U.S. patent application number 16/306235 was filed with the patent office on 2020-07-09 for substituted indazoles useful for treatment and prevention of allergic and/or inflammatory diseases in animals.
The applicant listed for this patent is BAYER ANIMAL HEALTH GMBH BAYER PHARMA AKTIENGESELLSCHAFT. Invention is credited to Gerald BEDDIES, Ulf BOMER, Ulrich BOTHE, Adrian FOSTER, Maria De Lourdes MOTTIER, Reinhard NUBBEMEYER, Nicole SCHMIDT.
Application Number | 20200216413 16/306235 |
Document ID | / |
Family ID | 56097024 |
Filed Date | 2020-07-09 |
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United States Patent
Application |
20200216413 |
Kind Code |
A1 |
BEDDIES; Gerald ; et
al. |
July 9, 2020 |
SUBSTITUTED INDAZOLES USEFUL FOR TREATMENT AND PREVENTION OF
ALLERGIC AND/OR INFLAMMATORY DISEASES IN ANIMALS
Abstract
The present application relates to the use of substituted
indazoles for treatment and/or prophylaxis of allergic and/or
inflammatory diseases in animals, especially for treatment and/or
prophylaxis of atopic dermatitis, Flea Allergy Dermatitis,
inflammatory bowel disease, osteoarthritis and inflammatory pain,
non-infectious recurrent airway disease, insect hypersensitivity,
asthma, respiratory disease, mastitis and endometritis in
animals.
Inventors: |
BEDDIES; Gerald;
(Leverkusen, DE) ; FOSTER; Adrian; (Sandwich,
GB) ; BOTHE; Ulrich; (Berlin, DE) ; SCHMIDT;
Nicole; (Wuppertal, DE) ; BOMER; Ulf;
(Glienicke, DE) ; NUBBEMEYER; Reinhard; (Berlin,
DE) ; MOTTIER; Maria De Lourdes; (Langenfeld,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAYER ANIMAL HEALTH GMBH
BAYER PHARMA AKTIENGESELLSCHAFT |
Leverkusen
Berlin |
|
DE
DE |
|
|
Family ID: |
56097024 |
Appl. No.: |
16/306235 |
Filed: |
May 29, 2017 |
PCT Filed: |
May 29, 2017 |
PCT NO: |
PCT/EP2017/062876 |
371 Date: |
November 30, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 11/06 20180101;
A61P 37/00 20180101; A61P 11/04 20180101; A61P 1/04 20180101; A61P
19/00 20180101; A61P 29/00 20180101; A61P 11/00 20180101; A61P
15/00 20180101; A61P 1/00 20180101; A61P 37/08 20180101; A61P 17/00
20180101; C07D 405/14 20130101; A61P 19/02 20180101; A61P 43/00
20180101; C07D 401/12 20130101 |
International
Class: |
C07D 401/12 20060101
C07D401/12; C07D 405/14 20060101 C07D405/14 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 1, 2016 |
EP |
16172544.5 |
Claims
1. A compound of the general formula (I) ##STR00062## in which:
R.sup.1 is C.sub.1-C.sub.6-alkyl, where the C.sub.1-C.sub.6-alkyl
group is unsubstituted or mono- or polysubstituted identically or
differently by halogen, hydroxyl, an unsubstituted or mono- or
poly-halogen-substituted C.sub.3-C.sub.6-cycloalkyl, or an R.sup.6,
R.sup.7SO.sub.2, R.sup.7SO or R.sup.8O group, or a group selected
from: ##STR00063## R.sup.2 and R.sup.3 always have the same
definition and are both either hydrogen or C.sub.1-C.sub.6-alkyl;
R.sup.4 is halogen, cyano, an unsubstituted or a singly or
multiply, identically or differently substituted
C.sub.1-C.sub.6-alkyl or an unsubstituted or a singly or multiply,
identically or differently substituted C.sub.3-C.sub.6-cycloalkyl,
and the substituents are selected from the group of halogen and
hydroxyl; R.sup.5 is hydrogen, halogen or an unsubstituted or mono-
or poly-halogen-substituted C.sub.1-C.sub.6-alkyl; R.sup.6 is an
unsubstituted or mono- or di-methyl-substituted monocyclic
saturated heterocycle having 4 to 6 ring atoms, which contains a
heteroatom or a heterogroup from the group of O, S, SO and
SO.sub.2; R.sup.7 is C.sub.1-C.sub.6-alkyl, where the
C.sub.1-C.sub.6-alkyl group is unsubstituted or mono- or
polysubstituted identically or differently by halogen, hydroxyl or
C.sub.3-C.sub.6-cycloalkyl; or R.sup.7 is
C.sub.3-C.sub.6-cycloalkyl; R.sup.8 is C.sub.1-C.sub.6-alkyl, where
the C.sub.1-C.sub.6-alkyl group is unsubstituted or mono- or
polysubstituted identically or differently by halogen; and a
diastereomers, an enantiomers, a metabolites, a salt, a solvate, a
solvate of the salts thereof, for use in the treatment and/or
prophylaxis of allergic and/or inflammatory diseases in an
animal.
2. The compound according to claim 1, where R.sup.1 is
C.sub.1-C.sub.6-alkyl, where the C.sub.1-C.sub.6-alkyl group is
unsubstituted or mono- or polysubstituted identically or
differently by fluorine, hydroxyl or an R.sup.6, R.sup.7SO.sub.2,
R.sup.7SO or R.sup.8O group; R.sup.2 and R.sup.3 always have the
same definition and are both either hydrogen or
C.sub.1-C.sub.3-alkyl; R.sup.4 is halogen, cyano or
C.sub.1-C.sub.3-alkyl, where the C.sub.1-C.sub.3-alkyl group is
unsubstituted or mono- or polysubstituted identically or
differently by halogen or hydroxyl; R.sup.5 is hydrogen, fluorine,
chlorine or C.sub.1-C.sub.3-alkyl; R.sup.6 is oxetanyl or
tetrahydrofuranyl; R.sup.7 is C.sub.1-C.sub.4-alkyl, where the
C.sub.1-C.sub.4-alkyl group is unsubstituted or monosubstituted by
hydroxyl or by cyclopropyl or substituted by three fluorine atoms;
R.sup.8 is an unsubstituted C.sub.1-C.sub.4-alkyl group or a
tri-fluorine-substituted C.sub.1-C.sub.4-alkyl group.
3. The compound according to claim 1, where R.sup.4 is
difluoromethyl, trifluoromethyl or methyl.
4. The compound according to claim 1, where R.sup.5 is hydrogen or
fluorine.
5. The compound according to claim 1, where R.sup.2 and R.sup.3 are
both either hydrogen or methyl.
6. The compound according to claim 2, where R.sup.1 is
C.sub.2-C.sub.6-alkyl, where the C.sub.2-C.sub.6-alkyl group is
unsubstituted, or the C.sub.2-C.sub.6-alkyl group is mono-, di- or
tri-fluorine-substituted or the C.sub.2-C.sub.6-alkyl group is
monosubstituted by hydroxyl, R.sup.6, R.sup.7SO.sub.2, or R.sup.8O,
or R.sup.1 is an oxetanyl-substituted C.sub.1-C.sub.3-alkyl group;
R.sup.2 and R.sup.3 always have the same definition and are both
either hydrogen or methyl; R.sup.4 is an unsubstituted or mono- or
poly-halogen-substituted C.sub.1-C.sub.3-alkyl group or a
C.sub.1-C.sub.3-alkyl group substituted by one hydroxyl group or a
C.sub.1-C.sub.3-alkyl group substituted by one hydroxyl group and
three fluorine atoms; R.sup.5 is hydrogen, fluorine or
C.sub.1-C.sub.3-alkyl; R.sup.7 is C.sub.1-C.sub.3-alkyl; R.sup.8 is
C.sub.1-C.sub.4-alkyl, where the C.sub.1-C.sub.4-alkyl group is
unsubstituted or mono-, di- or tri-fluorine-substituted.
7. The compound according to claim 6, in which R.sup.1 is a
C.sub.2-C.sub.5-alkyl group substituted by hydroxyl or
C.sub.1-C.sub.3-alkoxy or trifluoromethoxy or 2,2,2-trifluoroethoxy
or trifluoromethyl or is a methyl-SO.sub.2-substituted
C.sub.2-C.sub.4-alkyl group or is an oxetan-3-yl-substituted
C.sub.1-C.sub.2-alkyl group; R.sup.2 and R.sup.3 always have the
same definition and are both hydrogen or methyl; R.sup.4 is methyl,
ethyl, trifluoro-C.sub.1-C.sub.3-alkyl,
difluoro-C.sub.1-C.sub.3-alkyl, hydroxymethyl, 1-hydroxyethyl,
2-hydroxypropan-2-yl and 2,2,2-trifluoro-1-hydroxyethyl; R.sup.5 is
hydrogen, fluorine or methyl.
8. The compound according to claim 7, in which R.sup.1 is
4,4,4-trifluorobutyl, 3-hydroxy-3-methylbutyl, 3-hydroxybutyl,
3-methoxypropyl, 3-hydroxypropyl, 3-hydroxy-2-methylpropyl,
3-hydroxy-2,2-dimethylpropyl, 3-trifluoromethoxypropyl,
2-methoxyethyl, 2-hydroxyethyl, 2-(methyl sulphonyl)ethyl or
3-(methyl sulphonyl)propyl; R.sup.2 and R.sup.3 are both methyl or
hydrogen; R.sup.4 is difluoromethyl, trifluoromethyl or methyl;
R.sup.5 is hydrogen or fluorine.
9. The compound according to claim 8, in which R.sup.1 is
3-hydroxy-3-methylbutyl, 3-hydroxybutyl, 3-hydroxy-2-methylpropyl,
3-hydroxy-2,2-dimethylpropyl, 3-(methyl sulphonyl)propyl or
2-(methyl sulphonyl)ethyl; R.sup.2 and R.sup.3 are both methyl;
R.sup.4 is difluoromethyl or trifluoromethyl; R.sup.5 is
hydrogen.
10. The compound according to claim 8, in which R.sup.1 is
3-hydroxy-3-methylbutyl, 3-hydroxybutyl, 3-hydroxy-2-methylpropyl,
3-hydroxy-2,2-dimethylpropyl, 3-(methyl sulphonyl)propyl or
2-(methyl sulphonyl)ethyl; R.sup.2 and R.sup.3 are both methyl;
R.sup.4 is methyl; R.sup.5 is fluorine, where R.sup.5 is in the
ortho position to R.sup.4.
11. The compound according to claim 1 which is selected from the
group consisting of: 1)
N-[6-(2-Hydroxypropan-2-yl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-6-(triflu-
oromethyl)pyridine-2-carboxamide; 2)
N-[6-(Hydroxymethyl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-6-(trifluorometh-
yl)pyridine-2-carboxamide; 3)
N-[6-(2-Hydroxypropan-2-yl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(trifl-
uoromethyl)pyridine-2-carboxamide; 4)
N-[6-(Hydroxymethyl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(trifluoromet-
hyl)pyridine-2-carboxamide; 5)
N-[2-(2-Hydroxyethyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(triflu-
oromethyl)pyridine-2-carboxamide; 6)
N-[6-(2-Hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]-6-(trifl-
uoromethyl)pyridine-2-carboxamide; 7)
N-[2-(2-Hydroxyethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluorometh-
yl)pyridine-2-carboxamide; 8)
N-[6-(2-Hydroxypropan-2-yl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(tri-
fluoromethyl)pyridine-2-carboxamide; 9)
N-[6-(Hydroxymethyl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(trifluorom-
ethyl)pyridine-2-carboxamide; 10)
N-{6-(2-Hydroxypropan-2-yl)-2-[3-(methylsulphonyl)propyl]-2H-indazol-5-yl-
}-6-(trifluoromethyl)pyridine-2-carboxamide; 11)
N-[2-(3-Hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]--
6-(trifluoromethyl)pyridine-2-carboxamide; 12)
N-{6-(2-Hydroxypropan-2-yl)-2-[2-(methyl
sulphonyl)ethyl]-2H-indazol-5-yl}-6-(trifluoromethyl)pyridine-2-carboxami-
de; 13)
6-(Difluoromethyl)-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxyprop-
an-2-yl)-2H-indazol-5-yl]pyridine-2-carboxamide; 14)
6-(Difluoromethyl)-N-{6-(2-hydroxypropan-2-yl)-2-[2-(methyl
sulphonyl)ethyl]-2H-indazol-5-yl}pyridine-2-carboxamide; 15)
6-(Difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-ind-
azol-5-yl]pyridine-2-carboxamide; 16)
N-[6-(2-Hydroxypropan-2-yl)-2-(4,4,4-trifluorobutyl)-2H-indazol-5-yl]-6-(-
trifluoromethyl)pyridine-2-carboxamide; 17)
N-{6-(2-Hydroxypropan-2-yl)-2-[3-(trifluoromethoxy)propyl]-2H-indazol-5-y-
l}-6-(trifluoromethyl)pyridine-2-carb oxamide; 18)
N-{6-(2-Hydroxypropan-2-yl)-2-[3-(2,2,2-trifluoroethoxy)propyl]-2H-indazo-
l-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide; 19)
5-Fluoro-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indaz-
ol-5-yl]-6-methylpyridine-2-carboxamide; 20)
N-[2-(3-Hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]--
6-methylpyridine-2-carboxamide; 21)
6-(2-Hydroxypropan-2-yl)-N-[6-(2-hydroxypropan-2-yl)-2-(4,4,4-trifluorobu-
tyl)-2H-indazol-5-yl]pyridine-2-carboxamide; and 22)
N-{2-[2-(1-Hydroxycyclopropyl)ethyl]-6-(2-hydroxypropan-2-yl)-2H-indazol--
5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide and a diastereomer,
an enantiomer, a metabolite, a salt, a solvate, and a solvate of
the salt thereof.
12. Compound of the general formula (I) as defined in claim 1 for
use in the treatment and/or prophylaxis of allergic and/or
inflammatory diseases in domestic animals.
13. Compound of the general formula (I) as defined in claim 1 for
use in the treatment and/or prophylaxis of allergic and/or
inflammatory diseases in farm animals.
14. Compound of the general formula (I) as defined in claim 1, for
use in a method for treatment and/or prophylaxis of atopic
dermatitis, Flea Allergy Dermatitis, inflammatory bowel disease,
osteoarthritis and inflammatory pain, non-infectious recurrent
airway disease, insect hypersensitivity, asthma, respiratory
disease, mastitis and endometritis in animals.
15. Compound of the general formula (I) as defined in claim 1 for
use in a method for treatment and/or prophylaxis of Canine Atopic
Dermatitis, Flea Allergy Dermatitis in dogs or cats, inflammatory
bowel disease in dogs or cats, osteoarthritis and inflammatory pain
in dogs, cats, horses or cattle, non-infectious recurrent airway
disease in horses, insect hypersensitivity in horses, feline
asthma, bovine respiratory disease, mastitis in cattle,
endometritis in cattle, and swine respiratory disease.
16. Compound of the general formula (I) as defined in claim 1 for
use in a method for treatment and/or prophylaxis of Canine Atopic
Dermatitis and Flea Allergy Dermatitis in dogs or cats.
17. Compound of the general formula (I) as defined in claim 1 for
use in a method for treatment and/or prophylaxis of osteoarthritis
and inflammatory pain in cattle, bovine respiratory disease,
mastitis in cattle, endometritis in cattle, and swine respiratory
disease.
18. A method for the production of a medicament for treatment
and/or prophylaxis of allergic and/or inflammatory disease in an
animal, the method comprising producing the medicament with a
compound of the general formula (I) as defined in claim 1.
19. The method of claim 18, wherein the medicament is used for
treatment and/or prophylaxis in an animal of a disease or disorder
selected from the group consisting of atopic dermatitis, Flea
Allergy Dermatitis, inflammatory bowel disease, osteoarthritis and
inflammatory pain, non-infectious recurrent airway disease, insect
hypersensitivity, asthma, respiratory disease, mastitis and
endometritis.
20. The method of claim 18, wherein the animal is a domestic
animal.
21. The method of claim 18, wherein the animal is a farm
animal.
22. The method of claim 18 for treatment and/or prophylaxis of a
disease or disorder selected from the group consisting of Canine
Atopic Dermatitis, Flea Allergy Dermatitis in a dog or a cat,
inflammatory bowel disease in a dog or a cat, osteoarthritis and
inflammatory pain in a dog, a cat, a horse, or cattle,
non-infectious recurrent airway disease in a horse, insect
hypersensitivity in a horse, feline asthma, bovine respiratory
disease, mastitis in cattle, endometritis in cattle, and swine
respiratory disease.
23. The method of claim 18 for treatment and/or prophylaxis of
Canine Atopic Dermatitis or Flea Allergy Dermatitis in a dog or a
cat.
24. Medicament comprising a compound of the formula (I) as defined
in claim 1 in combination with an inert, non-toxic,
pharmaceutically suitable excipient, for use in a method of
treatment and/or prophylaxis of allergic and/or inflammatory
disease in an animal.
25. Method for treatment and/or prevention of allergic and/or
inflammatory disease in an animal by administering an effective
amount of at least a compound of the formula (I) of claim 1 to an
animal in need thereof.
26. A compound of the general formula (III) ##STR00064## in which
R.sup.1 is 4,4,4-trifluorobutyl, 3-hydroxy-3-methylbutyl,
3-methoxypropyl, 3-hydroxypropyl, 3-hydroxybutyl,
3-hydroxy-2-methylpropyl, 3-hydroxy-2,2-dimethylpropyl,
3-trifluoromethoxypropyl, 2-methoxyethyl, 2-hydroxyethyl, 2-(methyl
sulphonyl)ethyl, 3-(methyl sulphonyl)propyl or
2-(1-hydroxycyclopropyl)ethyl; R.sup.4 is difluoromethyl,
trifluoromethyl or methyl; and R.sup.5 is hydrogen or fluorine; and
a diastereomer, an enantiomer, a metabolite, a salt, a solvate or a
solvate of the salts thereof, for use in the treatment and/or
prophylaxis of allergic and/or inflammatory diseases in an
animal.
27. The Compound of claim 26 selected from the group consisting of:
methyl
5-{[(5-fluoro-6-methylpyridin-2-yl)carbonyl]amino}-2-(3-hydroxy-3--
methylbutyl)-2H-indazole-6-carboxylate, and methyl
2-(3-hydroxy-3-methylbutyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl-
}amino)-2H-indazole-6-carboxylate, for use in the treatment and/or
prophylaxis of allergic and/or inflammatory diseases in an animal.
Description
[0001] The present application relates to the use of novel
substituted indazoles for treatment and/or prophylaxis of allergic
and/or inflammatory diseases in animals and to the use thereof for
production of medicaments for treatment and/or prophylaxis of
allergic and/or inflammatory diseases in animals, especially of
atopic dermatitis and/or Flea Allergy Dermatitis, and especially in
domestic animals, particularly in dogs.
[0002] The present invention relates to the use of novel
substituted indazoles of the general formula (I) which inhibit
interleukin-1 receptor-associated kinase 4 (IRAK4).
[0003] Human IRAK4 (interleukin-1 receptor-associated kinase 4)
plays a key role in the activation of the immune system. Therefore,
this kinase is an important therapeutic target molecule for the
development of inflammation-inhibiting substances. IRAK4 is
expressed by a multitude of cells and mediates the signal
transduction of Toll-like receptors (TLRs), except TLR3, and
receptors of the interleukin (IL)-1.beta. family consisting of the
IL-1R (receptor), IL-18R, IL-33R and IL-36R (Janeway and Medzhitov,
Annu. Rev. Immunol., 2002; Dinarello, Annu. Rev. Immunol., 2009;
Flannery and Bowie, Biochemical Pharmacology, 2010).
[0004] Neither IRAK4 knockout mice nor human cells from patients
lacking IRAK4 react to stimulation by TLRs (except TLR3) and the
IL-1.beta. family (Suzuki, Suzuki, et al., Nature, 2002; Davidson,
Currie, et al., The Journal of Immunology, 2006; Ku, von Bernuth,
et al., JEM, 2007; Kim, Staschke, et al., JEM, 2007).
[0005] The binding of the TLR ligands or the ligands of the IL-I 3
family to the respective receptor leads to recruitment and binding
of MyD88 [Myeloid differentiation primary response gene (88)] to
the receptor. As a result, MyD88 interacts with IRAK4, resulting in
the formation of an active complex which interacts with and
activates the kinases IRAK1 or IRAK2 (Kollewe, Mackensen, et al.,
Journal of Biological Chemistry, 2004; Precious et al., J. Biol.
Chem., 2009). As a result of this, the NF (nuclear factor)-.kappa.B
signalling pathway and the MAPK (mitogen-activated protein kinase)
signal pathway is activated (Wang, Deng, et al., Nature, 2001). The
activation both of the NF-.kappa.B signal pathway and of the MAPK
signal pathway leads to processes associated with different immune
processes. For example, there is increased expression of various
inflammatory signal molecules and enzymes such as cytokines,
chemokines and COX-2 (cyclooxygenase-2), and increased mRNA
stability of inflammation-associated genes, for example COX-2, IL-6
(interleukin-6), IL-8 (Holtmann, Enninga, et al., Journal of
Biological Chemistry, 2001; Datta, Novotny, et al., The Journal of
Immunology, 2004). Furthermore, these processes may be associated
with the proliferation and differentiation of particular cell
types, for example monocytes, macrophages, dendritic cells, T cells
and B cells (Wan, Chi, et al., Nat Immunol, 2006; McGettrick and J.
O'Neill, British Journal of Haematology, 2007).
[0006] The central role of IRAK4 in the pathology of various
inflammatory disorders had already been shown by direct comparison
of wild-type (WT) mice with genetically modified animals having a
kinase-inactivated form of IRAK4 (IRAK4 KDKI). IRAK4 KDKI animals
have an improved clinical picture in the animal model of multiple
sclerosis, atherosclerosis, myocardial infarction and Alzheimer's
disease (Rekhter, Staschke, et al., Biochemical and Biophysical
Research Communication, 2008; Maekawa, Mizue, et al., Circulation,
2009; Staschke, Dong, et al., The Journal of Immunology, 2009; Kim,
Febbraio, et al., The Journal of Immunology, 2011; Cameron, Tse, et
al., The Journal of Neuroscience, 2012). Furthermore, it was found
that deletion of IRAK4 in the animal model protects against
virus-induced myocarditis by an improved anti-viral reaction with
simultaneously reduced systemic inflammation (Valaperti, Nishii, et
al., Circulation, 2013). It has also been shown that the expression
of IRAK4 correlates with the disease activity of
Vogt-Koyanagi-Harada syndrome (Sun, Yang, et al., PLoS ONE, 2014).
In addition, the high relevance of IRAK4 for immune
complex-mediated IFN.alpha. (interferon-alpha) production by
plasmacytoid dendritic cells, a key process in the pathogenesis of
systemic lupus erythematosus (SLE), has been shown (Chiang et al.,
The Journal of Immunology, 2010). Furthermore, the signalling
pathway is associated with obesity (Ahmad, R., P. Shihab, et al.,
Diabetology & Metabolic Syndrome, 2015). As well as the
essential role of IRAK4 in congenital immunity, there are also
hints that IRAK4 influences the differentiation of Th17 T cells,
components of adaptive immunity. In the absence of IRAK4 kinase
activity, fewer IL-17-producing T cells (Th17 T cells) are
generated compared to WT mice. The inhibition of IRAK4 enables the
prophylaxis and/or treatment of atherosclerosis, type 1 diabetes
mellitus, rheumatoid arthritis, spondyloarthritis (especially
psoriatic spondyloarthritis and Bekhterev's disease), lupus
erythematosus, psoriasis, vitiligo, giant cell arteritis, chronic
inflammatory bowel disorder and viral disorders, for example HIV
(human immunodeficiency virus), hepatitis virus (Staschke, et al.,
The Journal of Immunology, 2009; Marquez, et al., Ann Rheum Dis,
2014; Zambrano-Zaragoza, et al., International Journal of
Inflammation, 2014; Wang, et al., Experimental and Therapeutic
Medicine, 2015; Ciccia, et al., Rheumatology, 2015).
[0007] Due to the central role of IRAK4 in the MyD88-mediated
signal cascade of TLRs (except TLR3) and the IL-1 receptor family,
the inhibition of IRAK4 can be utilized for the prophylaxis and/or
treatment of disorders mediated by the receptors mentioned.
[0008] The prior art discloses a multitude of IRAK4 inhibitors
(see, for example, Annual Reports in Medicinal Chemistry (2014),
49, 117-133).
[0009] U.S. Pat. No. 8,293,923 and US20130274241 disclose IRAK4
inhibitors having a 3-substituted indazole structure. There is no
description of 2-substituted indazoles.
[0010] WO2013106254 and WO2011153588 disclose 2,3-disubstituted
indazole derivatives. WO2007091107 describes 2-substituted indazole
derivatives for the treatment of Duchenne muscular dystrophy. The
compounds disclosed do not have 6-hydroxyalkyl substitution.
[0011] WO2015091426 describes indazoles, such as Example 64,
substituted at the 2 position by a carboxamide side chain.
##STR00001##
Example 64
[0012] WO2015104662 discloses 2-substituted indazoles of the
following general formula:
##STR00002##
in which R.sub.2 is an alkyl or cycloalkyl group. There are
explicit descriptions of 2-substituted indazoles having a methyl,
2-methoxyethyl and cyclopentyl group at the 2 position (Examples 1,
4 and 76). Also described by Example 117 is an indazole derivative
having a hydroxyethyl substituent at the 1 position. However, no
indazole derivatives having a 3-hydroxy-3-methylbutyl substituent
at the 1 position or 2 position are described.
[0013] Indazoles having a hydroxyl-substituted alkyl group in the 2
position are encompassed generically by the general formula, but
are not disclosed explicitly in WO2015104662.
[0014] Indazoles having an alkyl group in the 2 position where the
alkyl group is additionally substituted by a methylsulphonyl group
are not encompassed by the general formula and the definitions of
the R.sub.2 substituents in WO2015104662.
[0015] In addition to the above-described substitution pattern on
the indazole in 1 and 2 positions, WO2015104662 describes indazoles
having substitution at the 6 position for which R.sub.1 is defined
as follows: alkyl, cyano, --NR.sub.aR.sub.b or optionally
substituted groups selected from cycloalkyl, aryl or heterocyclyl,
where the substituents are independently alkyl, alkoxy, halogen,
hydroxyl, hydroxyalkyl, amino, aminoalkyl, nitro, cyano, haloalkyl,
haloalkoxy, --OCOCH.sub.2--O-alkyl, --OP(O)(O-alkyl).sub.2 or
--CH.sub.2--OP(O)(O-alkyl).sub.2. For indazole compounds in which
R.sub.1 is an alkyl group, the effective filing date is 7 Jan. 2015
(international filing date of WO2015104662). The Indian
applications 146/CHE/2014 and 3018/CHE/2014 whose priority is
claimed do not disclose any indazole compounds for which R.sub.1 is
an alkyl group.
