U.S. patent application number 16/916168 was filed with the patent office on 2020-12-17 for tetrahydro-pyrido-pyrimidine derivatives.
This patent application is currently assigned to Novartis AG. The applicant listed for this patent is Novartis AG. Invention is credited to Nigel Graham Cooke, Paulo Fernandes Gomes Dos Santos, Nadege Graveleau, Christina Hebach, Klemens Hoegenauer, Gregory John Hollingworth, Alexander Baxter Smith, Nicolas Soldermann, Frank Stowasser, Ross Sinclair Strang, Nicola Tufilli, Anette von Matt, Romain Wolf, Frederic Zecri.
Application Number | 20200392132 16/916168 |
Document ID | / |
Family ID | 1000005051615 |
Filed Date | 2020-12-17 |
View All Diagrams
United States Patent
Application |
20200392132 |
Kind Code |
A1 |
Cooke; Nigel Graham ; et
al. |
December 17, 2020 |
Tetrahydro-Pyrido-Pyrimidine Derivatives
Abstract
The invention relates to substituted
tetrahydro-pyrido-pyrimidine derivatives of the formula (I),
##STR00001## wherein Y, R.sup.1, R.sup.2 and m are as defined in
the description. Such compounds are suitable for the treatment of a
disorder or disease which is mediated by the activity of the PI3K
enzymes.
Inventors: |
Cooke; Nigel Graham; (Basel,
CH) ; Fernandes Gomes Dos Santos; Paulo; (Basel,
CH) ; Graveleau; Nadege; (Basel, CH) ; Hebach;
Christina; (Basel, CH) ; Hoegenauer; Klemens;
(Basel, CH) ; Hollingworth; Gregory John;
(Horsham, GB) ; Smith; Alexander Baxter; (Basel,
CH) ; Soldermann; Nicolas; (Basel, CH) ;
Stowasser; Frank; (Basel, CH) ; Strang; Ross
Sinclair; (Basel, CH) ; Tufilli; Nicola;
(Basel, CH) ; von Matt; Anette; (Basel, CH)
; Wolf; Romain; (Basel, CH) ; Zecri; Frederic;
(Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novartis AG |
Basel |
|
CH |
|
|
Assignee: |
Novartis AG
Basel
CH
|
Family ID: |
1000005051615 |
Appl. No.: |
16/916168 |
Filed: |
June 30, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16163106 |
Oct 17, 2018 |
|
|
|
16916168 |
|
|
|
|
15613560 |
Jun 5, 2017 |
|
|
|
16163106 |
|
|
|
|
15285614 |
Oct 5, 2016 |
|
|
|
15613560 |
|
|
|
|
14151796 |
Jan 9, 2014 |
9499536 |
|
|
15285614 |
|
|
|
|
13175050 |
Jul 1, 2011 |
8653092 |
|
|
14151796 |
|
|
|
|
61361589 |
Jul 6, 2010 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/5377 20130101;
A61P 35/00 20180101; A61K 31/519 20130101; C07D 471/04
20130101 |
International
Class: |
C07D 471/04 20060101
C07D471/04; A61K 31/519 20060101 A61K031/519; A61K 31/5377 20060101
A61K031/5377 |
Claims
1.-20. (canceled)
21. A process or method for the manufacture of a
tetrahydro-pyrido-pyrimidine derivative of the formula (I) and/or
tautomers and/or N-oxides and/or pharmaceutically acceptable salts
thereof, ##STR00168## wherein Y is selected from O or NR.sup.3;
R.sup.1 is selected from phenyl, pyridyl, pyrimidinyl, pyrazinyl,
pyridazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, or
--C(O)--R.sup.4 wherein R.sup.4 is selected from
C.sub.1-C.sub.8-alkyl, halo-C.sub.1-C.sub.8-alkyl,
hydroxy-C.sub.1-C.sub.8-alkyl,
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl,
C.sub.1-C.sub.8-alkyl-sulfonyl-C.sub.1-C.sub.8-alkyl, heterocyclyl,
heterocyclyl-oxy, heterocyclyl-C.sub.1-C.sub.8-alkyl,
C.sub.3-C.sub.12-cycloalkyl,
C.sub.3-C.sub.12-cycloalkyl-C.sub.1-C.sub.8-alkyl, heteroaryl,
heteroaryl-oxy, heteroaryl-C.sub.1-C.sub.8-alkyl, hydroxy,
C.sub.1-C.sub.8-alkoxy, amino, N--C.sub.1-C.sub.8-alkyl-amino or
N,N-di-C.sub.1-C.sub.8-alkyl-amino, wherein `C.sub.1-C.sub.8-alkyl`
in N--C.sub.1-C.sub.8-alkyl-amino and
N,N-di-C.sub.1-C.sub.8-alkyl-amino may be unsubstituted or
substituted by halogen, hydroxy or C.sub.1-C.sub.4-alkoxy; wherein
`C.sub.3-C.sub.12-cycloalkyl` in C.sub.3-C.sub.12-cycloalkyl and
C.sub.3-C.sub.12-cycloalkyl-C.sub.1-C.sub.8-alkyl may be
unsubstituted or substituted by 1-5 substituents independently
selected from oxo, halogen, C.sub.1-C.sub.8-alkyl,
halo-C.sub.1-C.sub.8-alkyl, hydroxy-C.sub.1-C.sub.8-alkyl,
hydroxyl, C.sub.1-C.sub.8-alkoxy,
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl, amino,
N--C.sub.1-C.sub.8-alkyl-amino, N,N-di-C.sub.1-C.sub.8-alkyl-amino,
C.sub.1-C.sub.8-alkyl-carbonyl,
halo-C.sub.1-C.sub.8-alkyl-carbonyl,
hydroxy-C.sub.1-C.sub.8-alkyl-carbonyl or
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl-carbonyl; wherein
`heterocyclyl` is selected from oxiranyl, aziridinyl, oxetanyl,
thiethanyl, azetidinyl, pyrrolidinyl, tetrahydrofuranyl,
tetrahydrothiophenyl, 2,3-dihydrofuranyl, 2,5-dihydrofuranyl,
2,3-dihydrothiophenyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl,
tetrahydropyranyl, piperidinyl, tetrahydrothiopyranyl, morpholinyl,
thiomorpholinyl, piperazinyl, azepanyl, thiepanyl or oxepanyl; each
of which is unsubstituted or substituted by 1-5 substituents
independently selected from oxo, halogen, C.sub.1-C.sub.8-alkyl,
halo-C.sub.1-C.sub.8-alkyl, hydroxy-C.sub.1-C.sub.8-alkyl,
hydroxyl, C.sub.1-C.sub.8-alkoxy,
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl, amino,
N--C.sub.1-C.sub.8-alkyl-amino, N,N-di-C.sub.1-C.sub.8-alkyl-amino,
C.sub.1-C.sub.8-alkyl-carbonyl,
halo-C.sub.1-C.sub.8-alkyl-carbonyl,
hydroxy-C.sub.1-C.sub.8-alkyl-carbonyl or
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl-carbonyl; wherein
`heterocyclyl` can be attached at a heteroatom or a carbon atom and
where the N and/or S heteroatoms can also optionally be oxidized to
various oxidation states; wherein `heteroaryl` is selected from
furanyl, thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl,
isothiazolyl, oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-oxadiazolyl,
1,2,5-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,3-thiadiazolyl,
1,3,4-thiadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl,
1,2,5-triazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,
1,2,3-triazinyl, 1,2,4-triazinyl or 1,3,5-triazinyl; each of which
is unsubstituted or substituted by 1-5 substituents independently
selected from halogen, C.sub.1-C.sub.8-alkyl,
halo-C.sub.1-C.sub.8-alkyl, hydroxy-C.sub.1-C.sub.8-alkyl,
hydroxyl, C.sub.1-C.sub.8-alkoxy,
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl, amino,
N--C.sub.1-C.sub.8-alkyl-amino, N,N-di-C.sub.1-C.sub.8-alkyl-amino,
C.sub.1-C.sub.8-alkyl-carbonyl,
halo-C.sub.1-C.sub.8-alkyl-carbonyl,
hydroxy-C.sub.1-C.sub.8-alkyl-carbonyl or
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl-carbonyl; wherein
`heteroaryl` can be attached at a heteroatom or a carbon atom and
where the N and/or S heteroatoms can also optionally be oxidized to
various oxidation states; R.sup.2 is selected from phenyl,
naphthyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, quinolinyl
or isoquinolinyl, each of which is unsubstituted or substituted by
1-5 substituents independently selected from halogen, cyano, nitro,
C.sub.1-C.sub.8-alkyl, halo-C.sub.1-C.sub.8-alkyl,
hydroxy-C.sub.1-C.sub.8-alkyl, hydroxyl, C.sub.1-C.sub.8-alkoxy,
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl, amino,
N--C.sub.1-C.sub.8-alkyl-amino, N,N-di-C.sub.1-C.sub.8-alkyl-amino,
C.sub.1-C.sub.8-alkyl-carbonyl,
halo-C.sub.1-C.sub.8-alkyl-carbonyl,
hydroxy-C.sub.1-C.sub.8-alkyl-carbonyl or
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl-carbonyl; R.sup.3 is
selected from H, C.sub.1-C.sub.4-alkyl or
halo-C.sub.1-C.sub.4-alkyl; and m is selected from 0 or 1.
comprising the steps of method A: b) deprotecting the compound of
formula (E), ##STR00169## wherein PG.sup.2 represents a suitable
protecting group and R.sup.1, Y and m are as defined for a compound
of formula (I) c) followed by reaction with R.sup.2-Hal, wherein
R.sup.2 is as defined for a compound of formula (I) and Hal
represents halogen, under customary Buchwald-Hartwig conditions
using a ligand with a palladium catalyst, in the presence of a
base, in an organic solvent; and wherein compound of formula (E) is
prepared comprising the step of d) deprotecting PG.sup.1 from the
compound of formula (C), ##STR00170## wherein PG.sup.1 represents a
suitable protecting group, and the other substituents are as
defined above, e) followed by coupling reaction with R.sup.1-Act
wherein when R.sup.1 is --C(O)--R.sup.4, wherein R.sup.4 is as
defined for a compound of formula (I), and Act represents an
activating group or a hydroxy group, the coupling reaction is an
amide, urea or carbamic ester formation, or; wherein when R.sup.1
is selected from phenyl, pyridyl, pyrimidinyl, pyrazinyl,
pyridazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl or 1,3,5-triazinyl
and Act represents halogen, the coupling reaction is carried out in
the presence of an amine base or alternatively, the reaction is
carried out under customary Buchwald-Hartwig conditions using a
ligand with a palladium catalyst in the presence of a base; wherein
the compound of formula (C) is prepared comprising the step of a)
coupling a compound of formula (A), ##STR00171## wherein X
represents halogen and PG.sup.2 is as defined above; with a
compound of formula (B), ##STR00172## wherein the substituents are
as defined above, wherein when HY is OH and X represents halogen,
the reaction takes place in the presence of a suitable base, or
wherein when HY is NR.sup.3H and X represents halogen: the reaction
takes place in the presence of a suitable base, or wherein when HY
is NR.sup.3H and X represents hydroxy: a base promoted phosphonium
coupling reaction is employed; or, alternatively comprising the
steps of a), d) and e) of method A as defined above, starting from
a compound of formula (A) wherein PG.sup.1 represents R.sup.2; or,
alternatively comprising the steps of method B: d) deprotecting the
compound of formula (D), ##STR00173## wherein PG.sup.1 represents a
suitable protecting group, and the other substituents are as
defined above e) followed by coupling reaction with R.sup.1-Act,
wherein when R.sup.1 is --C(O)--R.sup.4, wherein R.sup.4 is as
defined for a compound of formula (I), and Act represents an
activating group or a hydroxy group, the coupling reaction is an
amide, urea or carbamic ester formation, or; wherein when R.sup.1
is selected from phenyl, pyridyl, pyrimidinyl, pyrazinyl,
pyridazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl or 1,3,5-triazinyl
and Act represents halogen, the coupling reaction is carried out in
the presence of an amine base or alternatively, the reaction is
carried out under customary Buchwald-Hartwig conditions using a
ligand with a palladium catalyst in the presence of a base; wherein
compound of formula (D) is prepared comprising the step of b)
deprotecting PG.sup.1 from the compound of formula (C),
##STR00174## wherein PG.sup.1 represents a suitable protecting
group, and the other substituents are as defined above, c) followed
by coupling reaction with R.sup.2-Hal, wherein R.sup.2 is as
defined for a compound of formula (I) and Hal represents halogen,
under customary Buchwald-Hartwig conditions using a ligand with a
palladium catalyst, in the presence of a base, in an organic
solvent; wherein the compound of formula (C) is prepared as
described above; or, alternatively comprising the steps a), b) and
c) of method B as defined above, starting from a compound of
formula (B) wherein PG.sup.1 represents R.sup.1, and, if desired,
transforming a compound of formula (I) into a different compound of
formula (I), transforming a salt of an obtainable compound of
formula (I) into the free compound or a different salt,
transforming an obtainable free compound of formula (I) into a salt
thereof, and/or separating an obtainable mixture of isomers of a
compound of formula I into individual isomers.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the preparation and use of new
tetrahydro-pyrido-pyrimidine derivatives as drug candidates in free
form or in pharmaceutically acceptable salt form with valuable
druglike properties, such as e.g. metabolic stability and suitable
pharmacokinetics, form for the modulation, notably the inhibition
of the activity or function of the phosphoinositide 3' OH kinase
family (hereinafter PI3K).
BACKGROUND OF THE INVENTION
[0002] The invention relates to the treatment, either alone or in
combination, with one or more other pharmacologically active
compounds, of PI3K-related diseases including but not limited to
autoimmune disorders, inflammatory diseases, allergic diseases,
airway diseases, such as asthma and COPD, transplant rejection,
cancers eg of hematopoietic origin or solid tumors. The invention
also relates to the treatment, either alone or in combination, with
one or more other pharmacologically active compounds, includes
methods of treating conditions, diseases or disorders in which one
or more of the functions of B cells such as antibody production,
antigen presentation, cytokine production or lymphoid organogenesis
are abnormal or are undesirable including rheumatoid arthritis,
pemphigus vulgaris, idiopathic thrombocytopenia purpura, systemic
lupus erythematosus, multiple sclerosis, myasthenia gravis,
Sjogren's syndrome, autoimmune hemolytic anemia, ANCA-associated
vasculitides, cryoglobulinemia, thrombotic thrombocytopenic
purpura, chronic autoimmune urticaria, allergy (atopic dermatitis,
contact dermatitis, allergic rhinitis), goodpasture's syndrome, AMR
(antibody-mediated transplant rejection), B cell-mediated
hyperacute, acute and chronic transplant rejection and cancers of
haematopoietic origin including but not limited to multiple
myeloma; acute myelogenous leukemia; chronic myelogenous leukemia;
lymphocytic leukemia; myeloid leukemia; non-Hodgkin lymphoma;
lymphomas; polycythemia vera; essential thrombocythemia;
myelofibrosis with myeloid metaplasia; and Walden stroem
disease.
SUMMARY OF THE INVENTION
[0003] In a first aspect, the invention relates to
tetrahydro-pyrido-pyrimidine compounds of the formula (I) and/or
pharmaceutically acceptable salts and/or solvates thereof,
##STR00002##
wherein Y is selected from O or NR.sup.3; R.sup.1 is selected from
phenyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,
1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, or [0004]
--C(O)--R.sup.4 [0005] wherein [0006] R.sup.4 is selected from
C.sub.1-C.sub.8-alkyl, halo-C.sub.1-C.sub.8-alkyl,
hydroxy-C.sub.1-C.sub.8-alkyl,
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl,
C.sub.1-C.sub.8-alkyl-sulfonyl-C.sub.1-C.sub.8-alkyl, heterocyclyl,
heterocyclyl-oxy, heterocyclyl-C.sub.1-C.sub.8-alkyl,
C.sub.3-C.sub.12-cycloalkyl,
C.sub.3-C.sub.12-cycloalkyl-C.sub.1-C.sub.8-alkyl, heteroaryl,
heteroaryl-oxy, heteroaryl-C.sub.1-C.sub.8-alkyl, hydroxy,
C.sub.1-C.sub.8-alkoxy, amino, N--C.sub.1-C.sub.8-alkyl-amino or
N,N-di-C.sub.1-C.sub.8-alkyl-amino, [0007] wherein
`C.sub.1-C.sub.8-alkyl` in N--C.sub.1-C.sub.8-alkyl-amino and
N,N-di-C.sub.1-C.sub.8-alkyl-amino may be unsubstituted or
substituted by halogen, hydroxy or C.sub.1-C.sub.4-alkoxy; [0008]
wherein `C.sub.3-C.sub.12-cycloalkyl` in
C.sub.3-C.sub.12-cycloalkyl and
C.sub.3-C.sub.12-cycloalkyl-C.sub.1-C.sub.8-alkyl may be
unsubstituted or substituted by 1-5 substituents independently
selected from oxo, halogen, C.sub.1-C.sub.8-alkyl,
hydroxy-C.sub.1-C.sub.8-alkyl, hydroxyl, C.sub.1-C.sub.8-alkoxy,
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl, amino,
N--C.sub.1-C.sub.8-alkyl-amino, C.sub.1-C.sub.8-alkyl-carbonyl,
halo-C.sub.1-C.sub.8-alkyl-carbonyl,
hydroxy-C.sub.1-C.sub.8-alkyl-carbonyl or
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl-carbonyl; [0009]
wherein `heterocyclyl` is selected from oxiranyl, aziridinyl,
oxetanyl, thiethanyl, acetitinyl, pyrrolidinyl, tetrahydrofuranyl,
tetrahydrothiophenyl, 2,3-dihydrofuranyl, 2,5-dihydrofuranyl,
2,3-dihydrothiophenyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl,
tetrahydropyranyl, piperidinyl, tetrahydrothiopyranyl, morpholinyl,
thiomorpholinyl, piperazinyl, azepanyl, thiepanyl or oxepanyl; each
of which is unsubstituted or substituted by 1-5 substituents
independently selected from oxo, halogen, C.sub.1-C.sub.8-alkyl,
hydroxy-C.sub.1-C.sub.8-alkyl, hydroxyl, C.sub.1-C.sub.8-alkoxy,
alkyl, amino, N--C.sub.1-C.sub.8-alkyl-amino,
C.sub.1-C.sub.8-alkyl-carbonyl,
halo-C.sub.1-C.sub.8-alkyl-carbonyl,
hydroxy-C.sub.1-C.sub.8-alkyl-carbonyl or
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl-carbonyl; [0010]
wherein `heterocyclyl` can be attached at a heteroatom or a carbon
atom and where the N and/or S heteroatoms can also optionally be
oxidized to various oxidation states; [0011] wherein `heteroaryl`
is selected from [0012] furanyl, thiophenyl, pyrrolyl, imidazolyl,
pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl,
1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,3-oxadiazolyl,
1,3,4-oxadiazolyl, 1,2,5-thiadiazolyl, 1,2,4-thiadiazolyl,
1,2,3-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,3-triazolyl,
1,2,4-triazolyl, 1,2,5-triazolyl, pyridyl, pyrimidinyl, pyrazinyl,
pyridazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl or 1,3,5-triazinyl;
each of which is unsubstituted or substituted by 1-5 substituents
independently selected from halogen, C.sub.1-C.sub.8-alkyl,
halo-C.sub.1-C.sub.8-alkyl, hydroxy-C.sub.1-C.sub.8-alkyl,
hydroxyl, C.sub.1-C.sub.8-alkoxy,
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl, amino,
N--C.sub.1-C.sub.8-alkyl-amino, N,N-di-C.sub.1-C.sub.8-alkyl-amino,
C.sub.1-C.sub.8-alkyl-carbonyl,
halo-C.sub.1-C.sub.8-alkyl-carbonyl,
hydroxy-C.sub.1-C.sub.8-alkyl-carbonyl or
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl-carbonyl; wherein
`heteroaryl` can be attached at a heteroatom or a carbon atom and
where the N and/or S heteroatoms can also optionally be oxidized to
various oxidation states; R.sup.2 is selected from phenyl,
naphthyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, quinolinyl
or isoquinolinyl, each of which is unsubstituted or substituted by
1-5 substituents independently selected from halogen, cyano, nitro,
C.sub.1-C.sub.8-alkyl, hydroxy-C.sub.1-C.sub.8-alkyl, hydroxyl,
C.sub.1-C.sub.8-alkoxy,
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl, amino,
N--C.sub.1-C.sub.8-alkyl-amino, alkyl-amino,
C.sub.1-C.sub.8-alkyl-carbonyl,
halo-C.sub.1-C.sub.8-alkyl-carbonyl,
hydroxy-C.sub.1-C.sub.8-alkyl-carbonyl or
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl-carbonyl; R.sup.3 is
selected from H, C.sub.1-C.sub.4-alkyl or
halo-C.sub.1-C.sub.4-alkyl; and m is selected from 0 or 1.
DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 discloses the X-ray Powder Diffraction Pattern of
Example 1 citrate salt
[0014] FIG. 2 discloses the X-ray Powder Diffraction Pattern of
Example 1 fumarate salt
[0015] FIG. 3 discloses the X-ray Powder Diffraction Pattern of
Example 1 napadisylate salt
[0016] FIG. 4 discloses the X-ray Powder Diffraction Pattern of
Example 67 phosphate salt
[0017] FIG. 5 discloses the: X-ray Powder Diffraction Pattern of
Example 67 HCl salt
[0018] FIG. 6 discloses the X-ray Powder Diffraction Pattern of
Example 67 hippurate salt
[0019] FIG. 7 discloses the X-ray Powder Diffraction Pattern of
Example 1 anhydrous form
FIG. 8 discloses the X-ray Powder Diffraction Pattern of Example 1
trihydrate FIG. 9 discloses the X-ray Powder Diffraction Pattern of
Example 67 anhydrous form
DETAILED DESCRIPTION OF THE INVENTION
[0020] Any formula given herein is intended to represent hydrates,
solvates, and polymorphs of such compounds, and mixtures
thereof.
[0021] Unless otherwise specified, the term "compounds of the
invention" refers to compounds of formula (I) and subformulae
thereof, salts of the compounds, hydrates or solvates of the
compounds, salts of the compounds as well as stereoisomers
(including diastereoisomers and enantiomers), tautomers and
isotopically labeled compounds (including deuterium
substitutions).
[0022] As used herein, the term "a", "an", "the" and similar terms
used in the context of the present invention, especially in the
context of the claims, are to be construed to cover both the
singular and plural unless otherwise indicated herein or clearly
contradicted by the context.
[0023] All methods described herein can be performed in any
suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language, e.g. "such as", provided herein is intended
merely to better illuminate the invention and does not pose a
limitation on the scope of the invention otherwise claimed.
[0024] The invention may be more fully appreciated by reference to
the following description, including the following glossary of
terms and the concluding examples. As used herein, the terms
"including", "containing" and "comprising" are used herein in their
open, non-limiting sense. Where compounds of formula I are
mentioned, this is meant to include also the tautomers and N-oxides
of the compounds of formula I. Tautomers, such as tautomers between
keto- and enol form, lactam- and lactim form, amid form and imidic
acid form or enamine form and imine form, can be present for
example in the R1 or R2 portion of compounds of formula I. The
nitrogen atoms of the tetrahydro-pyrido-pyrimidine core of the
compounds of formula I as well as nitrogen containing heterocyclyl
and heteroaryl residues can form N-oxides.
[0025] Where the plural form is used for compounds, salts, and the
like, this is taken to mean also a single compound, salt, or the
like.
[0026] The general terms used hereinbefore and hereinafter
preferably have within the context of this disclosure the following
meanings, unless otherwise indicated:
[0027] As used herein, the term "alkyl" refers to a fully saturated
branched, including single or multiple branching, or unbranched
hydrocarbon moiety having up to 20 carbon atoms. Unless otherwise
provided, alkyl refers to hydrocarbon moieties having 1 to 16
carbon atoms, 1 to 10 carbon atoms, 1 to 7 carbon atoms, or 1 to 4
carbon atoms. Representative examples of alkyl include, but are not
limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl,
sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl,
n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl,
n-heptyl, n-octyl, n-nonyl, n-decyl and the like. Typically, alkyl
groups have 1-7, more preferably 1-4 carbons.
[0028] As used herein, the term "halo-alkyl" refers to an alkyl as
defined herein, which is substituted by one or more halo groups as
defined herein. The halo-alkyl can be mono-halo-alkyl,
di-halo-alkyl or poly-halo-alkyl including per-halo-alkyl. A
mono-halo-alkyl can have one iodo, bromo, chloro or fluoro within
the alkyl group. Di-halo-alky and poly-halo-alkyl groups can have
two or more of the same halo atoms or a combination of different
halo groups within the alkyl. Typically the poly-halo-alkyl
contains up to 12, or 10, or 8, or 6, or 4, or 3, or 2 halo groups.
Non-limiting examples of halo-alkyl include fluoro-methyl,
di-fluoro-methyl, tri-fluoro-methyl, chloro-methyl,
di-chloro-methyl, tri-chloro-methyl, penta-fluoro-ethyl,
hepta-fluoro-propyl, di-fluoro-chloro-methyl,
di-chloro-fluoro-methyl, di-fluoro-ethyl, di-fluoro-propyl,
di-chloro-ethyl and dichloro-propyl. A per-halo-alkyl refers to an
alkyl having all hydrogen atoms replaced with halo atoms.
[0029] As used herein, the term "heterocyclyl" or "heterocyclic"
refers to a 3 to 7 membered monocyclic or 7 to 10 membered
saturated or partially saturated ring or ring system, which
contains at least one heteroatom selected from N, O and S, where
the N and S can also optionally be oxidized to various oxidation
states. `Heterocyclyl` can be attached at a heteroatom or a carbon
atom. `Heterocyclyl` can include fused or bridged rings as well as
spirocyclic rings. Examples of heterocycles include oxiranyl,
aziridinyl, oxetanyl, thiethanyl, acetitinyl, pyrrolidinyl,
tetrahydrofuranyl, tetrahydrothiophenyl, 2,3-dihydrofuranyl,
2,5-dihydrofuranyl, 2,3-dihydrothiophenyl, 1-pyrrolinyl,
2-pyrrolinyl, 3-pyrrolinyl, tetrahydropyranyl, piperidinyl,
tetrahydrothiopyranyl, morpholinyl thiomorpholinyl, piperazinyl,
azepanyl, thiepanyl and oxepanyl.
[0030] As used herein, the term "heteroaryl" refers to a 4-, 5-,
6-, or 7-membered monocyclic, 7-, 8-, 9-, 10-, 11-, or 12-membered
bicyclic or 10-, 11-, 12-, 13-, 14- or 15-membered tricyclic
unsaturated ring or ring system--carrying the highest possible
number of conjugated double bonds in the ring(s), which contains at
least one heteroatom selected from N, O and S, wherein the N and S
can also optionally be oxidized to various oxidation states.
`Heteroaryl` can be attached at a heteroatom or a carbon atom.
`Heteroaryl` can include fused or bridged rings as well as
spirocyclic rings. Examples of heteroaryl include furanyl,
thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl,
isothiazolyl, oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-oxadiazolyl,
1,2,5-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,3-thiadiazolyl,
1,3,4-thiadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl,
1,2,5-triazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,
1,2,3-triazinyl, 1,2,4-triazinyl and 1,3,5-triazinyl.
[0031] As used herein, the term "cycloalkyl" refers to saturated or
partially unsaturated monocyclic, bicyclic or tricyclic hydrocarbon
groups of 3-12 carbon atoms. Unless otherwise provided, cycloalkyl
refers to cyclic hydrocarbon groups having between 3 and 10 ring
carbon atoms or between 3 and 7 ring carbon atoms. Exemplary
bicyclic hydrocarbon groups include octahydroindyl,
decahydronaphthyl. Exemplary tricyclic hydrocarbon
bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1]heptenyl,
6,6-dimethylbicyclo[3.1.1]heptyl,
2,6,6-trimethylbicyclo[3.1.1]heptyl, bicyclo[2.2.2]octy. Exemplary
tetracyclic hydrocarbon groups include adamantyl.
[0032] As used herein, the term "oxy" refers to an --O-- linking
group.
[0033] As used herein, the term "carboxy" or "carboxyl" is
--COOH.
[0034] As used herein, all substituents are written in a way to
show the order of functional groups (groups) they are composed of.
The functional groups are defined herein above.
[0035] "Treatment" includes prophylactic (preventive) and
therapeutic treatment as well as the delay of progression of a
disease or disorder.
[0036] "Combination" refers to either a fixed combination in one
dosage unit form, or a kit of parts for the combined administration
where a compound of the formula (I) and a combination partner (e.g.
an other drug as explained below, also referred to as "therapeutic
agent" or "co-agent") may be administered independently at the same
time or separately within time intervals, especially where these
time intervals allow that the combination partners show a
cooperative, e.g. synergistic effect. The terms "co-administration"
or "combined administration" or the like as utilized herein are
meant to encompass administration of the selected combination
partner to a single subject in need thereof, e.g. a patient, and
are intended to include treatment regimens in which the agents are
not necessarily administered by the same route of administration or
at the same time. The term "pharmaceutical combination" as used
herein means a product that results from the mixing or combining of
more than one active ingredient and includes both fixed and
non-fixed combinations of the active ingredients. The term "fixed
combination" means that the active ingredients, e.g. a compound of
formula (I) and a combination partner, are both administered to a
patient simultaneously in the form of a single entity or dosage.
The term "non-fixed combination" means that the active ingredients,
e.g. a compound of formula (I) and a combination partner, are both
administered to a patient as separate entities either
simultaneously, concurrently or sequentially with no specific time
limits, wherein such administration provides therapeutically
effective levels of the two compounds in the body of the patient.
The latter also applies to cocktail therapy, e.g. the
administration of three or more active ingredients.
[0037] Various embodiments of the invention are described herein.
It will be recognized that features specified in each embodiment
may be combined with other specified features to provide further
embodiments.
[0038] The invention further relates to pharmaceutically acceptable
prodrugs of a compound of formula (I). Particularly, the present
invention also relates to pro-drugs of a compound of formula I as
defined herein that convert in vivo to the compound of formula I as
such. Any reference to a compound of formula I is therefore to be
understood as referring also to the corresponding pro-drugs of the
compound of formula I, as appropriate and expedient.
[0039] The invention further relates to pharmaceutically acceptable
metabolites of a compound of formula (I).
[0040] In one embodiment, the invention provides a compound of the
formula (I) and/or a pharmaceutically acceptable salt and/or a
solvate thereof, selected from a compound of the formula (Ia)
##STR00003##
wherein R.sup.1, R.sup.2 and Y are as defined above.
[0041] In one embodiment, the invention provides a compound of the
formula (I) and/or a pharmaceutically acceptable salt and/or a
solvate thereof, selected from a compound of the formula (Ia')
##STR00004##
wherein R.sup.1, R.sup.2 and Y are as defined above.
[0042] In another embodiment, the invention provides a compound of
the formula (I) and/or a pharmaceutically acceptable salt and/or a
solvate thereof, selected from a compound of the formula (Ib)
##STR00005##
wherein R.sup.1 and R.sup.2 are as defined above.
[0043] In another embodiment, the invention provides a compound of
the formula (I) and/or a pharmaceutically acceptable salt and/or a
solvate thereof, selected from a compound of the formula (Ib')
##STR00006##
wherein R.sup.1 and R.sup.2 are as defined above.
[0044] In another embodiment, the invention provides a compound of
the formula (I) and/or a pharmaceutically acceptable salt and/or a
solvate thereof, selected from a compound of the formula (Ic)
##STR00007##
wherein R.sup.1 and R.sup.2 are as defined above.
[0045] In another embodiment, the invention provides a compound of
the formula (I) and/or a pharmaceutically acceptable salt and/or a
solvate thereof, selected from a compound of the formula (Ic')
##STR00008##
wherein R.sup.1 and R.sup.2 are as defined above.
[0046] In another embodiment, the invention provides a compound of
the formula (I) and/or a pharmaceutically acceptable salt and/or a
solvate thereof, selected from a compound of the formula (Id)
##STR00009##
wherein R.sup.4 and R.sup.2 are as defined above.
[0047] In another embodiment, the invention provides a compound of
the formula (I) and/or a pharmaceutically acceptable salt and/or a
solvate thereof, selected from a compound of the formula (Id')
##STR00010##
wherein R.sup.4 and R.sup.2 are as defined above.
[0048] In another embodiment, the invention provides a compound of
the formula (I) and/or a pharmaceutically acceptable salt and/or a
solvate thereof, selected from a compound of the formula (Ie)
##STR00011##
wherein R.sup.4 and R.sup.2 are as defined above.
[0049] In another embodiment, the invention provides a compound of
the formula (I) and/or a pharmaceutically acceptable salt and/or a
solvate thereof, selected from a compound of the formula (Ie')
##STR00012##
wherein R.sup.4 and R.sup.2 are as defined above.
[0050] In another embodiment, the invention provides a compound of
the formulae (I), (Ia), (Ia'), (Ib), (Ib') (Ic), (Ic'), (Id),
(Id'), (Ie) or (Ie') and/or a pharmaceutically acceptable salt
and/or a solvate thereof, wherein R.sup.2 is selected from
naphthyl, pyridyl or pyrimidinyl; each of which is unsubstituted or
substituted by 1-3 substituents independently selected from
halogen, cyano, nitro, C.sub.1-C.sub.8-alkyl,
hydroxy-C.sub.1-C.sub.8-alkyl, hydroxyl, C.sub.1-C.sub.8-alkoxy,
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl, amino,
N--C.sub.1-C.sub.8-alkyl-amino, C.sub.1-C.sub.8-alkyl-carbonyl,
halo-C.sub.1-C.sub.8-alkyl-carbonyl,
hydroxy-C.sub.1-C.sub.8-alkyl-carbonyl or
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl-carbonyl.
[0051] In another embodiment, the invention provides a compound of
the formulae (I), (Ia), (Ia'), (Ib), (Ib') (Ic), (Ic'), (Id),
(Id'), (Ie) or (Ie') and/or a pharmaceutically acceptable salt
and/or a solvate thereof, wherein R.sup.2 is selected from
3-pyridyl or 5-pyrimidinyl; each of which is substituted by 1-2
substituents independently selected from halogen, cyano, nitro,
C.sub.1-C.sub.8-alkyl, halo-C.sub.1-C.sub.8-alkyl,
hydroxy-C.sub.1-C.sub.8-alkyl, hydroxyl, C.sub.1-C.sub.8-alkoxy,
amino, N--C.sub.1-C.sub.8-alkyl-amino,
C.sub.1-C.sub.8-alkyl-carbonyl, carbonyl,
hydroxy-C.sub.1-C.sub.8-alkyl-carbonyl or
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl-carbonyl, wherein one
substituents is located in the para position relative to the point
of connection of R.sup.2 to the core of the compound.
[0052] In another embodiment, the invention provides a compound of
the formulae (I), (Ia), (Ia'), (Ib), (Ib') (Ic), (Ic'), (Id),
(Id'), (Ie) or (Ie') and/or a pharmaceutically acceptable salt
and/or a solvate thereof, wherein R.sup.2 is selected from
3-pyridyl or 5-pyrimidinyl; each of which is substituted by 1-2
substituents independently selected from halogen, cyano,
C.sub.1-C.sub.4-alkyl, halo-C.sub.1-C.sub.4-alkyl,
C.sub.1-C.sub.4-alkoxy or amino, wherein one substituents is
located in the para position relative to the point of connection of
R.sup.2 to the core of the compound.
[0053] In another embodiment, the invention provides a compound of
the formulae (I), (Ia), (Ia'), (Ib), (Ib') (Ic), (Ic'), (Id),
(Id'), (Ie) or (Ie') and/or a pharmaceutically acceptable salt
and/or a solvate thereof, wherein R.sup.2 is selected from
3-pyridyl or 5-pyrimidinyl; each of which is substituted by 1-2
substituents independently selected from fluoro, chloro, cyano,
methyl, trifluoromethyl, methoxy or amino, wherein one substituents
is located in the para position relative to the point of connection
of R.sup.2 to the core of the compound.
[0054] In another embodiment, the invention provides a compound of
the formulae (I), (Ia) or (Ia') and/or a pharmaceutically
acceptable salt and/or a solvate thereof, wherein R.sup.3 is H.
[0055] In another embodiment, the invention provides a compound of
the formulae (I), (Ia), (Ia'), (Ib), (Ib'), (Ic) or (Ic') and/or a
pharmaceutically acceptable salt and/or a solvate thereof, wherein
R.sup.1 is selected from phenyl, pyridyl, pyrimidinyl, pyrazinyl,
pyridazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl or
1,3,5-triazinyl.
[0056] In another embodiment, the invention provides a compound of
the formulae (I), (Ia), (Ia'), (Ib), (Ib'), (Ic) or (Ic') and/or a
pharmaceutically acceptable salt and/or a solvate thereof, wherein
R.sup.1 is selected from pyridyl or pyrimidinyl.
[0057] In another embodiment, the invention provides a compound of
the formulae (I), (Ia), (Ia'), (Ib), (Ib'), (Ic) or (Ic') and/or a
pharmaceutically acceptable salt and/or a solvate thereof, wherein
R.sup.1 is --C(O)--R.sup.4, wherein R.sup.4 is as defined
above.
[0058] In another embodiment, the invention provides a compound of
the formulae (I), (Ia), (Ia'), (Ib), (Ib'), (Ic) or (Ic') and/or a
pharmaceutically acceptable salt and/or a solvate thereof, wherein
R.sup.1 is --C(O)--R.sup.4, wherein R.sup.4 is as defined
above.
[0059] In another embodiment, the invention provides a compound of
the formulae (I), (Ia), (Ia'), (Ib), (Ib'), (Ic) or (Ic') wherein
R.sup.1 is --C(O)--R.sup.4; or a compound of the formulae (Id),
(Id'), (Ie) or (Ie') and/or a pharmaceutically acceptable salt
and/or a solvate thereof, wherein [0060] R.sup.4 is selected from
C.sub.1-C.sub.8-alkyl,
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl, heterocyclyl,
heterocyclyl-C.sub.0-C.sub.12-cycloalkyl, heteroaryl,
C.sub.1-C.sub.8-alkoxy or N,N-di-C.sub.1-C.sub.8-alkyl-amino,
wherein `C.sub.1-C.sub.8-alkyl` in
N,N-di-C.sub.1-C.sub.8-alkyl-amino may be unsubstituted or
substituted by halogen, hydroxy or C.sub.1-C.sub.4-alkoxy; [0061]
wherein `C.sub.3-C.sub.12-cycloalkyl` in
C.sub.3-C.sub.12-cycloalkyl may be unsubstituted or substituted by
1-3 substituents independently selected from oxo, halogen,
C.sub.1-C.sub.8-alkyl, hydroxyl, C.sub.1-C.sub.8-alkoxy,
C.sub.1-C.sub.8-alkyl-carbonyl; [0062] wherein `heterocyclyl` is
selected from pyrrolidinyl, tetrahydropyranyl, piperidinyl,
tetrahydrothiopyranyl, morpholinyl or piperazinyl; each of which is
unsubstituted or substituted by 1-3 substituents independently
selected from oxo, halogen, C.sub.1-C.sub.8-alkyl, hydroxyl,
C.sub.1-C.sub.8-alkoxy, C.sub.1-C.sub.8-alkyl-carbonyl; [0063]
wherein `heterocyclyl` can be attached at a heteroatom or a carbon
atom and where the N and/or S heteroatoms can also optionally be
oxidized to various oxidation states;
[0064] wherein `heteroaryl` is selected from [0065] furanyl,
imidazolyl, pyrazolyl, thiazolyl, oxazolyl, isoxazolyl,
1,3,4-oxadiazolyl, pyridyl, pyrazinyl; each of which is
unsubstituted or substituted by 1-3 substituents independently
selected from halogen, C.sub.1-C.sub.8-alkyl, hydroxyl,
C.sub.1-C.sub.8-alkoxy, C.sub.1-C.sub.8-alkyl-carbonyl; [0066]
wherein `heteroaryl` can be attached at a heteroatom or a carbon
atom and where the N and/or S heteroatoms can also optionally be
oxidized to various oxidation states.
[0067] In another embodiment, the invention provides a compound of
the formulae (I), (Ia), (Ia'), (Ib), (Ib'), (Ic) or (Ic') wherein
R.sup.1--C(O)--R.sup.4; or a compound of the formulae (Id), (Id'),
(Ie) or (Ie') and/or a pharmaceutically acceptable salt and/or a
solvate thereof, wherein [0068] R.sup.4 is selected from
heterocyclyl, C.sub.4-C.sub.8-cycloalkyl or heteroaryl; [0069]
wherein `C.sub.3-C.sub.12-cycloalkyl` may be unsubstituted or
substituted by 1-3 substituents independently selected from fluoro,
C.sub.1-C.sub.4-alkyl, hydroxyl, C.sub.1-C.sub.4-alkoxy; [0070]
wherein `heterocyclyl` is selected from pyrrolidinyl,
tetrahydropyranyl, piperidinyl, tetrahydrothiopyranyl, morpholinyl
or piperazinyl; each of which is unsubstituted or substituted by
1-3 substituents independently selected from oxo, halogen,
C.sub.1-C.sub.4-alkyl, hydroxyl, C.sub.1-C.sub.4-alkyl-carbonyl;
[0071] wherein `heterocyclyl` can be attached at a heteroatom or a
carbon atom and where the N and/or S heteroatoms can also
optionally be oxidized to various oxidation states; [0072] wherein
`heteroaryl` is selected from [0073] furanyl, imidazolyl,
pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, 1,3,4-oxadiazolyl,
pyridyl, pyrazinyl; each of which is unsubstituted or substituted
by 1-3 substituents independently selected from
C.sub.1-C.sub.4-alkyl, hydroxyl; [0074] wherein `heteroaryl` can be
attached at a heteroatom or a carbon atom and where the N and/or S
heteroatoms can also optionally be oxidized to various oxidation
states.
[0075] In another embodiment, the invention provides a compound of
the formulae (I), (Ia), (Ia'), (Ib), (Ib'), (Ic) or (Ic') wherein
R.sup.1--C(O)--R.sup.4; or a compound of the formulae (Id), (Id'),
(Ie) or (Ie') and/or a pharmaceutically acceptable salt and/or a
solvate thereof, wherein [0076] R.sup.4 is selected from
heterocyclyl; [0077] wherein `heterocyclyl` is selected from
pyrrolidinyl, tetrahydropyranyl, piperidinyl,
tetrahydrothiopyranyl, morpholinyl or piperazinyl; each of which is
unsubstituted or substituted by 1-3 substituents independently
selected from oxo, halogen, C.sub.1-C.sub.4-alkyl, hydroxyl,
C.sub.1-C.sub.4-alkyl-carbonyl; [0078] wherein `heterocyclyl` can
be attached at a heteroatom or a carbon atom and where the N and/or
S heteroatoms can also optionally be oxidized to various oxidation
states.
[0079] In another embodiment, the invention provides a compound of
the formulae (I), (Ia), (Ia'), (Ib), (Ib'), (Ic) or (Ic') wherein
R.sup.1 is --C(O)--R.sup.4; or a compound of the formulae (Id),
(Id'), (Ie) or (Ie') and/or a pharmaceutically acceptable salt
and/or a solvate thereof, wherein [0080] R.sup.4 is selected from
C.sub.1-C.sub.8-alkyl, C.sub.1-C.sub.8-alkoxy or
N,N-di-C.sub.1-C.sub.8-alkyl-amino, [0081] wherein
`C.sub.1-C.sub.8-alkyl` in N,N-di-C.sub.1-C.sub.8-alkyl-amino may
be unsubstituted or substituted by halogen, hydroxy or
C.sub.1-C.sub.4-alkoxy.
[0082] In another embodiment, the invention provides a compound of
the formulae (I), (Ia), (Ia'), (Ib), (Ib'), (Ic) or (Ic') wherein
R.sup.1 is --C(O)--R.sup.4; or a compound of the formulae (Id),
(Id'), (Ie) or (Ie') and/or a pharmaceutically acceptable salt
and/or a solvate thereof, wherein [0083] R.sup.4 is selected from
C.sub.1-C.sub.8-alkyl.
[0084] In another embodiment, the invention provides a compound of
the formulae (I), (Ia), (Ia'), (Ib), (Ib'), (Ic) or (Ic') wherein
R.sup.1 is --C(O)--R.sup.4; or a compound of the formulae (Id),
(Id'), (Ie) or (Ie') and/or a pharmaceutically acceptable salt
and/or a solvate thereof, wherein [0085] R.sup.2 is selected from
3-pyridyl or 5-pyrimidinyl; each of which is substituted by 1-2
substituents independently selected from fluoro, chloro, cyano,
methyl, trifluoromethyl, methoxy or amino, wherein one substituents
is located in the para position relative to the point of connection
of R.sup.2 to the core of the compound and [0086] R.sup.4 is
selected from heterocyclyl; [0087] wherein `heterocyclyl` is
selected from pyrrolidinyl, tetrahydropyranyl, piperidinyl,
tetrahydrothiopyranyl, morpholinyl or piperazinyl; each of which is
unsubstituted or substituted by 1-3 substituents independently
selected from oxo, halogen, C.sub.1-C.sub.4-alkyl, hydroxyl,
C.sub.1-C.sub.4-alkyl-carbonyl; [0088] wherein `heterocyclyl` can
be attached at a heteroatom or a carbon atom and where the N and/or
S heteroatoms can also optionally be oxidized to various oxidation
states.
[0089] In another embodiment, the invention provides a compound of
the formulae (I), (Ia), (Ia'), (Ib), (Ib'), (Ic) or (Ic') wherein
R.sup.1 is --C(O)--R.sup.4; or a compound of the formulae (Id),
(Id'), (Ie) or (Ie') and/or a pharmaceutically acceptable salt
and/or a solvate thereof, wherein [0090] R.sup.2 is selected from
3-pyridyl or 5-pyrimidinyl; each of which is substituted by 1-2
substituents independently selected from fluoro, chloro, cyano,
methyl, trifluoromethyl, methoxy or amino, wherein one substituents
is located in the para position relative to the point of connection
of R.sup.2 to the core of the compound and [0091] R.sup.4 is
selected from C.sub.1-C.sub.8-alkyl,
C.sub.1-C.sub.8-alkoxy-C.sub.1-C.sub.8-alkyl,
C.sub.1-C.sub.8-alkoxy or N,N-di-C.sub.1-C.sub.8-alkyl-amino,
[0092] wherein `C.sub.1-C.sub.8-alkyl` in
N,N-di-C.sub.1-C.sub.8-alkyl-amino may be unsubstituted or
substituted by halogen, hydroxy or C.sub.1-C.sub.4-alkoxy.
[0093] In another embodiment individual compounds according to the
invention are those listed in the Examples section below.
