
gel. .sup.1H NMR spectra were recorded on a Bruker instrument
operating at 400 MHz. .sup.1H NMR spectra were obtained for
solutions in various deuterated solvents such as CDCl.sub.3,
(CD.sub.3).sub.2SO, CD.sub.3OD or (CD.sub.3).sub.2CO. The chemical
shift .delta. values were reported in ppm and corrected to the
signal of the deuterated solvents (7.26 ppm for CDCl.sub.3) or TMS
(0 ppm). .sup.19F NMR spectra were calibrated relative to
CFCl.sub.3 (.delta.=0 ppm) as external standard. .sup.19F NMR
spectra were recorded .sup.1H-decoupled. When peak multiplicities
are reported, the following abbreviations are used: s (singlet), d
(doublet), t (triplet), m (multiplet), quint (quintet), br
(broadened). Coupling constants, when given, are reported in Hertz
(Hz). MALDI-ToF Mass spectra (MS) have been obtained on a
Voyager-De.TM. Pro measured in m/z.
[0393] The following abbreviations are used hereinafter: BSA
(bovine serum albumin), DMSO (dimethyl sulfoxide), ESI
(electronspray ionization), HCl (hydrochloric acid), M (molar),
MALDI (Matrix-assisted Laser Desorption/Ionization), MS (mass
spectrometry), PBS (phosphate buffered saline), TLC (thin layer
chromatography), nd (not determined).
Example 1
Preparation of Intermediate Compounds and of Compounds of the
Invention
[0394] Preparation of Intermediate Compounds
[0395] The following methods were used to prepare the intermediates
compounds used to produce compounds of formula (I).
Method 1:
8-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-chloro-1,3,5-triazi-
n-2-yl)-3-oxa-8-azabicyclo[3.2.1]octane (i1)
##STR00057##
[0397] 3-Oxa-8-azabicyclo[3.2.1]octane.HCl (Advanced ChemBlocks
Inc, product number A-861, 2.00 g, 13.4 mmol, 2.0 eq.) and
N,N-diisopropylethylamine (4.80 mL, 27.6 mmol, 4.1 eq.) are charged
into a flask and dissolved in dichloromethane (20 mL). The flask is
placed in an ice bath and the solution subsequently cooled down to
0.degree. C. This solution is then added dropwise to a solution of
cyanuric chloride in dichloromethane (20 mL) at 0.degree. C. The
resulting reaction mixture is stirred overnight, while it is
allowed to warm up to room temperature. Additional dichloromethane
(100 mL) is added and the organic layer is washed with a saturated
aqueous solution of sodium bisulfate. The organic layer is then
dried over anhydrous sodium sulfate, filtered and the solvent is
evaporated under reduced pressure. Purification by flash
chromatography (cyclohexane/ethyl acetate 4:1) gives the desired
intermediate ii as a colorless solid (79% yield). .sup.1H NMR (400
MHz, CDCl.sub.3): .delta.4.70-4.54 (m, 4H), 3.80-3.58 (m, 8H),
2.14-1.89 (m, 8H); MS (MALDI): m/z=338.4 ([M+H]+).
[0398] Method 1 is also used for the preparation of the following
intermediate compounds i2 to i10, and intermediates i79 to i81 and
i90.
TABLE-US-00001 Reagent Structure NMR MS i2 ##STR00058##
##STR00059## .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 3.78 (m, 8
H), 3.70 (m, 8 H). MS (MALDI): m/z = 285.9 ([M + H].sup.+). i3
##STR00060## ##STR00061## .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 4.75-4.56 (m, 2 H), 4.34- 4.30 (m, 2 H), 3.94 (dd,
.sup.2J.sub.H,H = 12.0 Hz, .sup.3J.sub.H,H = 4.0 Hz, 2 H), 3.74 (d,
.sup.2J.sub.H,H = 12.0 Hz, 2 H), 3.63 (dd, .sup.2J.sub.H,H = 12.0
Hz, .sup.3J.sub.H,H = 4.0 Hz, 2 H), 3.49 (dt, .sup.2J.sub.H,H =
12.0 Hz, .sup.3J.sub.H,H = 4.0 Hz, 2 H), 3.25 (dt, .sup.2J.sub.H,H
= 12.0 Hz, .sup.3J.sub.H,H = 4.0 Hz, 2 H), 1.31 (d, .sup.3J.sub.H,H
= 8.0 Hz, 6 H). MS (MALDI): m/z = 314.4 ([M + H].sup.+). i4
##STR00062## ##STR00063## .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 3.81-3.72 (m, 8 H), 3.43 (s, 4 H), 1.43 (br s, 12 H). MS
(MALDI): m/z = 342.5 ([M + H].sup.+). i5 ##STR00064## ##STR00065##
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 4.75-4.56 (m, 2 H),
4.34- 4.30 (m, 2 H), 3.94 (dd, .sup.2J.sub.H,H = 12.0 Hz,
.sup.3J.sub.H,H = 4.0 Hz, 2 H), 3.74 (d, .sup.2J.sub.H,H = 12.0 Hz,
2 H), 3.63 (dd, .sup.2J.sub.H,H = 12.0 Hz, .sup.3J.sub.H,H = 4.0
Hz, 2 H), 3.49 (dt, .sup.2J.sub.H,H = 12.0 Hz, .sup.3J.sub.H,H =
4.0 Hz, 2 H), 3.25 (dt, .sup.2J.sub.H,H = 12.0 Hz, .sup.3J.sub.H,H
= 4.0 Hz, 2 H), 1.31 (d, .sup.3J.sub.H,H = 8.0 Hz, 6 H). MS
(MALDI): m/z = 314.3 ([M + H].sup.+). i6 ##STR00066## ##STR00067##
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 4.40-4.37 (m, 4 H), 3.74
(d, .sup.3J.sub.H,H = 11.6 Hz, 4 H), 3.53 (dd, .sup.3J.sub.H,H =
11.6 Hz, .sup.2J.sub.H,H = 4.0 Hz, 4 H), 1.26 (d, .sup.3J.sub.H,H =
6.9 Hz, 12 H). MS (MALDI): m/z = 342.8 ([M + H].sup.+). i7
##STR00068## ##STR00069## .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 4.53 (br s, 2 H), 4.36 (br s, 2 H), 4.12-4.06 (m, 8 H),
3.92-3.83 (m, 8 H). MS (MALDI): m/z = 370.3 ([M + H].sup.+). i8
##STR00070## ##STR00071## .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO): .delta. 4.36-4.21 (m, 4 H), 3.85-3.75 (m, 4
H), 3.48-3.45 (m, 2 H), 3.40-3.34 (m, 2 H), 3.14-3.09 (m, 2 H),
1.72 (m, 4 H), 0.82 (m, 6 H). MS (MALDI): m/z = 342.3 ([M].sup.+).
i9 ##STR00072## ##STR00073## .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO): .delta. 3.64 (m, 8 H), 3.351-3.48 (m, 4 H),
2.46- 2.38 (m, 4 H), 2.20-2.16 (m, 4 H), 1.73-1.66 (m, 4 H). MS
(MALDI): m/z = 366.7 ([M + H].sup.+). i10 ##STR00074## ##STR00075##
.sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 4.40-4.25 (m, 2
H), 4.20-4.05 (m, 2 H), 4.08 (m, 2 H), 3.95 (m, 2 H), 3.83 (m, 4
H), 3.08 (m, 2 H), 2.30 (m, 2 H), 0.98 (m, 6 H), 0.48 (m, 6 H). MS
(MALDI): m/z = 370.4 ([M + H].sup.+). i79 ##STR00076## ##STR00077##
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 4.59-4.31 (m, 4 H),
3.66- 3.46 (m, 4 H), 2.70 (m, 4 H), 1.14 (m, 12 H). MS (MALDI): m/z
= 342.4 ([M + H].sup.+). i80 ##STR00078## ##STR00079## .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta. 3.73-3.64 (m, 8 H), 3.57 (s, 2 H),
3.51 (s, 2 H), 1.14 (s, 12 H). MS (MALDI): m/z = 342.3 ([M +
H].sup.+). i81 ##STR00080## ##STR00081## .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 4.41 (br s, 4 H), 4.32- 4.16 (m, 4 H),
3.24-3.10 (m, 4 H), 1.99-1.84 (m, 4 H), 1.84-1.67 (m, 4 H). MS
(MALDI): m/z = 338.4 ([M + H].sup.+) i90 ##STR00082## ##STR00083##
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 4.20 (m, 4 H), 4.10 (m,
4 H), 3.66 (m, 4 H), 1.35 (d, .sup.3J.sub.H,H = 6.9 Hz, 12 H) MS
(MALDI): m/z = 342.8 ([M + H].sup.+)
Method 2: 2,4-dichloro-6-morpholino-1,3,5-triazine (ill)
##STR00084##
[0400] To a solution of cyanuric chloride (18.1 g, 0.100 mol, 1.0
eq.) in dichloromethane (200 mL) is dropwise added a solution of
morpholine (17.4 g, 0.200 mol, 2.0 eq.) at -78.degree. C. over 2
hours. The resulting mixture is allowed to warm to 0.degree. C.
with stirring and mixed with an ice cold saturated solution of
sodium bisulfate in water. The phases are separated and the organic
phase is washed with half concentrated brine dried over sodium
sulfate and evaporated to yield the title compound ill as a
colorless solid. .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta.3.90-3.86 (m, 4H), 3.77-3.72 (m, 4H).
Method 3:
8-(4-chloro-6-morpholino-1,3,5-triazin-2-yl)-3-oxa-8-azabicyclo--
[3.2.1]octane (i12)
##STR00085##
[0401] 3-Oxa-8-azabicyclo[3.2.1]octane.HCl (Advanced ChemBlocks
Inc, product number A-861, 200 mg, 1.34 mmol, 1.1 eq.) and
N,N-diisopropylethylamine (470 .mu.L, 2.69 mmol, 2.1 eq.) are
charged in a flask and dissolved in ethanol (3 mL). The flask is
placed in an ice bath. A solution of compound ill (300 mg, 1.28
mmol, 1.0 eq.) in ethanol (2 mL) is added to the above solution at
0.degree. C. The resulting mixture is stirred overnight, while
allowing it to warm up to room temperature. Deionized water (20 mL)
is added and the aqueous layer is extracted with ethyl acetate
(3.times.30 mL). The combined organic layer is dried over anhydrous
sodium sulfate, filtered and the solvent is evaporated under
reduced pressure. Purification by flash chromatography
(cyclohexane/ethyl acetate 9:1.fwdarw.8:2) gives the desired
intermediate i12 as a colorless solid (78% yield). .sup.1H NMR (400
MHz, CDCl.sub.3): .delta.4.69-4.56 (m, 2H), 3.86-3.59 (m, 12H),
2.12-1.91 (m, 4H); MS (MALDI): m/z=312.7 ([M+H].sup.+).
[0402] Method 3 is also used for the preparation of the following
intermediate compounds i13 to i16, and intermediates i87 and
i91.
TABLE-US-00002 Reagent Structure NMR i13 ##STR00086## ##STR00087##
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 4.71- 4.61 (m, 1 H),
4.34-4.31 (m, 1 H), 3.96- 3.92 (m, 1 H), 3.79-3.70 (m, 9 H), 3.65-
3.61 (m, 1 H), 3.51-3.45 (m, 1 H), 3.29- 3.21 (m, 1 H), 1.36-1.30
(d, .sup.3J.sub.H,H = 6.9 Hz, 3 H). i14 ##STR00088## ##STR00089##
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 3.79-3.71 (m, 12 H),
3.46 (m, 4 H), 1.48 (s, 9 H). i15 ##STR00090## ##STR00091## .sup.1H
NMR (400 MHz, CDCl.sub.3): .delta. 4.12- 3.98 (m, 4 H), 3.84-3.70
(m, 4 H), 3.70- 3.62 (m, 4 H), 2.66-2.56 (m, 4 H). i16 ##STR00092##
##STR00093## .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 3.77 (m, 4
H), 3.68-3.63 (m, 8 H), 3.44 (s, 2 H), 1.44 (s, 6 H). i87
##STR00094## ##STR00095## .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 4.52 (m, 1 H), 4.43 (m, 1 H), 3.93 (m, 2 H), 3.65 (m, 10
H), 2.48 (m, 1 H), 1.88-1.72 (m, 4 H), 1.57 (m, 1 H) i91
##STR00096## ##STR00097## .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta. 4.44 (m, 1 H), 4.32 (m, 1 H), 4.00 (m, 4 H), 3.74- 3.65 (m,
12 H);
Method 4:
(S)-4-(4,6-dichloro-1,3,5-triazin-2-yl)-3-methylmorpholine
(i17)
##STR00098##
[0404] To a solution of cyanuric chloride (450 mg, 2.44 mol, 1.0
eq.) in dichloromethane (4 mL) is slowly added a solution of
(S)-3-methylmorpholine (Activate Scientific, product number AS3424,
0.28 mL, 2.44 mol, 1.0 eq.) and triethylamine (0.35 mL, 2.51 mol,
1.02 eq.) in dichloromethane (2 mL) at -50.degree. C. The resulting
mixture is stirred for 2 hours at -50.degree. C., then allowed to
warm to 0.degree. C. with stirring and mixed with an ice cold
saturated solution of sodium bisulfate in water. The phases are
separated and the organic phase is washed with brine dried over
sodium sulfate and evaporated to yield the title compound i17 as a
colorless solid (95% yield). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta.4.78-4.69 (m, 1H), 4.43-4.39 (m, 1H), 3.98-3.96 (m, 1H),
3.78-3.76 (m, 1H), 3.67-3.65 (m, 1H), 3.51-3.47 (m, 1H), 3.40-3.37
(m, 1H), 1.36 (m, 3H).
Method 5:
8-(4-chloro-6-((S)-3-methylmorpholino)-1,3,5-triazin-2-yl)-3-oxa-
-8-azabicyclo[3.2.1]octane (i18)
##STR00099##
[0406] 3-Oxa-8-azabicyclo[3.2.1]octane.HCl (Advanced ChemBlocks
Inc, product number A-861, 383 mg, 2.55 mmol, 1.1 eq.) and
N,N-diisopropylethylamine (1.0 mL, 5.60 mmol, 2.4 eq.) are charged
in a flask and dissolved in ethanol (4 mL). The flask is placed in
an ice bath. A solution of compound i17 (580 mg, 2.33 mmol, 1.0
eq.) in ethanol (2 mL) is added to the above solution at 0.degree.
C. The resulting mixture is stirred for 4 hours, while allowing it
to warm up to room temperature. Deionized water (20 mL) is added
and the aqueous layer is extracted with ethyl acetate (3.times.30
mL). The combined organic layer is dried over anhydrous sodium
sulfate, filtered and the solvent is evaporated under reduced
pressure. Purification by flash chromatography (cyclohexane/ethyl
acetate 9:1.fwdarw.8:2) gives the desired intermediate i18 as a
colorless solid (88% yield). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta.4.75-4.52 (m, 3H), 4.37-4.24 (m, 1H), 3.95-3.92 (m, 1H),
3.73-3.70 (m, 3H), 3.64-3.61 (m, 3H), 3.52-3.42 (m, 1H), 3.29-3.17
(m, 1H), 2.11-1.89 (m, 4H), 1.31 (m, 3H).
Method 6: tert-butyl
4-(4,6-dichloro-1,3,5-triazin-2-yl)piperazine-1-carboxylate
(i19)
##STR00100##
[0408] To a cooled (-50.degree. C.) solution of cyanuric chloride
(1.0 g, 5.42 mmol, 1.0 eq.) in dichloromethane (4 mL) is added
dropwise a solution of tert-butyl piperazine-1-carboxylate (Sigma,
product number 343536, 1.02 g, 5.48 mmol, 1.01 eq.) and
triethylamine (0.767 mL, 5.53 mmol, 1.02 eq.) in dichloromethane (2
mL). The resulting reaction mixture is stirred at -50.degree. C.
for 4 hours. A saturated aqueous solution of sodium bisulfate (10
mL) and dichloromethane (20 mL) are added. The mixture is
transferred to a separating funnel. The organic layer is separated,
washed with a saturated aqueous solution of sodium bisulfate (20
mL), dried over anhydrous sodium sulfate, filtered and then the
solvent is evaporated under reduced pressure to give pure
intermediate i19 (80% yield). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta.3.88-3.85 (m, 4H), 3.53-3.51 (m, 4H), 1.49 (m, 9H).
Method 7: tert-butyl
4-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-chloro-1,3,5-triazin-2-yl)pi-
perazine-1-carboxylate (i20)
##STR00101##
[0410] 3-Oxa-8-azabicyclo[3.2.1]octane.HCl (Advanced ChemBlocks
Inc, product number A-861, 235 mg, 1.57 mmol, 1.0 eq.) and
N,N-diisopropylethylamine (592 .mu.L, 3.14 mmol, 2.1 eq.) are
charged in a flask and dissolved in ethanol (6 mL). The flask is
placed in an ice bath. A solution of compound i19 (500 mg, 1.5
mmol, 1.0 eq.) in ethanol (2 mL) is added to the above solution at
0.degree. C. The resulting mixture is stirred overnight, while
allowed to warm up to room temperature. Deionized water (10 mL) is
added and the aqueous layer is extracted with ethyl acetate
(3.times.30 mL). The combined organic layer is dried over anhydrous
sodium sulfate, filtered and the solvent is evaporated under
reduced pressure. Purification by flash chromatography
(cyclohexane/ethyl acetate 8:2) gave the desired intermediate i20
as a colorless solid (77% yield). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta.4.68-4.60 (m, 2H), 3.76-3.70 (m, 6H), 3.64-3.62
(m, 2H), 3.47-3.45 (m, 4H), 2.08-1.95 (m, 4H), 1.48 (br s, 9H); MS
(MALDI): m/z=411.8 ([M+H].sup.+).
[0411] Method 7 is also used for the preparation of the following
intermediate compound i21.
TABLE-US-00003 Reagent Structure NMR MS i21 ##STR00102##
##STR00103## .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 4.76-4.61
(m, 1 H), 4.35-4.30 (m, 1 H), 3.94 (dd, .sup.2J.sub.H,H = 12 Hz,
.sup.3J.sub.H,H = 4.0 Hz, 1 H), 3.76-3.72 (m, 5 H), 3.65 (dd,
.sup.2J.sub.H,H = 12 Hz, .sup.3J.sub.H,H = 4.0 Hz, 1 H), 3.51-3.44
(m, 5 H), 3.25 (dt, .sup.2J.sub.H,H = 12 Hz, .sup.3J.sub.H,H = 4.0
Hz, 1 H), 1.48 (s, 9 H), 1.30 (d, .sup.3J.sub.H,H = 8.0 Hz, 3 H).
MS (MALDI): m/z = 399.1 ([M + H].sup.+).
Method 8: 4,4'-(6-chloropyrimidine-2,4-diyl)dimorpholine (i22) and
4,4'-(2-chloropyrimidine-4,6-diyl)dimorpholine (i23)
##STR00104##
[0413] 2,4,6-Trichloropyrimidine (Manchester Organics, product
number Y17832, 11.2 g, 61 mmol, 1.0 eq.), N,N-diisopropylethylamine
(23.3 mL, 134.2 mmol, 2.2 eq.) and morpholine (11.7 mL, 134.2 mmol,
2.2 eq.) are charged in a flask and dissolved in ethanol (120 mL).
The flask is equipped with a refluxed condenser and placed in an
oil bath preheated at 100.degree. C. The reaction mixture is
stirred at this temperature for 18 hours. After this time, the
reaction mixture is cooled down to room temperature and volatiles
are removed under reduced pressure. The resulting mixture is
dissolved in dichloromethane (100 mL) and washed twice with an
aqueous solution of sodium bisulfate (2.times.80 mL). The organic
layer is dried over anhydrous sodium sulfate, filtered and
concentrated under reduced pressure using a rotary evaporator.
Products i22 and i23 are isolated by flash chromatography on silica
gel (cyclohexane/ethyl acetate 3:1 to 1:1). The product fractions
are pooled and evaporated to yield i22 as a colorless powder (13.8
g, 80%) and i23 as a colorless powder (2.2 g, 13% yield).
[0414] 4,4'-(6-chloropyrimidine-2,4-diyl)dimorpholine (i22):
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta.5.85 (s, 1H), 3.71-3.75
(m, 12H), 3.52-3.55 (m, 4H); MS (MALDI): m/z: 285.4
([M+H].sup.+).
[0415] 4,4'-(2-chloropyrimidine-4,6-diyl)dimorpholine (i23):
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta.5.38 (s, 1H), 3.73-3.76
(m, 8H), 3.52-3.54 (m, 8H); MS (MALDI): m/z: 285.2
([M+H].sup.+).
Method 9:
8-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-chloropyrimidin-2-y-
l)-3-oxa-8-azabicyclo[3.2.1]octane (i24)
##STR00105##
[0417] A solution of 2,4,6-trichloropyrimidine (0.676 mL, 5.88
mmol, 1.0 eq.), 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride (1.76
g, 11.8 mmol, 2.0 eq.), and N,N-diisopropylethylamine (4.10 mL,
23.5 mmol, 4.0 eq.) in ethyl acetate (18 volumes) is heated for 16
hours (100.degree. C.). Then, the solvent is removed under reduced
pressure and the residue is dissolved in dichloromethane (60
volumes) and washed with a saturated aqueous sodium bisulfate
(3.times.60 volumes). The organic layer is dried over anhydrous
sodium sulfate, filtered and concentrated under reduced pressure.
Purification by column chromatography on silica gel
(cyclohexane/ethyl acetate 3:1 to 1:1) affords the desired
intermediate i24 as a colorless solid (1.23 g, 62%). .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta.5.80 (s, 1H), 4.59 (s, 2H), 4.35 (m,
2H), 3.76 (t, .sup.2J.sub.H,H=10.8 Hz, 4H), 3.59 (d,
.sup.2J.sub.H,H=10.8 Hz, 4H), 2.03 (m, 8H); MS (MALDI): m/z=337.7
([M+H].sup.+).
[0418] Method 9 is also used for the preparation of the following
intermediate compound i25.
TABLE-US-00004 Reagent Structure NMR MS i25 ##STR00106##
##STR00107## .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 5.83 (s, 1
H), 4.64-4.57 (m, 1 H), 4.27 (dd, .sup.3J.sub.H,H = 2.4 Hz,
.sup.2J.sub.H,H = 13.5 Hz, 1 H), 4.20-4.11 (m, 1 H), 3.97-3.87 (m,
3 H), 3.77-3.63 (m, 4 H), 3.56-3.46 (m, 2 H), 3.26-3.15 (m, 2 H),
1.28 (d, .sup.3J.sub.H,H = 3.2 Hz, 3 H), 1.27 (d, .sup.3J.sub.H,H =
3.2 Hz, 3 H). MS (MALDI): m/z = 313.6 ([M + H].sup.+).
Method 10: 4-(4,6-dichloropyrimidin-2-yl)morpholine (i26) and
4-(2,6-dichloropyrimidin-4-yl)morpholine (i27)
##STR00108##
[0420] To a solution of 2,4,6-trichloropyrimidine (14.0 mL, 122
mmol, 1.0 eq.) in EtOH (150 mL) is added a solution of morpholine
(11.2 mL, 256 mmol, 2.1 eq.) and N,N-diisopropylethylamine (44.6
mL, 256 mmol, 2.1 eq.) in EtOH (150 mL) dropwise at 0.degree. C.
The reaction mixture is stirred overnight at room temperature and
the solvent is removed under reduced pressure. The crude product is
extracted with dichloromethane (3.times.100 mL) and the organic
phase is successively washed with saturated aqueous sodium
bisulfate (3.times.400 mL). The combined organic layers are dried
over anhydrous sodium sulfate, filtered and evaporated under
reduced pressure. The crude mixture is purified by flash column
chromatography (SiO.sub.2, cyclohexane/ethyl acetate 9:1 to 3:1) to
yield i26 (5.02 g, 18%) and i27 (16.7 g, 59%), both as colorless
solids.
[0421] 4-(4,6-dichloropyrimidin-2-yl)morpholine (i26): .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta.6.56 (s, 1H), 3.78 (m, 4H) 3.74 (m,
4H).