[0016] Thus, indazole compounds of the following general
formula:
##STR00003##
in which R.sub.1 is an optionally substituted alkyl group are
described for the first time on 7 Jan. 2015 and hence after the
priority date of the present application.
[0017] Examples of substituents at the 6 position described in
WO2015104662 for R.sub.1 are cyclopropyl, cyclohexyl, cyano,
3-fluorophenyl and saturated heterocyclic substituents. Indazoles
having a hydroxyl-substituted alkyl group at position 6 are not
described explicitly in WO2015104662.
[0018] The compounds used in the present invention are also
described in copending patent application PCT/EP2015/077596,
published as WO2016083433 on 2 Jun. 2016.
[0019] Current treatment options for allergic and/or inflammatory
diseases in animals, for example for allergic skin diseases,
typically include the use of steroids and cyclosporine--both are
associated with side effects. Recently a janus-kinase (JAK)
inhibitor has been approved for use in Canine Atopic Dermatitis
(CAD) that symptomatically provides relief from pruritus, however,
the dosing regimen may again be limited by side effects. The
treatment of CAD with a disease modifying agent and without
treatment-related side effects remains an unmet medical need.
[0020] The problem addressed by the present invention is that of
providing a better treatment option for inflammatory and/or
allergic diseases in animals.
[0021] The present IRAK4 inhibitors are especially suitable for
treatment and for prevention of inflammatory disorders in animals
characterized by an overreacting immune system. Particular mention
should be made here of Canine Atopic Dermatitis, Flea Allergy
Dermatitis in dogs and cats, inflammatory bowel disease in dogs and
cats, osteoarthritis and inflammatory pain in dogs, cats, horses
and cattle, non-infectious recurrent airway disease in horses (also
known as chronic obstructive pulmonary disease, heaves), insect
hypersensitivity in horses (also known as sweet itch, summer
eczema), feline asthma, bovine respiratory disease, mastitis and
endometritis in cattle, and swine respiratory disease.
[0022] Atopic dermatitis, for example, is a common disease in
companion animals, particularly in cats and dogs.
[0023] As one specific example, Canine atopic dermatitis (CAD) is
one of the commonest diseases of dogs. CAD can affect patients from
an early age, recurring throughout their lifetime. In a study of
Lund et al. 1999, that investigated 31,484 dogs in 52 private
practices in the US, the prevalence of CAD was 8.7%. CAD is the
second most common cause of canine pruritus after Flea Allergy
Dermatitis (FAD).
[0024] Canine atopic dermatitis can be defined as a `genetically
predisposed inflammatory and pruritic allergic skin disease with
characteristic clinical features associated with IgE, most commonly
directed against environmental allergens` (Halliwell, Veterinary
Immunology and Immunopathology, 2006), like dust mites and pollen,
which are incredibly difficult for pets to avoid, since dust mites
are virtually everywhere and pollen permeates the air outdoors.
[0025] Canine atopic dermatitis is a complex and multifactorial
disease involving immune dysregulation, allergic sensitisation,
skin barrier defects, microbial colonization and environmental
factors.
[0026] IgE is not a prerequisite for the development of the
clinical signs in all cases, and a separate clinical entity known
as atopic-like dermatitis was defined as `an inflammatory and
pruritic skin disease with clinical features identical to those
seen in Canine Atopic Dermatitis in which an IgE response to
environmental or other allergens cannot be documented` (Nuttall et
al., Vet. Record, 2013).
[0027] The most common symptoms of Canine Atopic Dermatitis include
itching, excessive scratching, rubbing on the carpet, hair loss,
greasy or flaky skin with a foul odor, excessive chewing on the
paws and areas such as the groin and armpits. Over time, the skin
that is scratched can develop hot spots--raw, inflamed areas--that
may become infected.
[0028] At present, the treatment of acute flares of atopic
dermatitis (AD) should involve the search for, and then elimination
of, the cause of the flares, bathing with mild shampoos, and
controlling pruritus and skin lesions with interventions that
include topical and/or oral glucocorticoids or oclacitinib. For
chronic CAD, the first steps in management are the identification
and avoidance of flare factors, as well as ensuring that there is
adequate skin and coat hygiene and care; this might include more
frequent bathing and possibly increasing essential fatty acid
intake. The medications currently most effective in reducing
chronic pruritus and skin lesions are topical and oral
glucocorticoids, oral ciclosporin, oral oclacitinib, and, where
available, injectable recombinant interferons. Allergen-specific
immunotherapy and proactive intermittent topical glucocorticoid
applications are the only interventions likely to prevent or delay
the recurrence of flares of AD. (Olivry et al., BMC Veterinary
Research, 2015)
[0029] As another specific example, Flea allergy dermatitis (FAD)
or flea bite hypersensitivity is the most common dermatologic
disease of domestic dogs (Scott et al., In: Muller and Kirk's Small
Animal Dermatology, 2001), caused by the by far most prevalent flea
on dogs and cats: Ctenocephalides felis (Beresford-Jones, J Small
Animal Practice, 1981; Chesney, Veterinary Record, 1995). Cats also
develop FAD, which is one of the major causes of feline miliary
dermatitis.
[0030] FAD is most prevalent in the summer, although in warm
climates flea infestations may persist throughout the year. In
north temperate regions, the close association of pets and their
fleas with human dwellings creates conditions that permit a
year-round problem. Temperature extremes and low humidity tend to
inhibit flea development.
[0031] When feeding, fleas inject saliva that contains a variety of
histamine-like compounds, enzymes, polypeptides, and amino acids
that span a wide range of sizes (40-60 kD) and induce Type I, Type
IV, and basophil hypersensitivity. Flea-naive dogs exposed
intermittently to flea bites develop either immediate (15 min) or
delayed (24-48 hr) reactions, or both, and detectable levels of
both circulating IgE and IgG antiflea antibodies. Dogs exposed
continuously to flea bites have low levels of these circulating
antibodies and either do not develop skin reactions or develop them
later and to a considerably reduced degree. This could indicate
that immunologic tolerance may develop naturally in dogs
continually exposed to flea bites. Although the pathophysiology of
FAD in cats is poorly understood, similar mechanisms may exist.
[0032] The cat flea (Ctencephalides felis) causes severe irritation
in animals and people, and is responsible for Flea Allergy
Dermatitis. Typical symptoms are: pruritus, inflammation of the
skin and skin lesions (erythema, scales, papules, crusts and
lichenification). These lesions are most commonly seen along the
back and at the base of the tail.
[0033] As the condition progresses there may be hair loss, broken
hairs, oozing or crusty sores, pimply bumps and general redness and
inflammation of the skin. The sores can be very painful. In severe
cases the skin becomes thickened and dark, predominantly in the
area on the dog's back at the base of the tail. The dog, itself,
causes the damage with self mutilation due to the severe
itching.
[0034] In general, prevention and treatment of flea infestion is
the treatment option of choice. Most commonly neonicotinoids, like
imidacloprid, or gamma-aminobutyric acid (GABA)-gated chloride
channel blockers, like fipronil, are used. In cases where symptoms
of skin allergic dermatitis do not resolve, current treatments
mentioned under CAD, like topical and oral glucocorticoids, oral
ciclosporin, oral oclacitinib are used.
[0035] The present invention provides compounds of the general
formula (I)
##STR00004##
in which: [0036] R.sup.1 is C.sub.1-C.sub.6-alkyl, where the
C.sub.1-C.sub.6-alkyl group is unsubstituted or mono- or
polysubstituted identically or differently by [0037] halogen,
hydroxyl, an unsubstituted or mono- or poly-halogen-substituted
C.sub.3-C.sub.6-cycloalkyl, or an R.sup.6, R.sup.7SO.sub.2,
R.sup.7SO or R.sup.8O group, [0038] or a group selected from:
[0038] ##STR00005## [0039] R.sup.2 and R.sup.3 always have the same
definition and are both either hydrogen or C.sub.1-C.sub.6-alkyl;
[0040] R.sup.4 is halogen, cyano, an unsubstituted or a singly or
multiply, identically or differently substituted
C.sub.1-C.sub.6-alkyl or an unsubstituted or a singly or multiply,
identically or differently substituted C.sub.3-C.sub.6-cycloalkyl,
and the substituents are selected from the group of halogen and
hydroxyl; [0041] R.sup.5 is hydrogen, halogen or an unsubstituted
or mono- or poly-halogen-substituted C.sub.1-C.sub.6-alkyl; [0042]
R.sup.6 is an unsubstituted or mono- or di-methyl-substituted
monocyclic saturated heterocycle having 4 to 6 ring atoms, which
contains a heteroatom or a heterogroup from the group of O, S, SO
and SO.sub.2; [0043] R.sup.7 is C.sub.1-C.sub.6-alkyl, where the
C.sub.1-C.sub.6-alkyl group is unsubstituted or mono- or
polysubstituted identically or differently by halogen, hydroxyl or
C.sub.3-C.sub.6-cycloalkyl, [0044] or R.sup.7 is
C.sub.3-C.sub.6-cycloalkyl; [0045] R.sup.8 is
C.sub.1-C.sub.6-alkyl, where the C.sub.1-C.sub.6-alkyl group is
unsubstituted or mono- or polysubstituted identically or
differently by halogen; and the diastereomers, enantiomers,
metabolites, salts, solvates or solvates of the salts thereof, for
use in the treatment and/or prophylaxis of allergic and/or
inflammatory diseases in animals.
[0046] In the case of the mentioned use of the synthesis
intermediates and working examples of the invention described
hereinafter, any compound specified in the form of a salt of the
corresponding base or acid is generally a salt of unknown exact
stoichiometric composition, as obtained by the respective
preparation and/or purification process. Unless specified in more
detail, additions to names and structural formulae, such as
"hydrochloride", "trifluoroacetate", "sodium salt" or "x HCl", "x
CF.sub.3COOH", "x Na.sup.+" should not therefore be understood in a
stoichiometric sense in the case of such salts, but have merely
descriptive character with regard to the salt-forming components
present therein.
[0047] This applies correspondingly if synthesis intermediates or
working examples or salts thereof were obtained in the form of
solvates, for example hydrates, of unknown stoichiometric
composition (if they are of a defined type) by the preparation
and/or purification processes described.
[0048] Present compounds are the compounds of the formula (I) and
the salts, solvates and solvates of the salts thereof, the
compounds that are encompassed by formula (I) and are of the
formulae mentioned below and the salts, solvates and solvates of
the salts thereof and the compounds that are encompassed by the
formula (I) and are mentioned below as embodiments and the salts,
solvates and solvates of the salts thereof if the compounds that
are encompassed by the formula (I) and are mentioned below are not
already salts, solvates and solvates of the salts.
[0049] Preferred salts in the context of the present invention are
physiologically acceptable salts of the present compounds. However,
the present disclosure also encompasses salts which themselves are
unsuitable for pharmaceutical applications but which can be used,
for example, for the isolation or purification of the present
compounds.
[0050] Physiologically acceptable salts of the present compounds
include acid addition salts of mineral acids, carboxylic acids and
sulphonic acids, for example salts of hydrochloric acid,
hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic
acid, ethanesulphonic acid, toluenesulphonic acid, benzenesulphonic
acid, naphthalenedisulphonic acid, acetic acid, trifluoroacetic
acid, propionic acid, lactic acid, tartaric acid, malic acid,
citric acid, fumaric acid, maleic acid and benzoic acid.
[0051] Physiologically acceptable salts of the present compounds
also include salts of conventional bases, by way of example and
with preference alkali metal salts (e.g. sodium and potassium
salts), alkaline earth metal salts (e.g. calcium and magnesium
salts) and ammonium salts derived from ammonia or organic amines
having 1 to 16 carbon atoms, by way of example and with preference
ethylamine, diethylamine, triethylamine, ethyldiisopropylamine,
monoethanolamine, diethanolamine, triethanolamine,
dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine,
N-methylmorpholine, arginine, lysine, ethylenediamine and
N-methylpiperidine.
[0052] Solvates in the context of the invention are described as
those forms of the present compounds which form a complex in the
solid or liquid state by coordination with solvent molecules.
Hydrates are a specific form of the solvates in which the
coordination is with water.
[0053] The present compounds may, depending on their structure,
exist in different stereoisomeric forms, i.e. in the form of
configurational isomers or else, if appropriate, of conformational
isomers (enantiomers and/or diastereomers, including those in the
case of atropisomers). The present invention therefore encompasses
the use of enantiomers and diastereomers, and the respective
mixtures thereof. The stereoisomerically homogeneous constituents
can be isolated from such mixtures of enantiomers and/or
diastereomers in a known manner; chromatography processes are
preferably used for this purpose, especially HPLC chromatography on
an achiral or chiral phase.
[0054] If the present compounds can occur in tautomeric forms, the
present invention encompasses the use of all the tautomeric
forms.
[0055] The present invention also encompasses the use of all
suitable isotopic variants of the present compounds. An isotopic
variant of an present compound is understood here as meaning a
compound in which at least one atom within the present compound has
been exchanged for another atom of the same atomic number, but with
a different atomic mass than the atomic mass which usually or
predominantly occurs in nature. Examples of isotopes which can be
incorporated into an present compound are those of hydrogen,
carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine, chlorine,
bromine and iodine, such as 2H (deuterium), 3H (tritium), 13C, 14C,
15N, 17O, 18O, 32P, 33P, 33S, 34S, 35S, 36S, 18F, 36Cl, 82Br, 123I,
124I, 129I and 131I. Particular isotopic variants of an present
compound, such as, in particular, those in which one or more
radioactive isotopes have been incorporated, may be beneficial, for
example, for the examination of the mechanism of action or of the
active ingredient distribution in the body; because of the
comparative ease of preparability and detectability, particularly
compounds labelled with 3H or 14C isotopes are suitable for this
purpose. In addition, the incorporation of isotopes, for example of
deuterium, may lead to particular therapeutic benefits as a
consequence of greater metabolic stability of the compound, for
example an extension of the half-life in the body or a reduction in
the active dose required; such modifications of the present
compounds may therefore in some cases also constitute a preferred
embodiment of the use of the present invention. Isotopic variants
of the present compounds can be prepared by the processes known to
those skilled in the art, for example by the methods described
further below and the procedures described in the working examples,
by using corresponding isotopic modifications of the respective
reagents and/or starting compounds.
[0056] The present invention further provides the use of all the
possible crystalline and polymorphous forms of the present
compounds, where the polymorphs may be present either as single
polymorphs or as a mixture of a plurality of polymorphs in all
concentration ranges.
[0057] The present invention additionally also encompasses the use
of prodrugs of the present compounds. The term "prodrugs" in this
context refers to compounds which may themselves be biologically
active or inactive but are converted (for example metabolically or
hydrolytically) to present compounds during their residence time in
the body.
[0058] In the context of the present invention, unless specified
otherwise, the substituents have the following meanings:
[0059] Alkyl in the context of the invention represents a
straight-chain or branched alkyl group having the particular number
of carbon atoms specified. Examples include methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, 1-methylpropyl,
2-methylpropyl, tert-butyl, n-pentyl, 1-ethylpropyl, 1-methylbutyl,
2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, n-hexyl,
1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl,
1-ethylbutyl and 2-ethylbutyl. Preference is given to methyl,
ethyl, n-propyl, n-butyl, 2-methylbutyl, 3-methylbutyl and
2,2-dimethylpropyl.
[0060] Cycloalkyl in the context of the invention is a monocyclic
saturated alkyl group having the number of carbon atoms specified
in each case. Preferred examples include cyclopropyl, cyclobutyl,
cyclopentyl and cyclohexyl.
[0061] Alkoxy in the context of the invention represents a
straight-chain or branched alkoxy group having the particular
number of carbon atoms specified. 1 to 6 carbon atoms are
preferred. Examples include methoxy, ethoxy, n-propoxy, isopropoxy,
1-methylpropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentoxy,
isopentoxy, 1-ethylpropoxy, 1-methylbutoxy, 2-methylbutoxy,
3-methylbutoxy and n-hexoxy. Particular preference is given to a
linear or branched alkoxy group having 1 to 4 carbon atoms.
Examples which may be mentioned as being preferred are methoxy,
ethoxy, n-propoxy, 1-methylpropoxy, n-butoxy and isobutoxy.
[0062] Halogen in the context of the invention is fluorine,
chlorine and bromine. Preference is given to fluorine.
[0063] Hydroxyl in the context of the invention is OH.
[0064] A monocyclic saturated heterocycle is a monocyclic saturated
heterocycle which has 4 to 6 ring atoms and contains a heteroatom
or a heterogroup from the group of O, S, SO and SO.sub.2. A
heterocycle having a heteroatom or a heterogroup from the group of
O, SO and SO.sub.2 is preferred.
[0065] Examples include: oxetane, tetrahydrofuran,
tetrahydro-2H-pyran-4-yl, 1,1-dioxidotetrahydro-2H-thiopyran-3-yl,
1,1-dioxidotetrahydro-2H-thiopyran-2-yl,
1,1-dioxidotetrahydro-2H-thiopyran-4-yl,
1,1-dioxidotetrahydrothiophen-3-yl,
1,1-dioxidotetrahydrothiophen-2-yl, 1,1-dioxidothietan-2-yl or
1,1-dioxidothietan-3-yl. Particular preference is given here to
oxetane and tetrahydrofuran. Very particular preference is given to
oxetan-3-yl.
[0066] A symbol * at a bond denotes the bonding site in the
molecule.
[0067] When groups in the present compounds are substituted, the
groups may be mono- or polysubstituted, unless specified otherwise.
In the context of the present invention, all groups which occur
more than once are defined independently of one another.
Substitution by one, two or three identical or different
substituents is preferred.
[0068] A preferred embodiment of R.sup.1 is a C.sub.2-C.sub.6-alkyl
group substituted by 1, 2 or 3 fluorine atoms.
[0069] Particular preference is given to 2,2,2-trifluoroethyl,
3,3,3-trifluoropropyl and 4,4,4-trifluorobutyl.
[0070] Very particular preference is given to a
4,4,4-trifluorobutyl group.
[0071] A further preferred embodiment of R.sup.1 is a
C.sub.2-C.sub.6-alkyl group substituted by one or two hydroxyl
group(s) or one C.sub.1-C.sub.3-alkoxy or a
tri-fluorine-substituted C.sub.1-C.sub.3-alkoxy. Particular
preference is given to a C.sub.2-C.sub.5-alkyl group substituted by
hydroxyl or C.sub.1-C.sub.3-alkoxy or trifluoromethoxy or
2,2,2-trifluoroethoxy. Very particular preference is given to
3-hydroxy-3-methylbutyl, 3-methoxypropyl, 3-hydroxypropyl,
3-trifluoromethoxypropyl, 2-methoxyethyl or 2-hydroxyethyl.
Especially preferred is the 3-hydroxy-3-methylbutyl group.
[0072] Further preferably, R.sup.1 is a C.sub.2-C.sub.6-alkyl group
substituted by a C.sub.1-C.sub.6-alkyl-SO.sub.2 group. A
methyl-SO.sub.2-substituted C.sub.2-C.sub.4-alkyl group is
particularly preferred. Especially preferred for R.sup.1 are
2-(methylsulphonyl)ethyl or 3-(methylsulphonyl)propyl. From the
latter group, 2-(methylsulphonyl)ethyl is particularly
preferred.
[0073] Additionally preferably, R.sup.1 is a C.sub.1-C.sub.3-alkyl
group substituted by oxetanyl, tetrahydrofuranyl,
tetrahydro-2H-pyran-4-yl, 1,1-dioxidotetrahydro-2H-thiopyran-3-yl,
1,1-dioxidotetrahydro-2H-thiopyran-2-yl,
1,1-dioxidotetrahydro-2H-thiopyran-4-yl,
1,1-dioxidotetrahydrothiophen-3-yl,
1,1-dioxidotetrahydrothiophen-2-yl, 1,1-dioxidothietan-2-yl or
1,1-dioxidothietan-3-yl. Particular preference is given to a
C.sub.1-C.sub.3-alkyl group substituted by an oxetane group.
Especially preferred for R.sup.1 is an oxetan-3-ylmethyl group.
[0074] For R.sup.2 and R.sup.3, which always have the same
definition, hydrogen or methyl are preferred. Methyl is
particularly preferred.
[0075] In the case of R.sup.4, preference is given to an
unsubstituted or mono- or poly-halogen-substituted
C.sub.1-C.sub.3-alkyl group or a C.sub.1-C.sub.3-alkyl group
substituted by one hydroxyl group or a C.sub.1-C.sub.3-alkyl group
substituted by one hydroxyl group and three fluorine atoms.
[0076] For R.sup.4, particular preference is given to the following
groups: methyl, ethyl, trifluoro-C.sub.1-C.sub.3-alkyl,
difluoro-C.sub.1-C.sub.3-alkyl, hydroxymethyl, 1-hydroxyethyl,
2-hydroxypropan-2-yl and 2,2,2-trifluoro-1-hydroxyethyl. For
R.sup.4, particular preference is given to the methyl,
trifluoromethyl and difluoromethyl groups. Particular preference is
given here to a trifluoromethyl group.
[0077] A preferred embodiment of R.sup.5 is hydrogen, fluorine,
chlorine or C.sub.1-C.sub.3-alkyl. More preferably, R.sup.5 is
hydrogen, fluorine or methyl. Most preferably, R.sup.5 is hydrogen
or fluorine.
[0078] Particular preference is also given to compounds in which
R.sup.4 is methyl or trifluoromethyl and R.sup.5 is fluorine. Very
particular preference is given to compounds in which R.sup.4 is
methyl and R.sup.5 is fluorine, where R.sup.5 is in the ortho
position to R.sup.4.
[0079] For R.sup.6, preferred embodiments include oxetanyl,
tetrahydrofuranyl, tetrahydro-2H-pyran-4-yl,
1,1-dioxidotetrahydro-2H-thiopyran-3-yl,
1,1-dioxidotetrahydro-2H-thiopyran-2-yl,
1,1-dioxidotetrahydro-2H-thiopyran-4-yl,
1,1-dioxidotetrahydrothiophen-3-yl,
1,1-dioxidotetrahydrothiophen-2-yl, 1,1-dioxidothietan-2-yl or
1,1-dioxidothietan-3-yl. Particular preference is given here to
oxetanyl. Very particular preference is given to oxetan-3-yl.
[0080] R.sup.7 is exclusively connected to the functional groups
--SO.sub.2-- and --SO--, i.e. is an R.sup.7-substituted
--SO.sub.2-- or SO group. In this connection, R.sup.7 is preferably
C.sub.1-C.sub.4-alkyl, where the C.sub.1-C.sub.4-alkyl group is
unsubstituted or monosubstituted by hydroxyl or by cyclopropyl or
substituted by three fluorine atoms. Additionally preferred for
R.sup.7 is a cyclopropyl group. Particularly preferred for R.sup.7
are methyl, ethyl or hydroxyethyl. Very particular preference is
given to methyl for R.sup.7.
[0081] This means that, in the case of a C.sub.1-C.sub.6-alkyl
group substituted by R.sup.7SO.sub.2-- or R.sup.7SO--, in the
context of R.sup.1, preference is given to a C.sub.1-C.sub.6-alkyl
substituted by a C.sub.1-C.sub.6-alkyl-SO.sub.2 or a
C.sub.1-C.sub.6-alkyl-SO. For R.sup.1, preference is given here
especially to methylsulphonylethyl and methylsulphonylpropyl. Very
particular preference is given here to methylsulphonylethyl.
[0082] For R.sup.8, preference is given to an unsubstituted
C.sub.1-C.sub.4-alkyl group or a tri-fluorine-substituted
C.sub.1-C.sub.4-alkyl group. Particular preference is given to
methyl, ethyl, trifluoromethyl or 2,2,2-trifluoroethyl. Very
particular preference is given to methyl, trifluoromethyl or
2,2,2-trifluoroethyl.
[0083] As described previously, the intracellular enzyme
interleukin-1 receptor-associated kinase 4 (IRAK4) plays an
integral part in the signaling pathway of receptors activated by
cytokines and TLR ligands that are implicated in inflammatory
processes. Besides inflammation, IRAK4 is also involved in the
signaling of allergic processes. Such allergic processes play an
important role in the pathogenesis of allergic skin diseases, like
atopic dermatitis.
[0084] For example, IL-33 a recent addition to the IL-1 family of
cytokines that also includes IL-18 and IL-1, binds to and activates
IL-33 receptors (IL-33R) that then associate with MyD88, IRAK4 and
TRF6 (Schmitz et al, Immunity, 2005). IRAK4 is an essential
component of this signaling pathway. IL-33R are strongly expressed
on T helper cell type 2 (Th2) cells, mast cells and eosinophils.
IL-33 activates these cells and promotes Th2 immune responses
(Schmitz et al, Immunity, 2005). These cell types are each involved
in the pathogenesis of atopic dermatitis. IL-33 levels in the serum
correlate with the severity of atopic dermatitis in man and
decrease on treatment with topical steroids & calcineurin
inhibitor (Tamagawa-Mineoka et al, J American Academy Dermatology,
2014). In models of acute Canine Atopic Dermatitis it has been
shown that the IL-33 gene was significantly up-regulated in skin
lesions (Schamber et al., G3 (Bethesda), 2014; Olivry et al,
Journal of Investigative Dermatology, 2016).
[0085] Furthermore, a second member of the IL-1 family of
cytokines, IL-18 has been implicated in atopic dermatitis. Serum
levels of IL-18 increase with severity of atopic dermatitis in
children (Sohn et al, Allergy and Asthma Proceedings, 2004). In
models of acute Canine Atopic Dermatitis it has been shown that the
IL-18 gene was significantly up-regulated in skin lesions (Schamber
et al., G3 (Bethesda), 2014; Olivry et al, Journal of Investigative
Dermatology, 2016). In addition, atopic dermatitis-like
inflammation & itching were initiated by over-release of IL-18
and accelerated by IL-1 in mice (Konishi et al, Proceedings of the
National Academy of Sciences, 2002). Again IRAK4 has been shown to
be an essential component of the IL-18 signaling cascade (Suzuki et
al, J Immunology, 2003). Similarly IRAK4 is critical for the
signaling of IL-1 and TLR ligands (Suzuki et al, Nature, 2002). TLR
agonists are known to induce itch (Liu et al, Neuroscience
bulletin, 2012), an important symptom of atopic dermatitis, and
anti-IL-1 therapies are used off-label to treat atopic dermatitis.
Moreover, polymorphisms in IRAK4 are associated with elevated total
IgE in allergic diseases such as asthma and chronic rhinosinusitis
(Tewfik et al, Allergy, 2009). IgE levels are also elevated in
atopic dermatitis.
[0086] Hence, as IRAK4 is a critical part of the signaling pathways
that are activated by a number of cytokine, TLR ligands and IRAK4
has polymorphisms associated with increased IgE level, inhibition
of IRAK4 is an important therapeutic strategy for the treatment of
allergic skin diseases such as atopic dermatitis. Moreover, in
companion animals (especially dogs and cats) both atopic dermatitis
and Flea Allergy Dermatitis are appropriate indications since both
diseases are comprised of Type I hypersensitivity that involves IgE
antibodies, Th2 cells, mast cells and eosinophils. In addition, FAD
can be comprised of Type IV hypersensitivity in which IL-1 and
IL-18 are involved.