[0094] In another embodiment, the invention provides a compound of
the formula (I), selected from [0095]
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-
-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone;
[0096]
{3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[-
4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanon-
e; [0097]
{(S)-3-[6-(2,4-Dimethoxy-pyrimidin-5-yl)-5,6,7,8-tetrahydro-pyri-
do[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-metha-
none; [0098]
{3-[6-(2,4-Dimethoxy-pyrimidin-5-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyri-
midin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone;
[0099]
2-Methoxy-5-{4-[(S)-1-(tetrahydro-pyran-4-carbonyl)-pyrrolidin-3-yloxy]-7-
,8-dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile; [0100]
2-Methoxy-5-{4-[1-(tetrahydro-pyran-4-carbonyl)-pyrrolidin-3-yloxy]-7,8-d-
ihydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile; [0101]
1-{(S)-3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]-
pyrimidin-4-yloxy]-pyrrolidin-1-yl}-propan-1-one; [0102]
1-{3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyri-
midin-4-yloxy]-pyrrolidin-1-yl}-propan-1-one; [0103]
{(S)-3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]py-
rimidin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone;
[0104]
{3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d-
]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone;
[0105]
2-Amino-5-{4-[(S)-1-(tetrahydro-pyran-4-carbonyl)-pyrrolidin-3-ylo-
xy]-7,8-dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile;
[0106]
2-Amino-5-{4-[1-(tetrahydro-pyran-4-carbonyl)-pyrrolidin-3-yloxy]-7,8-dih-
ydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile; [0107]
(S)-(3-(6-(5-Fluoro-6-methoxypyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d-
]pyrimidin-4-yloxy)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone;
[0108]
(3-(6-(5-Fluoro-6-methoxypyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,-
3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone;
[0109]
(S)-2-Methoxy-5-(4-(1-(2-methoxyacetyl)pyrrolidin-3-yloxy)-7,8-dih-
ydropyrido[4,3-d]pyrimidin-6(5H)-yl)nicotinonitrile; [0110]
2-Methoxy-5-(4-(1-(2-methoxyacetyl)pyrrolidin-3-yloxy)-7,8-dihydropyrido[-
4,3-d]pyrimidin-6(5H)-yl)nicotinonitrile; [0111]
(S)-5-(4-(1-(Cyclopentanecarbonyl)pyrrolidin-3-yloxy)-7,8-dihydropyrido[4-
,3-d]pyrimidin-6(5H)-yl)-2-methoxynicotinonitrile; [0112]
5-(4-(1-(Cyclopentanecarbonyl)pyrrolidin-3-yloxy)-7,8-dihydropyrido[4,3-d-
]pyrimidin-6(5H)-yl)-2-methoxynicotinonitrile; [0113]
(2,4-Dimethyl-oxazol-5-yl)-{(S)-3-[6-(6-methoxy-5-methyl-pyridin-3-yl)-5,-
6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanon-
e; [0114]
(2,4-Dimethyl-oxazol-5-yl)-{3-[6-(6-methoxy-5-methyl-pyridin-3-y-
l)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-met-
hanone; [0115]
Furan-3-yl-{(S)-3-[6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-
-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone; [0116]
Furan-3-yl-{3-[6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyr-
ido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone; [0117]
Furan-3-yl-{(S)-3-[6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-
-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone; [0118]
Furan-3-yl-{3-[6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyr-
ido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone; [0119]
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-
-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(3-methyl-3H-imidazol-4-yl)-methano-
ne; [0120]
{3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyri-
do[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(3-methyl-3H-imidazol-4-yl)-m-
ethanone; [0121]
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-
-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(2-methyl-oxazol-4-yl)-methanone;
[0122]
{3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[-
4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(2-methyl-oxazol-4-yl)-methanone-
; [0123]
(3-Methoxy-cyclobutyl)-{(S)-3-[6-(6-methoxy-5-methyl-pyridin-3-yl-
)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-meth-
anone; [0124]
(3-Methoxy-cyclobutyl)-{3-[6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-te-
trahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone;
[0125]
({(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-py-
rido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-oxazol-4-yl-methanone;
[0126]
({3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido-
[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-oxazol-4-yl-methanone;
[0127]
1-(4-{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-d]pyrimidin-4-yloxy]-pyrrolidine-1-carbonyl}-piperidin-1-yl)-ethanon-
e; [0128]
1-(4-{3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro--
pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidine-1-carbonyl}-piperidin-1-yl)-et-
hanone; [0129]
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-
-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(4-methyl-oxazol-5-yl)-methanone;
[0130]
{3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[-
4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(4-methyl-oxazol-5-yl)-methanone-
; [0131]
5-{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro--
pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidine-1-carbonyl}-1H-pyridin-2-one;
[0132]
5-{3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-d]pyrimidin-4-yloxy]-pyrrolidine-1-carbonyl}-1H-pyridin-2-one;
[0133]
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyr-
ido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(1-methyl-1H-imidazol-4-yl)--
methanone; [0134]
{3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]p-
yrimidin-4-yloxy]-pyrrolidin-1-yl}-(1-methyl-1H-imidazol-4-yl)-methanone;
[0135]
{(S)-3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4-
,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-oxazol-4-yl-methanone;
[0136]
{3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimi-
din-4-yloxy]-pyrrolidin-1-yl}-oxazol-4-yl-methanone; [0137]
{(S)-3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]py-
rimidin-4-yloxy]-pyrrolidin-1-yl}-oxazol-5-yl-methanone; [0138]
{3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimi-
din-4-yloxy]-pyrrolidin-1-yl}-oxazol-5-yl-methanone; [0139]
{(S)-3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]py-
rimidin-4-yloxy]-pyrrolidin-1-yl}-(2-methyl-oxazol-4-yl)-methanone;
[0140]
{3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimi-
din-4-yloxy]-pyrrolidin-1-yl}-(2-methyl-oxazol-4-yl)-methanone;
[0141]
{(S)-3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]py-
rimidin-4-yloxy]-pyrrolidin-1-yl}-(2,2-dimethyl-tetrahydro-pyran-4-yl)-met-
hanone; [0142]
{3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimi-
din-4-yloxy]-pyrrolidin-1-yl}-(2,2-dimethyl-tetrahydro-pyran-4-yl)-methano-
ne; [0143]
{(S)-3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(2,4-dimethyl-oxazol-5-yl)-met-
hanone; [0144]
{3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimi-
din-4-yloxy]-pyrrolidin-1-yl}-(2,4-dimethyl-oxazol-5-yl)-methanone;
[0145]
(4,4-Difluoro-cyclohexyl)-{(S)-3-[6-(5,6-dimethoxy-pyridin-3-yl)-5,6,7,8--
tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone;
[0146]
(4,4-Difluoro-cyclohexyl)-{3-[6-(5,6-dimethoxy-pyridin-3-yl)-5,6,7-
,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone;
[0147]
2-Methoxy-5-{4-[(S)-1-(2-tetrahydro-pyran-4-yl-acetyl)-pyrrolidin--
3-yloxy]-7,8-dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile;
[0148]
2-Methoxy-5-{4-[1-(2-tetrahydro-pyran-4-yl-acetyl)-pyrrolidin-3-yl-
oxy]-7,8-dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile;
[0149]
5-{4-[(S)-1-(2,4-Dimethyl-oxazole-5-carbonyl)-pyrrolidin-3-yloxy]-7,8-dih-
ydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-2-methoxy-nicotinonitrile;
[0150]
5-{4-[1-(2,4-Dimethyl-oxazole-5-carbonyl)-pyrrolidin-3-yloxy]-7,8-dihydro-
-5H-pyrido[4,3-d]pyrimidin-6-yl}-2-methoxy-nicotinonitrile; [0151]
5-{4-[(S)-1-(2,2-Dimethyl-tetrahydro-pyran-4-carbonyl)-pyrrolidin-3-yloxy-
]-7,8-dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-2-methoxy-nicotinonitrile;
[0152]
5-{4-[1-(2,2-Dimethyl-tetrahydro-pyran-4-carbonyl)-pyrrolidin-3-yl-
oxy]-7,8-dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-2-methoxy-nicotinonitrile-
; [0153]
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-py-
rido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(5-methyl-oxazol-4-yl)-meth-
anone; [0154]
{3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]p-
yrimidin-4-yloxy]-pyrrolidin-1-yl}-(5-methyl-oxazol-4-yl)-methanone;
[0155]
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyr-
ido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(5-methyl-isoxazol-4-yl)-met-
hanone; [0156]
{3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]p-
yrimidin-4-yloxy]-pyrrolidin-1-yl}-(5-methyl-isoxazol-4-yl)-methanone;
[0157]
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyr-
ido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(3-methyl-isoxazol-4-yl)-met-
hanone; [0158]
{3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]p-
yrimidin-4-yloxy]-pyrrolidin-1-yl}-(3-methyl-isoxazol-4-yl)-methanone;
[0159]
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyr-
ido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(3-methyl-isoxazol-4-yl)-met-
hanone; [0160]
{3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]p-
yrimidin-4-yloxy]-pyrrolidin-1-yl}-(3-methyl-isoxazol-4-yl)-methanone;
[0161]
Isoxazol-3-yl-{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8--
tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone;
[0162]
Isoxazol-3-yl-{3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetr-
ahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone;
[0163]
Isoxazol-5-yl-{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone;
[0164]
Isoxazol-5-yl-{3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro--
pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone; [0165]
2-Methoxy-5-{4-[(S)-1-(thiazole-4-carbonyl)-pyrrolidin-3-yloxy]-7,8-dihyd-
ro-5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile; [0166]
2-Methoxy-5-{4-[1-(thiazole-4-carbonyl)-pyrrolidin-3-yloxy]-7,8-dihydro-5-
H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile; [0167]
2-Methoxy-5-{4-[(S)-1-(1-methyl-1H-pyrazole-4-carbonyl)-pyrrolidin-3-ylox-
y]-7,8-dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile;
[0168]
2-Methoxy-5-{4-[1-(1-methyl-1H-pyrazole-4-carbonyl)-pyrrolidin-3-yloxy]-7-
,8-dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile; [0169]
2-Methoxy-5-{4-[(S)-1-(1-methyl-1H-pyrazole-3-carbonyl)-pyrrolidin-3-ylox-
y]-7,8-dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile;
[0170]
2-Methoxy-5-{4-[1-(1-methyl-1H-pyrazole-3-carbonyl)-pyrrolidin-3-yloxy]-7-
,8-dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile; [0171]
(2,2-Dimethyl-tetrahydro-pyran-4yl)-{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-
-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-
-methanone; [0172]
(2,2-Dimethyl-tetrahydro-pyran-4yl)-{3-[6-(6-Methoxy-5-methyl-pyridin-3-y-
l)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-met-
hanone; [0173]
(1,1-Dioxo-hexahydro-1lambda*6*-thiopyran-4-yl)-{(S)-3-[6-(6-methoxy-5-tr-
ifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yl-
oxy]-pyrrolidin-1-yl}-methanone; [0174]
(1,1-Dioxo-hexahydro-1lambda*6*-thiopyran-4-yl)-{3-[6-(6-methoxy-5-triflu-
oromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-
-pyrrolidin-1-yl}-methanone; [0175]
(S)-(2,4-Dimethyloxazol-5-yl)(3-(6-(6-methoxy-5-(trifluoromethyl)pyridin--
3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)met-
hanone; [0176]
(2,4-Dimethyloxazol-5-yl)(3-(6-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl-
)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)methano-
ne; [0177]
(S)-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-t-
etrahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(thiazol-5-yl)meth-
anone; [0178]
(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydropyrid-
o[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(thiazol-5-yl)methanone;
[0179]
(S)-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydrop-
yrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(1-methyl-1H-pyrazol-5-yl)me-
thanone; [0180]
(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydropyrid-
o[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(1-methyl-1H-pyrazol-5-yl)methan-
one; [0181]
4-((S)-3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydr-
opyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidine-1-carbonyl)pyrrolidin-2-one;
[0182]
4-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrah-
ydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidine-1-carbonyl)pyrrolidin-2-one-
; [0183]
(S)-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tet-
rahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(pyridin-3-yl)methan-
one; [0184]
(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydropyrid-
o[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(pyridin-3-yl)methanone;
[0185]
(S)-(1H-Imidazol-4-yl)(3-(6-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)-5-
,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)methanone;
[0186]
(1H-Imidazol-4-yl)(3-(6-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl-
)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)methano-
ne; [0187]
5-((S)-3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-
-tetrahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidine-1-carbonyl)pyrrolidi-
n-2-one; [0188]
5-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydropyr-
ido[4,3-d]pyrimidin-4-yloxy)pyrrolidine-1-carbonyl)pyrrolidin-2-one;
[0189]
(S)-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetr-
ahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(pyridin-4-yl)methano-
ne; [0190]
(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetra-
hydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(pyridin-4-yl)methanon-
e; [0191]
(S)-(1,3-Dimethyl-1H-pyrazol-4-yl)(3-(6-(6-methoxy-5-(trifluorom-
ethyl)pyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrro-
lidin-1-yl)methanone; [0192]
(1,3-Dimethyl-1H-pyrazol-4-yl)(3-(6-(6-methoxy-5-(trifluoromethyl)pyridin-
-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)me-
thanone; [0193]
(S)-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydrop-
yrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(1H-pyrazol-4-yl)methanone;
[0194]
(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahyd-
ropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(1H-pyrazol-4-yl)methanon-
e; [0195]
(S)-(3-(6-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-te-
trahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(5-methyl-1,3,4-oxa-
diazol-2-yl)methanone;
[0196]
(3-(6-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahyd-
ropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(5-methyl-1,3,4-oxadiazol-
-2-yl)methanone; [0197]
(S)-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydrop-
yrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(pyrazin-2-yl)methanone;
[0198]
(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahyd-
ropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(pyrazin-2-yl)methanone;
[0199]
(S)-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetr-
ahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(1-methyl-1H-imidazol-
-4-yl)methanone; [0200]
(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydropyrid-
o[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(1-methyl-1H-imidazol-4-yl)metha-
none; [0201]
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-p-
yrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(1-methyl-1H-pyrazol-4-yl)-
-methanone; [0202]
{3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(1-methyl-1H-pyrazol-4-yl)-met-
hanone; [0203]
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-p-
yrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-thiazol-4-yl-methanone;
[0204]
{3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydr-
o-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-thiazol-4-yl-methanone;
[0205]
{(S)-3-[6-(5-Chloro-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyr-
ido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-meth-
anone; [0206]
{3-[6-(5-Chloro-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]p-
yrimidin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone;
[0207]
(S)-(3-(6-(6-Amino-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrah-
ydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4--
yl)methanone; [0208]
(3-(6-(6-Amino-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydropyrido[-
4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanon-
e; [0209]
(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrah-
ydropyrido[4,3-d]pyrimidin-4-yloxy)azetidin-1-yl)(tetrahydro-2H-pyran-4-yl-
)methanone; [0210]
{(S)-3-[6-(2-Methoxy-pyrimidin-5-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyri-
midin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone;
[0211]
{3-[6-(2-Methoxy-pyrimidin-5-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidi-
n-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone;
[0212]
[(S)-3-(6-Quinolin-3-yl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy-
)-pyrrolidin-1-yl]-(tetrahydro-pyran-4-yl)-methanone; [0213]
[3-(6-Quinolin-3-yl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-py-
rrolidin-1-yl]-(tetrahydro-pyran-4-yl)-methanone; [0214]
(S)-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydrop-
yrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)me-
thanone; [0215]
(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydropyrid-
o[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)methan-
one; [0216]
(S)-1-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydr-
opyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)-3,3-dimethylbutan-1-one;
[0217]
1-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrah-
ydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)-3,3-dimethylbutan-1-on-
e; [0218]
1-{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-t-
etrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-propan-1-one;
[0219]
1-{3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-propan-1-one;
[0220]
2-Methoxy-5-[4-((S)-1-propionyl-pyrrolidin-3-yloxy)-7,8-dihydro-5H-pyrido-
[4,3-d]pyrimidin-6-yl]-nicotinonitrile; [0221]
2-Methoxy-5-[4-(1-propionyl-pyrrolidin-3-yloxy)-7,8-dihydro-5H-pyrido[4,3-
-d]pyrimidin-6-yl]-nicotinonitrile; [0222]
(S)-6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-4-(1-(pyridin-2-yl)pyrr-
olidin-3-yloxy)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine; [0223]
6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-4-(1-(pyridin-2-yl)pyrrolid-
in-3-yloxy)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine; [0224]
(S)-6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-4-(1-(pyrimidin-2-yl)py-
rrolidin-3-yloxy)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine; [0225]
6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-4-(1-(pyrimidin-2-yl)pyrrol-
idin-3-yloxy)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine; [0226]
(S)-1-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydr-
opyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)propan-1-one;
[0227]
1-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydropyr-
ido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)propan-1-one; [0228]
(S)-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydrop-
yrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)-
methanone; [0229]
(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydropyrid-
o[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)meth-
anone; [0230]
(S)-2-Methoxy-5-(4-(1-(tetrahydro-2H-pyran-4-carbonyl)pyrrolidin-3-ylamin-
o)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)nicotinonitrile;
[0231]
2-Methoxy-5-(4-(1-(tetrahydro-2H-pyran-4-carbonyl)pyrrolidin-3-ylamino)-7-
,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)nicotinonitrile; [0232]
(S)-1-(4-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrah-
ydropyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidine-1-carbonyl)piperidin-1-yl-
)ethanone; [0233]
1-(4-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydro-
pyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidine-1-carbonyl)piperidin-1-yl)eth-
anone; [0234]
(2,2-Dimethyltetrahydro-2H-pyran-4-yl)((S)-3-(6-(6-methoxy-5-(trifluorome-
thyl)pyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-ylamino)pyrr-
olidin-1-yl)methanone; [0235]
(2,2-Dimethyltetrahydro-2H-pyran-4-yl)(3-(6-(6-methoxy-5-(trifluoromethyl-
)pyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-ylamino)pyrrolid-
in-1-yl)methanone; [0236]
(S)-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydrop-
yrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)(oxazol-5-yl)methanone;
[0237]
(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahyd-
ropyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)(oxazol-5-yl)methanone;
[0238]
((S)-3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetr-
ahydropyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)((1s,4R)-4-methoxyc-
yclohexyl)methanone; [0239]
(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydropyrid-
o[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)((1s,4R)-4-methoxycyclohexyl)m-
ethanone; [0240]
((S)-3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydrop-
yrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)((1r,4S)-4-methoxycyclohex-
yl)methanone; [0241]
(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydropyrid-
o[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)((1r,4S)-4-methoxycyclohexyl)m-
ethanone; [0242]
((1s,4R)-4-Hydroxycyclohexyl)((S)-3-(6-(6-methoxy-5-(trifluoromethyl)pyri-
din-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1--
yl)methanone; [0243]
((1s,4R)-4-Hydroxycyclohexyl)(3-(6-(6-methoxy-5-(trifluoromethyl)pyridin--
3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)m-
ethanone; [0244]
((1r,4S)-4-Hydroxycyclohexyl)((S)-3-(6-(6-methoxy-5-(trifluoromethyl)pyri-
din-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1--
yl)methanone; [0245]
((1r,4S)-4-Hydroxycyclohexyl)(3-(6-(6-methoxy-5-(trifluoromethyl)pyridin--
3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)m-
ethanone; [0246]
(S)-(3-(6-(6-Methoxy-5-methylpyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d-
]pyrimidin-4-ylamino)pyrrolidin-1-yl)(1-methyl-1H-imidazol-4-yl)methanone;
[0247]
(3-(6-(6-Methoxy-5-methylpyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,-
3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)(1-methyl-1H-imidazol-4-yl)methano-
ne; [0248]
(S)-(3-(6-(6-Methoxy-5-methylpyridin-3-yl)-5,6,7,8-tetrahydropy-
rido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)(oxazol-5-yl)methanone;
[0249]
(3-(6-(6-Methoxy-5-methylpyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,-
3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)(oxazol-5-yl)methanone;
[0250]
(S)-(3-(6-(6-Methoxy-5-methylpyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d-
]pyrimidin-4-ylamino)pyrrolidin-1-yl)(oxazol-4-yl)methanone; [0251]
(3-(6-(6-Methoxy-5-methylpyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyr-
imidin-4-ylamino)pyrrolidin-1-yl)(oxazol-4-yl)methanone; [0252]
(2,2-Dimethyltetrahydro-2H-pyran-4-yl)((S)-3-(6-(6-methoxy-5-methylpyridi-
n-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl-
)methanone; [0253]
(2,2-Dimethyltetrahydro-2H-pyran-4-yl)(3-(6-(6-methoxy-5-methylpyridin-3--
yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)met-
hanone; [0254]
(S)-1-(3-(6-(6-Methoxy-5-methylpyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-
-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)propan-1-one; [0255]
1-(3-(6-(6-Methoxy-5-methylpyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]p-
yrimidin-4-ylamino)pyrrolidin-1-yl)propan-1-one; [0256]
(S)-(3-(6-(5-Chloro-6-methoxypyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d-
]pyrimidin-4-ylamino)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone;
[0257]
(3-(6-(5-Chloro-6-methoxypyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,-
3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanon-
e; [0258]
(S)-(3-(6-(6-Methoxy-5-methylpyridin-3-yl)-5,6,7,8-tetrahydropyr-
ido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)me-
thanone; [0259]
(3-(6-(6-Methoxy-5-methylpyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyr-
imidin-4-ylamino)pyrrolidin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone;
[0260]
(Tetrahydro-pyran-4-yl)-{(S)-3-{6-(5-(trifluoromethyl)pyridin-3-yl-
)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl}-meth-
anone; [0261]
(Tetrahydro-pyran-4-yl)-{3-{6-(5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-t-
etrahydropyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl}-methanone;
[0262]
(S)-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetr-
ahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(4-methylpiperazin-1--
yl)methanone; [0263]
(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydropyrid-
o[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(4-methylpiperazin-1-yl)methanon-
e; [0264]
(S)-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-te-
trahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(morpholino)methano-
ne; [0265]
(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetra-
hydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(morpholino)methanone;
[0266]
(S)-(4-Hydroxypiperidin-1-yl)(3-(6-(6-methoxy-5-(trifluoromethyl)p-
yridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-
-yl)methanone; [0267]
4-Hydroxypiperidin-1-yl)(3-(6-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)-
-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)methanon-
e; [0268]
(S)--N-(2-Hydroxyethyl)-3-(6-(6-methoxy-5-(trifluoromethyl)pyrid-
in-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yloxy)-N-methylpyrroli-
dine-1-carboxamide; [0269]
N-(2-Hydroxyethyl)-3-(6-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7-
,8-tetrahydropyrido[4,3-d]pyrimidin-4-yloxy)-N-methylpyrrolidine-1-carboxa-
mide; [0270]
(S)-1-(4-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrah-
ydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidine-1-carbonyl)piperazin-1-yl)e-
thanone; [0271]
1-(4-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydro-
pyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidine-1-carbonyl)piperazin-1-yl)ethan-
one; [0272]
(S)-2-Methoxy-5-(4-(1-(morpholine-4-carbonyl)pyrrolidin-3-yloxy)-7,8-dihy-
dropyrido[4,3-d]pyrimidin-6(5H)-yl)nicotinonitrile; [0273]
2-Methoxy-5-(4-(1-(morpholine-4-carbonyl)pyrrolidin-3-yloxy)-7,8-dihydrop-
yrido[4,3-d]pyrimidin-6(5H)-yl)nicotinonitrile; [0274]
(S)-(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydrop-
yrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(oxazol-4-yl)methanone:
[0275]
(3-(6-(6-Methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahyd-
ropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidin-1-yl)(oxazol-4-yl)methanone;
[0276]
1-(4-{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8--
tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidine-1-carbonyl}-piperid-
in-1-yl)-ethanone; [0277]
1-(4-{3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro--
pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidine-1-carbonyl}-piperidin-1-yl)-et-
hanone; [0278]
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-p-
yrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(3-methyl-3H-imidazol-4-yl-
)-methanone; [0279]
{3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(3-methyl-3H-imidazol-4-yl)-me-
thanone; [0280]
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-p-
yrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-oxazol-5-yl-methanone;
[0281]
{3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydr-
o-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-oxazol-5-yl-methanone;
[0282]
{(S)-3-[6-(6-Methoxy-pyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]-
pyrimidin-4-yloxy)pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone;
or [0283]
{3-[6-(6-Methoxy-pyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyri-
midin-4-yloxy)pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone.
[0284] Compounds of the formula (I) may have different isomeric
forms. As used herein, the term "an optical isomer" or "a
stereoisomer" refers to any of the various stereo isomeric
configurations which may exist for a given compound of the present
invention and includes geometric isomers. It is understood that a
substituent may be attached at a chiral center of a carbon atom.
Therefore, the invention includes enantiomers, diastereomers or
racemates of the compound. "Enantiomers" are a pair of
stereoisomers that are non-superimposable mirror images of each
other. A 1:1 mixture of a pair of enantiomers is a "racemic"
mixture. The term is used to designate a racemic mixture where
appropriate. "Diastereoisomers" are stereoisomers that have at
least two asymmetric atoms, but which are not mirror-images of each
other. The absolute stereochemistry is specified according to the
Cahn-Ingold-Prelog R-S system. When a compound is a pure enantiomer
the stereochemistry at each chiral carbon may be specified by
either R or S. Resolved compounds whose absolute configuration is
unknown can be designated (+) or (-) depending on the direction
(dextro- or levorotatory) which they rotate plane polarized light
at the wavelength of the sodium D line. Certain of the compounds
described herein contain one or more asymmetric centers or axes and
may thus give rise to enantiomers, diastereomers, and other
stereoisomeric forms that may be defined, in terms of absolute
stereochemistry, as (R)- or (S)-. The present invention is meant to
include all such possible isomers, including racemic mixtures,
optically pure forms and intermediate mixtures. Optically active
(R)- and (S)-isomers may be prepared using chiral synthons or
chiral reagents, or resolved using conventional techniques. If the
compound contains a double bond, the substituent may be E or Z
configuration. If the compound contains a disubstituted cycloalkyl,
the cycloalkyl substituent may have a cis- or trans-configuration.
All tautomeric forms are also intended to be included.
[0285] As used herein, the term "pharmaceutically acceptable salts"
refers to salts that retain the biological effectiveness and
properties of the compounds of this invention and, which typically
are not biologically or otherwise undesirable. In many cases, the
compounds of the present invention are capable of forming acid
and/or base salts by virtue of the presence of amino and/or
carboxyl groups or groups similar thereto.
[0286] Pharmaceutically acceptable acid addition salts can be
formed with inorganic acids and organic acids, e.g., acetate,
aspartate, benzoate, besylate, bromide/hydrobromide,
bicarbonate/carbonate, bisulfate/sulfate, camphorsulfornate,
chloride/hydrochloride, chlortheophyllonate, citrate,
ethandisulfonate, fumarate, gluceptate, gluconate, glucuronate,
hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate,
laurylsulfate, malate, maleate, malonate, mandelate, mesylate,
methylsulphate, naphthoate, napsylate, nicotinate, nitrate,
octadecanoate, oleate, oxalate, palmitate, pamoate,
phosphate/hydrogen phosphate/dihydrogen phosphate,
polygalacturonate, propionate, stearate, succinate,
sulfosalicylate, tartrate, tosylate and trifluoroacetate salts.
[0287] Inorganic acids from which salts can be derived include, for
example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, phosphoric acid, and the like.
[0288] Organic acids from which salts can be derived include, for
example, acetic acid, propionic acid, glycolic acid, oxalic acid,
maleic acid, malonic acid, succinic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, mandelic acid, methanesulfonic
acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic
acid, and the like. Pharmaceutically acceptable base addition salts
can be formed with inorganic and organic bases.
[0289] Inorganic bases from which salts can be derived include, for
example, ammonium salts and metals from columns 1 to 12 of the
periodic table. In certain embodiments, the salts are derived from
sodium, potassium, ammonium, calcium, magnesium, iron, silver,
zinc, and copper; particularly suitable salts include ammonium,
potassium, sodium, calcium and magnesium salts.
[0290] Organic bases from which salts can be derived include, for
example, primary, secondary, and tertiary amines, substituted
amines including naturally occurring substituted amines, cyclic
amines, basic ion exchange resins, and the like. Certain organic
amines include isopropylamine, benzathine, cholinate,
diethanolamine, diethylamine, lysine, meglumine, piperazine and
tromethamine.
[0291] The pharmaceutically acceptable salts of the present
invention can be synthesized from a parent compound, a basic or
acidic moiety, by conventional chemical methods. Generally, such
salts can be prepared by reacting free acid forms of these
compounds with a stoichiometric amount of the appropriate base
(such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate or the
like), or by reacting free base forms of these compounds with a
stoichiometric amount of the appropriate acid. Such reactions are
typically carried out in water or in an organic solvent, or in a
mixture of the two. Generally, use of non-aqueous media like ether,
ethyl acetate, ethanol, isopropanol, or acetonitrile is desirable,
where practicable. Lists of additional suitable salts can be found,
e.g., in "Remington's Pharmaceutical Sciences", 20th ed., Mack
Publishing Company, Easton, Pa., (1985); and in "Handbook of
Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and
Wermuth (Wiley-VCH, Weinheim, Germany, 2002).
[0292] For isolation or purification purposes it is also possible
to use pharmaceutically unacceptable salts, for example picrates or
perchlorates. For therapeutic use, only pharmaceutically acceptable
salts or free compounds are employed.
[0293] In view of the close relationship between the novel
compounds of the formula (I) in free form and those in the form of
their salts, including those salts that can be used as
intermediates, for example in the purification or identification of
the novel compounds, any reference to the compounds or a compound
of the formula (I) hereinbefore and hereinafter is to be understood
as referring to the compound in free form and/or also to one or
more salts thereof, as appropriate and expedient, as well as to one
or more solvates, e.g. hydrates.
[0294] Any formula given herein is also intended to represent
unlabeled forms as well as isotopically labeled forms of the
compounds. Isotopically labeled compounds have structures depicted
by the formulas given herein except that one or more atoms are
replaced by an atom having a selected atomic mass or mass number.
Examples of isotopes that can be incorporated into compounds of the
invention include isotopes of hydrogen, carbon, nitrogen, oxygen,
phosphorous, fluorine, and chlorine, such as .sup.2H, .sup.3H,
.sup.11C, .sup.13C, .sup.14C, .sup.15N, .sup.18F .sup.31P,
.sup.32P, .sup.35S, .sup.36Cl, .sup.125I respectively. The
invention includes various isotopically labeled compounds as
defined herein, for example those into which radioactive isotopes,
such as .sup.3H and .sup.14C or those into which non-radioactive
isotopes, such as .sup.2H and .sup.13C, are present. Such
isotopically labelled compounds are useful in metabolic studies
(with .sup.14C), reaction kinetic studies (with, for example
.sup.2H or .sup.3H), detection or imaging techniques, such as
positron emission tomography (PET) or single-photon emission
computed tomography (SPECT) including drug or substrate tissue
distribution assays, or in radioactive treatment of patients. In
particular, an .sup.18F or labeled compound may be particularly
desirable for PET or SPECT studies. Isotopically labeled compounds
of this invention and prodrugs thereof can generally be prepared by
carrying out the procedures disclosed in the schemes or in the
examples and preparations described below by substituting a readily
available isotopically labeled reagent for a non-isotopically
labeled reagent.
[0295] Further, substitution with heavier isotopes, particularly
deuterium (i.e., .sup.2H or D) may afford certain therapeutic
advantages resulting from greater metabolic stability, for example
increased in vivo half-life or reduced dosage requirements or an
improvement in therapeutic index. It is understood that deuterium
in this context is regarded as a substituent of a compound of the
formula (I). The concentration of such a heavier isotope,
specifically deuterium, may be defined by the isotopic enrichment
factor. The term "isotopic enrichment factor" as used herein means
the ratio between the isotopic abundance and the natural abundance
of a specified isotope. If a substituent in a compound of this
invention is denoted deuterium, such compound has an isotopic
enrichment factor for each designated deuterium atom of at least
3500 (52.5% deuterium incorporation at each designated deuterium
atom), at least 4000 (60% deuterium incorporation), at least 4500
(67.5% deuterium incorporation), at least 5000 (75% deuterium
incorporation), at least 5500 (82.5% deuterium incorporation), at
least 6000 (90% deuterium incorporation), at least 6333.3 (95%
deuterium incorporation), at least 6466.7 (97% deuterium
incorporation), at least 6600 (99% deuterium incorporation), or at
least 6633.3 (99.5% deuterium incorporation).
[0296] Isotopically-labeled compounds of the formula (I) can
generally be prepared by conventional techniques known to those
skilled in the art or by processes analogous to those described in
the accompanying Examples and Preparations using an appropriate
isotopically-labeled reagents in place of the non-labeled reagent
previously employed.
[0297] Pharmaceutically acceptable solvates in accordance with the
invention include those wherein the solvent of crystallization may
be isotopically substituted, e.g. D.sub.2O, d.sub.6-acetone,
d.sub.6-DMSO.
[0298] Compounds of the invention, i.e. compounds of the formula
(I) that contain groups capable of acting as donors and/or
acceptors for hydrogen bonds may be capable of forming co-crystals
with suitable co-crystal formers. These co-crystals may be prepared
from compounds of the formula (I) by known co-crystal forming
procedures. Such procedures include grinding, heating,
co-subliming, co-melting, or contacting in solution compounds of
the formula (I) with the co-crystal former under crystallization
conditions and isolating co-crystals thereby formed. Suitable
co-crystal formers include those described in WO 2004/078163. Hence
the invention further provides co-crystals comprising a compound of
the formula (I).
[0299] Any asymmetric atom (e.g., carbon or the like) of the
compound(s) of the present invention can be present in racemic or
enantiomerically enriched, for example the (R)-, (S)- or
(R,S)-configuration. In certain embodiments, each asymmetric atom
has at least 50% enantiomeric excess, at least 60% enantiomeric
excess, at least 70% enantiomeric excess, at least 80% enantiomeric
excess, at least 90% enantiomeric excess, at least 95% enantiomeric
excess, or at least 99% enantiomeric excess in the (R)- or
(S)-configuration. Substituents at atoms with unsaturated bonds
may, if possible, be present in cis- (Z)- or trans- (E)-form.
[0300] Accordingly, as used herein a compound of the present
invention can be in the form of one of the possible isomers,
rotamers, atropisomers, tautomers or mixtures thereof, for example,
as substantially pure geometric (cis or trans) isomers,
diastereomers, optical isomers (antipodes), racemates or mixtures
thereof.
[0301] Mixtures of isomers obtainable according to the invention
can be separated in a manner known to those skilled in the art into
the individual isomers; diastereoisomers can be separated, for
example, by partitioning between polyphasic solvent mixtures,
recrystallisation and/or chromatographic separation, for example
over silica gel or by e.g. medium pressure liquid chromatography
over a reversed phase column, and racemates can be separated, for
example, by the formation of salts with optically pure salt-forming
reagents and separation of the mixture of diastereoisomers so
obtainable, for example by means of fractional crystallisation, or
by chromatography over optically active column materials.
[0302] Any resulting racemates of final products or intermediates
can be resolved into the optical antipodes by known methods, e.g.,
by separation of the diastereomeric salts thereof, obtained with an
optically active acid or base, and liberating the optically active
acidic or basic compound. In particular, a basic moiety may thus be
employed to resolve the compounds of the present invention into
their optical antipodes, e.g., by fractional crystallization of a
salt formed with an optically active acid, e.g., tartaric acid,
dibenzoyl tartaric acid, diacetyl tartaric acid, di-O,O'-p-toluoyl
tartaric acid, mandelic acid, malic acid or camphor-10-sulfonic
acid. Racemic products can also be resolved by chiral
chromatography, e.g., high pressure liquid chromatography (HPLC)
using a chiral adsorbent. Compounds of the present invention are
either obtained in the free form, as a salt thereof, or as prodrug
derivatives thereof.
[0303] When both a basic group and an acid group are present in the
same molecule, the compounds of the present invention may also form
internal salts, e.g., zwitterionic molecules.
[0304] The present invention also provides pro-drugs of the
compounds of the present invention that converts in vivo to the
compounds of the present invention. A pro-drug is an active or
inactive compound that is modified chemically through in vivo
physiological action, such as hydrolysis, metabolism and the like,
into a compound of this invention following administration of the
prodrug to a subject. The suitability and techniques involved in
making and using pro-drugs are well known by those skilled in the
art. Prodrugs can be conceptually divided into two non-exclusive
categories, bioprecursor prodrugs and carrier prodrugs. See The
Practice of Medicinal Chemistry, Ch. 31-32 (Ed. Wermuth, Academic
Press, San Diego, Calif., 2001). Generally, bioprecursor prodrugs
are compounds, which are inactive or have low activity compared to
the corresponding active drug compound, which contain one or more
protective groups and are converted to an active form by metabolism
or solvolysis. Both the active drug form and any released metabolic
products should have acceptably low toxicity.
[0305] Carrier prodrugs are drug compounds that contain a transport
moiety, e.g. that improve uptake and/or localized delivery to a
site(s) of action. Desirably for such a carrier prodrug, the
linkage between the drug moiety and the transport moiety is a
covalent bond, the prodrug is inactive or less active than the drug
compound, and any released transport moiety is acceptably
non-toxic. For prodrugs where the transport moiety is intended to
enhance uptake, typically the release of the transport moiety
should be rapid. In other cases, it is desirable to utilize a
moiety that provides slow release, e.g., certain polymers or other
moieties, such as cyclodextrins. Carrier prodrugs can, for example,
be used to improve one or more of the following properties:
increased lipophilicity, increased duration of pharmacological
effects, increased site-specificity, decreased toxicity and adverse
reactions, and/or improvement in drug formulation (e.g., stability,
water solubility, suppression of an undesirable organoleptic or
physiochemical property). For example, lipophilicity can be
increased by esterification of (a) hydroxyl groups with lipophilic
carboxylic acids (e.g., a carboxylic acid having at least one
lipophilic moiety), or (b) carboxylic acid groups with lipophilic
alcohols (e.g., an alcohol having at least one lipophilic moiety,
for example aliphatic alcohols).
[0306] Exemplary prodrugs are, e.g., esters of free carboxylic
acids and S-acyl derivatives of thiols and O-acyl derivatives of
alcohols or phenols, wherein acyl has a meaning as defined herein.
Suitable prodrugs are often pharmaceutically acceptable ester
derivatives convertible by solvolysis under physiological
conditions to the parent carboxylic acid, e.g., lower alkyl esters,
cycloalkyl esters, lower alkenyl esters, benzyl esters, mono- or
di-substituted lower alkyl esters, such as the omega-(amino, mono-
or di-lower alkylamino, carboxy, lower alkoxycarbonyl)-lower alkyl
esters, the alpha-(lower alkanoyloxy, lower alkoxycarbonyl or
di-lower alkylaminocarbonyl)-lower alkyl esters, such as the
pivaloyloxymethyl ester and the like conventionally used in the
art. In addition, amines have been masked as arylcarbonyloxymethyl
substituted derivatives which are cleaved by esterases in vivo
releasing the free drug and formaldehyde (Bundgaard, J. Med. Chem.
2503 (1989)). Moreover, drugs containing an acidic NH group, such
as imidazole, imide, indole and the like, have been masked with
N-acyloxymethyl groups (Bundgaard, Design of Prodrugs, Elsevier
(1985)). Hydroxy groups have been masked as esters and ethers. EP
039,051 (Sloan and Little) discloses Mannich-base hydroxamic acid
prodrugs, their preparation and use.
[0307] Furthermore, the compounds of the present invention,
including their salts, can also be obtained in the form of their
hydrates, or include other solvents used for their crystallization.
The compounds of the present invention may inherently or by design
form solvates with pharmaceutically acceptable solvents (including
water); therefore, it is intended that the invention embrace both
solvated and unsolvated forms. The term "solvate" refers to a
molecular complex of a compound of the present invention (including
pharmaceutically acceptable salts thereof) with one or more solvent
molecules. Such solvent molecules are those commonly used in the
pharmaceutical art, which are known to be innocuous to the
recipient, e.g., water, ethanol, and the like. The term "hydrate"
refers to the complex where the solvent molecule is water.
[0308] The compounds of the present invention, including salts,
hydrates and solvates thereof, may inherently or by design form
polymorphs.
[0309] The invention relates in a second aspect to the manufacture
of a compound of formula I. The compounds of formula I or salts
thereof are prepared in accordance with processes known per se,
though not previously described for the manufacture of the
compounds of the formula I.
General Reaction Processes:
##STR00013##
[0311] In one embodiment, the invention relates to a process for
manufacturing a compound of formula I (Method A) comprising steps
a, d, e, b, and c.
[0312] The compound of formula I is obtained via the step b of
deprotecting PG.sup.2 from the compound of formula (E), wherein
PG.sup.2 represents a suitable protecting group, preferable a
benzyl group, and the other substituents are as defined above,
##STR00014##
followed by reaction step c with
R.sup.2-Hal,
[0313] Wherein R.sup.2 is defined above and Hal represents halogen,
particularly iodo or bromo; under customary Buchwald-Hartwig
conditions using a ligand such as X-Phos,
di-tert-butyl(2'-methylbiphenyl-2-yl)phosphine or
2-di-t-butylphosphino-2'-(N,N-dimethylamino)biphenyl with a
palladium catalyst such as Pd.sub.2(dba).sub.3 or
Pd.sub.2(dba).sub.3.CHCl.sub.3 or Pd(OAc).sub.2, preferably
Pd.sub.2(dba).sub.3 with X-Phos, in the presence of a base such as
preferably Cs.sub.2CO.sub.3 or tert-BuONa, in an organic solvent
such as an ether, preferably dioxane or THF. The reaction is
preferably stirred at a temperature of approximately 80-120.degree.
C., preferably 120.degree. C. The reaction is preferably carried
out under an inert gas such as nitrogen or argon.
[0314] The compound of formula (E) is prepared comprising the step
d of deprotecting PG.sup.1 from the compound of formula (C),
wherein PG.sup.1 represents a suitable protecting group, for
example BOC, and the other substituents are as defined above,
##STR00015##
followed by coupling reaction step e with
R.sup.1-Act,
[0315] step e1: Where R.sup.1 is --C(O)--R.sup.4, wherein R.sup.4
is defined above, and Act represents an activating group or a
hydroxy group: The coupling reaction is an amide, urea or carbamic
ester formation. There are many known ways of preparing amides urea
or carbamic ester. The coupling reaction step can be carried out
with Act representing an activating group, preferably in a one step
procedure or with Act representing a hydroxy group either involving
a one or two step procedure. For examples of amide bond formations,
see Mantalbetti, C.A.G.N and Falque, V., Amide bond formation and
peptide coupling, Tetrahedron, 2005, 61(46), pp 10827-10852 and
references cited therein. For examples of urea synthesis, see
Sartori, G.; Maggi, R. Acyclic and cyclic ureas, Science of
Synthesis (2005), 18, 665-758; Gallou, Isabelle. Unsymmetrical
ureas Synthetic methodologies and application in drug design,
Organic Preparations and Procedures International (2007), 39(4),
355-383. For examples of carbamate synthesis see Adams, Philip;
Baron, Frank A. Esters of carbamic acid, Chemical Reviews (1965),
65(5), 567-602. The examples provided herein are thus not intended
to be exhaustive, but merely illustrative; step e2: Where R.sup.1
is selected from phenyl, pyridyl, pyrimidinyl, pyrazinyl,
pyridazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl or 1,3,5-triazinyl
and Act represents halogen, particularly iodo or bromo: The
coupling reaction is carried out in the presence of an amine base
such as N,N-diisopropylethylamine. The reaction is carried out in
the presence of an organic solvent or, preferably without a solvent
under microwave heating e.g. at 160.degree. C. for 20 min.
Alternatively, the reaction can be carried out under customary
Buchwald-Hartwig conditions using a ligand such as X-Phos or
2-di-t-butylphosphino-2'-(N,N-dimethylamino)biphenyl with a
palladium catalyst such as Pd.sub.2(dba).sub.3 or
Pd.sub.2(dba).sub.3.CHCl.sub.3 or Pd(OAc).sub.2, preferably
Pd.sub.2(dba).sub.3 with X-Phos, in the presence of a base such as
preferably Cs.sub.2CO.sub.3 or tert-BuONa, in an organic solvent
such as an ether, preferably dioxane or THF. The reaction is
preferably stirred at a temperature of approximately 80-120.degree.
C., preferably 120.degree. C. The reaction is preferably carried
out under an inert gas such as nitrogen or argon.
[0316] The compound of formula (C) is prepared comprising the step
a of coupling a compound of formula (A), wherein PG.sup.2
represents a suitable protecting group, for example a benzyl group
and X represents halogen, particularly chloro, or hydroxy; with a
compound of formula (B) wherein PG.sup.1 represents a suitable
protecting group, for example BOC, and the other substituents are
as defined above,
##STR00016##
step a1: Where YH is OH and X represents halogen: The reaction
takes place in the presence of a suitable base such as sodium
hydroxide (NaH) or potassium t-butoxide (tBuOK) and polar organic
solvent such as THF, 2-methyltetrahydrofuran or Dioxane under inert
gas conditions at room temperature. step a2: Where YH is NR.sup.3H
and X represents halogen: The reaction takes place in the presence
of a suitable base such as for example potassium carbonate or a
suitable amine base such as triethylamine or
N,N-diisopropylethylamine at elevated temperature (e.g. 120.degree.
C.) for 20-48 h. Typical conditions comprise the use of 1.0
equivalent of a compound of formula (A), 1.0 equivalent of a
compound of formula (B) and 1.5 equivalents of the base at
120.degree. C. for 48 h. step a3: Where YH is NR.sup.3H and X
represents hydroxy: A base promoted phosphonium coupling reaction
is employed, whereby a compound of the formula (A) in a suitable
solvent such as acetonitrile is reacted with a phosphonium salt
such as benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP) in the presence of a base such as
1,8-diaza-7-bicyclo[5.4.0]undecene (DBU) followed by addition of a
compound of the formula (B). The reaction mixture is preferably
stirred at a temperature of 20.degree. C. to 90.degree. C. for
18-72 h. The reaction may preferably be carried out under an inert
gas, e.g. nitrogen or argon. Typical conditions comprise of 1
equivalent of a compound of the formula (A), 1.0-1.5 equivalents of
BOP, 2.0-4.0 equivalents of DBU and 2.0-3.0 equivalents of a
compound of the formula (B) in acetonitrile at 65.degree. C. for 72
hours under argon.
[0317] In another embodiment, the invention relates to a process
for manufacturing a compound of formula I, comprising steps a, d
and e as defined above, starting from a compound of formula (A)
wherein PG.sup.2 represents R.sup.2 (Method A-a).
##STR00017##
[0318] The process for manufacturing a compound of formula (A)
wherein PG.sup.2 represents R.sup.2 comprises the steps f, g, h, i
and optionally k.
step f) Quaternarization of compound of formula (F), R.sup.8=alkyl
e.g. benzyl with compound of general formula R.sup.9--Hal, wherein
R.sup.9 represents alkyl, particularly methyl and Hal represents
halogen, particularly iodo or bromo, under customary condition
using in particular acetone as organic solvent leads to the
formation of compound of general formula (G). step g) The compound
of formula (H) is prepared by reaction of R.sup.2--NH.sub.2 with
compound of general formula (G). The reaction is performed by using
a base such a in particular K.sub.2CO.sub.3 in an organic solvent
such as in particular a 2/1 mixture of ethanol and water and
heating the reaction mixture at 80-100.degree. C., in particular
80.degree. C.
[0319] step h) The compound of formula (J) is prepared by reaction
of a compound of formula (H) with base such as in particular NaH
and compound of general formula (R.sup.10O).sub.2CO, wherein
R.sup.10 represents alkyl, particularly methyl. The reaction
mixture is stirred under high temperature (90.degree. C.).
step i) The compound of formula (A), X.dbd.OH is prepared by
pyrimidine ring formation, reacting the compound of formula (J)
with formamidine acetate in the presence of a base such as sodium
methoxide and in an organic solvent such as methanol at elevated
temperature such as 90.degree. C. for 2-18 h. step k) The compound
of formula (A), X.dbd.Cl is prepared by reaction of a compound of
formula (A), X.dbd.OH with phosphoryl chloride in the presence of a
base such as triethylamine in an organic solvent such as toluene at
elevated temperature such as 100.degree. C. for 12-18 h.
[0320] In another embodiment, the invention relates to a process
for manufacturing a compound of formula I (Method B) comprising
steps a, b, c, d, and e.
[0321] The compound of formula I is obtained via the step d of
deprotecting PG.sup.1 from the compound of formula, (D), wherein
PG.sup.1 represents a suitable protecting group, preferable a BOC
group, and the other substituents are as defined above,
##STR00018##
followed by reaction step e with
R1-Act,
[0322] step e1: Where R.sup.1 is --C(O)--R.sup.4, wherein R.sup.4
is defined above, and Act represents an activating group or a
hydroxy group: The coupling reaction is an amide, urea or carbamic
ester formation. There are many known ways of preparing amides urea
or carbamic ester. The coupling reaction step can be carried out
with Act representing an activating group, preferably in a one step
procedure or with Act representing a hydroxy group either involving
a one or two step procedure. For examples of amide bond formations,
see Mantalbetti, C.A.G.N and Falque, V., Amide bond formation and
peptide coupling, Tetrahedron, 2005, 61(46), pp 10827-10852 and
references cited therein. For examples of urea synthesis, see
Sartori, G.; Maggi, R. Acyclic and cyclic ureas, Science of
Synthesis (2005), 18, 665-758; Gallou, Isabelle. Unsymmetrical
ureas Synthetic methodologies and application in drug design,
Organic Preparations and Procedures International (2007), 39(4),
355-383. For examples of carbamate synthesis see Adams, Philip;
Baron, Frank A. Esters of carbamic acid, Chemical Reviews (1965),
65(5), 567-602. The examples provided herein are thus not intended
to be exhaustive, but merely illustrative; step e2: Where R.sup.1
is selected from phenyl, pyridyl, pyrimidinyl, pyrazinyl,
pyridazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl or 1,3,5-triazinyl
and Act represents halogen, particularly iodo or bromo: The
coupling reaction is carried out in the presence of an amine base
such as N,N-diisopropylethylamine. The reaction is carried out in
the presence of an organic solvent or, preferably without a solvent
under microwave heating e.g. at 160.degree. C. for 20 min.
Alternatively, the reaction can be carried out under customary
Buchwald-Hartwig conditions using such a ligand such as X-Phos or
2-di-t-butylphosphino-2'-(N,N-dimethylamino)biphenyl with a
palladium catalyst such as Pd.sub.2(dba).sub.3 or
Pd.sub.2(dba).sub.3.CHCl.sub.3 or Pd(OAc).sub.2, preferably
Pd.sub.2(dba).sub.3 with X-Phos, in the presence of a base such as
preferably Cs.sub.2CO.sub.3 or tert-BuONa, in an organic solvent
such as an ether, preferably dioxane or THF. The reaction is
preferably stirred at a temperature of approximately 80-120.degree.
C., preferably 120.degree. C. The reaction is preferably carried
out under an inert gas such as nitrogen or argon.
[0323] The compound of formula (D) is prepared comprising the step
b of deprotecting PG.sup.2 from the compound of formula (C),
wherein PG.sup.2 represents a suitable protecting group, for
example a benzyl group, and the other substituents are as defined
above,
##STR00019##
followed by reaction step c with
R.sup.2-Hal,
[0324] Wherein R.sup.2 is defined above and Hal represents halogen,
particularly iodo or bromo; under customary Buchwald-Hartwig
conditions using such a ligand such as X-Phos or
2-di-t-butylphosphino-2'-(N,N-dimethylamino)biphenyl with a
palladium catalyst such as Pd.sub.2(dba).sub.3 or
Pd.sub.2(dba).sub.3.CHCl.sub.3 or Pd(OAc).sub.2, preferably
Pd.sub.2(dba).sub.3 with X-Phos, in the presence of a base such as
preferably Cs.sub.2CO.sub.3 or tert-BuONa, in an organic solvent
such as an ether, preferably dioxane or THF. The reaction is
preferably stirred at a temperature of approximately 80-120.degree.
C., preferably 120.degree. C. The reaction is preferably carried
out under an inert gas such as nitrogen or argon.
[0325] The compound of formula (C) is prepared as described above
for method A.
[0326] In another embodiment, the invention relates to a process
for manufacturing a compound of formula I, comprising steps a, b
and c as defined above, starting from a compound of formula (B)
wherein PG.sup.1 represents R.sup.1 (Method B-a).
[0327] The term "activating group" as used herein relates to a
group that can activate a carboxylic acid, carbonic acid or
carbamic acid derivative, for coupling with an amine moiety to form
an amide, urea or carbamic ester moiety, respectively. Such groups
are chlorides, or groups resulting from the reaction of the acid
derivative with an activating agent. Suitable activating agents are
known to the skilled person, examples of such activating reagents
are carbodiimide derivatives, pentafluorophenyl ester derivatives,
triazole derivatives, imidazole derivatives.
"Protecting Group":
[0328] In the methods describe above, functional groups which are
present in the starting materials and are not intended to take part
in the reaction, are present in protected form if necessary, and
protecting groups that are present are cleaved, whereby said
starting compounds may also exist in the form of salts provided
that a salt-forming group is present and a reaction in salt form is
possible. In additional process steps, carried out as desired,
functional groups of the starting compounds which should not take
part in the reaction may be present in unprotected form or may be
protected for example by one or more protecting groups. The
protecting groups are then wholly or partly removed according to
one of the known methods. Protecting groups, and the manner in
which they are introduced and removed are described, for example,
in "Protective Groups in Organic Chemistry", Plenum Press, London,
New York 1973, and in "Methoden der organischen Chemie",
Houben-Weyl, 4th edition, Vol. 15/1, Georg-Thieme-Verlag, Stuttgart
1974 and in Theodora W. Greene, "Protective Groups in Organic
Synthesis", John Wiley & Sons, New York 1981. A characteristic
of protecting groups is that they can be removed readily, i.e.
without the occurrence of undesired secondary reactions, for
example by solvolysis, reduction, photolysis or alternatively under
physiological conditions.
[0329] The invention further includes any variant of the present
processes, in which an intermediate product obtainable at any stage
thereof is used as starting material and the remaining steps are
carried out, or in which the starting materials are formed in situ
under the reaction conditions, or in which the reaction components
are used in the form of their salts or optically pure
antipodes.
[0330] Compounds of the invention and intermediates can also be
converted into each other according to methods generally known to
those skilled in the art.
[0331] Intermediates and final products can be worked up and/or
purified according to standard methods, e.g. using chromatographic
methods, distribution methods, (re-) crystallization, and the
like.
[0332] The following applies in general to all processes mentioned
herein before and hereinafter. All the above-mentioned process
steps can be carried out under reaction conditions that are known
to those skilled in the art, including those mentioned
specifically, in the absence or, customarily, in the presence of
solvents or diluents, including, for example, solvents or diluents
that are inert towards the reagents used and dissolve them, in the
absence or presence of catalysts, condensation or neutralizing
agents, for example ion exchangers, such as cation exchangers, e.g.
in the H+ form, depending on the nature of the reaction and/or of
the reactants at reduced, normal or elevated temperature, for
example in a temperature range of from about -100.degree. C. to
about 190.degree. C., including, for example, from approximately
-80.degree. C. to approximately 150.degree. C., for example at from
-80 to -60.degree. C., at room temperature, at from -20 to
40.degree. C. or at reflux temperature, under atmospheric pressure
or in a closed vessel, where appropriate under pressure, and/or in
an inert atmosphere, for example under an argon or nitrogen
atmosphere.
[0333] At all stages of the reactions, mixtures of isomers that are
formed can be separated into the individual isomers, for example
diastereoisomers or enantiomers, or into any desired mixtures of
isomers, for example racemates or mixtures of diastereoisomers, for
example analogously to the methods described herein above.
[0334] The solvents from which those solvents that are suitable for
any particular reaction may be selected include those mentioned
specifically or, for example, water, esters, such as lower
alkyl-lower alkanoates, for example ethyl acetate, ethers, such as
aliphatic ethers, for example diethyl ether, or cyclic ethers, for
example tetrahydrofuran or dioxane, liquid aromatic hydrocarbons,
such as benzene or toluene, alcohols, such as methanol, ethanol or
1- or 2-propanol, nitriles, such as acetonitrile, halogenated
hydrocarbons, such as methylene chloride or chloroform, acid
amides, such as dimethylformamide or dimethyl acetamide, bases,
such as heterocyclic nitrogen bases, for example pyridine or
N-methylpyrrolidin-2-one, carboxylic acid anhydrides, such as lower
alkanoic acid anhydrides, for example acetic anhydride, cyclic,
linear or branched hydrocarbons, such as cyclohexane, hexane or
isopentane, methycyclohexane, or mixtures of those solvents, for
example aqueous solutions, unless otherwise indicated in the
description of the processes. Such solvent mixtures may also be
used in working up, for example by chromatography or partitioning.