[0422] 4-(2,6-dichloropyrimidin-4-yl)morpholine (i27): .sup.1H NMR
(400 MHz, CDCl.sub.3): .delta.6.41 (s, 1H), 3.78 (m, 4H), 3.65 (m,
4H).
Method 11:
(S)-4-(2-chloro-6-morpholinopyrimidin-4-yl)-3-methylmorpholine
(i28)
##STR00109##
[0424] A solution of i27 (694 mg, 2.97 mmol, 1.0 eq.),
(S)-3-methylmorpholine (0.500 mL, 4.46 mmol, 1.5 eq.) and
N,N-diisopropylethylamine (1.29 mL, 7.43 mmol, 2.5 eq.) in EtOH
(5.0 mL) is heated to reflux for 3 days. Then, the solvent is
removed under reduced pressure. The residue is dissolved in
dichloromethane (60 volumes) and washed with saturated aqueous
sodium bisulfate (3.times.60 volumes). The organic layer is dried
over anhydrous sodium sulfate, filtered and concentrated under
reduced pressure. The crude mixture is purified by flash
chromatography (SiO.sub.2, cyclohexane/ethyl acetate 3:1 to 1:1) to
afford the title compound
(S)-4-(2-chloro-6-morpholinopyrimidin-4-yl)-3-methylmorpholine
(i28) as a colorless solid (425 mg, 48%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta.5.85 (s, 1H), 4.62 (dd, .sup.2J.sub.H,H=13.6
Hz, .sup.3J.sub.H,H 10=2.9 Hz, 1H), 4.25 (dd, .sup.2J.sub.H,H=13.6
Hz, .sup.3J.sub.H,H=2.9 Hz, 1H), 3.93 (dd, .sup.2J.sub.H,H=11.4 Hz,
.sup.3J.sub.H,H=3.8 Hz, 1H), 3.75, (t, .sup.3J.sub.H,H=5.0 Hz, 4H),
3.71 (s, 1H), 3.66 (dd, .sup.2J.sub.H,H=11.3 Hz,
.sup.3J.sub.H,H=3.2 Hz, 1H), 3.53 (m, 5H), 3.23 (m, 1H), 1.26 (d,
.sup.2J.sub.H,H=11.3 Hz, 3H); MS (MALDI): m/z=299.4
([M+H].sup.+).
[0425] Method 11 is also used for the preparation of the following
intermediate compound i29.
TABLE-US-00005 Reagent Structure NMR MS i29 ##STR00110##
##STR00111## .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 5.86 (s, 1
H), 4.60 (br s, 2 H), 3.80-3.72 (m, 6 H), 3.62-3.56 (m, 2 H),
3.56-3.50 (m, 4 H), 2.08- 1.90 (m, 4 H). MS (MALDI): m/z = 309.6
([M + H].sup.+).
Method 12:
(S)-4-(6-chloro-2-morpholinopyrimidin-4-yl)-3-methylmorpholine
(i30)
##STR00112##
[0427] A solution of (S)-3-methylmorpholine (194 mg, 1.32 mmol, 1.5
eq.), i26 (300 mg, 1.28 mmol, 1.0 eq.) and
N,N-diisopropylethylamine (3.0 eq.) in DMF (17 volumes) is heated
for 16 hours (130.degree. C.). Then, the solvent is removed under
reduced pressure. The residue is dissolved in dichloromethane (100
volumes) and washed with saturated aqueous sodium bisulfate
(3.times.100 volumes). The organic layer is dried over anhydrous
sodium sulfate, filtered and concentrated under reduced pressure.
The crude mixture is purified by flash chromatography (SiO.sub.2,
cyclohexane/ethyl acetate 5:1) to afford the title compound i30 as
a colorless solid (257 mg, 67%). .sup.1H NMR (400 MHz, CDCl.sub.3):
.delta.5.84 (s, 1H), 4.18 (m, 1H), 3.94 (m, 2H), 3.71 (m, 10H),
3.53, (dt, .sup.2J.sub.H,H=12.0 Hz, .sup.3J.sub.H,H=3.1 Hz, 1H),
3.20 (dt, .sup.2J.sub.H,H=12.8 Hz, .sup.3J.sub.H,H=3.8 Hz, 1H),
1.27 (d, .sup.3J.sub.H,H=6.8 Hz, 3H); MS (MALDI): m/z=298.4
([M].sup.+).
Method 14:
8-(4,6-dichloro-1,3,5-triazin-2-yl)-3-oxa-8-azabicyclo[3.2.1]oc-
tane (i32)
##STR00113##
[0429] A solution of cyanuric chloride (1.97 g, 10.7 mmol, 1.0 eq.)
in dichloromethane (10 mL) is cooled to -50.degree. C. A solution
of 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride (1.60 g, 10.7
mmol, 1.0 eq.) and N,N-diisopropylethylamine (3.73 mL, 21.4 mmol,
2.0 eq.) in dichloromethane (40 mL) is slowly added over a period
of 5 hours. The mixture is stirred for another 5 hours at this
temperature. Then, dichloromethane (20 mL) and saturated aqueous
sodium bisulfate (50 mL) are added and the mixture is allowed to
warm to room temperature.
[0430] The layers are separated and the organic layer is washed
with saturated aqueous sodium bisulfate (2.times.50 mL). The
organic layer is dried over anhydrous sodium sulfate and the
solvent is removed under reduced pressure. The crude mixture is
recrystallized from n-heptane/dichloromethane (20 mL/13 mL) to
afford the title compound
8-(4,6-dichloro-1,3,5-triazin-2-yl)-3-oxa-8-azabicyclo[3.2.1]octane
(i32) as a colorless solid (2.47 g, 47%). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta.4.74 (m, 2H), 3.72 (d, .sup.3J.sub.H,H=1.5 Hz,
4H), 2.08 (m, 4H). [0431] Method 14 is also used for the
preparation of the following intermediate compounds i33 and
i34.
TABLE-US-00006 [0431] Reagent Structure NMR i33 ##STR00114##
##STR00115## .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 4.54-4.60
(m, 1 H), 4.20 (dd, .sup.3J.sub.H,H = 2.9 Hz, .sup.2J.sub.H,H = 14
Hz, 1 H), 3.92 (dd, .sup.3J.sub.H,H = 3.4 Hz, .sup.2J.sub.H,H = 12
Hz, 1 H), 3.71 (d, .sup.2J.sub.H,H = 12 Hz, 1 H), 3.57 (dd,
.sup.3J.sub.H,H = 3.2 Hz, .sup.2J.sub.H,H = 12 Hz, 1 H), 3.42 (m, 1
H), 3.32 (m, 1 H), 1.27 (d, .sup.3J.sub.H,H = 6.9 Hz, 3 H). i34
##STR00116## ##STR00117## .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO): .delta. 3.88- 3.81 (m, 4 H), 3.51 (s, 2 H),
1.46 (s, 6 H).
Method 15:
9-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-chloro-1,3,5-triaz-
in-2-yl)-3,7-dioxa-9-azabicyclo[3.3.1]nonane (i35)
##STR00118##
[0433] To a solution of 3,7-dioxa-9-azabicyclo[3.3.1]nonane (184
mg, 0.700 mmol, 1.0 eq.) and N,N-diisopropylethylamine (0.170 mL,
0.970 mmol, 1.4 eq.) in 1,4-dioxane (1.0 mL) a solution of i32 (100
mg, 0.770 mmol, 1.1 eq.) in 1,4-dioxane (2.0 mL) is added. The
resulting mixture is heated for 1 hour at 70.degree. C. Then,
dichloromethane (50 mL) and water (50 mL) are added. The aqueous
layer is extracted with dichloromethane (3.times.50 mL), the
combined organic layers are dried over anhydrous sodium sulfate and
the solvent is evaporated. The crude mixture is purified by
automated flash chromatography on silica gel (cyclohexane/ethyl
acetate 2:1 to 0:1) to afford the title compound
9-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-chloro-1,3,5-triazin-2-yl)-3-
,7-dioxa-9-azabicyclo[3.3.1]nonane (i35) as a colorless solid (192
mg, 77%). .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.4.70
(m, 1H), 4.55 (m, 2H), 4.44 (m, 1H), 4.12 (m, 4H), 3.90 (m, 4H),
3.72 (m, 2H), 3.64 (m, 2H), 2.08 (m, 2H), 1.97 (m, 2H); MS (MALDI):
m/z=354.3 ([M].sup.+).
Method 16:
9-(4-chloro-6-((R)-3-methylmorpholino)-1,3,5-triazin-2-yl)-3,7--
dioxa-9-azabicyclo[3.3.1]nonane (i36)
##STR00119##
[0435] To a solution of 3,7-dioxa-9-azabicyclo[3.3.1]nonane (173
mg, 1.27 mmol, 1.05 eq.) and N,N-diisopropylethylamine (0.50 mL,
2.52 mmol, 2.1 eq.) in tetrahydrofuran (5 mL) a solution of i33
(300 mg, 2.52 mmol, 2.1 eq.) in 1,4-dioxane (2.0 mL) is added. The
resulting mixture is heated for 2 hours (70.degree. C.). Then,
ethyl acetate (20 mL) and saturated aqueous sodium bisulfate (20
mL) are added. The phases are separated and the organic layer is
washed with saturated aqueous sodium bisulfate (2.times.20 mL). The
organic layer is dried over anhydrous sodium sulfate and the
solvent is removed under reduced pressure. The crude mixture is
purified by automated flash chromatography (SiO.sub.2,
cyclohexane/ethyl acetate 2:1 to 0:1) to afford the title compound
i36 as a colorless solid (316 mg, 76%). .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO): .delta.4.55-4.53 (m, 1H), 4.42 (m, 1H), 4.32
(m, 1H), 4.25-4.16 (m, 1H), 4.01-3.97 (m, 4H), 3.87 (dd,
.sup.3J.sub.H,H=3.8 Hz, .sup.2J.sub.H,H=11.2 Hz, 1H), 3.73-3.65 (m,
5H), 3.53 (dd, .sup.3J.sub.H,H=3.0 Hz, .sup.2J.sub.H,H=11.6 Hz,
1H), 3.38 (m, 1H), 3.15 (m, 1H), 1.20 (d, .sup.3J.sub.H,H=6.9 Hz,
3H).
[0436] Method 16 is also used for the preparation of the following
intermediate compounds i37 to i53, intermediate i82 and
intermediates i85, i86, i92, i93, i94.
TABLE-US-00007 Reagent Structure NMR MS i37 ##STR00120##
##STR00121## .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.
4.58-4.50 (m, 1 H), 4.44-4.35 (m, 2 H), 4.25-4.12 (m, 1 H), 3.90-
3.86 (m, 1 H), 3.75- 3.65 (m, 3 H), 3.56- 3.49 (m, 3 H), 3.38 (m, 1
H), 3.16 (m, 1 H), 1.25 (d, .sup.3J.sub.H,H = 6.9 Hz, 6 H), 1.19
(d, .sup.3J.sub.H,H = 6.9 Hz, 3 H). MS (MALDI): m/z = 328.2 ([M +
H].sup.+). i38 ##STR00122## ##STR00123## .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO): .delta. 4.54-4.46 (m, 1 H), 4.18-4.13 (m, 1
H), 3.88 (m, 1 H), 3.80- 3.65 (m, 5 H), 3.54 (m, 1 H), 3.44-3.36
(m, 3 H), 3.18 (m, 1 H), 1.44 (s, 6 H), 1.21 (d, .sup.3J.sub.H,H =
6.9 Hz, 3 H). i39 ##STR00124## ##STR00125## .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO): .delta. 4.65-4.51 (m, 2 H), 4.31-4.20 (m, 2
H), 3.66 (m, 3 H), 3.69- 3.56 (m, 2 H), 3.54- 3.48 (m, 3 H), 3.42-
3.35 (m, 2 H), 3.31 (s, 3 H), 3.21-3.13 (m, 2 H), 1.21 (d,
.sup.3J.sub.H,H = 6.9 Hz, 3 H). MS (MALDI): m/z = 344.2 ([M +
H].sup.+). i40 ##STR00126## ##STR00127## .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO): .delta. 4.55-4.51 (m, 1 H), 4.42-4.35 (m, 2
H), 4.12-4.25 (m, 2 H), 4.04- 4.07 (m, 1 H), 3.86- 3.88 (m, 1 H),
3.78- 3.75 (m, 2 H), 3.69-3.65 (m, 1 H), 3.55-3.51 (m, 1 H), 3.38
(m, 1 H), 3.20- 3.13 (m, 1 H), 2.68 (m, 1 H), 1.81 (m, 1 H), 1.20
(d, .sup.3J.sub.H,H = 6.9 Hz, 3 H). i41 ##STR00128## ##STR00129##
.sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 4.69-4.53 (m, 3
H), 4.31-4.15 (m, 1 H), 3.93-3.78 (m, 3 H), 3.71- 3.53 (m, 4 H),
3.42- 3.35 (m, 1 H), 3.22- 3.16 (m, 1 H), 3.12-3.08 (m, 1 H), 1.81
(m, 1 H), 1.21 (d, .sup.3J.sub.H,H = 6.9 Hz, 3 H). i42 ##STR00130##
##STR00131## .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.
4.95-4.88 (m, 1 H), 4.64 (m, 1 H), 4.54 (m, 1 H), 4.31- 4.09 (m, 1
H), 3.89- 3.85 (m, 1 H), 3.75- 3.73 (m, 1 H), 3.66-3.63 (m, 2 H),
3.52 (m, 1 H), 3.45-3.32 (m, 3 H), 3.18- 3.12 (m, 1 H), 1.90- 1.83
(m, 2 H), 1.21 (d, .sup.3J.sub.H,H = 6.9 Hz, 3 H). MS (MALDI): m/z
= 312.2 ([M + H].sup.+). i43 ##STR00132## ##STR00133## .sup.1H NMR
(400 MHz, (CD.sub.3).sub.2SO): .delta. 4.94-4.88 (m, 1 H), 4.64 (m,
1 H), 4.54 (m, 1 H), 4.29- 4.12 (m, 1 H), 3.89- 3.85 (m, 1 H),
3.75- 3.73 (m, 2 H), 3.66-3.63 (m, 2 H), 3.52 (m, 1 H), 3.45-3.32
(m, 2 H), 3.18- 3.12 (m, 1 H), 1.90- 1.83 (m, 2 H), 1.21 (d,
.sup.3J.sub.H,H = 6.9 Hz, 3 H). MS (MALDI): m/z = 312.2 ([M +
H].sup.+). i53 ##STR00134## ##STR00135## .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO): .delta. 4.65 (m, 1 H), 4.55 (m, 1 H), 4.32 (m,
1 H), 4.22 (m, 2 H), 3.98 (m, 1 H), 3.86 (m, 2 H), 3.63 (m, 2 H),
3.55 (m, 1 H), 3.49- 3.34 (m, 4 H), 3.17 (m, 1 H), 3.12 (m, 1 H),
1.21 (d, .sup.3J.sub.H,H = 6.9 Hz, 3 H). MS (MALDI): m/z = 330.1
([M + H].sup.+). i82 ##STR00136## ##STR00137## .sup.1H NMR (400
MHz, (CD.sub.3).sub.2SO): .delta. 4.67-4.53 (m, 1 H), 4.45-4.34 (m,
2 H), 4.31-4.09 (m, 1 H), 3.88 (m, 1 H), 3.68 (m, 1 H), 3.55 (m, 3
H), 3.38 (m, 1 H), 3.13 (m, 1 H), 2.55 (m, 2 H), 1.20 (d,
.sup.3J.sub.H,H = 6.9 Hz, 3 H), 1.19 (d, .sup.3J.sub.H,H = 6.9 Hz,
6 H). MS (MALDI): m/z = 328.3 ([M + H].sup.+). i85 ##STR00138##
##STR00139## .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.
4.53 (m, 1 H), 4.22 (m, 3 H), 4.11- 4.08 (m, 2 H), 3.88 (m, 1 H),
3.66 (m, 3 H), 3.54 (m, 1 H), 3.36 (m, 1 H), 3.18 (m, 1 H), 1.33
(m, 6 H), 1.22 (d, .sup.3J.sub.H,H = 6.9 Hz, 3 H) MS (MALDI): m/z =
328.2 ([M + H].sup.+) i86 ##STR00140## ##STR00141## .sup.1H NMR
(400 MHz, (CD.sub.3).sub.2SO): .delta. 4.55 (m, 1 H), 4.22-4.07 (m,
5 H), 3.88 (m, 1 H), 3.70- 3.63 (m, 3 H), 3.54 (m, 1 H), 3.38 (m, 1
H), 3.19 (m, 1 H), 1.33 (m, 6 H), 1.21 (d, .sup.3J.sub.H,H = 6.9
Hz, 3 H) MS (MALDI): m/z = 328.5 ([M + H].sup.+). i92 ##STR00142##
##STR00143## .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.
4.54-4.15 (m, 4 H), 3.86 (m, 2 H), 3.77 (m, 1 H), 3.66 (m, 2 H),
3.55-3.46 (m, 2 H), 3.38 (m, 1 H), 3.14 (m, 2 H), 1.70 (m, 2 H),
1.22 (d, .sup.3J.sub.H,H = 6.9 Hz, 3 H), 0.86 (m, 3 H) MS (MALDI):
m/z = 328.6 ([M + H].sup.+). i93 ##STR00144## ##STR00145## .sup.1H
NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 4.54-4.15 (m, 4 H), 3.86
(m, 2 H), 3.77 (m, 1 H), 3.66 (m, 2 H), 3.55-3.46 (m, 2 H), 3.38
(m, 1 H), 3.14 (m, 2 H), 1.70 (m, 2 H), 1.22 (d, .sup.3J.sub.H,H =
6.9 Hz, 3 H), 0.86 (m, 3 H) MS (MALDI): m/z = 328.1 ([M +
H].sup.+). i94 ##STR00146## ##STR00147## .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO): .delta. 4.45 (m, 1 H), 4.11 (m, 1 H), 3.87 (m,
1 H), 3.66 (m, 5 H), 3.50 (m, 3 H), 3.38 (m, 1 H), 3.15 (m, 1 H),
2.44 (m, 2 H), 2.21 (m, 2H), 1.70 (m, 2 H), 1.19 (d,
.sup.3J.sub.H,H = 6.9 Hz, 3H) MS (MALDI): m/z = 340.6 ([M +
H].sup.+).
Method 17:
9-(4-chloro-6-(3,3-dimethylmorpholino)-1,3,5-triazin-2-yl)-3,7--
dioxa-9-azabicyclo[3.3.1]nonane (i54)
##STR00148##
[0437] 5 mg, 1.20 mmol, 1.05 eq.) ) in 1,4-dioxane (5 mL) a mL) is
added. The resulting mixture is heated for 2 hours (70.degree. C.).
Then ethyl acetate (20 mL) and saturated aqueous sodium bisulfate
(20 mL) are added. The phases are separated and the organic layer
is washed with saturated aqueous sodium bisulfate (2.times.20 mL).
The organic layer is dried over anhydrous sodium sulfate and the
solvent is removed under reduced pressure. The crude mixture is
purified by automated flash chromatography (SiO.sub.2,
cyclohexane/ethyl acetate 2:1 to 0:1) to afford the title compound
i54 as a colorless solid (178 mg, 44%). .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO): .delta.4.32 (m, 2H), 4.05-3.98 (m, 4H), 3.77
(m, 4H), 3.71 (m, 4H), 3.44 (m, 2H), 1.41 (s, 6H). MS (MALDI):
m/z=356.3 ([M+H].sup.+).
[0438] Method 17 is also used for the preparation of the following
intermediate compounds i55 to i64.
TABLE-US-00008 Reagent Structure NMR MS i55 ##STR00149##
##STR00150## .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.
4.36 (m, 2 H), 3.77-3.74 (m, 6 H), 3.55 (m, 2 H), 3.44 (m, 2 H),
1.44 (s, 6 H), 1.26 (d, .sup.3J.sub.H,H = 6.9 Hz, 6 H). MS (MALDI):
m/z = 342.9 ([M + H].sup.+). i56 ##STR00151## ##STR00152## .sup.1H
NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 4.52 (m, 1 H), 4.20 (m,
1 H), 3.90 (m, 2 H), 3.77 (m, 4 H), 3.65 (m, 1 H), 3.51-3.41 (m, 5
H), 3.28 (s, 3 H), 3.12 (m, 1 H), 1.44 (s, 3 H), 1.43 (s, 3 H). i57
##STR00153## ##STR00154## .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO): .delta. 4.98 (m, 1 H), 4.35 (m, 1 H), 4.18 (m,
1 H), 4.00 (m, 1 H), 3.87 (m, 1 H), 3.81-3.65 (m, 5 H), 3.51-3.35
(m, 5 H), 3.21-3.04 (m, 1 H), 1.44 (s, 3 H), 1.45 (s, 3 H). MS
(MALDI): m/z = 344.2 ([M + H].sup.+). i58 ##STR00155## ##STR00156##
.sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 3.77 (m, 4 H),
3.65 (m, 4 H), 3.44 (m, 2 H), 2.56 (m, 4 H), 1.64 (m, 1 H), 1.44
(s, 6 H), 0.44 (m, 2 H), 0.35 (m, 2 H). MS (MALDI): m/z = 353.0 ([M
+ H].sup.+). 159 ##STR00157## ##STR00158## .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO): .delta. 3.76 (m, 4 H), 3.68 (m, 4 H),
3.47-3.44 (m, 4 H), 3.24 (m, 3 H), 2.52- 2.45 (m, 6 H), 1.44 (s, 6
H). MS (MALDI): m/z = 371.1 ([M + H].sup.+).
Method 18: 4-(difluoromethyl)pyridin-2-amine (i65)
##STR00159##
[0440] Palladium acetate (275 mg, 1.22 mmol, 0.05 eq.) and
2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl
(Sigma-Aldrich, product number 638064, 1.17 g, 2.45 mmol, 0.10 eq.)
are dissolved in 1,4-dioxane (10 mL) under nitrogen atmosphere, and
the resulting mixture is allowed to stir at room temperature for 45
minutes. This solution is then added to a mixture of
tert-butylcarbamate (Sigma, product number 167398, 4.30 g, 36.7
mmol, 1.5 eq.), Cs.sub.2CO.sub.3 (15.9 g, 48.8 mmol, 2.0 eq.) and
2-chloro-4-difluoromethyl-pyridine (Manchester Organics, product
number U15343, 4.00 g, 24.5 mmol, 1.0 eq.) in 1,4-dioxane (80 mL)
under nitrogen atmosphere. The resulting reaction mixture is then
heated at 90.degree. C. for 3 hours, during which it turned
brownish. After this time, the mixture is allowed to cool to room
temperature. It is then diluted with ethyl acetate, washed with an
aqueous saturated solution of ammonium chloride (2.times.30 mL) and
deionized water. The organic layer is dried over anhydrous sodium
sulfate, filtered and the solvent is evaporated under reduced
pressure. The brownish residue is mixed with 4 M HCl in dioxane (50
mL, excess) and methanol (20 mL), and then heated at 80.degree. C.
for 45 minutes. Deionized water is added and the aqueous layer is
washed with ethyl acetate (3.times.). The aqueous layer is then
basified to pH=9, with solid sodium hydroxide. The aqueous layer is
extracted with ethyl acetate (3.times.). The combined organic layer
is dried over anhydrous sodium sulfate, filtered and concentrated
to dryness under reduced pressure. The desired product i65 is
obtained as a colorless solid, which is used in the next step
without further purification (98% yield). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta.8.16 (d, J.sub.H,H=5.2 Hz, 1H), 6.74 (d,
J.sub.H,H=4.8 Hz, 1H), 6.59 (s, 1H), 6.51 (t, .sup.2J.sub.H,F=56
Hz, 1H), 4.61 (br s, 2H); .sup.19F NMR (376 MHz, CDCl.sub.3):
.delta.-116.0 (s, 2 F).