[0087] The present compounds act as inhibitors of IRAK4 kinase and
therefore have an unforeseeable useful pharmacological activity
spectrum in the treatment and/or prophylaxis of allergic and/or
inflammatory diseases in animals.
[0088] Preference is given to compounds of the formula (I) in which
[0089] R.sup.1 is C.sub.1-C.sub.6-alkyl, where the
C.sub.1-C.sub.6-alkyl group is unsubstituted or mono- or
polysubstituted identically or differently by fluorine, hydroxyl or
an R.sup.6, R.sup.7SO.sub.2, R.sup.7SO or R.sup.8O group; [0090]
R.sup.2 and R.sup.3 always have the same definition and are both
either hydrogen or C.sub.1-C.sub.3-alkyl; [0091] R.sup.4 is
halogen, cyano or C.sub.1-C.sub.3-alkyl, where the
C.sub.1-C.sub.3-alkyl group is unsubstituted or mono- or
polysubstituted identically or differently by halogen or hydroxyl;
[0092] R.sup.5 is hydrogen, fluorine, chlorine or
C.sub.1-C.sub.3-alkyl; [0093] R.sup.6 is oxetanyl or
tetrahydrofuranyl; [0094] R.sup.7 is C.sub.1-C.sub.4-alkyl, where
the C.sub.1-C.sub.4-alkyl group is unsubstituted or monosubstituted
by hydroxyl or by cyclopropyl or substituted by three fluorine
atoms; [0095] R.sup.8 is unsubstituted C.sub.1-C.sub.4-alkyl or
tri-fluorine-substituted C.sub.1-C.sub.4-alkyl; and the
diastereomers, enantiomers, metabolites, salts, solvates or
solvates of the salts thereof, for use in the treatment and/or
prophylaxis of allergic and/or inflammatory diseases in
animals.
[0096] Preference is additionally given to compounds of the formula
(I) in which [0097] R.sup.1 is C.sub.2-C.sub.6-alkyl, where
C.sub.2-C.sub.6-alkyl is unsubstituted, or [0098]
C.sub.2-C.sub.6-alkyl is mono-, di- or tri-fluorine-substituted or
[0099] C.sub.2-C.sub.6-alkyl is monosubstituted by hydroxyl,
R.sup.6, R.sup.7SO.sub.2, or R.sup.8O, or in which R.sup.1 is an
oxetanyl-substituted C.sub.1-C.sub.3-alkyl; [0100] R.sup.2 and
R.sup.3 always have the same definition and are both either
hydrogen or methyl; [0101] R.sup.4 is an unsubstituted or mono- or
poly-halogen-substituted C.sub.1-C.sub.3-alkyl group or a
C.sub.1-C.sub.3-alkyl group substituted by one hydroxyl group or a
C.sub.1-C.sub.3-alkyl group substituted by one hydroxyl group and
three fluorine atoms; [0102] R.sup.5 is hydrogen, fluorine or
C.sub.1-C.sub.3-alkyl; [0103] R.sup.7 is C.sub.1-C.sub.3-alkyl;
[0104] R.sup.8 is C.sub.1-C.sub.4-alkyl, where the
C.sub.1-C.sub.4-alkyl group is unsubstituted or mono-, di- or
tri-fluorine-substituted; and the diastereomers, enantiomers,
metabolites, salts, solvates or solvates of the salts thereof, for
use in the treatment and/or prophylaxis of allergic and/or
inflammatory diseases in animals.
[0105] Particular preference is also given to compounds of the
general formula (I) in which [0106] R.sup.1 is a
C.sub.2-C.sub.5-alkyl group substituted by hydroxyl or
C.sub.1-C.sub.3-alkoxy or trifluoromethoxy or 2,2,2-trifluoroethoxy
or trifluoromethyl or [0107] is a methyl-SO.sub.2-substituted
C.sub.2-C.sub.4-alkyl group or [0108] is an oxetan-3-yl-substituted
C.sub.1-C.sub.2-alkyl group; [0109] R.sup.2 and R.sup.3 always have
the same definition and are both hydrogen or methyl; [0110] R.sup.4
is methyl, ethyl, trifluoro-C.sub.1-C.sub.3-alkyl,
difluoro-C.sub.1-C.sub.3-alkyl, hydroxymethyl, 1-hydroxyethyl,
2-hydroxypropan-2-yl and 2,2,2-trifluoro-1-hydroxyethyl and [0111]
R.sup.5 is hydrogen, fluorine or methyl; and the diastereomers,
enantiomers, metabolites, salts, solvates or solvates of the salts
thereof, for use in the treatment and/or prophylaxis of allergic
and/or inflammatory diseases in animals.
[0112] Very particular preference is given to compounds in which
[0113] R.sup.1 is 4,4,4-trifluorobutyl, 3-hydroxy-3-methylbutyl,
3-hydroxybutyl, 3-methoxypropyl, 3-hydroxypropyl,
3-hydroxy-2-methylpropyl, 3-hydroxy-2,2-dimethylpropyl,
3-trifluoromethoxypropyl, 2-methoxyethyl, 2-hydroxyethyl,
2-(methylsulphonyl)ethyl or 3-(methylsulphonyl)propyl; [0114]
R.sup.2 and R.sup.3 are both methyl or hydrogen and [0115] R.sup.4
is difluoromethyl, trifluoromethyl or methyl and [0116] R.sup.5 is
hydrogen or fluorine; and the diastereomers, enantiomers,
metabolites, salts, solvates or solvates of the salts thereof, for
use in the treatment and/or prophylaxis of allergic and/or
inflammatory diseases in animals.
[0117] Very particular preference is also given to compounds in
which [0118] R.sup.1 is 3-hydroxy-3-methylbutyl, 3-hydroxybutyl,
3-hydroxy-2-methylpropyl, 3-hydroxy-2,2-dimethylpropyl,
3-(methylsulphonyl)propyl or 2-(methylsulphonyl)ethyl; [0119]
R.sup.2 and R.sup.3 are both methyl; [0120] R.sup.4 is
difluoromethyl or trifluoromethyl; and [0121] R.sup.5 is hydrogen;
and the diastereomers, enantiomers, metabolites, salts, solvates or
solvates of the salts thereof, for use in the treatment and/or
prophylaxis of allergic and/or inflammatory diseases in
animals.
[0122] Particular preference is additionally also given to
compounds in which [0123] R.sup.1 is 3-hydroxy-3-methylbutyl,
3-hydroxybutyl, 3-hydroxy-2-methylpropyl,
3-hydroxy-2,2-dimethylpropyl, 3-(methylsulphonyl)propyl or
2-(methylsulphonyl)ethyl; [0124] R.sup.2 and R.sup.3 are both
methyl; [0125] R.sup.4 is methyl and [0126] R.sup.5 is fluorine,
where R.sup.5 is in the ortho position to R.sup.4; and the
diastereomers, enantiomers, metabolites, salts, solvates or
solvates of the salts thereof, for use in the treatment and/or
prophylaxis of allergic and/or inflammatory diseases in
animals.
[0127] The present invention especially provides the following
compounds: [0128] 1)
N-[6-(2-Hydroxypropan-2-yl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-
-6-(trifluoromethyl)pyridine-2-carboxamide [0129] 2)
N-[6-(Hydroxymethyl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-6-(trifluorometh-
yl)pyridine-2-carboxamide [0130] 3)
N-[6-(2-Hydroxypropan-2-yl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(trifl-
uoromethyl)pyridine-2-carboxamide [0131] 4)
N-[6-(Hydroxymethyl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(trifluoromet-
hyl)pyridine-2-carboxamide [0132] 5)
N-[2-(2-Hydroxyethyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(triflu-
oromethyl)pyridine-2-carboxamide [0133] 6)
N-[6-(2-Hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]-6-(trifl-
uoromethyl)pyridine-2-carboxamide [0134] 7)
N-[2-(2-Hydroxyethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluorometh-
yl)pyridine-2-carboxamide [0135] 8)
N-[6-(2-Hydroxypropan-2-yl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(tri-
fluoromethyl)pyridine-2-carboxamide [0136] 9)
N-[6-(Hydroxymethyl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(trifluorom-
ethyl)pyridine-2-carboxamide [0137] 10)
N-{6-(2-Hydroxypropan-2-yl)-2-[3-(methylsulphonyl)propyl]-2H-indazol-5-yl-
}-6-(trifluoromethyl)pyridine-2-carboxamide [0138] 11)
N-[2-(3-Hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]--
6-(trifluoromethyl)pyridine-2-carboxamide [0139] 12)
N-{6-(2-Hydroxypropan-2-yl)-2-[2-(methylsulphonyl)ethyl]-2H-indazol-5-yl}-
-6-(trifluoromethyl)pyridine-2-carboxamide [0140] 13)
6-(Difluoromethyl)-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl-
)-2H-indazol-5-yl]pyridine-2-carboxamide [0141] 14)
6-(Difluoromethyl)-N-{6-(2-hydroxypropan-2-yl)-2-[2-(methylsulphonyl)ethy-
l]-2H-indazol-5-yl}pyridine-2-carboxamide [0142] 15)
6-(Difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-ind-
azol-5-yl]pyridine-2-carboxamide [0143] 16)
N-[6-(2-Hydroxypropan-2-yl)-2-(4,4,4-trifluorobutyl)-2H-indazol-5-yl]-6-(-
trifluoromethyl)pyridine-2-carboxamide [0144] 17)
N-{6-(2-Hydroxypropan-2-yl)-2-[3-(trifluoromethoxy)propyl]-2H-indazol-5-y-
l}-6-(trifluoromethyl)pyridine-2-carboxamide [0145] 18)
N-{6-(2-Hydroxypropan-2-yl)-2-[3-(2,2,2-trifluoroethoxy)propyl]-2H-indazo-
l-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide [0146] 19)
5-Fluoro-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indaz-
ol-5-yl]-6-methylpyridine-2-carboxamide [0147] 20)
N-[2-(3-Hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]--
6-methylpyridine-2-carboxamide [0148] 21)
6-(2-Hydroxypropan-2-yl)-N-[6-(2-hydroxypropan-2-yl)-2-(4,4,4-trifluorobu-
tyl)-2H-indazol-5-yl]pyridine-2-carboxamide [0149] 22)
N-{2-[2-(1-Hydroxycyclopropyl)ethyl]-6-(2-hydroxypropan-2-yl)-2H-indazol--
5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide, for use in the
treatment and/or prophylaxis of allergic and/or inflammatory
diseases in animals.
[0150] The invention further provides compounds of the general
formula (III)
##STR00006##
in which [0151] R.sup.1 is 4,4,4-trifluorobutyl,
3-hydroxy-3-methylbutyl, 3-methoxypropyl, 3-hydroxypropyl,
3-hydroxybutyl, 3-hydroxy-2-methylpropyl,
3-hydroxy-2,2-dimethylpropyl, 3-trifluoromethoxypropyl,
2-methoxyethyl, 2-hydroxyethyl, 2-(methylsulphonyl)ethyl,
3-(methylsulphonyl)propyl or 2-(1-hydroxycyclopropyl)ethyl; [0152]
R.sup.4 is difluoromethyl, trifluoromethyl or methyl; and [0153]
R.sup.5 is hydrogen or fluorine; and the diastereomers,
enantiomers, metabolites, salts, solvates or solvates of the salts
thereof, for use in the treatment and/or prophylaxis of allergic
and/or inflammatory diseases in animals.
[0154] Preference is especially given to the following compounds of
the general formula (III): [0155] methyl
5-{[(5-fluoro-6-methylpyridin-2-yl)carbonyl]amino}-2-(3-hydroxy-3-methylb-
utyl)-2H-indazole-6-carboxylate and [0156] methyl
2-(3-hydroxy-3-methylbutyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl-
}amino)-2H-indazole-6-carboxylate, for use in the treatment and/or
prophylaxis of allergic and/or inflammatory diseases in
animals.
[0157] The compounds of the general formula (III) are suitable for
preparation of a portion of the compounds of the general formula
(I).
[0158] Furthermore, the compounds of the general formula (III) are
inhibitors of interleukin-1 receptor associated kinase-4
(IRAK4).
[0159] Compounds of the general formula (III) can be prepared from
compounds of the general formula (II)
##STR00007##
in which [0160] R.sup.1 is 4,4,4-trifluorobutyl,
3-hydroxy-3-methylbutyl, 3-methoxypropyl, 3-hydroxypropyl,
3-hydroxy-2-methylpropyl, 3-hydroxy-2,2-dimethylpropyl,
3-trifluoromethoxypropyl, 2-methoxyethyl, 2-hydroxyethyl,
2-(methylsulphonyl)ethyl, 3-(methylsulphonyl)propyl or
2-(1-hydroxycyclopropyl)ethyl; [0161] R.sup.4 is difluoromethyl,
trifluoromethyl or methyl; and [0162] R.sup.5 is hydrogen or
fluorine; by the reaction of (II) with appropriately substituted
alkyl halides or alkyl 4-methylbenzenesulphonates in the presence
of potassium carbonate.
[0163] Further, compounds of the general formula (I) can be
prepared from compounds of the formula (III)
##STR00008##
in which [0164] R.sup.1 is 4,4,4-trifluorobutyl,
3-hydroxy-3-methylbutyl, 3-hydroxybutyl, 3-methoxypropyl,
3-hydroxypropyl, 3-hydroxy-2-methylpropyl,
3-hydroxy-2,2-dimethylpropyl, 3-trifluoromethoxypropyl,
2-methoxyethyl, 2-hydroxyethyl, 3-(methylsulphonyl)propyl
2-(1-hydroxycyclopropyl)ethyl; [0165] R.sup.2 and R.sup.3 are
methyl; [0166] R.sup.4 is difluoromethyl, trifluoromethyl or
methyl; and [0167] R.sup.5 is hydrogen or fluorine; by a Grignard
reaction with methylmagnesium bromide.
[0168] The present compounds act as inhibitors of IRAK4 kinase and
have an unforeseeable useful pharmacological activity spectrum.
[0169] Further preference is given to compounds of the formula (I),
or the compounds particularly mentioned above, for use in the
treatment and/or prophylaxis of allergic and/or inflammatory
diseases in domestic animals, particularly in cats and dogs, and
more particularly in dogs.
[0170] The term "domestic animals" in this context includes, for
example, mammals, such as hamsters, guinea pigs, rats, mice,
chinchillas, ferrets or in particular dogs, cats; cage birds;
reptiles; amphibians or aquarium fish.
[0171] Further preference is given to compounds of the formula (I),
or the compounds particularly mentioned above, for use in the
treatment and/or prophylaxis of allergic dermatitis in domestic
animals, particularly canine and feline allergic dermatitis, and
more particularly canine allergic dermatitis. Further preference is
given to compounds of the formula (I), or the compounds
particularly mentioned above, for use in the treatment and/or
prophylaxis of allergic and/or inflammatory diseases in farm
animals, particularly in sheep, goats, horses, cattle and pigs, and
more particularly in cattle and pigs.
[0172] The term "farm animals" in this context includes, for
example, mammals, such as horses, sheep, goats, buffaloes,
reindeers, fallow deers or in particular cattle or pigs.
[0173] Further preference is given to compounds of the formula (I),
or the compounds particularly mentioned above, for use in a method
for treatment and/or prophylaxis of atopic dermatitis, Flea Allergy
Dermatitis, inflammatory bowel disease, osteoarthritis and
inflammatory pain, non-infectious recurrent airway disease, insect
hypersensitivity, asthma, respiratory disease, mastitis and
endometritis in animals, particularly of atopic dermatitis and Flea
Allergy Dermatitis.
[0174] Particular preference is given to compounds of the formula
(I), or the compounds particularly mentioned above, for use in a
method for treatment and/or prophylaxis of Canine Atopic
Dermatitis, Flea Allergy Dermatitis in dogs or cats, inflammatory
bowel disease in dogs or cats, osteoarthritis and inflammatory pain
in dogs, cats, horses or cattle, non-infectious recurrent airway
disease in horses, insect hypersensitivity in horses, feline
asthma, bovine respiratory disease, mastitis in cattle,
endometritis in cattle, and swine respiratory disease.
[0175] Very particular preference is given to compounds of the
formula (I), or the compounds particularly mentioned above, for use
in a method for treatment and/or prophylaxis of Canine Atopic
Dermatitis and Flea Allergy Dermatitis in dogs or cats, more
particularly in dogs.
[0176] Further very particular preference is given to compounds of
the formula (I), or the compounds particularly mentioned above, for
use in a method for treatment and/or prophylaxis of osteoarthritis
and inflammatory pain in cattle, bovine respiratory disease,
mastitis in cattle, endometritis in cattle, and swine respiratory
disease.
[0177] With regard to the compounds of formula (III), further
preference is given to compounds of the formula (III) for use in
the treatment and/or prophylaxis of allergic and/or inflammatory
diseases in domestic animals, particularly in cats and dogs, and
more particularly in dogs.
[0178] Further preference is given to compounds of the formula
(III) for use in the treatment and/or prophylaxis of allergic
dermatitis in domestic animals, particularly canine and feline
allergic dermatitis, and more particularly canine allergic
dermatitis.
[0179] Further preference is given to compounds of the formula
(III) for use in the treatment and/or prophylaxis of allergic
and/or inflammatory diseases in farm animals, particularly in
sheep, goats, horses, cattle and pigs, and more particularly in
cattle and pigs.
[0180] Further preference is given to compounds of the formula
(III) for use in a method for treatment and/or prophylaxis of
atopic dermatitis, Flea Allergy Dermatitis, inflammatory bowel
disease, osteoarthritis and inflammatory pain, non-infectious
recurrent airway disease, insect hypersensitivity, asthma,
respiratory disease, mastitis and endometritis in animals,
particularly of atopic dermatitis and Flea Allergy Dermatitis.
[0181] Particular preference is given to compounds of the formula
(III) for use in a method for treatment and/or prophylaxis of
Canine Atopic Dermatitis, Flea Allergy Dermatitis in dogs or cats,
inflammatory bowel disease in dogs or cats, osteoarthritis and
inflammatory pain in dogs, cats, horses or cattle, non-infectious
recurrent airway disease in horses, insect hypersensitivity in
horses, feline asthma, bovine respiratory disease, mastitis in
cattle, endometritis in cattle, and swine respiratory disease.
[0182] Very particular preference is given to compounds of the
formula (III) for use in a method for treatment and/or prophylaxis
of Canine Atopic Dermatitis and Flea Allergy Dermatitis in dogs or
cats, more particularly in dogs.
[0183] Further very particular preference is given to compounds of
the formula (III) for use in a method for treatment and/or
prophylaxis of osteoarthritis and inflammatory pain in cattle,
bovine respiratory disease, mastitis in cattle, endometritis in
cattle, and swine respiratory disease.
[0184] By way of example, compound examples 11, 12, 13, 19 (as
shown below) have been evaluated in an in vitro IRAK4 TR-FRET assay
detailed below using recombinant canine IRAK4 enzyme. IC50 values
of each compound have been calculated for the inhibition of canine
IRAK4. Example Compounds (11, 12, 13, 19) have been identified as
being useful in the treatment of allergic skin diseases in animals,
particularly dogs and cats, such as atopic dermatitis and Flea
Allergy Dermatitis. Example compounds 11, 12, 13, 19 were each
potent inhibitors of canine IRAK4 with IC50 values of 1.7, 9.2,
2.2, 7.6 nM, respectively. The IC50 values for each of these
compound examples were also similar to the IC50 values calculated
for inhibition of human IRAK4.
[0185] As a further example, example compound 12 has also been
evaluated in an in vitro assay to establish the effects of
compounds on lipopolysaccharide (LPS)-induced cytokine production
by canine peripheral blood mononuclear cells (PBMCs). Example
compound 12 inhibited the production of the pro-inflammatory
cytokine Tumor Necrosis factor alpha (TNF.alpha.) by canine PBMCs
induced by LPS, in a concentration-related manner. PBMCs include
cell types such as dendritics cells, T and B lymphocytes, as well
as monocytes each of which are implicated in atopic dermatitis and
TNF.alpha. is elevated in atopic dermatitis patients (Sumimoto et
al, Archives of Disease in Childhood, 1992). This example is also
illustrated by FIG. 7.
[0186] Hence, the present compounds demonstrate inhibition of
recombinant canine IRAK4 enzyme and cytokine production by canine
PBMCs indicating the potential therapeutic benefit of such compound
examples in Canine Atopic Dermatitis and Flea Allergy
Dermatitis.
[0187] In addition, example compound 12 has also been evaluated in
vivo in a further study to establish the effects of compounds in
the treatment of clinical signs associated with canine allergic
dermatitis, particularly Canine Atopic Dermatitis (CAD), in a House
Dust Mite model. Example compound 12 significantly reduced clinical
signs of CAD like skin edema and erythema. This example is also
illustrated by FIGS. 11 and 12.
[0188] Hence, the present compounds demonstrate reduction of
characteristic clinical signs of canine allergic dermatitis,
therefore indicating a therapeutic benefit of such compound
examples in canine allergic dermatitis, particularly in Canine
Atopic Dermatitis (CAD). Also, example compound 12 has been
evaluated in an in vivo model of canine Flea Allergy Dermatitis
(FAD) to establish the anti-pruritic effects of compounds.
Treatment with Example compound 12 substantially reduced pruritus
associated with allergic diseases like Flea Allergy Dermatitis.
This example is also illustrated by FIG. 13.
[0189] Hence, the present compounds demonstrate reduction of
associated pathognomonic clinical signs of allergic dermatitis as
skin inflammation and pruritus therefore indicating a therapeutic
benefit of such compound examples in canine allergic dermatitis,
particularly in Flea Allergy Dermatitis (FAD) and Canine Atopic
Dermatitis (CAD).
[0190] The term "canine allergic dermatitis" in this context
includes particularly Canine Atopic Dermatitis (CAD) and Flea
Allergy Dermatitis (FAD).
[0191] As a further example, example compound 12 has also been
evaluated in an ex vivo assay to establish the effects of compounds
on lipopolysaccharide (LPS)-induced cytokine production by bovine
peripheral blood mononuclear cells (PBMCs). Example compound 12
inhibited the production of the pro-inflammatory cytokine Tumor
Necrosis factor alpha (TNF.alpha.) by bovine PBMCs induced by LPS,
in a concentration-related manner. PBMCs include cell types such as
dendritic cells, T and B lymphocytes, as well as monocytes each of
which are implicated in inflammatory and infectious diseases with
overshooting pro-inflammatory immune response such as respiratory
diseases (Sterner-Kock, Haider, et al., Tropical Animal Health and
Production, 2016), enteric diseases (Pan, Rostagnio, et al.,
Veterinary Immunology and Immunopathology, 2015), and mastitis
(Zheng, Xu, et al., Free Radical Biology and Medicine, 2016) in
which TNF.alpha. is elevated in these patients. This example is
also illustrated by FIGS. 8 and 9.
[0192] Hence, the present compounds demonstrate inhibition of
cytokine production by bovine PBMCs indicating the potential
therapeutic benefit of such compound examples in inflammatory
and/or infectious diseases such as respiratory diseases, enteric
diseases and mastitis.
[0193] As a further example, example compound 12 has also been
evaluated in an ex vivo assay to establish the effects of compounds
on lipopolysaccharide (LPS)-induced cytokine production by porcine
peripheral blood mononuclear cells (PBMCs). Example compound 12
inhibited the production of the pro-inflammatory cytokine Tumor
Necrosis factor alpha (TNF.alpha.) by porcine PBMCs induced by LPS.
PBMCs include cell types such as dendritic cells, T and B
lymphocytes, as well as monocytes each of which are implicated in
inflammatory and infectious diseases with overshooting
pro-inflammatory immune response such as respiratory diseases and
enteric diseases in which TNF.alpha. is elevated in these patients.
This example is also illustrated by FIG. 10.
[0194] Hence, the present compounds demonstrate inhibition of
cytokine production by porcine PBMCs indicating the potential
therapeutic benefit of such compound examples in inflammatory
and/or infectious diseases such as respiratory diseases and enteric
diseases.
[0195] The prophylaxis and/or treatment of pruritus and pain,
especially of acute, chronic, inflammatory and neuropathic pain in
animals, is also provided by the present compounds.
[0196] In addition, the present compounds are suitable for the
treatment and/or prophylaxis of pain disorders, especially of
acute, chronic, inflammatory and neuropathic pain in animals. This
preferably includes hyperalgesia, allodynia, pain from arthritis
(such as osteoarthritis, rheumatoid arthritis and
spondyloarthritis), premenstrual pain, endometriosis-associated
pain, post-operative pain, pain from interstitial cystitis, CRPS
(complex regional pain syndrome), trigeminal neuralgia, pain from
prostatitis, pain caused by spinal cord injuries,
inflammation-induced pain, lower back pain, cancer pain,
chemotherapy-associated pain, HIV treatment-induced neuropathy,
burn-induced pain and chronic pain.
[0197] The present invention further also provides a method for
treatment and/or prevention of disorders in animals, especially the
disorders mentioned above, using an effective amount of at least
one of the presented compounds.
[0198] Preference is given to a method for treatment and/or
prevention of allergic and/or inflammatory diseases in animals by
administering an effective amount of at least a compound of the
present formula (I) as defined above to an animal in need
thereof.
[0199] In the context of the present invention, the term
"treatment" or "treating" includes inhibition, retardation,
checking, alleviating, attenuating, restricting, reducing,
suppressing, repelling or healing of a disease, a condition, a
disorder, an injury or a health problem, or the development, the
course or the progression of such states and/or the symptoms of
such states. The term "therapy" is understood here to be synonymous
with the term "treatment".
[0200] The terms "prevention", "prophylaxis" and "preclusion" are
used synonymously in the context of the present invention and refer
to the avoidance or reduction of the risk of contracting,
experiencing, suffering from or having a disease, a condition, a
disorder, an injury or a health problem, or a development or
advancement of such states and/or the symptoms of such states.
[0201] The treatment or prevention of a disease, a condition, a
disorder, an injury or a health problem may be partial or
complete.
[0202] The present compounds can be used alone or, if required, in
combination with other active ingredients. The present invention
further provides medicaments containing at least one of the present
compounds and one or more further active ingredients, for treatment
and/or prevention of allergic and/or inflammatory diseases in
animals. Preferred examples of active ingredients suitable for
combinations include:
[0203] General mention may be made of active ingredients such as
antibacterial (e.g. penicillins, vancomycin, ciprofloxacin),
antiviral (e.g. aciclovir, oseltamivir) and antimycotic (e.g.
naftifin, nystatin) substances and gamma globulins,
immunomodulatory and immunosuppressive compounds such as
cyclosporin, Methotrexat.RTM., TNF antagonists (e.g. Humira.RTM.,
Etanercept, Infliximab), IL-I inhibitors (e.g. Anakinra,
Canakinumab, Rilonacept), phosphodiesterase inhibitors (e.g.