The compounds, including their salts, may also be obtained in the
form of hydrates, or their crystals may, for example, include the
solvent used for crystallization. Different crystalline forms may
be present.
[0335] The invention relates also to those forms of the process in
which a compound obtainable as an intermediate at any stage of the
process is used as starting material and the remaining process
steps are carried out, or in which a starting material is formed
under the reaction conditions or is used in the form of a
derivative, for example in a protected form or in the form of a
salt, or a compound obtainable by the process according to the
invention is produced under the process conditions and processed
further in situ.
[0336] All starting materials, building blocks, reagents, acids,
bases, dehydrating agents, solvents and catalysts utilized to
synthesize the compounds of the present invention are either
commercially available or can be produced by organic synthesis
methods known to one of ordinary skill in the art (Houben-Weyl
4.sup.th Ed. 1952, Methods of Organic Synthesis, Thieme, Volume
21).
[0337] Members of the phosphoinositide-3 kinase (PI3K) family are
involved in cell growth, differentiation, survival, cytoskeletal
remodeling and the trafficking of intracellular organelles in many
different types of cells (Okkenhaug and Wymann, Nature Rev.
Immunol. 3:317 (2003).
[0338] To date, eight mammalian PI3Ks have been identified, divided
into three main classes (I, II and III) on the basis of their
genetic sequence, structure, adapter molecules, expression, mode of
activation, and preferred substrate.
[0339] PI3K.delta. is a lipid kinase belonging to the class I PI3K
family (PI3K .alpha., .beta., .gamma. and .delta.) that generates
second messenger signals downstream of tyrosine kinase-linked
receptors.
[0340] PI3K.delta. is a heterodimer composed of an adaptor protein
and a p110.delta. catalytic subunit which converts
phosphatidylinositol-4,5-bis-phosphate (PtdInsP2) to
phosphatidylinositol-3,4,5-tri-phosphate (PtdInsP3). Effector
proteins interact with PtdInsP3 and trigger specific signaling
pathways involved in cell activation, differentiation, migration,
and cell survival.
[0341] Expression of the p110.delta. and p110.gamma. catalytic
subunits is preferential to leukocytes. Expression is also observed
in smooth muscle cells, myocytes and endothelial cells. In
contrast, p110.alpha. and p110.beta. are expressed by all cell
types (Marone et al. Biochimica et Biophysica Acta 1784:159
(2008)).
[0342] PI3K.delta. is associated with B cell development and
function (Okkenhaug et al. Science 297:1031 (2002)).
[0343] B cells play also a critical role in the pathogenesis of a
number of autoimmune and allergic diseases as well as in the
process of transplant rejection (Martin and Chan, Annu. Rev.
Immunol. 24:467 (2006)).
[0344] Chemotaxis is involved in many autoimmune or inflammatory
diseases, in angiogenesis, invasion/metastasis, neurodegeneration
or wound healing (Gerard et al. Nat. Immunol. 2:108 (2001)).
Temporarily distinct events in leukocyte migration in response to
chemokines are fully dependent on PI3K.delta. and PI3K.gamma. (Liu
et al. Blood 110:1191 (2007)).
[0345] PI3K.alpha. and PI3K.beta. play an essential role in
maintaining homeostasis and pharmacological inhibition of these
molecular targets has been associated with cancer therapy (Maira et
al. Expert Opin. Ther. Targets 12:223 (2008)).
[0346] PI3K.alpha. is involved in insulin signaling and cellular
growth pathways (Foukas et al. Nature 441:366 (2006)). PI3K.delta.
isoform-selective inhibition is expected to avoid potential side
effects such as hyperglycemia, and metabolic or growth
disregulation.
[0347] The invention relates in a third aspect to the use of
compounds of the present invention as pharmaceuticals.
Particularly, the compounds of formula I have valuable
pharmacological properties, as described hereinbefore and
hereinafter. The invention thus provides: [0348] a compound of the
formula (I) as defined herein, as pharmaceutical/for use as
pharmaceutical; [0349] a compound of the formula (I) as defined
herein, as medicament/for use as medicament; [0350] a compound of
the formula (I) as defined herein, for use in therapy; [0351] a
compound of the formula (I) as defined herein, for the prevention
and/or treatment of conditions, diseases or disorders which are
mediated by the activity of the PI3K enzymes, preferably by the
activity of the PI3K.delta. isoform; [0352] the use of a compound
of formula (I) as defined herein, for the manufacture of a
medicament for the prevention and/or treatment of conditions,
diseases or disorders which are mediated by the activity of the
PI3K enzymes, preferably by the activity of the PI3K.delta.
isoform; [0353] the use of a compound of formula (I) as defined
herein, for the prevention and/or treatment of conditions, diseases
or disorders which are mediated by the activity of the PI3K
enzymes, preferably by the activity of the PI3K.delta. isoform;
[0354] the use of a compound of formula I as defined herein for the
inhibition of the PI3K, enzymes, preferably of the PI3K.delta.
isoform; [0355] the use of a compound of formula (I) as defined
herein, for the treatment of a disorder or disease selected from
autoimmune disorders, inflammatory diseases, allergic diseases,
airway diseases, such as asthma and COPD, transplant rejection,
cancers eg of hematopoietic origin or solid tumors. [0356] the use
of a compound of formula (I) as defined herein, for the treatment
of a disorder or disease selected from antibody production, antigen
presentation, cytokine production or lymphoid organogenesis are
abnormal or are undesirable including rheumatoid arthritis,
pemphigus vulgaris, idiopathic thrombocytopenia purpura, systemic
lupus erythematosus, multiple sclerosis, myasthenia gravis,
Sjogren's syndrome, autoimmune hemolytic anemia, ANCA-associated
vasculitides, cryoglobulinemia, thrombotic thrombocytopenic
purpura, chronic autoimmune urticaria, allergy (atopic dermatitis,
contact dermatitis, allergic rhinitis), goodpasture's syndrome, AMR
(antibody-mediated transplant rejection), B cell-mediated
hyperacute, acute and chronic transplant rejection and cancers of
haematopoietic origin including but not limited to multiple
myeloma; acute myelogenous leukemia; chronic myelogenous leukemia;
lymphocytic leukemia; myeloid leukemia; non-Hodgkin lymphoma;
lymphomas; polycythemia vera; essential thrombocythemia;
myelofibrosis with myeloid metaplasia; and Walden stroem disease.
[0357] the use of a compound of formula (I) as defined herein, for
the treatment of a disorder or disease selected from rheumatoid
arthritis (RA), pemphigus vulgaris (PV), idiopathic
thrombocytopenia purpura (ITP), thrombotic thrombocytopenic purpura
(TTP), autoimmune hemolytic anemia (AIHA), acquired hemophilia type
A (AHA), systemic lupus erythematosus (SLE), multiple sclerosis
(MS), myasthenia gravis (MG), Sjogren's syndrome (SS),
ANCA-associated vasculitides, cryoglobulinemia, chronic autoimmune
urticaria (CAU), allergy (atopic dermatitis, contact dermatitis,
allergic rhinitis), goodpasture's syndrome, transplant rejection
and cancers of haematopoietic origin. [0358] a method of modulating
the activity of the PI3K enzymes, preferably the PI3K.delta.
isoform, in a subject, comprising the step of administering to a
subject a therapeutically effective amount of a compound of formula
I as defined herein; [0359] a method for the treatment of a
disorder or disease mediated by the PI3K enzymes, preferably by the
PI3K.delta. isoform. comprising the step of administering to a
subject a therapeutically effective amount of a compound of formula
(I) as defined herein; [0360] a method for inhibition of the PI3K
enzymes, preferably the PI3K.delta. isoform, in a cell, comprising
contacting said cell with an effective amount of a compound of
formula I as defined herein.
[0361] As used herein, the term "subject" refers to an animal.
Typically the animal is a mammal. A subject also refers to for
example, primates (e.g., humans), cows, sheep, goats, horses, dogs,
cats, rabbits, rats, mice, fish, birds and the like. In certain
embodiments, the subject is a primate. In yet other embodiments,
the subject is a human.
[0362] As used herein, the term "inhibit", "inhibition" or
"inhibiting" refers to the reduction or suppression of a given
condition, symptom, or disorder, or disease, or a significant
decrease in the baseline activity of a biological activity or
process.
[0363] As used herein, the term "treat", "treating" or "treatment"
of any disease or disorder refers in one embodiment, to
ameliorating the disease or disorder (i.e., slowing or arresting or
reducing the development of the disease or at least one of the
clinical symptoms thereof). In another embodiment "treat",
"treating" or "treatment" refers to alleviating or ameliorating at
least one physical parameter including those which may not be
discernible by the patient. In yet another embodiment, "treat",
"treating" or "treatment" refers to modulating the disease or
disorder, either physically, (e.g., stabilization of a discernible
symptom), physiologically, (e.g., stabilization of a physical
parameter), or both. In yet another embodiment, "treat", "treating"
or "treatment" refers to preventing or delaying the onset or
development or progression of the disease or disorder.
[0364] As used herein, a subject is "in need of" a treatment if
such subject would benefit biologically, medically or in quality of
life from such treatment.
[0365] The term "administration" or "administering" of the subject
compound means providing a compound of the invention and prodrugs
thereof to a subject in need of treatment. Administration "in
combination with" one or more further therapeutic agents includes
simultaneous (concurrent) and consecutive administration in any
order, and in any route of administration.
[0366] The invention relates to the use of new
tetrahydro-pyrido-pyrimidine derivates for the prevention and/or
treatment of conditions, diseases or disorders which are mediated
by the activity of the PI3K enzymes.
[0367] Suitably, the invention relates to the treatment, either
alone or in combination, with one or more other pharmacologically
active compounds, of PI3K-related diseases including but not
limited to autoimmune disorders, inflammatory diseases, allergic
diseases, airway diseases, such as asthma and COPD, transplant
rejection, cancers eg of hematopoietic origin or solid tumors.
[0368] The invention also relates to the treatment, either alone or
in combination, with one or more other pharmacologically active
compounds, includes methods of treating conditions, diseases or
disorders in which one or more of the functions of B cells such as
antibody production, antigen presentation, cytokine production or
lymphoid organogenesis are abnormal or are undesirable including
rheumatoid arthritis, pemphigus vulgaris, idiopathic
thrombocytopenia purpura, systemic lupus erythematosus, multiple
sclerosis, myasthenia gravis, Sjogren's syndrome, autoimmune
hemolytic anemia, ANCA-associated vasculitides, cryoglobulinemia,
thrombotic thrombocytopenic purpura, chronic autoimmune urticaria,
allergy (atopic dermatitis, contact dermatitis, allergic rhinitis),
goodpasture's syndrome, AMR (antibody-mediated transplant
rejection), B cell-mediated hyperacute, acute and chronic
transplant rejection and cancers of haematopoietic origin including
but not limited to multiple myeloma; acute myelogenous leukemia;
chronic myelogenous leukemia; lymphocytic leukemia; myeloid
leukemia; non-Hodgkin lymphoma; lymphomas; polycythemia vera;
essential thrombocythemia; myelofibrosis with myeloid metaplasia;
and Walden stroem disease.
[0369] The invention includes methods of treating conditions,
diseases or disorders in which one or more of the functions of
neutrophils, such as superoxide release, stimulated exocytosis, or
chemoatractic migration are abnormal or are undesirable including
rheumatoid arthritis, sepsis, pulmonary or respiratory disorders
such as asthma, inflammatory dermatoses such as psoriasis and
others.
[0370] The invention includes methods of treating conditions,
diseases or disorders in which one or more of the functions of
basophil and mast cells such as chemoatractic migration or
allergen-IgE-mediated degranulation are abnormal or are undesirable
including allergic diseases (atopic dermatitis, contact dermatitis,
allergic rhinitis) as well as other disorders such as COPD, asthma
or emphysema.
[0371] The invention includes methods of treating conditions,
diseases or disorders in which one or more of the functions of T
cells such as cytokine production or cell-mediated cytotoxicity
abnormal or are undesirable including rheumatoid arthritis,
multiple sclerosis, acute or chronic rejection of cell tissue or
organ grafts or cancers of haematopoietic origin.
[0372] Further, the invention includes methods of treating
neurodegenerative diseases, cardiovascular diseases and platelet
aggregation.
[0373] Further, the invention includes methods of treating skin
diseases such as porphyria cutanea tarda, polymorphous light
eruption, dermatomyositis, solar urticaria, oral lichen planus,
panniculitis, scleroderma, urticarial vasculitis.
[0374] Further, the invention includes methods of treating chronic
inflammatory diseases such as sarcoidosis, granuloma annulare.
[0375] In other embodiments, the condition or disorder (e.g.
PI3K-mediated) is selected from the group consisting of:
polycythemia vera, essential thrombocythemia, myelofibrosis with
myeloid metaplasia, asthma, COPD, ARDS, Loffler's syndrome,
eosinophilic pneumonia, parasitic (in particular metazoan)
infestation (including tropical eosinophilia), bronchopulmonary
aspergillosis, polyarteritis nodosa (including Churg-Strauss
syndrome), eosinophilic granuloma, eosinophil-related disorders
affecting the airways occasioned by drug-reaction, psoriasis,
contact dermatitis, atopic dermatitis, alopecia areata, erythema
multiforme, dermatitis herpetiformis, scleroderma, vitiligo,
hypersensitivity angiitis, urticaria, bullous pemphigoid, lupus
erythematosus, pemphigus, epidermolysis bullosa acquisita,
autoimmune haematogical disorders (e.g. haemolytic anaemia,
aplastic anaemia, pure red cell anaemia and idiopathic
thrombocytopenia), systemic lupus erythematosus, polychondritis,
scleroderma, Wegener granulomatosis, dermatomyositis, chronic
active hepatitis, myasthenia gravis, Steven-Johnson syndrome,
idiopathic sprue, autoimmune inflammatory bowel disease (e.g.
ulcerative colitis and Crohn's disease), endocrine opthalmopathy,
Grave's disease, sarcoidosis, alveolitis, chronic hypersensitivity
pneumonitis, multiple sclerosis, primary biliary cirrhosis, uveitis
(anterior and posterior), interstitial lung fibrosis, psoriatic
arthritis, glomerulonephritis, cardiovascular diseases,
atherosclerosis, hypertension, deep venous thrombosis, stroke,
myocardial infarction, unstable angina, thromboembolism, pulmonary
embolism, thrombolytic diseases, acute arterial ischemia,
peripheral thrombotic occlusions, and coronary artery disease,
reperfusion injuries, retinopathy, such as diabetic retinopathy or
hyperbaric oxygen-induced retinopathy, and conditions characterized
by elevated intraocular pressure or secretion of ocular aqueous
humor, such as glaucoma.
[0376] In another embodiment, the compounds of the present
invention are useful in the treatment, prevention, or amelioration
of autoimmune disease and of inflammatory conditions, in particular
inflammatory conditions with an aetiology including an autoimmune
component such as arthritis (for example rheumatoid arthritis,
arthritis chronica progrediente and arthritis deformans) and
rheumatic diseases, including inflammatory conditions and rheumatic
diseases involving bone loss, inflammatory pain,
spondyloarhropathies including ankolsing spondylitis, Reiter
syndrome, reactive arthritis, psoriatic arthritis, and
enterophathics arthritis, hypersensitivity (including both airways
hypersensitivity and dermal hypersensitivity) and allergies.
Specific auto-immune diseases for which antibodies of the invention
may be employed include autoimmune haematological disorders
(including e.g. hemolytic anaemia, aplastic anaemia, pure red cell
anaemia and idiopathic thrombocytopenia), acquired hemophilia A,
cold agglutinin disease, cryoglobulinemia, thrombotic
thrombocytopenic purpura, Sjogren's syndrome, systemic lupus
erythematosus, inflammatory muscle disorders, polychondritis,
sclerodoma, anti-neutrophil cytoplasmic antibody-associated
vasculitis, IgM mediated neuropathy, opsoclonus myoclonus syndrome,
Wegener granulomatosis, dermatomyositis, chronic active hepatitis,
myasthenia gravis, psoriasis, Steven-Johnson syndrome, pemphigus
vulgaris, pemphigus foliacius, idio-pathic sprue, autoimmune
inflammatory bowel disease (including e.g. ulcerative colitis,
Crohn's disease and Irritable Bowel Syndrome), endocrine
ophthalmopathy, Graves' disease, sarcoidosis, multiple sclerosis,
neuromyelitis optica, primary biliary cirrhosis, juvenile diabetes
(diabetes mellitus type I), uveitis (anterior, intermediate and
posterior as well as panuveitis), keratoconjunctivitis sicca and
vernal keratoconjunctivitis, interstitial lung fibrosis, psoriatic
arthritis and glomerulonephritis (with and without nephrotic
syndrome, e.g. including idiopathic nephrotic syndrome or minimal
change nephropathy), tumors, inflammatory disease of skin and
cornea, myositis, loosening of bone implants, metabolic disorders,
such as atherosclerosis, diabetes, and dislipidemia.
[0377] In another embodiment, the compounds of the present
invention are useful in the treatment of conditions or disorders
selected from the group consisting of, primary cutaneous B-cell
lymphoma, immunobullous disease, pemphigus vulgaris, pemphigus
foliaceus, paraneoplastic pemphigus, bullous pemphigoid, mucous
membrane pemphigoid, epidermolysis bullosa acquisita, chronic graft
versus host disease, dermatomyositis, systemic lupus erythematosus,
vasculitis, small vessel vasculitis, hypocomplementemic urticarial
vasculitis, antineutrophil cytoplasmic antibody-vasculitis,
cryoglobulinemia, Schnitzler syndrome, Waldenstrom's
macroglobulinemia, angioedema, vitiligo, systemic lupus
erythematosus, idiopathic thrombocytopenic purpura, multiple
sclerosis, cold agglutinin disease, autoimmune hemolytic anemia,
antineutrophil cytoplasmic antibody--associated vasculitis, graft
versus host disease, cryoglobulinemia and thrombotic
thrombocytopenic.
[0378] In a further embodiment, the invention relates to a process
or a method for the treatment of one of the disorders or diseases
mentioned hereinabove, especially a disease which responds to the
inhibition of the PI3K enzymes. The compounds of formula I, or a
pharmaceutically acceptable salt thereof, can be administered as
such or in the form of pharmaceutical compositions,
prophylactically or therapeutically, preferably in an amount
effective against the said diseases, to a warm-blooded animal, for
example a human, requiring such treatment, the compounds especially
being used in the form of pharmaceutical compositions.
[0379] In a further embodiment, the invention relates to the use of
a compound of formula I, or a pharmaceutically acceptable salt
thereof, as such or in the form of a pharmaceutical composition
with at least one pharmaceutically acceptable carrier, for the
therapeutic and also prophylactic management of one or more of the
diseases mentioned hereinabove, mediated by the PI3K enzymes.
[0380] In a further embodiment, the invention relates to the use of
a compound of formula I, or a pharmaceutically acceptable salt
thereof, especially a compound of formula I which is said to be
preferred, or a pharmaceutically acceptable salt thereof, for the
preparation of a pharmaceutical composition for the therapeutic and
also prophylactic management of one or more of the diseases
mentioned hereinabove, especially a disorder or disease selected
from autoimmune disorders, inflammatory diseases, allergic
diseases, airway diseases, such as asthma and COPD, transplant
rejection, cancers eg of hematopoietic origin or solid tumors.
[0381] The invention relates in a fourth aspect to pharmaceutical
compositions comprising a compound of the present invention. The
invention thus provides [0382] a pharmaceutical composition
comprising (i.e. containing or consisting of) a compound as defined
herein and one or more carriers/excipients; [0383] a pharmaceutical
composition comprising a therapeutically effective amount of a
compound of formula I as defined herein, and one or more
pharmaceutically acceptable carriers/excipients.
[0384] As used herein, the term "pharmaceutically acceptable
carrier" includes any and all solvents, dispersion media, coatings,
surfactants, antioxidants, preservatives (e.g., antibacterial
agents, antifungal agents), isotonic agents, absorption delaying
agents, salts, preservatives, drugs, drug stabilizers, binders,
excipients, disintegration agents, lubricants, sweetening agents,
flavoring agents, dyes, and the like and combinations thereof, as
would be known to those skilled in the art (see, for example,
Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing
Company, 1990, pp. 1289-1329). Except insofar as any conventional
carrier is incompatible with the active ingredient, its use in the
therapeutic or pharmaceutical compositions is contemplated.
[0385] The present invention provides a pharmaceutical composition
comprising a compound of the present invention and a
pharmaceutically acceptable carrier. The pharmaceutical composition
can be formulated for particular routes of administration such as
oral administration, parenteral administration, and rectal
administration, etc. In addition, the pharmaceutical compositions
of the present invention can be made up in a solid form (including
without limitation capsules, tablets, pills, granules, powders or
suppositories), or in a liquid form (including without limitation
solutions, suspensions or emulsions). The pharmaceutical
compositions can be subjected to conventional pharmaceutical
operations such as sterilization and/or can contain conventional
inert diluents, lubricating agents, or buffering agents, as well as
adjuvants, such as preservatives, stabilizers, wetting agents,
emulsifiers and buffers, etc.
[0386] Typically, the pharmaceutical compositions are tablets or
gelatin capsules comprising the active ingredient together with
[0387] a) diluents, e.g., lactose, dextrose, sucrose, mannitol,
sorbitol, cellulose and/or glycine; [0388] b) lubricants, e.g.,
silica, talcum, stearic acid, its magnesium or calcium salt and/or
polyethyleneglycol; for tablets also [0389] c) binders, e.g.,
magnesium aluminum silicate, starch paste, gelatin, tragacanth,
methylcellulose, sodium carboxymethylcellulose and/or
polyvinylpyrrolidone; if desired [0390] d) disintegrants, e.g.,
starches, agar, alginic acid or its sodium salt, or effervescent
mixtures; and/or [0391] e) absorbents, colorants, flavors and
sweeteners.
[0392] Tablets may be either film coated or enteric coated
according to methods known in the art. Suitable compositions for
oral administration include an effective amount of a compound of
the invention in the form of tablets, lozenges, aqueous or oily
suspensions, dispersible powders or granules, emulsion, hard or
soft capsules, or syrups or elixirs. Compositions intended for oral
use are prepared according to any method known in the art for the
manufacture of pharmaceutical compositions and such compositions
can contain one or more agents selected from the group consisting
of sweetening agents, flavoring agents, coloring agents and
preserving agents in order to provide pharmaceutically elegant and
palatable preparations. Tablets may contain the active ingredient
in admixture with nontoxic pharmaceutically acceptable excipients
which are suitable for the manufacture of tablets. These excipients
are, for example, inert diluents, such as calcium carbonate, sodium
carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example, corn starch, or
alginic acid; binding agents, for example, starch, gelatin or
acacia; and lubricating agents, for example magnesium stearate,
stearic acid or talc. The tablets are uncoated or coated by known
techniques to delay disintegration and absorption in the
gastrointestinal tract and thereby provide a sustained action over
a longer period. For example, a time delay material such as
glyceryl monostearate or glyceryl distearate can be employed.
Formulations for oral use can be presented as hard gelatin capsules
wherein the active ingredient is mixed with an inert solid diluent,
for example, calcium carbonate, calcium phosphate or kaolin, or as
soft gelatin capsules wherein the active ingredient is mixed with
water or an oil medium, for example, peanut oil, liquid paraffin or
olive oil.
[0393] Certain injectable compositions are aqueous isotonic
solutions or suspensions, and suppositories are advantageously
prepared from fatty emulsions or suspensions. Said compositions may
be sterilized and/or contain adjuvants, such as preserving,
stabilizing, wetting or emulsifying agents, solution promoters,
salts for regulating the osmotic pressure and/or buffers. In
addition, they may also contain other therapeutically valuable
substances. Said compositions are prepared according to
conventional mixing, granulating or coating methods, respectively,
and contain about 0.1-75%, or contain about 1-50%, of the active
ingredient.
[0394] Suitable compositions for transdermal application include an
effective amount of a compound of the invention with a suitable
carrier. Carriers suitable for transdermal delivery include
absorbable pharmacologically acceptable solvents to assist passage
through the skin of the host. For example, transdermal devices are
in the form of a bandage comprising a backing member, a reservoir
containing the compound optionally with carriers, optionally a rate
controlling barrier to deliver the compound of the skin of the host
at a controlled and predetermined rate over a prolonged period of
time, and means to secure the device to the skin.
[0395] Suitable compositions for topical application, e.g., to the
skin and eyes, include aqueous solutions, suspensions, ointments,
creams, gels or sprayable formulations, e.g., for delivery by
aerosol or the like. Such topical delivery systems will in
particular be appropriate for dermal application, e.g., for the
treatment of skin cancer, e.g., for prophylactic use in sun creams,
lotions, sprays and the like. They are thus particularly suited for
use in topical, including cosmetic, formulations well-known in the
art. Such may contain solubilizers, stabilizers, tonicity enhancing
agents, buffers and preservatives.
[0396] As used herein a topical application may also pertain to an
inhalation or to an intranasal application. They may be
conveniently delivered in the form of a dry powder, either alone,
as a mixture, for example a dry blend with lactose, or a mixed
component particle, for example with phospholipids, from a dry
powder inhaler or an aerosol spray presentation from a pressurised
container, pump, spray, atomizer or nebuliser, with or without the
use of a suitable propellant.
[0397] The present invention further provides anhydrous
pharmaceutical compositions and dosage forms comprising the
compounds of the present invention as active ingredients, since
water may facilitate the degradation of certain compounds.
[0398] Anhydrous pharmaceutical compositions and dosage forms of
the invention can be prepared using anhydrous or low moisture
containing ingredients and low moisture or low humidity conditions.
An anhydrous pharmaceutical composition may be prepared and stored
such that its anhydrous nature is maintained. Accordingly,
anhydrous compositions are packaged using materials known to
prevent exposure to water such that they can be included in
suitable formulary kits. Examples of suitable packaging include,
but are not limited to, hermetically sealed foils, plastics, unit
dose containers, e.g., vials, blister packs, and strip packs.
[0399] The invention further provides pharmaceutical compositions
and dosage forms that comprise one or more agents that reduce the
rate by which the compound of the present invention as an active
ingredient will decompose. Such agents, which are referred to
herein as "stabilizers," include, but are not limited to,
antioxidants such as ascorbic acid, pH buffers, or salt buffers,
etc.
[0400] Examples of physiologically acceptable carriers include
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid; low molecular weight (less
than about 10 residues) polypeptide; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides, and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as TWEEN.RTM., polyethylene glycol (PEG), and
PLURONICS.RTM..
[0401] Suitable excipients/carriers may be any solid, liquid,
semi-solid or, in the case of an aerosol composition, gaseous
excipient that is generally available to one of skill in the
art.
[0402] Solid pharmaceutical excipients include starch, cellulose,
talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, magnesium stearate, sodium stearate, glycerol
monostearate, sodium chloride, dried skim milk and the like.
[0403] Liquid and semisolid excipients may be selected from
glycerol, propylene glycol, water, ethanol and various oils,
including those of petroleum, animal, vegetable or synthetic
origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil,
etc. Preferred liquid carriers, particularly for injectable
solutions, include water, saline, aqueous dextrose, and
glycols.
[0404] Compressed gases may be used to disperse a compound of the
formula (I) in aerosol form. Inert gases suitable for this purpose
are nitrogen, carbon dioxide, etc. Other suitable pharmaceutical
excipients and their formulations are described in Remington's
Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing
Company, 18th ed., 1990).
[0405] The dosage of the active ingredient depends upon the disease
to be treated and upon the species, its age, weight, and individual
condition, the individual pharmacokinetic data, and the mode of
administration. The amount of the compound in a formulation can
vary within the full range employed by those skilled in the art.
Typically, the formulation will contain, on a weight percent (wt %)
basis, from about 0.01-99.99 wt % of a compound of formula (I)
based on the total formulation, with the balance being one or more
suitable pharmaceutical excipients.
[0406] Pharmaceutical compositions comprising a compound of formula
(I) as defined herein in association with at least one
pharmaceutical acceptable carrier (such as excipient a and/or
diluent) may be manufactured in conventional manner, e.g. by means
of conventional mixing, granulating, coating, dissolving or
lyophilising processes.
[0407] In a further embodiment, the invention relates to a
pharmaceutical composition for administration to a warm-blooded
animal, especially humans or commercially useful mammals suffering
from a disease which responds to an inhibition of the PI3K enzymes,
comprising an effective quantity of a compound of formula I for the
inhibition of the PI3K enzymes, or a pharmaceutically acceptable
salt thereof, together with at least one pharmaceutically
acceptable carrier.
[0408] In a further embodiment, the invention relates to a
pharmaceutical composition for the prophylactic or especially
therapeutic management of a disorder or disease selected from
autoimmune disorders, inflammatory diseases, allergic diseases,
airway diseases, such as asthma and COPD, transplant rejection,
cancers eg of hematopoietic origin or solid tumors; of a
warm-blooded animal, especially a human or a commercially useful
mammal requiring such treatment.
[0409] The invention relates in a fifth aspect to combinations
comprising a compound of formula I and one or more additional
active ingredients. The invention thus provides [0410] a
combination in particular a pharmaceutical combination, comprising
a therapeutically effective amount of a compound of formula I and
one or more therapeutically active agents, e.g. an
immunosuppressant, immunomodulatory, anti-inflammatory or
chemotherapeutic agent, e.g. as indicated below; [0411] a combined
pharmaceutical composition, adapted for simultaneous or sequential
administration, comprising a therapeutically effective amount of a
compound of formula (I) as defined herein; therapeutically
effective amount(s) of one or more combination partners e.g. an
immunosuppressant, immunomodulatory, anti-inflammatory or
chemotherapeutic agent, e.g. as indicated below; one or more
pharmaceutically acceptable excepients; [0412] a combined
pharmaceutical composition as defined herein (i) as pharmaceutical,
(ii) for use in the treatment of a disease mediated by the PI3K
enzymes, (iii) in a method of treatment of a disease mediated by
the PI3K enzymes.
[0413] By "combination", there is meant either a fixed combination
in one dosage unit form, or a kit of parts for the combined
administration where a compound of the formula (I) and a
combination partner may be administered independently at the same
time or separately within time intervals that especially allow that
the combination partners show a cooperative, e.g. synergistic
effect.
[0414] The term "a therapeutically effective amount" of a compound
of the present invention refers to an amount of the compound of the
present invention that will elicit the biological or medical
response of a subject, for example, reduction or inhibition of an
enzyme or a protein activity, or ameliorate symptoms, alleviate
conditions, slow or delay disease progression, or prevent a
disease, etc. In one non-limiting embodiment, the term "a
therapeutically effective amount" refers to the amount of the
compound of the present invention that, when administered to a
subject, is effective to (1) at least partially alleviating,
inhibiting, preventing and/or ameliorating a condition, or a
disorder or a disease (i) mediated by the dysregulation of PI3K
delta, or (ii) associated with the dysregulation of PI3K delta, or
(iii) characterized by the dysregulation of the PI3K delta; or (2)
reducing or inhibiting the activity of the PI3K delta. In another
non-limiting embodiment, the term "a therapeutically effective
amount" refers to the amount of the compound of the present
invention that, when administered to a cell, or a tissue, or a
non-cellular biological material, or a medium, is effective to at
least partially reducing or inhibiting PI3K delta.
[0415] The compounds of formula I may be administered as the sole
active ingredient or in conjunction with, e.g. as an adjuvant to,
other drugs e.g. immunosuppressive or immunomodulating agents or
other anti-inflammatory agents, e.g. for the treatment or
prevention of alto- or xenograft acute or chronic rejection or
inflammatory or autoimmune disorders, or a chemotherapeutic agent,
e.g a malignant cell anti-proliferative agent. For example, the
compounds of formula I may be used in combination with a
calcineurin inhibitor, e.g. cyclosporin A or FK 506; a mTOR
inhibitor, e.g. rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI779,
ABT578, AP23573, TAFA-93, biolimus-7 or biolimus-9; an ascomycin
having immuno-suppressive properties, e.g. ABT-281, ASM981, etc.;
corticosteroids; cyclophosphamide; azathioprene; methotrexate;
leflunomide; mizoribine; mycophenolic acid or salt; mycophenolate
mofetil; 15-deoxyspergualine or an immunosuppressive homologue,
analogue or derivative thereof; a PKC inhibitor, e.g. as disclosed
in WO 02/38561 or WO 03/82859, e.g. the compound of Example 56 or
70; a JAK3 kinase inhibitor, e.g.
N-benzyl-3,4-dihydroxy-benzylidene-cyanoacetamide
.alpha.-cyano-(3,4-dihydroxy]N-benzylcinnamamide (Tyrphostin AG
490), prodigiosin 25-C(PNU156804),
[4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline] (WHI-P131),
[4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline]
(WHI-P154), [4-(3',5'-dibromo-4'-hydroxylphenyl)-amino-6,7-di
methoxyquinazoline] WHI-P97, KRX-211,
3-{(3R,4R)-4-methyl-3-[methyl-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-amino]-pi-
peridin-1-yl}-3-oxo-propionitrile, in free form or in a
pharmaceutically acceptable salt form, e.g. mono-citrate (also
called CP-690,550), or a compound as disclosed in WO 04/052359 or
WO 05/066156; immunosuppressive monoclonal antibodies, e.g.,
monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3,
CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD52, CD58, CD80, CD86 or
their ligands; other immunomodulatory compounds, e.g. a recombinant
binding molecule having at least a portion of the extracellular
domain of CTLA4 or a mutant thereof, e.g. an at least extracellular
portion of CTLA4 or a mutant thereof joined to a non-CTLA4 protein
sequence, e.g. CTLA4Ig (for ex. designated ATCC 68629) or a mutant
thereof, e.g. LEA29Y; adhesion molecule inhibitors, e.g. LFA-1
antagonists, ICAM-1 or -3 antagonists, VCAM-4 antagonists or VLA-4
antagonists; or antihistamines; or antitussives, or a
bronchodilatory agent; or an angiotensin receptor blockers; or an
anti-infectious agent.
[0416] Where the compounds of formula I are administered in
conjunction with other immunosuppressive/immunomodulatory,
anti-inflammatory, chemotherapeutic or anti-infectious therapy,
dosages of the co-administered immunosuppressant, immunomodulatory,
anti-inflammatory, chemotherapeutic or anti-infectious compound
will of course vary depending on the type of co-drug employed, e.g.
whether it is a steroid or a calcineurin inhibitor, on the specific
drug employed, on the condition being treated and so forth.
[0417] A compound of the formula (I) may also be used to advantage
in combination with each other or in combination with other
therapeutic agents, especially other antiproliferative agents. Such
antiproliferative agents include, but are not limited to, aromatase
inhibitors; antiestrogens; topoisomerase I inhibitors;
topoisomerase II inhibitors; microtubule active agents; alkylating
agents; histone deacetylase inhibitors; compounds, which induce
cell differentiation processes; cyclooxygenase inhibitors; MMP
inhibitors; mTOR inhibitors; antineoplastic antimetabolites; platin
compounds; compounds targeting/decreasing a protein or lipid kinase
activity and further anti-angiogenic compounds; compounds which
target, decrease or inhibit the activity of a protein or lipid
phosphatase; gonadorelin agonists; anti-androgens; methionine
aminopeptidase inhibitors; bisphosphonates; biological response
modifiers; antiproliferative antibodies; heparanase inhibitors;
inhibitors of Ras oncogenic isoforms; telomerase inhibitors;
proteasome inhibitors; agents used in the treatment of hematologic
malignancies; compounds which target, decrease or inhibit the
activity of Flt-3; Hsp90 inhibitors; temozolomide (TEMODAL.RTM.);
and leucovorin.
[0418] The term "aromatase inhibitor", as used herein, relates to a
compound which inhibits the estrogen production, i.e., the
conversion of the substrates androstenedione and testosterone to
estrone and estradiol, respectively. The term includes, but is not
limited to, steroids, especially atamestane, exemestane and
formestane; and, in particular, non-steroids, especially
aminoglutethimide, roglethimide, pyridoglutethimide, trilostane,
testolactone, ketoconazole, vorozole, fadrozole, anastrozole and
letrozole. Exemestane can be administered, e.g., in the form as it
is marketed, e.g., under the trademark AROMASIN. Formestane can be
administered, e.g., in the form as it is marketed, e.g., under the
trademark LENTARON. Fadrozole can be administered, e.g., in the
form as it is marketed, e.g., under the trademark AFEMA.
Anastrozole can be administered, e.g., in the form as it is
marketed, e.g., under the trademark ARIMIDEX. Letrozole can be
administered, e.g., in the form as it is marketed, e.g., under the
trademark FEMARA or FEMAR. Aminoglutethimide can be administered,
e.g., in the form as it is marketed, e.g., under the trademark
ORIMETEN. A combination of the invention comprising a
chemotherapeutic agent which is an aromatase inhibitor is
particularly useful for the treatment of hormone receptor positive
tumors, e.g., breast tumors.
[0419] The term "anti-estrogen", as used herein, relates to a
compound which antagonizes the effect of estrogens at the estrogen
receptor level. The term includes, but is not limited to,
tamoxifen, fulvestrant, raloxifene and raloxifene hydrochloride.
Tamoxifen can be administered, e.g., in the form as it is marketed,
e.g., under the trademark NOLVADEX. Raloxifene hydrochloride can be
administered, e.g., in the form as it is marketed, e.g., under the
trademark EVISTA. Fulvestrant can be formulated as disclosed in
U.S. Pat. No. 4,659,516 or it can be administered, e.g., in the
form as it is marketed, e.g., under the trademark FASLODEX. A
combination of the invention comprising a chemotherapeutic agent
which is an antiestrogen is particularly useful for the treatment
of estrogen receptor positive tumors, e.g., breast tumors.
[0420] The term "anti-androgen", as used herein, relates to any
substance which is capable of inhibiting the biological effects of
androgenic hormones and includes, but is not limited to,
bicalutamide (CASODEX), which can be formulated, e.g., as disclosed
in U.S. Pat. No. 4,636,505.
[0421] The term "gonadorelin agonist", as used herein, includes,
but is not limited to, abarelix, goserelin and goserelin acetate.
Goserelin is disclosed in U.S. Pat. No. 4,100,274 and can be
administered, e.g., in the form as it is marketed, e.g., under the
trademark ZOLADEX. Abarelix can be formulated, e.g., as disclosed
in U.S. Pat. No. 5,843,901.
[0422] The term "topoisomerase I inhibitor", as used herein,
includes, but is not limited to, topotecan, gimatecan, irinotecan,
camptothecian and its analogues, 9-nitrocamptothecin and the
macromolecular camptothecin conjugate PNU-166148 (compound A1 in WO
99/17804). Irinotecan can be administered, e.g., in the form as it
is marketed, e.g., under the trademark CAMPTOSAR. Topotecan can be
administered, e.g., in the form as it is marketed, e.g., under the
trademark HYCAMTIN.
[0423] The term "topoisomerase II inhibitor", as used herein,
includes, but is not limited to, the anthracyclines, such as
doxorubicin, including liposomal formulation, e.g., CAELYX;
daunorubicin; epirubicin; idarubicin; nemorubicin; the
anthraquinones mitoxantrone and losoxantrone; and the
podophillotoxines etoposide and teniposide. Etoposide can be
administered, e.g., in the form as it is marketed, e.g., under the
trademark ETOPOPHOS. Teniposide can be administered, e.g., in the
form as it is marketed, e.g., under the trademark VM 26-BRISTOL.
Doxorubicin can be administered, e.g., in the form as it is
marketed, e.g., under the trademark ADRIBLASTIN or ADRIAMYCIN.
Epirubicin can be administered, e.g., in the form as it is
marketed, e.g., under the trademark FARMORUBICIN. Idarubicin can be
administered, e.g., in the form as it is marketed, e.g., under the
trademark ZAVEDOS. Mitoxantrone can be administered, e.g., in the
form as it is marketed, e.g., under the trademark NOVANTRON.
[0424] The term "microtubule active agent" relates to microtubule
stabilizing, microtubule destabilizing agents and microtublin
polymerization inhibitors including, but not limited to, taxanes,
e.g., paclitaxel and docetaxel; vinca alkaloids, e.g., vinblastine,
especially vinblastine sulfate; vincristine, especially vincristine
sulfate and vinorelbine; discodermolides; cochicine; and
epothilones and derivatives thereof, e.g., epothilone B or D or
derivatives thereof. Paclitaxel may be administered, e.g., in the
form as it is marketed, e.g., TAXOL. Docetaxel can be administered,
e.g., in the form as it is marketed, e.g., under the trademark
TAXOTERE. Vinblastine sulfate can be administered, e.g., in the
form as it is marketed, e.g., under the trademark VINBLASTIN R.P.
Vincristine sulfate can be administered, e.g., in the form as it is
marketed, e.g., under the trademark FARMISTIN. Discodermolide can
be obtained, e.g., as disclosed in U.S. Pat. No. 5,010,099. Also
included are epothilone derivatives which are disclosed in WO
98/10121, U.S. Pat. No. 6,194,181, WO 98/25929, WO 98/08849, WO
99/43653, WO 98/22461 and WO 00/31247. Especially preferred are
epothilone A and/or B.
[0425] The term "alkylating agent", as used herein, includes, but
is not limited to, cyclophosphamide, ifosfamide, melphalan or
nitrosourea (BCNU or Gliadel).
[0426] Cyclophosphamide can be administered, e.g., in the form as
it is marketed, e.g., under the trademark CYCLOSTIN. Ifosfamide can
be administered, e.g., in the form as it is marketed, e.g., under
the trademark HOLOXAN.
[0427] The term "histone deacetylase inhibitors" or "HDAC
inhibitors" relates to compounds which inhibit the histone
deacetylase and which possess antiproliferative activity. This
includes compounds disclosed in WO 02/22577, especially
N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]-amino]methyl]ph-
enyl]-2E-2-propenamide,
N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]--
2E-2-propenamide and pharmaceutically acceptable salts thereof. It
further especially includes suberoylanilide hydroxamic acid (SAHA).
The term "antineoplastic antimetabolite" includes, but is not
limited to, 5-fluorouracil or 5-FU; capecitabine; gemcitabine; DNA
demethylating agents, such as 5-azacytidine and decitabine;
methotrexate and edatrexate; and folic acid antagonists, such as
pemetrexed. Capecitabine can be administered, e.g., in the form as
it is marketed, e.g., under the trademark XELODA. Gemcitabine can
be administered, e.g., in the form as it is marketed, e.g., under
the trademark GEMZAR. Also included is the monoclonal antibody
trastuzumab which can be administered, e.g., in the form as it is
marketed, e.g., under the trademark HERCEPTIN.
[0428] The term "platin compound", as used herein, includes, but is
not limited to, carboplatin, cis-platin, cisplatinum and
oxaliplatin. Carboplatin can be administered, e.g., in the form as
it is marketed, e.g., under the trademark CARBOPLAT. Oxaliplatin
can be administered, e.g., in the form as it is marketed, e.g.,
under the trademark ELOXATIN.
[0429] The term "compounds targeting/decreasing a protein or lipid
kinase activity; or a protein or lipid phosphatase activity; or
further anti-angiogenic compounds", as used herein, includes, but
is not limited to, protein tyrosine kinase and/or serine and/or
threonine kinase inhibitors or lipid kinase inhibitors, e.g.,
[0430] a) compounds targeting, decreasing or inhibiting the
activity of the platelet-derived growth factor-receptors (PDGFR),
such as compounds which target, decrease or inhibit the activity of
PDGFR, especially compounds which inhibit the PDGF receptor, e.g.,
a N-phenyl-2-pyrimidine-amine derivative, e.g., imatinib, SU101,
SU6668 and GFB-111; [0431] b) compounds targeting, decreasing or
inhibiting the activity of the fibroblast growth factor-receptors
(FGFR); [0432] c) compounds targeting, decreasing or inhibiting the
activity of the insulin-like growth factor receptor I (IGF-IR),
such as compounds which target, decrease or inhibit the activity of
IGF-IR, especially compounds which inhibit the IGF-IR receptor,
such as those compounds disclosed in WO 02/092599; [0433] d)
compounds targeting, decreasing or inhibiting the activity of the
Trk receptor tyrosine kinase family; [0434] e) compounds targeting,
decreasing or inhibiting the activity of the Axl receptor tyrosine
kinase family; [0435] f) compounds targeting, decreasing or
inhibiting the activity of the c-Met receptor; [0436] g) compounds
targeting, decreasing or inhibiting the activity of the Kit/SCFR
receptor tyrosine kinase; [0437] h) compounds targeting, decreasing
or inhibiting the activity of the C-kit receptor tyrosine
kinases--(part of the PDGFR family), such as compounds which
target, decrease or inhibit the activity of the c-Kit receptor
tyrosine kinase family, especially compounds which inhibit the
c-Kit receptor, e.g., imatinib; [0438] i) compounds targeting,
decreasing or inhibiting the activity of members of the c-Abl
family and their gene-fusion products, e.g., BCR-Abl kinase, such
as compounds which target decrease or inhibit the activity of c-Abl
family members and their gene fusion products, e.g., a
N-phenyl-2-pyrimidine-amine derivative, e.g., imatinib, PD180970,
AG957, NSC 680410 or PD173955 from ParkeDavis; [0439] j) compounds
targeting, decreasing or inhibiting the activity of members of the
protein kinase C (PKC) and Raf family of serine/threonine kinases,
members of the MEK, SRC, JAK, FAK, PDK and Ras/MAPK family members,
or PI(3) kinase family, or of the PI(3)-kinase-related kinase
family, and/or members of the cyclin-dependent kinase family (CDK)
and are especially those staurosporine derivatives disclosed in
U.S. Pat. No. 5,093,330, e.g., midostaurin; examples of further
compounds include, e.g., UCN-01; safingol; BAY 43-9006; Bryostatin
1; Perifosine; Ilmofosine; RO 318220 and RO 320432; GO 6976; Isis
3521; LY333531/LY379196; isochinoline compounds, such as those
disclosed in WO 00/09495; FTIs; PD184352; or QAN697 (a PI3K
inhibitor); [0440] k) compounds targeting, decreasing or inhibiting
the activity of protein-tyrosine kinase inhibitors, such as
compounds which target, decrease or inhibit the activity of
protein-tyrosine kinase inhibitors include imatinib mesylate
(GLEEVEC) or tyrphostin. A tyrphostin is preferably a low molecular
weight (Mr<1500) compound, or a pharmaceutically acceptable salt
thereof, especially a compound selected from the
benzylidenemalonitrile class or the S-arylbenzenemalonirile or
bisubstrate quinoline class of compounds, more especially any
compound selected from the group consisting of Tyrphostin
A23/RG-50810, AG 99, Tyrphostin AG 213, Tyrphostin AG 1748,
Tyrphostin AG 490, Tyrphostin B44, Tyrphostin B44 (+) enantiomer,
Tyrphostin AG 555, AG 494, Tyrphostin AG 556, AG957 and adaphostin
(4-{[(2,5-dihydroxyphenyl)methyl]amino}-benzoic acid adamantyl
ester, NSC 680410, adaphostin; and [0441] l) compounds targeting,
decreasing or inhibiting the activity of the epidermal growth
factor family of receptor tyrosine kinases (EGFR, ErbB2, ErbB3,
ErbB4 as homo- or hetero-dimers), such as compounds which target,
decrease or inhibit the activity of the epidermal growth factor
receptor family are especially compounds, proteins or antibodies
which inhibit members of the EGF receptor tyrosine kinase family,
e.g., EGF receptor, ErbB2, ErbB3 and ErbB4 or bind to EGF or EGF
related ligands, and are in particular those compounds, proteins or
monoclonal antibodies generically and specifically disclosed in WO
97/02266, e.g., the compound of Example 39, or in EP 0 564 409; WO
99/03854; EP 0520722; EP 0 566 226; EP 0 787 722; EP 0 837 063;
U.S. Pat. No. 5,747,498; WO 98/10767; WO 97/30034; WO 97/49688; WO
97/38983 and, especially, WO 96/30347, e.g., compound known as CP
358774; WO 96/33980, e.g., compound ZD 1839; and WO 95/03283, e.g.,
compound ZM105180, e.g., trastuzumab (HERCEPTIN), cetuximab,
Iressa, Tarceva, OSI-774, CI-1033, EKB-569, GW-2016, E1.1, E2.4,
E2.5, E6.2, E6.4, E2.11, E6.3 or E7.6.3; and
7H-pyrrolo-[2,3-d]pyrimidine derivatives which are disclosed in WO
03/013541.
[0442] Further anti-angiogenic compounds include compounds having
another mechanism for their activity, e.g., unrelated to protein or
lipid kinase inhibition, e.g., thalidomide (THALOMID) and TN
P-470.
[0443] Compounds which target, decrease or inhibit the activity of
a protein or lipid phosphatase are, e.g., inhibitors of phosphatase
1, phosphatase 2A, PTEN or CDCl.sub.25, e.g., okadaic acid or a
derivative thereof.
[0444] Compounds which induce cell differentiation processes are
e.g. retinoic acid, .alpha.- .gamma.- or .delta.-tocopherol or
.alpha.- .gamma.- or .delta.-tocotrienol.
[0445] The term cyclooxygenase inhibitor, as used herein, includes,
but is not limited to, e.g., Cox-2 inhibitors, 5-alkyl substituted
2-arylaminophenylacetic acid and derivatives, such as celecoxib
(CELEBREX), rofecoxib (VIOXX), etoricoxib, valdecoxib or a
5-alkyl-2-arylaminophenylacetic acid, e.g.,
5-methyl-2-(2'-chloro-6'-fluoroanilino)phenyl acetic acid or
lumiracoxib. The term "bisphosphonates", as used herein, includes,
but is not limited to, etridonic, clodronic, tiludronic,
pamidronic, alendronic, ibandronic, risedronic and zoledronic acid.
"Etridonic acid" can be administered, e.g., in the form as it is
marketed, e.g., under the trademark DIDRONEL. "Clodronic acid" can
be administered, e.g., in the form as it is marketed, e.g., under
the trademark BONEFOS. "Tiludronic acid" can be administered, e.g.,
in the form as it is marketed, e.g., under the trademark SKELID.
"Pamidronic acid" can be administered, e.g., in the form as it is
marketed, e.g., under the trademark AREDIA.TM.' "Alendronic acid"
can be administered, e.g., in the form as it is marketed, e.g.,
under the trademark FOSAMAX. "Ibandronic acid" can be administered,
e.g., in the form as it is marketed, e.g., under the trademark
BONDRANAT. "Risedronic acid" can be administered, e.g., in the form
as it is marketed, e.g., under the trademark ACTONEL. "Zoledronic
acid" can be administered, e.g., in the form as it is marketed,
e.g., under the trademark ZOMETA.
[0446] The term "mTOR inhibitors" relates to compounds which
inhibit the mammalian target of rapamycin (mTOR) and which possess
antiproliferative activity, such as sirolimus (Rapamune),
everolimus (Certican.TM.), CCI-779 and ABT578.
[0447] The term "heparanase inhibitor", as used herein, refers to
compounds which target, decrease or inhibit heparin sulphate
degradation. The term includes, but is not limited to, PI-88.
[0448] The term "biological response modifier", as used herein,
refers to a lymphokine or interferons, e.g., interferon
.gamma..
[0449] The term "inhibitor of Ras oncogenic isoforms", e.g., H-Ras,
K-Ras or N-Ras, as used herein, refers to compounds which target,
decrease or inhibit the oncogenic activity of Ras, e.g., a
"farnesyl transferase inhibitor", e.g., L-744832, DK8G557 or
R115777 (Zarnestra).
[0450] The term "telomerase inhibitor", as used herein, refers to
compounds which target, decrease or inhibit the activity of
telomerase. Compounds which target, decrease or inhibit the
activity of telomerase are especially compounds which inhibit the
telomerase receptor, e.g., telomestatin.