Method 19: 5-bromo-4-(difluoromethyl)pyridin-2-amine (i66)
##STR00160##
[0442] To a solution of compound i65 (3.00 g, 20.8 mmol, 1.0 eq.)
in tetrahydrofuran (60 mL) is added N-bromosuccinimide (3.89 g,
21.9 mmol, 1.05 eq.) at 0.degree. C. in an ice bath. The resulting
mixture is stirred overnight, while it is allowed to warm up to
room temperature. Ethyl acetate is added and the organic layer is
washed with aqueous sodium carbonate (8%). The organic layer is
then separated and acidified with an aqueous 3 M HCl-solution. The
aqueous layer is washed with ethyl acetate (3.times.50 mL) and then
basified to pH=10, with solid sodium hydroxide. The aqueous layer
is extracted with ethyl acetate (3.times.50 mL). The combined
organic layer is dried over anhydrous sodium sulfate, filtered and
concentrated to dryness under reduced pressure. The desired product
i66 is obtained as a brownish solid, which is used in the next step
without further purification (79% yield). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta.8.20 (s, 1H), 6.75 (s, 1H), 6.71 (t,
.sup.2J.sub.H,F=54 Hz, 1H); 4.62 (br s, 2H); .sup.19F NMR (376 MHz,
CDCl.sub.3): .delta.-118.9 (s, 2 F).
Method 20:
N'-(5-bromo-4-(difluoromethyl)pyridin-2-yl)-N,N-dimethylformimi-
damide (i67)
##STR00161##
[0444] To a solution of compound i66 (3.68 g, 16.5 mmol, 1.0 eq.)
in tetrahydrofuran (50 mL) is added N,N-dimethylformamide dimethyl
acetal (Manchester Organics, product number 005030, 3.30 mL, 24.8
mmol, 1.5 eq.) and the resulting mixture is stirred at 60.degree.
C. for 3 hours. The mixture is allowed to cool to room temperature
and the solvent is evaporated under reduced pressure. The crude
product is purified by column chromatography on silica gel
(cyclohexane/ethyl acetate 1:1) to afford the desired product i67
as a yellowish solid (82% yield). .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta.8.43 (s, 1H), 8.34 (br s, 1H), 7.17 (s, 1H),
6.73 (t, .sup.2J.sub.H,F=54 Hz, 1H), 3.12 (s, 3H), 3.10 (s, 3H);
.sup.19F NMR (376 MHz, CDCl.sub.3): .delta.-118.6 (s, 2 F); MS
(MALDI): m/z=278.5 ([M+H].sup.+).
Method 21:
N'-(4-(difluoromethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborola-
n-2-yl)pyridin-2-yl)-N,N-dimethylformimidamide (i68)
##STR00162##
[0446] To a 2 M solution of isopropylmagnesium chloride (Sigma,
product number 230111, 3.10 mL, 6.20 mmol, 1.15 eq.) in
tetrahydrofuran (6 mL) is slowly added a solution of compound i67
(1.50 g, 5.39 mmol, 1.0 eq.) in tetrahydrofuran (5 mL) at 0.degree.
C. The resulting brownish mixture is stirred at 0.degree. C. for 45
minutes and then at room temperature for 15 minutes. After this
time, TLC monitoring (cyclohexane/ethyl acetate 1:1) showed
complete consumption of starting material.
2-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (Manchester
Organics, product number W23343, 1.43 mL, 7.00 mmol, 1.3 eq.) is
added and the mixture is heated at 60.degree. C. for 3 hours. The
mixture is then placed in an Erlenmeyer flask, cooled to 0.degree.
C. with an ice bath and quenched with a 15% aqueous solution of
ammonium chloride. The layers are separated and the aqueous layer
is extracted with ethyl acetate (3.times.40 mL). The combined
organic layers are dried over anhydrous sodium sulfate, filtered
and the solvent is evaporated under reduced pressure. Heptane is
added and the organic layer is washed with a saturated aqueous
solution of sodium bicarbonate, dried over anhydrous sodium
sulfate, filtered and then concentrated to dryness under reduced
pressure. The desired product i68 is obtained as a brownish oil,
which is used in the next step without further purification (94%
yield). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.8.66 (s, 1H),
8.51 (s, 1H), 7.34-7.04 (m, 2H), 3.12 (s, 3H), 3.12 (s, 3H), 1.34
(s, 12H); .sup.19F NMR (376 MHz, CDCl.sub.3): .delta.-115.6 (s, 2
F); MS (MALDI): m/z=326.0 ([M+H].sup.+).
Method 22: 4-(difluoromethyl)pyrimidin-2-amine (i69)
##STR00163##
[0448] To a solution of ethyl vinyl ether (4.00 mL, 41.8 mmol, 1.0
eq.) in a mixture of pyridine (4.10 mL, 50.7 mmol, 1.2 eq.) and
dichloromethane (40 mL), is added dropwise a solution of
2,2-difluoroacetic anhydride (Manchester Organics, (product number
L24754, 5.90 mL, 50.1 mmol, 1.2 eq.) in dichloromethane (5 mL) at
-70.degree. C. in a dry ice/isopropanol bath. The resulting
solution is allowed to warm up to room temperature overnight. The
mixture is then washed with deionized water, dried over anhydrous
sodium sulfate, filtered and the solvent is evaporated under
reduced pressure to afford an orange oil.
[0449] At the same time, a suspension of guanidine.HCl (Sigma,
product number 50940, 4.80 g, 50.2 mmol, 1.2 eq.) in ethanol (20
mL) is stirred at room temperature for 1 hour. To this solution are
added sodium hydroxide pellets (2.00 g, 50.0 mmol, 1.2 eq.) in one
portion. The resulting suspension is stirred at room temperature
overnight.
[0450] The orange oil is diluted with dichloromethane (20 mL) and
added dropwise over 1 hour to the ethanol suspension. The resulting
suspension is stirred at room temperature for 2 hours.
Dichloromethane is evaporated under reduced pressure. Deionized
water (25 mL) is added to the residue. The resulting mixture is
stirred vigorously for 2 hours and is then allowed to stand at room
temperature overnight. The formed solid is filtered off, washed
with deionized water (2.times.) and heptane (1.times.) and then
dried in vacuo. The desired product i69 is obtained as a colorless
solid (65% yield). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.8.43
(d, J.sub.H,H=4.8 Hz, 1H), 7.02 (br s, 2H), 6.76 (d,
.sup.2J.sub.H,H=5.2 Hz, 1H), 6.67 (t, .sup.2J.sub.H,F=55 Hz, 1H);
.sup.19F NMR (376 MHz, CDCl.sub.3): .delta.-120.5 (s, 2 F).
Method 23: 5-bromo-4-(difluoromethyl)pyrimidin-2-amine (i70)
##STR00164##
[0452] To a solution of compound i69 (3.00 g, 20.7 mmol, 1.0 eq.)
in tetrahydrofuran (90 mL) is added N-bromosuccinimide (3.86 g,
21.7 mmol, 1.0 eq.) portionwise at 0.degree. C. The reaction
mixture is allowed to warm up to room temperature overnight. After
this time, the solvent is evaporated under reduced pressure. The
residue is taken up in ethyl acetate (200 mL), washed with an
aqueous saturated solution of sodium carbonate (4.times.), dried
over anhydrous sodium sulfate, filtered and then concentrated to
dryness under reduced pressure. The desired product i70 is obtained
as a yellowish solid, which is used in the next step without
further purification (98% yield). .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO): .delta.8.50 (s, 1H), 7.30 (br s, 2H), 6.87 (t,
.sup.2J.sub.H,F=53 Hz, 1H); .sup.19F NMR (376 MHz,
(CD.sub.3).sub.2SO):8-121.4 (s, 2 F).
Method 24: N-tert-butyl
carboxylate-N-(5-bromo-4-(difluoromethyl)pyrimidin-2-yl)-carbamate
(i71)
##STR00165##
[0454] Compound i70 (4.35 g, 19.4 mmol, 1.0 eq.) and
4-(dimethylamino)pyridine (480 mg, 3.92 mmol, 0.20 eq.) are
dissolved in tetrahydrofuran (50 mL). N,N-Diisopropylethylamine
(7.50 mL, 42.1 mmol, 2.2 eq.) and di-tert-butyl dicarbonate (9.33
g, 42.7 mmol, 2.2 eq.) are then added at 0.degree. C. and the
resulting solution is allowed to warm up to room temperature
overnight. The solvent is evaporated under reduced pressure. The
crude product is purified by column chromatography on silica gel
(cyclohexane/ethyl acetate 9:1.fwdarw.4:1) to afford the desired
product i71 as a colorless solid (85% yield).
[0455] .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.8.92 (s, 1H), 6.73
(t, .sup.2J.sub.H,F=53 Hz, 1H), 1.47 (s, 18H); .sup.19F NMR (376
MHz, CDCl.sub.3): .delta.-120.4 (s, 2 F).
##STR00166##
[0456] Substituted monochloro-triazine or substituted
monochloro-pyrimidine (1.0 eq.), compound i68 (1.1 eq.), potassium
phosphate tribasic (2.0 eq.) and
chloro(2-dicyclohexyl-phosphino-2',4',6'-triisopropyl-1,1'-biphenyl)
[2-(2'-amino-1,1'-biphenyl)]-palladium(II) (Sigma-Aldrich, product
number 741825, 0.05 eq.) are charged in a flask. Under nitrogen
atmosphere, 1,4-dioxane (30 volumes) and deionized water (1.5
volume) are added and the resulting mixture is then directly placed
into an oil bath pre-heated at 95.degree. C. The reaction mixture
is stirred at this temperature for 2 hours. A 5 M aqueous
HCl-solution (20 eq.) is added. The resulting mixture is heated to
60.degree. C. overnight. The pH of the resulting mixture is
adjusted to 8-9 by addition of a 2 M aqueous solution of sodium
hydroxide, the mixture is then extracted with ethyl acetate
(3.times.20 volumes). The combined organic layers are dried over
anhydrous sodium sulfate, filtered and the solvent is evaporated
under reduced pressure. Purification by flash chromatography
affords the desired products of structure (I).
##STR00167##
[0457] Compound i71 (1.0 eq.),
4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane)
(Manchester Organics, product number M23170, 1.5 eq.), potassium
acetate (3.0 eq.) and
[1,1'-bis(diphenylphosphino)-ferrocene]-dichloropalladium(II)
(Sigma-Aldrich, product number 697230, 0.099 eq.) are dissolved in
1,4-dioxane (12.5 volumes) under nitrogen atmosphere. The resulting
mixture is heated at 100.degree. C. for 15 minutes (solution turned
black). TLC monitoring (cyclohexane/ethyl acetate 3:1) is used to
show complete consumption of starting material.
[0458] To the resulting mixture, substituted chloro-triazine or
substituted chloropyrimidine (1.1 eq.), an aqueous solution of
potassium carbonate (2 M, 3.0 eq.) and a previously mixed solution
of triphenylphosphine (0.12 eq.) and palladium acetate (0.04 eq.)
in tetrahydrofuran (100 volumes) are added. The resulting mixture
is heated at 60.degree. C. for 2 hours and subsequently allowed to
cool to room temperature.
[0459] A 5 M aqueous HCl-solution (20 eq.) is added. The resulting
mixture is heated to 60.degree. C. overnight. The pH of the
resulting mixture is adjusted to 8-9 by addition of a 2 M aqueous
solution of sodium hydroxide, the mixture is then extracted with
ethyl acetate (3.times.20 volumes). The combined organic layers are
dried over anhydrous sodium sulfate, filtered and the solvent is
evaporated under reduced pressure. Purification by flash
chromatography affords the desired products.
Method 27: tert-butyl
N-tert-butoxycarbonyl-N-(5-(4-chloro-6-morpholino-1,3,5-triazin-2-yl)-4-(-
difluoromethyl)pyrimidin-2-yl)carbamate (i74)
##STR00168##
[0461] Intermediate i71 (2.00 g, 4.71 mmol, 1.0 eq.),
bis(pinacolato)diboron (1.80 g, 7.09 mmol, 1.5 eq.), KOAc (1.60 g,
16.3 mmol, 3.4 eq.) and
[1,1'-bis(diphenylphosphino)ferrocene]-dichloropalladium(II) (350
mg, 478 .mu.mol, 0.10 eq.) are mixed in 1,4-dioxane under nitrogen
atmosphere and heated at 95.degree. C. for 45 minutes. A
pre-catalyst solution of palladium(II) acetate (43.0 mg, 192
.mu.mol, 0.04 eq.) and triphenylphosphine 148 mg, 564 .mu.mol, 0.12
eq.) in tetrahydrofuran (2 mL) is also prepared and stirred at room
temperature for 1 hour. This solution is then added to the cooled
above solution at room temperature, followed by the addition of
4-(4,6-dichloro-1,3,5-triazin-2-yl)morpholine ill (1.65 g, 7.05
mmol, 1.5 eq.) and aqueous K.sub.2CO.sub.3-solution (2.4 M, 5.90
mL, 14.2 mmol, 3.0 eq.). The resulting mixture is heated at
55.degree. C. overnight. After this time, the mixture is poured
onto an aqueous NH.sub.4Cl-solution (15%) and extracted with ethyl
acetate (3.times.). The combined organic layer is dried over
anhydrous sodium sulfate, filtered and concentrated under reduced
pressure. Purification by column chromatography on silica gel
(cyclohexane/ethyl acetate 1:0 to 4:1) gives product i74 as a
colorless solid (36% yield).
[0462] .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.57 (s, 1H), 7.55
(t, .sup.2J.sub.H,F=54 Hz, 1H), 3.99-3.91 (m, 4H), 3.84-3.76 (m,
4H), 1.49 (s, 18H); .sup.19F NMR (376 MHz, CDCl.sub.3):
.delta.-121.0 (s, 2 F).
Method 32: (E)-4-ethoxy-1,1-difluoro-but-3-en-2-one (i83)
##STR00169##
[0464] To a cooled (-70.degree. C.) solution of pyridine (61.5 mL,
760.5 mmol, 1.2 eq) in dichloromethane (500 mL) is added ethyl
vinyl ether (60 mL, 626.5 mmol, 1 eq), followed by a solution of
difluoroacetic anhydride (88.5 mL, 760.5 mmol, 1.2 eq) in
dichloromethane (75 mL). Then the mixture is slowly warmed to room
temperature overnight. The mixture is transferred into a separating
funnel and the organic layer is washed with water (6.times.800 mL)
until the pH of the aqueous layer becomes neutral. The organic
layer is dried over sodium sulfate and solvent is removed under
reduced pressure to afford the desired product i83 as an orange oil
(76.7 g, 81%). .sup.1'H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.
7.92 (d, .sup.3J.sub.H,H=12.5 Hz, 1H), 6.34 (t,
.sup.2J.sub.H,F=53.6 Hz, 1H), 5.87 (d, .sup.3J.sub.H,H=12.5 Hz,
1H), 4.14 (q, .sup.3J.sub.H,H=7.1 Hz, 2H), 1.28 (t,
.sup.3J.sub.H,H=7.1 Hz, 3H); .sup.19F NMR (400 MHz,
(CD.sub.3).sub.2SO): 6-127.39 (s, 2F).
Method 33:
(E)-3-(difluoromethyl)-5-ethoxy-3-hydroxy-pent-4-enenitrile
(i84)
##STR00170##
[0466] To a cooled (-70.degree. C.) solution of n-butyl lithium
2.5M (102.9 mL, 256.7 mmol, 1 eq) in tetrahydrofuran (435 mL) is
added acetonitrile (13.4 mL, 256.7 mmol, 1 eq). A white suspension
is formed and is stirred at -70.degree. C. for 1.5 hours. A
solution of (E)-4-ethoxy-1,1-difluoro-but-3-en-2-one (i83) (38.5 g,
256.7 mmol, 1 eq) in tetrahydrofuran (65 mL) is added to the white
suspension (mixture becomes an orange solution). The mixture is
stirred at -70.degree. C. for 1 hour and slowly warmed to room
temperature. Water (400 mL) is added. Then ethyl acetate (600 mL)
is added. Layers are separated and aqueous layer is extracted with
ethyl acetate (3.times.600 mL). Combined organic layers are dried
over sodium sulfate and solvent is evaporated under reduced
pressure. Filtration on a short pad of silica gel, using a mixture
of cyclohexane/ethyl acetate (3:1) as eluent, gives the desired
product i84 as a dark orange oil (43.4 g, 88%)..sup.1H NMR (400
MHz, (CD.sub.3).sub.2SO): .delta. 6.66 (d, .sup.3J.sub.H,H=12.8 Hz,
1H), 6.20 (s, 1H), 5.79 (t, .sup.2J.sub.H,F=55.8 Hz, 1H), 4.75 (d,
.sup.3J.sub.H,H=12.8 Hz, 1H), 3.74 (q, .sup.3J.sub.H,H=7.0 Hz, 2H),
2.88 (d, .sup.3J.sub.H,H=16.8 Hz, 1H), 2.81 (d,
.sup.3J.sub.H,H=16.8 Hz, 1H), 1.21 (t, .sup.3J.sub.H,H=7.0 Hz, 3H);
.sup.19F NMR (400 MHz, (CD.sub.3).sub.2SO): 6-129.32 (d,
.sup.2J.sub.F,F=311.2 Hz, 1F), -130.05 (d, .sup.2J.sub.F,F=311.2
Hz, 1F).
Method 34: 4-(difluoromethyl)pyridin-2-amine (i65)
##STR00171##
[0468] To a solution of
(E)-3-(difluoromethyl)-5-ethoxy-3-hydroxy-pent-4-enenitrile (i84)
(8.1 g, 42.4 mmol, 1 eq) in acetic acid (80 mL) is added
O-methylhydroxylamine hydrochloride (Fluorochem, product number
078603) (10.6 g, 127.2 mmol, 3 eq). Mixture is stirred at
50.degree. C. for 7 hours. Then reaction mixture is cooled down to
room temperature and hydrobromic acid in acetic acid (33%) (14.2
mL, 84.8 mmol, 2 eq) is added. Reaction mixture is stirred at
90.degree. C. overnight. Reaction mixture is degassed and placed
under nitrogen. Reaction mixture is maintained at room temperature
with a water bath with ice while zinc powder (8.12 g, 127.2 mmol, 3
eq) is added portionwise. Reaction mixture is stirred 3 h at room
temperature. Mixture is filtered over a short pad of celite and the
cake is washed with ethyl acetate. Then the major part of the
solvent is removed under reduced pressure. 60 mL of aqueous
ammonium hydroxide (28%) is added. Aqueous layer is extracted with
dichloromethane (3.times.150 mL). Combined organic layers are dried
over sodium sulfate. Compound i65 is recrystallized from
dichloromethane and heptane as anti-solvent (solvent switch at the
rotavap). Compound i65 is collected, as a light yellow solid, by
filtration (5.12 g, 84%).
Method 35:
9-[4-chloro-6-(3-oxa-9-azabicyclo[3.3.1]nonan-9-yl)-1,3,5-triaz-
in-2-yl]-3,7-dioxa-9-azabicyclo[3.3.1]nonane (i89)
##STR00172##
[0470] To a solution of 3-oxa-9-azabicyclo[3.3.1]nonane
hydrochloride (176 mg, 1.20 mmol, 1.05 eq.) and
N,N-diisopropylethylamine (0.42 mL, 2.40 mmol, 2.1 eq.) in
1,4-dioxane (5 mL) a solution of i88 (300 mg, 1.14 mmol, 1 eq.) in
1,4-dioxane (1 mL) is added. The resulting mixture is heated for 3
hours (75.degree. C.). Then, ethyl acetate (20 mL) and saturated
aqueous sodium bisulfate (20 mL) are added. The phases are
separated and the organic layer is washed with saturated aqueous
sodium bisulfate (2.times.20 mL). The organic layer is dried over
anhydrous sodium sulfate and the solvent is removed under reduced
pressure. The crude mixture is purified by automated flash
chromatography (SiO.sub.2, cyclohexane/ethyl acetate 2:1 to 0:1) to
afford the title compound i89 as a colorless solid (297 mg, 75%).
.sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.4.58 (m, 1H),
4.44 (m, 1H), 4.40 (m, 1H), 4.32 (m, 1H), 4.00-3.97 (m, 4H),
3.94-3.90 (m, 2H), 3.72-3.64 (m, 6H), 2.46 (m, 1H), 1.90-1.70 (m,
4H), 1.53 (m, 1H). MS (MALDI): m/z=368.0 ([M+H].sup.+).
Preparation of Compounds of the Invention
Compound 1:
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyridin-2-amine
(1)
##STR00173##
[0472] According to general procedure 1, compound 1 is obtained
from starting materials i2 and i68 in 73% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.02 (s, 1H), 7.65
(t, .sup.2J.sub.H,F=55 Hz, 1H), 6.83 (s, 1H), 4.85 (br s, 2H),
3.89-3.79 (m, 8H), 3.77-3.72 (m, 8H); .sup.19F NMR (376 MHz,
CDCl.sub.3): .delta.-115.9 (s, 2 F); MS (MALDI): m/z=393.9
([M+H].sup.+).
Compound 2:
4-(difluoromethyl)-5-(4,6-dimorpholino-1,3,5-triazin-2-yl)pyrimidin-2-ami-
ne (2)
##STR00174##
[0474] According to general procedure 2, compound 2 is obtained
from starting materials i2 and i71 in 74% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.20 (s, 1H), 7.62
(t, .sup.2J.sub.H,F=54 Hz, 1H), 5.97 (br s, 2H), 3.91-3.68 (m,
16H); .sup.19F NMR (376 MHz, CDCl.sub.3): .delta.-121.5 (s, 2 F);
MS (MALDI): m/z=395.2 ([M+H].sup.+).
Compound 3:
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(3-oxa-8-azabicyclo[3.2.1]oc-
tan-8-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine
(3)
##STR00175##
[0476] According to general procedure 1, compound 3 is obtained
from starting materials i1 and i68 in 75% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.04 (s, 1H), 7.71
(t, .sup.2J.sub.H,F=55 Hz, 1H), 6.83 (s, 1H), 4.89 (br s, 2H),
4.71-4.64 (m, 4H), 3.79-3.76 (m, 4H), 3.67-3.62 (m, 4H), 2.09-1.98
(m, 8H); .sup.19F NMR (376 MHz, CDCl.sub.3): .delta.-115.4-(-117.3)
(m, 2 F); MS (MALDI): m/z=446.3 ([M+H].sup.+).
Compound 4:
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1,3,5-triazin-2-y-
l)-4-(difluoromethyl)pyridin-2-amine (4)
##STR00176##
[0478] According to general procedure 1, compound 4 is obtained
from starting materials i12 and i68 in 57% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.03 (s, 1H), 7.68
(m, 1H), 6.83 (s, 1H), 4.94 (br s, 2H), 4.70-4.65 (m, 2H),
3.93-3.57 (m, 12H), 2.14-1.92 (m, 4H); .sup.19F NMR (376 MHz,
CDCl.sub.3): .delta.-116.0-(-116.2) (m, 2 F); MS (MALDI): m/z=420.6
([M+H].sup.+).
Compound 5:
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1,3,5-triazin-2-y-
l)-4-(difluoromethyl)pyrimidin-2-amine (5)
##STR00177##
[0480] According to general procedure 2, compound 5 is obtained
from starting materials i71 and i12 in 50% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.23 (s, 1H), 7.65
(t, 2J.sub.H,F=54 Hz, 1H), 5.66 (br s, 2H), 4.68 (m, 2H), 3.90-3.61
(m, 12H), 2.13-1.92 (4H); .sup.19F NMR (376 MHz, CDCl.sub.3):
.delta.-120.4-(-121.5) (m, 2 F); MS (MALDI): m/z=420.9
([M+H].sup.+).
Compound 6:
5-(4,6-bis((S)-3-methylmorpholino)-1,3,5-triazin-2-yl)-4-(difluoromethyl)-
pyridin-2-amine (6)
##STR00178##
[0482] According to general procedure 1, compound 6 is obtained
from starting materials i3 and i68 in 79% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.8.87 (s, 1H), 7.70
(t, .sup.2J.sub.H,F=55 Hz, 1H), 6.86 (s, 1H), 5.48 (br s, 2H),
4.73-4.72 (m, 2H), 4.41-4.38 (m, 2H), 3.98 (dd, J.sub.H,H=11.6, 3.8
Hz, 2H), 3.78 (d, J.sub.H,H=12 Hz, 2H), 3.67 (dd, J.sub.H,H=12, 3.2
Hz, 2H), 3.52 (td, J.sub.H,H=12, 3.0 Hz, 2H), 3.27 (td,
J.sub.H,H=13, 3.8 Hz, 2H), 1.33 (d, .sup.3J.sub.H,H=6.8 Hz, 6H);
.sup.19F NMR (376 MHz, CDCl.sub.3): .delta.-115.4-(-116.2) (m, 2
F); MS (MALDI): m/z=421.9 ([M+H].sup.+).