Apremilast), Jak/STAT inhibitors (e.g. Tofacitinib, Baricitinib,
GLPG0634), leflunomid, cyclophosphamide, rituximab, belimumab,
tacrolimus, rapamycin, mycophenolate mofetil, interferons,
corticosteroids (e.g. prednisone, prednisolone, methylprednisolone,
hydrocortisone, betamethasone), cyclophosphamide, azathioprine and
sulfasalazine; paracetamol, non-steroidal anti-inflammatory
substances (NSAIDS) (aspirin, ibuprofen, naproxen, etodolac,
celecoxib, colchicine).
[0204] In addition to those mentioned above, the inventive IRAK4
inhibitors can also be combined with the following active
ingredients:
substances for treatment of pulmonary disorders, for example
beta-2-sympathomimetics (e.g. salbutamol), anticholinergics (e.g.
glycopyrronium), methylxanthines (e.g. theophylline), leukotriene
receptor antagonists (e.g. montelukast), PDE-4 (phosphodiesterase
type 4) inhibitors (e.g. roflumilast), methotrexate, IgE
antibodies, azathioprine and cyclophosphamide, cortisol-containing
preparations; substances for treatment of osteoarthritis such as
non-steroidal anti-inflammatory substances (NSAIDs). In addition to
the two therapies mentioned, methotrexate and biologics for B-cell
and T-cell therapy (e.g. rituximab, abatacept) should be mentioned
for rheumatoid disorders, for example rheumatoid arthritis,
spondyloarthritis and juvenile idiopathic arthritis. Neurotrophic
substances such as acetylcholinesterase inhibitors (e.g.
donepezil), MAO (monoaminooxidase) inhibitors (e.g. selegiline),
interferons und anticonvulsives (e.g. gabapentin); active
ingredients for treatment of cardiovascular disorders such as
beta-blockers (e.g. metoprolol), ACE inhibitors (e.g. benazepril),
angiotensin receptor blockers (e.g. losartan, valsartan), diuretics
(e.g. hydrochlorothiazide), calcium channel blockers (e.g.
nifedipine), statins (e.g. simvastatin, fluvastatin); anti-diabetic
drugs, for example metformin, glinides (e.g. nateglinide), DPP-4
(dipeptidyl peptidase-4) inhibitors (e.g. linagliptin, saxagliptin,
sitagliptin, vildagliptin), SGLT2 (sodium/glucose cotransporter 2)
inhibitors/gliflozin (e.g. dapagliflozin, empagliflozin), incretin
mimetics (hormone glucose-dependent insulinotropic peptide (GIP)
and glucagon-like peptid 1 (GLP-1) analogues/agonists) (e.g.
exenatide, liraglutide, lixisenatide), .alpha.-glucosidase
inhibitors (e.g. acarbose, miglitol, voglibiose) and sulphonylureas
(e.g. glibenclamide, tolbutamide), insulin sensitizers (e.g.
pioglitazone) and insulin therapy (e.g. NPH insulin, insulin
lispro) Active ingredients such as mesalazine, sulfasalazine,
azathioprine, 6-mercaptopurine or methotrexate, probiotic bacteria
(Mutaflor, VSL #3.RTM., Lactobacillus GG, Lactobacillus plantarum,
L. acidophilus, L. casei, Bifidobacterium infantis 35624,
Enterococcus fecium SF68, Bifidobacterium longum, Escherichia coli
Nissle 1917), antibiotics, for example ciprofloxacin and
metronidazole, anti-diarrhoea drugs, for example loperamide, or
laxatives (bisacodyl) for treatment of chronic inflammatory bowel
diseases. Immunosuppressants such as glucocorticoids and
non-steroidale anti-inflammatory substances (NSAIDs), cortisone,
chloroquine, cyclosporine, azathioprine, belimumab, rituximab,
cyclophosphamide for treatment of lupus erythematosus. Vitamin D3
analogues, for example calcipotriol, tacalcitol or calcitriol,
salicylic acid, urea, ciclosporine, methotrexate, efalizumab for
dermatological disorders.
[0205] Mention should also be made of medicaments comprising at
least one of the present compounds and one or more further active
ingredients for the inventive use, especially EP4 inhibitors
(prostaglandin E2 receptor 4 inhibitors), P2X3 inhibitors (P2X
purinoceptor 3), PTGES inhibitors (prostaglandin E synthase
inhibitors) or AKR1C3 inhibitors (aldo-keto reductase family 1
member C3 inhibitors), for treatment and/or prevention of the
aforementioned disorders.
[0206] The present compounds can act systemically and/or locally.
For this purpose, they can be administered in a suitable manner,
for example by the oral, parenteral, pulmonal, nasal, sublingual,
lingual, buccal, rectal, dermal, transdermal or conjunctival route,
via the ear or as an implant or stent.
[0207] The present compounds can be administered in administration
forms suitable for these administration routes.
[0208] Suitable administration forms for oral administration are
those which work according to the prior art and release the present
compounds rapidly and/or in a modified manner and which contain the
present compounds in crystalline and/or amorphous and/or dissolved
form, for example tablets (uncoated or coated tablets, for example
with gastric juice-resistant or retarded-dissolution or insoluble
coatings which control the release of the present compound),
tablets or films/oblates which disintegrate rapidly in the oral
cavity, films/lyophilizates, capsules (for example hard or soft
gelatin capsules), sugar-coated tablets, chewables (for example
soft chewables), granules, pellets, powders, emulsions,
suspensions, aerosols or solutions.
[0209] Parenteral administration can be accomplished with avoidance
of a resorption step (for example by an intravenous, intraarterial,
intracardiac, intraspinal or intralumbar route) or with inclusion
of a resorption (for example by an intramuscular, subcutaneous,
intracutaneous, percutaneous or intraperitoneal route).
Administration forms suitable for parenteral administration include
preparations for injection and infusion in the form of solutions,
suspensions, emulsions, lyophilizates or sterile powders.
[0210] For the other administration routes, suitable examples are
inhalable medicament forms (including powder inhalers, nebulizers),
nasal drops, solutions or sprays, tablets, films/oblates or
capsules for lingual, sublingual or buccal administration,
suppositories, ear or eye preparations, vaginal capsules, aqueous
suspensions (lotions, shaking mixtures), lipophilic suspensions,
ointments, creams, pour-ons, transdermal therapeutic systems (e.g.
patches), milk, pastes, foams, sprinkling powders, implants or
stents.
[0211] Preference is given to oral or parenteral administration,
especially oral administration.
[0212] The present compounds can be converted to the administration
forms mentioned. This can be accomplished in a manner known per se
by mixing with inert, nontoxic, pharmaceutically suitable
excipients. These excipients include carriers (for example
microcrystalline cellulose, lactose, mannitol), solvents (e.g.
liquid polyethylene glycols), emulsifiers and dispersing or wetting
agents (for example sodium dodecylsulphate, polyoxysorbitan
oleate), binders (for example polyvinylpyrrolidone), synthetic and
natural polymers (for example albumin), stabilizers (e.g.
antioxidants, for example ascorbic acid), colorants (e.g. inorganic
pigments, for example iron oxides) and flavour and/or odour
correctants.
[0213] The present invention further provides medicaments which
comprise at least one present compound, typically together with one
or more inert, nontoxic, pharmaceutically suitable excipients, for
use in a method for treatment and/or prophylaxis of allergic and/or
inflammatory diseases in animals.
[0214] In general, it has been found to be advantageous in the case
of parenteral administration to administer amounts of about 0.001
to 1 mg/kg, preferably about 0.01 to 0.5 mg/kg, of body weight to
achieve effective results. In the case of oral administration the
dosage is about 0.01 to 100 mg/kg, preferably about 0.01 to 20
mg/kg and most preferably 0.1 to 10 mg/kg of body weight.
[0215] It may nevertheless be necessary in some cases to deviate
from the stated amounts, specifically as a function of the body
weight, route of administration, individual response to the active
ingredient, nature of the preparation and time or interval over
which administration takes place. Thus in some cases it may be
sufficient to manage with less than the abovementioned minimum
amount, while in other cases the upper limit mentioned must be
exceeded. In the case of administration of greater amounts, it may
be advisable to divide them into several individual doses over the
day.
[0216] The working examples which follow illustrate the invention.
The invention is not restricted to the examples.
[0217] Unless stated otherwise, the percentages in the tests and
examples which follow are percentages by weight; parts are parts by
weight. Solvent ratios, dilution ratios and concentration data for
the liquid/liquid solutions are based in each case on volume.
PREPARATION OF THE COMPOUNDS
[0218] The preparation of the present compounds is illustrated by
the synthesis schemes which follow.
[0219] Starting materials used for synthesis of the present
compounds are carboxylic acids (Intermediate V3), which are
commercially available or can be prepared by routes known from the
literature or analogously to routes known from the literature (see,
for example, European Journal of Organic Chemistry 2003, 8,
1559-1568, Chemical and Pharmaceutical Bulletin, 1990, 38, 9,
2446-2458, Synthetic Communications 2012, 42, 658-666, Tetrahedron,
2004, 60, 51, 11869-11874) (see, for example, Synthesis Scheme 1).
Some carboxylic acids V3 can be prepared proceeding from carboxylic
esters (Intermediate V2) by hydrolysis (cf., for example, the
reaction of ethyl 6-(hydroxymethyl)pyridine-2-carboxylate with
aqueous sodium hydroxide solution in methanol, WO2004113281) or--in
the case of a tert-butyl ester--by reaction with an acid, for
example hydrogen chloride or trifluoroacetic acid (cf., for
example, Dalton Transactions, 2014, 43, 19, 7176-7190). The
carboxylic acids V3 can also be used in the form of their alkali
metal salts. The Intermediates V2 can optionally also be prepared
from the Intermediates V1 which bear a chlorine, bromine or iodine
as substituent X.sup.1 by reaction in a carbon monoxide atmosphere,
optionally under elevated pressure, in the presence of a phosphine
ligand, for example 1,3-bis(diphenylphosphino)propane, a palladium
compound, for example palladium(II) acetate, and a base, for
example triethylamine, with addition of ethanol or methanol in a
solvent, for example dimethyl sulphoxide (for preparation methods
see, for example, WO2012112743, WO 2005082866, Chemical
Communications (Cambridge, England), 2003, 15, 1948-1949,
WO200661715). The Intermediates V1 are either commercially
available or can be prepared by routes known from the literature.
Illustrative preparation methods are detailed in WO 2012061926,
European Journal of Organic Chemistry, 2002, 2, 327-330, Synthesis,
2004, 10, 1619-1624, Journal of the American Chemical Society,
2013, 135, 32, 12122-12134, Bioorganic and Medicinal Chemistry
Letters, 2014, 24, 16, 4039-4043, US2007185058, WO2009117421.
##STR00009##
[0220] X.sup.1 is chlorine, bromine or iodine.
[0221] R.sup.d is methyl, ethyl, benzyl or tert-butyl.
[0222] R.sup.4, R.sup.5 are each as defined in the general formula
(I).
[0223] Methyl 5-amino-1H-indazole-6-carboxylate (Intermediate 2)
can be obtained proceeding from methyl 1H-indazole-6-carboxylate
(Intermediate 0) according to Synthesis Scheme 2 by nitration and
reduction of the nitro group of Intermediate 1 with hydrogen in the
presence of palladium on charcoal analogously to WO 2008/001883.
For preparation of the Intermediates 3 proceeding from Intermediate
2, it is possible to use various coupling reagents known from the
literature (Amino Acids, Peptides and Proteins in Organic
Chemistry, Vol. 3--Building Blocks, Catalysis and Coupling
Chemistry, Andrew B. Hughes, Wiley, Chapter 12--Peptide-Coupling
Reagents, 407-442; Chem. Soc. Rev., 2009, 38, 606). For example, it
is possible to use 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride in combination with 1-hydroxy-1H-benzotriazole
hydrate (HOBt, WO2012107475; Bioorg. Med. Chem. Lett., 2008, 18,
2093),
(1H-benzotriazol-1-yloxy)(dimethylamino)-N,N-dimethylmethaniminium
tetrafluoroborate (TBTU, CAS 125700-67-6),
(dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yloxy)met-
hanaminium hexafluorophosphate (HATU, CAS 148893-10-1),
propanephosphonic anhydride (as solution in ethyl acetate or DMF,
CAS68957-94-8) or di-1H-imidazol-1-ylmethanone (CDI) as coupling
reagents, with addition of a base such as triethylamine or
N-ethyl-N-isopropylpropan-2-amine in each case to the reaction
mixture. Preference is given to the use of TBTU and
N-ethyl-N-isopropylpropan-2-amine in THF.
##STR00010##
[0224] The substituents R.sup.4, R.sup.5 are each as defined in the
general formula (I).
[0225] Proceeding from the Intermediates 3, it is possible to
prepare 2-substituted indazole derivatives (Intermediate 4) (see
synthesis scheme 3). Useful reactions for this purpose include
those with optionally substituted alkyl chlorides, alkyl bromides,
alkyl iodides or alkyl 4-methylbenzenesulphonates. The alkyl
halides or alkyl 4-methylbenzenesulphonates used are commercially
available or can be prepared analogously to routes known from
literature (for the preparation of alkyl
4-methylbenzenesulphonates, one example is the reaction of an
appropriate alcohol with 4-methylbenzenesulphonyl chloride in the
presence of triethylamine or pyridine; see, for example, Bioorganic
and Medicinal Chemistry, 2006, 14, 12 4277-4294). Optionally, in
the case of use of alkyl chlorides or alkyl bromides, it is also
possible to add an alkali metal iodide such as potassium iodide or
sodium iodide. Bases used may, for example, be potassium carbonate,
caesium carbonate or sodium hydride. In the case of reactive alkyl
halides, it is also possible in some cases to use
N-cyclohexyl-N-methylcyclohexanamine. Useful solvents include, for
example, 1-methylpyrrolidin-2-one, DMF, DMSO or THF. Optionally,
the alkyl halides or alkyl 4-methylbenzenesulphonates used may have
functional groups which have optionally been protected with a
protecting group beforehand (see also P. G. M. Wuts, T. W. Greene,
Greene's Protective Groups in Organic Synthesis, Fourth Edition,
ISBN: 9780471697541). If, for example, alkyl halides or alkyl
4-methylbenzenesulphonates having one or more hydroxyl groups are
used, these hydroxyl groups may optionally be protected by a
tert-butyl(dimethyl)silyl group or a similar silicon-containing
protecting group familiar to those skilled in the art.
Alternatively, the hydroxyl groups may also be protected by the
tetrahydro-2H-pyran (THP) group or by the acetyl or benzoyl group.
The protecting groups used can then be detached subsequently to the
synthesis of Intermediate 4, or else after the synthesis of (I).
If, for example, a tert-butyl(dimethylsilyl) group is used as
protecting group, it can be detached using tetrabutylammonium
fluoride in a solvent such as THF, for example. A THP protecting
group can be detached, for example, using 4-methylbenzenesulphonic
acid (optionally in monohydrate form). Acetyl groups or benzoyl
groups can be detached by treatment with aqueous sodium hydroxide
solution.
[0226] Optionally, the alkyl halides or alkyl
4-methylbenzenesulphonates used may contain functional groups which
can be converted by oxidation or reduction reactions known to those
skilled in the art (see, for example, Science of Synthesis, Georg
Thieme Verlag). If, for example, the functional group is a sulphide
group, this can be oxidized by methods known in the literature to a
sulphoxide or sulphone group. In the case of a sulphoxide group,
this can likewise be oxidized to a sulphone group. For these
oxidation steps, it is possible to use, for example,
3-chloroperbenzoic acid (CAS 937-14-4) (in this regard, see also,
for example, US201094000 for the oxidation of a
2-(methylsulphanyl)ethyl-1H-pyrazole derivative to a
2-(methylsulphinyl)ethyl-1H-pyrazole derivative and the oxidation
of a further 2-(methylsulphanyl)ethyl-1H-pyrazole derivative to a
2-(methylsulphonyl)ethyl-1H-pyrazole derivative). If the alkyl
halides or tosylates used contain a keto group, this can be reduced
by reduction methods known to those skilled in the art to an
alcohol group (see, for example, Chemische Berichte, 1980, 113,
1907-1920 for the use of sodium borohydride). These oxidation or
reduction steps can be effected subsequently to the synthesis of
Intermediate 4, or else after the synthesis of the present
compounds of the general formula (I). Alternatively, Intermediate 4
can be prepared via Mitsunobu reaction (see, for example, K. C. K.
Swamy et. al. Chem. Rev. 2009, 109, 2551-2651) of Intermediate 3
with optionally substituted alkyl alcohols. It is possible to
utilize various phosphines such as triphenylphosphine,
tributylphosphine or 1,2-diphenylphosphinoethane in combination
with diisopropyl azodicarboxylate (CAS 2446-83-5) or further
diazene derivatives mentioned in the literature (K. C. K. Swamy et.
al. Chem. Rev. 2009, 109, 2551-2651). Preference is given to the
use of triphenylphosphine and diisopropyl azodicarboxylate. If the
alkyl alcohol bears a functional group it is possible--as in the
case of the abovementioned reactions with alkyl halides--for known
protecting group strategies (further pointers can be found in P. G.
M. Wuts, T. W. Greene, Greene's Protective Groups in Organic
Synthesis, Fourth Edition, ISBN: 9780471697541) and--as in the case
of the abovementioned reactions with alkyl halides--for oxidation
or reduction steps to be effected correspondingly to the synthesis
of Intermediate 4, or else after the synthesis of the present
compounds of the general formula (I). Proceeding from Intermediate
4, present compounds of the general formula (I) where R.sup.2 and
R.sup.3 are defined as C.sub.1-C.sub.6-alkyl (where R.sup.2 and
R.sup.3 have the same definition) may be obtained by a Grignard
reaction (cf., for example, the reaction of a methyl
1H-indazole-6-carboxylate derivative with methylmagnesium bromide
in EP 2489663). For the Grignard reaction, it is possible to use
alkylmagnesium halides. Particular preference is given to
methylmagnesium chloride or methylmagnesium bromide in THF or
diethyl ether, or else in mixtures of THF and diethyl ether.
Alternatively, proceeding from Intermediate 4, present compounds of
the general formula (I) where R.sup.2 and R.sup.3 are defined as
C.sub.1-C.sub.6-alkyl (where R.sup.2 and R.sup.3 have the same
definition) may be obtained by a reaction with an alkyllithium
reagent (cf., for example, the reaction of a methyl
2-amino-4-chloro-1-methyl-1H-benzimidazole-7-carboxylate derivative
with isopropyllithium or tert-butyllithium in WO2006116412).
Proceeding from Intermediate 4, it is possible to prepare present
compounds of the general formula (I) where R.sup.2 and R.sup.3 are
defined as H by reduction with lithium aluminium hydride in THF,
lithium borohydride in THF or sodium borohydride in THF, optionally
with addition of methanol, or mixtures of lithium borohydride and
sodium borohydride.
##STR00011##
[0227] The substituents R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5
are each as defined in the general formula (I).
[0228] Proceeding from Intermediate 3, Intermediate 5 where R.sup.2
and R.sup.3 are defined as C.sub.1-C.sub.6-alkyl (where R.sup.2 and
R.sup.3 have the same definition) may be obtained by a Grignard
reaction (cf., for example, Synthesis Scheme 4). For this purpose,
it is possible to use suitable alkylmagnesium halides, for example
methylmagnesium chloride or methylmagnesium bromide in THF or in
diethyl ether or else in mixtures of THF and diethyl ether.
[0229] Proceeding from Intermediate 5, it is then possible to
prepare a portion (I-a) of the present compounds (I) where R.sup.2
and R.sup.3 are defined as C.sub.1-C.sub.6-alkyl (where R.sup.2 and
R.sup.3 have the same definition). For this purpose, analogously to
Synthesis Scheme 3 (preparation of Intermediate 3), useful
reactions are those of Intermediate 5 with optionally substituted
alkyl chlorides, alkyl bromides, alkyl iodides or alkyl
4-methylbenzenesulphonates. It is possible to use protecting group
strategies analogously to those described in Synthesis Scheme
3.
[0230] Alternatively, for preparation of a portion (I-a) of the
present compounds (I) where R.sup.2 and R.sup.3 are defined as
C.sub.1-C.sub.6-alkyl (where R.sup.2 and R.sup.3 have the same
definition), it is possible to use the Mitsunobu reaction of
Intermediate 5 with optionally substituted alkyl alcohols
(analogously to Synthesis Scheme 3).
[0231] If R.sup.1 in the compounds of the formula (I-a) includes a
suitable functional group, it is optionally possible subsequently,
in analogy to Synthesis Scheme 3, to use oxidation or reduction
reactions for preparation of further present compounds.
##STR00012##
[0232] The substituents R.sup.1, R.sup.4, R.sup.5 are each as
defined in the general formula (I). R.sup.2 and R.sup.3 always have
the same definition and are both C.sub.1-C.sub.6-alkyl.
[0233] Proceeding from Intermediate 1, it is possible to prepare
Intermediate 4 in an alternative manner (see Synthesis Scheme 5).
First of all, Intermediate 1 is converted to Intermediate 6 by
methods as in Synthesis Scheme 3 (preparation of Intermediate 4
from Intermediate 3).
[0234] Intermediate 6 can then be converted to Intermediate 7 by
reduction of the nitro group. For example, the nitro group can be
reduced with palladium on carbon under a hydrogen atmosphere (cf.,
for example, WO2013174744 for the reduction of
6-isopropoxy-5-nitro-1H-indazole to
6-isopropoxy-1H-indazol-5-amine) or by the use of iron and ammonium
chloride in water and ethanol (see, for example, also Journal of
the Chemical Society, 1955, 2412-2419), or by the use of tin(II)
chloride (CAS 7772-99-8). The use of iron and ammonium chloride in
water and ethanol is preferred. The preparation of Intermediate 4
from Intermediate 7 can be effected analogously to Synthesis Scheme
2 (preparation of Intermediate 3 from Intermediate 2).
[0235] As described for Synthesis Scheme 3, it is optionally
possible to use protecting group strategies in the case of
Synthesis Scheme 5 as well. Optionally, it is additionally
possible, proceeding from Intermediate 6 or Intermediate 7, as
described for Synthesis Scheme 3, to conduct oxidation or reduction
reactions known to those skilled in the art (cf., for example
Science of Synthesis, Georg Thieme Verlag).
##STR00013##
[0236] The substituents R.sup.1, R.sup.4, R.sup.5 are each as
defined in the general formula (I).
Synthesis of the Example Compounds
Abbreviations and Elucidations
TABLE-US-00001 [0237] DMF N,N-dimethylformamide DMSO dimethyl
sulphoxide THF tetrahydrofuran RT room temperature HPLC
high-performance liquid chromatography h hour(s) HCOOH formic acid
MeCN acetonitrile min minute(s) UPLC ultrahigh-performance liquid
chromatography DAD diode array detector ELSD evaporating light
scattering detector ESI electrospray ionization SQD single
quadrupole detector CPG core-pulled precision glass NH.sub.3
ammonia
[0238] The term sodium chloride solution always means a saturated
aqueous sodium chloride solution.
[0239] The chemical names of the intermediates and examples were
generated using the ACD/LABS (Batch Version 12.01.) software.
Methods
[0240] In some cases, the present compounds and precursors and/or
intermediates thereof were analysed by LC-MS.
[0241] Method A1: UPLC (MeCN--HCOOH):
[0242] Instrument: Waters Acquity UPLC-MS SQD 3001; column: Acquity
UPLC BEH C18 1.7 50.times.2.1 mm; eluent A: water+0.1% by vol. of
formic acid (99%), eluent B: acetonitrile; gradient: 0-1.6 min
1-99% B, 1.6-2.0 min 99% B; flow rate 0.8 ml/min; temperature:
60.degree. C.; injection: 2 .mu.l; DAD scan: 210-400 nm; MS ESI+,
ESI-, scan range 160-1000 m/z; ELSD.
[0243] Method A2: UPLC (MeCN--NH.sub.3):
[0244] Instrument: Waters Acquity UPLC-MS SQD 3001; column: Acquity
UPLC BEH C18 1.7 50.times.2.1 mm; eluent A: water+0.2% by vol. of
ammonia (32%), eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B,
1.6-2.0 min 99% B; flow rate 0.8 ml/min; temperature: 60.degree.
C.; injection: 2 .mu.l; DAD scan: 210-400 nm; MS ESI+, ESI-, scan
range 160-1000 m/z; ELSD.
[0245] Method A3: (LC-MS)
[0246] Instrument: Agilent 1290 Infinity LC; column: Acquity UPLC
BEH C18 1.7 50.times.2.1 mm; eluent A: water+0.05% by vol. of
formic acid, eluent B: acetonitrile+0.05% by vol. of formic acid;
gradient: 0-1.7 min 2-90% B, 1.7-2.0 min 90% B; flow rate 1.2
ml/min; temperature: 60.degree. C.; injection: 2 .mu.l; DAD scan:
190-390 nm; MS: Agilent TOF 6230.
[0247] Method A4: (LC-MS)
[0248] Instrument: Waters Acquity; column: Kinetex (Phenomenex),
50.times.2 mm; eluent A: water+0.05% by vol. of formic acid, eluent
B: acetonitrile+0.05% by vol. of formic acid; gradient: 0-1.9 min
1-99% B, 1.9-2.1 min 99% B; flow rate 1.5 ml/min; temperature:
60.degree. C.; injection: 0.5 .mu.l; DAD scan: 200-400 nm.
[0249] In some cases, the present compounds and the precursors
and/or intermediates thereof were purified by the following
illustrative preparative HPLC methods:
[0250] Method P1: system: Waters Autopurification system: Pump
2545, Sample Manager 2767, CFO, DAD 2996, ELSD 2424, SQD; column:
XBridge C18 5 .mu.m 100.times.30 mm; eluent A: water+0.1% by vol.
of formic acid, eluent B: acetonitrile; gradient: 0-8 min 10-100%
B, 8-10 min 100% B; flow: 50 ml/min; temperature: room temperature;
solution: max. 250 mg/max. 2.5 ml DMSO or DMF; injection:
1.times.2.5 ml; detection: DAD scan range 210-400 nm; MS ESI+,
ESI-, scan range 160-1000 m/z.
[0251] Method P2: system: Waters Autopurification system: Pump 254,
Sample Manager 2767, CFO, DAD 2996, ELSD 2424, SQD 3100; column:
XBridge C18 5 .mu.m 10.times.30 mm; eluent A: water+0.2% by vol. of
ammonia (32%), eluent B: methanol; gradient: 0-8 min 30-70% B;
flow: 50 ml/min; temperature: room temperature; detection: DAD scan
range 210-400 nm; MS ESI+, ESI-, scan range 160-1000 m/z; ELSD.
[0252] Method P3: system: Labomatic, pump: HD-5000, fraction
collector: LABOCOL Vario-4000, UV detector: Knauer UVD 2.1S;
column: XBridge C18 5 .mu.m 100.times.30 mm; eluent A: water+0.2%
by vol. of ammonia (25%), eluent B: acetonitrile; gradient: 0-1 min
15% B, 1-6.3 min 15-55% B, 6.3-6.4 min 55-100% B, 6.4-7.4 min 100%
B; flow: 60 ml/min; temperature: room temperature; solution: max.