[0451] The term "methionine aminopeptidase inhibitor", as used
herein, refers to compounds which target, decrease or inhibit the
activity of methionine aminopeptidase. Compounds which target,
decrease or inhibit the activity of methionine aminopeptidase are,
e.g., bengamide or a derivative thereof.
[0452] The term "proteasome inhibitor", as used herein, refers to
compounds which target, decrease or inhibit the activity of the
proteasome. Compounds which target, decrease or inhibit the
activity of the proteasome include, e.g., PS-341 and MLN 341.
[0453] The term "matrix metalloproteinase inhibitor" or "MMP
inhibitor", as used herein, includes, but is not limited to,
collagen peptidomimetic and nonpeptidomimetic inhibitors,
tetracycline derivatives, e.g., hydroxamate peptidomimetic
inhibitor batimastat and its orally bioavailable analogue
marimastat (BB-2516), prinomastat (AG3340), metastat (NSC 683551)
BMS-279251, BAY 12-9566, TAA211, MM1270B or AAJ996.
[0454] The term "agents used in the treatment of hematologic
malignancies", as used herein, includes, but is not limited to,
FMS-like tyrosine kinase inhibitors, e.g., compounds targeting,
decreasing or inhibiting the activity of FMS-like tyrosine kinase
receptors (Flt-3R); interferon, 1-b-D-arabinofuransylcytosine
(ara-c) and bisulfan; and ALK inhibitors, e.g., compounds which
target, decrease or inhibit anaplastic lymphoma kinase.
[0455] Compounds which target, decrease or inhibit the activity of
FMS-like tyrosine kinase receptors (Flt-3R) are especially
compounds, proteins or antibodies which inhibit members of the
Flt-3R receptor kinase family, e.g., PKC412, midostaurin, a
staurosporine derivative, SU11248 and MLN518.
[0456] The term "HSP90 inhibitors", as used herein, includes, but
is not limited to, compounds targeting, decreasing or inhibiting
the intrinsic ATPase activity of HSP90; degrading, targeting,
decreasing or inhibiting the HSP90 client proteins via the
ubiquitin proteasome pathway. Compounds targeting, decreasing or
inhibiting the intrinsic ATPase activity of HSP90 are especially
compounds, proteins or antibodies which inhibit the ATPase activity
of HSP90, e.g., 17-allylamino, 17-demethoxygeldanamycin (17AAG), a
geldanamycin derivative, other geldanamycin related compounds,
radicicol and HDAC inhibitors.
[0457] The term "antiproliferative antibodies", as used herein,
includes, but is not limited to, trastuzumab (Herceptin.TM.),
Trastuzumab-DM1, erlotinib (Tarceva.TM.), bevacizumab
(Avastin.TM.), rituximab (Rituxan.RTM.), PRO64553 (anti-CD40) and
2C4 antibody. By antibodies is meant, e.g., intact monoclonal
antibodies, polyclonal antibodies, multispecific antibodies formed
from at least two intact antibodies, and antibodies fragments so
long as they exhibit the desired biological activity.
[0458] For the treatment of acute myeloid leukemia (AML), compounds
of formula (I) can be used in combination with standard leukemia
therapies, especially in combination with therapies used for the
treatment of AML. In particular, compounds of formula (I) can be
administered in combination with, e.g., farnesyl transferase
inhibitors and/or other drugs useful for the treatment of AML, such
as Daunorubicin, Adriamycin, Ara-C, VP-16, Teniposide,
Mitoxantrone, Idarubicin, Carboplatinum and PKC412.
[0459] The structure of the active agents identified by code nos.,
generic or trade names may be taken from the actual edition of the
standard compendium "The Merck Index" or from databases, e.g.,
Patents International, e.g., IMS World Publications.
[0460] The above-mentioned compounds, which can be used in
combination with a compound of the formula (I), can be prepared and
administered as described in the art, such as in the documents
cited above.
[0461] A compound of the formula (I) may also be used to advantage
in combination with known therapeutic processes, e.g., the
administration of hormones or especially radiation. A compound of
formula (I) may in particular be used as a radiosensitizer,
especially for the treatment of tumors which exhibit poor
sensitivity to radiotherapy.
[0462] By "combination", there is meant either a fixed combination
in one dosage unit form, or a kit of parts for the combined
administration where a compound of the formula (I) and a
combination partner may be administered independently at the same
time or separately within time intervals that especially allow that
the combination partners show a cooperative, e.g., synergistic,
effect or any combination thereof. The terms "co-administration" or
"combined administration" or the like as utilized herein are meant
to encompass administration of the selected combination partner to
a single subject in need thereof (e.g. a patient), and are intended
to include treatment regimens in which the agents are not
necessarily administered by the same route of administration or at
the same time. The term "pharmaceutical combination" as used herein
means a product that results from the mixing or combining of more
than one active ingredient and includes both fixed and non-fixed
combinations of the active ingredients. The term "fixed
combination" means that the active ingredients, e.g. a compound of
formula I and a combination partner, are both administered to a
patient simultaneously in the form of a single entity or dosage.
The term "non-fixed combination" means that the active ingredients,
e.g. a compound of formula (I) and a combination partner, are both
administered to a patient as separate entities either
simultaneously, concurrently or sequentially with no specific time
limits, wherein such administration provides therapeutically
effective levels of the two compounds in the body of the patient.
The latter also applies to cocktail therapy, e.g. the
administration of three or more active ingredients.
Experimental Details:
[0463] Insofar as the production of the starting materials is not
particularly described, the compounds are known or may be prepared
analogously to methods known in the art or as described
hereafter.
[0464] The following examples are illustrative of the invention
without any limitation.
Abbreviations
[0465] AcOH acetic acid [0466] aq aqueous [0467] Ar aryl [0468] BOC
tert-butyl-carbonate [0469] BOP
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate [0470] br.s. broad singlet [0471] CDCl.sub.3
chloroform-d [0472] CDI 1,1'-carbonyldiimidazole [0473]
CH.sub.2Cl.sub.2 dichloromethane [0474] CH.sub.3CN acetonitrile
[0475] Cs.sub.2CO.sub.3 cesium carbonate [0476] d doublet [0477] dd
doublet of doublets [0478] DIPEA N-ethyldiisopropylamine [0479] DME
1,4-dimethoxyethane [0480] DMF N,N-dimethylformamide [0481] DBU
1,8-diaza-7-bicyclo[5.4.0]undecene [0482] DMSO dimethylsulfoxide
[0483] dt doublet of triplets [0484] EDC 1-ethyl-3-(3-di
methylaminopropyl)carbodiimide hydrochloride [0485] eq. equivalent
[0486] EtOAc ethyl acetate [0487] FCC flash column chromatography
[0488] h hour [0489] HBTU
(2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate [0490] HCl hydrochloric acid [0491] HOBT
benztriazol-1-ol [0492] HPLC high pressure liquid chromatography
[0493] HT high throughput [0494] H.sub.2O water [0495] Hyflo Hyflo
Super Cel Medium [0496] Isolute.RTM. SCX-2 polymer supported
sulfonic acid macroporous polystyrene [0497] K kelvin [0498]
K.sub.2CO.sub.3 potassium carbonate [0499] LC liquid chromatography
[0500] M molar [0501] MeCN acetonitrile [0502] MeOD methanol-d4
[0503] MeOH methanol [0504] 2-Me-THF 2-methyltetrahydrofuran [0505]
MgSO4 magnesium sulfate [0506] MHz mega herz [0507] MS mass
spectroscopy [0508] m multiplet [0509] mBar millibar [0510] mL
millilitre [0511] mm millimeter [0512] mM millimolar [0513] min.
minute [0514] mw microwave [0515] NaOH sodium hydroxide [0516]
Na.sub.2SO.sub.4 sodium sulfate [0517] NaHCO.sub.3 sodium hydrogen
carbonate [0518] NaO.sup.tBu sodium tert-butoxide [0519] NEt.sub.3
triethylamine [0520] NH.sub.3 ammonia [0521] NH.sub.4OH
concentrated solution of ammonia in water possessing a specific
gravity of 0.88 [0522] NMP N-methylpyrrolidinone [0523] NMR nuclear
magnetic resonance [0524] OBD optimum bed density [0525]
Pd(OAc).sub.2 palladium acetate [0526] Pd(OH) 2/C palladium
hydroxide on carbon [0527] Pd.sub.2(dba).sub.3
tris(dibenzylideneacetone)dipalladium [0528]
Pd.sub.2(dba).sub.3.CHCl.sub.3
tris(dibenzylideneacetone)dipalladium chloroform complex [0529]
PL-HCO.sub.3 MP polymer supported hydrogen carbonate macroporous
polystyrene [0530] PL-SO.sub.3H MP polymer supported sulfonic acid
macroporous polystyrene [0531] rt room temperature [0532] Rt
retention time [0533] s singulet [0534] SCX-2 polymer supported
sulfonic acid macroporous polystyrene [0535] t triplet [0536] TBME
tert-butylmethyl ether [0537] tBuOK potassium tert-butoxide [0538]
tert-BuONa sodium tert-butoxide [0539] TFA trifluoroacetic acid
[0540] THF tetrahydrofuran [0541] UPLC ultra performance liquid
chromatography [0542] X-Phos
dicyclohexyl(2',4',6'-triisopropylbiphenyl-2-yl)phosphine
[0543] Microwave equipment used is a Biotage Initiator.RTM.
[0544] All compounds are named using AutoNom.
LCMS Methods Used:
[0545] LC method 1 (Rt.sup.(1)): The retention times (Rt) were
obtained on a Agilent HPLC system with an Ascentis.RTM. Express
column C18 30.times.2.1 mm, 2.7 .mu.m (Supelco) applying a gradient
(H.sub.2O+0.05% formic acid+3.75 mM Ammonium
acetate)/(CH.sub.3CN+0.04% formic acid) 90/10 to 5/95 over 3.7 min
and 1.2 mL/min as solvent flow and then 5/95 over 0.7 min with 1.4
mL/min as solvent flow and 40.degree. C. for the oven temperature.
Detection method UV 220-400 nm-MS. LC method 2 (Rt.sup.(2)): The
retention times (Rt) were obtained on a Agilent HPLC system with an
Ascentis.RTM. Express column C18 30.times.2.1 mm, 2.7 .mu.m
(Supelco) applying a gradient (H.sub.2O+0.05% formic acid+3.75 mM
Ammonium acetate)/(CH.sub.3CN+0.04% formic acid) 95/5 to 5/95 over
3.7 min and 1.2 mL/min as solvent flow and then 5/95 over 0.7 min
with 1.4 mL/min as solvent flow and 40.degree. C. for the oven
temperature. Detection method UV 220-400 nm-MS.
LC Method 3 (Rt.sup.(3)):
[0546] The retention times (Rt) were obtained on a Agilent HPLC
system with an Ascentis.RTM. Express column C18 30.times.2.1 mm,
2.7 .mu.m (Supelco) applying a gradient (H.sub.2O+0.05% formic
acid+3.75 mM Ammonium acetate)/(CH.sub.3CN+0.04% formic acid) 99/1
over 0.5 min and 1.2 mL/min as solvent flow then 99/1 to 5/95 over
1.7 min and 1.2 mL/min as solvent flow and then 5/95 over 0.7 min
with 1.4 mL/min as solvent flow and 40.degree. C. for the oven
temperature. Detection method UV 220-400 nm-MS.
LC method 4 (Rt.sup.(4)): The retention times (Rt) were obtained on
a Agilent HPLC system with an Ascentis.RTM. Express column C18
30.times.2.1 mm, 2.7 .mu.m (Supelco) applying a gradient
(H.sub.2O+0.05% formic acid+3.75 mM Ammonium
acetate)/(CH.sub.3CN+0.04% formic acid) 90/10 to 5/95 over 1.7 min
and 1.2 mL/min as solvent flow and then 5/95 over 0.7 min with 1.4
mL/min as solvent flow and 40.degree. C. for the oven temperature.
Detection method UV 220-400 nm-MS. LC method 5 (Rt.sup.(5)):
[0547] The retention times (Rt) were obtained on a Agilent HPLC
system with an Ascentis.RTM. Express column C18 30.times.2.1 mm,
2.7 .mu.m (Supelco) applying a gradient (H.sub.2O+0.05%
TFA)/(CH.sub.3CN+0.04% TFA) 95/5 to 5/95 over 3.7 min and 1.2
mL/min as solvent flow and then 5/95 over 0.7 min with 1.4 mL/min
as solvent flow and 40.degree. C. for the oven temperature.
Detection method UV 220-400 nm-MS.
LC method 6 (Rt.sup.(6)):
[0548] The retention times (Rt) were obtained on a Agilent HPLC
system with an Ascentis.RTM. Express column C18 30.times.2.1 mm,
2.7 .mu.m (Supelco) applying a gradient
(H.sub.2O+TFA)/(CH.sub.3CN+0.04% TFA) 99/1 over 0.5 min and 1.2
mL/min as solvent flow then 99/1 to 5/95 over 1.7 min and 1.2
mL/min as solvent flow and then 5/95 over 0.7 min with 1.4 mL/min
as solvent flow and 40.degree. C. for the oven temperature.
Detection method UV 220-400 nm-MS.
LC method 7 (Rt.sup.(7)):
[0549] The retention times (Rt) were obtained on a Waters Agilent
HPLC system with an Ascentis.RTM. Express column C18 30.times.2.1
mm, 2.7 .mu.m (Supelco) applying a gradient (H.sub.2O+0.05%
TFA)/(CH.sub.3CN+0.04% TFA) 90/10 to 5/95 over 1.7 min and 1.2
mL/min as solvent flow and then 5/95 over 0.7 min with 1.4 mL/min
as solvent flow and 40.degree. C. for the oven temperature.
Detection method UV 220-400 nm-MS.
LC method 8 (Rt.sup.(8)):
[0550] The retention times (Rt) were obtained on a Waters HPLC
alliance-HT system with an XTerra column MS C18, 50.times.4.6 mm, 5
.mu.m, reverse phase, applying a gradient (H.sub.2O+0.1%
TFA)/(CH.sub.3CN+0.1% TFA) 95/5 to 0/100 over 8.0 min and 2.0
mL/min as solvent flow and 45.degree. C. for the oven temperature.
Detection method UV 220-400 nm-MS.
Purification Method:
Preparative Reverse Phase Gilson HPLC
[0551] Method A: Column SunFire prep C18 OBD 5 .mu.m, 30.times.100
mm from WATERS, with H.sub.2O+0.1% TFA and Acetonitrile+0.1% TFA as
mobile phase. Detection method UV 220-400 nm [0552] Method B:
Column Atlantis prep T3 OBD 5 .mu.m, 30.times.150 mm from WATERS,
with H.sub.2O+0.1% TFA and Acetonitrile+0.1% TFA as mobile phase.
Detection method UV 220-400 nm [0553] Method C: Column XTerra RP18
OBD 5 .mu.m, 19.times.50 mm from WATERS, with H.sub.2O+0.1% TFA and
Acetonitrile+0.1% TFA as mobile phase. Detection method UV 220-400
nm
X-ray Powder Diffraction
Instrumentation:
Method X1
TABLE-US-00001 [0554] Instrument Bruker AXS, D8 Advance Irradiation
CuK.alpha. (30 kV, 40 mA) Detector PSD (Vantec) detector Scan range
2.degree.-40.degree. (2 theta value)
Method X2
TABLE-US-00002 [0555] Instrument Bruker D8 GADDS Discover
Irradiation CuK.alpha. (40 kV, 40 mA) Detector HI-STAR Area
detector Scan range 6.degree.-40.degree. (2 theta value)
Preparation of intermediate compounds
##STR00020##
Intermediate 1: 5-Bromo-2-methoxy-3-trifluoromethyl-pyridine
[0556] To 2-methoxy-3-(trifluoromethyl)pyridine (20.0 g, 113.0
mmol) and 1,3-dibromo-5,5-dimethylimidazolidine-2,4-dione (43.6 g,
152.0 mmol) was added TFA (80 mL) and the resulting mixture stirred
at rt for 18 h under argon. The TFA was removed in vacuo (50 mbar,
45.degree. C.) and the residue suspended in tert-butyl methyl ether
(200 mL). The resulting colourless solid was removed by filtration
and washed with tert-butyl methyl ether (50 mL).
[0557] The filtrate was concentrated in vacuo and suspended in
EtOAc (50 mL) The insoluble colourless solid was removed by
filtration and washed with EtOAc (50 mL). The filtrate was
concentrated in vacuo, diluted with heptane/tert-butyl methyl ether
(5/1, 20 mL) and the insoluble colourless solid was removed by
filtration. The filtrate was purified by column chromatography on
silica gel with heptane/EtOAc, 100/0 to 90/10. The crude product
was filtered through a plug of NaHCO.sub.3 (20 g) and the filtrate
evaporated in vacuo to give a golden oil (27.9 g). The oil was
dissolved in heptanes (20 mL) and purified by filtered through a
plug of silica gel (80 g), eluting with heptane to give
5-bromo-2-methoxy-3-(trifluoromethyl)pyridine as a colourless oil
(22.5 g, 74% yield). .sup.1H-NMR (400 MHz, DMSO-d.sub.6, 298 K): 8
ppm 4.03 (s, 3H) 7.95 (d, 1H) 8.4 (d, 1H).
##STR00021##
Intermediate 2: 1-((S)-3-Hydroxy-pyrrolidin-1-yl)-propan-1-one
[0558] (S)-Pyrrolidin-3-ol (10.0 g, 81.0 mmol), triethylamine (23.6
mL, 170.0 mmol) and CH.sub.2Cl.sub.2 (150 mL) were combined in a
pear-shaped flask to give a beige suspension. The mixture was
cooled to -10.degree. C. and propionyl chloride (7.06 mL, 81.0
mmol) was added dropwise over 15 min, maintaining the temperature
between -10 to 0.degree. C. The resulting beige suspension was
stirred for 2 h at 0.degree. C. MeOH (9.8 mL) was added and the
mixture allowed to warm to room temperature then stirred for 1 h to
give a brown solution. The mixture was evaporated in vacuo to give
a beige residue which was stirred in diethylether (200 mL) and
filtered. The filtrate was evaporated in vacuo to give
1-((S)-3-hydroxy-pyrrolidin-1-yl)-propan-1-one as a yellow oil
(11.23 g, 95% yield). .sup.1H-NMR (400 MHz, DMSO-d.sub.6, 298 K):
.delta. ppm 0.92-1.02 m, 3H) 1.67-1.97 (m, 2H) 2.13-2.28 (m, 2H)
3.18-3.52 (m, 4H) 4.17-4.32 (m, 1H) 4.85-4.97 (m, 1H). LCMS:
[M+H]+=144.0
##STR00022##
Intermediate 3:
((S)-3-Hydroxy-pyrrolidin-1-yl)-(tetrahydro-pyran-4-yl)-methanone
[0559] The (S)-pyrrolidin-3-ol hydrochloride (3.69 g, 29.9 mmol)
and triethylamine (6.65 g, 9.16 mL, 65.7 mmol) were put in
CH.sub.2Cl.sub.2 (15 mL). The suspension was cooled at
.about.3.degree. C. To this mixture, a solution of
tetrahydro-pyran-4-carbonyl chloride (4.67 g, 29.9 mmol) in
CH.sub.2Cl.sub.2 (15 mL) was added slowly. Then the resulting
reaction mixture was stirred for 1.5 h at 3-10.degree. C. The
reaction mixture was then concentrated to give a powder. To this
powder, addition of EtOAc (100 mL). The solid was filtered and
washed with EtOAc. The recovered filtrate was then concentrated to
give
((S)-3-hydroxy-pyrrolidin-1-yl)-(tetrahydro-pyran-4-yl)-methanone
as beige powder. (6.77 g, 98% yield). .sup.1H-NMR (400 MHz,
Methanol-d.sub.4, 298 K): .delta. ppm 1.59-2.15 (m, 6H) 2.69-2.86
(m, 1H) 3.43-3.75 (m, 6H) 3.94-4.00 (m, 2H) 4.37-4.48 (m, 1H).
LCMS: [M+H]+=199.9, Rt.sup.(6)=0.86 min
##STR00023##
Intermediate 4:
[(S)-1-(Tetrahydro-pyran-4-carbonyl)-pyrrolidin-3-yl]-carbamic acid
tert-butyl ester
[0560] To a vigorously stirring solution of
tetrahydro-2H-pyran-4-carbonyl chloride (0.455 g, 3.06 mmol) in
CH.sub.2Cl.sub.2 (10 mL) was added simultaneously portionwise sat.
NaHCO.sub.3(aq) (10 mL) and a solution of the
(S)-pyrrolidin-3-yl]-carbamic acid tert-butyl ester (570 mg, 3.06
mmol) at rt. The resulting biphasic mixture was stirred vigorously
at rt for 3 h. The organic layer was separated by filtration
through a phase separation tube, concentrated in vacuo and purified
by flash chromatography on silica gel with CH.sub.2Cl.sub.2/MeOH to
give [(S)-1-(tetrahydro-pyran-4-carbonyl)-pyrrolidin-3-yl]-carbamic
acid tert-butyl ester as a colourless gum (0.623 g, 68% yield)
LCMS: [M+H]+=299.6, Rt.sup.(7)=0.73 min.
##STR00024##
Intermediate 5:
(S)-3-Amino-pyrrolidin-1-yl-(tetrahydro-pyran-4-yl)-methanone
[0561] To
(S)-1-(tetrahydro-pyran-4-carbonyl)-pyrrolidin-3-yl]-carbamic acid
tert-butyl ester (intermediate 4) (0.623 g, 2.09 mmol) in
CH.sub.2Cl.sub.2 (2.0 mL) was added TFA (2.0 mL) and the resulting
mixture stood at rt for 8 h. Evaporated in vacuo and eluted through
an Isolute.RTM. SCX-2 cartridge, eluting with methanol, then with
2M ammonia in methanol. Basic fractions were concentrated in vacuo
to give
[(S)-3-amino-pyrrolidin-1-yl-(tetrahydro-pyran-4-yl)-methanone as a
colourless solid (0.34 g, 82% yield) LCMS: [M+H]+=199.0,
Rt.sup.(3)=0.1 min.
##STR00025##
Intermediate 6:
3-(4-Acetyl-piperazine-1-carbonyl)-1-methyl-3H-imidazol-3-ium
iodide
[0562] 1-(Piperazin-1-yl)ethanone (143 mg, 1.12 mmol) and CDI (199
mg, 1.23 mmol) were refluxed in THF (10 mL) under argon overnight.
Cooled to room temperature, diluted with CH.sub.2Cl.sub.2 (20 mL)
and water (5 mL) and the organic layer filtered through a phase
separation tube and concentrated in vacuo. Dissolved in
acetonitrile (5 mL) in a glass vial and methyl iodide (0.279 mL,
4.46 mmol) was added. The vial was capped and stood at room
temperature for 24 h. The solvent was evaporated in vacuo and the
residue triturated with heptane/EtOAc, 10/1 (10 mL) to give
3-(4-acetyl-piperazine-1-carbonyl)-1-methyl-3H-imidazol-3-ium
iodide as a colourless gum (400 mg) which was used without further
purification.
##STR00026##
Intermediate 7:
(S)-3-(5,6,7,8-Tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidine-1-c-
arboxylic acid tert-butyl ester
[0563] Pd(OH).sub.2/C (1.2 g, 1.71 mmol) was flushed with argon,
(S)-3-(6-Benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrol-
idine-1-carboxylic acid tert-butyl ester (10.95 g, 26.7 mmol)
dissolved in methanol (25 mL) was added followed by the addition of
ammonium formate (1.68 g, 26.7 mmol). The reaction mixture was
refluxed for 1 h, cooled down to room temperature, filtered through
a celite pad and concentrated under vacuum. Purification by flash
chromatography on silica gel (CH.sub.2Cl.sub.2 then TBME then
TBME/MeOH 100/0 to 90/10 then TBME/MeOH/NH.sub.4OH 85/15/5) gave
(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidine-1-c-
arboxylic acid tert-butyl ester (7.39 g, 87% yield) as a yellow
sticky oil. .sup.1H NMR (400 MHz, methanol-d4, 298K) .delta. ppm
1.46-1.46 (m, 9H) 2.10-2.30 (m, 2H) 2.78-2.83 (m, 2H) 3.11-3.14 (m,
2H) 3.41-3.60 (m, 3H) 3.65-3.72 (m, 1H) 3.78 (s, 2H) 5.68 (m, 1H)
8.52 (s, 1H). LCMS: [M+H].sup.+=321.2, Rt.sup.(2)=0.87 min
Alternative Synthesis for Intermediate 7:
[0564] Pd(OH).sub.2/C (1.54 g, 2.2 mmol) was flushed with nitrogen,
(S)-3-(6-benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrol-
idine-1-carboxylic acid tert-butyl ester (8.5 g, 20.67 mmol)
dissolved in methanol (50 mL) was added followed by the addition of
triethylammonium formate (7.9 g, 53.7 mmol). The reaction mixture
was refluxed for 1 h, cooled down to room temperature, filtered
through a celite pad and the filtrate was partitioned between
2-Me-THF (50 mL) and water (20 mL). The upper organic phase was
collected and the bottom aqueous phase was re-extracted with
2-Me-THF (10 mL). All the organic layers were combined and
concentrated under vacuum to provide
(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidine-1-c-
arboxylic acid tert-butyl ester (6.2 g, 94% yield) as a yellow
gum.
##STR00027##
(S)-3-(6-Benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrol-
idine-1-carboxylic acid tert-butyl ester
[0565] To a solution of (S)-3-hydroxypyrrolidine-1-carboxylic acid
tert-butyl ester (0.94 g, 5.01 mmol) in THF (20 mL) was added under
argon NaH (0.23 g, 5.78 mmol). The mixture was stirred at rt for 25
min., then
6-benzyl-4-chloro-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine (1 g,
3.85 mmol) was added and stirring continued at rt for 4 h. The
mixture was quenched with H.sub.2O, extracted with
CH.sub.2Cl.sub.2. The organic layer was filtered and evaporated to
dryness. Purification by flash chromatography on silica gel
(heptanes/ethyl acetate, 1/1) gave the
(S)-3-(6-benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrol-
idine-1-carboxylic acid tert-butyl ester (1.35 g, 85% yield) as a
yellow gum. .sup.1H NMR (400 MHz, DMSO-d6, 298K) .delta. ppm 1.39
(s, 9H) 2.00-2.20 (m, 2H) 2.35-2.81 (m, 4H) 3.36-3.63 (m, 6H) 3.70
(br.s, 2H) 5.50-5.59 (m, 1H) 7.25-7.37 (m, 5H) 8.56 (s, 1H). LCMS:
[M+H]+=411.6, Rt.sup.(7)=1.00 min
Alternative synthesis for
(S)-3-(6-Benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrol-
idine-1-carboxylic acid tert-butyl ester
[0566] To a solution of (S)-3-hydroxypyrrolidine-1-carboxylic acid
tert-butyl ester (6.21 g, 33.16 mmol) and
6-benzyl-4-chloro-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine (9 g,
34.65 mmol) in 2-Me-THF (100 mL) was added under nitrogen tBuOK
(8.17 g, 72.95 mmol). The mixture was stirred at rt for 25 min. The
mixture was quenched with H.sub.2O. The organic layer was washed
with brine. The resulting organic solution was concentrated in
vacuo to provide
(S)-3-(6-benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrol-
idine-1-carboxylic acid tert-butyl ester (12.6 g, 89% yield) as a
yellow gum.
##STR00028##
Intermediate 8:
6-(2,4-Dimethoxy-pyrimidin-5-yl)-4-((S)-pyrrolidin-3-yloxy)-5,6,7,8-tetra-
hydro-pyrido[4,3-d]pyrimidine
[0567] 5-Bromo-2,4-dimethoxy-pyrimidine (89 mg, 0.41 mmol), X-Phos
(46 mg, 0.09 mmol) bis(dibenzylideneacetone)palladium(0) (29 mg,
0.03 mmol), cesium carbonate (203 mg, 0.62 mmol) were combined and
flushed 10 min with Argon. To this mixture was added
(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidine-1-c-
arboxylic acid tert-butyl ester (intermediate 7) (100 mg, 0.31
mmol) in dioxane (4 mL), the vial was capped and the reaction
mixture was stirred at 120.degree. C. for 4.5 h. The mixture was
allowed to cool to rt and filtered through a celite pad. The
filtrate was diluted with EtOAc (20 mL) and washed with sat.
NaHCO.sub.3(aq) (10 mL), brine (10 mL), dried (Na.sub.2SO.sub.4)
and concentrated in vacuo. Dissolved in dioxane (4 mL) and added to
a glass vial containing 5-bromo-2,4-dimethoxy-pyrimidine (89 mg,
0.41 mmol), X-Phos (46 mg, 0.09 mmol)
tris(dibenzylideneacetone)dipalladium(0) (29 mg, 0.03 mmol), cesium
carbonate (203 mg, 0.62 mmol). The vial was capped and the reaction
mixture was stirred at 120.degree. C. for 4.5 h. The mixture was
allowed to cool to rt and filtered through a celite pad. The
filtrate was diluted with EtOAc (20 mL) and washed with sat.
NaHCO.sub.3(aq) (10 mL) then brine (10 mL), dried
(Na.sub.2SO.sub.4) and concentrated in vacuo to give
(S)-3-(6-(2,4-dimethoxy-pyrimidin-5-yl)-5,6,7,8-tetrahydropyrido[4,3-d]py-
rimidin-4-yloxy)pyrrolidine-1-carboxylic acid tert-butyl ester
which was used without further purification.
(S)-3-(6-(2,4-dimethoxy-pyrimidin-5-yl)-5,6,7,8-tetrahydropyrido[4,3-d]py-
rimidin-4-yloxy)pyrrolidine-1-carboxylic acid tert-butyl ester was
dissolved in CH.sub.2Cl.sub.2 (2.0 mL) and TFA added (1 mL). The
resulting mixture was stirred for 30 min. at room temperature. The
reaction mixture was concentrated in vacuo. Purification by
preparative reverse phase Gilson HPLC and subsequent neutralization
of the combined fractions by PL-HCO3 cartridge & lyophilisation
gave
6-(2,4-dimethoxy-pyrimidin-5-yl)-4-((S)-pyrrolidin-3-yloxy)-5,6,7,8-tetra-
hydro-pyrido[4,3-d]pyrimidine as a yellow powder (11 mg, 10% yield
over 2 steps). LCMS: [M+H]+=359.1, Rt.sup.(2)=0.79 min
##STR00029##
Intermediate 9:
2-Amino-5-[4-((S)-pyrrolidin-3-yloxy)-7,8-dihydro-5H-pyrido[4,3-d]pyrimid-
in-6-yl]nicotinonitrile
[0568]
(S)-3-(5,6,7,8-Tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidi-
ne-1-carboxylic acid tert-butyl ester (intermediate 7) (84 mg,
0.263 mmol), imidodicarbonic acid,
2-[5-bromo-3-(cyano)-2-pyridinyl]-, 1,3-bis(1,1-dimethylethyl)
ester (115 mg, 0.289 mmol), X-Phos (376 mg, 0.079 mmol),
tris(dibenzylideneacetone)dipalladium(0) (24 mg, 0.026 mmol),
cesium carbonate (171 mg, 0.526 mmol) were combined in a glass vial
and flushed 10 min with Argon. To this mixture was added dioxane
(4.0 mL), the vial was capped and the reaction mixture stirred at
120.degree. C. for 1.5 h. The reaction was allowed to cool to rt
and filtered through a celite pad, The filtrate was diluted with
EtOAc (20 mL) and washed with sat. NaHCO.sub.3(aq) (10 mL) and
brine (10 mL), dried (Na.sub.2SO.sub.4) and concentrated in vacuo
to give (S)-tert-butyl
3-(6-(6-(bis(tert-butoxycarbonyl)amino)-5-(cyano)pyridin-3-yl)-5,6,7,8-te-
trahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidine-1-carboxylate
which was used without further purification. (S)-tert-butyl
3-(6-(6-(bis(tert-butoxycarbonyl)amino)-5-(cyano)pyridin-3-yl)-5,6,7,8-te-
trahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidine-1-carboxylate
was dissolved in CH.sub.2Cl.sub.2 (2.0 mL) and TFA added (1 mL).
The resulting mixture was stirred for 30 min at room temperature.
The reaction mixture was concentrated in vacuo. Purification by
preparative reverse phase Gilson HPLC and subsequent neutralization
of the combined fractions by PL-HCO3 cartridge & lyophilisation
gave
2-amino-5-[4-((S)-pyrrolidin-3-yloxy)-7,8-dihydro-5H-pyrido[4,3-d]pyrimid-
in-6-yl]nicotinonitrile as a yellow powder (17 mg, 19% yield over 2
steps). LCMS: [M+H]+=338.3, Rt.sup.(3)=1.16 min.
##STR00030##
Imidodicarbonic acid, 2-[5-bromo-3-(cyano)-2-pyridinyl]-,
1,3-bis(1,1-dimethylethyl) ester
[0569] To 2-amino-5-bromonicotinonitrile (0.785 g, 3.96 mmol),
triethylamine (0.553 mL, 3.96 mmol) and 4-dimethylaminoyridine (20
mg, 0.164 mmol) in CH.sub.2Cl.sub.2 (25 mL) was added
di-tert-butyl-dicarbonate (2.16 g, 9.91 mmol) and the resulting
mixture stirred at room temperature for 18 h. Evaporated to dryness
in vacuo and triturated in heptane (25 mL) for 72 h. The resulting
precipitate was filtered and washed with heptane (10 mL) to give
imidodicarbonic acid, 2-[5-bromo-3-(cyano)-2-pyridinyl]-,
1,3-bis(1,1-dimethylethyl) ester as a beige solid (1.1 g, 70%
yield). .sup.1H NMR (400 Mhz, CDCl.sub.3, 298K) 1.51 (s, 18H) 8.16
(d, 1H) 8.77 (d, 1H). LCMS: [M+H]+=398/400.1, Rt.sup.(4)=1.43
min.
##STR00031##
Intermediate 10:
(S)-3[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyri-
midin-4-yloxy]-pyrrolidine-1-carboxylic acid tert-butyl ester
[0570] To a glass vial was added
(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidine-1-c-
arboxylic acid tert-butyl ester (intermediate 7) (1.00 g, 3.12
mmol), 5-bromo-2,3-dimethoxypyridine (0.82 g, 3.75 mmol), sodium
tert-butoxide (0.46 g, 4.68 mmol),
tris(dibenzylideneacetone)dipalladium(0) (0.11 g, 0.13 mmol),
2-di-t-butylphosphino-2'-(N,N-dimethylamino)biphenyl (0.06 g, 0.18
mmol) and anhydrous toluene (10 mL). The vial was flushed with a
stream of argon for 15 sec and capped. The mixture was heated with
stirring for 18 h at 80.degree. C. Allowed to cool and filtered
through a celite pad. The filtrate was diluted with EtOAc (50 mL)
and washed with brine (20 mL). The organic layer was separated,
dried (Na.sub.2SO.sub.4) and concentrated in vacuo. Purified by
flash column chromatography on silica gel with EtOAc/MeOH, 98/2 to
92/18 to give
(S)-3-[6-(5,6-dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyr-
imidin-4-yloxy]-pyrrolidine-1-carboxylic acid tert-butyl ester as a
pale yellow foam (1.05 g, 74% yield). LCMS: [M+H]+=458.1,
Rt.sup.(4)=1.02 min.
##STR00032##
Intermediate 11:
(S)-3-[6-(5-Cyano-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d-
]pyrimidin-4-yloxy]-pyrrolidine-1-carboxylic acid tert-butyl
ester
[0571] To a glass vial was added
(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidine-1-c-
arboxylic acid tert-butyl ester (intermediate 7) (630 mg, 1.97
mmol), 5-bromo-2-methoxynicotinonitrile (419 mg, 1.97 mmol), cesium
carbonate (1281.0 mg, 3.93 mmol),
tris(dibenzylideneacetone)dipalladium(0) (180 mg, 0.20 mmol),
X-Phos (319 mg, 0.67 mmol) and anhydrous dioxane (10.0 mL). The
vial was flushed with a stream of argon for 15 sec and capped. The
mixture was heated with stirring for 1 h at 110.degree. C. and then
stirred at room temperature for 18 h. Diluted with CH.sub.2Cl.sub.2
(100 mL) and water (30 mL) and filtered through a celite pad. The
organic phase was separated by filtering through a phase separation
tube and concentrated in vacuo. Purified by flash chromatography on
silica gel with heptanes/EtOAc, 80/20 to 0/100 to give
(S)-3-[6-(5-cyano-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d-
]pyrimidin-4-yloxy]-pyrrolidine-1-carboxylic acid tert-butyl ester
as a brown gum (350 mg, 39% yield) LCMS: [M+H]+=453.6,
Rt.sup.(7)=1.29 min.
##STR00033##
Intermediate 12:
(S)-3-[6-(5-Fluoro-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3--
d]pyrimidin-4-yloxy]-pyrrolidine-1-carboxylic acid tert-butyl
ester
[0572] To a glass vial was added
(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidine-1-c-
arboxylic acid tert-butyl ester (intermediate 7) (150 mg, 0.47
mmol), 5-bromo-3-fluoro-2-methoxypyridine (96 mg, 0.47 mmol),
cesium carbonate (305 mg, 0.94 mmol),
tris(dibenzylideneacetone)dipalladium(0) (43 mg, 0.05 mmol), X-Phos
(76 mg, 0.16 mmol) and anhydrous dioxane (2.0 mL). The vial was
flushed with a stream of argon for 15 sec and capped. The mixture
was heated with stirring for 1.5 h at 110.degree. C. and then
stirred at room temperature for 18 h. Diluted with CH.sub.2Cl.sub.2
(25 mL), filtered through a celite pad and concentrated in vacuo.
Purified by reverse phase Gilson HPLC (Method A) to give
(S)-3-[6-(5-fluoro-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3--
d]pyrimidin-4-yloxy]-pyrrolidine-1-carboxylic acid tert-butyl ester
trifluoroacetate as a brown gum (45 mg, 17% yield) LCMS:
[M+H]+=446.4, Rt.sup.(4)=1.41 min.
##STR00034##
Intermediate 13:
(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-py-
rido[4,3-d]pyrimidin-4-yloxy]-pyrrolidine-1-carboxylic acid
tert-butyl ester
[0573] To a glass vial was added
(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidine-1-c-
arboxylic acid tert-butyl ester (intermediate 7) (150 mg, 0.47
mmol) 5-bromo-2-methoxy-3-(trifluoromethyl)pyridine (intermediate
1)(120 mg, 0.47 mmol), cesium carbonate (305 mg, 0.94 mmol),
tris(dibenzylideneacetone)dipalladium(0) (43 mg, 0.05 mmol), X-Phos
(76 mg, 0.16 mmol) and anhydrous dioxane (2.0 mL). The vial was
flushed with a stream of argon for 15 sec and capped. The mixture
was heated with stirring for 1 h at 110.degree. C. and then stirred
at room temperature for 18 h. Diluted with CH.sub.2Cl.sub.2 (10 mL)
and water (2 mL), filtered through a celite pad. The organic phase
was separated by filtering through a phase separation tube and
concentrated in vacuo. Purified by reverse phase Gilson HPLC
(Method A) to give (S)-tert-butyl
3-(6-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydropyrido-
[4,3-d]pyrimidin-4-yloxy)pyrrolidine-1-carboxylate trifluoroacetate
(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-py-
rido[4,3-d]pyrimidin-4-yloxy]-pyrrolidine-1-carboxylic acid
tert-butyl ester trifluoroacetate as a brown gum (90 mg, 32% yield)
LCMS: [M+H]+=496.5, Rt.sup.(7)=1.43 min.
##STR00035##
Intermediate 14:
4-Methoxy-6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4-
,3-d]pyrimidine
[0574] To a glass vial was added
4-methoxy-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine (WO
2008/130481, p 47) (0.570 g, 3.45 mmol),
5-bromo-2-methoxy-3-methylpyridine (0.697 g, 3.45 mmol), cesium
carbonate (2.25 g, 6.90 mmol),
tris(dibenzylideneacetone)dipalladium(0) (0.316 g, 0.345 mmol),
X-Phos (0.493 g, 1.04 mmol) and anhydrous dioxane (5 mL). The vial
was flushed with a stream of argon for 15 sec and capped. The
mixture was heated with stirring for 1 h 45 min at 110.degree. C.
then allowed to cool to room temperature and stirred at RT for 3
days. The reaction mixture was filtered through a celite pad and
concentrated in vacuo. Purified by flash chromatography on silica
gel with heptane/EtOAc, 100/0 to 0/100 then EtOAc/MeOH, 90/10 to
give
4-methoxy-6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4-
,3-d]pyrimidine as a brown gum (0.36 g, 36% yield) LCMS:
[M+H]+=287.0, Rt.sup.(7)=0.80 min.
##STR00036##
Intermediate 15:
6-(5-Chloro-6-methoxy-pyridin-3-yl)-4-methoxy-5,6,7,8-tetrahydro-pyrido[4-
,3-d]pyrimidine
[0575] To a glass vial was added
4-methoxy-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine (WO
2008/130481, p 47) (0.273 g, 1.65 mmol),
5-bromo-3-chloro-2-methoxypyridine (0.368 g, 1.65 mmol), sodium
tert-butoxide (318 mg, 3.31 mmol), diacetoxypalladium (0.037 g,
0.17 mmol), X-Phos (0.079 g, 0.17 mmol) and anhydrous
toluene/tert-butanol, 5/1 (6 mL). The vial was flushed with a
stream of argon for 15 sec and capped. The mixture was heated with
stirring for 2 h at 110.degree. C. then allowed to cool to room
temperature and stirred at rt for 5 days. Diluted with
CH.sub.2Cl.sub.2 (10 mL) and water (2 mL), filtered through a
celite pad. The organic phase was separated by filtering through a
phase separation tube and concentrated in vacuo. Purified by flash
chromatography on silica gel with heptane/EtOAc 100/0 to 0/100 to
give
6-(5-chloro-6-methoxy-pyridin-3-yl)-4-methoxy-5,6,7,8-tetrahydro-pyrido[4-
,3-d]pyrimidine as a yellow solid (95 mg, 19% yield) LCMS:
[M+H]+=307.0/308.9, Rt.sup.(3)=1.62 min.
##STR00037##
Intermediate 16:
4-Methoxy-6-(5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,-
3-d]pyrimidine
[0576] To a glass vial was added
4-methoxy-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine (0.273 g, 1.65
mmol), 3-bromo-5-(trifluoromethyl)pyridine (0.373 g, 1.65 mmol),
cesium carbonate (1.08 g, 3.31 mmol),
tris(dibenzylideneacetone)dipalladium(0) (0.076 g, 0.083 mmol),
X-Phos (0.079 g, 0.165 mmol) and anhydrous dioxane (5 mL). The vial
was flushed with a stream of argon for 15 sec and capped. The
mixture was heated with stirring for 1.5 h at 110.degree. C.
Filtered through a celite pad, concentrated in vacuo and purified
by flash chromatography on silica gel with heptanes/EtOAc, 100/0 to
0/100 to give
4-methoxy-6-(5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyri-
do[4,3-d]pyrimidine as an orange gum (195 mg, 34% yield) .sup.1H
NMR (DMSO-d6, 298K) 2.95 (t, 2H) 3.77 (t, 2H) 4.02 (s, 3H) 4.37 (s,
2H) 7.67-7.71 (m, 1H) 8.30-8.34 (m, 1H) 8.63 (s, 1H) 8.67-8.71 (1H,
m) LCMS: [M+H]+=311.2, Rt.sup.(4)=0.94 min.
##STR00038##
Intermediate 17:
6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrim-
idin-4-ol
[0577] To
4-methoxy-6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-
-pyrido[4,3-d]pyrimidine (intermediate 14)(360 mg, 1.26 mmol) in
MeOH (2.0 mL) in a glass vial was added 2M NaOH(aq) (2.0 mL). The
vial was capped and heated at 90.degree. C. for 24 h. Acidified
with glacial AcOH to pH 6, evaporated in vacuo and the residue
extracted with CH.sub.2Cl.sub.2 (2.times.30 mL). With each
extraction, the CH.sub.2Cl.sub.2 layer was decanted from the solid
residue. The CH.sub.2Cl.sub.2 layers were combined and eluted
through an Isolute.RTM. SCX-2 cartridge, eluting with methanol,
then with 2M ammonia in methanol. Basic fractions were concentrated
in vacuo to give
6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-
-d]pyrimidin-4-ol as a brown gum (260 mg, 76% yield) LCMS:
[M+H]+=273.1, Rt.sup.(3)=1.33 min.
##STR00039##
Intermediate 18:
6-(5-Chloro-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrim-
idin-4-ol
[0578] To
6-(5-chloro-6-methoxy-pyridin-3-yl)-4-methoxy-5,6,7,8-tetrahydro-
-pyrido[4,3-d]pyrimidine (intermediate 15)(95 mg, 0.31 mmol) in
MeOH (5.0 mL) in a glass vial was added 2M NaOH(aq) (3.0 mL). The
vial was capped and heated at 90.degree. C. for 24 h. Acidified
with glacial AcOH to pH 6, evaporated in vacuo and the residue
extracted with CH.sub.2Cl.sub.2 (1.times.50 mL with stirring). With
each extraction, the CH.sub.2Cl.sub.2 layer was decanted from the
solid residue. The CH.sub.2Cl.sub.2 layers were combined. The solid
residue was then washed with water (10 mL) and filtered. This
filtered solid was combined with the CH.sub.2Cl.sub.2 layers and
evaporated in vacuo to give
6-(5-chloro-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrim-
idin-4-ol as a yellow solid (90 mg, 107% yield) LCMS:
[M+H]+=293.0/294.8, Rt.sup.(3)=1.38 min.
##STR00040##
Intermediate 19:
6-(5-Trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimi-
din-4-ol
[0579] To
4-methoxy-6-(5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro--
pyrido[4,3-d]pyrimidine (intermediate 16) (190 mg, 0.612 mmol) in
MeOH (2.0 mL) in a glass vial was added 2M NaOH(aq) (2.0 mL). The
vial was capped and heated at 90.degree. C. for 24 h. Acidified
with glacial AcOH to pH 6, evaporated in vacuo and the residue
extracted with CH.sub.2Cl.sub.2 (2.times.30 mL with sonication).
With each extraction, the CH.sub.2Cl.sub.2 layer was decanted from
the solid residue. The CH.sub.2Cl.sub.2 layers were combined and
eluted through an Isolute.RTM. SCX-2 cartridge, eluting with
methanol, then with 2M ammonia in methanol. Basic fractions were
concentrated in vacuo to give
6-(5-(trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrim-
idin-4-ol as a yellow solid (167 mg) LCMS: [M+H]+=297.2,
Rt.sup.(4)=0.69 min.
##STR00041##
Intermediate 20:
(S)-3[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d-
]pyrimidin-4-ylamino)-pyrrolidine-1-carboxylic acid tert-butyl
ester
[0580] To
6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro--
pyrido[4,3-d]pyrimidin-4-ol (intermediate 17) (178 mg, 0.654 mmol)
in acetonitrile (2.0 mL) was added BOP (376 mg, 0.854 mmol) and DBU
(0.197 mL, 1.31 mmol). The resulting solution was stood at rt for 2
min then added (S)-tert-butyl 3-aminopyrrolidine-1-carboxylate (365
mg, 1.96 mmol) in acetonitrile (2.0 mL) and heated the mixture at
75.degree. C. for 72 h. The reaction mixture was evaporated in
vacuo and purified by reverse phase Gilson HPLC (Method A) to give
(S)-3-[6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3--
d]pyrimidin-4-ylamino)-pyrrolidine-1-carboxylic acid tert-butyl
ester trifluoroacetate (60 mg, 17% yield) as a brown gum. LCMS:
[M+H]+=441.2, Rt.sup.(3)=1.50 min
##STR00042##
Intermediate 21:
(S)-3[6-(5-Chloro-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d-
]pyrimidin-4-ylamino]-pyrrolidine-1-carboxylic acid tert-butyl
ester
[0581] To
6-(5-chloro-6-methoxypyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-
-d]pyrimidin-4-ol (intermediate 18) (90 mg, 0.31 mmol) in
acetonitrile (3.0 mL) was added BOP (177 mg, 0.40 mmol) and DBU
(0.15 mL, 0.99 mmol). The resulting solution was stood at rt for 2
min then added (S)-tert-butyl 3-aminopyrrolidine-1-carboxylate
(0.17, 0.93 mmol) and heated the mixture at 70.degree. C. for 96 h.
The reaction mixture was evaporated in vacuo and purified by
reverse phase Gilson HPLC (Method A) to give
(S)-3-[6-(5-chloro-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyr-
ido[4,3-d]pyrimidin-4-ylamino]-pyrrolidine-1-carboxylic acid
tert-butyl ester trifluoroacetate (50 mg, 35% yield) as a brown
gum. LCMS: [M+H]+=461.1/463.0, Rt.sup.(4)=0.93 min.
##STR00043##
Intermediate 22:
(S)-3-(6-Benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-ylamino)-pyrr-
olidine-1-carboxylic acid tert-butyl ester
[0582] 6-Benzyl-4-chloro-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine
(5.0 g, 19.06 mmol), (S)-tert-butyl
3-aminopyrrolidine-1-carboxylate (4.11 g, 20.96 g) and
triethylamine (3.98 mL, 28.6 mmol) were heated in a sealed vial at
120.degree. C. for 42 h. The mixture was allowed to cool, diluted
with tert-butyl methyl ether (100 mL) and the resulting suspension
stirred for 10 min. The mixture was diluted with water (50 mL) and
the organic layer separated. The organic layer was washed with
brine (20 mL), dried (Na.sub.2SO.sub.4) and evaporated in vacuo to
give a brown gum. The residue was purified by column chromatography
on silica gel with EtOAc/MeOH, 98/2 to 82/18 to give
(S)-3-(6-benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-ylamino)-pyrr-
olidine-1-carboxylic acid tert-butyl ester as a pale yellow foam
(7.36 g, 93% yield). .sup.1H-NMR (400 MHz, CDCl.sub.3, 298 K):
.delta. ppm 1.48 (s, 9H) 2.10-2.31 (m, 2H) 2.80-2.96 (m, 4H)
3.15-3.87 (m, 8H) 4.44-4.77 (m, 1H) 5.62-5.73 (m, 1H) 7.29-7.45 (m,
5H) 8.50 (s, 1H). LCMS: [M+H]+=410.0, Rt.sup.(6)=1.39 min.
Alternative Synthesis for Intermediate 22:
[0583] (S)-tert-Butyl-3-aminopyrrolidine-1-carboxylate (50 g, 192.5
mmol) was added to
6-benzyl-4-chloro-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine
(39.440 g, 211.8 mmol) in NMP (200 mL) solution followed by the
addition of K.sub.2CO.sub.3 (39.9 g, 288.8 mmol). The mixture was
heated to 120.degree. C. for 20 h. The mixture was allowed to cool,
partitioned between water (300 mL) and ethylacetate (500 mL). the
bottom aqueous phase was discarded and the upper organic phase was
washed with brine (150 mL) and concentrated in vacuo to provide
crude
(S)-3-(6-benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-ylamino)-pyrr-
olidine-1-carboxylic acid tert-butyl ester as a pale yellow foam
(76.44 g, 97% yield).