Compound 7:
5-(4,6-bis((S)-3-methylmorpholino)-1,3,5-triazin-2-yl)-4-(difluoromethyl)-
pyrimidin-2-amine (7)
##STR00179##
[0484] According to general procedure 2, compound 7 is obtained
from starting materials i71 and i3 in 52% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.24 (s, 1H), 7.66
(t, .sup.2J.sub.H,F=54 Hz, 1H), 5.77 (br s, 2H), 4.73 (br s, 2H),
4.45-4.32 (m, 2H), 3.98 (dd, J.sub.H,H=12, 3.6 Hz, 2H), 3.78 (d,
J.sub.H,H=12 Hz, 2H), 3.67 (dd, J.sub.H,H=11, 2.8 Hz, 2H), 3.52
(td, J.sub.H,H=12, 2.8 Hz, 2H), 3.27 (td, J.sub.H,H=13, 3.2 Hz,
2H), 1.33 (d, .sup.3J.sub.H,H=6.8 Hz, 6H); .sup.19F NMR (376 MHz,
CDCl.sub.3): .delta.-120.5-(-122.7) (m, 2 F); MS (MALDI): m/z=423.3
([M+H].sup.+).
Compound 8:
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyridin-2-amine (8)
##STR00180##
[0486] According to general procedure 1, compound 8 is obtained
from starting materials i13 and i68 in 47% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.03 (s, 1H), 7.70
(t, .sup.2J.sub.H,F=55 Hz, 1H), 6.84 (s, 1H), 4.78 (br s, 2H), 4.75
(m, 1H), 4.42-4.38 (m, 1H), 4.00-3.96 (m, 1H), 3.84-3-66 (m, 10H),
3.55-3.50 (m, 1H), 3.30-3.25 (m, 1H), 1.33 (d, 3J.sub.H,H=6.8 Hz,
3H); .sup.19F NMR (376 MHz, CDCl.sub.3): .delta.-116.1-(-115.9) (m,
2 F); MS (MALDI): m/z=408.9 ([M+H].sup.+).
Compound 9:
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triaz-
in-2-yl)pyrimidin-2-amine (9)
##STR00181##
[0488] According to general procedure 2, compound 9 is obtained
from starting materials i71 and i13 in 60% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.24 (s, 1H), 7.66
(t, .sup.2J.sub.H,F=54 Hz, 1H), 5.67 (br s, 2H), 4.74 (m, 1H),
4.41-4.38 (m, 1H), 4.00-3.97 (m, 1H), 3.90-3.72 (m, 9H), 3.68-3.36
(m, 1H), 3.56-3.49 (m, 1H), 3.32-3.25 (m, 1H), 1.33 (d,
3J.sub.H,H=6.9 Hz, 3H); .sup.19F NMR (376 MHz, CDCl.sub.3):
.delta.-121.3-(-121.6) (m, 2 F); MS (MALDI): m/z=409.4
([M+H].sup.+).
Compound 10:
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-((S)-3-methylmorpholino)-1,3-
,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine (10)
##STR00182##
[0490] According to general procedure 1, compound 10 is obtained
from starting materials i18 and i68 in 42% yield as a colorless
solid. 1H NMR (400 MHz, CDCl.sub.3): .delta.9.04 (s, 1H), 7.69 (t,
.sup.2J.sub.H,F=55 Hz, 1H), 6.84 (s, 1H), 4.85 (br s, 2H),
4.71-4.65 (m, 3H), 4.42-4.39 (m, 1H), 3.98-3.95 (m, 1H), 3.79-3.76
(m, 3H), 3.70-3.65 (m, 3H), 3.56-3.53 (m, 1H), 3.30-3.27 (m, 1H),
2.10-1.99 (m, 4H), 1.33 (m, 3H); .sup.19F NMR (376 MHz,
CDCl.sub.3): .delta.-115.9-(-116.2) (m, 2 F); MS (MALDI): m/z=434.2
([M+H]*).
Compound 11:
5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-((S)-3-methylmorpholino)-1,3-
,5-triazin-2-yl)-4-(difluoromethyl)pyrimidin-2-amine (11)
##STR00183##
[0492] According to general procedure 2, compound 11 is obtained
from starting materials i71 and i18 in 46% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.25 (s, 1H), 7.68
(t, .sup.2J.sub.H,F=55 Hz, 1H), 5.81 (br s, 2H), 4.71-4.65 (m, 3H),
4.42-4.38 (m, 1H), 4.00-3.96 (m, 1H), 3.81-3.60 (m, 6H), 3.55-3.50
(m, 1H), 3.31-3.24 (m, 1H), 2.11-2.00 (m, 4H), 1.37-1.28 (m, 3H);
.sup.19F NMR (376 MHz, CDCl.sub.3): .delta.-121.5-(-121.7) (m, 2
F); MS (MALDI): m/z=434.6 ([M+H].sup.+).
Compound 12:
4-(difluoromethyl)-5-(4-morpholino-6-(piperazin-1-yl)-1,3,5-triazin-2-yl)-
pyridin-2-amine (12)
##STR00184##
[0494] According to general procedure 1, compound 12 is obtained
from starting materials i68 and i14 in 86% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.85 (s,
1H), 7.74 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.84 (s, 2H), 6.75 (s,
1H), 3.82-3.70 (m, 8H), 3.69-3.60 (m, 4H), 2.88-2.80 (m, 4H); 19F
NMR (376 MHz, (CD.sub.3).sub.2SO): .delta.-115.4 (s, 2 F); MS
(MALDI): m/z=393.8 ([M+H].sup.+).
Compound 13:
4-(difluoromethyl)-5-(4-morpholino-6-(piperazin-1-yl)-1,3,5-triazin-2-yl)-
pyrimidin-2-amine (13)
##STR00185##
[0496] According to general procedure 2, compound 13 is obtained
from starting materials i71 and i14 in 55% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.23 (s, 1H), 7.64
(t, 2J.sub.H,F=55 Hz, 1H), 5.60 (br s, 2H), 3.83-3.75 (m, 12H),
2.94-2.88 (m, 4H); .sup.19F NMR (376 MHz, CDCl.sub.3):
.delta.-111.4 (s, 2 F); MS (MALDI): m/z=394.1 ([M+H].sup.+).
Compound 14:
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-(piperazin-1-yl)-1,3,5-
-triazin-2-yl)pyridin-2-amine (14)
##STR00186##
[0498] According to general procedure 1, compound 14 is obtained
from starting materials i21 and i68 in 47% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.02 (s, 1H), 7.67
(t, .sup.2J.sub.H,F=56 Hz, 1H), 6.84 (s, 1H), 4.90 (br s, 2H), 4.74
(s, 1H), 4.40 (d, J.sub.H,H=16 Hz, 1H), 3.98 (dd, J.sub.H,H=4.0 Hz,
12 Hz, 1H), 3.91 (m, 4H), 3.78 (d, J.sub.H,H=12 Hz, 1H), 3.68 (dd,
J.sub.H,H=4.0, 12 Hz, 1H), 3.56 (t, J.sub.H,H=4.0 Hz, 1H), 3.26
(dt, J.sub.H,H=4.0, 12 Hz, 1H), 2.99 (t, J.sub.H,H=4.0 Hz, 4H),
1.32 (d, J.sub.H,H=8.0 Hz, 3H);.sup.19F NMR (376 MHz, CDCl.sub.3):
.delta.-115.9 (s, 2 F); MS (MALDI): m/z=407.2 ([M+H].sup.+).
Compound 15:
(S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-(piperazin-1-yl)-1,3,5-
-triazin-2-yl)pyrimidin-2-amine (15)
##STR00187##
[0500] According to general procedure 2, compound 15 is obtained
from starting materials i71 and i21 in 30% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.24 (s, 1H), 7.66
(t, .sup.2J.sub.H,F=56 Hz, 1H), 5.69 (br s, 2H), 4.74 (s, 1H), 4.40
(d, J.sub.H,H=16 Hz, 1H), 4.38 (dd, J.sub.H,H=4.0, 12 Hz, 1H), 3.83
(m, 4H), 3.78 (d, J.sub.H,H=12 Hz, 1H), 3.68 (dd, J.sub.H,H=4.0, 12
Hz, 1H), 3.54 (dt, J.sub.H,H=4.0, 12 Hz, 1H), 3.28 (dt,
J.sub.H,H=4.0, 12 Hz, 1H), 2.92 (t, J.sub.H,H=8.0 Hz, 4H), 1.33 (t,
J.sub.H,H=8.0 Hz, 3H); .sup.19F NMR (376 MHz, CDCl.sub.3):
.delta.-121.4 (s, 2 F); MS (MALDI): m/z=408.7 ([M+H].sup.+).
Compound 16:
4-(difluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridin-2-amine
(16)
##STR00188##
[0502] According to general procedure 1, compound 16 is obtained
from starting materials i22 and i68 in 73% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.8.31 (s, 1H), 7.30
(t, .sup.2J.sub.H,F=55 Hz, 1H), 6.85 (s, 1H), 6.04 (s, 1H), 4.73
(br s, 2H), 3.81-3.72 (m, 12H), 3.65-3.59 (m, 4H); .sup.19F NMR
(376 MHz, CDCl.sub.3): .delta.-115.1 (s, 2 F); MS (MALDI):
m/z=393.3 ([M+H].sup.+).
Compound 17:
4'-(difluoromethyl)-2,6-dimorpholino-[4,5'-bipyrimidin]-2'-amine
(17)
##STR00189##
[0504] According to general procedure 2, compound 17 is obtained
from starting materials i71 and i22 in 7% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.8.60 (s, 1H), 7.11
(t, .sup.2J.sub.H,F=55 Hz, 1H), 6.02 (s, 1H), 5.46 (br s, 2H),
3.80-3.74 (m, 12H), 3.64-3.60 (m, 4H); .sup.19F NMR (376 MHz,
CDCl.sub.3): .delta.-119.5 (s, 2 F); MS (MALDI): m/z=394.3
([M+H].sup.+).
Compound 18:
4-(difluoromethyl)-5-(4,6-dimorpholinopyrimidin-2-yl)pyridin-2-amine
(18)
##STR00190##
[0506] According to general procedure 1, compound 18 is obtained
from starting materials i23 and i68 in 89% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.8.94 (s, 1H), 7.61
(t, .sup.2J.sub.H,F=55 Hz, 1H), 6.83 (s, 1H), 5.50 (s, 1H), 4.74
(br s, 2H), 3.82-3.78 (m, 8H), 3.61-3.57 (m, 8H);.sup.19F NMR (376
MHz, CDCl.sub.3): .delta.-115.4 (s, 2 F); MS (MALDI): m/z=393.3
([M+H].sup.+).
Compound 19:
4'-(difluoromethyl)-4,6-dimorpholino-[2,5'-bipyrimidin]-2'-amine
(19)
##STR00191##
[0508] According to general procedure 2, compound 19 is obtained
from starting materials i71 and i23 in 7% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.16 (s, 1H), 7.58
(t, .sup.2J.sub.H,F=55 Hz, 1H), 5.75 (br s, 2H), 5.50 (s, 1H),
3.82-3.79 (m, 8H), 3.61-3.58 (m, 8H); .sup.19F NMR (376 MHz,
CDCl.sub.3): .delta.-121.1 (s, 2 F); MS (MALDI): m/z=395.3
([M+H].sup.+).
Compound 20:
4-(difluoromethyl)-5-(4-morpholino-6-thiomorpholino-1,3,5-triazin-2-yl)py-
ridin-2-amine (20)
##STR00192##
[0510] According to general procedure 1, compound 20 is obtained
from starting materials i15 and i68 in 77% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.02 (s, 1H), 7.65
(t, .sup.2J.sub.H,F=55 Hz, 1H), 6.84 (s, 1H), 4.83 (br s, 2H),
4.23-4.07 (m, 4H), 3.90-3.79 (m, 4H), 3.79-3.71 (m, 4H), 2.71-2.62
(m, 4H); .sup.19F NMR (376 MHz, CDCl.sub.3): .delta.-116.0 (s, 2
F); MS (MALDI): m/z=410.3 ([M+H].sup.+).
Compound 21:
4-(difluoromethyl)-5-(4-morpholino-6-thiomorpholino-1,3,5-triazin-2-yl)py-
rimidin-2-amine (21)
##STR00193##
[0512] According to general procedure 2, compound 21 is obtained
from starting materials i71 and i15 in 70% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.21 (s, 1H), 7.60
(t, .sup.2J.sub.H,F=54 Hz, 1H), 5.90 (br s, 2H), 4.22-4.06 (m, 4H),
3.91-3.78 (m, 4H), 3.78-3.71 (m, 4H), 2.71-2.62 (m, 4H); .sup.19F
NMR (376 MHz, CDCl.sub.3): .delta.-120.5-(-121.5) (m, 2 F); MS
(MALDI): m/z=411.2 ([M+H].sup.+).
Compound 22:
5-(6-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-2-(3-oxa-8-azabicyclo[3.2.1]oc-
tan-8-yl)pyrimidin-4-yl)-4-(difluoromethyl)pyridin-2-amine (22)
##STR00194##
[0514] According to general procedure 1, compound 22 is obtained
from starting materials i24 and i68 in 61% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.34 (s,
1H), 7.55 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.76 (s, 1H), 6.60 (br s,
2H), 6.36 (s, 1H), 4.64-4.47 (m, 4H), 3.67-3.49 (m, 4H), 3.56-3.49
(m, 4H), 1.98-1.79 (m, 8H); .sup.19F NMR (376 MHz,
(CD.sub.3).sub.2SO): .delta.-114.9-(-115.2) (m, 2 F); MS (MALDI):
m/z=445.3 ([M+H].sup.+).
Compound 23:
5-(2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-morpholinopyrimidin-4-yl)-4--
(difluoromethyl)pyridin-2-amine (23)
##STR00195##
[0516] According to general procedure 1, compound 23 is obtained
from starting materials i29 and i68 in 54% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.8.30 (s, 1H), 7.30
(t, .sup.2J.sub.H,F=55 Hz, 1H), 6.84 (s, 1H), 6.04 (s, 1H), 4.85
(br s, 2H), 4.62 (br s, 2H), 3.82-3.74 (m, 6H), 3.65-3.56 (m, 6H),
2.09-2.00 (m, 2H), 2.00-1.91 (m, 2H); .sup.19F NMR (376 MHz,
CDCl.sub.3): .delta.-115.2-(-116.2) (m, 2 F); MS (MALDI): m/z=419.0
([M+H].sup.+).
Compound 24:
2-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-4'-(difluoromethyl)-6-morpholino--
[4,5'-bipyrimidin]-2'-amine (24)
##STR00196##
[0518] According to general procedure 2, compound 24 is obtained
from starting materials i29 and i71 in 72% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.71 (s,
1H), 7.35 (s, 2H), 7.32 (t, .sup.2J.sub.H,F=54 Hz, 1H), 6.45 (s,
1H), 4.54 (br s, 2H), 3.71-3.50 (m, 12H), 1.95-1.78 (m, 4H);
.sup.19F NMR (376 MHz, (CD.sub.3).sub.2SO): .delta.-119.2 (s, 2 F);
MS (MALDI): m/z=420.6 ([M+H].sup.+).
Compound 25:
5-(2,6-bis((S)-3-methylmorpholino)pyrimidin-4-yl)-4-(difluoromethyl)pyrid-
in-2-amine (25)
##STR00197##
[0520] According to general procedure 1, compound 25 is obtained
from starting materials i25 and i68 in 57% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.31 (s,
1H), 7.52 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.76 (s, 1H), 6.59 (br s,
2H), 6.30 (s, 1H), 4.60-4.50 (m, 1H), 4.44-4.33 (m, 1H), 4.24-4.15
(m, 1H), 4.12-4.04 (m, 1H), 3.94-3.83 (m, 2H), 3.74-3.64 (m, 2H),
3.59-3.51 (m, 2H), 3.45-3.35 (m, 2H), 3.14-3.02 (m, 2H), 1.18 (t,
.sup.3J.sub.H,H=7.2 Hz, 6H); .sup.19F NMR (376 MHz,
(CD.sub.3).sub.2SO): .delta.-113.7-(-115.9) (m, 2 F); MS (MALDI):
m/z=421.1 ([M+H].sup.+).
Compound 26:
4'-(difluoromethyl)-2.6-bis((S)-3-methylmorpholino)-[4,5'-bipyrimidin]-2'-
-amine (26)
##STR00198##
[0522] According to general procedure 2, compound 26 is obtained
from starting materials i25 and i71 in 56% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.8.60 (s, 1H), 7.14
(t, .sup.2J.sub.H,F=54 Hz, 1H), 5.98 (s, 1H), 5.48 (br s, 2H),
4.71-4.62 (m, 1H), 4.34-4.23 (m, 2H), 4.08-3.92 (m, 3H), 3.83-3.65
(m, 4H), 3.61-3.49 (m, 2H), 3.25 (dt, .sup.2J.sub.H,H=13 Hz,
.sup.3J.sub.H,H=3.6 Hz, 2H), 1.33-1.27 (m, 6H); .sup.19F NMR (376
MHz, CDCl.sub.3): .delta.-119.5 (s, 1 F), 119.7 (m, 1 F); MS
(MALDI): m/z=422.2 ([M+H].sup.+).
Compound 27:
(S)-4-(difluoromethyl)-5-(6-(3-methylmorpholino)-2-morpholinopyrimidin-4--
yl)pyridin-2-amine (27)
##STR00199##
[0524] According to general procedure 1, compound 27 is obtained
from starting materials i30 and i68 in 74% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.8.31 (s, 1H), 7.30
(t, .sup.2J.sub.H,F=55 Hz, 1H), 6.85 (s, 1H), 6.02 (s, 1H), 4.75
(br s, 2H), 4.35-4.25 (m, 1H), 4.06-3.96 (m, 2H), 3.83-3.69 (m,
10H), 3.58 (dt, .sup.2J.sub.H,H=12 Hz, .sup.3J.sub.H,H=3.2 Hz, 1H),
3.25 (dt, .sup.2J.sub.H,H=13 Hz, .sup.3J.sub.H,H=3.8 Hz, 1H), 1.31
(d, .sup.3J.sub.H,H=6.8 Hz, 3H); .sup.19F NMR (376 MHz,
CDCl.sub.3): .delta.-114.9-(-115.0) (m, 2 F); MS (MALDI): m/z=407.1
([M+H].sup.+).
Compound 28:
(S)-4'-(difluoromethyl)-6-(3-methylmorpholino)-2-morpholino-[4,5'-bipyrim-
idin]-2'-amine (28)
##STR00200##
[0526] According to general procedure 2, compound 28 is obtained
from starting materials i30 and i71 in 53% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.8.60 (s, 1H), 7.13
(t, .sup.2J.sub.H,F=54 Hz, 1H), 6.01 (s, 1H), 5.47 (brs, 2H),
4.71-4.63 (m, 1H), 4.31 (dd, .sup.2J.sub.H,H=14 Hz,
.sup.3J.sub.H,H=2.4 Hz, 1H), 3.97 (dd, .sup.2J.sub.H,H=11 Hz,
.sup.3J.sub.H,H=3.4 Hz, 1H), 3.79 (t, .sup.3J.sub.H,H=4.6 Hz, 4H),
3.72-3.66 (m, 2H), 3.65-3.58 (m, 3H), 3.58-3.50 (m, 2H), 3.30-3.21
(m, 1H), 1.30 (d, .sup.3J.sub.H,H=6.8 Hz, 3H); .sup.19F NMR (376
MHz, CDCl.sub.3): .delta.-119.7 (br s, 2 F); MS (MALDI): m/z=408.9
([M+H].sup.+).
Compound 29:
5-(4-(8-Oxa-3-azabicyclo[3.2.1]octan-3-yl)-6-(8-oxa-3-azabicyclo[3.2.1l]o-
ctan-3-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine
(29)
##STR00201##
[0528] According to general procedure 1, compound 29 is obtained
from starting materials i68 and i81 in 89% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.9.03 (s, 1H), 7.69
(t, .sup.2J.sub.H,F=55 Hz, 1H), 6.83 (s, 1H), 4.85 (br s, 2H),
4.50-4.24 (m, 8H), 3.28-3.12 (m, 4H), 1.94 (br s, 4H), 1.86-1.71
(m, 4H); .sup.19F NMR (376 MHz, CDCl.sub.3): .delta.-115.1-(-117.2)
(m, 2 F); MS (MALDI): m/z=446.3 ([M+H].sup.+).
Compound 30:
5-[4,6-bis(2,2-dimethylmorpholin-4-yl)-1,3,5-triazin-2-yl]-4-(difluoromet-
hyl)pyridin-2-amine (30)
##STR00202##
[0530] According to general procedure 1, compound 30 is obtained
from starting materials i68 and i80 in 63% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.86 (s,
1H), 7.71 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.84 (br s, 2H), 6.76 (s,
1H), 3.81-3.56 (m, 12H), 1.14 (s, 12H); MS (MALDI): m/z=450.0
([M+H].sup.+).
Compound 31:
(S)-4-(difluoromethyl)-5-(2-(3-methylmorpholino)-6-morpholinopyrimidin-4--
yl)pyridin-2-amine (31)
##STR00203##
[0532] According to general procedure 1, compound 31 is obtained
from starting materials i28 and i68 in 58% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.31 (s,
1H), 7.52 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.74 (s, 1H), 6.59 (br s,
2H), 6.35 (s, 1H), 4.59-4.51 (m, 1H), 4.22-4.14 (m, 1H), 3.91-3.84
(m, 1H), 3.72-3.50 (m, 10H), 3.44-3.35 (m, 1H), 3.14-3.03 (m, 1H),
1.16 (d, .sup.3J.sub.H,H=6.7 Hz, 3H); .sup.19F NMR (376 MHz,
(CD.sub.3).sub.2SO): .delta.-113.7-(-115.3) (m, 2 F); MS (MALDI):
m/z=407.1 ([M+H].sup.+).
Compound 32:
(S)-4'-(difluoromethyl)-2-(3-methylmorpholino)-6-morpholino-[4,5'-bipyrim-
idin]-2'-amine (32)
##STR00204##
[0534] According to general procedure 2, compound 32 is obtained
from starting materials i28 and i71 in 63% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.8.60 (s, 1H), 7.13
(t, .sup.2J.sub.H,F=54 Hz, 1H), 5.99 (s, 1H), 5.46 (br s, 2H),
4.34-4.25 (m, 1H), 4.06-3.97 (m, 2H), 3.82-3.68 (m, 10H), 3.58 (dt,
.sup.2J.sub.H,H=12 Hz, .sup.3J.sub.H,H=3.2 Hz, 1H), 3.26 (dt,
.sup.2J.sub.H,H=13 Hz, .sup.3J.sub.H,H=3.7 Hz, 1H), 1.31 (d,
.sup.3J.sub.H,H=6.8 Hz, 3H); .sup.19F NMR (376 MHz,
(CD.sub.3).sub.2SO): .delta.-119.5 (s, 2 F); MS (MALDI): m/z=408.7
([M+H].sup.+).
Compound 33:
4-(difluoromethyl)-5-[4-[(2S,6R)-2,6-dimethylmorpholin-4-yl]-6-[(3R)-3-me-
thylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine (33)
##STR00205##
[0536] According to general procedure 1, compound 33 is obtained
from starting materials i68 and i82 in 71% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.87 (s,
1H), 7.74 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.83 (br s, 2H), 6.76 (s,
1H), 4.71-4.62 (m, 1H), 4.45-4.34 (m, 2H), 4.31-4.09 (m, 1H), 3.90
(m, 1H), 3.71 (m, 1H), 3.55 (m, 3H), 3.38 (m, 1H), 3.13 (m, 1H),
2.55 (m, 2H), 1.20 (d, .sup.3J.sub.H,H=6.9 Hz, 3H), 1.19 (d,
.sup.3J.sub.H,H=6.9 Hz, 6H); MS (MALDI): m/z=436.1
([M+H].sup.+).