250 mg/2 ml DMSO; injection: 2.times.2 ml; detection: UV 218 nm;
Software: SCPA PrepCon5.
[0253] Method P4: system: Labomatic, pump: HD-5000, fraction
collector: LABOCOL Vario-4000, UV detector: Knauer UVD 2.1S;
column: Chromatorex RP C18 10 .mu.m 125.times.30 mm; eluent A:
water+0.1% by vol. of formic acid, eluent B: acetonitrile;
gradient: 0-15 min 65-100% B; flow: 60 ml/min; temperature: room
temperature; solution: max. 250 mg/2 ml DMSO; injection: 2.times.2
ml; detection: UV 254 nm; Software: SCPA PrepCon5.
[0254] Method P5: system: Sepiatec: Prep SFC100, column: Chiralpak
IA 5 .mu.m 250.times.20 mm; eluent A: carbon dioxide, eluent B:
ethanol; gradient: isocratic 20% B; flow: 80 ml/min; temperature:
40.degree. C.; solution: max. 250 mg/2 ml DMSO; injection:
5.times.0.4 mL; detection: UV 254 nm.
[0255] Method P6: system: Agilent: Prep 1200, 2.times. prep pump,
DLA, MWD, Gilson: Liquid Handler 215; column: Chiralcel OJ-H 5
.mu.m 250.times.20 mm; eluent A: hexane, eluent B: ethanol;
gradient: isocratic 30% B; flow: 25 ml/min; temperature: 25.degree.
C.; solution: 187 mg/8 ml ethanol/methanol; injection: 8.times.1.0
ml; detection: UV 280 nm.
[0256] Method P7: system: Labomatic, pump: HD-5000, fraction
collector: LABOCOL Vario-4000, UV detector: Knauer UVD 2.1S;
column: XBridge C18 5 .mu.m 100.times.30 mm; eluent A: water+0.1%
by vol. of formic acid, eluent B: acetonitrile; gradient: 0-3 min:
65% B isocratic, 3-13 min: 65-100% B; flow: 60 ml/min; temperature:
room temperature; solution: max. 250 mg/2 ml DMSO; injection:
2.times.2 ml; detection: UV 254 nm.
[0257] Method P8: system: Agilent: Prep 1200, 2.times.prep pump,
DLA, MWD, Gilson: Liquid Handler 215; column: Chiralpak IF 5 .mu.m
250.times.20 mm; eluent A: ethanol, eluent B: methanol; gradient:
isocratic 50% B; flow: 25 ml/min; temperature: 25.degree. C.;
solution: 600 mg/7 ml N,N-dimethylformamide; injection:
10.times.0.7 ml; detection: UV 254 nm.
[0258] In some cases, substance mixtures were purified by column
chromatography on silica gel.
[0259] For preparation of some of the present compounds and the
precursors and/or intermediates thereof, a column chromatography
purification ("flash chromatography") was conducted on silica gel
using Isolera.RTM. devices from Biotage. This was done using
cartridges from Biotage, for example the "SNAP Cartridge, KP_SIL"
cartridge of different size and "Interchim Puriflash Silica HP 15UM
flash column" cartridges from Interchim of different size.
Starting Materials
Intermediate V2-1
Methyl 6-(2-hydroxypropan-2-yl)pyridine-2-carboxylate
##STR00014##
[0261] 2.00 g (9.26 mmol) of 2-(6-bromopyridin-2-yl)propan-2-ol
(CAS 638218-78-7) were dissolved in 20 ml of methanol and 20 ml of
DMSO. Subsequently, 250 mg of 1,3-bis(diphenylphosphino)propane,
130 mg of palladium(II) acetate and 3 ml of triethylamine were
added. The reaction mixture was purged three times with carbon
monoxide at room temperature and stirred under a 13 bar carbon
monoxide atmosphere for 30 min. The carbon monoxide atmosphere was
removed by applying a vacuum and the mixture was stirred under a 14
bar carbon monoxide atmosphere at 100.degree. C. for 24 h. The
autoclave was decompressed, water was added to the reaction
mixture, and the reaction mixture was extracted three times with
ethyl acetate, washed with saturated aqueous sodium
hydrogencarbonate solution and sodium chloride solution, filtered
through a hydrophobic filter and concentrated. This gave 1.60 g of
a crude product.
[0262] UPLC-MS (Method A1): R.sub.t=0.76 min (UV detector: TIC),
mass found 195.00.
Intermediate V3-1
Potassium 6-(2-hydroxypropan-2-yl)pyridine-2-carboxylate
##STR00015##
[0264] 1.60 g of the crude product of Intermediate 0-1 were
initially charged in 15 ml of methanol, 0.74 g of potassium
hydroxide was added and the mixture was stirred at 50.degree. C.
for 16.5 h. After concentration, this gave 2.1 g of a residue which
was used without further purification. UPLC-MS (Method A1):
R.sub.t=0.47 min (UV detector: TIC), mass found 181.00.
Intermediate 1-1
Methyl 5-nitro-1H-indazole-6-carboxylate
##STR00016##
[0266] 4.60 g (26.1 mmol) of methyl 1H-indazole-6-carboxylate (CAS
No: 170487-40-8) were dissolved in 120 ml of sulphuric acid (96%)
and cooled to -15.degree. C. in a three-neck flask having a CPG
stirrer, dropping funnel and internal thermometer. Over a period of
15 min, the nitrating acid (10 ml of 96% sulphuric acid in 5 ml of
65% nitric acid), which had been prepared and cooled beforehand,
was added dropwise to this solution. After the dropwise addition
had ended, the mixture was stirred for a further 1 h (internal
temperature at -13.degree. C.). The reaction mixture was added to
ice, and the precipitate was filtered off with suction, washed with
water and dried in a drying cabinet at 50.degree. C. under reduced
pressure. 5.49 g of the title compound were obtained.
[0267] UPLC-MS (Method A2): R.sub.t=0.75 min
[0268] MS (ESIpos): m/z=222(M+H).sup.+
[0269] .sup.1H NMR (400 MHz, DMSO-d6): .delta. [ppm]=3.87 (s, 3H),
7.96 (s, 1H), 8.44 (s, 1H), 8.70 (s, 1H), 13.98 (br. s., 1H).
Intermediate 2-1
Methyl 5-amino-1H-indazole-6-carboxylate
##STR00017##
[0271] 4.40 g (19.8 mmol) of methyl
5-nitro-1H-indazole-6-carboxylate (Intermediate 1-1) were dissolved
in 236 ml of methanol and hydrogenated with 1.06 g (0.99 mmol) of
palladium on activated carbon under standard hydrogen pressure at
25.degree. C. for 3 h. The reaction mixture was filtered through
Celite, the filter was washed with methanol, and the filtrate was
concentrated. 3.53 g of the title compound were obtained.
[0272] .sup.1H NMR (300 MHz, DMSO-d6): .delta. [ppm]=3.85 (s, 3H)
6.01 (s, 2H) 6.98 (s, 1H) 7.79-7.91 (m, 1H) 7.99 (s, 1H) 12.84 (br.
s., 1H).
Intermediate 3-1
Methyl
5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6--
carboxylate
##STR00018##
[0274] 4.95 g (25.9 mmol) of
6-(trifluoromethyl)pyridine-2-carboxylic acid were initially
charged in 45 ml of THF. 9.07 g (28.2 mmol) of
O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate and 4.92 ml (28.2 mmol) of
N-ethyl-N-isopropylpropan-2-amine were added and the mixture was
stirred at 25.degree. C. for 30 min. Subsequently, 4.50 g (23.5
mmol) of methyl 5-amino-1H-indazole-6-carboxylate (Intermediate
2-1) were added and the mixture was stirred at 25.degree. C. for 24
h. The reaction mixture was filtered with suction through a
membrane filter and the solids were washed with THF and with water,
and dried in a drying cabinet overnight. 7.60 g of the title
compound were obtained.
[0275] UPLC-MS (Method A2): R.sub.t=1.16 min
[0276] MS (ESIpos): m/z=365 (M+H).sup.+
[0277] .sup.1H NMR (400 MHz, DMSO-d6): .delta. [ppm]=3.97 (s, 3H),
8.13-8.27 (m, 2H), 8.30 (s, 1H), 8.33-8.45 (m, 1H), 8.45-8.51 (m,
1H), 9.15 (s, 1H), 12.57 (s, 1H), 13.44 (s, 1H).
Intermediate 3-2
Methyl
5-({[6-(difluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-c-
arboxylate
##STR00019##
[0279] 2.85 g (23.5 mmol) of
6-(difluoromethyl)pyridine-2-carboxylic acid were initially charged
in 30 ml of THF. 6.05 g (18.8 mmol) of
O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate and 3.3 ml of N-ethyl-N-isopropylpropan-2-amine
were added and the mixture was stirred at room temperature for 10
minutes. Subsequently, 3.00 g (15.7 mmol) of methyl
5-amino-1H-indazole-6-carboxylate were added and the mixture was
stirred at room temperature overnight. The reaction mixture was
admixed with water, and the precipitate was filtered off with
suction and washed repeatedly with water and dichloromethane. This
gave 1.53 g (27% of theory) of the title compound. The phases of
the filtrate were separated, the organic phase was concentrated,
admixed with a little dichloromethane and suspended in an
ultrasound bath, and the precipitate was filtered off with suction.
This gave a further 1.03 g of the title compound. 1H-NMR (first
product fraction, 300 MHz, DMSO-d6): .delta. [ppm]=3.99 (s, 3H),
7.09 (t, 1H), 8.00 (d, 1H), 8.21-8.40 (m, 4H), 9.14 (s, 1H), 12.53
(s, 1H), 13.44 (s, 1H).
Intermediate 3-3
Methyl
5-({[6-(2-hydroxypropan-2-yl)pyridin-2-yl]carbonyl}amino)-1H-indazo-
le-6-carboxylate
##STR00020##
[0281] 2.10 g of potassium
6-(2-hydroxypropan-2-yl)pyridine-2-carboxylate (Intermediate V3-1)
were initially charged in 15 ml of THF. 3.69 g (11.5 mmol) of
O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate and 2.00 ml of N-ethyl-N-isopropylpropan-2-amine
were added and the mixture was stirred at room temperature for 15
min. Subsequently, 1.83 g (9.58 mmol) of methyl
5-amino-1H-indazole-6-carboxylate (Intermediate 2-1) were added and
the mixture was stirred at room temperature for 19 h. The mixture
was admixed with water and ethyl acetate, the undissolved solids
were filtered off, the phases of the filtrate were separated, and
the aqueous phase was extracted twice with ethyl acetate, washed
with sodium chloride solution, filtered through a hydrophobic
filter, concentrated and purified by column chromatography on
silica gel (hexane/ethyl acetate). After the solvents had been
removed, 1.56 g of the title compound were obtained as a yellow
foam.
[0282] UPLC-MS (Method A1): R.sub.t=1.00 min (UV detector: TIC
Smooth), mass found 354.00.
[0283] 1H-NMR (500 MHz, DMSO-d6): .delta. [ppm]=1.63 (s, 6H), 3.97
(s, 3H), 5.37 (s, 1H), 7.90-7.95 (m, 1H), 8.03-8.07 (m, 2H), 8.23
(s, 1H), 8.29 (s, 1H), 9.19 (s, 1H), 12.79 (s, 1H), 13.41 (br.s.,
1H).
Intermediate 4-1
Methyl
2-(oxetan-3-ylmethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl-
}amino)-2H-indazole-6-carboxylate
##STR00021##
[0285] 1.00 g (2.66 mmol) of methyl
5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carbox-
ylate (Intermediate 3-1) was dissolved in 10 ml of DMF and, after
addition of 1.10 g (7.99 mmol) of potassium carbonate and 221 mg
(1.33 mmol) of potassium iodide, the mixture was stirred at
25.degree. C. for 30 min. 603 mg (3.99 mmol) of
3-bromomethyloxetane were added, and the mixture was stirred at
25.degree. C. for 24 h. The reaction mixture was partitioned
between water and ethyl acetate. The mixture was extracted twice
with ethyl acetate, and the combined organic phases were filtered
through a hydrophobic filter and concentrated. The residue was
purified by column chromatography on silica gel (hexane/ethyl
acetate). 260 mg of the title compound were obtained.
[0286] UPLC-MS (Method A2): R.sub.t=1.24 min
[0287] MS (ESIpos): m/z=435 (M+H).sup.+
[0288] .sup.1H NMR (400 MHz, DMSO-d6): .delta. [ppm]=3.49-3.64 (m,
1H), 3.95 (s, 3H), 4.49 (t, 2H), 4.68 (dd, 2H), 4.81 (d, 2H), 8.20
(dd, 1H), 8.35-8.41 (m, 1H), 8.43-8.49 (m, 2H), 8.55-8.58 (m, 1 H),
9.06 (s, 1H), 12.53 (s, 1H).
Intermediate 4-2
Methyl
2-(2-methoxyethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}am-
ino)-2H-indazole-6-carboxylate
##STR00022##
[0290] 1.00 g (2.75 mmol) of methyl
5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carbox-
ylate (Intermediate 3-1) was dissolved in 5 ml of DMF, and 387
.mu.l (4.12 mmol) of 2-bromoethyl methyl ether, 1.14 g (8.23 mmol)
of potassium carbonate and 228 mg (1.37 mmol) of potassium iodide
were added while stirring. The reaction mixture was stirred at
25.degree. C. for 24 h, diluted with water and extracted twice with
ethyl acetate. The combined organic phases were filtered through a
hydrophobic filter and concentrated. The residue was purified by
column chromatography on silica gel (hexane/ethyl acetate). 12 mg
of the title compound were obtained.
[0291] UPLC-MS (Method A1): R.sub.t=1.24 min
[0292] MS (ESIpos): m/z=423 (M+H).sup.+
[0293] .sup.1H NMR (400 MHz, DMSO-d6): .delta. [ppm]=3.24 (s, 3H),
3.86 (t, 2H), 3.96 (s, 3H), 4.65 (t, 2H), 8.21 (dd, 1H), 8.35-8.42
(m, 1H), 8.43-8.51 (m, 2H), 8.52 (d, 1H), 9.06 (s, 1H), 12.53 (s, 1
H).
Intermediate 4-3
Methyl
2-(3-methoxypropyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}a-
mino)-2H-indazole-6-carboxylate
##STR00023##
[0295] 1.00 g (2.75 mmol) of methyl
5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carbox-
ylate (Intermediate 3-1) was dissolved in 5 ml of DMF, and 460
.mu.l (4.12 mmol) of 1-bromo-3-methoxypropane, 1.14 g (8.23 mmol)
of potassium carbonate and 228 mg (1.37 mmol) of potassium iodide
were added while stirring. The reaction mixture was stirred at
25.degree. C. for 72 h, diluted with water and extracted twice with
ethyl acetate. The combined organic phases were filtered through a
hydrophobic filter and concentrated. The residue was purified by
column chromatography on silica gel (hexane/ethyl acetate). 28 mg
of the title compound were obtained.
[0296] UPLC-MS (Method A1): R.sub.t=1.29 min
[0297] MS (ESIpos): m/z=437 (M+H).sup.+
[0298] .sup.1H NMR (400 MHz, DMSO-d6): .delta. [ppm]=2.17 (quin,
2H), 3.24 (s, 3H), 3.33-3.36 (m, 2H), 3.96 (s, 3H), 4.53 (t, 2H),
8.21 (dd, 1H), 8.35-8.42 (m, 1H), 8.45-8.49 (m, 2H), 8.54 (d, 1H),
9.06 (s, 1H), 12.54 (s, 1H).
Intermediate 4-4
Methyl
2-(3-hydroxy-3-methylbutyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]ca-
rbonyl}amino)-2H-indazole-6-carboxylate
Preparation Method 1
##STR00024##
[0300] 930 mg (2.55 mmol) of methyl
5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carbox-
ylate (Intermediate 3-1), 1.06 g of potassium carbonate and 212 mg
of potassium iodide were initially charged in 9 ml of DMF and the
mixture was stirred for 15 min. Then 0.62 ml of
4-bromo-2-methylbutan-2-ol was added and the mixture was stirred at
60.degree. C. for 16 h. The mixture was admixed with water and
extracted twice with ethyl acetate, and the extract was washed
three times with saturated sodium chloride solution, filtered and
concentrated. Column chromatography purification on silica gel
(hexane/ethyl acetate) gave 424 mg of the title compound.
[0301] UPLC-MS (Method A2): R.sub.t=1.21 min (UV detector: TIC),
mass found 450.00.
[0302] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. [ppm]=1.16 (s,
6H) 2.02-2.11 (m, 2H) 3.96 (s, 3H) 4.51-4.60 (m, 3H) 8.20 (dd,
J=7.83, 1.01 Hz, 1H) 8.39 (s, 1H) 8.45 (s, 2H) 8.55 (d, J=0.76 Hz,
1H) 9.05 (s, 1H) 12.52 (s, 1H)
Preparation Method 2
[0303] 1.95 g (7.03 mmol) of methyl
5-amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylate
(Intermediate 7-1) were initially charged in 30 ml of THF. 1.48 g
(7.73 mmol) of 6-(trifluoromethyl)pyridine-2-carboxylic acid, 2.71
g (8.44 mmol) of O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate and 1.47 ml (8.44 mmol) of
N-ethyl-N-isopropylpropan-2-amine were added and the mixture was
stirred at 25.degree. C. for 20.5 h. Water was added, the mixture
was extracted three times with ethyl acetate and the extracts were
washed with sodium chloride solution, filtered through a
hydrophobic filter and concentrated. The residue was separated by
column chromatography on silica gel (hexane/ethyl acetate
gradient). 2.79 g of the title compound were obtained.
[0304] UPLC-MS (Method A1): R.sub.t=1.23 min (UV detector: TIC),
mass found 450.00.
Intermediate 4-5
Methyl
2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-5-({[6-(trifluoromethyl-
)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate
##STR00025##
[0306] 1.00 g (2.66 mmol, 97%) of methyl
5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carbox-
ylate (Intermediate 3-1) was initially charged in 50 ml of DMF,
1.10 g (7.99 mmol) of potassium carbonate and 221 mg (1.33 mmol) of
potassium iodide were added while stirring, and the mixture was
stirred at 25.degree. C. for 30 min. Subsequently, 857 .mu.l (3.99
mmol) of (2-bromoethoxy)(tert-butyl)dimethylsilane were added and
the mixture was stirred at 25.degree. C. for 24 h. The reaction
mixture was diluted with water and extracted with ethyl acetate.
The combined organic phases were filtered through a hydrophobic
filter and concentrated. The residue was purified by column
chromatography on silica gel (hexane/ethyl acetate). 400 mg of the
title compound were obtained.
[0307] UPLC-MS (Method A1): R.sub.t=1.58 min
[0308] MS (ESIpos): m/z=523 (M+H).sup.+
[0309] .sup.1H NMR (300 MHz, DMSO-d6): .delta. [ppm]=-0.18--0.13
(m, 6H), 0.74 (s, 9H), 3.96 (s, 3H), 4.08 (t, 2H), 4.57 (t, 2H),
8.15-8.25 (m, 1H), 8.32-8.43 (m, 1H), 8.43-8.52 (m, 3H), 9.07 (s,
1H), 12.53 (s, 1H).
Intermediate 4-6
Methyl
2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-5-({[6-(trifluoromethy-
l)pyridin-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate
##STR00026##
[0311] Analogously to Intermediate 4-5, 1.00 g (2.75 mmol) of
methyl
5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carbox-
ylate (Intermediate 3-1) was dissolved in 10 ml of DMF, 1.14 g
(8.24 mmol) of potassium carbonate and 228 mg (1.37 mmol) of
potassium iodide were added while stirring, and the mixture was
stirred at 25.degree. C. for 30 min. Subsequently, 1.04 g (4.12
mmol) of (3-bromopropoxy)(tert-butyl)dimethylsilane were added and
the mixture was stirred at 25.degree. C. for 24 h. The reaction
mixture was filtered and the filtercake was washed with ethyl
acetate. The reaction mixture was partitioned between water and
ethyl acetate and the aqueous phase was extracted twice with ethyl
acetate. The combined organic phases were filtered through a
hydrophobic filter and concentrated. Purification of the residue by
preparative HPLC gave 428 mg of the title compound.
[0312] UPLC-MS (Method A1): R.sub.t=1.63 min
[0313] MS (ESIpos): m/z=537 (M+H).sup.+
[0314] .sup.1H NMR (400 MHz, DMSO-d6): .delta. [ppm]=-0.02-0.06 (m,
6H), 0.87 (s, 9H), 2.14 (quin, 2H), 3.62 (t, 2H), 3.96 (s, 3H),
4.54 (t, 2H), 8.20 (d, 1H), 8.35-8.42 (m, 1H), 8.43-8.48 (m, 3H),
8.49-8.53 (m, 1H), 9.06 (s, 1H).
Intermediate 4-7
Methyl
5-({[6-(2-hydroxypropan-2-yl)pyridin-2-yl]carbonyl}amino)-2-(4,4,4--
trifluorobutyl)-2H-indazole-6-carboxylate
##STR00027##
[0316] 300 mg (0.80 mmol) of methyl
5-({[6-(2-hydroxypropan-2-yl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-c-
arboxylate (Intermediate 3-3) were initially charged in 4.5 ml of
DMF. 287 mg (1.21 mmol) of 1,1,1-trifluoro-4-iodobutane and 333 mg
of potassium carbonate were added and the mixture was stirred at
100.degree. C. for 23 h. Water was added, and the mixture was
extracted three times with ethyl acetate. The mixture was
concentrated and the product was purified by preparative HPLC. This
gave 72 mg of the title compound.
[0317] UPLC-MS (Method A1): R.sub.t=1.26 min (UV detector: TIC),
mass found 464.17.
Intermediate 4-8
Methyl
5-{[(5-fluoro-6-methylpyridin-2-yl)carbonyl]amino}-2-(3-hydroxy-3-m-
ethylbutyl)-2H-indazole-6-carboxylate
##STR00028##
[0319] 195 mg (0.46 mmol) of methyl
5-amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylate
(Intermediate 7-1) were reacted with 78 mg (0.50 mmol) of
5-fluoro-6-methylpyridine-2-carboxylic acid analogous to
Intermediate 4-4 (Preparation Method 2) within 19.5 h. 228 mg of a
crude product were obtained after analogous aqueous workup.
[0320] UPLC-MS (Method A1): R.sub.t=1.20 min (UV detector: TIC),
mass found 414.00.
Intermediate 4-9
Methyl
2-(3-hydroxy-3-methylbutyl)-5-{[(6-methylpyridin-2-yl)carbonyl]amin-
o}-2H-indazole-6-carboxylate
##STR00029##
[0322] 195 mg (0.45 mmol) of methyl
5-amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylate
(Intermediate 7-1) were reacted with 70 mg (0.50 mmol) of
6-methylpyridine-2-carboxylic acid analogously to preparation of
Intermediate 4-4 (Preparation Method 2) within 19.5 h. 278 mg of
the title compound as crude product were obtained after analogous
aqueous workup.
[0323] UPLC-MS (Method A1): R.sub.t=1.14 min (UV detector: TIC),
mass found 396.00.
Intermediate 4-10
Methyl
2-[3-(2,2,2-trifluoroethoxy)propyl]-5-({[6-(trifluoromethyl)pyridin-
-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate
##STR00030##
[0325] A mixture of 250 mg (0.58 mmol) of methyl
5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carbox-
ylate (Intermediate 3-1), 193 mg (0.88 mmol) of 3-bromopropyl
2,2,2-trifluoroethyl ether, 242 mg of potassium carbonate and 145
mg of potassium iodide in 3 ml of DMF was stirred at 100.degree. C.
for 20 h. Water was added, the mixture was extracted with ethyl
acetate and the extract was washed with sodium chloride solution
and concentrated.
[0326] Purification by preparative HPLC gave 52 mg of the title
compound.
[0327] UPLC-MS (Method A1): R.sub.t=1.39 min (UV detector: TIC),
mass found 504.12.
Intermediate 4-11
Methyl
5-({[6-(difluoromethyl)pyridin-2-yl]carbonyl}amino)-2-(3-hydroxy-3--
methylbutyl)-2H-indazole-6-carboxylate
##STR00031##
[0329] 2.00 g of methyl
5-amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylate
(Intermediate 7-1) were initially charged in 40 ml of THF. 1.50 g
of 6-(difluoromethyl)pyridine-2-carboxylic acid, 2.78 g of
O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TBTU, CAS Number 125700-67-6) and 1.5 ml of
N-ethyl-N-isopropylpropan-2-amine were added and the mixture was
stirred at RT for 24 h. Water was added, the mixture was extracted
three times with ethyl acetate, and the combined organic phases
were washed with sodium chloride solution and filtered through a
hydrophobic filter. The mixture was concentrated and the residue
was purified by column chromatography on silica gel (hexane/ethyl
acetate). This gave 3.05 g of the title compound as a yellow
solid.
[0330] UPLC-MS (Method A1): Rt=1.15 min (UV detector TIC), mass
found 432.00.
[0331] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. [ppm]=1.17 (s,
6H), 2.04-2.11 (m, 2H), 3.99 (s, 3H), 4.52-4.60 (m, 3H), 7.10 (t,
1H), 8.00 (dd, 1H), 8.28-8.38 (m, 2H), 8.44-8.47 (m, 1H), 8.56 (d,
1H), 9.05 (s, 1H), 12.49 (s, 1H).
Intermediate 5-1
N-[6-(2-Hydroxypropan-2-yl)-1H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-
-carboxamide
##STR00032##
[0333] To a solution, cooled in an ice-water cooling bath, of 1.50
g (4.12 mmol) of methyl
5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carbox-
ylate (Intermediate 3-1) in 20 ml of THF were cautiously added 6.9
ml (5 equivalents) of a 3M methylmagnesium bromide solution in
diethyl ether. The mixture was stirred while cooling with an ice
bath for 1 h and at room temperature for 19.5 h. Another 2
equivalents of methylmagnesium bromide solution were added and the
mixture was stirred at room temperature for a further 24 h.
Saturated aqueous ammonium chloride solution was added and the
mixture was stirred and extracted three times with ethyl acetate.
The combined organic phases were washed with sodium chloride
solution, filtered through a hydrophobic filter and concentrated.
The residue was purified by column chromatography on silica gel
(hexane/ethyl acetate). 763 mg of the title compound were
obtained.
[0334] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. [ppm]=1.63 (s,
6H), 5.99 (s, 1H), 7.49 (s, 1H), 8.06 (s, 1H), 8.14-8.19 (m, 1H),
8.37 (t, 1H), 8.46 (d, 1H), 8.78 (s, 1H), 12.32 (s, 1H), 12.97 (s,
1H).