##STR00044##
Intermediate 23:
(S)-3-(5,6,7,8-Tetrahydro-pyrido[4,3-d]pyrimidin-4-ylamino)-pyrrolidine-1-
-carboxylic acid tert-butyl ester
[0584] To a solution of
(S)-3-(6-benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-ylamino)-pyrr-
olidine-1-carboxylic acid tert-butyl ester (intermediate 22) (30.1
g, 73.5 mmol) in MeOH (100 mL) was added 20% palladium hydroxide on
carbon (3.3 g) then ammonium formate (4.63 g, 73.5 mmol) and the
mixture heated at reflux for 1 h. Added ammonium formate (0.38 g,
6.02 mmol) and continued heating at reflux for 30 min. The reaction
mixture was allowed to cool and filtered through a celite pad,
washing with MeOH (50 mL) then CH.sub.2Cl.sub.2 (50 mL). The
filtrate was evaporated in vacuo to give a brown oil. Dissolved in
CH.sub.2Cl.sub.2 (100 mL), added solid NaHCO.sub.3 (10 g) and
filtered through a celite pad. The filtrate was evaporated in vacuo
to give a brown oil. Dissolved in EtOAc (50 mL) and a solid
precipitated which was filtered to give
(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-ylamino)-pyrrolidine-1-
-carboxylic acid tert-butyl ester as a beige solid (15.55 g, 66%
yield). .sup.1H-NMR (400 MHz, DMSO-d.sub.6, 298 K): .delta. ppm
1.40 (s, 9H) 1.81-1.98 (m, 1H) 2.05-2.17 (m, 1H) 2.92 (t, 2H)
3.10-3.46 (m, 5H) 3.49-3.63 (m, 3H) 4.47-4.63 (m, 1H) 6.46 (d, 1H,
N--H) 8.25 (s, 1H). LCMS: [M+H]+=320.0, Rt.sup.(6)=1.29 min.
Alternative Synthesis for Intermediate 23:
[0585] Pd(OH).sub.2/C (6.60 g, 5.3 mmol) was flushed with nitrogen,
(S)-3-(6-benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-ylamino)-pyrr-
olidine-1-carboxylic acid tert-butyl ester (intermediate 22)
dissolved in methanol (164 mL) was added followed by the addition
of triethylammonium formate (28.4 g, 188.0 mmol). The reaction
mixture was refluxed for 1 h, cooled down to room temperature,
filtered through a celite pad and the filtrate was concentrated
under vacuum. the residue was recrystallized with methyl tert-butyl
ether (200 mL) and heptanes (50 mL) to provide
(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-ylamino)-pyrrolidine-1-
-carboxylic acid tert-butyl ester as a beige solid (25.7 g, 85%
yield).
##STR00045##
Intermediate 24:
(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-py-
rido[4,3-d]pyrimidin-4-ylamino]-pyrrolidine-1-carboxylic acid
tert-butyl ester
[0586] To a glass vial was added
(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-ylamino)-pyrrolidine-1-
-carboxylic acid tert-butyl ester (intermediate 23) (3.5 g, 10.96
mmol), 5-bromo-2-methoxy-3-(trifluoromethyl)pyridine (intermediate
1) (3.09 g, 12.05 mmol), sodium tert-butoxide (1.58 g, 16.44 mmol),
tris(dibenzylideneacetone)dipalladium(0) (0.502 g, 0.548 mmol),
2-di-t-butylphosphino-2'-(N,N-dimethylamino)biphenyl (0.225 g,
0.657 mmol) and anhydrous tert-butanol (6 mL). The vial was flushed
with a stream of argon for 15 sec and capped. The mixture was
heated with stirring for 5 h at 100.degree. C. Allowed to cool and
partitioned between EtOAc (100 mL) and water (20 mL) and filtered
the biphasic mixture through a celite pad. The organic layer was
separated, dried (MgSO.sub.4) and concentrated in vacuo. Purified
by flash column chromatography through Biotage.RTM. amino silica
gel eluting with heptane/EtOAc, 100/0 to 0/100 then EtOAc/MeOH
(90/10) to give
(S)-3-[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-py-
rido[4,3-d]pyrimidin-4-ylamino]-pyrrolidine-1-carboxylic acid
tert-butyl ester as a yellow foam (4.00 g, 74% yield). LCMS:
[M+H]+=495.2, Rt.sup.(3)=1.59 min.
Alternative Synthesis for Intermediate 24:
[0587] To a glass flask was added
(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-ylamino)-pyrrolidine-1-
-carboxylic acid tert-butyl ester (intermediate 23) (6.331 g, 15.86
mmol), 5-bromo-2-methoxy-3-(trifluoromethyl)pyridine (intermediate
1) (4.465 g, 17.442 mmol), sodium tert-butoxide (2.29 g, 23.78
mmol), tris(dibenzylideneacetone)dipalladium(0) (0.726 g, 0.793
mmol), di-tert-butyl(2'-methylbiphenyl-2-yl)phosphine (0.297 g,
0.951 mmol) and anhydrous tert-butanol (30 mL). The flask was
flushed with a stream of nitrogen for 15 sec and capped. The
mixture was heated with stirring for 4 h under reflux. The mixture
was allowed to cool to rt and partitioned between EtOAc (100 mL)
and water (20 mL). The biphasic mixture was filtered the through a
celite pad. The organic layer was separated and concentrated in
vacuo to give crude
(S)-3-[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-py-
rido[4,3-d]pyrimidin-4-ylamino]-pyrrolidine-1-carboxylic acid
tert-butyl ester as a yellow foam (7.46 g, 95% yield).
##STR00046##
Intermediate 25:
(S)-3[6-(5-Cyano-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]-
pyrimidin-4-ylamino]-pyrrolidine-1-carboxylic acid tert-butyl
ester
[0588] To a glass vial was added
(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-ylamino)-pyrrolidine-1-
-carboxylic acid tert-butyl ester (intermediate 23) (566 mg, 1.77
mmol), 5-bromo-2-methoxynicotinonitrile (453 mg, 2.13 mmol), cesium
carbonate (1155 mg, 3.54 mmol),
tris(dibenzylideneacetone)dipalladium(0) (162 mg, 0.18 mmol),
X-Phos (287 mg, 0.60 mmol) and anhydrous tert-butanol (5 mL). The
vial was flushed with a stream of argon for 15 sec and capped. The
mixture was heated with stirring for 18 h at 110.degree. C. Allowed
to cool and partitioned between CH.sub.2Cl.sub.2 (20 mL) and water
(10 mL) and filtered the biphasic mixture through a celite pad. The
organic layer was separated by filtering through a phase separation
tube and concentrated in vacuo. Purified by flash column
chromatography on silica gel with heptane/EtOAc, 100/0 to 0/100
then EtOAc/MeOH (90/10) to give
(S)-3[6-(5-cyano-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]-
pyrimidin-4-ylamino]-pyrrolidine-1-carboxylic acid tert-butyl ester
as a brown gum (234 mg, 29% yield). LCMS: [M+1-1]+=452.1,
Rt.sup.(4)=0.90 min.
Preparation of Examples
##STR00047##
[0590] a)
(S)-3-(6-Benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-ylox-
y)-pyrrolidine-1-carboxylic acid tert-butyl ester III is firstly
prepared by reacting
6-benzyl-4-chloro-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine with
(S)-3-hydroxy-pyrrolidine-1-carboxylic acid tert-butyl ester in the
presence of a suitable base such as sodium hydride (NaH) and polar
organic solvent such as THF or dioxane under inert gas conditions
at room temperature. b) N-debenzylation is performed under
customary transfer hydrogenation conditions, using among the
possible palladium catalysts, preferably palladium hydroxide on
carbon Pd(OH).sub.2/C and among the possible formate salt
preferably ammonium formate and organic solvent such as preferably
methanol. The reaction is preferably carried out under refluxing
conditions. c) Buchwald-Hartwig cross coupling between
(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidine-1-c-
arboxylic acid tert-butyl ester IV and aryl bromide of the general
formula R.sup.2--X where X=Bromo or Iodo is performed under
customary Buchwald-Hartwig conditions using such a ligand such as
X-Phos or 2-di-t-butylphosphino-2'-(N,N-dimethylamino)biphenyl with
a palladium catalyst such as Pd.sub.2(dba).sub.3 or
Pd.sub.2(dba).sub.3.CHCl.sub.3 or Pd(OAc).sub.2, preferably
Pd.sub.2(dba).sub.3 with X-Phos, base such as preferably
Cs.sub.2CO.sub.3 or preferably tert-BuONa, and organic solvent such
as preferably dioxane or preferably THF. The reaction is preferably
stirred at a temperature of approximately 80-120.degree. C.,
preferably 120.degree. C. The reaction may preferably carry out
under an inert gas such as nitrogen or argon. d) N--BOC
deprotection is performed under customary BOC deprotection
conditions using among the possible acid preferably
trifluoro-acetic acid or HCl and suitable organic solvent such as
CH.sub.2Cl.sub.2 or diethyl ether. The reaction is preferably
performed at room temperature. e)
[0591] Reaction of compounds of general formula VI with an acid
chloride of formula R.sup.4C(O)Cl or carboxylic acid of formula
R.sup.4C(O)OH. Those skilled in the art will appreciate that there
are many known ways of preparing amides. For example, see
Mantalbetti, C.A.G.N and Falque, V., Amide bond formation and
peptide coupling, Tetrahedron, 2005, 61(46), pp 10827-10852 and
references cited therein. The examples provided herein are thus not
intended to be exhaustive, but merely illustrative.
[0592] The following general methods i-v have been used.
[0593] i. To a vigorously stirring solution of the acid chloride
(1.3 eq.) in CH.sub.2Cl.sub.2 was added simultaneously portionwise
excess sat. NaHCO.sub.3(aq) and a solution of the amine of general
formula VI (1.0 eq.) in CH.sub.2Cl.sub.2 at rt. The resulting
biphasic mixture was stirred vigorously at rt for 2 h. The organic
layer was separated, dried (MgSO.sub.4), concentrated in vacuo and
purified by either reverse phase chromatography, normal phase
chromatography or crystallisation.
[0594] ii. To the amine of general formula VI (1.0 eq.) in
CH.sub.2Cl.sub.2 was added the acid chloride (1.1 eq.) and
triethylamine (3.0 eq.) at rt. The reaction mixture was stirred at
room temperature for 1 h. The reaction mixture was concentrated
under vacuum and subsequently partitioned between water and a
suitable organic solvent and purified either reverse phase
chromatography, normal phase chromatography or crystallisation.
[0595] iii. To the carboxylic acid (1.0 eq.) and HBTU (1.2 eq.) in
DMF was added triethylamine (4.0 eq.). The mixture was stirred for
20 min and then the amine of general formula VI (1.0 eq.) in DMF
was added. The mixture was allowed to stir overnight at room
temperature and subsequently partitioned between water and a
suitable organic solvent. The organic phase was separated, dried
(MgSO.sub.4), concentrated in vacuo and purified by either reverse
phase chromatography, normal phase chromatography or
crystallisation.
[0596] iv. To the carboxylic acid (1.0 eq.) and the amine general
formula VI (1.0 eq.) in DMF was added DCC (1.2 eq.) in DMF. The
reaction mixture was stirred at rt for 18 h and concentrated in
vacuo and purified by either reverse phase chromatography, normal
phase chromatography or crystallisation.
[0597] v. To the carboxylic acid (1.1 eq.) and the amine general
formula VI (1.0 eq.) in CH.sub.2Cl.sub.2 was added benztriazol-1-ol
(1.1 eq.) and EDC (1.6 eq.). The reaction mixture was stirred at rt
for 18 h and subsequently partitioned between water and a suitable
organic solvent. The organic phase was separated, dried
(MgSO.sub.4), concentrated in vacuo and purified by either reverse
phase chromatography, normal phase chromatography or
crystallization.
##STR00048##
[0598] a) N--BOC deprotection is performed under customary BOC
deprotection conditions using among the possible acid preferably
trifluoro-acetic acid and organic solvent preferably
CH.sub.2Cl.sub.2. The reaction is preferably performed at room
temperature. b) Reaction of compound of general formula IX with an
acid chloride of formula R.sup.4C(O)Cl or carboxylic acid of
formula R.sup.4C(O)OH using general methods i-v as described in
Scheme 1, step e. Those skilled in the art will appreciate that
there are many known ways of preparing amides. For example, see
Mantalbetti, C.A.G.N and Falque, V., Amide bond formation and
peptide coupling, Tetrahedron, 2005, 61(46), pp 10827-10852 and
references cited therein. The examples provided herein are thus not
intended to be exhaustive, but merely illustrative.
[0599] c) Removal of the benzyl protecting group is performed using
standard methodology as described in "Protecting groups in Organic
Synthesis" by T. W. Greene and P. Wutz, 3.sup.rd edition, 1999,
John Wiley and Sons. Typical conditions comprise of 1.0 eq. of
compound of general formula X (8.0 eq. of ammonium formate and 20%
(w/w) palladium hydroxide Pd(OH).sub.2/C (catalyst) heated under
reflux in methanol. d) Buchwald-Hartwig cross coupling between
compound of general formula XI and compounds of general formula
R.sup.2--X where X=Bromo or Iodo is performed under customary
Buchwald-Hartwig conditions using such a ligand such as X-Phos or
2-di-t-butylphosphino-2'-(N,N-dimethylamino)biphenyl with a
palladium catalyst such as Pd.sub.2(dba).sub.3 or
Pd.sub.2(dba).sub.3.CHCl.sub.3 or Pd(OAc).sub.2, preferably
Pd.sub.2(dba).sub.3 with X-Phos, base such as preferably
Cs.sub.2CO.sub.3 or preferably tert-BuONa, and organic solvent such
as preferably dioxane or preferably THF. The reaction is preferably
stirred at a temperature of approximately 80-150.degree. C.,
preferably 120.degree. C. The reaction may preferably be carried
out under an inert gas such as nitrogen or argon.
##STR00049##
[0600] Compounds of general formula XVII can be prepared in a
similar manner as described for steps a-e in Scheme 1, starting
from 6-benzyl-4-chloro-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine
(I) and tert-butyl 3-hydroxyazetidine-1-carboxylate (XII).
##STR00050##
[0601] a)
(S)-3-(6-Benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-ylox-
y)-pyrrolidine-1-carboxylic acid tert-butyl ester XIX is firstly
prepared by reacting
6-benzyl-4-chloro-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine with
(S)-3-Amino-pyrrolidine-1-carboxylic acid tert-butyl ester in the
presence of a suitable base such as triethylamine or
N,N-diisopropylethylamine at elevated temperature (e.g. 120.degree.
C.) for 24-48 h. Typical conditions comprise of 1.0 eq. of
6-benzyl-4-chloro-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine, 1.0
eq. of (S)-3-amino-pyrrolidine-1-carboxylic acid tert-butyl ester
and 1.5 eq. of triethylamine at 120.degree. C. for 48 h. b) Removal
of the benzyl protecting group is performed using standard
methodology as described in "Protecting groups in Organic
Synthesis" by T.W. Greene and P. Wutz, 3.sup.rd edition, 1999, John
Wiley and Sons. Typical conditions comprise of 1.0 eq. of
(S)-3-(6-benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrol-
idine-1-carboxylic acid tert-butyl ester XIX, 1.1-8.0 eq. of
ammonium formate and 20% (w/w) palladium hydroxide Pd(OH).sub.2/C
(catalyst) heated under reflux in methanol. c)
(S)-3-(5,6,7,8-Tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidine-1-c-
arboxylic acid tert-butyl ester XX is reacted with halide
R.sup.2--X (where R2 is defined above and X is halo and preferably
bromo or iodo), in the presence of a suitable base such as sodium
tert-butoxide or cesium carbonate and a suitable catalyst system
such as Pd.sub.2(dba).sub.3 with
2-di-t-butylphosphino-2'-(N,N-dimethylamino)biphenyl or
Pd.sub.2(dba).sub.3 with X-Phos in a suitable solvent such as
anhydrous tert-butanol or anhydrous dioxane, heated at elevated
temperature (e.g. 100.degree. C.). The reaction may preferably be
carried out under an inert gas such as nitrogen or argon. Typical
conditions comprise of 1 eq. of
(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidine--
1-carboxylic acid tert-butyl ester XX, 1-1.5 eq. of R2-X, 1.5-2.0
eq. of sodium tert-butoxide, 5-10 mol % Pd.sub.2(dba).sub.3 and
5-10 mol % 2-di-t-butylphosphino-2'-(N,N-dimethylamino)biphenyl in
anhydrous tert-butanol at 100.degree. C. for 5-24 hours under an
atmosphere of argon. d) N-Boc deprotection is performed under
customary Boc deprotection conditions with a suitable acid such as
trifluoroactetic acid in a suitable solvent such as
CH.sub.2Cl.sub.2 at room temperature. Typical conditions comprise
of 1 eq. of compound of general formula XII in excess
trifluoroacetic acid in CH.sub.2Cl.sub.2 at room temperature for
1-3 h. e) Reaction of compounds of general formula XXII with an
acid chloride of formula R.sup.4C(O)Cl or carboxylic acid of
formula R.sup.4C(O)OH using general methods i-v as described in
Scheme 1, step e. Those skilled in the art will appreciate that
there are many known ways of preparing amides. For example, see
Mantalbetti, C.A.G.N and Falque, V., Amide bond formation and
peptide coupling, Tetrahedron, 2005, 61(46), pp 10827-10852 and
references cited therein. The examples provided herein are thus not
intended to be exhaustive, but merely illustrative.
##STR00051##
[0602] a) 4-Methoxy-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine (WO
2008/130481, p 47) is reacted with halide R.sup.2--X (where R2 is
defined above and X is halo and preferably bromo or iodo), in the
presence of a suitable base such as cesium carbonate or sodium
tert-butoxide and a suitable catalyst system such as
Pd.sub.2(dba).sub.3 with X-Phos or Pd(OAc).sub.2 with X-Phos in a
suitable solvent such as dioxane or THF, heated at elevated
temperature (e.g. 110.degree. C.). The reaction may preferably be
carried out under an inert gas such as nitrogen or argon. Typical
conditions comprise of 1 eq. of
4-methoxy-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine, 1-1.5 eq. of
R2-X, 1.5-2.0 eq. of cesium carbonate, 5-10 mol %
Pd.sub.2(dba).sub.3 and 5-10 mol % X-Phos in dioxane at 110.degree.
C. for 5-24 hours under an atmosphere of argon. b) Compounds of
general formula XXV is reacted with aqueous sodium hydroxide in a
suitable solvent such as methanol or dioxane at elevated
temperature (e.g. 100.degree. C.) for 18-24 h. Typical conditions
comprise of 1 eq. of compounds of general formula XXV in excess 2N
sodium hydroxide(aq) in methanol at 100.degree. C. for 18 h. c)
Compounds of general formula XXI can be prepared using a base
promoted phosphonium coupling reaction whereby compounds of general
formula XXVI in a suitable solvent such as acetonitrile is reacted
with a phosphonium salt such as
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP) in the presence of a base such as
1,8-diaza-7-bicyclo[5.4.0]undecene (DBU) followed by addition of
(S)-tert-butyl 3-aminopyrrolidine-1-carboxylate. The reaction
mixture is preferably stirred at a temperature of 20.degree. C. to
90.degree. C. for 18-72 h. The reaction may preferably be carried
out under an inert gas, e.g. nitrogen or argon. Typical conditions
comprise of 1 equivalent of compounds of general formula XXVI,
1.0-1.5 eq. of BOP, 2.0-4.0 eq. of DBU and 2.0-3.0 eq. of
(S)-tert-butyl 3-aminopyrrolidine-1-carboxylate in acetonitrile at
65.degree. C. for 72 hours under argon. Steps d) and e) can be
carried out in a similar manner described for steps d) and e) in
Scheme 1. Step f) can be carried out using a base promoted
phosphonium coupling reaction in a similar manner as step c) in
Scheme 5. Typical conditions comprise of 1 eq. of compounds of
general formula XXVI, 1.0-1.5 eq. of BOP, 2.0-4.0 eq. of DBU and
2.0-3.0 eq. of amine of general formula XVII in acetonitrile at
90.degree. C. for 24 hours under argon.
##STR00052##
a) Alcohol of general formula XXVIII is reacted with the
6-benzyl-4-chloro-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine under
customary conditions by deprotonation of the secondary alcohol
using sodium hydride (NaH) and organic solvent THF under inert gaz
conditions at room temperature. b) N-debenzylation is performed
under customary transfer hydrogenation conditions, using among the
possible palladium catalysts, preferably palladium hydroxide
Pd(OH).sub.2 and among the possible formate salt preferably
ammonium formate and organic solvent such as preferably methanol.
The reaction is preferably carried out under refluxing conditions.
c) Buchwald-Hartwig cross coupling between compound of general
formula XI and compounds of general formula R.sup.2--X is performed
under customary Buchwald-Hartwig conditions using such a ligand
such as X-Phos or
2-di-t-butylphosphino-2'-(N,N-dimethylamino)biphenyl with a
palladium catalyst such as Pd.sub.2(dba).sub.3 or
Pd.sub.2(dba).sub.3.CHCl.sub.3 or Pd(OAc).sub.2, preferably
Pd.sub.2(dba).sub.3 with X-Phos, base such as preferably
Cs.sub.2CO.sub.3 or preferably tert-BuONa, and organic solvent such
as preferably dioxane or preferably THF. The reaction is preferably
stirred at a temperature of approximately 80-150.degree. C.,
preferably 120.degree. C. The reaction may preferably carried out
under an inert gas such as nitrogen or argon.
##STR00053##
[0603] a) Compounds of general formula VI is reacted with phosgene
in a suitable solvent such as CH.sub.2Cl.sub.2 in the presence of a
suitable base such as triethylamine or N,N-diisopropylethylamine at
a temperature of 0.degree. C. to 25.degree. C. for 1-2 hours. The
reaction may preferably be carried out under an inert gas such as
nitrogen or argon. Typical conditions comprise 1.0 eq. of compound
of general formula VI, 1.0-5.0 eq. of phosgene, 3.0-4.0 eq. of
triethylamine in CH.sub.2Cl.sub.2 under argon for 1 hour. b)
Compound of general formula XXIX is reacted with amine R5R6NH in
the presence of a suitable base such as triethylamine or
N,N-diisopropylethylamine in a suitable solvent such as
CH.sub.2Cl.sub.2 or N,N-dimethylformamide at a temperature of
10.degree. C. to 30.degree. C. for 1-18 h. The reaction may
preferably be carried out under an inert gas such as nitrogen or
argon. Typical conditions comprise 1.0 eq. of compound of general
formula XXIX, 1.0-1.2 eq. of R.sup.5R.sup.6NH, 3.0-4.0 eq. of
triethylamine in CH.sub.2Cl.sub.2 under argon for 2 hours. c)
Compounds of general formula VI is reacted with carbamoyl chloride
R.sup.5R.sup.6NCOCl in the presence of a suitable base such as
triethylamine or N,N-diisopropylethylamine in a suitable solvent
such as CH.sub.2Cl.sub.2 or N,N-dimethylformamide at a temperature
of 0.degree. C. to 25.degree. C. for 1-18 hours. The reaction may
preferably be carried out under an inert gas such as nitrogen or
argon. Typical conditions comprise 1.0 eq. of compound of general
formula VI, 1.0-1.2 eq. of R.sup.5R.sup.6NCOCl, 3.0-4.0 eq. of
triethylamine in CH.sub.2Cl.sub.2 under argon for 18 hours. d)
Compounds of general formula VI is reacted with compounds of
general formula XXXI in the presence of a suitable base such as
triethylamine or N,N-diisopropylethylamine in a suitable solvent
such as CH.sub.2Cl.sub.2 or N,N-dimethylformamide at a temperature
of 0.degree. C. to 25.degree. C. for 1-18 hours. The reaction may
preferably be carried out under an inert gas such as nitrogen or
argon. Typical conditions comprise 1.0 eq. of compound of general
formula VI, 1.0-1.2 eq. of compound of general formula XXXI,
1.0-2.0 eq. of triethylamine in CH.sub.2Cl.sub.2 under argon for 18
hours. e) Compounds of general formula VI is reacted with compounds
of formula R.sup.7OCOCl in the presence of a suitable base such as
triethylamine or N,N-diisopropylethylamine in a suitable solvent
such as CH.sub.2Cl.sub.2 or N,N-dimethylformamide at a temperature
of 0.degree. C. to 25.degree. C. for 1-18 hours. The reaction may
preferably be carried out under an inert gas such as nitrogen or
argon. Typical conditions comprise 1.0 eq. of compound of general
formula VI, 1.0-1.2 eq. of compounds of general formula
R.sup.7OCOCl, 3.0-4.0 eq. of triethylamine in CH.sub.2Cl.sub.2
under argon for 18 hours.
##STR00054##
[0604] a) Quaternarization of the tertiary amine of general formula
XXXIII (where R.sup.8=alkyl e.g. benzyl) with compound of general
formula R.sup.9--X (where R.sup.9=alkyl e.g. methyl and X=Bromo or
Iodo) under customary conditions using in particular acetone as
organic solvent. b) Alkylation of amine of general formula
R.sup.2--NH2 with quaternary amine XXXIV was performed by using
base such a in particular K.sub.2CO.sub.3 and organic solvent such
as in particular a 2/1 mixture of ethanol and water and heating the
reaction mixture at 80-100.degree. C., in particular 80.degree. C.
c) Compound of general formula XXXV was reacted with base such as
in particular NaH and compound of general formula
(R.sup.10O).sub.2CO (where R.sup.10=alkyl e.g. carbonic acid
dimethyl ester). The reaction mixture is stirred under high
temperature (90.degree. C.). d) Pyrimidine ring formation was
obtained by reacting the compound of general formula XXXVI with
formamidine acetate with a base such as sodium methoxide and
organic solvent such as methanol at elevated temperature such as
90.degree. C. for 2-18 h. e) Compound of general formula XXVI was
reacted with phosphoryl chloride in presence of base such as
triethylamine in organic solvent such as toluene at elevated
temperature such as 100.degree. C. for 12-18 h. f) Alcohol of
general formula XXVIII is reacted with Compound of general formula
XXXVII under customary conditions by deprotonation of the secondary
alcohol using sodium hydride (NaH) and organic solvent THF under
inert gas conditions at room temperature.
[0605] Where it is stated that compounds were prepared in the
manner described for an earlier example, the skilled person will
appreciate that reaction times, number of equivalents of reagents
and reaction temperatures may be modified for each specific
reaction, and that it may nevertheless be necessary or desirable to
employ different work-up or purification conditions.
##STR00055##
Example 1:
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-
-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone
Synthesis of Example 1--Method 1a (According to Scheme 8)
[0606] Sodium hydride (60% in dispersion oil, 17.88 mg, 0.447 mmol)
was added under argon to a solution of intermediate 3 (75 mg, 0.378
mmol) in 2 mL of dry THF. The suspension was stirred under an
atmosphere of argon at ambient temperature for 15 min.
4-Chloro-6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,-
3-d]pyrimidine (100 mg, 0.344 mmol) was added and stirred at rt for
an additional 3 hours. The reaction mixture was quenched with
H.sub.2O, extracted with CH.sub.2Cl.sub.2. The organic layer was
washed with brine, dried over Na.sub.2SO.sub.4, filtered and
evaporated to dryness. Purification by flash-chromatography on
silica gel (CH.sub.2Cl.sub.2/MeOH 95/5) gave
{(S)-3[6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3--
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone
as a light yellow gum (115 mg, 74% yield). .sup.1H-NMR (400 MHz,
methanol-d.sub.4, 298K) .delta. ppm 1.59-1.87 (m, 4H) 2.20 (s, 3H)
2.27-2.43 (m, 2H) 2.74-2.91 (m, 1H) 2.97-3.03 (m, 2H) 3.42-4.14 (m,
15H) 5.75-5.86 (m, 1H) 7.39-7.43 (m, 1H) 7.63-7.68 (m, 1H)
8.57-8.61 (m, 1H). LCMS: [M+H].sup.+=454.2, Rt.sup.(3)=1.46
min.
##STR00056##
4-Chloro-6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,-
3-d]pyrimidine
[0607] A mixture of
6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrim-
idin-4-ol (650 mg, 2.387 mmol), phosphoroxy chloride (0.334 mL,
3.58 mmol), triethylamine (0.665 mL, 4.77 mmol) and toluene (12 mL)
was heated at 100.degree. C. for 16 h. The mixture was neutralized
with the addition of solid sodium bicarbonate, filtered and the
solution was concentrated in vacuum. The remaining black residue
was taken up in CH.sub.2Cl.sub.2 and water, the layers were
separated and the organic phase washed with brine, dried over
sodium sulfate, filtered and concentrated to give a dark brown
solid. The solid was triturated in ethylacetate, filtered and dried
under high vacuum to yield
4-chloro-6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,-
3-d]pyrimidine (630 mg, 91% yield) as a tan solid. .sup.1H-NMR (400
MHz, DMSO-d6, 298K) .delta. ppm 2.15 (s, 3H) 3.03 (t, 2H) 3.53 (t,
2H) 3.82 (s, 3H) 4.26 (s, 2H) 7.49 (dd, 1H) 7.74 (d, 1H) 8.85 (s,
1H). LCMS: [M+H].sup.+=291.1, Rt.sup.(4)=0.97 min.
##STR00057##
6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrim-
idin-4-ol
[0608] A mixture of
6'-methoxy-5'-methyl-4-oxo-3,4,5,6-tetrahydro-2H-[1,3']bipyridinyl-3-carb-
oxylic acid methyl ester (900 mg, 3.23 mmol), formamidine acetate
(521 mg, 4.85 mmol), sodium methoxide (5.4 Molar) in methanol
(2.395 mL, 12.94 mmol) and methanol (4 mL) was heated to 90.degree.
C. for 3 h. The mixture was allowed to cool down to rt, diluted in
CH.sub.2Cl.sub.2, neutralized with acetic acid (0.741 mL, 12.94
mmol) and quenched with H.sub.2O. The layers were separated and
aqueous was washed twice with CH.sub.2Cl.sub.2, organics were
combined, washed with brine, dried over sodium sulfate, filtered
and evaporated to give a yellow solid. The solid was triturated in
ethylacetate to yield
6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrim-
idin-4-ol (669 mg, yield 76%) as a white powder. .sup.1H-NMR (400
MHz, DMSO-d6, 298K) .delta. ppm 2.14 (s, 3H) 2.72 (t, 2H) 3.39 (t,
2H) 3.81 (s, 3H) 3.90 (s, 2H) 7.42 (d, 1H) 7.67 (d, 1H) 8.07 (s,
1H) 12.46 (br.s., 1H). LCMS: [M+H].sup.+=273.1, Rt.sup.(3)=1.30
min.
##STR00058##
6'-Methoxy-5'-methyl-4-oxo-3,4,5,6-tetrahydro-2H-[1,3']bipyridinyl-3-carb-
oxylic acid methyl ester
[0609] To a stirred suspension of sodium hydride (60%, 153 mg, 6.36
mmol) in dimethyl carbonate (3.82 mL, 45.4 mmol) at room
temperature was slowly added
6'-methoxy-5'-methyl-2,3,5,6-tetrahydro-[1,3']bipyridinyl-4-one (1
g, 4.54 mmol). The reaction mixture was heated to reflux
(90.degree. C.) for 1 h and then cooled to room temperature. The
mixture was partitioned between CH.sub.2Cl.sub.2 and water and a
solution of 1N HCl was added cautiously. The aqueous layer was
separated and washed with an addition portion of CH.sub.2Cl.sub.2.
The combined organic extracts were washed with brine, dried over
sodium sulfate, filtered and evaporated to give the crude product,
which was purified by flash-chromatography on silica gel
(heptane/ethylacetate 3/1) to afford
6'-methoxy-5'-methyl-4-oxo-3,4,5,6-tetrahydro-2H-[1,3']bipyridinyl-3-carb-
oxylic acid methyl ester (975 mg, yield 77%) as a white solid.
.sup.1H-NMR (400 MHz, DMSO, 298K) (mixture of keto and enol
tautomers observed) .delta. ppm 2.12 (s, 6H) 2.36-2.69 (m, 4H)
3.26-3.96 (m, 20H) 7.34-7.77 (m, 4H) 11.84 (s, 1H). LCMS:
[M+H].sup.+=279.1, Rt.sup.(3)=1.51 min (tautomer 1) and 1.70 min
(tautomer 2).
##STR00059##
6'-Methoxy-5'-methyl-2,3,5,6-tetrahydro-[1,3']bipyridinyl-4-one
[0610] A slurry of iodide salt 1-benzyl-1-methyl-4-oxo-piperidinium
(Ref: Tortolani, R.; Org. Lett., Vol. 1, No 8, 1999) (3.61 g, 10.86
mmol) in water (10 mL) was added slowly to a refluxing solution of
2-methoxy-5-amino-3-picolin (1 g, 7.24 mmol) and potassium
carbonate (0.140 g, 1.013 mmol) in ethanol (20 mL). The reaction
mixture was heated to reflux for an additional 3 h. The reaction
mixture was cooled to rt and partitioned between CH.sub.2Cl.sub.2
and water. The organic layer was separated and washed with an
addition portion of CH.sub.2Cl.sub.2. The combined organic layers
were washed with brine, dried over sodium sulfate, filtered and
concentrated to give the crude product which was purified by
flash-chromatography on silica gel (heptane/ethylacetate 1/1) to
afford
6'-methoxy-5'-methyl-2,3,5,6-tetrahydro-[1,3']bipyridinyl-4-one
(1.15 g, yield 72%) as a light yellow gum. .sup.1H-NMR (400 MHz,
DMSO, 298K) .delta. ppm 2.12 (s, 3H) 2.42 (t, 4H) 3.46 (t, 4H) 3.80
(s, 3H) 7.40 (d, 1H) 7.71 (d, 1H). LCMS: [M+H].sup.+=221.1,
Rt.sup.(3)=1.41 min.
Synthesis of Example 1--Method 1b (According to Scheme 1)
Step 3
[0611] To a mixture
6-(6-methoxy-5-methyl-pyridin-3-yl)-4-((S)-pyrrolidin-3-yloxy)-5,6,7,8-te-
trahydro-pyrido[4,3-d]pyrimidine (639 mg, 1.87 mmol) in
CH.sub.2Cl.sub.2 (5 mL) was added the acid chloride
tetrahydo-2H-pyran-4-carbonyl chloride (306 mg, 2.06 mmol) and
triethylamine (0.522 mL, 3.74 mmol) at rt. The reaction mixture was
stirred at rt for 10 min. The reaction mixture was concentrated
under vacuum. Purification by preparative reverse phase Gilson HPLC
and subsequent neutralization of the combined fractions by
extraction with CH.sub.2Cl.sub.2/1N NaOH, separation of the organic
phase through a phase separation tube and evaporated gave
{(S)-3-[6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-
-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone
(432 mg, 51% yield) as a white powder. .sup.1H-NMR (400 MHz,
DMSO-d6, 298K) .delta. ppm 1.50-1.65 (m, 4H) 2.10-2.32 (m, 5H)
2.62-2.78 (m, 1H) 2.85-2.95 (m, 2H) 3.30-3.95 (m, 13H) 4.0-4.20 (m,
2H) 5.61-5.72 (m, 1H) 7.42 (br, 1H) 7.68 (m, 1H) 8.60-8.61 (m, 1H).
LCMS: [M+H].sup.+=454.2, Rt.sup.(1)=1.42 min.
##STR00060##
6-(6-Methoxy-5-methyl-pyridin-3-yl)-4-((S)-pyrrolidin-3-yloxy)-5,6,7,8-te-
trahydro-pyrido[4,3-d]pyrimidine
Step 2
[0612]
(S)-3[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido-
[4,3-d]pyrimidin-4-yloxy]-pyrrolidine-1-carboxylic acid tert-butyl
ester (2.05 g, 4.63 mmol) was dissolved in TFA/CH.sub.2Cl.sub.2
(1/2) and stirred at rt for 1 h. The reaction mixture was
concentrated under vacuum, the residue was diluted with
CH.sub.2Cl.sub.2, the organic layer washed with NaOH 1N then brine,
dried over Na.sub.2SO.sub.4, filtered and evaporated to give
6-(6-methoxy-5-methyl-pyridin-3-yl)-4-((S)-pyrrolidin-3-yloxy)-5,6,7,8-te-
trahydro-pyrido[4,3-d]pyrimidine. .sup.1H NMR (400 MHz, CDCl.sub.3,
298K) .delta. ppm 2.20-2.30 (m, 2H), 2.22 (s, 3H), 3.00-3.06 (t,
2H), 3.09-3.18 (m, 1H), 3.22-3.37 (m, 3H), 3.45-3.50 (t, 2H), 3.95
(s 3H), 4.10 (s, 2H), 4.20-4.65 (br.s 1H), 5.63-5.69 (m, 1H),
7.21-7.252 (m, 1H), 7.70-7.74 (m, 1H), 8.60 (s, 1H). LCMS:
[M+H].sup.+=341.9, Rt.sup.(7)=0.61 min.
##STR00061##
(S)-3[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d-
]pyrimidin-4-yloxy]-pyrrolidine-1-carboxylic acid tert-butyl
ester
Step 1
[0613] X-Phos (0.96 g, 2.01 mmol, 0.3 eq.), Pd.sub.2(dba).sub.3
(0.615 g, 0.672 mmol, 0.1 eq.), Cs.sub.2CO.sub.3 (4.38 g, 13.44
mmol, 2 eq.) were combined and flushed 10 min with Argon. To this
mixture, a solution of
(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidine-1-c-
arboxylic acid tert-butyl ester (intermediate 7) (2.15 g, 6.72
mmol) in dioxane (6 mL) and 5-bromo-2-methoxy-3-methylpyridine
(1.76 g, 8.73 mmol) were added at rt and the reaction mixture was
stirred at 120.degree. C. for 2 h. The reaction was cooled down to
rt, the reaction mixture filtered over Hyflo, AcOEt was added and
the organic layer was washed with brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The
residue was dissolved in dioxane (6 mL) and added to a glass vial
containing 5-bromo-2-methoxy-3-methylpyridine (1.76 g, 8.73 mmol),
X-Phos (0.96 g, 2.01 mmol), Pd.sub.2(dba).sub.3 (0.615 g, 0.672
mmol), Cs.sub.2CO.sub.3 (4.38 g, 13.44 mmol). The vial was capped
and the reaction mixture was stirred at 120.degree. C. for 2 h. The
reaction was cooled down to rt, the reaction mixture filtered over
Hyflo, AcOEt was added and the organic layer was washed with brine,
dried over Na.sub.2SO.sub.4, filtered and concentrated under
vacuum. Purification by flash chromatography on silica gel
(CH.sub.2Cl.sub.2 then TBME then TBME/MeOH 99/1 to 90/10) gave
(S)-3[6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d-
]pyrimidin-4-yloxy]-pyrrolidine-1-carboxylic acid tert-butyl ester
as a yellow foam (2.05 g, 69% yield). .sup.1H NMR (400 MHz,
DMSO-d6, 298K) .delta. ppm 1.35-1.44 (br.s., 9H) 2.07-2.23 (m, 2H),
2.14 (s, 3H), 2.87-2.93 (m, 2H), 3.39-3.68 (m, 6H), 3.81 (s, 3H),
4.03-4.08 (m, 2H), 5.56-5.63 (m, 1H), 7.41-7.46 (m, 1H), 7.67-7.73
(m, 1H), 8.60 (s, 1H). LCMS: [M+H].sup.+=342.2, Rt.sup.(2)=0.94
min.
Crystallization of Example 1 by Heating and Cooling in
Acetonitrile
[0614] 1 part of Example 1 (eg. 100 mg) was mixed with 5 parts of
acetonitrile (0.5 mL for each 100 mg of compound) with stirring. A
solution was obtained by heating up to 40-60.degree. C. The mixture
was then allowed to slowly cooled down to RT. After further cooling
overnight (5.degree. C.), precipitation was observed. In case no
precipitation is not observed, the volume of ethanol can be reduce
using a nitrogen stream and repeating the overnight cooling step.
The mixture was centrifuged to remove the ethanol. The solid was
dried under vacuum at 25.degree. C. and 70 mbar. A crystalline
anhydrous form of Example 1 with a MP of 131.degree. C. was
obtained. This crystalline form was also observed under other
methods and/or solvents, such as heating and cooling in ethanol,
acetone, ethyl acetate, isopropanol, by slurry in heptane, or by
antisolvent addition in THF or 3-methyl-1-butanol using heptane as
antisolvent. These results show the reproducibility and scalability
of the crystalline form as well as suggests that the same form can
be prepared under different experimental conditions than the ones
described above.
[0615] List of most significant peaks from X-ray Powder Diffraction
Pattern of Example 1 anhydrous form (Method X2):
TABLE-US-00003 2-Theta in deg Intensity in % 7.5 56 10.9 12.5 11.7
25.1 14.3 23.8 15.1 100 15.8 40.9 16.7 22.1 17.7 65.1 18.9 28.9
20.5 24.7 21.8 26 22.5 28.3 23.3 31.3 24.2 76.1 24.6 51.8 25.0 41.3
25.6 20.4 26.2 20.8 27.0 14.2 28.0 17.5 29.1 16.1 32.8 14 34.6
11.4
Crystallization of Trihydrate Form of Example 1 by Slurry in
Water
[0616] Slurry of Example 1 in water e.g., 1 part of Example 1 in 10
parts of water, at RT produced a trihydrate form of Example 1. The
crystals were separated by centrifugation and dried at room
environment.
[0617] List of most significant peaks from X-ray Powder Diffraction
Pattern of Example 1 trihydrate form (Method X2):
TABLE-US-00004 2-Theta in deg Intensity in % 6.6 24.3 8.9 7.9 13.3
100 14.5 18.3 15.0 12.6 16.5 12.4 17.5 15.7 17.7 17.2 18.2 9.8 20.0
10.7 21.6 11.7 22.6 20.3 23.8 11.4 24.4 15.2 26.7 26.5 27.5 18.7
27.8 16.6 29.2 9.8 33.3 9 33.9 7.6 35.7 8.2 38.8 7
Preparation of Citrate Salt of Example 1
[0618] 0.5 g of Example 1 (assay 91.8%) were dissolved in 5 mL of
methylethylketone and 0.25 mL of water and heated at 60.degree. C.
213 mg of citric acid were added at 50.degree. C. and the mixture
was allowed to cool down to RT within 30 min. Crystallization
occurs at 45.degree. C. The mixture was stirred for 16 h at RT. The
crystals were collected by filtration. The filter cake was washed 3
times with 1 mL of methylethylketone and afterwards dried for 16 h
at 50.degree. C. and ca. 10 mbar vacuum. Elementary analysis of the
citrate salt showed a 1:1 (monohydrate) form. List of most
significant peaks from X-ray Powder Diffraction Pattern of Example
1 citrate salt (Method X1):
TABLE-US-00005 2-Theta in deg Intensity in % 5.7 62 11.5 100 12.1 4
14.3 4 15.4 12 17.2 21 17.9 31 19.3 25 20.2 37 20.7 8 21.9 5 23.3
11 23.9 36 25.5 28 27.0 5 27.7 6 29.8 8 30.3 7
Preparation of Fumarate Salt of Example 1
[0619] 0.5 g Example 1 (assay 91.8%) were dissolved in 15 mL of
acetonitrile and 0.2 mL of water and heated at 76.degree. C. 129 mg
of fumaric acid were added at 60.degree. C. The solution was
allowed to cool down to RT within 30 min. The salt precipitated and
the suspension was stirred for 16 h at RT. The crystals were
collected by filtration. The filter cake was washed 3 times with 1
mL of acetonitrile and afterwards dried for 16 h at 50.degree. C.
and ca. 10 mbar vacuum. Elementary analysis of the fumarate salt
showed a 1:1 (monohydrate) form.
[0620] List of most significant peaks from X-ray Powder Diffraction
Pattern of Example 1 fumarate salt (Method X1):
TABLE-US-00006 2-Theta in deg Intensity in % 6.0 100 6.5 12 9.8 8
12.3 10 13.1 14 15.6 22 17.7 16 19.1 21 19.7 27 23.9 40 24.7 6 24.9
10 25.2 5 26.4 11 27.0 4
Preparation of Napadisylate Salt of Example 1
[0621] 0.5 g Example 1 (assay 91.8%) were dissolved in 5 mL of
ethanol absolute and 0.25 mL of water at 60.degree. C. 250 mg of
naphthalendisulfonic acid were added at 50.degree. C. and the
mixture was allowed to cool down to RT within 30 min.
Crystallization occurs at 40.degree. C. The mixture was stirred for
16 h at RT. The crystals were collected by filtration. The filter
cake was washed 3 times with 1 mL of ethanol and afterwards dried
for 16 h at 50.degree. C. and ca. 10 mbar vacuum. Elementary
analysis of the napadisylate salt showed a 2:1 (monohydrate) form.
List of most significant peaks from X-ray Powder Diffraction
Pattern of Example 1 napadisylate salt (Method X1):
TABLE-US-00007 2-Theta in deg Intensity in % 4.3 100 8.5 3 9.4 6
12.2 12 12.9 12 13.5 37 15.0 26 15.6 12 16.0 11 17.7 28 18.9 23
19.3 11 20.0 11 20.8 3 21.2 5 22.0 9 23.0 41 24.5 39 26.5 20
[0622] Examples 2-9 were prepared using procedures analogous to
those used in Example 1 (method 1b) using appropriate starting
materials.
TABLE-US-00008 Rt.sup.(1) (min.) MS: [M + H]+ Example 2
##STR00062## 471.3 1.21 Name:
{(S)-3-[6-(2,4-Dimethoxy-pyrimidin-5-yl)-5,6,7,8-tetrahydro-pyrido[4-
,3-d]pyrimidin-4-
yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone
Purification method: Reverse phase method C Prepared using process
step 3 of method 1b from intermediate 8 and tetrahydro-pyran-4-
carbonyl chloride .sup.1H NMR (400 MHz, methanol-d4, 298K) .delta.
ppm 1.50-1.86 (m, 4H) 2.20-2.45 (m, 2H) 2.70- 2.87 (m, 1H)
2.96-2.99 (m, 2H) 3.35-4.14 (m, 18H) 5.69-5.85 (m, 1H) 7.96 (m, 1H)
8.58 (m, 1H) Rt.sup.(1) (min.) MS: [M + H]+ Example 3 ##STR00063##
1.42 465.2 Name:
2-Methoxy-5-{4-[(S)-1-(tetrahydro-pyran-4-carbonyl)-pyrrolidin-3-ylo-
xy]-7,8-dihydro- 5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile
Purification method: Reverse phase method A Prepared using process
steps 2-3 of method 1b from intermediate 11 and tetrahydro-
pyran-4-carbonyl chloride .sup.1H NMR (400 MHz, DMSO-d6, 298K)
.delta. ppm 1.50-1.64 (m, 4H) 2.10-2.31 (m, 2H) 2.62- 2.77 (m, 1H)
2.87-2.95 (m, 2H) 3.29-3.96 (m, 13H) 4.08-4.21 (m, 2H) 5.58-5.73
(m, 1H) 8.06-8.09 (m, 1H) 8.23-8.27 (m, 1H) 8.60-8.64 (m, 1H)
Rt.sup.(1) (min.) MS: [M + H]+ Example 4 ##STR00064## 1.27 414.2
Name:
1-{(S)-3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4-
,3-d]pyrimidin-4- yloxy]-pyrrolidin-1-yl}-propan-1-one Purification
method: Reverse phase method A Prepared using process steps 2-3 of
method 1b from intermediate 10 and propionyl chloride .sup.1H NMR
(400 MHz, CDCl.sub.3-d, 298K) .delta. ppm 1.10-1.20 (m, 3H)
2.19-2.49 (m, 4H) 3.02-3.08 (m, 2H) 3.45-3.52 (m, 2H) 3.56-3.68 (m,
2H) 3.72-3.90 (m, 2H) 3.91 (s, 3H) 3.99 (s, 3H) 4.07-4.12 (m, 2H)
5.75-5.78 (m, 1H) 6.89-7.01 (m, 1H) 7.44-7.46 (m, 1H) 8.60-8.62 (m,
1H) Rt.sup.(1) (min.) MS: [M + H]+ Example 5 ##STR00065## 1.25
470.2 Name:
{(S)-3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-
-d]pyrimidin-4-
yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone
Purification method: Reverse phase method A Prepared using process
steps 2-3 of method 1b from intermediate 10 and tetrahydro-
pyran-4-carbonyl chloride .sup.1H NMR (400 MHz, CDCl.sub.3-d, 298K)
.delta. ppm 1.56-1.68 (m, 2H) 1.88-2.00 (m, 2H) 2.20-2.38 (m, 2H)
2.53-2.70 (m, 1H) 3.05-3.10 (m, 2H) 3.39-3.54 (m, 4H) 3.59-3.82 (m,
4H) 3.91 (s, 3H) 3.99 (s, 3H) 4.01-4.10 (m, 4H) 5.62-5.78 (m, 1H)
6.89-6.90 (m, 1H) 7.40-7.43 (m, 1H) 8.60-8.65 (m, 1H) Rt.sup.(1)
(min.) MS: [M + H]+ Example 6 ##STR00066## 1.10 450.2 Name:
2-Amino-5-{4-[(S)-1-(tetrahydro-pyran-4-carbonyl)-pyrrolidin-3-yloxy-
]-7,8-dihydro- 5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile
Prepared using process step 3 of method 1b from intermediate 9 and
tetrahydro-pyran-4- carbonyl chloride Purification method: 1-Normal
phase chromatography CH.sub.2Cl.sub.2/MeOH as solvent 2-Reverse
phase method A .sup.1H NMR (400 MHz, CDCl.sub.3-d, 298K) .delta.
ppm 1.45-1.75 (m, 2H) 1.86-2.02 (m, 2H) 2.20-2.40 (m, 2H) 2.50-2.75
(m, 1H) 3.02-3.09 (m, 2H) 3.38-4.20 (m, 12H) 4.96 (s, 1H) 5.70-5.78
(m, 1H) 7.39 (m, 1H) 8.13-8.14 (m, 1H) 8.62-8.64 (m, 1H) Rt.sup.(1)
(min.) MS: [M + H]+ Example 7 ##STR00067## 1.41 458.1 Name:
{(S)-3-[6-(5-Fluoro-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone
Purification method: Biotage 11 g KP--NH cartridge eluting with
Heptane/EtOAc 100/0 to 0/100 Prepared using process steps 2-3 of
method 1b from intermediate 12 and tetrahydro- pyran-4-carbonyl
chloride .sup.1H NMR (400 MHz, CDCl.sub.3, 298K) .delta. ppm
1.56-1.74 (m, 2H) 1.87-2.02 (m, 2H) 2.19-2.42 (m, 2H) 2.51-2.74 (m,
1H) 3.01-3.09 (m, 2H) 3.39-4.20 (m, 15H) 5.70-5.79 (m, 1H) 7.13-
7.20 (m, 1H) 7.63-7.69 (m, 1H) 8.59-8.66 (m, 1H) Rt.sup.(1) (min.)
MS: [M + H]+ Example 8 ##STR00068## 1.35 425.1 Name:
2-Methoxy-5-{4-[(S)-1-(2-methoxy-acetyl)-pyrrolidin-3-yloxy]-7,8-dih-
ydro-5H- pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile Purification
method: Reverse phase method A Prepared using process steps 2-3 of
method 1b from intermediate 11 and methoxy acetyl chloride .sup.1H
NMR (400 MHz, DMSO-d6, 298K) .delta. ppm 2.11-2.32 (m, 2H)
2.88-2.95 (m, 2H) 3.26- 3.32 (m, 3H) 3.46-3.84 (m, 6H) 3.91-3.95
(m, 3H) 3.98-4.08 (m, 2H) 4.13-4.19 (m, 2H) 5.59-5.71 (m, 1H)
8.07-8.10 (m, 1H) 8.25-8.28 (m, 1H) 8.61-8.62 (m, 1H) Rt.sup.(8)
(min.) MS: [M + H]+ Example 9 ##STR00069## 3.79 449.1 Name:
5-[4-((S)-1-Cyclopentanecarbonyl-pyrrolidin-3-yloxy)-7,8-dihydro-5H--
pyrido[4,3- d]pyrimidin-6-yl]-2-methoxy-nicotinonitrile
Purification method: Reverse phase method A Prepared using process
steps 2-3 of method 1b from intermediate 11 and
cyclopentanecarbonyl chloride
##STR00070##
Example 10:
(2,4-Dimethyl-oxazol-5-yl)-{(S)-3[6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6-
,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone
Step 1
[0623] A mixture of 2,4-dimethyl-oxazole-5-carboxylic acid (36.4
mg, 0.258 mmol), HTBU (98 mg, 0.258 mmol), DIPEA (86 .mu.l, 0.49
mmol) in DMF (5 mL) was stirred at rt for 20 min. then a solution
of
6-(6-methoxy-5-methyl-pyridin-3-yl)-4-((S)-pyrrolidin-3-yloxy)-5,6,7,8-te-
trahydro-pyrido[4,3-d]pyrimidine (prepared in example 1, method 1b,
step 2) (80 mg, 0.23 mmol) and DIPEA (86 .mu.l, 0.49 mmol) in DMF
(0.4 mL) was added. The reaction mixture was stirred 30 min at rt.