Compound 34:
5-[4,6-bis[(2R,6S)-2,6-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(dif-
luoromethyl)pyridin-2-amine (34)
##STR00206##
[0538] According to general procedure 1, compound 34 is obtained
from starting materials i68 and i79 in 75% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.86 (s,
1H), 7.71 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.83 (br s, 2H), 6.76 (s,
1H), 4.64-4.46 (m, 4H), 3.60-3.48 (m, 4H), 2.63 (m, 4H), 1.14 (m,
12H); MS (MALDI): m/z=450.0 ([M+H].sup.+).
Compound 37:
5-[4,6-bis(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-1,3,5-triazin-2-yl]-4-
-(difluoromethyl)pyridin-2-amine (37)
##STR00207##
[0540] According to general procedure 1, compound 37 is obtained
from starting materials i7 and i68 in 39% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.85 (s,
1H), 7.68 (t, .sup.3J.sub.H,F=55 Hz, 1H), 6.87 (br s, 2H), 6.74 (s,
1H), 4.51 (br s, 2H), 4.45 (br s, 2H), 4.07-3.93 (m, 8H), 3.79-3.67
(m, 8H); .sup.19F NMR (376 MHz, (CD.sub.3).sub.2SO): .delta.-115.8
(s, 2 F); MS (MALDI): m/z=478.1 ([M+H].sup.+).
Compound 38:
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-(3-ox-
a-8-azabicyclo[3.2.1]octan-8-yl)-1,3,5-triazin-2-yl]pyridin-2-amine
(38)
##STR00208##
[0542] According to general procedure 1, compound 38 is obtained
from starting materials i35 and i68 in 67% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.87 (s,
1H), 7.73 (t, .sup.3J.sub.H,F=55 Hz, 1H), 6.87 (br s, 2H), 6.75 (s,
1H), 4.70-4.54 (m, 2H), 4.53-4.43 (m, 2H), 4.05-3.97 (m, 4H),
3.79-3.67 (m, 4H), 3.63-3.55 (m, 4H) 2.00-1.83 (m, 4H); .sup.19F
NMR (376 MHz, (CD.sub.3).sub.2SO): .delta.-115.8 (s, 1 F), -115.9
(s, 1 F); MS (MALDI): m/z=462.1 ([M+H].sup.+).
Compound 39:
5-[4,6-bis(3,3-dimethylmorpholin-4-yl)-1,3,5-triazin-2-yl]-4-(difluoromet-
hyl)pyridin-2-amine (39)
##STR00209##
[0544] According to general procedure 1, compound 39 is obtained
from starting materials i4 and i68 in 28% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.78 (s,
1H), 7.70 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.82 (br s, 2H), 6.77 (s,
1H), 3.87-3.75 (m, 8H), 3.45 (br s, 4H), 1.49 (s, 12H); .sup.19F
NMR (376 MHz, (CD.sub.3).sub.2SO): .delta.-114.9-(-115.1) (n, 2 F);
MS (MALDI): m/z=450.1 ([M+H].sup.+).
Compound 40:
5-[4,6-bis[(3R,5S)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(dif-
luoromethyl)pyridin-2-amine (40)
##STR00210##
[0546] According to general procedure 1, compound 40 is obtained
from starting materials i6 and i68 in 42% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.90 (s,
1H), 7.82 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.84 (br s, 2H), 6.77 (s,
1H), 4.59-4.43 (m, 4H), 3.82-3.73 (m, 4H), 3.60-3.51 (m, 4H), 1.29
(d, .sup.2J.sub.H,H=6.9 Hz, 12H); .sup.19F NMR (376 MHz,
(CD.sub.3).sub.2SO): .delta.-114.9-(-115.0) (m, 2 F); MS (MALDI):
m/z=450.2 ([M+H].sup.+).
Compound 41:
5-[4,6-bis[(3R)-3-methylmorpholin-4-yl]-135-triazin-2-yl]-4-(difluorometh-
yl)pyridin-2-amine (41)
##STR00211##
[0548] According to general procedure 1, compound 41 is obtained
from starting materials i5 and i68 in 98% yield as a colorless
solid. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 9.04 (s, 1H),
7.70 (t, .sup.2J.sub.H,F=52.0 Hz, 1H), 6.84 (s, 1H), 4.88 (br s,
2H), 4.77-4.72 (m, 2H), 4.41 (d, .sup.2J.sub.H,H=12.0 Hz, 2H), 3.98
(dd, .sup.2J.sub.H,H=12.0 Hz, .sup.3J.sub.H,H=4.0 Hz, 2H), 3.78 (d,
.sup.2J.sub.H,H=12.0 Hz, 2H), 3.68 (dd, .sup.2J.sub.H,H=12.0 Hz,
.sup.3J.sub.H,H=4.0 Hz, 2H), 3.53 (dt, .sup.2J.sub.H,H=12.0 Hz,
.sup.3J.sub.H,H=4.0 Hz, 2H), 3.28 (dt, .sup.2J.sub.H,H=12.0 Hz,
.sup.3J.sub.H,H=4.0 Hz, 2H), 1.33 (d, .sup.2J.sub.H,H=8.0 Hz, 6H);
.sup.19F NMR (376 MHz, CDCl.sub.3): .delta.-115.9 (s, 1 F), -116.0
(s, 1 F); MS (MALDI): m/z=421.7 ([M+H].sup.+).
Compound 42:
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-morpholino-1,3,5-t-
riazin-2-yl]pyridin-2-amine (42)
##STR00212##
[0550] According to general procedure 1, compound 42 is obtained
from starting materials i16 and i68 in 35% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.83 (s,
1H), 7.73 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.84 (br s, 2H), 6.76 (s,
1H), 3.85-3.76 (m, 4H), 3.76-3.63 (m, 8H), 3.45 (br s, 2H), 1.49
(s, 6H); .sup.19F NMR (376 MHz, (CD.sub.3).sub.2SO): .delta.-116
(s, 2 F); MS (MALDI): m/z=422.1 ([M+H].sup.+).
Compound 44:
4-(difluoromethyl)-5-[4-[(3R,5S)-3,5-dimethylmorpholin-4-yl]-6-[(3R)-3-me-
thylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine (44)
##STR00213##
[0552] According to general procedure 1, compound 44 is obtained
from starting materials i37 and i68 in 75% yield as a colorless
solid. 1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.89 (s, 1H),
7.79 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.83 (br s, 2H), 6.76 (s, 1H),
4.65 (br s, 1H), 4.50 (br s, 2H), 4.37-4.25 (m, 1H), 3.93 (dd,
.sup.3J.sub.H,H=11 Hz, .sup.3J.sub.H,H=3.2 Hz, 1H), 3.79-3.67 (m,
3H), 3.59-3.51 (m, 3H), 3.45-3.36 (m, 1H), 3.22-3.11 (m, 1H), 1.30
(d, .sup.3J.sub.H,H=6.7 Hz, 6H), 1.24 (d, .sup.3J.sub.H,H=6.7 Hz,
3H); 19F NMR (376 MHz, (CD.sub.3).sub.2SO): .delta.-115.0 (br s, 2
F); MS (MALDI): m/z=436.1 ([M+H].sup.+).
Compound 45:
4-(difluoromethyl)-5-4-(3,3-dimethylmorpholin-4-yl)-6-[(3R)-3-methylmorph-
olin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine (45)
##STR00214##
[0554] According to general procedure 1, compound 45 is obtained
from starting materials i38 and i68 in 71% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.84 (s,
1H), 7.74 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.83 (br s, 2H), 6.76 (s,
1H), 4.58 (br s, 1H), 4.31-4.19 (m, 1H), 3.93 (dd,
.sup.2J.sub.H,H=12 Hz, .sup.3J.sub.H,H=3.9 Hz, 1H), 3.84-3.81 (m,
4H), 3.76-3.69 (m, 1H), 3.58 (dd, .sup.2J.sub.H,H=11 Hz,
.sup.3J.sub.H,H=3.2 Hz, 1H), 3.46-3.38 (m, 3H), 3.23-3.13 (m, 1H),
1.50 (br s, 6H), 1.23 (d, .sup.3J.sub.H,H=6.7 Hz, 3H); .sup.19F NMR
(376 MHz, (CD.sub.3).sub.2SO): .delta.-114.8-(-115.5) (m, 2 F); MS
(MALDI): m/z=436.0 ([M+H].sup.+).
Compound 46:
4-(difluoromethyl)-5-[4-[(3R)-3-(methoxymethyl)morpholin-4-yl]-6-[(3R)-3--
methylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine (46)
##STR00215##
[0556] According to general procedure 1, compound 46 is obtained
from starting materials i39 and i68 in 67% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.87 (s,
1H), 7.77 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.84 (br s, 2H), 6.76 (s,
1H), 4.67 (br s, 2H), 4.44-4.24 (m, 2H), 3.96-3.83 (m, 3H),
3.75-3.63 (m, 2H), 3.60-3.36 (m, 5H), 3.31 (s, 3H), 3.21-3.04 (m,
2H), 1.23 (d, .sup.3J.sub.H,H=6.7 Hz, 3H); .sup.19F NMR (376 MHz,
(CD.sub.3).sub.2SO): .delta.-115.0 (br s, 2 F); MS (MALDI):
m/z=452.3 ([M+H].sup.+).
Compound 47:
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-[(3R)-
-3-methylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine
(47)
##STR00216##
[0558] According to general procedure 1, compound 47 is obtained
from starting materials i36 and i68 in 85% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.86 (s,
1H), 7.72 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.84 (br s, 2H), 6.75 (s,
1H), 4.64 (br s, 1H), 4.53-4.42 (m, 2H), 4.37-4.25 (m, 1H),
4.05-3.96 (m, 4H), 3.92-3.84 (m, 1H), 3.77-3.66 (m, 5H), 3.60-3.52
(m, 1H), 3.44-3.35 (m, 1H), 3.22-3.10 (m, 1H), 1.23 (d,
.sup.3J.sub.H,H=6.7 Hz, 3H); .sup.19F NMR (376 MHz,
(CD.sub.3).sub.2SO): .delta.-114.9-(-117.1) (m, 2 F); MS (MALDI):
m/z=450.0 ([M+H].sup.+).
Compound 50:
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-(3-oxa-6-azabicyc-
lo[3.1.1]heptan-6-yl)-1,3,5-triazin-2-yl]pyridin-2-amine (50)
##STR00217##
[0560] According to general procedure 1, compound 50 is obtained
from starting materials i40 and i68 in 52% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.90 (s,
1H), 7.82 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.87 (br s, 2H), 6.76 (s,
1H), 4.55-4.51 (m, 1H), 4.34-4.14 (m, 3H), 4.12-4.25 (m, 2H),
3.92-3.80 (m, 1H), 3.76-3.68 (m, 3H), 3.55-3.51 (m, 1H), 3.38 (m,
1H), 3.20-3.13 (m, 1H), 2.68 (m, 1H), 1.78 (m, 1H), 1.20 (d,
.sup.3J.sub.H,H=6.9 Hz, 3H); .sup.19F NMR (376 MHz,
(CD.sub.3).sub.2SO): .delta.-115.0 (br s, 2 F); MS (MALDI):
m/z=420.6 ([M+H].sup.+).
Compound 51:
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-(6-oxa-3-azabicyc-
lo[3.1.1]heptan-3-yl)-1,3,5-triazin-2-yl]pyridin-2-amine (51)
##STR00218##
[0562] According to general procedure 1, compound 51 is obtained
from starting materials i41 and i68 in 36% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.99 (s,
1H), 7.89 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.84 (br s, 2H), 6.77 (s,
1H), 4.69 (m, 3H), 4.37 (m, 1H), 3.91-3.85 (m, 3H), 3.75-3.53 (m,
4H), 3.42-3.35 (m, 1H), 3.22-3.15 (m, 1H), 3.12-3.08 (m, 1H), 1.85
(m, 1H), 1.24 (d, .sup.3J.sub.H,H=6.9 Hz, 3H); .sup.19F NMR (376
MHz, (CD.sub.3).sub.2SO): .delta.-116.0 (br s, 2 F); MS (MALDI):
m/z=420.6 ([M+H].sup.+).
Compound 52:
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-[(1R,4R)-2-oxa-5--
azabicyclo[2.2.1]heptan-5-yl]-1,3,5-triazin-2-yl]pyridin-2-amine
(52)
##STR00219##
[0564] According to general procedure 1, compound 52 is obtained
from starting materials i42 and i68 in 44% yield as a colorless
solid (1:1 mixture of rotamers). .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO): .delta.8.89 (m, 1H), 7.77 (m, 1H), 6.84 (br s,
2H), 6.76 (s, 1H), 5.02-4.97 (m, 1H), 4.68-4.66 (m, 2H), 4.31 (m,
1H), 3.89-3.85 (m, 1H), 3.79-3.57 (m, 3H), 3.57-3.44 (m, 4H), 3.22
(m, 1H), 1.90-1.83 (m, 2H), 1.21 (d, .sup.3J.sub.H,H=6.9 Hz, 3H);
.sup.19F NMR (376 MHz, (CD.sub.3).sub.2SO): .delta.-115.5 (br s, 2
F); MS (MALDI): m/z=420.2 ([M+H].sup.+).
Compound 53:
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-[(1S,4S)-2-oxa-5--
azabicyclo[2.2.1]heptan-5-yl]-1,3,5-triazin-2-yl]pyridin-2-amine
(53)
##STR00220##
[0566] According to general procedure 1, compound 53 is obtained
from starting materials i43 and i68 in 53% yield as a colorless
solid (1:1 mixture of rotamers). .sup.1H NMR (400 MHz,
(CD.sub.3).sub.2SO): .delta.8.90 (m, 1H), 7.77 (m, 1H), 6.84 (br s,
2H), 6.76 (s, 1H), 5.02-4.96 (m, 1H), 4.68-4.62 (m, 2H), 3.90 (m,
1H), 3.80 (m, 1H), 3.70 (m, 2H), 3.57 (m, 2H), 3.45 (m, 3H), 3.20
(m, 1H), 1.90-1.83 (m, 2H), 1.21 (d, .sup.3J.sub.H,H=6.9 Hz, 3H);
.sup.19F NMR (376 MHz, (CD.sub.3).sub.2SO): .delta.-115.0 (br s, 2
F); MS (MALDI): m/z=420.2 ([M+H].sup.+).
Compound 54:
5-[4,6-bis[(3R)-3-ethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(difluoromet-
hyl)pyridin-2-amine (54)
##STR00221##
[0568] According to general procedure 1, compound 54 is obtained
from starting materials i8 and i68 in 61% yield as a colorless
solid. 1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.87 (s, 1H),
7.77 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.83 (br s, 2H), 6.76 (s, 1H),
4.47 (m, 4H), 3.89-3.81 (m, 4H), 3.51-3.34 (m, 4H), 3.12 (m, 2H),
1.71 (m, 4H), 0.86 (m, 6H). .sup.19F NMR (376 MHz,
(CD.sub.3).sub.2SO): .delta.-115.0 (br s, 2 F); MS (MALDI):
m/z=450.3 ([M+H].sup.+).
Compound 55:
5-[4,6-bis(8-oxa-5-azaspiro[3.5]nonan-5-yl)-1,3,5-triazin-2-yl]-4-(difluo-
romethyl)pyridin-2-amine (55)
##STR00222##
[0570] According to general procedure 1, compound 55 is obtained
from starting materials i9 and i68 in 59% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.74 (s,
1H), 7.65 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.81 (br s, 2H), 6.75 (s,
1H), 3.68 (m, 8H), 3.49 (m, 4H), 2.46-2.38 (m, 4H), 2.25-2.16 (m,
4H), 1.72-1.66 (m, 4H); .sup.19F NMR (376 MHz, (CD.sub.3).sub.2SO):
.delta. -115.5 (br s, 2 F); MS (MALDI): m/z=474.3
([M+H].sup.+).
Compound 56:
5-[4,6-bis(3R)-3-isopropylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(difluoro-
methyl)pyridin-2-amine (56)
##STR00223##
[0572] According to general procedure 1, compound 56 is obtained
from starting materials i10 and i68 in 59% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.87 (s,
1H), 7.76 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.82 (br s, 2H), 6.76 (s,
1H), 4.50 (m, 2H), 4.29 (m, 2H), 4.02-3.84 (m, 4H), 3.40 (m, 4H),
3.08 (m, 2H), 2.34 (m, 2H), 1.02 (m, 6H), 0.77 (m, 6H); .sup.19F
NMR (376 MHz, (CD.sub.3).sub.2SO): .delta.-115.0 (br s, 2 F); MS
(MALDI): m/z=478.4 ([M+H].sup.+).
Compound 66:
4-(difluoromethyl)-5-[4-(3.3-dimethylmorpholin-4-yl)-6-[(3R,5S)-3,5-dimet-
hylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine (66)
##STR00224##
[0574] According to general procedure 1, compound 66 is obtained
from starting materials i55 and i68 in 61% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.87 (s,
1H), 7.77 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.83 (br s, 2H), 6.76 (s,
1H), 4.46 (m, 2H), 3.81-3.77 (m, 6H), 3.55 (m, 2H), 3.44 (m, 2H),
1.49 (s, 6H), 1.28 (d, .sup.3J.sub.H,H=6.9 Hz, 6H); .sup.19F NMR
(376 MHz, (CD.sub.3).sub.2SO): .delta.-115.0 (br s, 2 F); MS
(MALDI): m/z=450.4 ([M+H].sup.+).
Compound 67:
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-[(3R)-3-(methoxyme-
thyl)morpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine (67)
##STR00225##
[0576] According to general procedure 1, compound 67 is obtained
from starting materials i56 and i68 in 37% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.84 (s,
1H), 7.89 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.85 (br s, 2H), 6.76 (s,
1H), 4.60 (m, 1H), 4.31 (m, 1H), 3.92 (m, 2H), 3.83 (m, 4H), 3.65
(m, 1H), 3.51-3.41 (m, 5H), 3.28 (s, 3H), 3.12 (m, 1H), 1.49 (s,
3H), 1.48 (s, 3H); .sup.19F NMR (376 MHz, (CD.sub.3).sub.2SO):
.delta.-115.5 (br s, 2 F); MS (MALDI): m/z=466.4 ([M+H].sup.+).
Compound 68:
[(3R)-4-[4-[6-amino-4-(difluoromethyl)-3-pyridyl]-6-(3.3-dimethylmorpholi-
n-4-yl)-1,3,5-triazin-2-yl]morpholin-3-yl]methanol (68)
##STR00226##
[0578] According to general procedure 1, compound 68 is obtained
from starting materials i57 and i68 in 58% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.83 (s,
1H), 7.77 (m, 1H), 6.84 (br s, 2H), 6.76 (s, 1H), 4.91 (m, 1H),
4.35 (m, 2H), 4.05 (m, 1H), 3.97-3.70 (m, 6H), 3.54-3.38 (m, 5H),
3.12 (m, 1H), 1.49 (s, 3H), 1.48 (s, 3H); .sup.19F NMR (376 MHz,
(CD.sub.3).sub.2SO): .delta.-115.5 (br s, 2 F); MS (MALDI):
m/z=452.2 ([M+H].sup.+).
Compound 69:
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-(3,7-dioxa-9-azabi-
cyclo[3.3.1]nonan-9-yl)-1,3,5-triazin-2-yl]pyridin-2-amine (69)
##STR00227##
[0580] According to general procedure 1, compound 69 is obtained
from starting materials i54 and i68 in 57% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.83 (s,
1H), 7.69 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.85 (br s, 2H), 6.76 (s,
1H), 4.47-4.37 (m, 2H), 4.01 (m, 4H), 3.80-3.71 (m, 8H), 3.45 (m,
2H), 1.48 (s, 6H); .sup.19F NMR (376 MHz, (CD.sub.3).sub.2SO):
.delta.-115.7 (br s, 2 F); MS (MALDI): m/z=464.3 ([M+H].sup.+).
Compound 70:
5-[4-(4-cyclopropylpiperazin-1-yl)-6-(3,3-dimethylmorpholin-4-yl)-1,3,5-t-
riazin-2-yl]-4-(difluoromethyl)pyridin-2-amine (70)
##STR00228##
[0582] According to general procedure 1, compound 70 is obtained
from starting materials i58 and i68 in 12% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.82 (s,
1H), 7.72 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.83 (br s, 2H), 6.76 (s,
1H), 3.82 (m, 4H), 3.71 (m, 4H), 3.44 (m, 2H), 2.58 (m, 4H), 1.64
(m, 1H), 1.44 (s, 6H), 0.45 (m, 2H), 0.36 (m, 2H); .sup.19F NMR
(376 MHz, (CD.sub.3).sub.2SO): .delta.-115.4 (br s, 2 F); MS
(MALDI): m/z=460.4 ([M].sup.+).
Compound 71:
4-(difluoromethyl)-5-[4-(3,3-dimethylmorpholin-4-yl)-6-[4-(2-methoxyethyl-
)piperazin-1-yl]-1,3,5-triazin-2-yl]pyridin-2-amine (71)
##STR00229##
[0584] According to general procedure 1, compound 71 is obtained
from starting materials i59 and i68 in 42% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta.8.82 (s,
1H), 7.73 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.83 (br s, 2H), 6.76 (s,
1H), 3.88-3.69 (m, 10H), 3.47-3.44 (m, 4H), 3.24 (m, 3H), 2.52-2.45
(m, 4H), 1.44 (s, 6H); .sup.19F NMR (376 MHz, (CD.sub.3).sub.2SO):
.delta. -115.4 (br s, 2 F); MS (MALDI): m/z=478.4 ([M].sup.+).
Compound 77:
[(3R)-4-[4-[6-amino-4-(difluoromethyl)-3-pyridyl]-6-[(3R)-3-methylmorphol-
in-4-yl]-1,3,5-triazin-2-yl]morpholin-3-yl]methanol (77)
##STR00230##
[0586] According to general procedure 1, compound 77 is obtained
from starting materials i53 and i68 in 31% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.88 (s,
1H), 7.78 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.84 (br s, 2H), 6.76 (s,
1H), 4.96 (m, 1H), 4.73 (m, 1H), 4.58-4.24 (m, 3H), 4.05 (m, 1H),
3.90 (m, 2H), 3.72 (m, 2H), 3.59 (m, 1H), 3.51-3.36 (m, 4H),
3.23-3.02 (m, 2H), 1.23 (d, .sup.3J.sub.H,H=6.9 Hz, 3H); MS
(MALDI): m/z=438.3 ([M+H].sup.+).
Compound 78:
4-(difluoromethyl)-5-[4-[(3R,5R)-3,5-dimethylmorpholin-4-yl]-6-[(3R)-3-me-
thylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine (78)
##STR00231##
[0588] According to general procedure 1, compound 78 is obtained
from starting materials i85 and i68 in 71% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.90 (s,
1H), 7.82 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.84 (br s, 2H), 6.76 (s,
1H), 4.66 (m, 1H), 4.32 (m, 3H), 4.15-4.11 (m, 2H), 3.92 (m, 1H),
3.70 (m, 3H), 3.57 (m, 1H), 3.40 (m, 1H), 3.18 (m, 1H), 1.37 (m,
6H), 1.24 (d, .sup.3J.sub.H,H=6.9 Hz, 3H); .sup.19F NMR (376 MHz,
(CD.sub.3).sub.2SO): .delta.-114.9 (br s, 2 F); MS (MALDI):
m/z=435.4 ([M].sup.+).