Intermediate 5-2
6-(Difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]pyridine-2--
carboxamide
##STR00033##
[0336] Analogously to the preparation of Intermediate 5-1, 2.40 g
(6.93 mmol) of methyl
5-({[6-(difluoromethyl)pyridin-2-yl]carbonyl}amino)-1H-indazole-6-carboxy-
late (Intermediate 3-2) in 10 ml of THF were reacted with three
portions of 3M methylmagnesium bromide solution in diethyl ether
(6.9 ml, then stirring at room temperature for 45 min; 11.6 ml,
then stirring at room temperature for 2 h; 6.9 ml, then stirring at
room temperature for 2 h). After the workup as for Intermediate
5-1, 2.39 g of a crude product were obtained, which were used
further without further purification.
Intermediate 6-1
Methyl
2-(3-hydroxy-3-methylbutyl)-5-nitro-2H-indazole-6-carboxylate
##STR00034##
[0338] 5.00 g (22.6 mmol) of methyl
5-nitro-1H-indazole-6-carboxylate (Intermediate 1-1) were initially
charged in 40 ml of DMF. 5.65 g (33.9 mmol) of
4-bromo-2-methylbutan-2-ol, 9.37 g (67.8 mmol) of potassium
carbonate and 5.63 g (33.9 mmol) of potassium iodide were added and
the mixture was stirred at 100.degree. C. for 20 h. Water was
added, the mixture was extracted three times with ethyl acetate and
the extracts were washed with sodium chloride solution, filtered
through a hydrophobic filter and concentrated. The residue was
purified by column chromatography on silica gel (hexane/ethyl
acetate). The solids obtained were stirred with diethyl ether,
filtered off with suction, washed with diethyl ether and dried.
2.49 g of the title compound were obtained.
[0339] UPLC-MS (Method A1): R.sub.t=0.93 min (UV detector: TIC),
mass found 307.00.
[0340] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. [ppm]=1.15 (s,
6H), 2.02-2.11 (m, 2H), 3.84 (s, 3H), 4.54 (s, 1H), 4.58-4.65 (m,
2H), 8.05 (s, 1H), 8.69 (s, 1H), 8.86 (s, 1H).
Intermediate 7-1
Methyl
5-amino-2-(3-hydroxy-3-methylbutyl)-2H-indazole-6-carboxylate
##STR00035##
[0342] 4.53 g of iron and 217 mg of ammonium chloride were added to
2.49 g (8.10 mmol) of methyl
2-(3-hydroxy-3-methylbutyl)-5-nitro-2H-indazole-6-carboxylate
(Intermediate 6-1) in 30 ml of ethanol and 10 ml of water, and the
mixture was stirred at 90.degree. C. for 21.5 h. The mixture was
filtered through Celite and washed through with ethanol three
times, and the filtrate was concentrated and the residue was
admixed with water. Extraction was effected three times with ethyl
acetate (to improve the phase separation, sodium chloride solution
was added). The combined organic phases were washed with sodium
chloride solution, filtered through a hydrophobic filter and
concentrated.
[0343] This gave 1.95 g (85% of theory) of the title compound.
[0344] UPLC-MS (Method A1): R.sub.t=0.67 min (UV detector: TIC),
mass found 277.00.
[0345] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. [ppm]=1.14 (s,
6H), 1.96-2.08 (m, 2H), 3.85 (s, 3H), 4.39-4.51 (m, 3H), 5.81 (s,
2H), 6.80 (s, 1H), 8.05 (s, 1H), 8.18 (s, 1H).
WORKING EXAMPLES
Example 1
N-[6-(2-Hydroxypropan-2-yl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-6-(trifluo-
romethyl)pyridine-2-carboxamide
##STR00036##
[0347] 75 mg (0.18 mmol) of methyl
2-(2-methoxyethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-2-
H-indazole-6-carboxylate (Intermediate 4-2) were dissolved in 500
.mu.l of THF and admixed with 887 .mu.l (0.89 mmol) of a 1 M
methylmagnesium bromide solution in THF. The reaction mixture was
stirred at 25.degree. C. for 60 min. Subsequently, 1 ml of a
saturated aqueous ammonium chloride solution was added cautiously
and the mixture was filtered. The aqueous phase was extracted twice
with ethyl acetate, and the organic phases were combined, filtered
through a hydrophobic filter and concentrated. The residue was
dissolved in 3 ml of DMSO and purified by preparative HPLC. The
product-containing fractions were freeze-dried. 20 mg of the title
compound were obtained.
[0348] UPLC-MS (Method A1): R.sub.t=1.08 min
[0349] MS (ESIpos): m/z=423 (M+H).sup.+
[0350] .sup.1H NMR (300 MHz, DMSO-d6): .delta. [ppm]=1.62 (s, 6H),
3.22 (s, 3H), 3.82 (t, 2H), 4.55 (t, 2H), 5.96 (s, 1H), 7.57 (s,
1H), 8.16 (dl H), 8.29-8.42 (m, 2H), 8.42-8.50 (m, 1H), 8.71 (s,
1H), 12.36 (s, 1H)
Example 2
N-[6-(Hydroxymethyl)-2-(2-methoxyethyl)-2H-indazol-5-yl]-6-(trifluoromethy-
l)pyridine-2-carboxamide
##STR00037##
[0352] 13 mg (0.36 mmol) of lithium aluminium hydride were
suspended in 1 ml of THF and the mixture was cooled to 0.degree. C.
75 mg (0.17 mmol) of methyl
2-(2-methoxyethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}a-
mino)-2H-indazole-6-carboxylate (Intermediate 4-2) dissolved in 500
.mu.l of THF were added dropwise and the mixture was stirred at
25.degree. C. for 60 min. The mixture was diluted with water and
extracted twice with ethyl acetate, and the combined organic phases
were washed with sodium chloride solution, filtered through a
hydrophobic filter, concentrated and dried under reduced pressure.
This gave 13 mg of the title compound.
[0353] UPLC-MS (Method A2): R.sub.t=0.99 min
[0354] MS (ESIpos): m/z=394 (M+H).sup.+
[0355] .sup.1H NMR (400 MHz, DMSO-d6): .delta. [ppm]=3.23 (s, 3H),
3.83 (t, 2H), 4.56 (t, 2H), 4.69 (d, 2H), 5.77 (t, 1H), 7.57 (s,
1H), 8.19 (d, 1H), 8.33-8.41 (m, 2H), 8.43-8.47 (m, 1H), 8.51 (s,
1H), 11.20 (s, 1H)
Example 3
N-[6-(2-Hydroxypropan-2-yl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(triflu-
oromethyl)pyridine-2-carboxamide
##STR00038##
[0357] 75 mg (0.17 mmol) of methyl
2-(3-methoxypropyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)--
2H-indazole-6-carboxylate (Intermediate 4-3) were dissolved in 500
.mu.l of THF and admixed with 859 .mu.l (0.86 mmol) of a 1 M
methylmagnesium bromide solution in THF. The reaction mixture was
stirred at 25.degree. C. for 60 min. Subsequently, 1 ml of a
saturated ammonium chloride solution was added cautiously and the
mixture was filtered. The aqueous phase was extracted twice with
ethyl acetate, and the organic phases were combined, filtered
through a hydrophobic filter and concentrated. The residue was
dissolved in 3 ml of DMSO and purified by preparative HPLC. The
product-containing fractions were freeze-dried. 25 mg of the title
compound were obtained.
[0358] UPLC-MS (Method A1): R.sub.t=1.13 min
[0359] MS (ESIpos): m/z=437 (M+H).sup.+
[0360] .sup.1H NMR (400 MHz, DMSO-d6): .delta. [ppm]=1.62 (s, 6H),
2.14 (quin, 2H), 3.23 (s, 3H), 3.26-3.32 (m, 2H), 4.44 (t, 2H),
5.95 (s, 1H), 7.58 (s, 1H), 8.16 (d, 1H), 8.31-8.40 (m, 2H),
8.43-8.48 (m, 1H), 8.72 (s, 1H), 12.36 (s, 1H).
Example 4
N-[6-(Hydroxymethyl)-2-(3-methoxypropyl)-2H-indazol-5-yl]-6-(trifluorometh-
yl)pyridine-2-carboxamide
##STR00039##
[0362] 13 mg of lithium aluminium hydride were suspended in THF and
the mixture was cooled to 0.degree. C. 75 mg (0.17 mmol) of methyl
2-(3-methoxypropyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)--
2H-indazole-6-carboxylate (Intermediate 4-3) in THF were added
dropwise and the mixture was allowed to come to room temperature
within 30 min. The mixture was diluted with water and filtered, the
residue was washed with ethyl acetate and the filtrate was
extracted with ethyl acetate. The combined ethyl acetate phases
were washed with sodium chloride solution, filtered through a
hydrophobic filter and concentrated. The residue was purified by
preparative HPLC.
[0363] .sup.1H NMR (300 MHz, DMSO-d6): .delta. [ppm]=2.14 (quin,
2H), 3.23 (s, 3H), 3.29 (t, 2H), 4.45 (t, 2 H), 4.68 (d, 2H), 5.77
(t, 1H), 7.58 (s, 1H), 8.18 (d, 1H), 8.32-8.48 (m, 3H), 8.51 (s,
1H), 11.21 (s, 1H).
Example 5
N-[2-(2-Hydroxyethyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(trifluo-
romethyl)pyridine-2-carboxamide
Stage A
Preparation of
N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(2-hydroxypropan-2-yl)-2-
H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide
##STR00040##
[0365] 100 mg (0.19 mmol) of methyl
2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-5-({[6-(trifluoromethyl)pyrid-
in-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate (Intermediate
4-5) were dissolved in 1 ml of THF and admixed with 669 .mu.l (0.67
mmol) of a 1 M methylmagnesium bromide solution in THF. The
reaction mixture was stirred at 25.degree. C. for 60 min. Another
287 .mu.l (0.29 mmol) of a 1 M methylmagnesium bromide solution in
THF were added and the mixture was stirred at 25.degree. C. for 3
h. Subsequently, 20 ml of a saturated ammonium chloride solution
were added cautiously and the mixture was filtered. The aqueous
phase was extracted twice with ethyl acetate, and the organic
phases were combined, dried over magnesium sulphate, filtered,
concentrated and dried under reduced pressure. This gave 50 mg of
N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(2-hydroxypropan-2-yl-
)-2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide.
[0366] UPLC-MS (Method A2): R.sub.t=1.51 min
[0367] MS (ESIpos): m/z=523 (M+H).sup.+
[0368] .sup.1H NMR (300 MHz, DMSO-d6): .delta. [ppm]=-0.17--0.09
(m, 6H), 0.78 (s, 9H), 1.62 (s, 6H), 4.04 (t, 2H), 4.47 (t, 2H),
5.98 (s, 1H), 7.57 (s, 1H), 8.16 (d, 1H), 8.29 (s, 1H), 8.37 (t,
1H), 8.45 (d, 1H), 8.73 (s, 1H), 12.38 (s, 1H).
Stage B
##STR00041##
[0370] 50 mg (96 .mu.mol) of
N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(hydroxymethyl)-2H-indaz-
ol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide were dissolved
in 1.0 ml of THF and admixed with 144 .mu.l (0.14 mmol) of a 1 M
solution of tetrabutylammonium fluoride in THF. The reaction
mixture was stirred at room temperature for 1 h. The mixture was
diluted with water and extracted twice with ethyl acetate, and the
combined organic phases were washed with saturated sodium chloride
solution, filtered through a hydrophobic filter and concentrated.
This gave 36 mg of
N-[2-(2-hydroxyethyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-(triflu-
oromethyl)pyridine-2-carboxamide (Example 5).
[0371] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): d [ppm]=1.62 (s, 6H),
3.86 (q, 2H), 4.43 (t, 2H), 4.95 (t, 1H), 5.94 (s, 1H), 7.57 (s,
1H), 8.16 (dd, 1H), 8.30 (s, 1H), 8.37 (t, 1H), 8.45 (d, 1H), 8.72
(s, 1H), 12.36 (s, 1H).
[0372] UPLC-MS (Method A2): R.sub.t=0.97 min (UV detector: TIC),
mass found 408.00.
Example 6
N-[6-(2-Hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]-6-(triflu-
oromethyl)pyridine-2-carboxamide
Stage A
Preparation of
N-[2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-6-(2-hydroxypropan-2-yl)--
2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide
##STR00042##
[0374] 50 mg (0.09 mmol) of methyl
2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-5-({[6-(trifluoromethyl)pyri-
din-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate (Intermediate
4-6) were dissolved in 500 .mu.l of THF and admixed with 326 .mu.l
(0.33 mmol) of a 1 M methylmagnesium bromide solution in THF. The
reaction mixture was stirred at 25.degree. C. for 60 min.
Subsequently, 20 ml of a saturated ammonium chloride solution were
added cautiously and the mixture was extracted twice with ethyl
acetate. The combined organic phases were filtered through a
hydrophobic filter, concentrated and dried under reduced pressure.
The residue was purified by preparative HPLC. 40 mg of
N-[2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-6-(2-hydroxypropan-2-yl)--
2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide were
obtained.
[0375] UPLC-MS (Method A1): R.sub.t=1.58 min
[0376] MS (ESIpos): m/z=537 (M+H).sup.+
[0377] .sup.1H NMR (300 MHz, DMSO-d6): .delta. [ppm]=0.02-0.05 (m,
6H), 0.84-0.91 (m, 9H), 1.62 (s, 6 H), 2.02-2.18 (m, 2H), 3.55-3.62
(m, 2H), 4.45 (t, 2H), 5.96 (s, 1H), 7.57 (s, 1H), 8.16 (d, 1 H),
8.31 (s, 1H), 8.33-8.42 (m, 1H), 8.45 (d, 1H), 8.72 (s, 1H), 12.37
(s, 1H).
Stage B
##STR00043##
[0379] 37 mg (0.07 mmol) of
N-[2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-6-(2-hydroxypropan-2-yl)--
2H-indazol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide were
dissolved in 500 .mu.l of THF and admixed with 207 .mu.l (0.21
mmol) of a 1 M solution of tetrabutylammonium fluoride in THF. The
reaction mixture was stirred at 25.degree. C. for 2 h. The mixture
was diluted with water and extracted twice with ethyl acetate, and
the combined organic phases were washed with saturated sodium
chloride solution, filtered and concentrated. After purification by
preparative HPLC, 10 mg of
N-[6-(2-hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-indazol-5-yl]-6-(trifl-
uoromethyl)pyridine-2-carboxamide (Example 6, contained secondary
component) were obtained.
[0380] UPLC-MS (Method A2): R.sub.t=1.00 min
[0381] MS (ESIpos): m/z=423 (M+H).sup.+
[0382] .sup.1H NMR selected signals (400 MHz, DMSO-d6): .delta.
[ppm]=1.61 (s), 2.00-2.12 (m), 3.38 (t, 2H), 4.44 (t, 2H), 4.62
(br. s., 1H), 5.93 (br. s., 1H), 7.55 (s, 1H), 8.13 (d, 1H),
8.27-8.38 (m, 2H), 8.43 (d, 1H), 8.71 (s, 1H), 12.30 (br. s.,
1H).
Example 7
N-[2-(2-Hydroxyethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluoromethy-
l)pyridine-2-carboxamide
Stage A
N-[2-(2-{[tert-Butyl(dimethyl)silyl]oxy}ethyl)-6-(hydroxymethyl)-2H-indazo-
l-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide
##STR00044##
[0384] 100 mg (0.19 mmol) of methyl
2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-5-({[6-(trifluoromethyl)pyrid-
in-2-yl]carbonyl}amino)-2H-indazole-6-carboxylate (Intermediate
4-5) were dissolved in 1 ml of THF and admixed with 191 .mu.l (0.38
mmol) of a 2 M lithium borohydride solution. The mixture was left
to stir at 25.degree. C. for 24 h. 14 mg (0.38 mmol) of sodium
borohydride and 500 .mu.l of methanol were added, and the mixture
was stirred at 25.degree. C. for 4 h. Another 14 mg (0.38 mmol) of
sodium borohydride were added, and the mixture was stirred at
25.degree. C. for 24 h. Water was added cautiously to the reaction
mixture and the organic phase was removed. The mixture was then
extracted twice with ethyl acetate, and the combined organic phases
were washed with saturated sodium chloride solution, filtered
through a hydrophobic filter and concentrated. The residue was
taken up in 2 ml of DMSO and purified by preparative HPLC. This
gave 30 mg of
N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(hydroxymethyl)-2H-indaz-
ol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide.
[0385] UPLC-MS (Method A2): R.sub.t=1.44 min
[0386] MS (ESIpos): m/z=495 (M+H).sup.+
[0387] .sup.1H NMR (300 MHz, DMSO-d6): .delta. [ppm]=-0.16--0.12
(m, 6H), 0.75-0.79 (m, 9H), 4.05 (t, 2 H), 4.48 (t, 2H), 4.69 (d,
2H), 5.75-5.77 (m, 1H), 7.57 (s, 1H), 8.18 (dd, 1H), 8.30-8.33 (m,
1 H), 8.38 (t, 1H), 8.45 (d, 1H), 8.51 (s, 1H), 11.20 (s, 1H).
Stage B
##STR00045##
[0389] 33 mg (0.07 mmol) of
N-[2-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-6-(hydroxymethyl)-2H-indaz-
ol-5-yl]-6-(trifluoromethyl)pyridine-2-carboxamide were dissolved
in 1 ml of THF and admixed with 100 .mu.l (0.10 mmol) of a 1 M
solution of tetrabutylammonium fluoride in THF. The reaction
mixture was stirred at 25.degree. C. for 1 h. The mixture was
diluted with water and extracted twice with ethyl acetate, and the
combined organic phases were washed with saturated sodium chloride
solution, filtered through a hydrophobic filter, concentrated and
dried under reduced pressure. 25 mg of
N-[2-(2-hydroxyethyl)-6-(hydroxymethyl)-2H-indazol-5-yl]-6-(trifluorometh-
yl)pyridine-2-carboxamide (Example 7) were obtained.
[0390] UPLC-MS (Method A2): R.sub.t=0.87 min
[0391] MS (ESIpos): m/z=381 (M+H).sup.+
[0392] .sup.1H NMR (300 MHz, DMSO-d6): .delta. [ppm]=3.87 (q, 2H),
4.44 (t, 2H), 4.69 (d, 2H), 4.98 (t, 1H), 5.70-5.81 (m, 1H), 7.57
(s, 1H), 8.11-8.23 (m, 1H), 8.31-8.42 (m, 2H), 8.43-8.49 (m, 1H),
8.51 (s, 1H), 11.20 (s, 1H).
Example 8
N-[6-(2-Hydroxypropan-2-yl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(trif-
luoromethyl)pyridine-2-carboxamide
##STR00046##
[0394] 50 mg (0.12 mmol) of methyl
2-(oxetan-3-ylmethyl)-5-([6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino)-
-2H-indazole-6-carboxylate (Intermediate 4-1) were dissolved in 500
.mu.l of THF and admixed with 576 .mu.l (0.58 mmol) of a 1 M
methylmagnesium bromide solution in THF. The reaction mixture was
stirred at 25.degree. C. for 60 min. Subsequently, 20 ml of a
saturated aqueous ammonium chloride solution were added cautiously
and the mixture was concentrated. The aqueous phase was extracted
twice with ethyl acetate, and the organic phases were combined,
dried over magnesium sulphate, filtered and concentrated. The
residue was dissolved in 2.0 ml of DMSO and purified by preparative
HPLC. The product-containing fractions were freeze-dried. 30 mg of
the title compound were obtained.
[0395] UPLC-MS (Method A2): R.sub.t=1.03 min
[0396] MS (ESIpos): m/z=435 (M+H).sup.+
[0397] .sup.1H NMR (400 MHz, DMSO-d6): .delta. [ppm]=1.62 (s, 6H),
3.45-3.61 (m, 1H), 4.48 (t, 2H), 4.66 (dd, 2H), 4.72 (d, 2H), 5.94
(s, 1H), 7.57 (s, 1H), 8.16 (d, 1H), 8.33-8.42 (m, 2H), 8.42-8.47
(m, 1H), 8.72 (s, 1H), 12.36 (s, 1H).
Example 9
N-[6-(Hydroxymethyl)-2-(oxetan-3-ylmethyl)-2H-indazol-5-yl]-6-(trifluorome-
thyl)pyridine-2-carboxamide
##STR00047##
[0399] 75 mg (0.17 mmol) of methyl
2-(oxetan-3-ylmethyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl}amino-
)-2H-indazole-6-carboxylate (Intermediate 4-1) were dissolved in 1
ml of a mixture of THF/methanol (1:1), and 8 mg (0.21 mmol) of
sodium borohydride were added. The mixture was left to stir at
25.degree. C. for 60 min. The reaction mixture was concentrated,
and the residue was admixed with water. The suspension was stirred
vigorously for 15 min, and the solids were filtered off with
suction, washed twice with water and twice with diethyl ether, and
dried under reduced pressure. 48 mg of the title compound were
obtained.
[0400] UPLC-MS (Method A2): R.sub.t=0.94 min
[0401] MS (ESIpos): m/z=407 (M+H).sup.+
[0402] .sup.1H NMR (300 MHz, DMSO-d6): .delta. [ppm]=3.55 (s, 1H),
4.48 (t, 2H), 4.61-4.77 (m, 6H), 7.57 (s, 1H), 8.18 (dd, 1H),
8.33-8.49 (m, 3H), 8.51 (s, 1H), 11.21 (s, 1H).
Example 10
N-{6-(2-Hydroxypropan-2-yl)-2-[3-(methylsulphonyl)propyl]-2H-indazol-5-yl}-
-6-(trifluoromethyl)pyridine-2-carboxamide
##STR00048##
[0404] A mixture of 500 mg (1.32 mmol) of
N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]-6-(trifluoromethyl)pyridine--
2-carboxamide (Intermediate 5-1), 569 mg of potassium carbonate and
114 mg of potassium iodide in 5.0 ml of DMF was stirred at room
temperature for 15 min. 414 mg of
1-bromo-3-(methylsulphonyl)propane were added and the mixture was
stirred at room temperature overnight. Water was added, the mixture
was twice extracted with ethyl acetate and the extracts were washed
with sodium chloride solution and concentrated. The residue was
purified by column chromatography (dichloromethane/methanol
gradient). The product fraction was stirred with diethyl ether,
filtered and dried. 59 mg of the title compound were obtained.
[0405] UPLC-MS (Method A2): R.sub.t=1.02 min
[0406] MS (ESIpos): m/z=485 (M+H).sup.+
[0407] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. [ppm]=1.63 (s,
6H), 2.26-2.42 (m, 2H), 2.99 (s, 3H), 3.06-3.16 (m, 2H), 4.55 (t,
2H), 5.96 (s, 1H), 7.60 (s, 1H), 8.16 (d, 1H), 8.33-8.48 (m, 3H),
8.73 (s, 1H), 12.37 (s, 1H).
Example 11
N-[2-(3-Hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-
-(trifluoromethyl)pyridine-2-carboxamide
##STR00049##
[0408] Preparation Method 1
[0409] 705 mg (1.57 mmol) of methyl
2-(3-hydroxy-3-methylbutyl)-5-({[6-(trifluoromethyl)pyridin-2-yl]carbonyl-
}amino)-2H-indazole-6-carboxylate (Intermediate 4-4) were initially
charged in 10 ml of THF and cooled in an ice-water cooling bath.
2.6 ml (5.0 equivalents) of 3M methylmagnesium bromide solution (in
diethyl ether) were added and the mixture was left to stir while
cooling with an ice bath for 1 h and at room temperature for 4.5 h.
Another 1 equivalent of the methylmagnesium bromide solution was
added and the mixture was left to stir at room temperature for 20.5
h. Another 1 equivalent again of the methylmagnesium bromide
solution was added and the mixture was left to stir at room
temperature for 22 h. The reaction mixture was admixed with
saturated aqueous ammonium chloride solution, stirred and extracted
three times with ethyl acetate. The combined organic phases were
washed with sodium chloride solution, filtered through a
hydrophobic filter and concentrated. This gave 790 mg of a residue
which was purified by means of preparative HPLC. This gave 234 mg
of the title compound and 164 mg of a product fraction which was
stirred with diethyl ether. After filtration with suction followed
by drying, a further 146 mg of the title compound were
obtained.
[0410] UPLC-MS (Method A1): R.sub.t=1.10 min (UV detector: TIC),
mass found 450.00.
[0411] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. [ppm]=1.14 (s,
6H), 1.61 (s, 6H), 1.99-2.08 (m, 2H), 4.42-4.55 (m, 3H), 5.93 (s,
1H), 7.56 (s, 1H), 8.15 (dd, 1H), 8.32-8.39 (m, 2H), 8.41-8.47 (m,
1H), 8.70 (s, 1H), 12.34 (s, 1H).
Preparation Method 2
[0412] A mixture of 500 mg (1.37 mmol) of
N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]-6-(trifluoromethyl)pyridine--
2-carboxamide (Intermediate 5-1), 569 mg of potassium carbonate and
114 mg of potassium iodide in 5 ml of DMF was stirred at room
temperature for 15 min. 344 mg (1.5 equivalents) of
4-bromo-2-methylbutan-2-ol were added and the mixture was heated to
100.degree. C. for 2 h. Another 0.5 equivalent of
4-bromo-2-methylbutan-2-ol was added and the mixture was stirred at
room temperature for 16 h. The mixture was admixed with water and
extracted twice with ethyl acetate, and the combined organic phases
were washed with saturated sodium chloride solution and filtered
through a hydrophobic filter and concentrated. The residue was
purified by column chromatography purification on silica gel
(hexane/ethyl acetate). This gave 100 mg of a product fraction
which was stirred with diethyl ether. The solid was filtered and
dried. 60 mg of the title compound were obtained.
[0413] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. [ppm]=1.14 (s,
6H), 1.61 (s, 6H), 1.99-2.07 (m, 2H), 4.43-4.52 (m, 3H) 5.94 (s,
1H) 7.57 (s, 1H) 8.15 (dd, 1H) 8.33-8.40 (m, 2H), 8.42-8.48 (m,
1H), 8.71 (s, 1H), 12.35 (s, 1H)
Example 12
N-{6-(2-Hydroxypropan-2-yl)-2-[2-(methylsulphonyl)ethyl]-2H-indazol-5-yl}--
6-(trifluoromethyl)pyridine-2-carboxamide
##STR00050##
[0415] 160 mg (0.44 mmol) of
N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]-6-(trifluoromethyl)pyridine--
2-carboxamide (Intermediate 5-1) were suspended together with 182
mg of potassium carbonate and 36 mg of potassium iodide in 1.0 ml
of DMF, and the mixture was stirred at room temperature for 15 min.
Then 123 mg of 2-bromoethyl methyl sulphone (0.66 mmol) were added
and the mixture was stirred at room temperature overnight. Water
was added, the mixture was extracted twice with ethyl acetate and
the extracts were washed with saturated aqueous sodium chloride
solution, filtered through a hydrophobic filter and
concentrated.
[0416] Purification of the residue by preparative HPLC gave 20 mg
of the title compound.