The reaction mixture was directly purified by preparative reverse
phase Gilson HPLC and subsequent neutralization of the combined
fractions over PL-HCO.sub.3 MP gave
(2,4-dimethyl-oxazol-5-yl)-{(S)-3[6-(6-methoxy-5-methyl-pyridin-3-
-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-m-
ethanone (91 mg, 84% yield) as a white lyophilized powder.
.sup.1H-NMR (400 MHz, methanol-d4, 298K) .delta. ppm 2.17 (s, 3H)
2.27-2.52 (m, 8H) 2.95-3.03 (m, 2H) 3.44-3.55 (m, 2H) 3.70-4.26 (m,
9H) 5.76-5.92 (m, 1H) 7.40 (br. s., 1H) 7.64 (br. s., 1H) 8.55-8.62
(m, 1H), LCMS: [M+H]+=465.2, Rt.sup.(1)=1.51 min.
[0624] Examples 11-49 and 51-53 were prepared using procedures
analogous to those used in Example 10, step 1 using appropriate
starting materials.
TABLE-US-00009 Rt.sup.(1) (min.) MS: [M + H]+ Example 11
##STR00071## 1.57 436.2 Name:
Furan-3-yl-{(S)-3-[6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetra-
hydro- pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone
Purification method: Reverse phase method A Prepared using
furan-3-carboxylic acid .sup.1H NMR (400 MHz, methanol-d4, 298K)
.delta. ppm 2.17 (s, 3H) 2.30-2.45 (m, 2H) 2.93-3.05 (m, 2H)
3.45-3.54 (m, 2H) 3.72-4.21 (m, 9H) 5.79-5.86 (m, 1H) 6.78-6.82 (m,
1H) 7.37- 7.44 (m, 1H) 7.56-7.61 (m, 1H) 7.61-7.69 (m, 1H)
8.01-8.12 (m, 1H) 8.54-8.62 (m, 1H) Rt.sup.(1) (min.) MS: [M + H]+
Example 12 ##STR00072## 1.36 437.2 Name:
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3- d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-oxazol-5-yl-methanone
Purification method: Reverse phase method A Prepared using
oxazole-5-carboxylic acid .sup.1H NMR (400 MHz, methanol-d4, 298K)
.delta. ppm 2.17 (s, 3H) 2.30-2.39 (m, 1H) 2.41-2.50 (m, 1H)
2.95-3.03 (m, 2H) 3.45-3.52 (m, 2H) 3.76-4.32 (m, 9H) 5.79-5.94 (m,
1H) 7.40 (br. s., 1H) 7.62-7.66 (m, 1H) 7.75-7.82 (m, 1H) 8.34-8.40
(m, 1H) 8.56-8.61 (m, 1H) Rt.sup.(1) (min.) MS: [M + H]+ Example 13
##STR00073## 1.13 450.2 Name:
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o
[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(3-methyl-3H-imidazol-4-yl)-met-
hanone Purification method: Reverse phase method A Prepared using
3-methyl-3H-imidazole-4-carboxylic acid .sup.1H NMR (400 MHz,
methanol-d4, 298K) .delta. ppm 2.17 (s, 3H) 2.30-2.45 (m, 2H)
2.93-3.05 (m, 2H) 3.43-3.55 (m, 2H) 3.74-4.24 (m, 12H) 5.82 (br.
s., 1H) 7.35-7.56 (m, 2H) 7.66 (m, 1H) 7.76 (br. s., 1H) 8.55-8.60
(m, 1H) Rt.sup.(1) (min.) MS: [M + H]+ Example 14 ##STR00074## 1.49
451.2 Name:
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(2-methyl-oxazol-4-yl)-methanone
Purification method: Reverse phase method A Prepared using
2-methyl-oxazole-4-carboxylic acid .sup.1H NMR (400 MHz,
methanol-d4, 298K) .delta. ppm 2.17 (s, 3H) 2.27-2.43 (m, 2H)
2.43-2.50 (m, 3H) 2.95-3.02 (m, 2H) 3.45-3.53 (m, 2H) 3.72-4.33 (m,
9H) 5.78-5.89 (m, 1H) 7.37- 7.43 (m, 1H) 7.61-7.67 (m, 1H)
8.25-8.31 (m, 1H) 8.57-8.60 (m, 1H) Rt.sup.(1) (min.) MS: [M + H]+
Example 15 ##STR00075## 1.53 454.2 Name:
(3-Methoxy-cyclobutyl)-{(S)-3-[6-(6-methoxy-5-methyl-pyridin-3-yl)-5-
,6,7,8-
tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone
Purification method: Reverse phase method A Prepared using
3-methoxy-cyclobutanecarboxylic acid .sup.1H NMR (400 MHz,
methanol-d4, 298K) .delta. ppm 1.99-2.55 (m, 9H) 2.78-2.95 (m, 1H),
2.95- 3.02 (m, 2H) 3.20-3.23 (m, 3H) 3.47-3.52 (m, 2H) 3.52-4.10
(m, 10H) 5.73-5.81 (m, 1H) 7.38-7.42 (m, 1) 7.63-7.67 (m, 1H) 8.57
(s, 1H) Rt.sup.(1) (min.) MS: [M + H]+ Example 16 ##STR00076## 1.41
437.2 Name:
({(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyri-
do[4,3- d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-oxazol-4-yl-methanone
Purification method: Reverse phase method A Prepared using
oxazole-4-carboxylic acid .sup.1H NMR (400 MHz, methanol-d4, 298K)
.delta. ppm 2.17 (s, 3H) 2.29-2.37 (m, 1H) 2.37-2.44 (m, 1H)
2.94-3.03 (m, 2H) 3.45-3.53 (m, 2H) 3.75-4.38 (m, 9H) 5.79-5.89 (m,
1H) 7.38- 7.42 (m, 1H) 7.62-7.66 (m, 1H) 8.19-8.26 (m, 1H)
8.44-8.48 (m, 1H) 8.56-8.61 (m, 1H) Rt.sup.(1) (min.) MS: [M + H]+
Example 17 ##STR00077## 1.35 495.2 Name:
1-(4-{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro--
pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidine-1-carbonyl}-piperidin-1-yl)-ethanone
Purification method: Reverse phase method A Prepared using
1-acetyl-piperidine-4-carboxylic acid .sup.1H NMR ((400 MHz,
methanol-d4, 298K) .delta. ppm 1.49-1.89 (m, 4H) 2.06-2.13 (m, 3H)
2.18 (s, 3H) 2.23-2.43 (m, 2H) 2.61-2.93 (m, 2H) 2.95-3.04 (m, 2H)
3.15-3.25 (m, 1H) 3.42-3.53 (m, 2H) 3.55-4.12 (m, 10H) 4.46-4.59
(m, 1H) 5.74-5.86 (m, 1H) 7.38-7.45 (m, 1H) 7.62- 7.67 (m, 1H)
8.56-8.61 (m, 1H) Rt.sup.(1) (min.) MS: [M + H]+ Example 18
##STR00078## 1.47 451.2 Name:
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(4-methyl-oxazol-5-yl)-methanone
Purification method: Reverse phase method A Prepared using
4-methyl-oxazole-5-carboxylic acid .sup.1H NMR (400 MHz,
methanol-d4, 298K) .delta. ppm 2.17 (s, 3H) 2.29-2.47 (m, 5H)
2.95-3.3.03 (m, 2H) 3.45-3.52 (m, 2H) 3.73-4.30 (m, 9H) 5.79-5.90
(m, 1H) 7.41 (m, 1H) 7.65 (br.s., 1H) 8.19-8.24 (m, 1H) 8.55-8.61
(m, 1H) Rt.sup.(1) (min.) MS: [M + H]+ Example 19 ##STR00079## 1.25
463.1 Name:
5-{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyr-
ido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidine-1-carbonyl}-1H-pyridin-2-one
Purification method: Reverse phase method A Prepared using
6-oxo-1,6-dihydro-pyridine-3-carboxylic acid .sup.1H NMR (400 MHz,
methanol-d4, 298K) .delta. ppm 2.19 (s, 3H) 2.30-2.40 (m, 2H)
2.95-3.05 (m, 2H) 3.45-3.55 (m, 2H) 3.74-4.22 (m, 9H) 5.73-5.85 (m,
1H) 6.50-6.56 (m, 1H) 7.39- 7.45 (m, 1H) 7.60-7.70 (m, 1H)
7.78-7.90 (m, 2H) 8.50-8.60 (m, 1H) Rt.sup.(1) (min.) MS: [M + H]+
Example 20 ##STR00080## 1.22 450.2 Name:
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(1-methyl-1H-imidazol-4-yl)-methanon-
e Purification method: Reverse phase method A Prepared using
1-methyl-1H-imidazole-4-carboxylic acid .sup.1H NMR (400 MHz,
methanol-d4, 298K) .delta. ppm 2.17 (s, 3H) 2.28-2.41 (m, 2H)
2.94-3.02 (m, 2H) 3.45-3.52 (m, 2H) 3.73-4.35 (m, 12H) 5.80-5.85
(m, 1H) 7.38-7.43 (m, 1H) 7.60- 7.69 (m, 3H) 8.55-8.61 (m, 1H)
Rt.sup.(1) (min.) MS: [M + H]+ Example 21 ##STR00081## 1.23 453.1
Name:
{(S)-3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-
-d]pyrimidin-4- yloxy]-pyrrolidin-1-yl}-oxazol-4-yl-methanone
Purification method: Normal phase chromatography with EtOAc/MeOH as
solvent Prepared using intermediate 10 and method 1b of process
step 2 of example 1 followed by process step 1 of example 10 using
oxazole-4-carboxylic acid .sup.1H NMR (400 MHz, DMSO-d6, 298K)
.delta. ppm 2.10-2.37 (m, 2 H) 2.81-2.99 (m, 2 H) 3.46-4.27 (m, 14
H) 5.58-5.77 (m, 1 H) 7.08-7.20 (m, 1 H) 7.30-7.42 (m, 1 H) 8.43-
8.54 (m, 1 H) 8.55-8.69 (m, 2 H) Rt.sup.(1) (min.) MS: [M + H]+
Example 22 ##STR00082## 1.18 453.1 Name:
{(S)-3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-
-d]pyrimidin-4- yloxy]-pyrrolidin-1-yl}-oxazol-5-yl-methanone
Purification method: Reverse phase method A Prepared using
intermediate 10 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using oxazole-5-carboxylic acid
.sup.1H NMR (400 MHz, DMSO-d6, 373K) .delta. ppm 2.22-2.42 (m, 2 H)
2.80-3.00 (m, 2 H) 3.50- 4.30 (m, 14 H) 5.63-5.83 (m, 1 H)
7.06-7.09 (m, 1 H) 7.38-7.40 (m, 1 H) 7.69 (s, 1 H) 8.40 (s, 1 H)
8.57 (s, 1 H) Rt.sup.(1) (min.) MS: [M + H]+ Example 23
##STR00083## 1.3 467.2 Name:
{(S)-3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-
-d]pyrimidin-4-
yloxy]-pyrrolidin-1-yl}-(2-methyl-oxazol-4-yl)-methanone
Purification method: Reverse phase method A Prepared using
intermediate 10 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using 2-methyl-oxazole-4-carboxylic
acid .sup.1H NMR (400 MHz, DMSO-d6, 298K) .delta. ppm 2.11-2.36 (m,
2 H) 2.40-2.44 (m, 3 H) 2.81- 2.97 (m, 2 H) 3.40-4.28 (m, 14 H)
5.62-5.78 (m, 1 H) 7.11-7.21 (m, 1 H) 7.29-7.41 (m, 1 H) 8.42-8.52
(m, 1 H) 8.59-8.67 (m, 1 H) Rt.sup.(1) (min.) MS: [M + H]+ Example
24 ##STR00084## 1.38 498.3 Name:
{(S)-3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-
-d]pyrimidin-4-
yloxy]-pyrrolidin-1-yl}-(2,2-dimethyl-tetrahydro-pyran-4-yl)-methanone
Purification method: Reverse phase method A Prepared using
intermediate 10 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using
2,2-dimethyl-tetrahydro-pyran-4-carboxylic acid .sup.1H NMR (400
MHz, DMSO-d6, 298K) .delta. ppm 0.97-1.24 (m, 6 H) 1.28-1.58 (m, 4
H) 2.08- 2.34 (m, 2 H) 2.72-2.97 (m, 3 H) 3.43-4.12 (m, 16 H)
5.55-5.76 (m, 1 H) 7.14-7.20 (m, 1 H) 7.31-7.37 (m, 1 H) 8.59-8.64
(m, 1 H) Rt.sup.(1) (min.) MS: [M + H]+ Example 25 ##STR00085##
1.32 481.2 Name:
{(S)-3-[6-(5,6-Dimethoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-
-d]pyrimidin-4-
yloxy]-pyrrolidin-1-yl}-(2,4-dimethyl-oxazol-5-yl)-methanone
Purification method: Reverse phase method A Prepared using
intermediate 10 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using
2,2-dimethyl-tetrahydro-pyran-4-carboxylic acid .sup.1H NMR (400
MHz, DMSO-d6, 298K) .delta. ppm 2.11-2.48 (m, 8 H) 2.83-2.98 (m, 2
H) 3.43- 4.18 (m, 14 H) 5.56-5.89 (m, 1 H) 7.12-7.20 (m, 1 H)
7.32-7.40 (m, 1 H) 8.57-8.67 (m, 1 H) Rt.sup.(1) (min.) MS: [M +
H]+ Example 26 ##STR00086## 1.57 504.3
Name:
(4,4-Difluoro-cyclohexyl)-{(S)-3-[6-(5,6-dimethoxy-pyridin-3-yl)-5,6-
,7,8-tetrahydro-
pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone
Purification method: Reverse phase method A Prepared using
intermediate 10 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using
4,4-difluoro-cyclohexanecarboxylic acid .sup.1H NMR (400 MHz,
DMSO-d6, 298K) .delta. ppm 1.49-2.35 (m, 10 H) 2.61-2.67 (m, 1 H)
2.84-2.99 (m, 2 H) 3.42-3.83 (m, 12 H) 4.00-4.19 (m, 2 H) 5.57-5.78
(m, 1 H) 7.11- 7.25 (m, 1 H) 7.29-7.43 (m, 1 H) 8.52-8.68 (m, 1 H)
Rt.sup.(1) (min.) MS: [M + H]+ Example 27 ##STR00087## 1.46 479.2
Name:
2-Methoxy-5-{4-[(S)-1-(2-tetrahydro-pyran-4-yl-acetyl)-pyrrolidin-3--
yloxy]-7,8- dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile
Purification method: Reverse phase method A Prepared using
intermediate 11 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using
(tetrahydro-pyran-4-yl)-acetic acid .sup.1H NMR (400 MHz, DMSO-d6,
298K) .delta. ppm 1.11-1.28 (m, 2H) 1.49-1.64 (m, 2H) 1.87- 1.99
(m, 1H) 2.07-2.29 (m, 4H) 2.86-2.95 (m, 2H) 3.19-3.30 (m, 2H)
3.42-3.88 (m, 8H) 3.90-3.96 (m, 3H) 4.09-4.19 (m, 2H) 5.57-5.70 (m,
1H) 8.07-8.11 (m, 1H) 8.22-8.28 (m, 1H) 8.58-8.65 (m, 1H)
Rt.sup.(1) (min.) MS: [M + H]+ Example 28 ##STR00088## 1.51 476.2
Name:
5-{4-[(S)-1-(2,4-Dimethyl-oxazole-5-carbonyl)-pyrrolidin-3-yloxy]-7,-
8-dihydro-5H-
pyrido[4,3-d]pyrimidin-6-yl}-2-methoxy-nicotinonitrile Purification
method: Reverse phase method A Prepared using intermediate 11 and
process step 2, method 1b of example 1 followed by process step 1
of example 10 using 2,4-dimethyl-oxazole-5-carboxylic acid .sup.1H
NMR (400 MHz, DMSO-d6, 298K) .delta. ppm 2.16-2.46 (m, 5H) 2.30 (s,
3H) 2.85-2.96 (m, 2H) 3.50-4.20 (m, 8H) 3.92 (s, 3H) 5.64-5.80 (m,
1H) 8.04-8.12 (m, 1H) 8.22-8.30 (m, 1H) 8.62 (s, 1H) Rt.sup.(1)
(min.) MS: [M + H]+ Example 29 ##STR00089## 1.58 493.2 Name:
5-{4-[(S)-1-(2,2-Dimethyl-tetrahydro-pyran-4-carbonyl)-pyrrolidin-3--
yloxy]-7,8-
dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-2-methoxy-nicotinonitrile
Purification method: Reverse phase method A Prepared using
intermediate 11 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using
2,2-dimethyl-tetrahydro-pyran-4-carboxylic acid .sup.1H NMR (400
MHz, DMSO-d6, 298K) .delta. ppm 1.00-1.24 (m, 6H) 1.28-1.73 (m, 4H)
2.10- 2.34 (m, 2H) 2.62-2.97 (m, 3H) 3.43-3.84 (m, 8H) 3.94 (s, 3H)
4.09-4.20 (m, 2H) 5.58-5.75 (m, 1H) 8.05-8.11 (m, 1H) 8.20-8.29 (m,
1H) 8.59-8.65 (m, 1H) Rt.sup.(1) (min.) MS: [M + H]+ Example 30
##STR00090## 1.55 451.1 Name:
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(5-methyl-oxazol-4-yl)-methanone
Purification method: Reverse phase method A Prepared using
5-methyl-oxazole-4-carboxylic acid .sup.1H NMR (400 MHz, DMSO-d6,
298K) .delta. ppm 2.10-2.32 (m, 2H) 2.13 (s, 3H) 2.52.2.54 (m, 3H)
2.85-2.93 (m, 2H) 3.42-3.50 (m, 2H) 3.61-4.22 (m, 6H) 3.81(s, 3H)
5.64-5.72 (m, 1H) 7.41-7.45 (m, 1H) 7.67-7.71 (m, 1H) 8.27-8.33 (m,
1H) 8.59-8.64 (m, 1H) Rt.sup.(1) (min.) MS: [M + H]+ Example 31
##STR00091## 1.53 451.1 Name:
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(5-methyl-isoxazol-4-yl)-methanone
Purification method: Reverse phase method A Prepared using
5-methyl-isoxazole-4-carboxylic acid .sup.1H NMR (400 MHz, DMSO-d6,
298K) .delta. ppm 2.10-2.31(m, 2H) 2.14 (s, 3H) 2.54-2.59 (m, 3H)
2.83-2.97 (m, 2H) 3.41-3.53 (m, 2H) 3.59-4.15 (m, 6H) 3.81 (s, 3H)
5.65-5.73 (m, 1H) 7.40-7.48 (m, 1H) 7.67-7.74 (m, 1H) 8.56-8.66 (m,
1H) 8.83-8.95 (m, 1H) Rt.sup.(1) (min.) MS: [M + H]+ Example 32
##STR00092## 1.53 451.1 Name:
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(3-methyl-isoxazol-4-yl)-methanone
Purification method: Reverse phase method A Prepared using
3-methyl-isoxazole-4-carboxylic acid .sup.1H NMR (400 MHz, DMSO-d6,
298K) .delta. ppm 2.08-2.37 (m, 8H) 2.82-2.95 (m, 2H) 3.40- 3.53
(m, 2H) 3.55-4.16 (m, 9H) 5.65-5.75 (m, 1H) 7.41-7.48 (m, 1H)
7.68-7.73 (m, 1H) 8.57-8.65 (m, 1H) 9.28-9.40 (m, 1H) Rt.sup.(1)
(min.) MS: [M + H]+ Example 33 ##STR00093## 1.59 437.2 Name:
Isoxazol-3-yl-{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-te-
trahydro-
pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone
Purification method: Reverse phase method A Prepared using
isoxazole-3-carboxylic acid .sup.1H NMR (400 MHz, DMSO-d6, 298K)
.delta. ppm 2.13 (m, 3H) 2.21-2.31 (m, 2H) 2.86-2.94 (m, 2H)
3.43-3.50 (m, 2H) 3.66-4.15 (m, 9H) 5.67-5.73 (m, 1H) 6.84-6.91 (m,
1H) 7.42-7.46 (m, 1H) 7.67-7.74 (m, 1H) 8.57-8.64 (m, 1H) 9.05-9.13
(m, 1H) Rt.sup.(1) (min.) MS: [M + H]+ Example 34 ##STR00094## 1.5
437.2 Name:
Isoxazol-5-yl-{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-te-
trahydro-
pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone
Purification method: Reverse phase method A Prepared using
isoxazole-5-carboxylic acid .sup.1H NMR (400 MHz, DMSO-d6, 298K)
.delta. ppm 2.08-2.16 (m, 3H) 2.19-2.36 (m, 2H) 2.85- 2.95 (m, 2H)
3.42-3.49 (m, 2H) 3.66-4.23 (m, 9H) 5.66-5.78 (m, 1H) 7.06-7.13 (m,
1H) 7.41-7.46 (m, 1H) 7.68-7.74 (m, 1H) 8.59-8.64 (m, 1H) 8.73-8.79
(m, 1H) Rt.sup.(1) (min.) MS: [M + H]+ Example 35 ##STR00095## 1.5
464.1 Name:
2-Methoxy-5-{4-[(S)-1-(thiazole-4-carbonyl)-pyrrolidin-3-yloxy]-7,8--
dihydro-5H- pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile
Purification method: Reverse phase method A Prepared using
intermediate 11 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using thiazole-4-carboxylic acid
.sup.1H NMR (400 MHz, CDCl.sub.3-d, 298K) .delta. ppm 2.30-2.37 (m,
2H) 3.07-3.12 (m, 2H) 3.46-3.53 (m, 2H) 3.81-4.43 (m, 9H) 5.80-5.85
(m, 1H) 7.55-7.59 (m, 1H) 8.09-8.13 (m, 1H) 8.18- 8.23 (m, 1H)
8.63-8.69 (m, 1H) 8.75-8.85 (m, 1H) Rt.sup.(1) (min.) MS: [M + H]+
Example 36 ##STR00096## 1.35 461.2 Name:
2-Methoxy-5-{4-[(S)-1-(1-methyl-1H-pyrazole-4-carbonyl)-pyrrolidin-3-
-yloxy]-7,8-
dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile
Purification method: Reverse phase method A Prepared using
intermediate 11 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using
1-methyl-1H-imidazole-4-carboxylic acid .sup.1H NMR (400 MHz,
CDCl.sub.3-d, 298K) .delta. ppm 2.24-2.47 (m, 2H) 3.03-3.17 (m, 2H)
3.45-3.58 (m, 2H) 3.87-4.20 (m, 12H) 5.75-5.85 (m, 1H) 7.54-7.60
(m, 1H) 7.73-7.90 (m, 2H) 8.09- 8.14 (m, 1H) 8.61-8.68 (m, 1H)
Rt.sup.(1) (min.) MS: [M + H]+ Example 37 ##STR00097## 1.47 461.2
Name:
2-Methoxy-5-{4-[(S)-1-(1-methyl-1H-pyrazole-3-carbonyl)-pyrrolidin-3-
-yloxy]-7,8-
dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile
Purification method: Reverse phase method A Prepared using
intermediate 11 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using
1-methyl-1H-pyrazole-3-carboxylic acid .sup.1H NMR (400 MHz,
CDCl.sub.3-d, 298K) .delta. ppm 2.24-2.41 (m, 2H) 3.07-3.14 (m, 2H)
3.44-3.58 (m, 2H) 3.74-4.44 (m, 12H) 5.77-5.86 (m, 1H) 6.78-6.84
(m, 1H) 7.33-7.39 (m, 1H) 7.54- 7.59 (m, 1H) 8.08-8.14 (m, 1H)
8.63-8.70 (m, 1H) Rt.sup.(1) (min.) MS: [M + H]+ Example 38
##STR00098## 1.58 482.3 Name:
(2,2-Dimethyl-tetrahydro-pyran-4yl)-{(S)-3-[6-(6-Methoxy-5-methyl-py-
ridin-3-yl)-
5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methan-
one Purification method: Reverse phase method A Prepared using
2,2-dimethyl-tetrahydro-pyran-4-carboxylic acid .sup.1H NMR (400
MHz, DMSO-d6, 298K) .delta. ppm 1.02-1.21 (m, 6H) 1.27-1.71 (m, 4H)
2.08- 2.32 (m, 5H) 2.67-2.94 (m, 3H) 3.41-4.08 (m, 13H) 5.60-5.73
(m, 1H) 7.41-7.46 (m, 1H) 7.65-7.72 (m, 1H) 8.58-8.65 (m, 1H)
Rt.sup.(1) (min.) MS: [M + H]+ Example 39 ##STR00099## 1.60 556.1
Name:
(1,1-Dioxo-hexahydro-1lambda*6*-thiopyran-4-yl)-{(S)-3-[6-(6-methoxy-
-5-
trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4--
yloxy]-pyrrolidin-1- yl}-methanone Purification method: Reverse
phase method A Prepared using intermediate 13 and process step 2,
method 1b of example 1 followed by process step 1 of example 10
using 1,1-dioxo-hexahydro-1lambda*6*-thiopyran-4- carboxylic acid
.sup.1H NMR (400 MHz, DMSO-d6, 298K) .delta. ppm 1.90-2.37 (m, 6 H)
2.72-3.27 (m, 7 H) 3.43- 3.81 (m, 6 H) 3.89-3.97 (m, 3 H) 4.13-4.20
(m, 2 H) 5.61-5.75 (m, 1 H) 7.80-7.86 (m, 1 H) 8.15-8.22 (m, 1 H)
8.60-8.65 (m, 1 H) Rt.sup.(1) (min.) MS: [M + H]+ Example 40
##STR00100## 1.77 519.2 Name:
(2,4-Dimethyl-oxazol-5-yl)-{(S)-3-[6-(6-methoxy-5-trifluoromethyl-py-
ridin-3-yl)-
5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methan-
one Purification method: Reverse phase method A Prepared using
intermediate 13 and process step 2, method 1b of example 1 followed
by process step 1of example 10 using
2,4-dimethyl-oxazole-5-carboxylic acid .sup.1H NMR (400 MHz,
DMSO-d6, 298K) .delta. ppm 2.13-2.45 (m, 8H) 2.89-2.96 (m, 2H)
3.54- 4.21 (m, 11H) 5.64-5.79 (m, 1H) 7.81-7.85 (m, 1H) 8.218-8.22
(m., 1H) 8.61-8.65 (m, 1H) Rt.sup.(1) (min.) MS: [M + H]+
Example 41 ##STR00101## 1.69 507.1 Name:
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-thiazol-5-yl-methanone
Purification method: Reverse phase method A Prepared using
intermediate 13 and process step 2, method 1b of example 1 followed
by process step 1of example 10 using thiazole-5-carboxylic acid
.sup.1H NMR (400 MHz, DMSO-d6, 298K) .delta. ppm 2.15-2.42 (m, 2H)
2.88-2.97 (m, 2H) 3.53- 3.61 (m, 2H) 3.67-4.11 (m, 7 H) 4.15-4.24
(m, 2H) 5.67-5.79 (m, 1H) 7.81-7.88 (m, 1H) 8.18-8.23 (m, 1H)
8.35-8.45 (m, 1H) 8.60-8.66 (m, 1H) 9.22-9.29 (m, 1H) Rt.sup.(1)
(min.) MS: [M + H]+ Example 42 ##STR00102## 1.74 504.2 Name:
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(2-methyl-2H-pyrazol-3-yl)-methanone
Purification method: Reverse phase method A Prepared using
intermediate 13 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using
2-methyl-2H-pyrazole-3-carboxylic acid .sup.1H NMR (400 MHz,
DMSO-d6, 298K) .delta. ppm 2.15-2.35 (m, 2H) 2.88-2.97 (m, 2H)
3.51- 4.13 (m, 12 H) 4.13-4.25 (m, 2H) 5.63-5.74 (m, 1H) 6.63-6.74
(m, 1H) 7.43-7.52 (m, 1H) 7.81-7.89 (m, 1H) 8.17-8.25 (m, 1H)
8.57-8.67 (m, 1H) Rt.sup.(1) (min.) MS: [M + H]+ Example 43
##STR00103## 1.47 507.2 Name:
4-{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetra-
hydro-pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidine-1-carbonyl}-pyrrolidin-2-one
Purification method: Reverse phase method A Prepared using
intermediate 13 and process step 2, method 1b of example 1 followed
by process step 1of example 10 using 5-oxo-pyrrolidine-3-carboxylic
acid .sup.1H NMR (400 MHz, DMSO-d6, 298K) .delta. ppm 2.10-2.43 (m,
4H) 2.89-2.96 (m, 2H) 3.35- 3.79 (m, 9H) 3.90-3.94 (m, 3H)
4.15-4.20 (m, 2H) 5.60-5.73 (m, 1H) 7.53-7.62 (m, 1H) 7.81-7.87 (m,
1H) 8.17-8.22 (m, 1H) 8.60-8.64 (m, 1H) Rt.sup.(1) (min.) MS: [M +
H]+ Example 44 ##STR00104## 1.63 501.2 Name:
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-pyridin-3-yl-methanone
Purification method: Reverse phase method A Prepared using
intermediate 13 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using nicotinic acid .sup.1H NMR
(400 MHz, DMSO-d6, 298K) .delta. ppm 2.11-2.37 (m, 2H) 2.87-2.99
(m, 2H) 3.51- 4.13 (m, 9H) 4.13-4.29 (m, 2H) 5.60-5.75 (m, 1H)
7.43-7.53 (m, 1H) 7.81-8.04 (m, 2H) 8.17-8.28 (m, 1H) 8.53-8.82 (m,
3H) Rt.sup.(1) (min.) MS: [M + H]+ Example 45 ##STR00105## 1.36
490.2 Name:
(1H-Imidazol-4-yl)-{(S)-3-[6-(6-methoxy-5-trifluoromethyl-pyridin-3--
yl)-5,6,7,8-
tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methanone
Purification method: Reverse phase method A Prepared using
intermediate 13 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using 1H-imidazole-4-carboxylic
acid .sup.1H NMR (400 MHz, DMSO-d6, 298K) .delta. ppm 2.12-2.35 (m,
2H) 2.87-2.95 (m, 2H) 3.60- 4.31 (m, 11H) 5.63-5.76 (m, 1H)
7.57-7.65 (m, 1H) 7.70-7.78 (m, 1H) 7.80-7.85 (m, 1H) 8.16-8.21 (m,
1H) 8.61-8.65 (m, 1H) Rt.sup.(1) (min.) MS: [M + H]+ Example 46
##STR00106## 1.47 507.2 Name:
5-{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetra-
hydro-pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidine-1-carbonyl}-pyrrolidin-2-one
Purification method: Reverse phase method A Prepared using
intermediate 13 and process step 2, method 1b of example 1 followed
by process step 1of example 10 using 5-oxo-pyrrolidine-2-carboxylic
acid .sup.1H NMR (400 MHz, DMSO-d6, 298K) .delta. ppm 1.78-2.40 (m,
6H) 2.89-2.97 (m, 2H) 3.43- 3.86 (m, 6H) 3.90-3.94 (m, 3H)
4.15-4.20 (m, 2H) 4.30-4.45 (m, 1H) 5.60-5.75 (m, 1H) 7.70-7.89 (m,
2H) 8.16-8.22 (m, 1H) 8.61-8.63 (m, 1H) Rt.sup.(1) (min.) MS: [M +
H]+ Example 47 ##STR00107## 1.61 501.2 Name:
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-pyridin-4-yl-methanone
Purification method: Reverse phase method A Prepared using
intermediate 13 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using isonicotinic acid .sup.1H NMR
(400 MHz, DMSO-d6, 298K) .delta. ppm 2.12-2.36 (m, 2H) 2.86-2.99
(m, 2H) 3.46- 3.81 (m, 5H) 3.84-4.13 (m, 4H) 4.14-4.28 (m, 2H)
5.59-5.74 (m, 1H) 7.44-7.56 (m, 2H) 7.82-7.91 (m, 1H) 8.18-8.27 (m,
1H) 8.52-8.72 (m, 3H) Rt.sup.(1) (min.) MS: [M + H]+ Example 48
##STR00108## 1.62 518.2 Name:
(1,3-Dimethyl-1H-pyrazol-4-yl)-{(S)-3-[6-(6-methoxy-5-trifluoromethy-
l-pyridin-3-yl)-
5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-methan-
one Purification method: Reverse phase method A Prepared using
intermediate 13 and process step 2, method 1b of example 1 followed
by process step 1of example 10 using
1,3,-dimethyl-1H-pyrazole-4-carboxylic acid .sup.1H NMR (400 MHz,
DMSO-d6, 298K) .delta. ppm 2.13-2.35 (m, 5H) 2.89-2.97 (m, 2H)
3.53- 4.06 (m, 12H) 4.14-4.22 (m, 2H) 5.64-5.72 (m, 1H) 7.80-7.88
(m, 1H) 7.99-8.11 (m, 1H) 8.16-8.22 (m, 1H) 8.58-8.66 (m, 1H)
Rt.sup.(1) (min.) MS: [M + H]+ Example 49 ##STR00109## 1.51 490.1
Name:
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(1H-pyrazol-4-yl)-methanone
Purification method: Reverse phase method A Prepared using
intermediate 13 and process step 2, method 1b of example 1 followed
by process step 1of example 10 using 1H-pyrazole-4-carboxylic acid
.sup.1H NMR (400 MHz, DMSO-d6, 298K) .delta. ppm 2.12-2.39 (m, 2H)
2.87-2.97 (m, 2H) 3.43- 4.11 (m, 9H) 4.12-4.22 (m, 2H) 5.63-5.79
(m, 1H) 7.78-7.94 (m, 2H) 8.10-8.25 (m, 3H) 8.59-8.68 (m, 1H)
Rt.sup.(1) (min.) MS: [M + H]+ Example 51 ##STR00110## 1.72 506.2
Name:
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(5-methyl-[1,3,4]oxadiazol-2-yl)-met-
hanone Purification method: Reverse phase method A Prepared using
intermediate 13 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using
5-methyl-[1,3,4]oxadiazole-2-carboxylic acid Rt.sup.(1) (min.) MS:
[M + H]+ Example 52 ##STR00111## 1.71 502.1 Name:
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-pyrazin-2-yl-methanone
Purification method: Reverse phase method A Prepared using
intermediate 13 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using pyrazine-2-carboxylic acid
.sup.1H NMR (400 MHz, DMSO-d6, 298K) .delta. ppm 2.15-2.36 (m, 2H)
2.87-2.97 (m, 2H) 3.52- 3.64 (m, 2H) 3.66-4.11 (m, 7H) 4.12-4.24
(m, 2H) 5.67-5.75 (m, 1H) 7.81-7.87 (m, 1H) 8.17-8.25 (m, 1H)
8.56-8.80 (m, 3H) 8.97-9.02 (m, 1H) Rt.sup.(1) (min.) MS: [M + H]+
Example 53 ##STR00112## 1.45 504.2 Name:
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(1-methyl-1H-imidazol-4-yl)-methanon-
e Purification method: Reverse phase method A Prepared using
intermediate 13 and process step 2, method 1b of example 1 followed
by process step 1 of example 10 using
1-methyl-1H-imidazole-4-carboxylic acid .sup.1H NMR (400 MHz,
DMSO-d6, 298K) .delta. ppm 2.07-2.36 (m, 2 H) 2.82-3.05 (m, 2 H)
3.20- 4.43 (m, 14 H) 5.60-5.74 (m, 1 H) 7.59-7.72 (m, 2 H)
7.78-7.87 (m, 1 H) 8.14-8.21 (m, 1 H) 8.59-8.66 (m, 1 H)
##STR00113##
Example 54:
{(S)-3[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-py-
rido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(1-methyl-1H-pyrazol-4-yl)--
methanone
[0625] To a mixture of
6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-4-((S)-pyrrolidin-3-yloxy)-5-
,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine (prepared using step 1,
example 91 from intermediate 13) (44 mg, 0.11 mmol),
1-methyl-1H-pyrazole-4-carboxylic acid (15 mg, 0.12 mmol),
benztriazol-1-ol (19 mg, 0.12 mmol) in CH.sub.2Cl.sub.2 (1.0 mL)
was added EDC (34 mg, 0.18 mmol) and the resulting mixture was
stirred at rt for 18 h. Partitioned between CH.sub.2Cl.sub.2 (10
mL) and sat. NaHCO.sub.3(aq) (2.0 mL) and the organic layer
separated by filtering through a phase separation tube.
Concentrated in vacuo and purified by flash chromatography through
Biotage.RTM. amino silica gel eluting with cyclohexane/EtOAc, 100/0
to 0/100 to give
{(S)-3-[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-p-
yrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(1-methyl-1H-pyrazol-4-yl)-
-methanone as a white lyophilized powder (44 mg, 75% yield). 1H NMR
(400 MHz, CDCl.sub.3-d, 298K) .delta. ppm 2.26-2.45 (m, 2H)
3.04-3.10 (m, 2H) 3.49-3.57 (m, 2H) 3.89-4.00 (m, 7H) 4.01 (s, 3H)
4.10-4.18 (m, 2H) 5.78-5.83 (m, 1H) 7.60-7.62 (m, 1H) 7.76-7.89 (m,
2H) 8.04-8.07 (m, 1H) 8.61-8.66 (m, 1H) MS: [M+H].sup.+=504.2,
Rt.sup.(3)=1.59 min.
[0626] Example 55 was prepared using procedures analogous to those
used in example 54 using appropriate starting materials.
TABLE-US-00010 Structure Rt.sup.(1) (min.) MS: [M + H]+ Example 55
##STR00114## 1.77 507.2 Name:
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-thiazol-4-yl-methanone
Purification method: Flash chromatography on Biotage amino silica
gel eluting with cyclohexane/EtOAc 100/0 to 0/100 Prepared using
thiazole-4-carboxylic acid .sup.1H NMR (400 MHz, CDCl.sub.3-d,
298K) .delta. ppm 2.29-2.40 (m, 2H) 3.05-3.12 (m, 2H) 3.49-3.56 (m,
2H) 3.80-4.45 (m, 9H) 5.80-5.86 (m, 1H) 7.60-7.62 (m, 1H) 8.04-8.18
(m, 1H) 8.21- 8.25 (m, 1H) 8.64-8.68 (m, 1H) 8.79-8.84 (m, 1H)
##STR00115##
Example 56:
{(S)-3[6-(5-Chloro-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3--
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone
[0627] To a
6-(5-chloro-6-methoxy-pyridin-3-yl)-4-((S)-pyrrolidin-3-yloxy)-5,6,7,8-te-
trahydro-pyrido[4,3-d]pyrimidine (97 mg, 0.196 mmol) in
CH.sub.2Cl.sub.2 (5 mL) was added the acid chloride
tetrahydo-2H-pyran-4-carbonyl chloride (36.7 mg, 0.235 mmol) and
triethylamine (0.035 mL, 0.254 mmol) at temperature between
0-10.degree. C. The reaction mixture was stirred at -3.degree. C.
for 30 min. The reaction mixture was concentrated under vacuum.
Purification by preparative reverse phase Gilson HPLC and
subsequent neutralization of the combined fractions over
PL-HCO.sub.3 MP gave
{(S)-3[6-(5-chloro-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido-
[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methano-
ne (38 mg, 41% yield) as a white lyophilized powder. .sup.1H NMR
(400 MHz, CDCl.sub.3-d, 298 K) .delta. ppm 1.56-1.68 (m, 2H)
1.86-2.04 (m, 2H) 2.20-2.40 (m, 2H) 2.50-2.72 (m, 1H) 3.05-3.13 (m,
2H) 3.38-4.16 (m, 16H) 5.70-5.78 (m, 1H) 7.42-7.45 (m, 1H)
7.78-7.81 (m, 1H) 8.61-8.66 (m, 1H). LCMS: [M+H].sup.+=474.2,
Rt.sup.(2)=1.52 min.
##STR00116##
6-(5-Chloro-6-methoxy-pyridin-3-yl)-4-((S)-pyrrolidin-3-yloxy)-5,6,7,8-te-
trahydro-pyrido[4,3-d]pyrimidine
[0628]
(S)-3[6-(5-Chloro-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido-
[4,3-d]pyrimidin-4-yloxy]-pyrrolidine-1-carboxylic acid tert-butyl
ester (766.2 mg, 1.66 mmol) was dissolved in a TFA/CH.sub.2Cl.sub.2
(1/2) solution and stirred at rt for 1 h. The reaction mixture was
concentrated under vacuum, the residue was diluted with
CH.sub.2Cl.sub.2, the organic layer washed with NaOH 1N then brine,
dried over Na.sub.2SO.sub.4, filtered and evaporated to give
6-(5-chloro-6-methoxy-pyridin-3-yl)-4-((S)-pyrrolidin-3-yloxy)-5,6,7,8-te-
trahydro-pyrido[4,3-d]pyrimidine. LCMS: [M+H].sup.+=362.1,
Rt.sup.(3)=1.28 min.
##STR00117##
(S)-3[6-(5-Chloro-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d-
]pyrimidin-4-yloxy]-pyrrolidine-1-carboxylic acid tert-butyl
ester
[0629] X-Phos (0.073 g, 0.154 mmol), Pd.sub.2(dba).sub.3 (0.100 g,
0.110 mmol), NaOtBu (0.395 g, 4.11 mmol) and
5-bromo-3-chloro-2-methoxy-pyridine (0.732 g, 3.29 mmol) were
combined and flushed under a stream of argon for 10 min. To this
mixture, a solution of
(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidine-1-c-
arboxylic acid tert-butyl ester (Intermediate 7) (2.15 g, 6.72
mmol) in THF (6 mL) was added at rt and the reaction mixture was
stirred at 90.degree. C. for 3 h. The reaction was cooled down to
rt, EtOAc was added, the mixture filtered through a celite pad and
concentrated under vacuum. Purification by flash chromatography on
silica gel (CH.sub.2Cl.sub.2/MeOH 99/1 to 95/5) gave
(S)-3[6-(5-chloro-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d-
]pyrimidin-4-yloxy]-pyrrolidine-1-carboxylic acid tert-butyl ester
as yellow foam (0.766 g, 60% yield). LCMS: [M+H].sup.+=462.1,
Rt.sup.(3)=1.84 min
##STR00118##
Example 57:
{(S)-3-[6-(6-Amino-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyr-
ido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-meth-
anone
[0630] To a solution of (S)-tert-butyl
3-(6-(6-(bis(tert-butoxycarbonyl)amino)-5-(trifluoromethyl)pyridin-3-yl)--
5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidine-1 (120
mg, 0.18 mmol) in CH.sub.2Cl.sub.2 (2.0 mL), was added TFA (2.0 mL)
and the mixture stood at rt for 1 h. Concentrated in vacuo and
eluted through an Isolute.RTM. SCX-2 cartridge, eluting with
methanol, then with 2M ammonia in methanol. Basic fractions were
concentrated in vacuo to give
5-[4(S)-pyrrolidin-3-yloxy)-7,8-dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl]-3-
-(trifluoromethyl)pyridin-2-yl)amine (61 mg, 90% yield).
5-[4-((S)-pyrrolidin-3-yloxy)-7,8-dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl]-
-3-(trifluoromethyl)pyridin-2-yl)amine (30 mg, 0.079 mmol) was
dissolved in CH.sub.2Cl.sub.2 (2.0 mL) and was added simultaneously
portionwise with sat.NaHCO.sub.3(aq) (2.0 mL) to a vigorously
stirring solution of tetrahydro-2H-pyran-4-carbonyl chloride (15
mg, 0.10 mmol) in CH.sub.2Cl.sub.2 (2.0 mL) at rt. The resulting
biphasic mixture was stirred at rt for 1 h. Diluted with
CH.sub.2Cl.sub.2 (10 mL) and the organic layer was separated by
filtering through a phase separation tube and concentrated in
vacuo. Purification by reverse phase Gilson HPLC (Method A) and
subsequent neutralization of the combined fractions by elution
through an Isolute.RTM. SCX-2 cartridge, eluting with methanol,
then with 2M ammonia in methanol. Basic fractions were concentrated
in vacuo to give
{(S)-3[6-(6-amino-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyri-
do[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-metha-
none as a pale yellow powder (19 mg, 50% yield) .sup.1H NMR (400
MHz, CDCl.sub.3, 298K) .delta. ppm 1.56-1.72 (m, 2H) 1.87-2.03 (m,
2H) 2.23-2.74 (m, 3H) 3.04-3.14 (m, 2H) 3.48-4.13 (m, 12H)
5.15-5.43 (m, 2H, Ar--NH2) 5.73-5.79 (m, 1H) 7.55-7.64 (m, 1H)
7.93-8.02 (m, 1H) 8.61-8.67 (m, 1H) LCMS: [M+H]+=397.1,
Rt.sup.(3)=1.32 min.
##STR00119##
(S)-tert-butyl
3-(6-(6-(bis(tert-butoxycarbonyl)amino)-5-(trifluoromethyl)pyridin-3-yl)--
5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidine-1-carboxylate
[0631] To a glass vial was added
(S)-3-(5,6,7,8-Tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidine-1-c-
arboxylic acid tert-butyl ester (Intermediate 7) (100 mg, 0.312
mmol), imidodicarbonic acid,
2-[5-bromo-3-(trifluoromethyl)-2-pyridinyl]-,
1,3-bis(1,1-dimethylethyl) ester (138 mg, 0.312 mmol), cesium
carbonate (203 mg, 0.62 mmol),
tris(dibenzylideneacetone)dipalladium(0) (29 mg, 0.03 mmol), X-Phos
(51 mg, 0.11 mmol) and anhydrous dioxane (2 mL). The vial was
flushed with a stream of argon for 15 sec and capped. The mixture
was heated with stirring for 1 h at 110.degree. C. and then stirred
at room temperature for 18 h. Allowed to cool and partitioned
between CH.sub.2Cl.sub.2 (10 mL) and water (2 mL) and filtered the
biphasic mixture through a celite pad. The organic layer was
separated by filtering through phase separation tube and
concentrated in vacuo. Purification by reverse phase Gilson HPLC
(Method A) to give (S)-tert-butyl
3-(6-(6-(bis(tert-butoxycarbonyl)amino)-5-(trifluoromethyl)pyridin-3-yl)--
5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yloxy)pyrrolidine-1-carboxylate
trifluoroacetate (as a yellow gum (120 mg, 48% yield). LCMS:
[M+H]+=681.5, Rt.sup.(4)=1.49 min.
##STR00120##
Imidodicarbonic acid,
2-[5-bromo-3-(trifluoromethyl)-2-pyridinyl]-,1,3-bis(1,1-dimethylethyl)
ester
[0632] To 5-bromo-3-(trifluoromethyl)pyridin-2-amine (1.72 g, 7.14
mmol), triethylamine (0.995 mL, 7.14 mmol) and
4-dimethylaminoyridine (20 mg, 0.164 mmol) in CH.sub.2Cl.sub.2 (50
mL) was added di-tert-butyl-dicarbonate (3.89 g, 17.84 mmol) and
the resulting mixture stirred at room temperature for 18 h.
Evaporated to dryness in vacuo and triturated in heptane (25 mL)
for 72 h. The resulting precipitate was filtered and washed with
heptane (10 mL) to give Imidodicarbonic acid,
2-[5-bromo-3-(trifluoromethyl)-2-pyridinyl]-,
1,3-bis(1,1-dimethylethyl) ester as a beige solid (2.23 g, 71%
yield). .sup.1H NMR (400 Mhz, CDCl.sub.3, 298K) 1.35 (s, 18H) 8.15
(d, 1H) 8.76 (d, 1H) LCMS: [M+H]+=441/443.1, Rt.sup.(4)=1.46
min.
##STR00121##
Example 58:
{3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-d]pyrimidin-4-yloxy]-azetidin-1-yl}-(tetrahydro-pyran-4-yl)-methanon-
e
[0633]
3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro--
pyrido[4,3-d]pyrimidin-4-yloxy]-azetidine-1-carboxylic acid
tert-butyl ester (186 mg, 0.312 mmol) in CH.sub.2Cl.sub.2 (2.0 mL)
was added TFA (1.0 mL) and the mixture stirred at room temperature
for 1 h. Evaporated in vacuo to give
4-(azetidin-3-yloxy)-(6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,-
8-tetrahydro-pyrido[4,3-d]pyrimidine ditrifluoroacetate as a brown
gum (110 mg). To a vigorously stirring solution of
tetrahydro-2H-pyran-4-carbonyl chloride (19 mg, 0.128 mmol) in
CH.sub.2Cl.sub.2 was added simultaneously portionwise sat.
NaHCO.sub.3(aq) (2.0 mL) and a solution of
4-(azetidin-3-yloxy)-(6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,-
8-tetrahydro-pyrido[4,3-d]pyrimidine ditrifluoroacetate (60 mg,
0.099 mmol) in CH.sub.2Cl.sub.2 (2.0 mL) at rt. The resulting
biphasic mixture was stirred vigorously at rt for 1 h. Diluted with
CH.sub.2Cl.sub.2 (10 mL), the organic layer separated, dried
(MgSO.sub.4), concentrated in vacuo and purified by reverse phase
Gilson HPLC (Method A) and subsequent neutralization of the
combined fractions by eluting through an Isolute.RTM. SCX-2
cartridge, eluting with methanol, then with 2M ammonia in methanol.
Basic fractions were concentrated in vacuo to give
{3-[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-d]pyrimidin-4-yloxy]-azetidin-1-yl}-(tetrahydro-pyran-4-yl)-methanon-
e as a yellow solid (3.0 mg, 5% yield 2.sup.nd step).sup.1H NMR
(400 MHz, DMSO-d6, 298K) .delta. ppm 1.42-1.67 (m, 4H) 2.90-2.98
(m, 2H) 3.55-3.62 (m, 2H) 3.78-4.32 (m, 13H) 4.61-4.69 (m, 1H)
5.42-5.49 (m, 1H) 7.86-7.90 (m, 1H) 8.22-8.26 (m, 1H) 8.58-8.62 (s,
1H) LCMS: [M+H]+=494.6, Rt.sup.(7)=0.98 min.
##STR00122##
3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido-
[4,3-d]pyrimidin-4-yloxy]-azetidine-1-carboxylic acid tert-butyl
ester
[0634] To a glass vial was added
3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-azetidine-1-carboxy-
lic acid tert-butyl ester (110 mg, 0.359 mmol),
5-Bromo-2-methoxy-3-(trifluoromethyl)pyridine (92 mg, 0.359 mmol),
cesium carbonate (234 mg, 0.718 mmol),
tris(dibenzylideneacetone)dipalladium(0) (33 mg, 0.036 mmol),
X-Phos (58 mg, 0.122 mmol) and anhydrous dioxane (2.0 mL). The vial
was flushed with a stream of argon for 15 sec and capped. The
mixture was heated with stirring for 1.5 h at 110.degree. C. and
then stirred at room temperature for 18 h. Diluted with
CH.sub.2Cl.sub.2 (50 mL), filtered through a celite pad and
concentrated in vacuo. Purified by reverse phase Gilson HPLC
(Method A) to give the
3-[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido-
[4,3-d]pyrimidin-4-yloxy]-azetidine-1-carboxylic acid tert-butyl
ester trifluoroacetate as a brown gum (186 mg, 87% yield) LCMS:
[M+H]+=482.3, Rt.sup.(7)=1.56 min.