Compound 79:
4-(difluoromethyl)-5-[4-[(3S,5S)-3,5-dimethylmorpholin-4-yl]-6-[(3R)-3-me-
thylmorpholin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine (79)
##STR00232##
[0590] According to general procedure 1, compound 79 is obtained
from starting materials i86 and i68 in 65% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.91 (s,
1H), 7.82 (t, 2J.sub.H,F=55 Hz, 1H), 6.85 (br s, 2H), 6.76 (s, 1H),
4.66 (m, 1H), 4.32 (m, 3H), 4.15-4.11 (m, 2H), 3.92 (m, 1H), 3.70
(m, 3H), 3.57 (m, 1H), 3.40 (m, 1H), 3.19 (m, 1H), 1.37 (m, 6H),
1.24 (d, .sup.3J.sub.H,H=6.9 Hz, 3H); .sup.19F NMR (376 MHz,
(CD.sub.3).sub.2SO): .delta.-114.9 (br s, 2 F); MS (MALDI):
m/z=434.3 ([M].sup.+).
Compound 80:
4-(difluoromethyl)-5-[4-morpholino-6-(3-oxa-9-azabicyclo[3.3.1]nonan-9-yl-
)-1,3,5-triazin-2-yl]pyridin-2-amine (80)
##STR00233##
[0592] According to general procedure 1, compound 80 is obtained
from starting materials i87 and i68 in 57% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.85 (s,
1H), 7.73 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.84 (br s, 2H), 6.75 (s,
1H), 4.61-4.57 (m, 2H), 3.95 (m, 2H), 3.75-3.65 (m, 10H), 2.48 (m,
1H), 1.88-1.72 (m, 4H), 1.57 (m, 1H); .sup.19F NMR (376 MHz,
(CD.sub.3).sub.2SO): .delta.-115.4 (m, 2 F); MS (MALDI): m/z=434.3
([M+H].sup.+).
Compound 82:
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-(3-ox-
a-9-azabicyclo[3.3.1]nonan-9-yl)-1,3,5-triazin-2-yl]pyridin-2-amine
(82)
##STR00234##
[0594] According to general procedure 1, compound 82 is obtained
from starting materials i89 and i68 in 51% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.84 (s,
1H), 7.70 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.85 (br s, 2H), 6.75 (s,
1H), 4.62 (m, 1H), 4.54 (m, 1H), 4.52 (m, 1H), 4.44 (m, 1H),
4.04-3.92 (m, 6H), 3.75-3.62 (m, 6H), 2.45 (m, 1H), 1.89-1.75 (m,
4H), 1.57 (m, 1H); .sup.19F NMR (376 MHz, (CD.sub.3).sub.2SO):
.delta.-115.7 (m, 2 F); MS (MALDI): m/z=476.2 ([M+H].sup.+).
Compound 83:
5-[4,6-bis[(3S,5S)-3,5-dimethylmorpholin-4-yl]-1,3,5-triazin-2-yl]-4-(dif-
luoromethyl)pyridin-2-amine (83)
##STR00235##
[0596] According to general procedure 1, compound 83 is obtained
from starting materials i90 and i68 in 56% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.92 (s,
1H), 7.87 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.84 (br s, 2H), 6.77 (s,
1H), 4.32 (m, 4H), 4.14 (m, 4H), 3.70 (m, 4H), 1.39 (d,
.sup.3J.sub.H,H=6.9 Hz, 12H); .sup.19F NMR (376 MHz,
(CD.sub.3).sub.2SO): .delta.-115.5 (br s, 2 F); MS (MALDI):
m/z=448.3 ([M].sup.+).
Compound 84:
4-(difluoromethyl)-5-[4-(3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl)-6-morph-
olino-1,3,5-triazin-2-yl]pyridin-2-amine (84)
##STR00236##
[0598] According to general procedure 1, compound 84 is obtained
from starting materials i91 and i68 in 63% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.86 (s,
1H), 7.71 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.87 (br s, 2H), 6.75 (s,
1H), 4.49 (m, 2H), 4.02 (m, 4H), 3.74-3.65 (m, 12H); .sup.19F NMR
(376 MHz, (CD.sub.3).sub.2SO): .delta.-115.6 (br s, 2 F); MS
(MALDI): m/z=436.4 ([M+H].sup.+).
Compound 85:
4-(difluoromethyl)-5-[4-[(3S)-3-ethylmorpholin-4-yl]-6-[(3R)-3-methylmorp-
holin-4-yl]-1,3,5-triazin-2-pyridin-2-yl]pyridin-2-amine (85)
##STR00237##
[0600] According to general procedure 1, compound 85 is obtained
from starting materials i92 and i68 in 52% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.88 (s,
1H), 7.77 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.85 (br s, 2H), 6.76 (s,
1H), 4.70-4.25 (m, 4H), 3.90 (m, 3H), 3.72 (m, 1H), 3.60-3.45 (m,
4H), 3.16 (m, 2H), 1.73 (m, 2H), 1.22 (d, .sup.3J.sub.H,H=6.9 Hz,
3H), 0.86 (m, 3H); .sup.19F NMR (376 MHz, (CD.sub.3).sub.2SO):
.delta.-114.9 (br s, 2 F); MS (MALDI): m/z=436.9 ([M+H].sup.+).
Compound 86:
4-(difluoromethyl)-5-[4-[(3R)-3-ethylmorpholin-4-yl]-6-[(3R)-3-methylmorp-
holin-4-yl]-1,3,5-triazin-2-yl]pyridin-2-amine (86)
##STR00238##
[0602] According to general procedure 1, compound 86 is obtained
from starting materials i93 and i68 in 47% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.88 (s,
1H), 7.77 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.85 (br s, 2H), 6.76 (s,
1H), 4.65 (m, 1H), 4.49-4.30 (m, 3H), 3.93-3.82 (m, 3H), 3.72 (m,
1H), 3.57 (m, 1H), 3.50 (m, 1H), 3.43-3.37 (m, 2H), 3.19-3.14 (m,
2H), 1.73 (m, 2H), 1.22 (d, .sup.3J.sub.H,H=6.9 Hz, 3H), 0.86 (m,
3H); .sup.19F NMR (376 MHz, (CD.sub.3).sub.2SO): .delta.-115.3 (br
s, 2 F); MS (MALDI): m/z=436.9 ([M+H].sup.+).
Compound 88:
4-(difluoromethyl)-5-[4-[(3R)-3-methylmorpholin-4-yl]-6-(8-oxa-5-azaspiro-
[3.5]nonan-5-yl)-1,3,5-triazin-2-yl]pyridin-2-amine (88)
##STR00239##
[0604] According to general procedure 1, compound 88 is obtained
from starting materials i94 and i68 in 50% yield as a colorless
solid. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2SO): .delta. 8.82 (s,
1H), 7.71 (t, .sup.2J.sub.H,F=55 Hz, 1H), 6.84 (br s, 2H), 6.75 (s,
1H), 4.55 (m, 1H), 4.23 (m, 1H), 3.91 (m, 1H), 3.78 (m, 2H), 3.69
(m, 3H), 3.56 (m, 1H), 3.50 (m, 2H), 3.41 (m, 1H), 3.16 (m, 1H),
2.50 (m, 2H), 2.26 (m, 2H), 1.73 (m, 2H), 1.21 (d,
.sup.3J.sub.H,H=6.9 Hz, 3H); .sup.19F NMR (376 MHz,
(CD.sub.3).sub.2SO): .delta.-114.9 (br s, 2 F); MS (MALDI):
m/z=446.8 ([M+H].sup.+).
Example 2
In Vitro mTOR Binding Assay and in-Cell Western Blot
[0605] In Vitro mTOR Binding Assay
[0606] N-terminally GST-tagged mTOR (Cat. No. PR8683B; 0.45 mg/ml;
truncated version: amino acids 1360-2549), Alexa Fluor.RTM. 647
labeled kinase Tracer 314 (Cat. No. PV6087), LanthaScreen
Eu-anti-GST Tag antibody (Cat. No. PV5594) were purchased from Life
Technologies. The 1.times.mTOR Kinase Buffer consists of 50 mM
HEPES pH 7.5, 5 mM MgCl.sub.2, 1 mM EGTA, and 0.01% Pluronic F-127
(Sigma Cat. No. P2443-250G).
[0607] A 10-point 4-fold serial dilution (highest concentration at
10 .mu.mol/L and lowest concentration at 40 pmol/L) of each
compound was tested for mTOR binding in duplicate in a 384-well
plate. To perform the LanthaScreen kinase binding assay 5 .mu.l of
the test compounds concentrated 3.times. the final concentration, 5
.mu.l of 9 nM GST-mTOR/6 nM Eu-anti-GST antibody mixture and 5
.mu.l of 30 nM Tracer 314 solution were mixed together resulting to
a final concentration of 3 nM GST-mTOR, 2 nM Eu-anti-GST antibody
and 10 nM Tracer 314 per well. After 30 min incubation at RT,
time-resolved FRET was measured with a Synergy 4 multi-mode
microplate reader (Biotek Instruments) using the following
settings: 100 microsecs delay before data collection, 200 microsecs
time for data collection, 10 measurements per data point. Emission
filter: 665 nm/8 nm with sensitivity set to 190 and 620 nm/10 nm
with sensitivity set to 130; Excitation filter: 340 nm/30 nm;
Dichroic mirror 400 nm.
[0608] For data analysis, the mean background (wells with only mTOR
kinase buffer) was subtracted and the emission ratio calculated by
dividing the signal emitted at 665 nm from the acceptor (Alexa
Fluor.RTM. 647 labeled Tracer 314) by the signal emitted at 620 nm
from the donor (Eu-labeled antibody). IC.sub.50 values of each
compound were determined by plotting the emission ratio versus the
compound concentrations (in logarithmic scale) and then by fitting
a sigmoidal dose-response curve with variable slope to the data
using GraphPad.TM. Prism.
[0609] In-Cell Western Blot
[0610] A2058 cells are plated at 20,000 cells/well in a 96-well
plate (Perkin Elmer, Cat. No. 6005558) and 24 hours later treated
with different compounds for 1 hour. For each compound 7 different
concentrations are applied on cells (5 .mu.M, 1.25 .mu.M, 0.625
.mu.M, 0.3125 .mu.M, 0.155 .mu.M, 0.08 .mu.M and 0.04 .mu.M). Cells
are fixed with 4% paraformaldehyde for 30 minutes at room
temperature, washed 2 times with 1% BSA in PBS, permeabilized with
0.1% Triton X-100 in PBS/1% BSA for 30 minutes at room temperature
and blocked with 5% goat serum in PBS/1% BSA/0.1% Triton X-100 for
30 minutes at room temperature. Cells are stained with primary
antibody either with rabbit anti-pPKB S473 (1:500; Cell Signaling
Technology, Cat. No. 4058) combined with mouse anti-.alpha.-tubulin
(1:2000; used for normalization; Sigma, Cat. No. T9026) or with
rabbit anti-pS6 S235/S236 (1:500; Cell Signaling Technology, Cat.
No. 4856) combined with mouse anti-.alpha.-tubulin (1:2000; used
for normalization) over night at 4.degree. C. After 3 times 5
minutes wash with PBS/1% BSA/0.1% triton cells are treated with the
secondary antibodies goat-anti-mouse IRDye680 (LICOR, Cat. No.
926-68070) and goat-anti-rabbit IRDye800 (LICOR, 926-32211) (each
diluted 1:500 in PBS/1% BSA/0.1% triton) for 1 hour while shaking
in the dark. Cells are washed 3 times 5 minutes with PBS/1%
BSA/0.1% triton and plate scanned with the Odyssey Infrared
Scanning system using both 700 and 800 nm channels. As control for
0% inhibition vehicle (0.2% DMSO) is added to cells. To correct for
background staining in the data analysis wells are treated only
with secondary antibodies.
[0611] For data analysis the mean background signal from channel
700 nm and 800 nm are subtracted from each signal in channel 700 nm
and 800 nm, respectively. The signals in each channel are
normalized to the 0% inhibition and then signal ratio 800 nm over
700 nm is performed to obtain the values for either pPKB S473 or
pS6 S235/S236 normalized to .alpha.-Tubulin.
[0612] IC.sub.50 values of each compound are determined by plotting
the normalized pPBK S473 and pS6 S235/S236 signals, respectively,
versus the compound concentrations (in logarithmic scale) and then
by fitting a sigmoidal dose-response curve with variable slope to
the data using GraphPad.TM. Prism.
TABLE-US-00009 TABLE 1 Comparative biological activities This
invention This invention Compound 1 WO2010/052569 Compound 2
WO2010/052569 ##STR00240## ##STR00241## ##STR00242## ##STR00243##
pPKB 108 149 34 64 S473 IC.sub.50 [nM] pS6 196 340 80 650 S235/236
IC.sub.50 [nM] mTOR 8 190 59 199 IC.sub.50 [nM]
TABLE-US-00010 TABLE 2 Comparative biological activities This
invention This invention Compound 6 WO2010/052569 Compound 7
WO2010/052569 ##STR00244## ##STR00245## ##STR00246## ##STR00247##
pPKB 155 255 59 118 S473 IC.sub.50 [nM] pS6 215 433 97 224 S235/236
IC.sub.50 [nM] mTOR 23 nd 71 nd IC.sub.50 [nM]
TABLE-US-00011 TABLE 3 Comparative biological activities This
invention This invention Compound 8 WO2010/052569 Compound 9
WO2010/052569 ##STR00248## ##STR00249## ##STR00250## ##STR00251##
pPKB 74 196 35 91 S473 IC.sub.50 [nM] pS6 68 90 72 164 S235/236
IC.sub.50 [nM] mTOR 10 nd 24 nd IC.sub.50 [nM]
TABLE-US-00012 TABLE 4 Comparative biological activities This
invention This invention Compound 12 WO2010052569 Compound 13
WO2010052569 ##STR00252## ##STR00253## ##STR00254## ##STR00255##
pPKB 208 302 43 116 S473 IC.sub.50 [nM] pS6 515 743 150 416
S235/236 IC.sub.50 [nM] mTOR 543 796 1015 2834 IC.sub.50 [nM]
TABLE-US-00013 TABLE 5 Comparative biological activities This
invention This invention Compound 16 WO2007/084786 Compound 14
WO2007/084786 ##STR00256## ##STR00257## ##STR00258## ##STR00259##
pPKB 207 263 90 194 S473 IC.sub.50 [nM] pS6 184 277 149 384
S235/236 IC.sub.50 [nM] mTOR 30 179 155 644 IC.sub.50 [nM]
TABLE-US-00014 TABLE 6 Comparative biological activities This
invention This invention Compound 18 WO2008/098058 Compound 19
WO2008/098058 ##STR00260## ##STR00261## ##STR00262## ##STR00263##
pPKB 243 555 78 175 S473 IC.sub.50 [nM] pS6 256 665 147 370
S235/236 IC.sub.50 [nM] mTOR 31 366 158 1925 IC.sub.50 [nM]
TABLE-US-00015 TABLE 7 Comparative biological activities This
invention This invention Compound 20 WO2010052569 Compound 21
WO2010052569 ##STR00264## ##STR00265## ##STR00266## ##STR00267##
pPKB 146 311 57 343 S473 IC.sub.50 [nM] pS6 250 559 216 996
S235/236 IC.sub.50 [nM] mTOR 13 118 54 394 IC.sub.50 [nM]
TABLE-US-00016 TABLE 8 Comparative biological activities This
invention This invention Compound 25 WO2007/084786 Compound 26
WO2007/084786 ##STR00268## ##STR00269## ##STR00270## ##STR00271##
pPKB 303 452 87 193 S473 IC.sub.50 [nM] pS6 294 553 191 617
S235/236 IC.sub.50 [nM] mTOR 32 152 47 287 IC.sub.50 [nM]
TABLE-US-00017 TABLE 9 Comparative biological activities This
invention This invention Compound 27 WO2007/084786 Compound 28
WO2007/084786 ##STR00272## ##STR00273## ##STR00274## ##STR00275##
pPKB 614 883 77 290 S473 IC.sub.50 [nM] pS6 766 1100 146 1027
S235/236 IC.sub.50 [nM] mTOR 65 376 23 1253 IC.sub.50 [nM]
TABLE-US-00018 TABLE 10 Comparative biological activities This
invention This invention Compound 23 WO2007/084786 Compound 24
WO2007/084786 ##STR00276## ##STR00277## ##STR00278## ##STR00279##
pPKB 285 564 84 340 S473 IC.sub.50 [nM] pS6 230 562 167 740
S235/236 IC.sub.50 [nM] mTOR 40 88 35 121 IC.sub.50 [nM]
TABLE-US-00019 TABLE 11 Comparative biological activities This
invention This invention Compound 31 WO2007/084786 Compound 32
WO2007/084786 ##STR00280## ##STR00281## ##STR00282## ##STR00283##
pPKB 146 248 100 191 S473 IC.sub.50 [nM] pS6 124 228 387 535
S235/236 IC.sub.50 [nM] mTOR 15 28 293 186 IC.sub.50 [nM]
TABLE-US-00020 TABLE 12 Results of in-cell Western Blot and mTOR
binding In-cell Western blot binding pPKB S473 pS6 S235/S236 mTOR
Compound IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] 1 108 196 8 2
34 80 59 3 231 105 8 4 178 135 nd 5 85 135 nd 6 155 215 23 7 59 97
71 8 74 68 10 9 35 72 24 10 138 93 nd 11 61 96 nd 12 219 407 543 13
37 120 1015 14 349.5 883 nd 15 49 286 nd 16 207 184 30 17 90 149
155 18 243 256 31 19 78 147 158 20 146 250 13 21 57 216 54 22 57
216 18 23 285 230 40 24 84 167 35 25 303 294 32 26 87 191 47 27 614
766 65 28 77 146 23 31 146 124 15 32 100 387 293 37 533 268 49 38
219 79 nd 39 106 47 1 40 252 160 5 41 436 261 22 42 54 45 3 44 197
87 5 45 234 93 7 46 956 426 36 47 469 176 29 50 1561 407 nd 51 875
352 nd 52 1050 332 nd 53 1318 612 nd 54 354 209 nd 55 942 526 nd 56
>10000 >10000 nd 66 244 139 4 67 787 395 nd 68 682 415 nd 69
244 140 21 70 914 906 nd 71 2337 3141 nd 77 476 nd 78 506 392 38 79
200 136 10 80 94 117 nd 82 329 169 40 83 379 294 32 84 116 146 nd
85 249 241 nd 86 231 236 nd 88 271 192 18
Example 3
Tolerability of Compound 3 and Compound 8 in Mice
[0613] Maximal Tolerated Dose (MTD) of Cpd. 3 and Cpd. 8 in BALB/c
Nude Mice
[0614] In order to find a dose that could be used for further
experiments and in order to show tolerability of compounds of the
present invention, female BALB/c nude mice were treated with Cpd. 3
and Cpd. 8 by per oral (p.o.) gavage twice for five days with a two
day dose holiday in between. Compounds were administered in the
following vehicle: 20% Hydroxypropyl-3-cyclodextrin (HPBCD), 10%
DMSO and 70% water. The major endpoint was to evaluate the body
weight loss and animal survival. The body weights were recorded
daily for the dosing days and at least twice weekly thereafter. The
animal death was checked daily for survival. Animals were to be
evaluated for 7 days post the final dose. The tolerated dose was
defined as the dose that results in less than 20% mean body-weight
loss, and no treatment related death during the study. The
randomized block design was used to assign experimental animals to
groups. First, the experimental animals were divided into
homogeneous blocks according to their initial body weight.
Secondly, within each block, randomization of experimental animals
to treatments was conducted.
[0615] Animal supplier: Shanghai Lingchang Bio-Technology Co. Ltd
(LC, Shanghai, China). Animal Certificate No.: 2013001803305. The
mice were kept in individual ventilated cage (IVC) systems at
constant temperature and humidity with 5 animals in each cage.
[0616] Diet: Mouse diet, C060 irradiation sterilized dry granule
food. Animals had free access during the entire study period.
Water: RO water, autoclaved before using. Animals had free access
to sterile drinking water.
[0617] OVCAR-3 and SUDHL/6 tumor bearing BALB/c nude mice were
treated with Cpd. 3, p.o. at 100 mg/kg for 21 or 15 consecutive
days, respectively under the same conditions. Body weight of the
mice was stable.
[0618] The following MTDs were determined in BALB/c mice: Cpd. 3:
100 mg/kg, Cpd. 8: 25 mg/kg. In conclusion, both Cpd. 3 and Cpd. 8
are well tolerated at therapeutically effective doses (see Example
4).
Example 4
Pharmacokinetics (PK) of Cpd. 3 and Cpd. 8
[0619] In order to determine distribution of compounds in the body,
PK of compounds was determined in rats and mice. For the treatment
of neurological disorders it is essential to employ compounds that
cross the blood brain barrier and reach efficacious concentrations
in the brain.
[0620] A. PK in Sprague Dawley (SD) Rats
[0621] A single oral administration of cpd. 3, 10 mg/kg was given
to female SD rats in a vehicle consisting of DMSO/HPBCD 20%
(10/90). Animals were housed in controlled environment and
enclosures provided sterile and adequate space with bedding
material, food and water, environmental and social enrichment
(group housing). Rats were randomized according to their individual
body weight, treated and blood, brain and liver were collected and
snap frozen at the following time points: 0.5, 2, 4 and 8 hours.
Blood (approximately 500-700 .mu.L, collected via cardiac puncture
under anesthesia) was immediately transferred into tubes containing
lithium-heparin as anticoagulant (Ref: T MLH, Venoject.RTM.,
Terumo). Tubes were centrifuged at 1,300 g for 10 minutes at
+4.degree. C. The resulting plasma was collected and snap frozen in
liquid nitrogen and stored at -80.degree. C. until analysis.
[0622] Compound concentrations were detected by HPLC-MS/MS. The
calibration curves were drawn using standards of known
concentrations.
[0623] Results are depicted in FIG. 1A. Cpd. 3 shows good oral
bioavailability and excellent penetration into the brain. A Cmax of
1.4 ng/ml in plasma and 1.3 ng/ml in brain indicate exposures that
are high enough for target engagement (table 1).
[0624] B. PK in B57BL/6J Mice
[0625] A single, oral administration of Cpd. 3 and Cpd. 8, 50 mg/kg
was administered to B57BL/6J mice by gavage. Animals were housed in
temperature controlled rooms with free access to food and water.
Test items were weighed in a glass vial and dissolved first in
DMSO. Tween 80 was added subsequently and finally, the formulation
was completed with HP.beta.CD 20%. The formulations were
administered via gavage at t=Oh, and at each of 8 time points,
three mice of each treatment group were anesthetized with
isoflurane for blood sampling via puncture of the retrobulbar
venous plexus. Blood was collected in tubes containing K3-EDTA and
stored on ice until centrifugation at 3000.times.g (10 min,
4.degree. C.). The plasma supernatant was separated and kept at
-20.degree. C. until being assayed. Thigh muscle and brain of mice
were snap frozen. The brain and muscle samples were homogenized in
PBS using the Precellys 24/Dual homogeniser.
[0626] Samples were analyzed by LC-MS. Calibration standards were
prepared by spiking 20 .mu.l of drug free blank plasma with
calibration solution. A volume of 40 .mu.l acetonitrile containing
the internal standard (150 ng/ml Diazepam for 2015PQR002 samples
and 300 ng/ml Griseofulvin for 2015PQR004 samples) was then added
to 20 .mu.l of brain or muscle homogenate, brain/muscle calibration
standard and brain/muscle QC sample. Samples were vigorously shaken
and centrifuged for 10 minutes at 6000 g and 20.degree. C. The
particle free supernatant was diluted with 1 volume of water. An
aliquot was transferred to 200 .mu.l sampler vials and subsequently
subjected to LC-MS with an injection volume of 1.5 .mu.l.
[0627] Both, Cpd. 3 and Cpd. 8 have good oral bioavailability in
mice (FIG. 1B). Distribution between plasma and brain indicates
penetration over the blood brain barrier. The half-life of the
compounds in mice has been estimated to be about 4.7-4.8 hours.
Pharmacological parameters are shown in table 1.