[0417] UPLC (Method A2): R.sub.t=1.01 min;
[0418] MS (ESIpos): m/z=471 (M+H)+
[0419] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. [ppm]=1.63 (s,
6H), 2.90 (s, 3H), 3.85 (t, 2H), 4.86 (t, 2H), 5.97 (s, 1H), 7.59
(s, 1H), 8.13-8.19 (m, 1H), 8.37 (s, 1H), 8.41-8.48 (m, 2H), 8.74
(s, 1H), 12.37 (s, 1H).
Example 13
6-(Difluoromethyl)-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-
-2H-indazol-5-yl]pyridine-2-carboxamide
##STR00051##
[0420] Preparation Method 1
[0421] A mixture of 250 mg of
6-(difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]pyridine-2-
-carboxamide (crude product of Intermediate 5-2), 144 mg of
potassium iodide and 239 mg of potassium carbonate in 2.5 ml of DMF
was stirred at room temperature for 15 min. 145 mg (0.87 mmol) of
4-bromo-2-methylbutan-2-ol were added, the mixture was stirred at
110.degree. C. for 3 h, another 96 mg of 4-bromo-2-methylbutan-2-ol
were added and the mixture was stirred at 110.degree. C. for 4 h.
Water was added, the mixture was extracted twice with ethyl acetate
and the extract was washed with semisaturated aqueous sodium
chloride solution, filtered through a hydrophobic filter and
concentrated. Purification was effected by column chromatography on
silica gel (hexane/ethyl acetate). 61 mg of the title compound were
obtained.
[0422] UPLC-MS (Method A1): R.sub.t=1.00 min (UV detector: TIC),
mass found 432.00.
[0423] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. [ppm]=1.14 (s,
6H), 1.63 (s, 6H), 1.97-2.08 (m, 2H), 4.41-4.55 (m, 3H), 5.99 (s,
1H), 7.03 (t, 1H), 7.56 (s, 1H), 7.94-8.00 (m, 1H), 8.24-8.38 (m,
3H), 8.71 (s, 1H), 12.49 (s, 1H).
Preparation Method 2
[0424] Analogously to the preparation of Example 11 (Preparation
Method 1), 3.00 g of methyl
5-({[6-(difluoromethyl)pyridin-2-yl]carbonyl}amino)-2-(3-hydroxy-3-methyl-
butyl)-2H-indazole-6-carboxylate (Intermediate 4-11) were reacted
with 3M methylmagnesium bromide solution (in diethyl ether). After
purification of the crude product by stirring with diethyl ether,
filtering followed by preparative HPLC, 1.37 g of the title
compound were obtained.
Example 14
6-(Difluoromethyl)-N-{6-(2-hydroxypropan-2-yl)-2-[2-(methylsulphonyl)ethyl-
]-2H-indazol-5-yl}pyridine-2-carboxamide
##STR00052##
[0426] A mixture of 250 mg of
6-(difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]pyridine-2-
-carboxamide (crude product of Intermediate 5-2), 144 mg of
potassium iodide and 239 mg of potassium carbonate in 2.5 ml of DMF
was stirred at room temperature for 15 min. 162 mg of 2-bromoethyl
methyl sulphone (0.87 mmol) were added and the mixture was stirred
at 110.degree. C. for 3 h. Water was added, the mixture was
extracted twice with ethyl acetate and the extract was washed with
semisaturated aqueous sodium chloride solution, filtered through a
hydrophobic filter and concentrated. The residue was purified by
preparative HPLC and the product fractions were additionally
purified by column chromatography purification on silica gel
(hexane/ethyl acetate). 40 mg of the title compound were
obtained.
[0427] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. [ppm]=1.65 (s,
6H), 2.90 (s, 3H), 3.85 (t, 2H), 4.85 (t, 2H), 6.03 (s, 1H), 7.04
(t, 1H), 7.59 (s, 1H), 7.98 (d, 1H), 8.25-8.36 (m, 2H), 8.43 (s,
1H), 8.75 (s, 1H), 12.52 (s, 1H).
Example 15
6-(Difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-2-(3-hydroxypropyl)-2H-inda-
zol-5-yl]pyridine-2-carboxamide
Stage A
Preparation of
N-[2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-6-(2-hydroxypropan-2-yl)--
2H-indazol-5-yl]-6-(difluoromethyl)pyridine-2-carboxamide
##STR00053##
[0429] A mixture of 250 mg of
6-(difluoromethyl)-N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]pyridine-2-
-carboxamide (Intermediate 5-2), 48 mg of potassium iodide and 239
mg of potassium carbonate in 2.5 ml of DMF was stirred at room
temperature for 15 min. 219 mg (0.87 mmol, 1.5 equivalents) of
(3-bromopropoxy)(tert-butyl)dimethylsilane were added and the
mixture was stirred at 110.degree. C. for 3 h. Another 1 equivalent
of (3-bromopropoxy)(tert-butyl)dimethylsilane was added and the
mixture was stirred at 100.degree. C. for 4 h. Water was added, the
mixture was extracted with ethyl acetate and the extract was washed
with aqueous sodium chloride solution, filtered through a
hydrophobic filter and concentrated. The residue was purified by
column chromatography (hexane/ethyl acetate). 92 mg of the title
compound were obtained.
Stage B
##STR00054##
[0431] Analogously to the preparation of Example 6, Stage B, 92 mg
of
N-[2-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-6-(2-hydroxypropan-2-yl)--
2H-indazol-5-yl]-6-(difluoromethyl)pyridine-2-carboxamide were
reacted with 0.53 ml of a 1 M solution of tetrabutylammonium
fluoride in THF within 1 h. Aqueous workup as in Example 6 and
purification by preparative HPLC gave 46 mg of the title
compound.
[0432] UPLC-MS (Method A1): R.sub.t=0.92 min (UV detector: TIC),
mass found 404.00.
[0433] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. [ppm]=1.64 (s,
6H), 2.05 (quin, 2H), 3.35-3.46 (m, 2H), 4.45 (t, 2H), 4.64 (t,
1H), 5.99 (s, 1H), 7.04 (t, 1H), 7.57 (s, 1H), 7.95-7.99 (m, 1H),
8.25-8.36 (m, 3H), 8.73 (s, 1H), 12.50 (s, 1H).
Example 16
N-[6-(2-Hydroxypropan-2-yl)-2-(4,4,4-trifluorobutyl)-2H-indazol-5-yl]-6-(t-
rifluoromethyl)pyridine-2-carboxamide
##STR00055##
[0435] A mixture of 210 mg (0.58 mmol) of
N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]-6-(trifluoromethyl)pyridine--
2-carboxamide (Intermediate 5-1) in 3 ml of DMF was admixed with
0.11 ml (0.87 mmol) of 1,1,1-trifluoro-4-iodobutane and 239 mg of
potassium carbonate, and the mixture was stirred at 80.degree. C.
for 6 h. After addition of water, the mixture was extracted three
times with ethyl acetate, and the combined organic phases were
washed with saturated sodium chloride solution, filtered through a
hydrophobic filter and concentrated. The crude product was purified
by preparative HPLC. 19 mg of the title compound were obtained.
[0436] UPLC-MS (Method A1): R.sub.t=1.27 min (UV detector: TIC),
mass found 474.15.
[0437] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. [ppm]=1.62 (s,
6H), 2.10-2.33 (m), 4.49 (t, 2H), 5.94 (s, 1H), 7.59 (s, 1H),
8.13-8.18 (m, 1H), 8.32-8.41 (m, 2H), 8.41-8.47 (m, 1H), 8.72 (s,
1H), 12.35 (s, 1H).
Example 17
N-{6-(2-Hydroxypropan-2-yl)-2-[3-(trifluoromethoxy)propyl]-2H-indazol-5-yl-
}-6-(trifluoromethyl)pyridine-2-carboxamide
##STR00056##
[0439] 150 mg (0.33 mmol) of
N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]-6-(trifluoromethyl)pyridine--
2-carboxamide (Intermediate 5-1) were initially charged in 2 ml of
THF. 58 mg (0.40 mmol) of 3-(trifluoromethoxy)propan-1-ol, 131 mg
of triphenylphosphine and 71 .mu.l of diisopropyl azodicarboxylate
(DIAD, CAS 2446-83-5) were added and the mixture was stirred at
room temperature for 19 h. 0.83 ml of sodium hydroxide solution
(2M) was added and the mixture was stirred at 40.degree. C. for 5
h. The mixture was diluted with water and extracted three times
with ethyl acetate, and the combined organic phases were
concentrated and purified by preparative HPLC. 16 mg of the title
compound were obtained as a crude product.
[0440] UPLC-MS (Method A2): R.sub.t=1.26 min (UV detector: TIC),
mass found 490.14.
[0441] .sup.1H-NMR (400 MHz, DMSO-d.sub.6, selected signals):
.delta. [ppm]=1.61 (s, 6H), 1.84 (d, 1H), 2.32 (quint., 2H), 4.08
(t, 2H), 4.51 (t, 2H), 7.58 (s, 1H), 8.15 (d, 1H), 8.31-8.39 (m,
2H), 8.44 (d, 1H), 8.72 (s, 1H), 12.35 (s, 1H).
Example 18
N-{6-(2-Hydroxypropan-2-yl)-2-[3-(2,2,2-trifluoroethoxy)propyl]-2H-indazol-
-5-yl}-6-(trifluoromethyl)pyridine-2-carboxamide
##STR00057##
[0443] Analogously to the preparation of Example 11 (Preparation
Method 1), 52 mg (0.10 mmol) of methyl
2-[3-(2,2,2-trifluoroethoxy)propyl]-5-({[6-(trifluoromethyl)pyridin-2-yl]-
carbonyl}amino)-2H-indazole-6-carboxylate (Intermediate 4-10) in 3
ml of THF were reacted with 2.times.171 .mu.l of 3M magnesium
bromide solution in diethyl ether. Purification by preparative HPLC
gave 12 mg of the title compound.
[0444] UPLC-MS (Method A1): R.sub.t=1.25 min (UV detector: TIC),
mass found 504.16.
[0445] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): .delta. [ppm]=1.63 (s,
6H), 2.20 (quin, 2H), 3.58 (t, 2H), 4.05 (q, 2H), 4.47 (t, 2H),
5.94 (s, 1H), 7.58 (s, 1H), 8.15 (dd, 1H), 8.32 (s, 1H), 8.36 (t,
1H), 8.45 (d, 1H), 8.73 (s, 1H), 12.36 (s, 1H).
Example 19
5-Fluoro-N-[2-(3-hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazo-
l-5-yl]-6-methylpyridine-2-carboxamide
##STR00058##
[0447] 228 mg (0.31 mmol) of methyl
5-{[(5-fluoro-6-methylpyridin-2-yl)carbonyl]amino}-2-(3-hydroxy-3-methylb-
utyl)-2H-indazole-6-carboxylate (Intermediate 4-8) were initially
charged in 4.5 ml of THF and cooled with an ice cooling bath. 0.63
ml of 3M methylmagnesium bromide solution (in diethyl ether) was
added and the mixture was left to stir while cooling with an ice
bath for 2 h and at room temperature for 21 h. The reaction mixture
was admixed with saturated aqueous ammonium chloride solution and
extracted three times with ethyl acetate. The combined organic
phases were concentrated. The residue was purified by preparative
HPLC. 82 mg of the title compound were obtained.
[0448] UPLC-MS (Method A2): R.sub.t=1.03 min (UV detector: TIC),
mass found 414.21.
[0449] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. [ppm]=1.13 (s,
6H), 1.63 (s, 6H), 1.99-2.05 (m, 2H), 2.55-2.59 (m, 3H), 4.42-4.50
(m, 3H), 5.95 (s, 1H), 7.54 (s, 1H), 7.83 (t, 1H), 8.05 (dd, 1H),
8.31 (s, 1H), 8.68 (s, 1H), 12.33 (s, 1H).
Example 20
N-[2-(3-Hydroxy-3-methylbutyl)-6-(2-hydroxypropan-2-yl)-2H-indazol-5-yl]-6-
-methylpyridine-2-carboxamide
##STR00059##
[0451] 278 mg (0.48 mmol) of methyl
2-(3-hydroxy-3-methylbutyl)-5-{[(6-methylpyridin-2-yl)carbonyl]amino}-2H--
indazole-6-carboxylate (Intermediate 4-9) were initially charged in
5.0 ml of THF and cooled with an ice cooling bath. 0.97 ml of 3M
methylmagnesium bromide solution (in diethyl ether) was added and
the mixture was left to stir while cooling with an ice bath for 2 h
and at room temperature for 20.5 h. Another 0.48 ml of 3M
methylmagnesium bromide solution was added and the mixture was left
to stir at room temperature for 67 h. The mixture was admixed with
saturated aqueous ammonium chloride solution and extracted three
times with ethyl acetate, and the extracts were washed with sodium
chloride solution, filtered through a hydrophobic filter and
concentrated. The residue was purified by preparative HPLC. 111 mg
of the title compound were obtained.
[0452] UPLC-MS (Method A2): R.sub.t=0.97 min (UV detector: TIC),
mass found 396.22.
[0453] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. [ppm]=1.15 (s,
6H), 1.64 (s, 6H), 2.00-2.08 (m, 2H), 2.61 (s, 3H), 4.41-4.59 (m,
3H), 5.92 (s, 1H), 7.50 (dd, 1H), 7.56 (s, 1H), 7.90-7.99 (m, 2H),
8.33 (s, 1H), 8.70 (s, 1H), 12.39 (s, 1H).
Example 21
6-(2-Hydroxypropan-2-yl)-N-[6-(2-hydroxypropan-2-yl)-2-(4,4,4-trifluorobut-
yl)-2H-indazol-5-yl]pyridine-2-carboxamide
##STR00060##
[0455] A solution of 72 mg (0.16 mmol) of methyl
5-({[6-(2-hydroxypropan-2-yl)pyridin-2-yl]carbonyl}amino)-2-(4,4,4-triflu-
orobutyl)-2H-indazole-6-carboxylate (Intermediate 4-7) in 10 ml of
THF was cooled in an ice/water cooling bath. 0.26 ml of 3M
methylmagnesium bromide solution in diethyl ether was added and the
mixture was stirred for 2 h and then at room temperature for 20 h.
Another 1 equivalent of the 3M methylmagnesium bromide solution was
added and the mixture was stirred at room temperature for 24 h.
Saturated aqueous ammonium chloride solution was added, the mixture
was three times extracted with ethyl acetate and the extracts were
washed with sodium chloride solution and concentrated. Preparative
HPLC gave 22 mg (31% of theory) of the title compound.
[0456] UPLC-MS (Method A2): R.sub.t=1.15 min (UV detector: TIC),
mass found 464.20.
[0457] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta. [ppm]=1.56 (s,
6H), 1.64 (s, 6H), 2.07-2.34 (m, 4H), 4.49 (t, 2H), 5.32 (s, 1H),
6.05 (s, 1H), 7.60 (s, 1H), 7.87 (dd, 1H), 7.99-8.05 (m, 2H), 8.35
(s, 1H), 8.79 (s, 1H), 12.45 (s, 1H).
Example 22
N-{2-[2-(1-Hydroxycyclopropyl)ethyl]-6-(2-hydroxypropan-2-yl)-2H-indazol-5-
-yl}-6-(trifluoromethyl)pyridine-2-carboxamide
##STR00061##
[0459] 250 mg (0.69 mmol) of
N-[6-(2-hydroxypropan-2-yl)-1H-indazol-5-yl]-6-(trifluoromethyl)pyridine--
2-carboxamide (Intermediate 5-1) were initially charged in 5 ml of
DMSO. 159 mg (0.96 mmol) of 1-(2-bromoethyl)cyclopropanol, 285 mg
of potassium carbonate and 171 mg of potassium iodide were added
and the mixture was stirred at 100.degree. C. for 5 h. Water was
added and the mixture was extracted three times with ethyl acetate.
The combined organic phases were washed with sodium chloride
solution, filtered through a hydrophobic filter and concentrated.
The residue was purified by preparative HPLC (column: Waters
XBridge C18 5.mu. 100.times.30 mm, eluent A: water+0.1% by volume
of formic acid (99%), eluent B: acetonitrile).
[0460] Freeze-drying gave 45 mg of the title compound.
[0461] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): .delta. [ppm]=0.18-0.22
(m, 2H), 0.48-0.52 (m, 2H), 1.62 (s, 6H), 2.08 (t, 2H), 4.54-4.60
(m, 2H), 5.36 (s, 1H), 5.96 (s, 1H), 7.57 (s, 1H), 8.16 (dd, 1H),
8.34-8.39 (m, 2H), 8.45 (d, 1H), 8.72 (s, 1H), 12.36 (s, 1H).
Assessment of Physiological Efficacy
IRAK4 Kinase Assay
[0462] The IRAK4-inhibitory activity of the present substances was
measured in the IRAK4 TR-FRET assay (TR-FRET=Time Resolved
Fluorescence Resonance Energy Transfer) described hereinafter.
[0463] Recombinant fusion protein from N-terminal GST (glutathione
S-transferase) and human IRAK4, expressed in baculovirus-infected
insect cells (Hi5, BTI-TN-5B1-4, cell line purchased from
Invitrogen, catalogue No. B855-02) and purified via affinity
chromatography, was used as enzyme. The substrate used for the
kinase reaction was the biotinylated peptide
biotin-Ahx-KKARFSRFAGSSPSQASFAEPG (C-terminus in amide form) which
can be purchased, for example, from Biosyntan GmbH
(Berlin-Buch).
[0464] For the assay, 11 different concentrations in the range from
20 .mu.M to 0.073 nM were prepared from a 2 mM DMSO solution of the
test substance. 50 nl of the respective solution were pipetted into
a black low-volume 384-well microtitre plate (Greiner Bio-One,
Frickenhausen, Germany), 2 l of a solution of IRAK4 in assay buffer
[50 mM HEPES pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 30 .mu.M
activated sodium orthovanadate, 0.1% (w/v) of bovine gamma-globulin
(BGG) 0.04% (v/v) nonidet-P40 (Sigma)] were added and the mixture
was incubated for 15 min to allow prebinding of the substances to
the enzyme prior to the kinase reaction. The kinase reaction was
then started by addition of 3 .mu.l of a solution of adenosine
triphosphate (ATP, 1.67 mM=final concentration in 5 .mu.l of assay
volume: 1 mM) and peptide substrate (0.83 .mu.M=final concentration
in 5 .mu.l assay volume: 0.5 .mu.M) in assay buffer, and the
resulting mixture was incubated at 22.degree. C. for the reaction
time of 45 min. The concentration of the IRAK4 was adjusted to the
respective activity of the enzyme and set such that the assay was
carried out in the linear range. Typical concentrations were in the
order of about 0.2 nM. The reaction was stopped by addition of 5
.mu.l of a solution of TR-FRET detection reagents [0.1 .mu.M
streptavidin-XL665 (Cisbio Bioassays; France, catalogue No.
610SAXLG)] and 1.5 nM anti-phosphoserine antibody [Merck Millipore,
"STK Antibody", catalogue No. 35-002] and 0.6 nM LANCE
EU-W1024-labelled anti-mouse-IgG antibody (Perkin-Elmer, product
No. AD0077; alternatively, it is possible to use a terbium
cryptate-labelled anti-mouse-IgG antibody from Cisbio Bioassays) in
aqueous EDTA solution (100 mM EDTA, 0.4% [w/v] bovine serum albumin
[BSA] in 25 mM HEPES pH 7.5).
[0465] The resulting mixture was incubated at 22.degree. C. for 1 h
to allow formation of a complex of the biotinylated phosphorylated
substrate and the detection reagents. The amount of the
phosphorylated substrate was then evaluated by measuring the
resonance energy transfer from europium chelate-labelled
anti-mouse-IgG antibody to streptavidin-XL665. To this end, the
fluorescence emissions at 620 nm and 665 nm were measured after
excitation at 350 nm in a TR-FRET measuring instrument, for example
a Rubystar (BMG Labtechnologies, Offenburg, Germany) or a Viewlux
(Perkin-Elmer). The ratio of the emissions at 665 nm and 622 nm was
taken as a measure of the amount of phosphorylated substrate. The
data were normalized (enzyme reaction without test substance=0%
inhibition; all other assay components but no enzyme=100%
inhibition). Typically, the test substances were tested on the same
microtitre plates at 11 different concentrations in the range from
20 .mu.M to 0.073 nM (20 .mu.M, 5.7 .mu.M, 1.6 .mu.M, 0.47 .mu.M,
0.13 .mu.M, 38 nM, 11 nM, 3.1 nM, 0.89 nM, 0.25 nM and 0.073 nM).
The dilution series were prepared prior to the assay (2 mM to 7.3
nM in 100% DMSO) by serial dilutions. The IC.sub.50 values were
calculated by a 4-parameter fit.
TABLE-US-00002 TABLE 1 IC.sub.50 values of the example compounds in
the IRAK4 kinase assay IC.sub.50 Example [nM] 1 30.6 2 135.6 3 7.2
4 52.7 5 264.5 6 35.7 7 867.3 8 15.0 9 103.8 10 18.5 11 3.4 12 10.7
13 1.3 14 10.8 15 12.3 16 21.5 17 36.0 18 47.5 19 8.9 20 13.3 21
117.2 22 3.7
[0466] The inhibitory activity of the present substances of the
general formula (III) with respect to IRAK4 was likewise measured
in the IRAK4 TR-FRET assay described above. The following are
mentioned by way of example: the compound Intermediate 4-2 with an
IC.sub.50=21.7 nM, Intermediate 4-3 with an IC.sub.50=13.0 nM and
Intermediate 4-4 with an IC.sub.50=6.2 nM.
TNF-.alpha. Secretion in THP-1 Cells
[0467] This test is suited to test substances for their ability to
inhibit secretion of TNF-.alpha. (tumour necrosis factor alpha) in
THP-1 cells (human monocytic acute leukaemia cell line).
TNF-.alpha. is a cytokine involved in inflammatory processes. In
this test, TNF-.alpha. secretion is triggered by incubation with
bacterial lipopolysaccharide (LPS).
[0468] THP-1 cells were kept in continuous suspension cell culture
[RPMI 1460 medium with L-Glutamax (Gibco, Cat. No. 61870-044)
supplemented with foetal calf serum (FCS) 10% (Invitrogen, Cat. No.
10082-147), 1% penicillin/streptomycin (Gibco BRL, Cat. No.
15140-114)] and should not exceed a cell concentration of
1.times.10.sup.6 cells/ml. The assay was carried out in cell
culture medium (RPMI 1460 medium with L-Glutamax supplemented with
FCS 10%).
[0469] In each case 2-2.5 .mu.l of the cell suspension (corresponds
to 4000 cells) per well were dispensed into a 384-well test plate
(Greiner, Cat. No. 784076), in each of which 40-50 nl substance had
been dissolved in 100% DMSO. This was done using 10 different
concentrations in the range from 20 .mu.M to 0.073 nM for each
substance. The cells were incubated at room temperature for 15 min.
2-2.5 .mu.l of 0.1 .mu.g/ml LPS (Sigma, Escherichia coli 055:B5,
Cat. No. L5418) dissolved in cell culture medium (final
concentration 0.05 .mu.g/ml) were then dispensed into each well. As
neutral control, cells were treated with 0.05 .mu.g/ml LPS and 1%
DMSO and, as inhibitor control, with 1% DMSO only.
[0470] The plates were centrifuged at 80 g for 30 s and incubated
at 37.degree. C., 5% CO.sub.2 and 95% atmospheric humidity for 17
h. The amount of TNF-.alpha. was determined using the TNF-alpha
HTRF Detection Kit (Cisbio, Cat. No. 62TNFPEB/C). To this end, 2
.mu.l of the detection solution in each case, consisting of
anti-TNF-.alpha.-XL665 conjugate and anti-TNF-.alpha.-cryptate
conjugate dissolved in the reconstitution buffer in accordance with
the manufacturer's instructions, were added for the HTRF
(Homogeneous Time-Resolved Fluorescence) test. After the addition,
the mixture was incubated either at room temperature for 3 h or at
4.degree. C. overnight. The signals were then read at 620/665 nm
using an HTRF-enabled measuring instrument such as the BMG
PheraStar.
[0471] The activity of the substances is expressed as the ratio
between neutral and inhibitor control in percent. The IC.sub.50
values were calculated using a 4-parameter fit.
TABLE-US-00003 TABLE 2 IC.sub.50 values of the example compounds
with respect to the secretion of TNF-.alpha. in THP-1 cells
IC.sub.50 Example [.mu.M] 1 1.0 2 15.1 3 0.7 4 5.6 5 5.4 6 0.9 7
16.4 8 1.0 9 6.5 10 1.0 11 0.2 12 0.3 13 0.1 14 0.2 15 0.2 16 0.2
17 0.5 18 0.3 19 0.1 20 0.2 21 1.8
In Vitro LPS (Lipopolysaccharide)-Induced Cytokine Production in
Human PBMCs (Peripheral Blood Mononuclear Cells)
[0472] The effect of the present compounds of the general formula
(I) on induced cytokine production in human PBMCs was examined.
Cytokine production was induced here by LPS, a TLR4 ligand, which
leads to activation of the IRAK4-mediated signal path.
[0473] The human PBMCs were obtained from anti-coagulated human
whole blood. For this purpose, 15 ml of Ficoll-Paque (Biochrom,
Cat. No. L6115) were initially pipetted in Leucosep tubes and 20 ml
of human blood were added. After centrifugation of the blood at 800
g for 15 min at room temperature, the plasma including the
platelets was removed and discarded. The PBMCs were transferred
into centrifugation tubes and made up with PBS (phosphate-buffered
saline) (Gibco, Cat. No. 14190). The cell suspension was
centrifuged at room temperature at 250 g for 10 min and the
supernatant was discarded. The PBMCs were resuspended in complete
medium (RPMI 1640, without L-glutamine (PAA, Cat. No. E15-039), 10%
FCS; 50 U/ml penicillin, 50 .mu.g/ml streptomycin (PAA, Cat. No.
P11-010) and 1% L-glutamine (Sigma, Cat. No. G7513)).
[0474] The assay was also carried out in complete medium. The PBMCs
were seeded in 96-well plates at a cell density of
2.5.times.10.sup.5 cells/well. The present compounds were subjected
to serial dilution in a constant volume of 100% DMSO and used in
the assay at 8 different concentrations in the range from 10 .mu.M
to 3 nM such that the final DMSO concentration was 0.4% DMSO. Prior
to the actual stimulation, the cells were then pre-incubated
therewith for 30 min. To induce cytokine secretion, the cells were
stimulated with 0.1 .mu.g/ml LPS (Sigma, Escherichia coli 0128:B12,
Cat. No. L2887) for 24 hours. Cell viability was determined using
the CellTiter-Glo luminescent assay (Promega, Cat. No. G7571
(G755/G756A)) in accordance with the manufacturer's instructions.
The amount of secreted TNF-.alpha. in the cell culture supernatant
was determined using the Human ProInflammatory 9-Plex Tissue
Culture Kit (MSD, Cat. No. K15007B) in accordance with the
instructions of the manufacturer. By way of example, Example
Compound 11 and Example Compound 12 have activity .ltoreq.1
.mu.M.