##STR00123##
3-(5,6,7,8-Tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-azetidine-1-carboxy-
lic acid tert-butyl ester
[0635] To a solution of
3-(6-benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-azetidine--
1-carboxylic acid tert-butyl ester (425 mg, 1.07 mmol) in MeOH (20
mL) was added 20% palladium hydroxide on carbon (90 mg) then
ammonium formate (473 mg, 7.51 mmol) and the mixture heated at
reflux for 1 h. The reaction mixture was allowed to cool and
filtered through a celite pad, washing with MeOH (20 mL) then
CH.sub.2Cl.sub.2 (20 mL). The filtrate was evaporated in vacuo and
purified by flash chromatography on silica gel with
CH.sub.2Cl.sub.2/MeOH/0.88 NH.sub.4OH, 100/0/0 to 90/10/1 to give
3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-azetidine-1-carboxy-
lic acid tert-butyl ester as a yellow gum (220 mg, 67% yield) LCMS:
[M+H]+=307.3, Rt.sup.(4)=0.81 min.
##STR00124##
3-(6-Benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-azetidine--
1-carboxylic acid tert-butyl ester
[0636] 3-Hydroxy-azetidine-1-carboxylic acid tert-butyl ester (217
mg, 1.25 mmol) was dissolved under argon in THF (10 mL) and NaH (58
mg, 1.44 mmol) was added. The resulting suspension was stirred at
rt under argon for 15 min following by the addition of a solution
of 6-benzyl-4-chloro-5,6,7,8-tetrahydro-pyrido[4,3d]pyrimidine (250
mg, 0.963 mmol). The reaction mixture was stirred at rt for 18 h,
quenched with water (20 mL) and diluted with CH.sub.2Cl.sub.2. The
organic layer was filtered through a phase separation tube and
concentrated in vacuo. Purification by flash chromatography on
silica gel with heptane/CH.sub.2Cl.sub.2, 50/50 to 0/100 then EtOAc
to give
3-(6-benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-azetidine--
1-carboxylic acid tert-butyl ester as a yellow gum (425 mg, 111%
yield) LCMS: [M+H]+=397.4, Rt.sup.(4)=0.98 min.
[0637] Example 59 was prepared according the general procedure
described in scheme 2.
##STR00125##
Example 59:
{(S)-3[6-(2-Methoxy-pyrimidin-5-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrim-
idin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone
Step 4
[0638] A mixture of 5-bromo-2-methoxy-pyrimidine (0.218 mmol),
X-Phos (28.4 mg, 0.060 mmol), Pd.sub.2(dba).sub.3 (18.2 mg, 0.020
mmol) and Cs.sub.2CO.sub.3 (129 mg, 0.397 mmol) was flushed with
argon before the addition of a solution of
(tetrahydro-pyran-4-yl)-[(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-
-4-yloxy)-pyrrolidin-1-yl]-methanone in dioxane (2 mL). The
reaction mixture was heated at 120.degree. C. for 1 h in a sealed
vial, cooled down to rt and filtered over Hyflo, The recovered
organic phase was washed with NaHCO.sub.3 and brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated. Purification by
preparative reverse phase Gilson HPLC and neutralization of the
combined fractions by passing through a SCX-2 cartridge (The
cartridge was washed with acetonitrile, CH.sub.2Cl.sub.2 and MeOH,
then a solution of NH.sub.3 in MeOH 3.5 N was used to released the
expected product) gave
{(S)-3[6-(2-methoxy-pyrimidin-5-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrim-
idin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone
(18.7 mg, 21% yield) .sup.1H NMR (400 MHz, CDCl.sub.3-d, 298K)
.delta. ppm 1.62-1.70 (m, 2H) 1.87-2.01 (m, 2H) 2.20-2.41 (m, 2H)
2.49-2.71 (m, 1H) 3.07-3.19 (m, 2H) 3.37-4.19 (m, 16H) 5.76 (m, 1H)
8.32 (s, 2H) 8.65-8.67 (m, 1H). LCMS: [M+H]+=441.2, Rt.sup.(1)=1.12
min.
##STR00126##
(Tetrahydro-pyran-4-yl)-[(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-
-4-yloxy)-pyrrolidin-1-yl]-methanone
Step 3
[0639] A solution of
[(S)-3-(6-benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrro-
lidin-1-yl]-(tetrahydro-pyran-4-yl)-methanone (10.9 g, 25.8 mmol)
was dissolved in methanol (300 mL) and Pd(OH).sub.2 on Carbon (2 g,
14.24 mmol) and ammonium formate (3.35 g, 51.6 mmol) were added.
The reaction mixture was refluxed for 2 h. The reaction was cooled
down to rt, the reaction mixture was filtered and evaporated under
high vacuum for 2 h to yield
(tetrahydro-pyran-4-yl)-[(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyr-
imidin-4-yloxy)-pyrrolidin-1-yl]-methanone (8.45 g, 95% yield) as a
light yellow foam. .sup.1H NMR (400 MHz, DMSO-d6, 298K) .delta. ppm
1.44-1.67 (m, 4H) 2.08-2.32 (m, 2H) 2.55-2.83 (m, 3H) 2.96 (t, 2H)
3.22-3.96 (m, 11H) 5.53-5.68 (m, 1H) 8.49-8.59 (m, 1H). LCMS:
[M+H]+=333.5, Rt.sup.(6)=1.24 min.
##STR00127##
[(S)-3-(6-Benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrro-
lidin-1-yl]-(tetrahydro-pyran-4-yl)-methanone
Step 2
[0640] To a solution of
6-benzyl-4-((S)-pyrrolidin-3-yloxy)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrim-
idine (420 mg, 1.35 mmol) in 4 mL of CH.sub.2Cl.sub.2 was added
tetrahydro-pyran-4-carbonyl chloride (0.210 mL, 1.637 mmol) and
Et.sub.3N (0.380 mL, 2.73 mmol). The reaction mixture was stirred
at room temperature for 30 min then was quenched with H.sub.2O,
extracted with CH.sub.2Cl.sub.2, filtered and evaporated under
vacuum. Purification by flash-chromatography on silica gel
(CH.sub.2Cl.sub.2/MeOH 95/5) gave
[(S)-3-(6-benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrro-
lidin-1-yl]-(tetrahydro-pyran-4-yl)-methanone (420 mg, 73% yield)
as a yellow foam. .sup.1H NMR (400 MHz, DMSO-d6, 298K) .delta. ppm
1.37-1.64 (m, 4H) 1.95-2.29 (m, 2H) 2.56-2.83 (m, 4H) 3.28-3.91 (m,
13H) 5.54-5.68 (m, 1H) 7.24-7.36 (m, 5H) 8.54-8.59 (m, 1H). LCMS:
[M+H]+=423.6, Rt.sup.(7)=0.68.
##STR00128##
6-Benzyl-4((S)-pyrrolidin-3-yloxy)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimi-
dine
[0641] Step 1 To a solution of
(S)-3-(6-benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrol-
idine-1-carboxylic acid tert-butyl ester (560 mg, 1.364 mmol) in 2
mL of CH.sub.2Cl.sub.2 was added TFA (1.576 mL, 20.46 mmol). The
reaction mixture was stirred at rt for 1 h, concentrated and then
eluted through an Isolute SCX-2 cartridge (10 g) to remove excess
TFA with (i) MeOH (ii) NH.sub.3/MeOH and the basic fraction
evaporated in vacuum to give
6-benzyl-4-((S)-pyrrolidin-3-yloxy)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrim-
idine (420 mg, quantitative yield) as a yellow gum. LCMS:
[M+H]+=311.2, Rt.sup.(3)=0.11.
[0642] Examples 60-62 were prepared using procedures analogous to
those used in Example 59 using appropriate starting materials.
TABLE-US-00011 Rt.sup.(1) (min.) MS: [M + H]+ Example 60
##STR00129## 1.27 460.2 Name:
[(S)-3-(6-Quinolin-3-yl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4--
yloxy)-pyrrolidin- 1-yl]-(tetrahydro-pyran-4-yl)-methanone
Purification method: Reverse phase method A Prepared using
3-bromo-quinoline .sup.1H NMR (400 MHz, CDCl.sub.3-d, 298K) .delta.
ppm 1.60-1.72 (m, 3H) 1.89-2.04 (m, 2H) 2.24-2.46 (m, 2H) 2.53-2.73
(m, 1H) 3.08-3.21 (m, 2H) 3.39-3.52 (m, 2H) 3.67-4.11 (m, 8H) 4.20-
4.40 (m, 2H) 5.73-5.83 (m, 1H) 7.47-7.62 (m, 3H) 7.73-7.80 (m, 1H)
8.02-8.14 (m, 1H) 8.62-8.68 (m, 1H) 8.86-8.91 (m, 1H). Rt.sup.(7)
(min.) MS: [M + H]+ Example 61 ##STR00130## 1.17 508.7 Name:
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone
Purification method: Reverse phase method A Prepared using
intermediate 1 .sup.1H NMR (400 MHz, DMSO-d6, 298K) .delta. ppm
1.50-1.66 (m, 4H) 2.07-2.46 (m, 2H) 2.60- 2.80 (m, 1H) 2.88-2.97
(m, 2H) 3.30-3.95 (m, 13H) 4.08-4.23 (m, 2H) 5.59-5.74 (m, 1H)
7.79-7.85 (m, 1H) 8.16-8.23 (m, 1H) 8.60-8.65 (m, 1H) Rt.sup.(1)
(min.) MS: [M + H]+ Example 62 ##STR00131## 2.11 494.2 Name:
1-{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetra-
hydro-pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-3,3-dimethyl-butan-1-one
Purification method: Reverse phase method A Prepared using step 2
of example 59 and 3,3-dimethyl-butyryl chloride followed by steps
3-4 of example 59 using intermediate 1 .sup.1H NMR (400 MHz,
CDCl.sub.3, 298K) .delta. ppm 1.00-1.12 (m, 9H) 2.13-2.35 (m, 4H)
3.08-3.15 (m, 2H) 3.45-3.93 (m, 6H) 4.02 (s, 3H) 4.03-4.15 (m, 2H)
5.72-5.79 (m, 1H) 7.57-7.62 (m, 1H) 8.03-8.07 (m, 1H) 8.64-8.69 (m,
1H)
[0643] Example 63 was prepared according the general procedure
described in scheme 2.
##STR00132##
Example 63:
1-{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-
-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-propan-1-one
Step 3
[0644]
1-[(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrol-
idin-1-yl]-propan-1-one (47.8 mg, 0.173 mmol), X-Phos (28 mg, 0.059
mmol) and Pd.sub.2(dba).sub.3.CHCl.sub.3 (17.90 mg, 0.017 mmol)
were combined and flushed with argon during several min before
addition of degassed dioxane.
5-Bromo-2-methoxy-3-trifluoromethyl-pyridine (intermediate 1) (54.5
mg, 0.213 mmol) and Cs.sub.2CO.sub.3 (113 mg, 0.346 mmol) were then
added to the reaction mixture and the resulting mixture flushed
with argon and heated at 150.degree. C. for 30 min. in a sealed
tube. The reaction mixture was cooled to rt, filtered over Hyflo
and evaporated. Purification by preparative reverse phase Gilson
HPLC and subsequent neutralization of the combined fractions over
PL-HCO.sub.3 MP gave
1-{(S)-3-[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-
-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-propan-1-one (26
mg, 33% yield) .sup.1H NMR (400 MHz, DMSO-d6, 298K) .delta. ppm
0.94-1.00 (m, 3H) 2.05-2.17 (m, 4H) 2.95-3.0 (m, 2H) 3.45-3.97 (m,
9H) 4.07-4.11 (m, 2H) 5.58-5.72 (m, 1H) 7.81-7.86 (m, 1H) 8.18-8.23
(m, 1H) 8.62 (s, 1H). MS: [M+H]+=452.2, Rt.sup.(1)=1.74 min.
##STR00133##
1-[(S)-3-(5,6,7,8-Tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidin-1-
-yl]-propan-1-one
Step 2
[0645] Pd(OH).sub.2 (150 mg, 1.070 mmol) was put into a round flask
and flushed under argon for 5 minutes. A solution of
1-[(S)-3-(6-benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyr-
rolidin-1-yl]-propan-1-one (560 mg, 1.528 mmol) in 22 mL of MeOH
was added followed by ammonium formate (482 mg, 7.64 mmol). The
reaction mixture was stirred under reflux (70.degree. C.) for 2 h.
The mixture was filtered over a pad of celite and dried under high
vacuum to give
1-[(S)-3-(5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyrrolidin-1-
-yl]-propan-1-one. No further purification (m=420 mg, quantitative
yield). MS: [M+H]+=277.5 Rt.sup.(6)=0.71 min.
##STR00134##
1-[(S)-3-(6-Benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyr-
rolidin-1-yl]-propan-1-one
Step 1
[0646] To a solution of
1-((S)-3-hydroxy-pyrrolidin-1-yl)-propan-1-one (intermediate 2)
(358 mg, 2.503 mmol) in 5 mL of THF was added NaH (108 mg, 2.70
mmol) under Ar. The mixture was stirred at rt for 15 min, then
6-benzyl-4-chloro-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine (500
mg, 1.925 mmol) and 5 mL of THF were added and stirred at rt for 5
h. The reaction was quenched with H.sub.2O and extracted with
ethylacetate, the org. layer was washed with brine, dried over
MgSO.sub.4, filtered and evaporated to dryness. The residue was
purified by flash-chromatography using Isco Companion system (12 g
of SiO.sub.2) CH.sub.2Cl.sub.2/MeOH (95/5). The collected fractions
were combined, evaporated and dried over high vacuum to give
1-[(S)-3-(6-benzyl-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy)-pyr-
rolidin-1-yl]-propan-1-one. (m=560 mg, yield 78%) MS: [M+H]+=367.6,
Rt.sup.(7)=0.64 min.
[0647] Example 64 was prepared using procedures analogous to those
used in Example 63 using appropriate starting materials.
TABLE-US-00012 Rt.sup.(1) (min.) MS: [M + H]+ Example 64
##STR00135## 1.46 409.2 Name:
2-Methoxy-5-[4-((S)-1-propionyl-pyrrolidin-3-yloxy)-7,8-dihydro-5H-p-
yrido[4,3- d]pyrimidin-6-yl]-nicotinonitrile Purification method:
Reverse phase method A Prepared using step 3 of example 59 and
5-bromo-2-methoxynicotinonitrile .sup.1H NMR (400 MHz, DMSO-d6,
298K) .delta. ppm 0.95-1.02 (m, 3H) 2.10-2.35 (m, 5H) 2.89- 2.98
(m, 2H) 3.40-3.90 (m, 5H) 3.93 (s, 3H) 4.16 (s, 2H) 5.58-5.71 (m,
1H) 8.08-8.10 (m, 1H) 8.24-8.28 (m, 1H) 8.61 (m, 1H).
##STR00136##
Examples 65:
6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-4-((S)-1-pyridin-2-yl-pyrrol-
idin-3-yloxy)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine
[0648] To a glass vial was added
6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-4-((S)-pyrrolidin-3-yloxy)-5-
,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine dihydrochloride (prepared
using step 1 of example 91 from intermediate 13) (75 mg, 0.16
mmol), 2-bromopyridine (1 mL, 10.25 mmol) and
N,N-diisopropylethylamine (0.14 mL, 0.80 mmol). The vial was capped
and the mixture heated in the microwave at 160.degree. C. for 20
min. Purification by reverse phase Gilson HPLC (Method A) and
subsequent neutralization of the combined fractions over
PL-HCO.sub.3 MP to give
6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-4-((S)-1-pyridin-2-yl-pyrrol-
idin-3-yloxy)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine as a light
brown solid (19 mg, 25% yield) .sup.1H NMR (400 MHz, DMSO-d6, 298K)
.delta. ppm 2.24-2.44 (m, 2H) 2.92 (t, 2H) 3.47-3.69 (m, 5H)
3.77-3.85 (m, 1H) 3.88-3.93 (m, 3H) 4.12-17 (m, 2H) 5.73-5.81 (m,
1H) 6.40-6.52 (d, 1H) 6.56-6.58 (m, 1H) 7.43-7.54 (m, 1H) 7.77-7.84
(m, 1H) 8.02-8.09 (m, 1H) 8.13-8.20 (m, 1H) 8.61-8.66 (m, 1H) LCMS:
[M+H]+=473.0, Rt.sup.(4)=0.85 min.
##STR00137##
Examples 66:
6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-4-((S)-1-pyrimidin-2-yl-pyrr-
olidin-3-yloxy)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine
[0649] To a glass vial was added
6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-4-((S)-pyrrolidin-3-yloxy)-5-
,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine dihydrochloride (prepared
using step 1 of example 91 from intermediate 13) (75 mg, 0.16
mmol), 2-bromopyrimidine (55 mg, 0.342 mmol) and
N,N-diisopropylethylamine (0.15 mL, 0.85 mmol). The vial was capped
and the mixture heated in the microwave at 160.degree. C. for 20
min. Purification by reverse phase Gilson HPLC (Method A) and
subsequent neutralization of the combined fractions over
PL-HCO.sub.3 MP to give
6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-4-((S)-1-pyrimidin-2-yl-pyrr-
olidin-3-yloxy)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine as a
brown solid (17 mg, 21% yield) .sup.1H NMR (400 MHz, DMSO-d6, 298K)
8 ppm 2.23-2.43 (m, 2H) 2.85-2.99 (t, 2H) 3.22-3.94 (m, 9H)
4.08-4.27 (m, 2H) 5.70-5.80 (m, 1H) 6.56-6.66 (t, 1H) 7.76-7.87 (m,
1H) 8.12-8.27 (m, 1H) 8.28-8.42 (m, 2H) 8.59-8.68 (m, 1H) LCMS:
[M+H]+=474.2, Rt.sup.(1)=1.91 min.
[0650] Example 67 was prepared according the general procedure
described in scheme 4
##STR00138##
Example 67:
1-{(S)-3[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro--
pyrido[4,3-d]pyrimidin-4-ylamino]-pyrrolidin-1-yl}-propan-1-one
[0651] To a solution of
(S)-3[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyr-
ido[4,3-d]pyrimidin-4-ylamino]-pyrrolidine-1-carboxylic acid
tert-butyl ester (intermediate 24) (13.4 g, 27.1 mmol) in
CH.sub.2Cl.sub.2 (100 mL), was added TFA (41.8 mL) and the mixture
stirred at rt for 1 h. Concentrated in vacuo and partitioned
between 2M NaOH(aq) (300 mL) and CH.sub.2Cl.sub.2 (200 mL). The
organic phase was separated and the aqueous phase extracted with
CH.sub.2Cl.sub.2 (2.times.200 mL). The organic phases were
combined, dried (MgSO.sub.4) and evaporated in vacuo to give a
brown foam. The foam was dissolved in CH.sub.2Cl.sub.2 (50 mL) and
was added simultaneously portionwise with sat.NaHCO.sub.3(aq) (50
mL) to a vigorously stirring solution of propionyl chloride (2.63
g, 28.5 mmol) in CH.sub.2Cl.sub.2 (50 mL) at rt. The resulting
biphasic mixture was stirred at rt for 1 h. Further propionyl
chloride (0.566 g, 6.12 mmol) was added and continued stirring
vigorously for 20 min. The organic layer was separated and the
aqueous layer extracted with CH.sub.2Cl.sub.2 (100 mL). The organic
layers were combined, dried (MgSO.sub.4) and concentrated in vacuo
to give a brown gum. The gum was stirred in EtOAc (100 mL) and the
resulting solid filtered (9.4 g). The mother liquors were
concentrated in vacuo and purified by column chromatography through
a Biotage.RTM. amino silica gel eluting with EtOAc/MeOH, 100/0 to
90/10 to give a yellow foam which was then stirred in EtOAc (20 mL)
and the resulting solid filtered (870 mg). Both batches of solids
were combined and stirred in refluxing EtOAc (50 mL) for 1 h.
Filtered to give
1-{(S)-3[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro--
pyrido[4,3-d]pyrimidin-4-ylamino]-pyrrolidin-1-yl}-propan-1-one as
a colourless solid (9.42 g, 76% yield). .sup.1H NMR (400 MHz,
DMSO-d6, 298K) .delta. ppm 0.95-1.05 (m, 3H) 1.87-2.32 (m, 4H)
2.77-2.86 (m, 2H) 3.25-3.88 (m, 6H) 3.93 (s, 3H) 3.98 (s, 2H)
4.55-4.80 (m, 1H) 6.70-6.80 (m, 1H, N--H) 7.86-7.92 (m, 1H)
8.27-8.33 (m, 1H) 8.33-8.37 (m, 1H) LCMS: [M+H]+=451.0,
Rt.sup.(6)=1.49 min.
Alternative Synthesis for Example 67
[0652] A solution of
(S)-3[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyr-
ido[4,3-d]pyrimidin-4-ylamino]-pyrrolidine-1-carboxylic acid
tert-butyl ester (intermediate 24) (29.04 g, 58.73 mmol) in
2-Me-THF (100 mL) was dropwise added into aqueous HCl solution (150
mL, 31%) over 15 min. The reaction mixture was partitioned between
water (300 mL) and isopropyl acetate (100 mL) and the upper organic
phase was discarded. The aqueous phase was partitioned between 25%
NaOH (aq) (200 g) and 2-Me-THF (200 mL), and the organic phase was
collected and dried. Triethylamine (16.32 mL, 117.48 mmol) was
added into the organic phase followed by dropwise addition of
propionyl chloride (6.0 g, 64.6 mmol) at 0.degree. C. The resulting
mixture was stirred at 0.degree. C. for 1 h. The reaction mixture
was washed with water (110 mL) and the resulting organic phase was
concentrated in vacuo to give a brown gum. The residue was
recrystallized with isopropanol and methyl tert-butyl ether to give
1-{(S)-3-[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-
-pyrido[4,3-d]pyrimidin-4-ylamino]-pyrrolidin-1-yl}-propan-1-one as
a colourless solid (17.2 g, 65% yield).
Crystallization of Example 67 by Heating in Acetonitrile/Water
[0653] 2.0 g of Example 67 (4.440 mol) were dissolved in 10 mL of
acetonitrile and 0.5 mL of water at 75.degree. C. The solution was
allowed to cool down to rt within 30 min resulting in a suspension.
The mixture was stirred for 16 h at rt. The crystals were collected
by filtration. The filter cake was washed 2 times with 1 mL of
acetonitrile and afterwards dried for 16 h at 24.degree. C. and ca.
10 mbar vacuum. Elementary analysis of the material showed a
waterless form. List of most significant peaks from X-ray Powder
Diffraction Pattern of Example 67 anhydrous form Method X1):
TABLE-US-00013 2-Theta in deg Intensity in % 7.9 31 9.6 88 11.5 29
13.4 8 15.2 7 15.9 100 16.8 57 17.6 9 18.7 20 20.0 8 20.6 40 22.0
32 22.4 53 22.7 26 23.4 17 23.9 23 24.5 41 25.1 20 25.8 13 26.7
31
Preparation of Phosphate Salt of Example 67
[0654] 2.0 g of Example 67 (4.440 mol) were dissolved in 10 mL of
acetonitrile and 0.5 mL of water at 75.degree. C. 512 mg of
ortho-phosphoric acid 85% (4.440 mol) were added at 70.degree. C.
Crystallization occurs quickly at 70.degree. C. The suspension was
allowed to cool down to rt within 30 min. The suspension was
diluted with 10 ml acetonitrile and stirred for 16 h at rt. The
crystals were collected by filtration. The filter cake was washed 3
times with 1 mL of acetonitrile and afterwards dried for 16 h at
24.degree. C. and ca. 10 mbar vacuum. Elementary analysis of the
phosphate salt showed a 1:1 (waterless) form List of most
significant peaks from X-ray Powder Diffraction Pattern of Example
67 phosphate salt (Method X1):
TABLE-US-00014 2-Theta in deg Intensity in % 5.2 51 9.8 56 10.3 19
11.6 100 14.9 14 15.5 48 15.9 11 16.6 65 19.5 54 20.7 62 21.5 10
22.1 21 23.3 57 25.8 18 26.4 29 27.2 20 28.2 13
Preparation of Hydrochloride Salt of Example 67
[0655] 2.0 g of Example 67 (4.440 mol) were dissolved in 20 mL of
acetonitrile and 1.0 mL of water at 70.degree. C. 459 mg of
hydrochloric acid 37% (4.440 mol) were added at 70.degree. C.
Crystallization occurs quickly at 70.degree. C. The suspension was
allowed to cool down to rt within 30 min and stirred for 16 h at
rt. The crystals were collected by filtration. The filter cake was
washed 3 times with 1 mL of acetonitrile and afterwards dried for
16 h at 24.degree. C. and ca. 10 mbar vacuum. Elementary analysis
of the HCl salt showed a 1:1 (waterless) form List of most
significant peaks from X-ray Powder Diffraction Pattern of Example
67 hydrochloride salt (Method X1):
TABLE-US-00015 2-Theta in deg Intensity in % 5.6 100 11.0 18 11.3
42 11.8 12 14.7 33 17.1 13 18.7 19 19.4 29 22.0 23 22.6 28 23.1 50
23.7 28 24.9 29 25.5 15
Preparation of hippurate salt of Example 67
[0656] 0.4 g of Example 67 (0.888 mmol) were dissolved in 8 mL of
acetonitrile and 0.2 mL of water at 70.degree. C. 167 mg of
hippuric acid (0.888 mmol) were added at 70.degree. C. The solution
was allowed to cool down to rt within 30 min. Crystallization
occurs at 40.degree. C. The suspension was stirred for 16 h at rt.
The crystals were collected by filtration. The filter cake was
washed 3 times with 1 mL of acetonitrile and afterwards dried for
16 h at 50.degree. C. and ca. 10 mbar vacuum List of most
significant peaks from X-ray Powder Diffraction Pattern of Example
67 hippurate salt (Method X1):
TABLE-US-00016 2-Theta in deg Intensity in % 5.2 76 7.5 100 10.3 60
10.9 63 11.8 9 13.1 16 16.1 44 16.7 26 17.7 49 18.4 38 21.2 49 23.2
74 24.2 67 26.2 28
[0657] Examples 68-69 were prepared using procedures analogous to
those used in example 67 using appropriate starting materials.
TABLE-US-00017 Rt.sup.(1) MS: (min.) [M + H]+ Example 68
##STR00139## 1.26 507.2 Name:
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-
d]pyrimidin-4-ylamino]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone
Purification method: Reverse phase HPLC Method A Prepared using
tetrahydro-pyran-4-carbonyl chloride .sup.1H NMR (400 MHz, DMSO-d6,
298K) .delta. ppm 1.48-1.67 (m, 4H) 1.88-2.35 (m, 2H) 2.59- 2.87
(m, 3H) 3.26-4.03 (m, 15H) 4.56-4.83 (m, 1H) 6.82-6.92 (m, 1H,
N--H) 7.86-7.90 (m, 1H) 8.26--8.32 (m, 1H) 8.37-8.42 (m, 1H)
Rt.sup.(1) MS: (min.) [M + H]+ Example 69 ##STR00140## 1.06 464.2
Name:
2-Methoxy-5-{4-[(S)-1-(tetrahydro-pyran-4-carbonyl)-pyrrolidin-3-yla-
mino]-7,8- dihydro-5H-pyrido[4,3-d]pyrimidin-6-yl}-nicotinonitrile
Purification method: Reverse phase HPLC Method A Prepared using
intermediate 25 and tetrahydro-pyran-4-carbonyl chloride .sup.1H
NMR (400 MHz, MeOH-d4, 298K) .delta. ppm 1.59-1.86 (m, 4H)
2.07-2.47 (m, 2H) 2.75- 2.98 (m, 3H) 3.44-4.13 (m, 15H) 4.64-5.24
(m, 1H, signal masked by water peak) 7.94- 7.99 (m, 1H) 8.20-8.26
(m, 1H) 8.33-8.39 (m, 1H)
##STR00141##
Example 70:
1-(4-{(S)-3-[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-d]pyrimidin-4-ylamino]-pyrrolidine-1-carbonyl}-piperidin-1--
yl)-ethanone
[0658]
(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-d]pyrimidin-4-ylamino]-pyrrolidine-1-carboxylic acid
tert-butyl ester (intermediate 24) (160 mg, 0.32 mmol) was
dissolved in CH.sub.2Cl.sub.2 (2.0 mL) and TFA (1.0 mL) added. The
resulting mixture was stirred at room temperature for 1 h then
evaporated in vacuo to give
[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4-
,3-d]pyrimidin-4-yl]-(S)-pyrrolidin-3-yl-amine ditrifluoroacetate
as a brown gum (160 mg), which was used without further
purification. To
[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4-
,3-d]pyrimidin-4-yl]-(S)-pyrrolidin-3-yl-amine ditrifluoroacetate
(40 mg, 0.06 mmol) was added 1-acetylpiperidine-4-carboxylic acid
(12 mg, 0.07 mmol)), N,N-diisopropylethylamine (0.05 mL, 0.26
mmol), CH.sub.2Cl.sub.2 (3.0 mL) and then HBTU (29 mg, 0.08 mmol).
The mixture was allowed to stir at room temperature for 18 h and
then partitioned between CH.sub.2Cl.sub.2 (10 mL) and water (5 mL).
The organic phase was filtered through a phase separation tube and
evaporated in vacuo. Purification by reverse phase Gilson HPLC
(Method A) and subsequent neutralization of the combined fractions
by elution through an Isolute.RTM. SCX-2 cartridge, eluting with
methanol, then with 2M ammonia in methanol. Basic fractions were
concentrated in vacuo to give
1-(4-{(S)-3[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahyd-
ro-pyrido[4,3-d]pyrimidin-4-ylamino]-pyrrolidine-1-carbonyl}-piperidin-1-y-
l)-ethanone as a pale yellow solid (19 mg, 50% yield for 2.sup.nd
step) .sup.1H NMR (400 MHz, DMSO-d6, 298K) .delta. ppm 1.20-1.70
(m, 4H) 1.79-2.35 (m, 5H) 2.53-2.85 (m, 4H) 3.04-3.14 (m, 1H)
3.35-4.79 (m, 14H) 6.80-6.87 (m, 1H, N--H) 7.87-7.91 (m, 1H)
8.26-8.31 (m, 1H) 8.35-8.41 (m, 1H) LCMS: [M+H]+=548.2,
Rt.sup.(1)=1.22 min.
[0659] Examples 71-80 were prepared using procedures analogous to
those used in example 70 using appropriate starting materials
TABLE-US-00018 Rt.sup.(1) MS: (min.) [M + H]+ Example 71
##STR00142## 1.40 535.3 Name:
(2,2-Dimethyl-tetrahydro-pyran-4-yl)-{(S)-3-[6-(6-methoxy-5-trifluor-
omethyl-
pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-ylamino]-pyrroli-
din-1-yl}- methanone Purification method: Reverse phase HPLC Method
A Prepared using 2,2-dimethyl-tetrahydro-pyran-4-carboxylic acid
.sup.1H NMR (400 MHz, DMSO-d6, 298K) .delta. ppm 1.05-1.20 (m, 6H)
1.30-1.58 (m, 4H) 1.86- 2.35 (m, 2H) 2.70-2.90 (m, 3H) 3.34-4.03
(m, 13H) 4.55-4.80 (m, 1H) 6.67-6.76 (m, 1H, N--H) 7.86-7.89 (m,
1H) 8.26-8.31 (m, 1H) 8.32-8.37 (m, 1H) Rt.sup.(1) MS: (min.) [M +
H]+ Example 72 ##STR00143## 1.22 490.1 Name:
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-
d]pyrimidin-4-ylamino]-pyrrolidin-1-yl}-oxazol-5-yl-methanone
Purification method: Reverse phase HPLC Method A Prepared using
oxazole-5-carboxylic acid .sup.1H NMR (400 MHz, DMSO-d6, 298K)
.delta. ppm 1.89-2.39 (m, 2H) 2.78-2.86 (m, 2H) 3.50- 4.20 (m, 11H)
4.65-4.84 (m, 1H) 6.75-6.83 (m, 1H, N--H) 7.75-7.83 (m, 1H)
7.86-7.92 (m, 1H) 8.26-8.32 (m, 1H) 8.35-8.38 (m, 1H) 8.55-8.60 (m,
1H) Rt.sup.(1) MS: (min.) [M + H]+ Example 73 ##STR00144## 1.38
(Isomer 1) 535.2 (Isomer 1) Example 74 ##STR00145## 1.48 (Isomer 2)
Cis/trans not assigned 535.2 (Isomer 2) Name:
((S)-3-(6-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrah-
ydropyrido[4,3-
d]pyrimidin-4-ylamino)pyrrolidin-1-yl)((1R,4R)-4-methoxycyclohexyl)methano-
ne
((S)-3-(6-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydropy-
rido[4,3-
d]pyrimidin-4-ylamino)pyrrolidin-1-yl)((1R,4S)-4-methoxycyclohexyl)methano-
ne Purification method: Reverse phase HPLC Method A Prepared using
4-methoxy-cyclohexanecarboxylic acid (mixture of cis/trans) .sup.1H
NMR (Isomer 1 cis/trans not assigned) (400 MHz, DMSO-d6, 298K)
.delta. ppm 1.04-1.47 (m, 4H) 1.64-2.45 (m, 7H) 2.77-2.86 (m, 2H)
3.00-3.77 (m, 10H) 3.87-4.03 (m, 5H) 4.53- 4.80 (m, 1H) 6.67-6.78
(m, 1H, N--H) 7.85-7.91 (m, 1H) 8.26-8.32 (m, 1H) 8.33-8.38 (m, 1H)
.sup.1H NMR (Isomer 2 cis/trans not assigned) (400 MHz, DMSO-d6,
298K) .delta. ppm 1.32-1.48 (m, 4H) 1.55-2.50 (m, 7H) 2.78-2.84 (m,
2H) 3.01-3.77 (m, 10H) 3.87-4.03 (m, 5H) 4.53- 4.80 (m, 1H)
6.67-6.78 (m, 1H, N--H) 7.85-7.91 (m, 1H) 8.26-8.32 (m, 1H)
8.33-8.38 (m, 1H) Rt.sup.(1) MS: (min.) [M + H]+ Example 75
##STR00146## 1.18 (Isomer 1) 521.2 (Isomer 1) Example 76
##STR00147## 1.29 (Isomer 2) Cis/trans not assigned 521.2 (Isomer
2) Name:
((1S,4R)-4-hydroxycyclohexyl)((S)-3-(6-(6-methoxy-5-(trifluoromethyl-
)pyridin-3-
yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)met-
hanone
((1R,4S)-4-hydroxycyclohexyl)((S)-3-(6-(6-methoxy-5-(trifluoromethyl)pyrid-
in-3-yl)-
5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl)methano-
ne Purification method: Reverse phase HPLC Method A Prepared using
4-hydroxy-cyclohexanecarboxylic acid (mixture of cis/trans) .sup.1H
NMR (Isomer 1 cis/trans not assigned) (400 MHz, DMSO-d6, 298K)
.delta. ppm 1.06-1.44 (m, 4H) 1.57-2.86 (m, 9H) 3.01-3.76 (m, 7H)
3.88-4.03 (m, 5H) 4.50-4.78 (m, 2H) 6.68- 6.78 (m, 1H, N--H)
7.86-7.91 (m, 1H) 8.26-8.32 (m, 1H) 8.33-8.39 (m, 1H) 1H NMR
(Isomer 1 cis/trans not assigned) (400 MHz, DMSO-d6, 298K) .delta.
ppm 1.28-1.52 (m, 4H) 1.59-2.85 (m, 9H) 3.03-3.83 (m, 8H) 3.88-4.03
(m, 5H) 4.55-4.79 (m, 1H) 6.68- 6.77 (m, 1H, N--H) 7.85-7.91 (m,
1H) 8.26-8.32 (m, 1H) 8.33-8.39 (m, 1H) Rt.sup.(1) MS: (min.) [M +
H]+ Example 77 ##STR00148## 0.89 449.2 Name:
-{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyri-
do[4,3-
d]pyrimidin-4-ylamino]-pyrrolidin-1-yl}-(1-methyl-1H-imidazol-4-yl)-methan-
one Purification method: Reverse phase HPLC Method A Prepared using
intermediate 20 and 1-methyl-1H-imidazole-4-carboxylic acid .sup.1H
NMR (400 MHz, MeOH-d4, 298K) .delta. ppm 2.07-2.44 (m, 5H)
2.86-2.95 (m, 2H) 3.44- 4.43 (m, 14H) 4.77-4.87 (m, 1H) 7.44-7.48
(m, 1H) 7.60-7.70 (m, 2H) 7.72-7.79 m, 1H) 8.32-8.41 (m, 1H)
Rt.sup.(1) MS: (min.) [M + H]+ Example 78 ##STR00149## 0.99 436.2
Name:
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-
d]pyrimidin-4-ylamino]-pyrrolidin-1-yl}-oxazol-5-yl-methanone
Purification method: Reverse phase HPLC Method A Prepared using
intermediate 20 and oxazole-5-carboxylic acid .sup.1H NMR (400 MHz,
DMSO-d6, 298K) .delta. ppm 1.95-2.35 (m, 5H) 2.74-2.83 (m, 2H)
3.35- 4.20 (m, 11H) 4.62-4.83 (m, 1H) 6.73-6.81 (m, 1H) 7.44-7.49
(m, 1H) 7.75-7.85 (m, 2H) 8.33-8.38 (m, 1H) 8.54-8.59 (m, 1H)
Rt.sup.(1) MS: (min.) [M + H]+ Example 79 ##STR00150## 1.01 436.2
Name:
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-
d]pyrimidin-4-ylamino]-pyrrolidin-1-yl}-oxazol-4-yl-methanone
Purification method: Reverse phase HPLC Method A Prepared using
intermediate 20 and oxazole-4-carboxylic acid .sup.1H NMR (400 MHz,
DMSO-d6, 298K) .delta. ppm 1.93-2.35 (m, 5H) 2.75-2.82 (m, 2H)
3.38- 4.27 (m, 11H) 4.61-4.78 (m, 1H) 6.74-6.80 (m, 1H) 7.45-7.49
(m, 1H) 7.78-7.84 (m, 1H) 8.32-8.37 (m, 1H) 8.47-8.53 (m, 1H)
8.61-8.66 (m, 1H) Rt.sup.(1) MS: (min.) [M + H]+ Example 80
##STR00151## 1.18 481.3 Name:
(2,2-Dimethyl-tetrahydro-pyran-4-yl)-{(S)-3-[6-(6-methoxy-5-methyl-p-
yridin-3-yl)-
5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-ylamino]-pyrrolidin-1-yl}-meth-
anone Purification method: Reverse phase HPLC Method A Prepared
using intermediate 20 and
2,2-dimethyl-tetrahydro-pyran-4-carboxylic acid .sup.1H NMR (400
MHz, DMSO-d6, 298K) .delta. ppm 1.05-1.22 (m, 6H) 1.30-1.58 (m, 4H)
1.90- 2.29 (m, 5H) 2.75-2.85 (m, 3H) 3.35-3.77 (m, 7H) 3.82 (s, 3H)
3.87-3.97 (m, 3H) 4.54- 4.79 (m, 1H) 6.66-6.75 (m, 1H) 7.47 (d, 1H)
7.81 (d, 1H) 8.35 (d, 1H)
##STR00152##
Example 81:
1-{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4-
,3-d]pyrimidin-4-ylamino]-pyrrolidin-1-yl}-propan-1-one
[0660] To a solution of
(S)-3[6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d-
]pyrimidin-4-ylamino]-pyrrolidine-1-carboxylic acid tert-butyl
ester trifluoroacetate (intermediate 20) (60 mg, 0.11 mmol) in
CH.sub.2Cl.sub.2 (2.0 mL), was added TFA (2.0 mL) and the mixture
stirred at rt for 1 h. Concentrated in vacuo to give
[6-(6-methoxy-5-methylpyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimi-
din-4-yl)]-(S)-pyrrolidin-3-yl)amine ditrifluoroacetate (60 mg).
[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyri-
midin-4-yl]-(S)-pyrrolidin-3-yl)amine ditrifluoroacetate (30 mg,
0.053 mmol) was dissolved in CH.sub.2Cl.sub.2 (2.0 mL) and was
added simultaneously portionwise with sat.NaHCO.sub.3(aq) (2.0 mL)
to a vigorously stirring solution of propionyl chloride (7 mg, 0.07
mmol) in CH.sub.2Cl.sub.2 (2.0 mL) at rt. The resulting biphasic
mixture was stirred at rt for 45 min. Diluted with CH.sub.2Cl.sub.2
(10 mL) and sat.NaHCO.sub.3(aq) (2.0 mL). The organic layer was
separated by filtering through phase separation tube and
concentrated in vacuo. Purification by reverse phase Gilson HPLC
(Method A) and subsequent neutralization of the combined fractions
by elution through an Isolute.RTM. SCX-2 cartridge, eluting with
methanol, then with 2M ammonia in methanol. Basic fractions were
concentrated in vacuo gave
1-{(S)-3[6-(6-methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,-
3-d]pyrimidin-4-ylamino]-pyrrolidin-1-yl}-propan-1-one as a
colourless powder (7 mg, 21% yield) .sup.1H NMR (400 MHz,
MeOH-d.sub.4, 298K) .delta. ppm 1.20-1.28 (m, 3H) 2.04-2.44 (m, 7H)
2.88-2.94 (m, 2H) 3.48-4.04 (m, 11H) 4.73-4.88 (m, 1H) 7.44-7.48
(m, 1H) 7.73-7.77 (m, 1H) 8.34-8.38 (m, 1H) LCMS: [M+H]+=397.1,
Rt.sup.(3)=1.32 min.
[0661] Examples 82-83 were prepared using procedures analogous to
those used in Example 81 using appropriate starting materials.
TABLE-US-00019 Rt.sup.(1) MS: (min.) [M + H]+ Example 82
##STR00153## 1.13 473.2 Name:
{(S)-3-[6-(5-Chloro-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-
d]pyrimidin-4-ylamino]-pyrrolidin-1-yl}-tetrahydro-pyran-4-yl)-methanone
Purification method: Reverse phase HPLC Method A Prepared using
intermediate 21 and tetrahydro-pyran-4-carbonyl chloride .sup.1H
NMR (400 MHz, MeOH-d4, 298K) .delta. ppm 1.58-1.87 (m, 4H)
2.04-2.45 (m, 2H) 2.73- 2.96 (m, 3H) 3.39-4.14 (m, 15H) 4.71-4.90
(m, 1H) 7.67-7.74 (m, 1H) 7.88-7.93 (m, 1H) 8.34-8.39 (m, 1H)
Rt.sup.(7) MS: (min.) [M + H]+ Example 83 ##STR00154## 0.86 453.6
Name:
{(S)-3-[6-(6-Methoxy-5-methyl-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrid-
o[4,3-
d]pyrimidin-4-ylamino]-pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone
Purification method: Reverse phase HPLC Method A then Method C
Prepared using intermediate 20 and tetrahydro-pyran-4-carbonyl
chloride .sup.1H NMR (400 MHz, MeOD-d6, 298K) .delta. ppm 1.57-1.88
(m, 4H) 2.04-2.45 (m, 5H) 2.73- 2.96 (m, 3H) 3.37-4.12 (m, 15H)
4.73-4.88 (m, 1H) 7.45-7.48 (m, 1H) 7.73-7.77 (m, 1H) 8.36-8.39 (m,
1H)
##STR00155##
Example 84:
(Tetrahydro-pyran-4-yl)-{(S)-3-{6-(5-(trifluoromethyl)pyridin-3-yl)-5,6,7-
,8-tetrahydropyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl}-methanone
[0662] To
6-(5-(trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-
-d]pyrimidin-4-ol (intermediate 19) (59 mg) in acetonitrile (1.0
ml) was added BOP (114 mg, 0.258 mmol) and DBU (0.060 ml, 0.398
mmol). The resulting solution was stood at RT for 1 min then added
[(S)-3-Amino-pyrrolidin-1-yl-(tetrahydro-pyran-4-yl)-methanone
(intermediate 5) (79 mg, 0.398 mmol) in acetonitrile (1.0 ml) and
heated the mixture at 85.degree. C. for 25 h. The reaction mixture
was evaporated in vacuo and purified by reverse phase Gilson HPLC
and subsequent neutralization of the combined fractions by eluting
through an Isolute.RTM. SCX-2 cartridge, eluting with methanol,
then with 2M ammonia in methanol. Basic fractions were concentrated
in vacuo gave crude title compound which was further purified by
flash chromatography on silica gel with EtOAc/MeOH 100/0 to 80/20
to give
(Tetrahydro-pyran-4-yl)-{(S)-3-{6-(5-(trifluoromethyl)pyridin-3-yl)-5,6,7-
,8-tetrahydropyrido[4,3-d]pyrimidin-4-ylamino)pyrrolidin-1-yl}-methanone
(19 mg, 6% yield) as a colourless solid. .sup.1H NMR (400 MHz,
DMSO-d6, 298K) .delta. ppm 1.47-1.69 (m, 4H) 1.83-2.37 (m, 2H)
2.58-2.89 (m, 3H) 3.23-4.20 (m, 12H) 4.56-4.82 (m, 1H) 6.75-6.89
(m, 1H, N--H) 7.68-7.79 (m, 1H) 8.28-8.42 (m, 2H) 8.74-8.83 (m,
1H). LCMS: [M+H]+=477.6, Rt.sup.(7)=0.84 min.
##STR00156##
Example 85:
{(S)-3[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-py-
rido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(4-methyl-piperazin-1-yl)-m-
ethanone
[0663] To a solution of
6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-4-((S)-pyrrolidin-3-yloxy)-5-
,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine (prepared using step 1,
example 91 from intermediate 13) (23.0 mg, 0.058 mmol) and
triethylamine (0.016 mL, 0.116 mmol) in CH.sub.2Cl.sub.2 (2 mL) was
added 4-methylpiperazine-1-carbonyl chloride hydrochloride (11.6
mg, 0.058 mmol) and the mixture stirred at rt for 18 h. Diluted
with CH.sub.2Cl.sub.2 (10 mL) and washed with sat. NaHCO.sub.3(aq)
(2 mL). The organic layer was filtered through a phase separation
tube and evaporated in vacuo. Purification was performed by reverse
phase Gilson HPLC (Method A) and subsequent neutralization of the
combined fractions by elution through an Isolute.RTM. SCX-2
cartridge, eluting with methanol, then with 2M ammonia in methanol.
Basic fractions were concentrated in vacuo to give
{(S)-3[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahyd-
ro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(4-methyl-piperazin-1--
yl)-methanone (25 mg, 58% yield) as a yellow powder. .sup.1H NMR
(400 MHz, CDCl.sub.3, 298K) .delta. ppm 2.17-2.27 (m, 2H) 2.55 (s,
3H) 2.65-2.78 (m, 4H) 3.07 (t, 2H) 3.45-3.73 (m, 9H) 3.86-3.95 (m,
1H) 4.02 (s, 3H) 4.13 (s, 2H) 5.66-5.73 (m, 1H) 7.62 (d, 1H) 8.06
(d, 1H) 8.64 (s, 1H) LCMS: [M+H]+=522.3, Rt.sup.(1)=1.21 min.
[0664] Example 86 was prepared using procedures analogous to those
used in Example 85 using appropriate starting materials.
TABLE-US-00020 Rt.sup.(1) MS: (min.) [M + H]+ Example 86
##STR00157## 1.71 509.2 Name:
-{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrah-
ydro-pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-morpholin-4-yl-methanone
Purification method: Reverse phase HPLC Method A Prepared using
morpholine 4-carbonyl chloride .sup.1H NMR (400 MHz, MeOH-d4, 298K)
.delta. ppm 2.21-2.31 (m, 2H) 3.03 (t, 2H) 3.23-3.42 (m, 4H)
3.48-3.81 (m, 9H) 3.82-3.88 (m, 1H) 3.99 (s, 3H) 4.17 (s, 2H)
5.72-5.77 (m, 1H) 7.79 (d, 1H) 8.13 (d, 1H) 8.59 (s, 1H)
##STR00158##
Example 87:
(4-Hydroxy-piperidin-1-yl)-{(S)-3-[6-(6-methoxy-5-trifluoromethyl-pyridin-
-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-
-methanone
[0665] To CH.sub.2Cl.sub.2 (5 mL) in a round bottomed flask was
added phosgene (20% solution in toluene, 0.20 mL, 0.379 mmol) and
the resulting solution cooled to 5.degree. C. under argon. A
solution of
6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-4-((S)-pyrrolidin-3-yloxy)-5-
,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine (prepared using step 1,
example 91 from intermediate 13) (50.0 mg, 0.126 mmol) and
triethylamine (0.053 mL, 0.380 mmol) in CH.sub.2Cl.sub.2 (1.0 mL)
was added and the mixture allowed to warm to room temperature with
stirring under argon over 1 h. Evaporated to dryness by bubbling a
stream of argon into the mixture to give a brown gum. Dissolved in
CH.sub.2Cl.sub.2 (3 mL) to give
(S)-3-[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-py-
rido[4,3-d]pyrimidin-4-yloxy]-pyrrolidine-1-carbonyl chloride as a
solution in CH.sub.2Cl.sub.2. LCMS: 458.4 [M+1]+, Rt.sup.(7)=1.38
min. This solution was used without further purification. 1.5 mL of
this solution was added to a solution of piperidin-4-ol (6.4 mg,
0.063 mmol) and triethylamine (0.053 mL, 0.380 mmol) in
CH.sub.2Cl.sub.2 and the mixture stirred at room temperature under
argon for 1 h. N,N-dimethylformamide (0.5 mL) was added and
stirring continued for 2 h. Diluted with CH.sub.2Cl.sub.2 (2 mL)
and washed with sat. NaHCO.sub.3(aq) (2 mL). The organic layer was
filtered through a phase separation tube and evaporated in vacuo.
Purification by reverse phase Gilson HPLC (Method A) and subsequent
neutralization of the combined fractions by elution through an
Isolute.RTM. SCX-2 cartridge, eluting with methanol, then with 2M
ammonia in methanol. Basic fractions were concentrated in vacuo to
give
(4-hydroxy-piperidin-1-yl)-{(S)-3[6-(6-methoxy-5-trifluoromethyl-pyridin--
3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}--
methanone as a pale yellow powder (22 mg, 64% yield). .sup.1H NMR
(400 MHz, MeOH-d.sub.4, 298K) .delta. ppm 1.35-1.57 (m, 2H)
1.80-1.94 (m, 2H) 2.20-2.31 (m, 2H) 2.94-3.09 (m, 4H) 3.45-3.87 (m,
10H) 3.98 (s, 3H) 4.17 (s, 2H) 5.70-5.76 (m, 1H) 7.78 (d, 1H) 8.13
(d, 1H) 8.58 (s, 1H) LCMS: [M+H]+=523.2, Rt.sup.(1)=1.58 min.
[0666] Example 88 was prepared using procedures analogous to those
used in Example 87 using appropriate starting materials.