TABLE-US-00021 TABLE 1 PK parameters of Cpd. 3 and Cpd. 8 after a
single, oral application to rats or mice. Species rat mouse Route
of administration po po Dose (mg/kg) 10 50 Plasma C.sub.max
(.mu.g/ml) 1.4 4.8 Brain C.sub.max (.mu.g/g) 1.3 7.7 Plasma
T.sub.max (hr) 0.5 0.5 Brain T.sub.max (hr) 0.5 0.5 Plasma
t.sub.1/2 (hr) 5.3 Brain t.sub.1/2 (hr) 5.0 Plasma AUC (h*.mu.g/ml)
0.4 20.5 Brain AUC (h*.mu.g/ml) 2.5 30.6
Example 5
Target Engagement of Cpd. 3 and Cpd. 8 in Brain and Muscle
[0628] Brain and thigh muscle was sampled from B57BL/6J mice used
for PK analysis (EXAMPLE 4). Tissue was snap frozen in liquid
nitrogen and stored at -80.degree. C. Tissue was thawed and lysed
in RIPA buffer 1 ml/half brain. Complete protease inhibitor and
PhosStop phosphatase inhibitor (Roche) was added to the buffer
before lysis. Tissue was homogenized manually and centrifuged twice
at 16000 rpm, 4.degree. C. for 20 minutes. Supernatant was frozen
with 10% glycerol at -80.degree. C. Protein concentration was
performed using Bradford reagent. For western blot analysis of AKT
and S6rP phosphorylation, 30 g of protein was denatured with
(3-mercaptoethanol at 95.degree. C. and separated on SDS page gels
at 100 V. Then proteins were transferred to a nitrocellulose
membrane (Bio-Rad, USA) at 80 V. Non-specific binding was blocked
with 5% bovine serum albumin (BSA) (Sigma-Aldrich, USA). Membranes
were incubated over night with primary antibody dissolved in TBST,
5% BSA at 4.degree. C. and with secondary antibody coupled to
horseradish peroxidase (GE Healthcare, UK) for 1 hour at RT and
washed. Imaging of protein bands was performed using Luminol
(Biozym, Hamburg, Germany. On a Li-Cor Odyssey FC reader.
Quantification of S6-phosphorylation was determined with the same
imaging system.
[0629] The following antibodies were used:
[0630] AKT, P-AKT (S473), S6rP, P-S6rP(S235/236): Cell Signaling
(UK)
[0631] .beta.-actin: Sigma Aldrich, USA
[0632] Target engagement of Cpd. 3 and Cpd. 8 could be shown in
brain and thigh muscle after a single oral administration to mice
(FIG. 2). Compounds cross the blood brain barrier and inhibit mTOR
signaling cascade as shown by significantly reduced phosphorylation
of S6rP and AKT. Signaling was altered at 30 min after
administration and lasted for at least 8 hours.
Example 6
mTOR Pathway Activation in Epileptic Mice
[0633] To evaluate whether mTOR signaling was activated in the
epileptic model used for this study, chronic epilepsy was induced
by administration of pilocarpine, an agonist at muscarinic
acetylcholine receptors. Systemic injections of pilocarpine in rats
or mice result in a generalized convulsive status epilepticus (SE)
with subsequent development of spontaneous epileptic seizures
within the following couple of weeks. Six weeks after SE induction
with pilocarpine almost 100% of the mice have developed chronic
epilepsy.
[0634] A ramping-up dosing protocol was used which has the
advantage that pilocarpine can be individually applied according to
the susceptibility of each mouse. Pilocarpine (Sigma-Aldrich,
Germany) was administered to female NMRI mice intraperitoneally at
a dose of 100 mg/kg (Pilo-SE group). If mice did not display SE,
further injections of 100 mg/kg pilocarpine were performed every 20
min until SE developed. As soon as a mouse showed generalized
seizure activity, pilocarpine injections were stopped. To reduce
peripheral side effects of pilocarpine, methylscopolamine
(Sigma-Aldrich, Germany) was administered 30 minutes prior to the
first injection of pilocarpine. Animals were continuously monitored
and duration of convulsive SE was registered [referred to Racine's
scale. SE onset was defined as continuous ongoing seizure activity
following occurrence of one or two generalized tonic-clonic
seizures [stage 4 or 5 on the Racine scale]. SE was characterized
by head nodding in an upright (sitting) body position, Straub tail,
and slight to moderate convulsions of forelimbs, sometimes
interrupted by further generalized tonic-clonic seizures. In order
to reduce mortality, SE was terminated by diazepam (10 mg/kg i.p.;
diazepam-ratiopharm 10, injection solution) 90 min after SE onset.
The following 2-5 days mice were injected with 0.9% sodium chloride
solution twice daily and fed with baby pap because most of the mice
do not eat or drink by themselves during the first days after SE.
In the sham-SE group mice were treated with the same treatment
scheme as the pilo-SE group but pilocarpine was replaced by 0.9%
sodium chloride solution. The mortality in the pilo-SE model in
mice is relatively high (overall about 50%) and can vary markedly
between experimental groups/days. Therefore we had to start with a
large number of mice for SE induction in order to reliably obtain a
group size of 20-22 animals for the MEST.
[0635] A. mTOR Signaling in Brain Samples
[0636] Brain samples from pilocarpine treated mice and untreated
mice were snap frozen and tissue was lysed in buffer containing 25
mM Tris-HCl, pH 8, 50 mM NaCl, 0.5% (w/v) sodium deoxycholate
(DOC), and 0.5% (w/v) Triton X-100 and supplemented with complete
protease inhibitor (Roche, Mannheim, Germany) and Phosphostop (NEB,
USA). Disruption of cell lysates was performed by drawing up the
cell suspension twenty times into a syringe with a small gauge
needle (21G) on ice. Protein concentrations in the lysates were
determined by using the Pierce BCA Protein Assay kit (Thermo
Scientific, Bonn, Germany) according to the manufacturer's
instructions. Equal amounts of total protein were separated on
4-20% SDS-PAGE gels and transferred to PVDF membranes which were
blocked overnight in 5% milk in phosphate buffered saline
supplemented with Tween-20 (PBST: 137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4, 1.4 mM KH2PO4, pH 7.3, 0.05% (w/v) Tween-20) at 4.degree.
C. Membranes were incubated with primary antibodies anti S6 1:1000,
anti S6 Phospho Serin (240/244) 1:1000, anti S6 Phospho Serin
(235/236) 1:2000 (NEB) and anti-Actin 1:100 (Sigma-Aldrich) for 1 h
in 2% milk in PBST at room temperature (RT) and washed three times
for 10 min in PBS-T. Secondary antibodies anti-rabbit-HRP 1:1000
(Dako, Hamburg, Germany) were incubated for 1 h in 2% milk in PBST
at RT and washed three times for 10 min in PBST. Proteins were
detected by enhanced chemiluminescence using SuperSignal West Femto
Chemiluminescent Substrate (Thermo Scientific) and the ChemiDoc
system (Bio-Rad, Munich, Germany) with QuantityOne software
(Bio-Rad) according to the manufacturer's protocol. Relative
protein expressions were quantified densitometrically with
QuantityOne (Bio-Rad) software and calculated by normalization to
the reference signals of actin with GraphPad Prism software
(GraphPad, San Diego, Calif., USA).
[0637] Epilepsy-induced mice showed significantly elevated levels
of total S6-protein as well as P-S6rP (Ser 240/244) (FIG. 3) in
brain samples. As S6rP phosphorylation is a downstream event of
mTOR activation, mTOR signaling was hyperactivated in mice that had
been pre-treated with pilocarpine to induce epileptic seizures. In
conclusion, the data indicate that mTOR overactivation is involved
in epileptogenesis of the pilocarpine model.
[0638] B. mTOR Signaling in Brain Samples after Administration of
Cpd. 3, Cpd. 8 and Everolimus
[0639] Pilocarpine pre-treated and naive mice were treated with a
single oral dose of Cpd. 3 (40 mg/kg), Cpd. 8 (25 mg/kg),
everolimus (5 mg/kg) or vehicle. 5 mice per group were utilized and
sacrificed after 3 hours. Brain lysates were generated and analyzed
by western blot as described under EXAMPLE 6 A. S6 phosphorylation
(S235/236) was quantified with QuantityOne software (Bio-Rad)
according to the manufacturer's protocol.
[0640] In both, naive mice and epileptic mice, S6 phosphorylation
(S235/236) was significantly decreased versus control by Cpd. 3
(4.6 fold; 4.3 fold, respectively) Cpd. 8 (10.9 fold; 2.8 fold,
respectively) (FIG. 3B). No significant changes in mTOR signaling
were observed in everolimus treated mice indicating that both, Cpd.
3 and Cpd. 8 penetrate into the brain at effective doses and
inhibit mTOR signaling in brain tissue in epileptic as well as in
naive mice. Everolimus does seem not reach effective concentrations
in the brain under the conditions used as mTOR signaling is not
inhibited by the compound.
Example 7
Cpd. 3 and Cpd. 8 Inhibit Seizures in the Maximal Electroshock
Seizure Threshold Test (MEST)
[0641] The antiepileptic effect of Cpd. 3 and Cpd. 8 was tested
using the maximal electroshock seizure threshold test (MEST), a
mouse epilepsy model. It has been shown before, that rapamycin
increases the seizure threshold in this model (Macias, M., et al.,
PLoS One, 2013. 8(5): p. e64455.).
[0642] A total of 166 adult female NMRI mice were used obtained
from Charles River (Sulzfeld, Germany) at a weight range of 21-25 g
(mice). Animals were kept under the following conditions: Housing:
Animals were housed in groups of max. 8 mice under controlled
conditions (temperature: 22.+-.1.degree. C.; humidity: 50% 60%),
under a 12-h light-dark cycle (lights on at 6:00 a.m.). Feeding:
Standard laboratory chow (Altromin 1324 standard diet, Altromin
Spezialfutter GmbH, Lage, Germany) was provided ad libitum.
Drinking water: Tap water was provided ad libitum.
[0643] Epilepsy was induced in half of the animals by pilocarpine
administration (see EXAMPLE 4). The MEST determines a population
seizure threshold in groups of -20 animals and not the threshold
for an individual animal. In the present study, the MEST was
determined by a staircase procedure as previously described. A
stimulator (BMT Medizintechnik, Berlin, Germany) that delivered a
constant current (adjustable from 1-200 mA regardless of the
impedance of the animal) with sinusoidal pulses (50/sec) for 0.2
sec was used.
[0644] Current administration was performed via bilateral
transcorneal stimulation (using copper electrodes). Before
transcorneal stimulation, a drop of tetracaine solution (2%) was
administered to the eyes of the mouse for local anesthesia. Two
minutes later, the mouse was restrained by hand to press the copper
electrodes on both corneas while the stimulus was applied by
stepping on a foot pedal switch connected with the stimulator.
Electrodes were covered with soft leather and soaked with saline
before each current application. Directly after stimulation, the
mouse was released from restraint in order to permit observation of
seizures exhibited. The stimulus intensity was varied by an
up-and-down method in which the current was lowered or raised by
0.06 mA log intervals according to whether the preceding mouse did
or did not exert a tonic hindlimb extension. The first stimulation
was started with a current near to the control threshold.
Drug solutions were prepared freshly before every experiment:
[0645] Phenobarbital (sodium salt): 15 mg in 10 ml distilled water
[0646] Levetiracetam: 50 mg in 10 ml distilled water [0647]
Rapamycin: 5 mg in 10 ml vehicle (4% ethanol (100%), then 5% PEG
400 and 5% Tween 80) [0648] Everolimus: 5/10 mg in 10 ml vehicle
(8% ethanol, 10% PEG400, and 10% Tween 80) [0649] Cpd. 3 (HCl):
40/100 mg in 10 ml (Suspension; 5% PEG 400 and 2% Tween 80, HPBCD
(10%)) [0650] Cpd. 8 (HCl): 12.5/25 mg in 10 ml (5% PEG 400 and 2%
Tween 80, HPBCD (10%))
[0651] The data generated from groups of 20-22 nonepileptic control
(sham-SE) and 20-25 epileptic mice (pilo-SE) were used to calculate
the CC50 (convulsant current that induces a tonic hindlimb seizure
in 50% of the mice per group with confidence limits for 95%
probability). Statistical analysis were calculated using Student's
t-test. All tests were used two-sided; a P<0.05 was considered
significant.
[0652] Experiments were performed in groups of 20-25 mice. Average
CC50 control values (without vehicle injection) were 15.8.+-.0.36
mA in nonepileptic mice (mean.+-.SEM of 5 threshold determinations
in 3 different groups of mice) and 15.6.+-.0.45 mA in epileptic
mice (mean.+-.SEM of 5 threshold determinations in 3 different
groups of mice). Phenobarbital produced the most pronounced
anticonvulsant effect of the compounds tested in this study. The
CC50 was 25.7 mA in nonepileptic and 26.56 mA in epileptic mice
which is a threshold increase of 69% and 66%, respectively. There
was no significant difference between nonepileptic and epileptic
mice (FIG. 4). Levetiracetam (50 mg/kg) significantly increased the
CC50 in nonepileptic mice by 21% while there was no effect in
epileptic mice (Table 2, FIG. 4).
[0653] 5 mg/kg rapamycin significantly increased the seizure
threshold in MEST by 13% in nonepileptic and only by 5% in
epileptic mice (FIG. 4) as shown before by Hartman et al. [25].
After intraperitoneal injections rapamycin increased the threshold
only in nonepileptic mice by 20%, while there was no effect in
epileptic mice (FIG. 4). 10 mg/kg and 5 mg/kg everolimus
significantly increased the CC50 by 10% and 13% in nonepileptic
mice, respectively. In epileptic mice, only the lower dose
increased the CC50 by 16% while 10 mg/kg was not effective (Table 3
and FIG. 4).
[0654] When 40 mg/kg Cpd. 3 was applied only 1 h before threshold
determination, CC50 was significantly decreased in both, epileptic
and nonepileptic mice. When pretreatment time was prolonged to 3 h,
CC50 was significantly increased by 6% only in nonepileptic mice
while CC50 in epileptic mice was no longer decreased but remained
on control level. Application of a higher dose (100 mg/kg) led to a
significant increase of the CC50 by 9% in epileptic but not in
nonepileptic mice (Tab. 2 and FIG. 4).
[0655] Overall, Cpd. 3 exhibited a slight but inconsistent
anticonvulsant effect with the parameters tested in this study. It
seems that prolongation of the pretreatment time has a favourable
effect. The use of a suspension could account for the marginal
effect and the apparent inconsistency of the data.
[0656] Cpd. 8 exhibited a pronounced dose-dependent anticonvulsant
effect in nonepileptic mice. CC50 was increased by 30% (25 mg/kg)
and 8% (12.5 mg/kg). This is comparable to the anticonvulsant
effect of levetiracetam. Reduction of the pretreatment time to 1 h
reduced the anticonvulsant effect. When the pretreatment time was
increased to 24 h the anticonvulsant effect vanished (Tab. 3 and
FIG. 4). In epileptic mice Cpd. 8 did not exert an anticonvulsant
effect with any dose or pretreatment time tested. When the
pretreatment time was reduced to 1 h, Cpd. 8 decreased the CC50 by
9% similar to Cpd. 3 (Tab. 3 and FIG. 4 A-D).
[0657] We performed no special tests for the determination of
adverse effects. Mice were observed about 30 min after injection in
their home cages for obvious sedation or reduction of wellbeing.
Five minutes before electrical stimulation mice were again observed
in their home cages and two minutes before stimulation during
handling procedure for treatment with local anaesthesia. Every
obvious variation from normal behavior was noted. Only
phenobarbital induced noticeable sedation which lasted for more
than 60 min. All other test drugs (including vehicle solutions)
were tolerated without any obvious adverse effects.
TABLE-US-00022 TABLE 2 Seizure thresholds of the Cpd. 3 versus
everolimus/levetiracetam. For every threshold the current necessary
to induce a seizure with full hindlimb tonus in 50% of the mice of
the respective group (CC50) and the standard deviation is
indicated. Further, the threshold change of the drug tests in
percent in relation to the vehicle threshold and the threshold
difference of the nonepileptic and epileptic mice in percent for
each test are shown. 50 mg/kg Vehicle p.o. 40 mg/kg 40 mg/kg
Levetiracetam 5 mg/kg 10% HP-.beta.-CD Cpd. 3 p.o. 100 mg/kg Cpd.
Cpd. 3 p.o. i.p. everolimus p.o. 5% PEG400 pretreatment 3 p.o.
pretreatment pretreatment pretreatment 3 h 2.5% Tween80 3 h
pretreatment 3 h 1 h 1 h Interval to SE 9 weeks after SE 10 weeks
after SE 13 weeks after 14 weeks 15 weeks 16 weeks after induction
SE SE CC50 Sham-SE 18.62 .+-. 1.1* 16.44 .+-. 1.17 17.49 .+-. 1.20*
16.72 .+-. 1.72 15.14 .+-. 1.06* 19.95 .+-. 1.22* (nonepileptic)
[mA] CC50 Pilo-SE 22.28 .+-. 2.16* 19.20 .+-. 1.28 18.20 .+-. 1.35
20.85 .+-. 1.38* 17.99 .+-. 1.39* 19.15 .+-. 1.79 (epileptic) [mA]
Threshold 20% 17% 4% 25% 19% -4% difference between nonepileptic
and epileptic [%] Threshold nonepileptic 13% 6% 2% -8% 21%
difference epileptic 16% 5% 9% -6% 0% between vehicle and drug
threshold [%] *p < 0.05; ANOVA + post hoc Dunnett's test always
compared to vehicle group (yellow). Significant differences between
nonepileptic and epileptic mice are not indicated.
TABLE-US-00023 TABLE 3 Seizure thresholds of the Cpd. 8 versus
everolimus. For every threshold the current necessary to induce a
seizure with full hindlimb tonus in 50% of the mice of the
respective group (CC50) and the standard deviation is indicated.
Further, the threshold change of the drug tests in percent in
relation to the vehicle threshold and the threshold difference of
the nonepileptic and epileptic mice in percent for each test are
shown. 25 mg/kg Cpd. Vehicle p.o. 12.5 mg/kg 25 mg/kg 10 mg/kg 25
mg/kg 8 p.o. 10% HP-.beta.-CD Cpd. 8 p.o. Cpd. 8 p.o. everolimus
p.o. Cpd. 8 p.o. pretreatment 5% PEG400 pretreatment pretreatment
pretreatment pretreatment 3 h 2.5% Tween80 3 h 1 h 3 h 24 h
Interval to SE 9 weeks after SE 10 weeks 11 weeks 12 weeks 13 weeks
after 14 weeks induction SE CC50 Sham-SE 22.91 .+-. 3.08* 15.62
.+-. 1.19 16.84 .+-. 1.29 17.71 .+-. 1.20* 17.14 .+-. 1.11* 15.62
.+-. 1.10 (nonepileptic) [mA] CC50 Pilo-SE 17.78 .+-. 1.38 16.84
.+-. 1.14 16.67 .+-. 1.19 15.25 .+-. 1.17* 16.98 .+-. 1.06* 16.47
.+-. 1.14 (epileptic) [mA] Threshold difference -22% 8% -1% -14% 1%
5% between nonepileptic and epileptic [%] Threshold Nonepileptic
30% 8% 13% 10% 0% difference epileptic 6% -1% -9% 1% -2% between
vehicle and drug threshold [%] *p < 0.05; ANOVA + post hoc
Dunnett's test always compared to vehicle group (yellow).
Significant differences between nonepileptic and epileptic mice are
not indicated.
Example 8
Viability and Protein Synthesis of STHdh Cells Treated with mTOR
Inhibitors
[0658] STHdh cells are immortalized striatal cells derived from a
knock-in mouse model expressing full-length HTT with 111
CAG-repeats (STHdh.sup.Q111/Q111) Control STHdh.sup.Q7/Q7 cells
were generated from wildtype mouse embryos. Cells can be
differentiated into neuronal cells with a dopamine containing
cocktail.
A. LDH Assay
[0659] LDH is a cytoplasmic enzyme that is excreted upon damage to
the plasma membrane, e.g. during apoptosis. Cytotoxicity of test
compounds was measured using the lactate dehydrogenase (LDH) assay
from Roche, Switzerland according to the manufacturer's
instructions.
[0660] STHdh.sup.Q111/Q111 and STHdh.sup.Q7/Q7 (Coriell Institute
for Medical Research, USA) were cultured in DMEM (Invitrogen, USA)
supplemented with FBS (Invitrogen, USA) 10%, Geniticine (G418,
Biochrom, Germany)) 1% and antibiotic antimycotic (Invitrogen, USA)
1% in a humidified incubator at 37.degree. C. and 5% CO2. For all
examples described for this invention, STHdh cells were
differentiated at the time of compound addition using a final
concentration of 50 .mu.M Forskolin, 750 .mu.M IBMX, 200 nM TPA, 10
.mu.M dopamine, 10 .mu.g/.mu.l .alpha.-FGF in culture media.
10.sup.4 STHdh.sup.Q7/Q7 and STHdh.sup.Q111/Q111 cells per well
were seeded in a 96 well plate incubated with Cpd. 3 (130 nM, 1230
nM), Cpd. 8 (400 nM and 1230 nM), INK128 (100 nM) and rapamycin
(400 nM) and LDH was measured after 8h, 24h, 34h, 48h and 72h (FIG.
5 A, B).
[0661] None of the compounds tested induced increased LDH activity
in the media indicating that no cytotoxicity was induced by the
mTOR inhibitors. At higher concentrations of Cpd. 3 and Cpd. 8 the
LDH activity was decreased compared to control. mTOR inhibitors
were well tolerated by STHdh cells and the number of apoptotic
cells was not increased by cell treatment of up to 72 hours. In
conclusion, compounds of this invention are suitable for assessment
in these cell lines and, furthermore, compounds do not appear to
have a direct effect on general the metabolic activity of neuronal
cells in general.
B. PrestoBlue Assay
[0662] Cell viability/metabolic activity was detected using the
PrestoBlue viability reagent (Invitrogen, USA) according to the
manufacturer's instructions. 10.sup.4 STHdh.sup.Q7/Q7 and
STHdh.sup.Q111/Q111 cells per well were seeded in a 96 well plate
incubated with Cpd. 3 (130 nM, 1230 nM), Cpd. 8 (400 nM and 1230
nM), INK128 (100 nM) and rapamycin (400 nM) and PrestoBlue was
measured after 8h, 24h, 34h, 48h and 72h. Non-toxic concentrations
determined were transferred to primary neurons used later on in
this study. Cells did not show changes in metabolic activity at 8h,
24h, 34h and 48 h for each of the compounds tested (FIG. 5 C, D).
At lower concentration of Cpd. 3 and Cpd. 8 even a slight increase
in mitochondrial activity could be detected. At 72 hours decreased
metabolic activity was detected after treatment with 1230 nM of
Cpd. 3 and (not significantly) also Cpd. 8 in STHdh.sup.Q111/Q111.
This effect has not been observed in STHdh.sup.Q7/Q7 cells
indicating that 1. Wild type cells are less sensitive to mTOR
inhibition and 2. Compounds are well tolerated and therefore
suitable for testing as autophagy-inducers in these cell lines.
Example 9
Cpd. 3 and Cpd. 8 Inhibit mTOR Signaling in STHdh.sup.q7/q7 and
STHdh.sup.Q111/Q111 Cells
[0663] In order to test whether Cpd. 3 and Cpd. 8 inhibit mTOR
signaling and induce autophagy in neuronal cell models of HD,
treatment of STHdh.sup.Q111/Q111 and STHdh.sup.Q71/Q7 cells was
employed. STHdh.sup.Q111/Q111 and STHdh.sup.Q7/Q7 cells were
maintained as described under EXAMPLE 6. Cells were seeded in 10 cm
dishes. When cells were about 80% confluent, cells were
differentiated and treated with compounds at the same time. Cells
were treated with Cpd. 3 (200 nM, 400 nM, 1230 nM), Cpd. 8 (130 nM,
1230 nM), INK128 (100 nM), rapamycin (400 nM) or DMSO control for 4
hours. At the end of incubation time cells were washed with cold
PBS buffer and lysed with 200 .mu.l of RIPA buffer supplemented
with 4% Complete protease inhibitor (Roche, Switzerland) and 10%
PhosStop (Roche, Switzerland). After 30 min of incubation on ice,
lysates were vortexed, centrifuged (20 min, 4.degree. C., 16000
rpm) and supernatants were collected. Protein concentration was
determined as described above. 30..mu.g of protein per sample was
analyzed by western blot as described above using the following
primary antibodies:
TABLE-US-00024 4E-PB1 (1:1000), P-4E-BP1 (T37/46) (1:1000), Cell
Signaling, UK mTOR (1:1000), P-mTOR(S2448) (1:1000), S6rP (1:1000),
P-S6rP (S235/236) (1:1000) beta-actin (1:50000) Sigma-Aldrich, USA
LC3 (1:200) Nanotools, Germany
[0664] Cpd. 3 inhibited mTOR signaling as indicated by reduced S6rP
phosphorylation and 4E-BP phosphorylation in both,
STHdh.sup.Q111/Q111 and STHdh.sup.Q7/Q7 cells in a concentration
dependent manner. Reference compounds/positive controls INK128 and
rapamycin showed a similar degree of pathway inhibition (FIG. 6A).