TABLE-US-00021 Rt.sup.(1) MS: (min.) [M + H]+ Example 88
##STR00159## 1.58 497.2 Name:
(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahyd-
ro-pyrido[4,3- d]pyrimidin-4-yloxy]-pyrrolidine-1-carboxylic acid
(2-hydroxy-ethyl)-methyl-amide Purification method: Reverse phase
HPLC Method A Prepared using 2-methylamino-ethanol .sup.1H NMR (400
MHz, MeOH-d4, 298K) .delta. ppm 2.20-2.32 (m, 2H) 2.97 (s, 3H)
2.98-3.06 (t, 2H) 3.27-3.38 (m, 1H) 3.40-3.80 (m, 8H) 3.82-3.89 (m,
1H) 3.98 (s, 3H) 4.18 (s, 2H) 5.71-5.76 (m, 1H) 7.78 (d, 1H) 8.14
(d, 1H) 8.58 (s, 1H)
##STR00160##
Example 89:
1-(4-{(S)-3[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahyd-
ro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidine-1-carbonyl}-piperazin-1-yl)-
-ethanone
[0667] To a solution of
6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-4-((S)-pyrrolidin-3-yloxy)-5-
,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine (prepared using step 1,
example 91 from intermediate 13) (25 mg, 0.063 mmol) and
triethylamine (0.013 mL, 0.095 mmol) in CH.sub.2Cl.sub.2 (2 mL) was
added 3-(4-acetyl-piperazine-1-carbonyl)-1-methyl-3H-imidazol-3-ium
iodide (Intermediate 6) (15 mg, 0.063 mmol) and the mixture stirred
at room temperature under argon for 18 h. Partitioned between
CH.sub.2Cl.sub.2 (10 mL) and sat. NaHCO.sub.3(aq) (2 mL) and the
organic layer was filtered through a phase separation tube and
evaporated in vacuo. Purification by reverse phase Gilson HPLC
(Method A) and subsequent neutralization of the combined fractions
by elution through an Isolute.RTM. SCX-2 cartridge, eluting with
methanol, then with 2M ammonia in methanol. Basic fractions were
concentrated in vacuo to give
1-(4-{(S)-3[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahyd-
ro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidine-1-carbonyl}-piperazin-1-yl)-
-ethanone as a pale yellow powder (9 mg, 25% yield). .sup.1H NMR
(400 MHz, MeOH-d.sub.4, 298K) .delta. ppm 2.14 (s, 3H) 2.24-2.33
(m, 2H) 3.04 (t, 2H) 3.25-3.91 (m, 13H) 3.99 (s, 3H) 4.18 (s, 2H)
5.74-5.78 (m, 1H) 7.79 (d, 1H) 8.14 (d, 1H) 8.60 (s, 1H) LCMS:
[M+H]+=550.2, Rt.sup.(1)=1.58 min.
##STR00161##
Example 90:
(S)-3[6-(5-cyano-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]-
pyrimidin-4-yloxy]-pyrrolidine-1-carboxylic acid methyl ester
[0668] To a solution of
2-methoxy-5-[4-((S)-pyrrolidin-3-yloxy)-7,8-dihydro-5H-pyrido[4,3-d]pyrim-
idin-6-yl]-nicotinonitrile (prepared using intermediate 11 and
method 1 b, process step 2, example 1) (25.0 mg, 0.071 mmol) and
triethylamine (0.04 mL, 0.29 mmol) in CH.sub.2Cl.sub.2 (2 mL) was
added methyl carbonochloridate (0.006 mL, 0.078 mmol) and the
mixture stirred at room temperature for 18 h. Diluted with
CH.sub.2Cl.sub.2 (2 mL) and washed with sat. NaHCO.sub.3(aq) (1
mL). The organic layer was filtered through a phase separation tube
and evaporated in vacuo.
[0669] Purification was performed by reverse phase Gilson HPLC
(Method A) and subsequent neutralization of the combined fractions
by elution through an Isolute.RTM. SCX-2 cartridge, eluting with
methanol, then with 2M ammonia in methanol. Basic fractions were
concentrated in vacuo gave
(S)-3-[6-(5-cyano-6-methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d-
]pyrimidin-4-yloxy]-pyrrolidine-1-carboxylic acid methyl ester (10
mg, 35% yield) as a yellow powder. .sup.1H NMR (400 MHz, DMSO-d6,
298K) .delta. ppm 2.09-2.31 (m, 2H) 2.91 (t, 2H) 3.45-3.75 (m, 9H)
3.93 (s, 3H) 4.17 (s, 2H) 5.58-5.65 (m, 1H) 8.09 (d, 1H) 8.27 (d,
1H) 8.61 (s, 1H) LCMS: [M+H]+=411.1, Rt.sup.(1)=1.58 min.
##STR00162##
Example 91:
{(S)-3[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-py-
rido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-oxazol-4-yl-methanone
Step 2
[0670] To the oxazole-4-carboxylic acid (27 mg, 0.24 mmol)) and
HBTU (89 mg, 0.24 mmol) in DMF (1 mL) was added
N,N-diisopropylethylamine (0.08 mL, 0.45 mmol). The mixture was
stirred for 20 min and then
6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-4-((S)-pyrrolidin-3-yloxy)-5-
,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine dihydrochloride (100 mg,
0.214 mmol) and additional N,N-diisopropylethylamine (0.08 mL, 0.45
mmol) were added. The mixture was allowed to stir at room
temperature for 30 min. Purified by reverse phase Gilson HPLC
(Method A) and subsequent neutralization of the combined fractions
over PL-HCO3 MP to give
{(S)-3[6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahydro-py-
rido[4,3-d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-oxazol-4-yl-methanone
as a yellow solid (38 mg, 36% yield) .sup.1H NMR (400 MHz, DMSO-d6,
298K) 8 ppm 2.11-2.39 (m, 2H) 2.80-3.01 (m, 2H) 3.22-4.29 (m, 11H)
5.59-5.80 (m, 1H) 7.72-7.94 (m, 1H) 8.10-8.29 (m, 1H) 8.41-8.55 (m,
1H) 8.57-8.77 (m, 2H) LCMS: [M+H]+=491.1, Rt.sup.(1)=1.69 min.
##STR00163##
6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-4-((S)-pyrrolidin-3-yloxy)-5-
,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine dihydrochloride
Step 1
[0671]
(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidine-1-carboxylic acid
tert-butyl ester (1.0 g, 1.69 mmol) in CH.sub.2Cl.sub.2 (5 mL) was
added 2M anhydrous HCl in diethyl ether (25.3 mL, 50.5 mmol) and
the mixture stirred at rt for 3 h. The resulting precipitate was
filtered and washed with diethyl ether to give
6-(6-methoxy-5-trifluoromethyl-pyridin-3-yl)-4-((S)-pyrrolidin-3-yloxy)-5-
,6,7,8-tetrahydro-pyrido[4,3-d]pyrimidine dihydrochloride as a
yellow solid (1.01 g, 128% yield). [M+H]+=396.0, Rt.sup.(4)=0.71
min. The free base can be generated by partitioning the
dihydrochloride salt between dichloromethane and 1N sodium
hydroxide solution (aq), separating the organic phase and
evaporating in vacuo. [M+H]+=396.0, Rt.sup.(4)=0.71 min.
[0672] Example 92 was prepared using procedures analogous to those
used in Example 91 using appropriate starting materials.
TABLE-US-00022 Rt.sup.(1) (min.) MS: [M + H]+ Example 92
##STR00164## 1.58 549.2 Name:
1-(4-{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-te-
trahydro-
pyrido[4,3-d]pyrimidin-4-yloxy]-pyrrolidine-1-carbonyl}-piperidin-1-yl)-et-
hanone Purification method: Reverse phase method A Prepared using
1-acetyl-piperidine-4-carboxylic acid .sup.1H NMR (400 MHz,
DMSO-d6, 298K) .delta. ppm 1.21-1.77 (m, 4 H) 1.92-2.02 (m, 3 H)
2.08- 2.36 (m, 2 H) 2.42-2.80 (m, 2 H) 2.88-2.98 (m, 2H) 3.00-3.18
(m, 1 H) 3.39-4.24 (m, 13 H) 5.60-5.74 (m, 1 H) 7.80-7.87 (m, 1 H)
8.15-8.22 (m, 1 H) 8.59-8.65 (m, 1 H) Rt.sup.(1) (min.) MS: [M +
H]+ Example 93 ##STR00165## 1.37 504.1 Name:
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-(3-methyl-3H-imidazol-4-yl)-methanon-
e Purification method: Reverse phase method A Prepared using
3-methyl-3H-imidazole-4-carboxylic acid .sup.1H NMR (400 MHz,
DMSO-d6, 298K) .delta. pm 2.14-2.38 (m, 2 H) 2.78-3.08 (m, 2 H)
3.44- 4.04 (m, 12 H) 4.08-4.27 (m, 2 H) 5.66-5.73 (m, 1 H)
7.32-7.57 (m, 1 H) 7.70-7.97 (m, 2 H) 8.13-8.28 (m, 1 H) 8.56-8.69
(m, 1 H) Rt.sup.(1) (min.) MS: [M + H]+ Example 94 ##STR00166##
0.95 490.9 Name:
{(S)-3-[6-(6-Methoxy-5-trifluoromethyl-pyridin-3-yl)-5,6,7,8-tetrahy-
dro-pyrido[4,3-
d]pyrimidin-4-yloxy]-pyrrolidin-1-yl}-oxazol-5-yl-methanone
Purification method: Reverse phase method A Prepared using
oxazole-5-carboxylic acid .sup.1H NMR (400 MHz, DMSO-d6, 298K)
.delta. ppm 2.28-2.43 (m, 2 H) 2.86-3.00 (m, 2 H) 3.39-4.27 (m, 11
H) 5.60-5.85 (m, 1 H) 7.72-7.91 (m, 2 H) 8.15-8.30 (m, 1 H) 8.53-
8.68 (m, 2 H)
[0673] Example 95 was prepared using procedures analogous to those
used in Example 1, method 1a using appropriate starting materials
according to scheme 8.
TABLE-US-00023 Structure Rt.sup.(3) (min.) MS: [M + H]+ Example 95
##STR00167## 1.39( 440.1 Name:
{(S)-3-[6-(6-Methoxy-pyridin-3-yl)-5,6,7,8-tetrahydro-pyrido[4,3-d]p-
yrimidin-4-
yloxy)pyrrolidin-1-yl}-(tetrahydro-pyran-4-yl)-methanone
Purification method: Flash-chromatography on silica gel with
CH.sub.2Cl.sub.2/ MeOH Prepared using
1-benzyl-1-methyl-4-oxo-piperidinium iodide (Ref: Tortolani, R.;
Org. Lett., Vol. 1, No 8, 1999) and 2-methoxypyridine .sup.1H NMR
(400 MHz, DMSO-d6, 298K) .delta. ppm 1.48-1.65 (m, 4H) 2.05-2.30
(m, 2H) 2.59- 2.78 (m, 1H) 2.85-2.93 (m, 2H) 3.25-4.11 (m, 15H)
5.59-5.73 (m, 1H) 6.73-6.79 (m, 1H) 7.53-7.59 (m, 1H) 7.86-7.89 (m,
1H) 8.58-8.64 (m, 1H).
[0674] The pharmaceutical composition or combination of the present
invention can be in unit dosage of about 1-2000 mg of active
ingredient(s) for a subject of about 50-70 kg, or about 1-500 mg or
about 1-250 mg or about 1-150 mg or about 0.5-100 mg, or about 1-50
mg of active ingredients. The therapeutically effective dosage of a
compound, the pharmaceutical composition, or the combinations
thereof, is dependent on the species of the subject, the body
weight, age and individual condition, the disorder or disease or
the severity thereof being treated. A physician, clinician or
veterinarian of ordinary skill can readily determine the effective
amount of each of the active ingredients necessary to prevent,
treat or inhibit the progress of the disorder or disease.
[0675] The above-cited dosage properties are demonstrable in vitro
and in vivo tests using advantageously mammals, e.g., mice, rats,
dogs, monkeys or isolated organs, tissues and preparations thereof.
The compounds of the present invention can be applied in vitro in
the form of solutions, e.g., aqueous solutions, and in vivo either
enterally, parenterally, advantageously intravenously, e.g., as a
suspension or in aqueous solution. The dosage in vitro may range
between about 10.sup.-3 molar and 10.sup.-9 molar concentrations. A
therapeutically effective amount in vivo may range depending on the
route of administration, between about 0.1-500 mg/kg, or between
about 1-100 mg/kg.
Biological Evaluation
[0676] The activity of a compound according to the present
invention can be assessed by the following in vitro & in vivo
methods.
Biological Assays
Determination of Enzymatic PI3K Alpha and PI3K Delta Isoform
Inhibition
1.1 Test of Lipid Kinase Activity
[0677] The efficacy of the compounds of examples 1-117 as PI3
kinase inhibitors can be demonstrated as follows:
[0678] The kinase reaction is performed in a final volume of 50
.mu.l per well of a half area COSTAR, 96 well plate. The final
concentrations of ATP and phosphatidyl inositol in the assay are 5
.mu.M and 6 .mu.g/mL, respectively. The reaction is started by the
addition of PI3 kinase, e.g. PI3 kinase 8.
[0679] p110.delta.. The components of the assay are added per well
as follows: [0680] 10 .mu.l test compound in 5% DMSO per well in
columns 2-1. [0681] Total activity is determined by addition 10
.mu.l of 5% vol/vol DMSO in the first 4 wells of column 1 and the
last 4 wells of column 12. [0682] The background is determined by
addition of 10 .mu.M control compound to the last 4 wells of column
1 and the first 4 wells of column 12. [0683] 2 mL `Assay mix` are
prepared per plate: [0684] 1.912 mL of HEPES assay buffer [0685]
8.33 .mu.l of 3 mM stock of ATP giving a final concentration of 5
.mu.M per well [0686] 1 .mu.l of [.sup.33P]ATP on the activity date
giving 0.05 .mu.Ci per well [0687] 30 .mu.l of 1 mg/mL PI stock
giving a final concentration of 6 .mu.g/mL per well [0688] 5 .mu.l
of 1 M stock MgCl.sub.2 giving a final concentration of 1 mM per
well [0689] 20 .mu.l of the assay mix are added per well. [0690] 2
mL `Enzyme mix` are prepared per plate (x* .mu.l PI3 kinase p110(3
in 2 mL of kinase buffer). The `Enzyme mix` is kept on ice during
addition to the assay plates. [0691] 20 .mu.l `Enzyme mix` are
added/well to start the reaction. [0692] The plate is then
incubated at room temperature for 90 minutes. [0693] The reaction
is terminated by the addition of 50 .mu.l WGA-SPA bead (wheat germ
agglutinin-coated Scintillation Proximity Assay beads) suspension
per well. [0694] The assay plate is sealed using TopSeal-S (heat
seal for polystyrene microplates, PerkinElmer LAS [Deutschland]
GmbH, Rodgau, Germany) and incubated at room temperature for at
least 60 minutes. [0695] The assay plate is then centrifuged at
1500 rpm for 2 minutes using the Jouan bench top centrifuge (Jouan
Inc., Nantes, France). [0696] The assay plate is counted using a
Packard TopCount, each well being counted for 20 seconds. * The
volume of enzyme is dependent on the enzymatic activity of the
batch in use.
[0697] In a more preferred assay, the kinase reaction is performed
in a final volume of 10 .mu.l per well of a low volume non-binding
CORNING, 384 well black plate (Cat. No. #3676). The final
concentrations of ATP and phosphatidyl inositol (PI) in the assay
are 1 .mu.M and 10 .mu.g/mL, respectively. The reaction is started
by the addition of ATP.
[0698] The components of the assay are added per well as
follows:
[0699] 50 nl test compounds in 90% DMSO per well, in columns 1-20,
8 concentrations (1/3 and 1/3.33 serial dilution step) in single.
[0700] Low control: 50 nl of 90% DMSO in half the wells of columns
23-24 (0.45% in final). [0701] High control: 50 nl of reference
compound (e.g. compound of Example 7 in WO 2006/122806) in the
other half of columns 23-24 (2.5 .mu.M in final). [0702] Standard:
50 nl of reference compound as just mentioned diluted as the test
compounds in columns 21-22. [0703] 20 mL `buffer` are prepared per
assay: [0704] 200 .mu.l of 1M TRIS HCl pH7.5 (10 mM in final)
[0705] 60 .mu.l of 1M MgCl.sub.2 (3 mM in final) [0706] 500 .mu.l
of 2M NaCl (50 mM in final) [0707] 100 .mu.l of 10% CHAPS (0.05% in
final) [0708] 200 .mu.l of 100 mM DTT (1 mM in final) [0709] 18.94
mL of nanopure water [0710] 10 mL `PI` are prepared per assay:
[0711] 200 .mu.l of 1 mg/mL I-alpha-Phosphatidylinositol (Liver
Bovine, Avanti Polar Lipids Cat. No. 840042C MW=909.12) prepared in
3% OctylGlucoside (10 .mu.g/mL in final) [0712] 9.8 mL of `buffer`
[0713] 10 mL `ATP` are prepared per assay: [0714] 6.7 .mu.l of 3 mM
stock of ATP giving a final concentration of 1 .mu.M per well 10 mL
of `buffer` [0715] 2.5 mL of each PI3K construct are prepared per
assay in `PI` with the following final concentration: [0716] 10 nM
PI3K alfa EMV B1075 [0717] 25 nM beta EMV BV949 [0718] 10 nM delta
EMV BV1060 [0719] 150 nM gamma EMV BV950 [0720] 5 .mu.l of
`PI/PI3K` are added per well. [0721] 5 .mu.l `ATP` are added per
well to start the reaction. [0722] The plates are then incubated at
room temperature for 60 minutes (alfa, beta, delta) or 120 minutes
(gamma). [0723] The reaction is terminated by the addition of 10
.mu.Kinase-Glo (Promega Cat. No. #6714). [0724] The assay plates
are read after 10 minutes in Synergy 2 reader (BioTek, Vermont USA)
with an integration time of 100 milliseconds and sensitivity set to
191. [0725] Output: The High control is around 60,000 counts and
the Low control is 30,000 or lower [0726] This luminescence assay
gives a useful Z' ratio between 0.4 and 0.7 The Z' value is a
universal measurement of the robustness of an assay. A Z' between
0.5 and 1.0 is considered an excellent assay.
[0727] For this assay, the PI3K constructs mentioned are prepared
as follows:
1.2 Generation of Gene Constructs
[0728] Two different constructs, BV 1052 and BV 1075, are used to
generate the PI3 Kinase .alpha. proteins for compound
screening.
PI3K.alpha. BV-1052 p85(iSH2)-Gly Linker-p110a(D20aa)-C-Term His
Tag
[0729] PCR products for the inter SH2 domain (iSH2) of the p85
subunit and for the p110-a subunit (with a deletion of the first 20
amino acids) are generated and fused by overlapping PCR.
[0730] The iSH2 PCR product is generated from first strand cDNA
using initially primers
TABLE-US-00024 gwG130-p01 (SEQ ID NO: 1)
(5'-CGAGAATATGATAGATTATATGAAGAAT-3') and gwG130-p02 (SEQ ID NO: 2)
(5'-TGGTTT-AATGCTGTTCATACGTTTGTCAAT-3').
[0731] Subsequently in a secondary PCR reaction, Gateway
(Invitrogen AG, Basel, Switzerland) recombination AttB1 sites and
linker sequences are added at the 5'end and 3'end of the p85 iSH2
fragment respectively, using primers
TABLE-US-00025 gwG130-p03 (SEQ ID NO: 3)
(5'-GGGACAAGTTTGTACAAAAAAGCAGGCTACGAAGGAGATATAC
ATAT-GCGAGAATATGATAGATTATATGAAGAAT-3') and gwG152-p04 (SEQ ID NO:
4) (5'-TACCATAATTCCACCACCACCACCGGAAATTCCCCCTGGTTT-
AATGCTGTTCATACGTTTGTCAAT-3').
[0732] The p110-a fragment is also generated from first strand
cDNA, initially using primers
TABLE-US-00026 gwG152-p01 (SEQ ID NO: 5)
(5'-CTAGTGGAATGTTTACTACCAAATGG-3') and gwG152-p02 (SEQ ID NO: 6)
(5'-GTTCAATG-CATGCTGTTTAATTGTGT-3').
[0733] In a subsequent PCR reaction, linker sequence and a
Histidine tag are added at the 5'end and 3'end of the p110-a
fragment respectively, using primers
TABLE-US-00027 gw152-p03 (SEQ ID NO: 7)
(5'-GGGGGAATTTCCGGTGGTGGTGGTGGAATTATGGTAC-
TAGTGGAATGTTTACTACC-AAATGGA-3') and gwG152-p06 (SEQ ID NO: 8)
(5'-AGCTCCGTGATGGTGATGGTGATGTGCTCCGTTCAATG-
CATGCTGTTTAATTGTGT-3').
[0734] The p85-iSH2/p110-a fusion protein is assembled in a third
PCR reaction by the overlapping linkers at the 3'end of the iSH2
fragment and the 5'end of the p110-a fragment, using the above
mentioned gwG130-p03 primer and a primer containing an overlapping
Histidine tag and the AttB2 recombination sequences
TABLE-US-00028 (SEQ ID NO: 9)
(5'-GGGACCACTTTGTACAAGAAAGCTGGGTTTAAGCTCCGTGATGGT
GATGGTGAT-GTGCTCC-3').
[0735] This final product is recombined in a (Invitrogen) OR
reaction into the donor vector pDONR201 to generate the ORF318
entry clone. This clone is verified by sequencing and used in a
Gateway LR reaction to transfer the insert into the Gateway adapted
pBlueBac4.5 (Invitrogen) vector for generation of the baculovirus
expression vector LR410.
PI3K.alpha. BV-1075 p85(iSH2)-12 XGly Linker-p110a(D20aa)-C-Term
his Tag
[0736] The construct for Baculovirus BV-1075 is generated by a
three-part ligation comprised of a p85 fragment and a p110-a
fragment cloned into vector pBlueBac4.5. The p85 fragment is
derived from plasmid p1661-2 digested with Nhe/Spe. The p110-a
fragment derived from LR410 (see above) as a SpeI/HindIII fragment.
The cloning vector pBlueBac4.5 (Invitrogen) is digested with
Nhe/HindIII. This results in the construct PED 153.8
[0737] The p85 component (iSH2) is generated by PCR using ORF 318
(described above) as a template and one forward primer KAC1028
(5'-GCTAGCATGCGAGAATATGATAGATTATATGAAGAATATACC) (SEQ ID NO: 10) and
two reverse primers,
TABLE-US-00029 KAC1029 (SEQ ID NO: 11)
(5'-GCCTCCACCACCTCCGCCTGGTTTAATGCTGTTCATACGTTTGTC) and KAC1039 (SEQ
ID NO: 12) (5'-TACTAGTCCGCCTCCACCACCTCCGCCTCCACCACCTCCGCC).
[0738] The two reverse primers overlap and incorporate the
12.times. Gly linker and the N-terminal sequence of the p110a gene
to the SpeI site. The 12.times. Gly linker replaces the linker in
the BV1052 construct. The PCR fragment is cloned into pCR2.1 TOPO
(Invitrogen). Of the resulting clones, p1661-2 is determined to be
correct. This plasmid is digested with Nhe and SpeI and the
resulting fragment is gel-isolated and purified for
sub-cloning.
[0739] The p110-a cloning fragment is generated by enzymatic digest
of clone LR410 (see above) with Spe I and HindIII. The SpeI site is
in the coding region of the p110a gene. The resulting fragment is
gel-isolated and purified for sub-cloning.
[0740] The cloning vector, pBlueBac4.5 (Invitrogen) is prepared by
enzymatic digestion with Nhe and HindIII. The cut vector is
purified with Qiagen (Quiagen N.V, Venlo, Netherlands) column and
then dephosphorylated with Calf Intestine alkaline phosphatase
(CIP) (New England BioLabs, Ipswich, Mass.). After completion of
the CIP reaction the cut vector is again column purified to
generate the final vector. A 3 part ligation is performed using
Roche Rapid ligase and the vendor specifications.
PI3K.beta. BV-949 p85(iSH2)-Gly Linker-p110b(Full-Length)-C-Term
His Tag
[0741] PCR products for the inter SH2 domain (iSH2) of the p85
subunit and for the full-length p110-b subunit are generated and
fused by overlapping PCR.
[0742] The iSH2 PCR product is generated from first strand cDNA
initially using primers
TABLE-US-00030 gwG130-p01 (SEQ ID NO: 1)
(5'-CGAGAATATGATAGATTATATGAAGAAT-3') and gwG130-p02 (SEQ ID NO: 2)
(5'-TGGTTT-AATGCTGTTCATACGTTTGTCAAT-3').
[0743] Subsequently, in a secondary PCR reaction Gateway
(Invitrogen) recombination AttB1 sites and linker sequences are
added at the 5'end and 3'end of the p85 iSH2 fragment respectively,
using primers
TABLE-US-00031 gwG130-p03 (SEQ ID NO: 3)
(5'-GGGACAAGTTTGTACAAAAAAGCAGGCTACGAAGGAGATA-
TACATATGCGAGAATATGATAGATTATATGAAGAAT-3') and gwG130-p05 (SEQ ID NO:
13) (5'-ACTGAAGCATCCTCCTCCTCCTCCTCCTGGTTTAAT-
GCTGTTCATACGTTTGTC-3').
[0744] The p110-b fragment is also generated from first strand cDNA
initially using primers
TABLE-US-00032 gwG130-p04 (SEQ ID NO: 4)
(5'-ATTAAACCAGGAGGAGGAGGAGGAGGATGCTTCAGTTTCATAA
TGCC-TCCTGCT-3')
which contains linker sequences and the 5'end of p110-b and
TABLE-US-00033 gwG130-p06 (SEQ ID NO: 14)
(5'-AGCTCCGTGATGGTGATGGTGATGTGCTCCAGATCTGTAGTCTTT-
CCGAACTGTGTG-3')
which contains sequences of the 3'end of p110-b fused to a
Histidine tag.
[0745] The p85-iSH2/p110-b fusion protein is assembled by an
overlapping PCR a reaction of the linkers at the 3'end of the iSH2
fragment and the 5'end of the p110-b fragment, using the above
mentioned gwG130-p03 primer and a primer containing an overlapping
Histidine tag and the AttB2 recombination sequences
(5'-GGGACCACTTTGTACAAGAAAGCTGGGTTT-AAGCTCCGTGATGGTGATGGTGATGTGCTCC-3')
(SEQ ID NO: 15).
[0746] This final product is recombined in a Gateway (Invitrogen)
OR reaction into the donor vector pDONR201 to generate the ORF253
entry clone. This clone is verified by sequencing and used in a
Gateway LR reaction to transfer the insert into the Gateway adapted
pBlueBac4.5 (Invitrogen) vector for generation of the baculovirus
expression vector LR280.
PI3K.delta. BV-1060 p85(iSH2)-Gly Linker-p110d(Full-Length)-C-Term
His Tag
[0747] PCR products for the inter SH2 domain (iSH2) of the p85
subunit and for the full-length p110-d subunit are generated and
fused by overlapping PCR.
[0748] The iSH2 PCR product is generated from first strand cDNA
using initially primers
TABLE-US-00034 gwG130-p01 (SEQ ID NO: 1)
(5'-CGAGAATATGATAGATTATATGAAGAAT-3') and gwG130-p02 (SEQ ID NO: 2)
(5'-TGGTTT-AATGCTGTTCATACGTTTGTCAAT-3').
[0749] Subsequently, in a secondary PCR reaction Gateway
(Invitrogen) recombination AttB1 sites and linker sequences are
added at the 5'end and 3'end of the p85 iSH2 fragment respectively,
using primers
TABLE-US-00035 gwG130-p03 (SEQ ID NO: 3)
(5'-GGGACAAGTTTGTACAAAAAAGCAGGCTACGAAGGAGATAT
ACAT-ATGCGAGAATATGATAGATTATATGAAGAAT-3') and gwG154-p04 (SEQ ID NO:
16) (5'-TCCTCCTCCTCCTCCTCCTGGTTTAATGCTGTTCATACGTT TGTC-3').
[0750] The p110-a fragment is also generated from first strand cDNA
using initially primers
TABLE-US-00036 gwG154-p01 (SEQ ID NO: 17)
(5'-ATGCCCCCTGGGGTGGACTGCCCCAT-3') and gwG154-p02 (SEQ ID NO: 18)
(5'-CTACTG-CCTGTTGTCTTTGGACACGT-3').
[0751] In a subsequent PCR reaction linker sequences and a
Histidine tag is added at the 5'end and 3'end of the p110-d
fragment respectively, using primers
TABLE-US-00037 gw154-p03 (SEQ ID NO: 19)
(5'-ATTAAACCAGGAGGAGGAGGAGGAGGACCCCCTGGGGTGGAC- TGCCCCATGGA-3') and
gwG154-p06 (SEQ ID NO: 20) (5'-AGCTCCGTGATGGTGAT-GGTGATGTGCT-
CCCTGCCTGTTGTCTTTGGACACGTTGT-3').
[0752] The p85-iSH2/p110-d fusion protein is assembled in a third
PCR reaction by the overlapping linkers at the 3'end of the iSH2
fragment and the 5'end of the p110-d fragment, using the above
mentioned gwG130-p03 primer and a primer containing an overlapping
Histidine tag and the Gateway (Invitrogen) AttB2 recombination
sequences
(5'-GGGACCACTTTGTA-CAAGAAAGCTGGGTTT-AAGCTCCGTGATGGTGATGGTGATGTGCTCC-3')
(SEQ ID NO: 21).
[0753] This final product is recombined in a Gateway (Invitrogen)
OR reaction into the donor vector pDONR201 to generate the ORF319
entry clone. This clone is verified by sequencing and used in a
Gateway LR reaction to transfer the insert into the Gateway adapted
pBlueBac4.5 (Invitrogen) vector for generation of the baculovirus
expression vector LR415.
PI3 K.gamma.BV-950 p110g(D144aa)-C-Term His Tag
[0754] This construct is obtained from Roger Williams lab, MRC
Laboratory of Molecular Biology, Cambridge, UK (November, 2003).
Description of the construct in: Pacold M. E. et al. (2000) Cell
103, 931-943.
1.3 Protein Expression and Purification
Methods to Generate Recombinant Baculovirus and Protein for PI3K
Isoforms:
[0755] The pBlue-Bac4.5 (for a, b, and d isoforms) or pVL1393 (for
g) plasmids containing the different PI3 kinase genes are
co-transfected with BaculoGold WT genomic DNA (BD Biosciences,
Franklin Lakes, N.J., USA) using methods recommended by the vendor.
Subsequently, the recombinant baculovirus obtained from the
transfection is plaque-purified on Sf9 insect cells to yield
several isolates expressing recombinant protein. Positive clones
are selected by anti-HIS or anti-isoform antibody western. For PI3K
alpha and delta isoforms, a secondary plaque-purification is
performed on the first clonal virus stocks of PI3K. Amplification
of all baculovirus isolates is performed at low multiplicity of
infection (moi) to generate high-titer, low passage stock for
protein production. The baculoviruses are designated BV1052
(.alpha.) and BV1075 (.alpha.), BV949 (.beta.), BV1060 (.delta.)
and BV950 (.gamma.).
[0756] Protein production involves infection (passage 3 or lower)
of suspended Tn5 (Trichoplusia ni) or TiniPro (Expression Systems,
LLC, Woodland, Calif., USA) cells in protein-free media at moi of
2-10 for 39-48 hours in 2 l glass Erlenmeyer flasks (110 rpm) or
wave-bioreactors (22-25 rpm). Initially, 10 I working volume
wave-bioreactors are seeded at a density of 3e5 cells/mL at half
capacity (5 L). The reactor is rocked at 15 rpm during the cell
growth phase for 72 hours, supplemented with 5% oxygen mixed with
air (0.2 I per minute). Immediately prior to infection, the
wave-reactor cultures are analyzed for density, viability and
diluted to approximately 1.5e6 cell/mL. 100-500 mL of high titer,
low passage virus is added following 2-4 hours of additional
culture. Oxygen is increased to 35% for the 39-48 hour infection
period and rocking platform rpm increased to 25. During infection,
cells are monitored by Vicell viability analyzer (Beckman Coulter,
Inc, Fullerton, Calif., USA) bioprocess for viability, diameter and
density. Nova Bioanalyzer (NOVA Biomedical Corp., Waltham, Mass.,
USA) readings of various parameters and metabolites (pH, O.sub.2
saturation, glucose, etc.) are taken every 12-18 hours until
harvest. The wave-bioreactor cells are collected within 40 hours
post infection. Cells are collected by centrifugation (4 degrees C.
at 1500 rpm), and subsequently maintained on ice during pooling of
pellets for lysis and purification. Pellet pools are made with
small amounts of cold, un-supplemented Grace's media (w/o protease
inhibitors).
PI3K Alpha Purification Protocol for HTS (BV1052)
[0757] PI3K alpha is purified in three chromatographic steps:
immobilized metal affinity chromatography on a Ni Sepharose resin
(GE Healthcare, belonging to General Electric Company, Fairfield,
Conn., USA), gel filtration utilizing a Superdex 200 26/60 column
(GE Healthcare), and finally a cation exchange step on a SP-XL
column (GE Healthcare). All buffers are chilled to 4.degree. C. and
lysis is performed chilled on ice. Column fractionation is
performed rapidly at room temperature.
[0758] Typically frozen insect cells are lysed in a hypertonic
lysis buffer and applied to a prepared IMAC column. The resin is
washed with 3-5 column volumes of lysis buffer, followed by 3-5
column volumes wash buffer containing 45 mM imidazole, and the
target protein is then eluted with a buffer containing 250 mM
imidazole. Fractions are analyzed by Coomassie stained SDS-PAGE
gels, and fractions containing target protein are pooled and
applied to a prepared GFC column. Fractions from the GFC column are
analyzed by Coomassie stained SDS-PAGE gels, and fractions
containing target protein are pooled. The pool from the GFC column
is diluted into a low salt buffer and applied to a prepared SP-XL
column. The column is washed with low salt buffer until a stable
A280 baseline absorbance is achieved, and eluted using a 20 column
volume gradient from 0 mM NaCl to 500 mM NaCl. Again, fractions
from the SP-XL column are analyzed by Coomassie stained SDS-PAGE
gels, and fractions containing the target protein are pooled. The
final pool is dialyzed into a storage buffer containing 50%
glycerol and stored at -20.degree. C. The final pool is assayed for
activity in a phosphoinosititol kinase assay.
PI3K Beta Purification Protocol for HTS (BV949)
[0759] PI3K beta is purified in two chromatographic steps:
immobilized metal affinity chromatography (IMAC) on a Ni Sepharose
resin (GE Healthcare) and gel filtration (GFC) utilizing a Superdex
200 26/60 column (GE Healthcare). All buffers are chilled to
4.degree. C. and lysis is performed chilled on ice. Column
fractionation is performed rapidly at room temperature.
[0760] Typically frozen insect cells are lysed in a hypertonic
lysis buffer and applied to a prepared IMAC column. The resin is
washed with 3-5 column volumes of lysis buffer, followed by 3-5
column volumes wash buffer containing 45 mM imidazole, and the
target protein is then eluted with a buffer containing 250 mM
imidazole. Fractions are analyzed by Coomassie stained SDS-PAGE
gels, and fractions containing target protein are pooled and
applied to a prepared GFC column. Fractions from the GFC column are
analyzed by Coomassie stained SDS-PAGE gels, and fractions
containing target protein are pooled. The final pool is dialyzed
into a storage buffer containing 50% glycerol and stored at
-20.degree. C. The final pool is assayed for activity in the
phosphoinostitol kinase assay.
PI3K Gamma Purification Protocol for HTS (BV950)
[0761] PI3K gamma is purified in two chromatographic steps:
immobilized metal affinity chromatography (IMAC) on a Ni Sepharose
resin (GE Healthcare) and gel filtration (GFC) utilizing a Superdex
200 26/60 column (GE Healthcare). All buffers are chilled to
4.degree. C. and lysis is performed chilled on ice. Column
fractionation is performed rapidly at room temperature. Typically
frozen insect cells are lysed in a hypertonic lysis buffer and
applied to a prepared IMAC column. The resin is washed with 3-5
column volumes of lysis buffer, followed by 3-5 column volumes wash
buffer containing 45 mM imidazole, and the target protein is then
eluted with a buffer containing 250 mM imidazole. Fractions are
analyzed by Coomassie stained SDS-PAGE gels, and fractions
containing target protein are pooled and applied to a prepared GFC
column. Fractions from the GFC column are analyzed by Coomassie
stained SDS-PAGE gels, and fractions containing target protein are
pooled. The final pool is dialyzed into a storage buffer containing
50% glycerol and stored at -20.degree. C. The final pool is assayed
for activity in the phosphoinostitol kinase assay.
PI3K Delta Purification Protocol for HTS (BV1060)
[0762] PI3K delta is purified in three chromatographic steps:
immobilized metal affinity chromatography on a Ni Sepharose resin
(GE Healthcare), gel filtration utilizing a Superdex 200 26/60
column (GE Healthcare), and finally a anion exchange step on a Q-HP
column (GE Healthcare). All buffers are chilled to 4.degree. C. and
lysis is performed chilled on ice. Column fractionation is
performed rapidly at room temperature. Typically frozen insect
cells are lysed in a hypertonic lysis buffer and applied to a
prepared IMAC column. The resin is washed with 3-5 column volumes
of lysis buffer, followed by 3-5 column volumes wash buffer
containing 45 mM imidazole, and the target protein is then eluted
with a buffer containing 250 mM imidazole. Fractions are analyzed
by Coomassie stained SDS-PAGE gels, and fractions containing the
target protein are pooled and applied to a prepared GFC column.
Fractions from the GFC column are analyzed by Coomassie stained
SDS-PAGE gels, and fractions containing the target protein are
pooled. The pool from the GFC column is diluted into a low salt
buffer and applied to a prepared Q-HP column. The column is washed
with low salt buffer until a stable A280 baseline absorbance is
achieved, and eluted using a 20 column volume gradient from 0 mM
NaCl to 500 mM NaCl. Again, fractions from the Q-HP column are
analyzed by Coomassie stained SDS-PAGE gels, and fractions
containing the target protein are pooled. The final pool is
dialyzed into a storage buffer containing 50% glycerol and stored
at -20.degree. C. The final pool is assayed for activity in the
phosphoinostitol kinase assay.
[0763] IC.sub.50 is determined by a four parameter curve fitting
routine that comes along with "excel fit". A four parameter
logistic equation is used to calculate IC.sub.50 values (IDBS
XLfit) of the percentage inhibition of each compound at 8
concentrations (usually 10, 3.0, 1.0, 0.3, 0.1, 0.030, 0.010 and
0.003 .mu.M). Alternatively, IC.sub.50 values are calculated using
idbsXLfit model 204, which is a 4 parameter logistic model.
[0764] Yet alternatively, for an ATP depletion assay, compounds of
the formula I to be tested are dissolved in DMSO and directly
distributed into a white 384-well plate at 0.5 .mu.l per well. To
start the reaction, 10 .mu.l of 10 nM PI3 kinase and 5 .mu.g/mL
1-alpha-phosphatidylinositol (P1) are added into each well followed
by 10 .mu.l of 2 .mu.M ATP. The reaction is performed until approx
50% of the ATP is depleted, and then stopped by the addition of 20
.mu.l of Kinase-Glo solution (Promega Corp., Madison, Wis., USA).
The stopped reaction is incubated for 5 minutes and the remaining
ATP is then detected via luminescence. IC.sub.50 values are then
determined.
[0765] Some of the compounds of examples 1-49 and 51-95 show a
certain level of selectivity against the different paralogs PI3K
.alpha., .beta., .gamma. and .delta..
[0766] Suitably, the compounds of examples 1-49 and 51-95 show a
certain level of selectivity for the isoform PI3K.delta., e.g. as
indicated in in vitro and in vivo tests against the different
paralogs PI3K.alpha. and .quadrature..beta..
[0767] The range of activity, expressed as IC.sub.50, in these
assays, is preferably between 1 nM and 5000 nM, more preferably
between 1 nM and about 1000 nM.
2. Cellular Assays
2.1 Phosphoinositide-3 Kinase (PI3K)-Mediated Akt 1/2 (S473)
Phosphorylation in Rat-1 Cells
[0768] Rat-1 cells stably overexpressing a myristoylated form of
the catalytic subunit of human phosphoinositide-3 kinase (PI3K)
alpha, beta or delta were plated in 384-well plates at a density of
7500 (PI3K alpha), 6200 (PI3K beta), or 4000 (PI3K delta) cells in
30 ul complete growth medium (Dulbecco's modified Eagle's medium
(DMEM high glucose) supplemented with 10% (v/v) fetal bovine serum,
1% (v/v) MEM non essential amino acids, 10 mM HEPES, 2 mM
L-glutamine, 10 .mu.g/mL puromycin and 1% (v/v)
Penicillin/Streptomycin) and were incubated at 37% C/5%
CO.sub.2/95% humidity for 24 h. Compounds were diluted in 384-well
compound plates to obtain 8-point serial dilutions for 40 test
compounds in 90% DMSO, as well as 4 reference compounds plus 16
high controls and 16 low (inhibited) controls. Predilution plates
were prepared by dispensing pipetting 250 nl of compound solutions
into 384-well polypropylen plates using a Hummingwell nanoliter
dispensor. Compounds were prediluted by the addition of 49.75 ul
complete growth medium. 10 ul of prediluted compound solution were
transferred to the cell plate using a 384-well pipettor, resulting
in a final DMSO concentration of 0.11%. Cells were incubated for 1
h at 37% C/5% CO.sub.2/95% humidity. The supernatant was removed,
the cells were lysed in 20 ul of lysis buffer for AlphaScreen.RTM.
SureFire.RTM. detection.
[0769] For detection of p-AKT(Ser473), the SureFire.RTM. p-Akt 1/2
(Ser473) Assay Kit (PerkinElmer, U.S.A) was used. 5 ul of cell
lysate was transferred to 384-well low volume Proxiplates for
detection using a 384-well pipettor. Addition of AlphaScreen.RTM.
SureFire.RTM. reagents was done according to the manufacturer's
protocol. First, 5 ul of reaction buffer plus activation buffer mix
containing AlphaScreen.RTM. acceptor beads was added, the plate was
sealed, and incubated on a plate shaker for 2 hours at room
temperature. Second, 2 ul of dilution buffer containing
AlphaScreen.RTM. donor beads was added, and the plate was incubated
on plate shaker as above for a further 2 hours. The plate was read
on an AlphaScreen.RTM. compatible plate reader, using standard
AlphaScreen.RTM. settings.
2.2 Determination of Murine B Cell Activation
[0770] PI3K.delta. has been recognized to modulate B cell function
when cells are stimulated through the B cell receptor (BCR)
(Okkenhaug et al. Science 297:1031 (2002). For assessing the
inhibitory property of compounds on B cell activation, the
upregulation of activation markers CD86 and CD69 on murine B cells
derived from mouse spleen antibody is measured after stimulation
with anti-IgM. CD69 is a well known activation marker for B and T
cells (Sancho et al. Trends Immunol. 26:136 (2005). CD86 (also
known as B7-2) is primarily expressed on antigen-presenting cells,
including B cells. Resting B cells express CD86 at low levels, but
upregulate it following stimulation of e.g. the BCR or IL-4
receptor. CD86 on a B cell interacts with CD28 on T cells. This
interaction is required for optimal T cell activation and for the
generation of an optimal IgG1 response (Carreno et al. Annu Rev
Immunol. 20:29 (2002)).
[0771] Spleens from Balb/c mice were collected, splenocytes were
isolated and washed twice with RPMI containing 10% foetal bovine
serum (FBS), 10 mM HEPES, 100 Units/mL penicilline/streptomycine.
RPMI supplemented in this way is subsequently referred to as
medium. The cells were adjusted to 2.5.times.10.sup.6 cells/mL in
medium and 200 .mu.l cell suspension (5.times.10.sup.6 cells) were
added to the appropriate wells of 96 well plates.
[0772] Then the cells were stimulated by adding 50 .mu.l anti-IgM
mAb in medium (final concentration: 30 .mu.g/mL). After incubation
for 24 hours at 37.degree. C., the cells were stained with the
following antibody cocktails: anti-mouse CD86-FITC, anti-mouse
CD69-PerCP-Cy5.5, anti-mouse CD19-PerCP for the assessment of B
cells, and anti-mouse CD3-FITC, anti-mouse CD69-PE for the
assessment of T cells (2 .mu.l of each antibody/well). After one
hour at room temperature (RT) in the dark the cells were
transferred to 96 Deepwell plates. The cells were washed once with
1 mL PBS containing 2% FBS and after re-suspension in 200 .mu.l the
samples were analyzed on a FACS Calibur flow cytometer. Lymphocytes
were gated in the FSC/SSC dot plot according to size and
granularity and further analyzed for expression of CD19, CD3 and
activation markers (CD86, CD69). Data were calculated from dot
blots as percentage of cells positively stained for activation
markers within the CD19+ or CD3+ population using BD CellQest
Software.
[0773] For assessing the inhibitory property of compounds,
compounds were first dissolved and diluted in DMSO followed by a
1:50 dilution in medium. Splenocytes from Balb/c mice were
isolated, re-suspended and transferred to 96 well plates as
described above (200 .mu.l/well). The diluted compounds or solvent
were added to the plates (25 .mu.l) and incubated at 37.degree. C.
for 1 hour. Then the cultures were stimulated with 25 .mu.l
anti-IgM mAb/well (final concentration 30 .mu.g/mL) for 24 hours at
37.degree. C. and stained with anti-mouse CD86-FITC and anti-mouse
CD19-PerCP (2 .mu.l of each antibody/well). CD86 expression on CD19
positive B cells was quantified by flow cytometry as described
above.
3 Determination of Antibody Production to Sheep Red Blood Cells
(SRBC).
[0774] In brief, OFA rats were injected i.v. with sheep
erythrocytes on d0 and treated orally on four consecutive days (d0
to d3) with the compounds under investigation. Spleen cell
suspensions were prepared on d4 and lymphocytes were plated onto
soft agar in presence of indicator cells (SRBC) and complement.
Lysis of the indicator cells due to secretion of SRBC-specific
antibody (predominantly of the IgM subclass) and presence of
complement yielded plaques. The number of plaques per plate were
counted and expressed as number of plaques per spleen.
Immunization:
[0775] Groups of five female OFA rats were immunized on day 0 with
2.times.10.sup.8/ml SRBC (obtained from Laboratory Animal Services
LAS, Novartis Pharma AG) in a volume of 0.5 ml per rat by i.v.
injection.
Compound Treatment:
[0776] Animals were treated with compound suspended in 0.5% CMC,
0.5% Tween80 in for 4 consecutive days (days 0, 1, 2 and 3)
starting on the day of immunization. Compound was administered
orally twice daily with 12 hours intervalls between doses in an
application volume of 5 ml/kg body weight.
Preparation of Spleen Cell Suspensions:
[0777] On day 4, animals were euthanized with CO.sub.2 Spleens were
removed, weighed, and deposited in plastic tubes containing 10 ml
of cold (4.degree. C.) Hank's balanced salt solution (HBSS; Gibco,
pH 7.3, containing 1 mg Phenolred/100 ml) for each rat spleen.
Spleens were homogenized with a glass potter, left on ice for 5
minutes and 1 ml supernatant was transferred into a new tube. Cells
were washed once in 4 ml HBSS then supernatants were discarded and
pellets re-suspended in 1 ml of HBSS. Lymphocyte numbers per spleen
were determined by automated cell counter and spleen cell
suspensions were adjusted to a cell concentration of
30.times.10.sup.6/ml.
Plaque Forming Assay:
[0778] Soft agar petri dishes were prepared with 0.7% agarose
(SERVA) in HBSS.
[0779] In addition, one ml of 0.7% agarose was prepared in plastic
tubes and kept at 48.degree. C. in a water bath. Some 50 .mu.l of a
30.times.10.sup.6/ml spleen cell suspension and 50 .mu.l of SRBC at
40.times.10.sup.8/ml were added, mixed rapidly (Vortex) and poured
onto the prepared agarose dishes. Petri dishes were slightly tilted
to achieve even distribution of cell mixture on agarose layer. The
dishes were left at room temperature for 15 minutes and were then
incubated at 37.degree. C. for 60 minutes. Then, 1.4 ml guinea pig
complement (Harlan; 10%) was added and the incubation continued for
another 60 minutes at 37.degree. C. SRBC-specific antibodies
released by the plated-out B cells bound to the antigen (SRBC) in
their vicinity. These antigen-antibody complexes activated
complement and led to the lysis of the SRBC leaving a bright spot
(plaque) within the red erythrocyte layer. Plaques were counted
with a microscope.
[0780] The following formula for determination of inhibition of
plaque formation was used:
% Inhibition=C*100/V-100
with: V=mean number of plaques/spleen for vehicle group; C=mean
number of plaques/spleen for compound treated group
REFERENCES
[0781] N. K. Jerne & A. A. Nordin (1963) Plaque formation in
agar by single antibody-producing cells. Science 140:405. [0782] N.
K. Jerne, A. A. Nordin & C. Henry (1963) The agar plaque
technique for recognizing antibody-producing cells. In: "Cell Bound
Antibodies", B. Amos & H. Koprowski, Eds., Wistar Inst. Press,
Philadelphia pp. 109-125.
Biological Data
Enzymatic Assay
TABLE-US-00038 [0783] Example PI3K alpha (uM) PI3K delta (uM) 1
2.0378 0.015 2 3.391 0.009 3 2.386 0.015 4 1.764 0.033 5 0.749
0.020 6 0.987 0.044 7 1.973 0.013 8 2.494 0.027 9 2.906 0.009 10
0.668 0.009 11 1.199 0.011 12 0.952 0.012 13 1.802 0.013 14 1.832
0.013 15 1.631 0.014 16 1.684 0.016 17 7.678 0.017 18 0.871 0.033
19 3.056 0.033 20 1.839 0.048 21 0.320 0.008 22 0.580 0.008 23
0.129 0.010 24 0.374 0.009 25 0.820 0.026 26 0.368 0.021 27 3.410
0.040 28 1.214 0.004 29 2.585 0.011 30 2.831 0.040 31 3.024 0.021
32 2.036 0.023 33 1.967 0.018 34 1.648 0.014 35 4.232 0.049 36
4.103 0.025 37 7.021 0.031 38 3.306 0.016 39 0.434 0.009 40 0.260
0.006 41 0.515 0.014 42 0.863 0.013 43 0.728 0.016 44 1.189 0.016
45 0.860 0.018 46 0.803 0.027 47 0.656 0.025 48 0.518 0.029 49
0.388 0.034 51 0.912 0.044 52 1.024 0.046 53 0.504 0.006 54 0.384
0.005 55 0.661 0.005 56 0.860 0.013 57 0.590 0.025 58 3.060 0.030
59 9.100 0.028 60 3.333 0.045 61 0.589 0.012 62 0.489 0.023 63
0.791 0.051 64 2.331 0.032 65 0.738 0.023 66 1.280 0.014 67 0.262
0.023 68 0.043 0.007 69 0.056 0.003 70 0.121 0.006 71 0.057 0.003
72 0.093 0.004 73 0.054 0.004 74 0.113 0.004 75 0.118 0.004 76
0.106 0.007 77 1.290 0.044 78 0.384 0.012 79 0.781 0.017 80 0.430
0.016 81 0.651 0.02 82 0.066 0.003 83 0.432 0.017 84 0.058 0.009 85
0.569 0.021 86 1.330 0.020 87 0.452 0.012 88 1.336 0.034 89 1.189
0.029 90 1.991 0.038 91 0.924 0.011 92 2.545 0.009 93 0.872 0.024
94 1.714 0.021 95 0.757 0.053
Cellular Assays
TABLE-US-00039 [0784] Cell PI3K.delta./ mCD86/IC50 Example IC50
[umol I-1] CD86 [nmol I-1] 1 0.153 94.9 5 0.455 125 20 0.2538 120
28 0.268 71.6 65 0.191 570 67 0.047 48.3 68 0.053 14.9 71 0.035
24.7 81 0.246 86.5 82 0.116 46.2
SRBC Assay
TABLE-US-00040 [0785] Plaques/spleen Example 1 12608 .+-. 4986 10
mg/kg bid Vehicle (0.5% CMC 168363 .+-. 49142 0.5% Tween80)
* * * * *