Cpd. 8 also inhibited mTOR signaling. An effect could be observed
already at a low concentration of 130 nM in STHdh.sup.Q7/Q7 cells
(FIG. 6B).
[0665] In order to detect levels of LC3-II, an autophagy marker in
autophagosomes, cell treatment was performed in the presence and
absence of Bafilomycin A, an inhibitor of autophagosome
degradation. Thus compounds lead to accumulation of LC3-II levels
when autophagy is induced. Cpd. 3 (FIG. 6C) and Cpd. 8 (FIG. 6D)
induced autophagy in striatal cells. For Cpd. 3 a stronger effect
on autophagy was observed in STHdh.sup.Q111/Q111 cells. Autophagy
induction was not significant in the non-mutHTT counterpart, but
signs of LC3II-elevation were observed in these cells as well. Cpd.
8 also induced autophagy in striatal cell lines. in a level
comparable to INK 128.
[0666] Data indicate that Cpd. 3 and Cpd. 8 inhibit mTOR signaling
in striatal cell lines that carry mutHTT or unmutated-Q7 extended
huntingtin. Inhibition of the mTOR pathway leads to induction of
autophagy. The basic mechanism for huntingtin aggregate clearance
via macroautophagy was induced by the mTOR or PI3K/mTOR inhibitor
Cpd. 3 and Cpd. 8 in a neuronal environment.
Example 10
Cpd. 3 and Cpd. 8 Inhibit Aggregate Formation in HEK Cells
Expressing Exon1 of mutHTT
[0667] HEK cells (DSMZ, Germany) were maintained in DMEM/Glutamax
(Invitrogen, USA), 1% Antibiotic/Antimycotic (Invitrogen, USA), 10%
FBS in a humidified incubator at 37.degree. C. and 5% CO2. HEK
cells were transiently transfected with pcDNA3.1/V5-His vector from
Clontech (Mountain View, USA) containing the expression sequence
for Exon1 of HTT with a 19Q (no aggregation) or 51Q extension and
enhanced green fluorescence protein (eGFP) tag using Attractene
Transfection Reagent (Qiagen, Netherlands). 1.times.10.sup.6 cells
were seeded in a 6 well plate. On the next day 2 .mu.g DNA were
mixed with 95.5 .mu.l transfection media and 4.5 .mu.l transfection
reagent. After an incubation time of 15 min the mixture was added
to the cells. After 72 hours cells were harvested or fixed for
analysis.
[0668] A. Cpd. 3 and Cpd. 8 Reduce Aggregate Formation in
Transfected HEK Cells Shown in a Filter Trap Assay
[0669] HEK cells transfected with 19Q- or 51Q-HTT-Exon1-eGFP were
treated with DMSO control. 51Q-HTT-Exon1-eGFP HEK cells form
aggregates. These cells were treated with Cpd. 3 (400, 1230 nM),
Cpd. 8 (130, 1230 nM), INK128 (100 nM) or Rapamycin (400 nM) for 8
hours and then lysed with RIPA buffer. After 30 min incubation on
ice, cells were homogenized (Dounce Homogenizer, Thermo Fisher,
USA). 50 .mu.g of protein solution in PBS was supplemented with 2%
SDS (Roth, Germany). Samples were sucked through a nitrocellulose
membrane (0.45 .mu.m) and washed twice with PBS buffer. Aggregated
proteins do not dissolve in SDS solution and bind to the membrane.
mutHTT was detected on the membrane using a ployQ antibody (1C2,
Millipore, Germany) diluted 1:1000 in TBST supplemented with 5%
milk powder and a HRP conjugated secondary anti-mouse antibody
(FIG. 7A, B) Bands were quantified with the Odyssey LI-CORE system
(Li-Core, USA) (FIG. 7B).
[0670] Data demonstrates that treatment of 51Q-HTT-Exon1 HEK cells
with mTOR inhibitors (Cpd. 3, Cpd.8, INK128 and Rapamycin) leads to
significant reduction of cytotoxic mutHTT aggregates in these
cells. 130 nM of Cpd. 8 was not sufficient to induce significant
aggregate reduction. Aggregate clearance led to a reduction of
aggregates that did not reach total clearance (compared to
19Q-HTT). As induction of autophagy has been demonstrated before,
it can be assumed that aggregate clearance is performed by
macroautophagy.
[0671] B. Cpd. 3 and Cpd. 8 Reduce Aggregate Formation in
Transfected HEK Cells Shown by Immune Cvtochemistry
[0672] HEK cells were seeded in poly-L-lysine (Sigma-Aldrich, USA)
coated coverslips in 24 well plates at 10.sup.4 cells per well and
transiently transfected with 19Q- or 51Q-HTT-Exon1-eGFP. Cells were
treated with Compounds as described under A for 8 or 24 hours. This
incubation ended 72 hours after transfection. Cells were fixed with
4% (paraformaldehyde) PFA. Coverslips were fixed glass slides using
mounting media that contained 4',6-Diamidin-2-phenylindol (DAPI)
(FIG. 7C). Nuclei and aggregates containing HTT-Exon1-eGFP were
counted manually. 10 000 cells were counted per sample (FIG.
7D).
[0673] Since the fluorescence of eGFP labeled HTT is very bright,
samples treated with DMSO appear darker overall. mTOR inhibitor
reduced number of mutHTT aggregates in HEK cells by macroautophagy
indicating that this mechanism might also clear huntingtin
aggregates in neurons of animal models or patients. The effect was
more pronounced after 24 hours (77% reduction, Cpd. 8, 130 nM; 73%
reduction, Cpd. 3, 400 nM) as compared to 8 hours (55% reduction
Cpd. 8, 130 nM; 66% reduction Cpd. 3, 400 nM) of treatment. In
terms of dose finding, lower concentrations of Cpd. 3 (400 nM) and
Cpd. 8 (130 nM) appear to be at least as efficient as high
concentrations. So it might be possible to treat patients at lower,
non-toxic doses.
Example 11
Cpd. 3 and Cpd. 8 Induce Autophagy in Brain of Wt Mice
[0674] In order to investigate whether mTOR inhibition by Cpd. 3
and Cpd. 8 induces autophagy--the essential mechanism for
huntingtin aggregate clearance--in the brain, BALB/c nude mice were
treated with Cpd. 3 and Cpd. 8 as described in sample 5. Brain
lysates were analyzed by western blot as described in example 5
using a LC3 antibody (1:20, Nanotools, Germany) and a p62 antibody
(SQTS1/p62 1:1000, Cell Signaling, USA) and the according secondary
HRP-coupled antibodies (FIG. 8 A, B).
[0675] Autophagy induction was proven by time-dependent increase in
the autophagic marker LC3II (upper band) and decrease in the
autophagic marker p62 after a single oral administration of both,
Cpd. 3 and Cpd. 8. Data indicate that concentration of compounds in
the brain is sufficient to induce autophagy in neuronal cells. As
induction of autophagy has been shown to lead to clearance of
huntingtin aggregates in cell models, Cpd. 3 and Cpd. 3 as well as
other mTOR inhibitors are likely to induce autophagic reduction of
mutHTT aggregates in animals or humans.
Example 12
Effect of Chronic Treatment of Cpd. 3 and Cpd.8 on Electrographic
Seizures in TSC1GFAP Ko Mice
[0676] This study was done to test the effect of Cpd. 3, Cpd. 8 and
a reference compound (Cpd. R, CAS-No 1225037-39-7) on spontaneous
seizures and mortality as compared to vehicle treatment in Tsc1GFAP
knockout mice. Tsc1 (Tsc1.sup.flox/flox-GFAP-Cre (Tsc1.sup.GFAP
conditional knockout) is a mouse model of TSC with conditional
inactivation of the Tsc1 gene in GFAP-positive cells (Tsc1GFAPCKO
mice), which develops progressive epilepsy, encephalopathy, and
premature death, as well as cellular and molecular brain
abnormalities likely contributing to epileptogenesis.
[0677] A: Animals and Treatment
[0678] Tsc1 mice belonging to either sex were acclimated to the
environment, examined, handled, and weighed prior to initiation of
the study to assure adequate health and suitability and to minimize
non-specific stress associated with human handling. During the
course of the study, 12/12 light/dark cycles were maintained. The
room temperature was maintained between 20 and 23.degree. C. with a
relative humidity maintained around 50%. Food and water were
provided ad libitum for the duration of the study. Each mouse was
randomly assigned to designated treatment groups. The dosing was
performed during the animals' light cycle phase.
[0679] Aseptic technique was used throughout the surgical
procedures described below. Mice were anesthetized with isoflurane
(3% for induction and 1-2% for maintenance with oxygen as a carrier
gas; approximate flow rate, 1 liter per minute) and placed on a
homeostatic heating pad to maintain the core temperature at
37.+-.1.degree. C. Adequate plane of anesthesia was insured by the
lack of a withdrawal response to toe pinch and a visible change in
the breathing rate. The mouse head was immobilized in a stereotaxic
frame with a nose piece that supplied the anesthetic continuously.
Ophthalmic ointment was applied to the eyes to prevent drying of
the cornea. The area for surgical incision was identified and
scrubbed with chlorhexidine followed by an alcohol scrub. A dorsal
skin incision was made on the head in a rostro-caudal direction and
the skin was reflected to reveal the skull. Subcutaneous tissues
were blunt dissected away with a sterile cotton-tipped applicator
and saline. The skull was cleaned to reveal bregma. Using a dental
drill or a 20-23 gauge needle, a small hole was made to allow
implantation of the indwelling electrode wire or skull screws. An
8201-EEG head mount (Pinnacle Technology, Inc., Lawrence, Kans.)
with bi-hemispheric leads in the frontal and parietal cortices and
an indwelling local field potential electrode targeting the region
above the CA1 were used. Initial fixation of this mount to the
skull was done by super glue. The headmount was then cemented onto
the skull with dental acrylic. The headmount and screws were
covered with dental cement. Application of the dental cement also
serves to close the wound. Typically, the skin is pulled taught
around the implant/headmount and only skin caudal to the implant
remains unopposed. Once the dental acrylic cured, isoflurane was
turned off and the animal was removed from the stereotaxic
apparatus.
[0680] After surgery, the animal was placed in a clean recovery
cage placed on a heated warm water circulating heating pad under
half of the cage until it is ambulatory. Before, during, and after
surgery, animals were administered fluids, nutrition, antibiotics,
and analgesics as required/recommended by the Program of Veterinary
Care (PVC) team and IACUC, in concert with the Attending
Veterinarian and/or according to IACUC Guidelines. Animals were
checked daily by Animal Care staff and during the 7 day post-op
period. Only animals that were fully recovered from surgery
(healthy-looking and displaying normal behaviors such as eating,
grooming, exploring and nesting) were used in experiments.
Post-surgery, mice were single-housed. Animals were housed
individually to prevent other animals from contaminating the
surgery site or damaging the implant. Mice were implanted with
electrodes at the age of P22 to P27 and allowed to recover for up
to 4 weeks of age (P35). A total of 56 mice were implanted to yield
four groups of .about.10 mice for the study.
[0681] EEG was recorded continuously using the Pinnacle Technology
8206 data conditioning and acquisition system (DCAS), which
performs secondary amplification and filtering before sending data
to Pinnacle's Sirenia.RTM. Acquisition software for collection via
a USB connection. EEG was recorded using 10.times. or 100.times.
gain pre-amplifiers. For seizure activity, which elicits large
spikes in amplitude the 10.times. gain is typically optimal. The
pre-amplified tether was connected to a low-torque commutator
mounted above the cage and allows for unencumbered freedom of
movement and reduced movement artifacts. Real-time visualization of
all EEG channels from all mice was observed using Sirenia or
PAL-8400 software. Synchronized video recordings were collected for
the duration of the EEG recordings.
[0682] Compounds were dissolved in vehicle consisting of 20%
Dexolve-7 (Davos Pharma, Liberty, Mo., USA) in water, pH 3. Mice in
the study were randomly assigned to one of the following treatment
groups: vehicle 10 ml/kg (group D; post natal day (PND) 21-53; p.o.
q.d.), Cpd. R 50 mg/kg (group C; PND21-53; p.o. q.d.), Cpd. 8 25
mg/kg (group A; PND21-53; p.o. q.d.), Cpd. 3 100 mg/kg (group B;
PND21-53; p.o. q.d.).
[0683] B: Electroencephalographic Recording and Analysis
[0684] After postoperative recovery was complete, mice were
tethered to the pinnacle recording system individually in their
home cages and spontaneous EEG was recorded for the following two
weeks (PND 35-48; weeks 6 and 7). Simultaneously, videographic
recording was made through this period to provide a data stream for
behavioral evaluation of the mice as needed. After the recording
period was completed, individual EEG traces were evaluated for
aberrant EEG activity. Electrographic seizures were identified by
their characteristic pattern of discrete periods of rhythmic spike
discharges that evolved in frequency and amplitude lasting at least
10 seconds, typically ending with repetitive burst discharges and
voltage suppression. These were defined as having very
stereotypical beginning, end, and evolution in the middle, starting
with low amplitude fast activity and high frequency spikes (tonic),
which gradually evolved into a slower, bursting (clonic) phase
followed by a severe voltage suppression often with rhythmic
artifact superimposed, which represents respirations and is picked
up clearly because the EEG itself is suppressed. Most typical
seizures in the TS mice last at least 30-40 seconds. Note that
these mice have abnormal EEG patterns often featuring frequent runs
of interictal spikes, which can also be called spike trains, but we
did not consider them seizures based on the above criteria (usually
there is no stereotypical evolution). Seizure incidence was
tabulated for each mouse and summarized for each treatment group
for week 6 (PND36), week 7, and week 8 (PND55).
[0685] Data were analyzed by analysis of variance (ANOVA). If one
or more of these factors were significant (P<0.05), further
post-tests (Dunnet's or Wilcoxon) were done to identify which
specific contrasts (i.e. Vehicle v. Cpd. R for week 6) were
significant. P values were adjusted for multiple comparisons and a
p<0.05 was deemed significant. Summary data are reported as
group mean.+-.standard error of the mean (SEM).
[0686] Body Weight
[0687] The body weight of individual subjects assigned to different
treatment groups prior to any treatment is shown in FIG. 9A. A
one-way ANOVA revealed no statistical difference between groups.
Body weight of animals treated with Cpd. 3 and Cpd. 8 was higher
towards the end of the study. At this time the normal life span of
TSC mice is close to the end. They will lose more weight and die.
As treated animals show higher weight they appear to have improved
health status and therefore might have an extended life span.
[0688] Electrographic Seizures and Mortality
[0689] Whereas Tsc1 genotype mice treated with vehicle daily from
PND21-55 suffered robust electrographic seizures (n=150), mice
treated with 25 mg/kg Cpd. 8 from PND21-53 showed significantly
lower numbers of seizures (n=8) and mice treated with 100 mg/kg
Cpd. 3 from PND21-53 showed significantly lower numbers of seizures
(n=2) over the same period. A reference group that received Cpd. R
from PND21-55 showed a modest reduction in the number of seizures
(n=76) that was significantly different from vehicle when across
all time points. Pairwise Wilcoxon tests were conducted to identify
which specific contrasts were significant (FIG. 9B). The incidence
in seizures was significantly different between vehicle and Cpd.
8/Cpd. 8 at weeks 6 (PND35-42; p=0.0009 and p=0.0005 respectfully),
at weeks 7 (PND43-48; p=0.0427 and p=0.0023), and at weeks 8
(PND49-55; p=0.0017 and p=0.0002) (FIG. 9B).
[0690] The protective effect of Cpd. 3 and Cpd. 8 against seizures
in the Tsc1 GFAP conditional knockout mice could be shown. Cpd. R
has a weaker seizure-suppressive effect than the new compounds
tested. Likely, this can be explained by the shorter half-life of
Cpd. R (1 1 hour) compared to Cpd. 3 (5 hours) and Cpd. 8 (5
hours).
Example 13
Treatment of R6/2 Mice and zQ175 Mice
[0691] R6/2 mice are a B6CBA-Tg(HDexon1)62Gbp/1J mouse model
expressing exon 1 of the human huntingtin gene with an extension of
160.+-.5 CAG repeats. Mice have a severe phenotype that develops
between 4 and 12 weeks. A PK/PD study in R6/2 mice is being
performed in order to determine a dose that is as low as possible
and still engages the target in different brain regions (striatum,
cortex, cerebellum). R6/2 mice and their wt counterparts are
treated in the following treatment groups consisting of 19 animals:
1. Wt, vehicle; 2. R6/2 vehicle; 3. R6/2 Cpd. 3; 4. R6/2 Cpd.8. Per
oral dosing is performed for 5 days with a following drug holiday
of 2 days for a total of 8 weeks. After 4 and 8 weeks animals are
tested in two phenotypic tests: LabMaster and Rotarod.
Consecutively, mice are sacrificed and brains are analyzed for mTOR
signaling and induction of autophagy using western blot analysis as
well as for levels of soluble and aggregated mHTT using TR-FRET
assays.
[0692] zQ175 knock-in (KI) mice exhibit extensive behavioral,
histopathological, and molecular phenotypes reminiscent of human
disease. These mice exhibit age-associated increase in mHTT
inclusions from 2-12 months of age in the striatum and cortex. 10
mice per group will be treated with 1. Vehicle; 2. Cpd. 3; 3. Cpd.
8 from 3 months to 5 months of age. Mice will be given peroral
dosing of compounds or vehicle for 5 consecutive days followed by 2
days of drug holiday. Key readouts are 1) Determine mHTT aggregate
formation in homogenate from striatum and muscle quadriceps using
Singulex (central versus peripheral effects).
[0693] Compound levels in plasma, brain and quadriceps muscle on
the last day of dosing (2h post dose) from experimental animals.
Satellite animals will be used on day one of study to determine PK
and basal HTT levels.
Example 14
Treatment of R6/2 Mice in an In Vivo Model of Huntington's
Disease
[0694] An in vivo model of Huntington's disease was used to
evaluate the efficacy of Cpd. 3 and cpd. 8 in reducing mutHTT
aggregate formation in the striatum. R6/2 mice are a
B6CBA-Tg(HDexon1)62Gbp/3J Huntington's disease mouse model
expressing exon 1 of the human huntingtin gene with an extension of
160.+-.5 CAG repeats. Mice have a severe phenotype that develops
between 4 and 12 weeks. As the animals are extremely fragile, doses
had to be reduced to 65% of the doses used in more robust animal
models.
[0695] B6CBA-Tg(HDexon1)62Gpb/3J (R6/2) mice were obtained from 40
breeding pairs of wildtype males with ovarian transplant females
purchased from Charles River (Charles River Laboratories, Sulzfeld,
Germany). The pups were transgenic for the human N-terminal
fragment of the HTT gene. For this study male mice were used and
were kept in separate cages to reduce the stress of hierarchy
fights from weaning on. Standard cages with a 12 hours light/dark
cycle were used. Standard chow and water were supplied ad
libitum.
[0696] R6/2 Pharmacokinetics
[0697] In order to test exposure of brain areas, R6/2 mice were
treated with a single oral dose of Cpd. 3 (75 mg/kg, 50 mg/kg, 25
mg/kg) or Cpd. 8 (25 mg/kg, 16 mg/kg, 8 mg/kg), 3 animals per
group. After one hour plasma was collected and animals were
sacrificed. Striatum, cerebellum and cortex was separately snap
frozen and analyzed for compound concentration using LC/MS/MS.
[0698] Tissue concentrations reached were well in the efficacious
range for all Cpd. 3 concentrations. For Cpd. 8 sufficient levels
were reached at all concentrations, although 8 mg/kg might give a
slightly too short target coverage (FIG. 10A).
[0699] Compound Longterm Treatment of R6/2 Mice
[0700] Treatment groups were established from genotype and
measurement of weight, rotarod performance and litter. They were
distributed equally over the four groups, each group contained 12
animals. R6/2 mice and their wt counterparts were dosed in the
following treatment groups: 1. Wt, vehicle (20% SBECD in water, pH
3); 2. R6/2 vehicle (20% SBECD in water, pH 3); 3. R6/2 Cpd. 3 (65
mg/kg); 4. R6/2 Cpd.8 (16.25 mg/kg). Per oral dosing was performed
for 6 days with a following drug holiday of 1 day for a total of
11.5 weeks.
[0701] Immunohistochemistry
[0702] To detect mutHTT aggregates, immunostaining of striata was
performed. Brains were fixed in 4% paraformaldehyde (SAV LP GmbH,
Flintsbach am Inn, Germany). Before embedding in O.C.T (Sakura
Finetek Germany GmbH, Staufen im Breisgau, Germany)), whole brains
were soaked in sucrose solution (30% w/v) for 3 days and cut
serially into 25 m coronal sections on a cryostat (Leica CM-3050-S,
Leica Biosystems Nussloch GmbH, Germany). Sections were stored in
PBS, supplemented with 0.03% sodium acetate at 4.degree. C. For
free-floating staining, striatal sections were placed into fresh
PBS. All steps were performed at room temperature. Blocking was
performed in 0.5% sodium borohydride in PBS for 30 minutes and
followed by washing. Primary antibody incubation with EM48
(MAB5374; Merck Chemicals GmbH, Darmstadt, Germany) diluted 1:1000
was performed overnight. On the following day sections were washed
with TBST and incubated with biotinylated goat anti-mouse IG-G
(Vecta BA9200, Vector Laboratories, Burlingame, Ca) at a 1:1000
dilution for 2 hours. Avidin-biotin complex (Vectastain.RTM. Elite
ABC Kit, Vector Laboratories, Burlingame, Ca) was used at a 1:400
dilution. Incubation time was 1 hour. To further enhance the
signal, ABC incubation was repeated after biotinylated tyramine,
supplemented with 0.001% H202 was administered for 8 minutes. For
color development, sections were incubated for 4 minutes in a
nickel-DAB-H202 containing buffer (0.6% nickel, 0.01% DAB and
0.001% H202 in 0.05 M Tris, 0.05 M imidazole). Reaction was stopped
by placing the sections in TI buffer (0.05 M Tris, 0.05 M
imidazole) and mounted in water, free floating. Sections were
dehydrated by ethanol and xylol dilution row and sealed under
coverslips with mounting medium (CV, Leica).
[0703] Images were acquired using a Zeiss Axioplan microscope
(Plan-NEOFLUAR .times.40/0.75 objective, AxioCam MRc) and
Axiovision 4.8 software (Carl Zeiss Microscopy GmbH, Jena,
Germany). Per animal three subsequent sections of the striatum were
analyzed, 4 animals were analysed The EM48 positive structures were
analyzed with the ImageJ built in particle analysis, with a fixed
threshold for all pictures (ImageJ 1.47v; NIH, Bethesda, Md., USA).
Statistical analysis was performed with GraphPad Prism 6 (GraphPad
Software Inc., La Jolla, Calif., USA). One-way ANOVA was performed
with multiple comparisons to the placebo group and Dunnet's
correction method.
[0704] Image analysis of stained striata showed specificity of
staining as no aggregates were detected in wt animals (FIG. 10B).
Furthermore, the number of aggregates counted was not changed in
Cpd. 3 or Cpd. 8 treated R6/2 mice versus vehicle controls. But the
seize of mutHTT aggregates was slightly (although not
significantly) reduced in Cpd. 8 treated animals and decreased by
40% in Cpd. 3 treated animals (FIG. 10B). This reduction in
aggregates could influence disease progression in HD patients.
* * * * *