In Vitro TLR-4/TLR-7-Induced Interleukin (IL)-23 Secretion of Human
Dendritic Cells (DCs)
[0475] The effect of the present compounds of the general formula
(I) on the induced production of the pro-inflammatory cytokine
IL-23 which plays an essential role for the generation of TH-17
cells was examined in human DCs. It is stated that TH-17 cells play
a crucial role in the pathogenesis of disorders such as rheumatoid
arthritis, psoriatic arthritis, Bekhterev's disease (ankylosing
spondylitis) or else multiple sclerosis (Lubberts, Nat. Rev.
Rheumatol., 2015; Marinoni et al., Auto. Immun. Highlights, 2014;
Isailovic et al., J. Autoimmun., 2015; Staschke et al., J Immunol.,
2009). To detect the effect of the present compounds on IL-23
production, human primary monocytes (isolated from human PBMCs
using magnetic separation [Miltenyi Biotech, Monocyte Isolation
Kit, Cat. No. 130-091-153] and by the addition of growth factors
(recombinant human GM-CSF [PeproTech, Cat. No. 300-03] and IL-4
[PeproTech, Cat. No. 200-04]) in complete medium (VLE (very low
endotoxin) RPMI 1640 [Biochrom AG, Cat. No. FG1415], 10% Fetal
Bovine Serum (FBS) [Gibco, Cat-No. 10493-106]; 50 .mu.M
.beta.-mercaptoethanol (Gibco, Cat. No. 31350], 50 U/ml penicillin
and streptomycin [Gibco, Cat. No. 15140-114]) were differentiated
in culture over 6 days to DCs. After the DCs had been harvested,
they were resuspended in complete medium and seeded in a cell
density of 2.times.10.sup.5 cells/well in a 96-well plate (Costar,
Cat. No. 3599). The present compounds were subjected to serial
dilution in a constant volume of 100% DMSO and used in the assay at
9 different concentrations in the range from 10 .mu.M to 1 nM. It
was ensured here that the DMSO concentration present was always
0.1% DMSO for each of the 9 concentrations used. There was a
30-minute preincubation of the DCs with the present compounds.
Thereafter, the DCs were stimulated to produce IL-23 by the
addition of 10 ng/ml LPS (Sigma, Escherichia coli serotype 0127:B8,
Cat. No. L3129) (TLR4 ligand) and 2.5 .mu.g/ml of TLR-7/8 ligand
R848 (Invivogen, Cat. No. tlrl-r848-5), both activate the
IRAK4-mediated signalling pathway, in an incubator (37.degree. C.,
95% rH, 5% CO.sub.2) for 24 hours. After this incubation time of 24
hours, the supernatants were harvested and analysed using a
commercially available hIL-23 ELISA (eBiosciences, Cat. No.
88-7237-88), which was conducted according to the manufacturer's
instructions. The results of the inhibition of IL-23 in human DCs
are shown by way of example for Example Compound 12 in FIG. 1.
In Vitro TLR-7/8- or TLR-9-Induced IFN.alpha. Production of Human
Plasmacytoid Dendritic Cells (pDCs)
[0476] With the aid of this test, the effect of the present
compounds of the general formula (I) on the production of
IFN.alpha. (interferon-alpha) in human pDCs, a key cytokine in the
pathogenesis of systemic lupus erythematosus (Mathian et al.,
Arthritis Rheum, 2009; Crow M. K., Rheum Dis Clin N Am, 2010), can
be studied. For this purpose, human PBMCs were isolated from whole
blood as described above and the plasmacytoid DCs (pDCs) were
isolated therefrom using a commercially available cell separation
kit (Miltenyi Biotech, Plasmacytoid Dendritic Cell Isolation Kit
II, Cat. No. 130-097-415). The obtained pDCs were resuspended in
complete medium (RPMI 1640+GlutaMax [Gibco, Cat. No. 61870-010]
supplemented with 10% FBS [Gibco, Cat. No. 10493-106] and 50 U
penicillin/streptomycin [Gibco, Cat. No. 15140-114]) and seeded at
a cell density of 5.times.10.sup.4 cells/well in a 96-well
microtitre plate (Costar, Cat. No. 3599). The present compounds
were subjected to serial dilution in a constant volume of 100% DMSO
and used in the assay at 9 different concentrations in the range
from 10 .mu.M to 1 nM. It was ensured that the DMSO concentration
present was always 0.1% DMSO for each of the 9 concentrations
tested. There was a 30-minute preincubation of the pDCs with the
present compounds. The pDCs were stimulated either with a TLR7/8
ligand (imiquimod, R837, Invivogen, Cat. No. tlrl-imq) or with a
TLR-9 ligand (CPG-A, ODN2216, Invivogen, Cat. No. tlrl-2216-1) and
this led to activation of the IRAK4-mediated signalling pathways.
After incubation for 24 hours, the cell culture supernatants were
removed and analysed using a commercially available human
IFN.alpha. ELISA (IFNalpha Multi-Subtype ELISA Kit, pbl Assay
Science, Cat. No. 41105-1). The results of the inhibition of
IFN.alpha. in human plasmacytoid DCs are shown by way of example
for Example Compound 12 in FIG. 2.
In Vivo Model of TLR-Mediated Inflammation
[0477] The present compounds of the general formula (I) were
examined for their in vivo efficacy in a model of in vivo
TLR-mediated inflammation. This mechanistic model particularly
shows the potential effect of the present compounds on
TLR4-mediated disorders, since an LPS-mediated inflammation model
was used. In this model, female Balb/c mice (about 8 weeks old;
Charles River Laboratories, Germany) were divided into groups of 5
animals each. The control group was treated with the vehicle in
which the substance had been dissolved (substance vehicle) and also
with the vehicle in which the LPS had been dissolved. The substance
treatment groups as well as the positive control group received 0.2
mg LPS/kg body weight (Sigma, Cat. No. L4391) (lipopolysaccharides
from E. coli 0111:B4) intraperitoneally (i.p.). In addition, the
positive control group was treated with the substance vehicle
described above. The substance was administered orally 16 hours
before induction of inflammation by administration of LPS. To
examine the effect of the present compounds on the inflammation,
blood samples were taken from the animals after 1.5 hours. The
concentration of particular cytokines in the plasma was determined
using the Mouse ProInflammatory 7-Plex Tissue Culture Kit (MSD,
Cat. No. K15012B) in accordance with the manufacturer's
instructions. IRAK4 inhibitors are effective in the TLR-mediated
inflammation model. FIG. 3 shows the amount of TNF-.alpha. in the
plasma, which is reduced in a dose-dependent manner by
administration of Example Compound 11 in comparison with the
LPS-induced concentration.
In Vivo Model of IL-1.beta.-Mediated Inflammation
[0478] To evaluate the potential efficacy of the present compounds
of the general formula (I) in IL-1.beta.-mediated disorders,
IL-1.beta. was administered i.p. to female Balb/c mice (about 8
weeks old, Charles River Laboratories, Germany) and the effect of
the present compounds on IL-1.beta.-mediated cytokine secretion was
examined. There were 5 animals in each group. The control group was
treated with the vehicles used for dissolving the substance and the
IL-1.beta.. The substance treatment groups and the positive control
group were each administered 90 .mu.g IL-1.beta./kg body weight
i.p. (R&D, Cat. No. 401-ML/CF). The substance or its vehicle in
the positive control group was administered 6 hours before the
administration of IL-1.beta.. 2 hours after administration of the
IL-1.beta., TNF-.alpha. was determined in the plasma isolated from
the blood using the Mouse ProInflammatory 7-Plex Tissue Culture Kit
(MSD, Cat. No. K15012B) in accordance with the manufacturer's
instructions. Administration of IL-1.beta. led to an elevated
TNF-.alpha. plasma concentration which was inhibited by treatment
with Example Compounds 11 and 12. This is illustrated by FIG.
4.
In Vivo Adjuvant-Induced Arthritis Model
[0479] To determine the anti-inflammatory activity of the present
compounds of the general formula (I), they were examined for their
in vivo efficacy in an arthritis model. For this purpose, male
Lewis rats (about 100-125 g, Charles River Laboratories, Germany)
were each administered 100 .mu.l of a complete Freund's adjuvant
(CFA) solution (M. tuberculosis H37Ra [Difo Lab, Cat. No. -231141]
dissolved in Incomplete Freund's adjuvant [Difco Lab, Cat. No.
-263910]) into the tailhead subcutaneously on day 0. There were n=8
rats in each group. Both a healthy control group and a disease
control group were included in the study. Each control group was
given p.o. treatment only with the vehicle of the test substance.
The treatment with different dosages of the test substance was
conducted in a preventative manner, i.e. starting from day 0, by
oral administration. On day 0, the starting condition of the
animals was additionally determined in terms of the disease
activity scores (rating of the severity of arthritis based on a
points system). Here, points were awarded according to the extent
of joint inflammation from 0 to 4 for the presence of an erythema
including joint swelling (0=none; 1=slight; 2=moderate; 3=distinct;
4=severe) for both hind paws and added up. To determine the
anti-inflammatory efficacy of the compounds, the disease activity
of the animals was scored by means of disease activity scoring
starting from day 8, when the animals first exhibit signs of
arthritis, and subsequently 3 times per week, until the end (day
20). Statistical analysis was performed using single-factor
variance analysis (ANOVA) and by comparison with the control group
by means of multiple comparative analysis (Dunnett's test).
[0480] The s.c. administration of CFA in rats leads to acute
arthritis with distinct joint inflammation in rats. This induced
arthritis was inhibited by the treatment with Example Compound 11.
This is illustrated by FIG. 5.
In Vivo Collagen Antibody-Induced Arthritis Model in Mice
[0481] The anti-inflammatory effect of the present compounds of the
general formula (I) was examined in a further murine arthritis
model. For this purpose, female Balb/c mice (about 9 weeks old,
Charles River Laboratories, Kingston, Canada) were each injected
intravenously on day 0 with 200 .mu.l of a collagen antibody
cocktail (10 mg/ml; ArthritoMab, MD Bioproducts) into the tail vein
(except for the healthy control group included in the study). On
day 6, these mice then each received a further intraperitoneal
injection of 200 .mu.l of LPS. There were n=10 mice in each group.
Both a healthy control group and a disease control group were
included in the study. Each control group was given p.o. treatment
only with the vehicle of the test substance. The treatment with
different dosages of the test substance was conducted in a
preventative manner, i.e. starting from day 0, by oral
administration. Over the course of the experiment, the extent of
disease was scored on the basis of a point award system for the
disease activity score on all four paws. In this awarding of
points, no points are awarded for a healthy paw, whereas points
from 1 [mild inflammation, for example, of the toe(s)] to 4 [severe
inflammation extending over the entire paw] are awarded in each
case for the particular extent of joint inflammation that has
arisen from the toes through the metatarsal joint to the ankle
joint, as explained as follows: [0482] 0=normal [0483] 1=erythema
and mild swelling limited to the tarsal or ankle or toes [0484]
2=erythema and mild swelling extending from the ankle to the
metatarsus (2 segments) [0485] 3=erythema and moderate swelling
extending from the ankle as far as the metatarsal joints [0486]
4=erythema and severe swelling encompassing the metatarsus, foot
and toes
[0487] For this parameter, the starting condition was determined
beforehand one day before the start of the experiment (day -1) and
this disease activity score was subsequently scored three times per
week from day 8 onwards. Statistical analysis was performed using
single-factor variance analysis (ANOVA) and by comparison with the
control group by means of multiple comparative analysis (Dunnett's
test).
[0488] The i.v. administration of a collagen antibody cocktail
including the subsequent i.p. administration of LPS in mice leads
to acute arthritis with distinct joint inflammation. This induced
arthritis was inhibited by the treatment with Example Compound 12.
This is illustrated by FIG. 6.
In Vivo NASH Mouse Model
[0489] To experimentally induce NASH, 200 .mu.g streptozotocin
(STZ; Sigma-Aldrich, USA) is each injected subcutaneously in 45
male 2-day-old C57BL/6 mice. Starting at 4 weeks of age, these
animals are fed ad libitum with a high-fat diet (HFD; 57 kcal %
fat, # HFD32 from CLEA, Japan). At an age of 6 weeks, the animals
are randomized into 3 groups (15 animals per group). While one of
the groups does not receive any treatment, the other 2 groups are
daily orally treated either with vehicle or the test substance over
4 weeks. After the 4-week treatment, all animals are sacrificed
painlessly under anaesthesia, and the livers are removed and fixed
for the histological study in Bouin's solution (H. Denk, "Fixierung
histologischer Praparate" [Fixing of Histological Preparations],
in: P. Bock (ed.): "Romeis Mikroskopische Technik" [Romei's
Microscopy Techniques], Urban & Schwarzenberg,
Munich-Vienna-Baltimore 1989, 17th edition, page 97, ISBN
3-541-11227-1). Thereafter, the liver samples are embedded in
paraffin and 5 .mu.m-thick paraffin sections are produced.
Histological sections of each liver are stained a) for the
determination of the NAFLD activity score (NAS) with
haematoxylin-eosin (HC), and b) for the determination of liver
fibrosis with Picro-Sirius red (Waldeck, Germany). The NAFLD
activity score is determined in the haematoxylin-eosin sections on
the basis of the criteria recommended by D. E. Kleiner et al.,
Hepatology 41 (2005), 1313-1321 (Table 1). For the histological
quantification of fibrotic areas, 5 digital photos (DFC280; Leica,
Germany) are taken for each section under 200-fold microscope
enlargement and the percentage of fibrosis is determined using the
ImageJ Software (National Institute of Health, USA).
In Vivo db/db Mouse Model
[0490] 30 male 8-week-old db/db mice are used. This model is a well
accepted model for obesity, insulin resistance and type 2 diabetes
(Aileen J F King; The use of animal models in diabetes research;
British Journal of Pharmacology 166 (2012), 877-894). During the
experiment, the animals receive a standard diet (RM1(E) 801492,
SDS) and tap water ad libitum. The animals are randomized into 3
groups (10 animals per group) and treated orally with the test
substance over 6 weeks. During the study period, blood is taken
from the animals at different time points (before start of
treatment, 3 weeks after start of treatment and 2 days before the
end of treatment) to determine insulin sensitivity parameters (e.g.
HbA1c, glucose content, insulin content). In addition, an OGTT
(oral glucose tolerance test) as a parameter for determination of
insulin sensitivity is conducted 1 day before start of treatment
and 2 days after the end of treatment. In addition, the HOMA-IR
index (fasting insulin level (mU/l)*fasting glucose level
(mmol/l)/22.5) is calculated.
In Vivo B-Cell Lymphoma-Associated Xenotransplantation Model
[0491] The anti-tumour activity of the present compounds of the
general formula (I) is studied in murine xenotransplantation
models. For this purpose, female C.B-17 SCID mice are implanted
subcutaneously with human B-cell lymphoma cell lines, e.g. TMD-8.
At a mean tumour size of 20-30 mm.sup.2, oral monotherapeutic
treatment is started with an present compound or by administration
of an present compound in combination with a standard therapy, each
administered orally. However, the animals are randomized
beforehand. The treatment is ended as soon as the untreated control
group has large tumours. The tumour size and body weight are
determined three times per week. Decreases in body weight are a
measure of treatment-related toxicity (>10%=critical, stoppage
in treatment until recovery, >20%=toxic, termination). The
tumour area is detected by an electronic caliper gauge [length
(mm).times.width (mm)]. At the end of the study, the tumour weight
is also determined. The anti-tumour efficacy defines the ratio of
tumour weight of treatment vs. control (T/C) [tumour weight of the
treatment group on day x/tumour weight of the control group on day
x] or the ratio of the tumour area of treatment vs. control [tumour
area of the treatment group on day x/tumour area of the control
group on day x]. A compound having a T/C greater than 0.5 is
defined as active (effective). Statistical analysis is preformed
using single-factor ANOVA and by comparison with the control group
by means of pair-by-pair comparative analysis (Dunnett's test).
Canine IRAK4 Kinase Assay
[0492] The IRAK4-inhibitory activity of the present compounds on
canine IRAK4 was measured in the Irak4 TR-FRET assay (TR-FRET=Time
Resolved Fluorescence Resonance Energy Transfer) described
hereinafter.
[0493] Recombinant fusion protein from N-terminal HIS
(Poly-histidine) and canine Irak4, expressed in
baculovirus-infected insect cells (Hi5, BTI-TN-5B1-4, cell line
purchased from Invitrogen, catalogue No. B855-02) and purified via
affinity chromatography, was used as enzyme. The substrate used for
the kinase reaction was the biotinylated peptide
biotin-Ahx-KKARFSRFAGSSPSQASFAEPG (C-terminus in amide form) which
can be purchased, for example, from Biosyntan GmbH
(Berlin-Buch).
[0494] For the assay, 11 different concentrations in the range from
20 .mu.M to 0.073 nM were prepared from a 2 mM solution of the test
substance in DMSO. 50 nl of the respective solution were pipetted
into a black low-volume 384-well microtitre plate (Greiner Bio-One,
Frickenhausen, Germany), 2 .mu.l of a solution of Irak4 in assay
buffer [50 mM HEPES pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 30
.mu.M activated sodium orthovanadate, 0.1% (w/v) of bovine
gamma-globulin (BGG) 0.04% (v/v) nonidet-P40 (Sigma)] were added
and the mixture was incubated for 15 min to allow prebinding of the
substances to the enzyme prior to the kinase reaction. The kinase
reaction was then started by addition of 3 .mu.l of a solution of
adenosine triphosphate (ATP, 1.67 mM=final concentration in 5 .mu.l
of assay volume: 1 mM) and peptide substrate (0.83 .mu.M=final
concentration in 5 .mu.l assay volume: 0.5 .mu.M) in assay buffer,
and the resulting mixture was incubated at 22.degree. C. for the
reaction time of 45 min. The concentration of the Irak4 was
adjusted to the respective activity of the enzyme and set such that
the assay was carried out in the linear range. Typical
concentrations were in the order of about 0.1 nM. The reaction was
stopped by addition of 5 .mu.l of a solution of TR-FRET detection
reagents [0.1 .mu.M streptavidin-XL665 (Cisbio Bioassays; France,
catalogue No. 610SAXLG)] and 1.5 nM anti-phosphoserine antibody
[Merck Millipore, "STK Antibody", catalogue No. 35-002] and 0.6 nM
LANCE EU-W1024-labelled anti-mouse-IgG antibody (Perkin-Elmer,
product No. AD0077; alternatively, it is possible to use a terbium
cryptate-labelled anti-mouse-IgG antibody from Cisbio Bioassays) in
aqueous EDTA solution (100 mM EDTA, 0.4% [w/v] bovine serum albumin
[BSA] in 25 mM HEPES pH 7.5).
[0495] The resulting mixture was incubated at 22.degree. C. for 1 h
to allow formation of a complex of the biotinylated phosphorylated
substrate and the detection reagents. The amount of the
phosphorylated substrate was then evaluated by measuring the
resonance energy transfer from europium chelate-labelled
anti-mouse-IgG antibody to streptavidin-XL665. To this end, the
fluorescence emissions at 620 nm and 665 nm were measured after
excitation at 350 nm in a TR-FRET measuring instrument, for example
a Rubystar (BMG Labtechnologies, Offenburg, Germany) or a Viewlux
(Perkin-Elmer). The ratio of the emissions at 665 nm and 622 nm was
taken as a measure of the amount of phosphorylated substrate. The
data were normalized (enzyme reaction without test substance=0%
inhibition; all other assay components but no enzyme=100%
inhibition). Typically, the test substances were tested on the same
microtitre plates at 11 different concentrations in the range from
20 .mu.M to 0.073 nM (20 .mu.M, 5.7 .mu.M, 1.6 .mu.M, 0.47 .mu.M,
0.13 .mu.M, 38 nM, 11 nM, 3.1 nM, 0.89 nM, 0.25 nM and 0.073 nM).
The dilution series were prepared prior to the assay (2 mM to 7.3
nM in 100% DMSO) by serial dilutions. The IC.sub.50 values were
calculated by a 4-parameter fit.
TABLE-US-00004 TABLE 3 IC.sub.50-values from two experiments of
example compounds in the IRAK4 canine kinase assay Example
IC.sub.50 Compound [nM] 11 1.48 1.86 12 8.99 9.34 13 1.75 2.68 19
8.46 6.75
In Vitro Lipopolysaccharide (LPS)-Induced Cytokine Production by
Canine Peripheral Blood Mononuclear Cells (PBMCs)
[0496] The effect of the present compounds of the general formula
(I) on induced cytokine production in canine PBMCs was examined.
Cytokine production was induced here by LPS, a TLR4 ligand, which
leads to activation of the IRAK4-mediated signal path.
[0497] The canine PBMCs were obtained from anti-coagulated dog
whole blood. For this purpose, canine leucocyte rich plasma was
prepared from 15 ml dog blood by centrifugation at 400 g for 15 min
at 4.degree. C., followed by harvest and then suspension of the
canine PBMC buffy coat in plasma. Seven (7) ml Ficoll-Paque Plus
(Fischer Scientific, Cat. No. 11778538) were pipetted in a
centrifugation tube and 7 ml canine leucocyte rich plasma then
layered on top of the Ficoll-Paque Plus. After centrifugation of
the tube at 400 g for 20 min at 4.degree. C., canine PBMCs were
harvested from the interface of the canine plasma and Ficoll-Paque
Plus. PBMCs were transferred into a fresh centrifugation tube and
made up with Hanks' Balanced Salt Solution 1.times. (HBSS) without
Ca.sup.2+/Mg.sup.2+ (Sigma-Aldrich, Cat. No. H9394). The cell
suspension was centrifuged at 400 g for 5 min at 4.degree. C. and
the supernatant was discarded. The cell pellet was then
re-suspended in 0.2% hypotonic saline to lyse any remaining red
blood cells. After 30 seconds the cell suspension was made isotonic
and centrifuged at 400 g for 5 min at 4.degree. C. The cell pellet
was then re-suspended in HBSS without Ca.sup.2+/Mg.sup.2+ for a
final wash and centrifuged at 400 g for 5 min at 4.degree. C. The
PBMCs were then re-suspended in complete medium (RPMI 1640 with
GlutaMAX (Sigma-Aldrich, Cat. No. R0883), 10% FCS; 50 U/ml
penicillin, 50 .mu.g/ml streptomycin (Sigma-Aldrich, Cat. No.
P4333)).
[0498] The assay was also carried out in complete medium. The PBMCs
were seeded in 96-well plates at a cell density of
2.5.times.10.sup.5 cells/well. The present compounds were dissolved
in DMSO and subjected to serial dilution in complete medium. The
compound examples were used in the assay at 8 different
concentrations in the range from 3 nM to 10 .mu.M such that the
final DMSO concentration was 0.0003-0.4%. To induce cytokine
secretion, the cells were stimulated with 0.1 .mu.g/ml LPS
(Sigma-Aldrich, Escherichia coli 0111:B4, Cat. No. L3024) for 24
hours. Cell viability was determined using 0.2% trypan blue
(Sigma-Aldrich, Cat. No. T8154). The amount of secreted TNF-.alpha.
in the cell culture supernatant was determined using canine
TNF.alpha. DuoSet Elisa (R&D Systems, Cat. No. DY1507) in
accordance with the instructions of the manufacturer. By way of
example, Example Compound 12 inhibited the production of TNF.alpha.
by canine PBMCs stimulated with LPS. This is illustrated by FIG.
7.
In Vitro Lipopolysaccharide (LPS)-Induced Cytokine Production by
Bovine Peripheral Blood Mononuclear Cells (PBMCs)
[0499] The effect of the present compounds of the general formula
(I) on induced cytokine production in bovine PBMCs was examined.
Cytokine production was induced here by LPS, a TLR4 ligand, which
leads to activation of the IRAK4-mediated signal path.
[0500] The bovine PBMCs were obtained from anti-coagulated cattle
whole blood. For this purpose, bovine leucocyte rich plasma was
prepared from 500 ml cattle blood by centrifugation at 1000 g for
20 min at room temperature (RT), followed by harvest and then
suspension of the bovine PBMC buffy coat in equal volume of PBS/5
mM EDTA (RT). Thirty (30) ml Ficoll-Paque Plus (Fischer Scientific,
Cat. No. 11778538) were pipetted in a Leucosep tube and 30 ml
bovine PBMC buffy coat/PBS/EDTA mixture then layered on top of the
Ficoll-Paque Plus. After centrifugation of the tube at 800 g for 25
min at RT, bovine PBMCs were harvested from the interface of the
bovine plasma and Ficoll-Paque Plus. PBMCs were transferred into a
fresh centrifugation tube and made up with cold PBS/5 mM EDTA. The
cell suspension was centrifuged at 350 g for 10 min at 4.degree. C.
and the supernatant was discarded. The cell pellet was then
re-suspended in 0.2% hypotonic saline to lyse any remaining red
blood cells. After 30 seconds the cell suspension was made isotonic
and centrifuged at 500 g for 5 min at 4.degree. C. The PBMC cell
pellet was then resuspended in complete medium (DMEM with GlutaMAX
(ThermoFisher, Cat. No. 32430100), 10% horse serum (ATCC.RTM.
30-2040.TM.), 20 .mu.M .beta.-mercaptoethanol (ThermoFisher Cat.
No. 31350010 [stock solution: 50 mM]).
[0501] The assay was also carried out in complete medium. The PBMCs
were seeded in 24-well plates at a cell density of 1.times.10.sup.6
cells/well. The present compounds were dissolved in DMSO and
subjected to serial dilution in complete medium. The compound
examples were used in the assay at 8 different concentrations in
the range from 0.003 .mu.M to 10 .mu.M such that the final DMSO
concentration was 0.5%. To induce cytokine secretion, the cells
were stimulated with 1 .mu.g/ml (FIG. 8) and 0.1 .mu.g/ml LPS (FIG.
9) (LPS from E. coli K12; Invivogen # tlrl-eklps) for 24 hours.
Cell viability was determined using Turk solution (Merck Millipore
#1092770100).
[0502] The amount of secreted TNF.alpha. in the cell culture
supernatant of LPS-exposed bovine PBMCs was determined using a
rabbit anti-bovine TNF.alpha. antibody based ELISA read-out. The
ELISA assay was performed in 384 well ELISA plates, which were
coated with 5 .mu.g/ml rabbit anti-bovine TNF.alpha. antibody
(BioRad, AHP2383) in 50 mM Na.sub.2CO.sub.3/NaHCO.sub.3 pH 9.6
buffer in 10 .mu.l/well overnight at 4.degree. C. After removal of
antibody and rinsing of wells for three times with 50 .mu.l of wash
buffer (PBS, 0.05% (v/v) Tween 20), the wells were incubated for 90
min at 37.degree. C. with 40 .mu.l blocking buffer (PBS, 0.05%
(v/v) Tween 20, 1% (w/v) bovine serum albumin). Thereafter,
blocking buffer was removed and culture supernatant samples were
added (20 .mu.l/well). After an incubation for 90 min at 37.degree.
C., the samples were removed and the wells were rinsed for three
times with 50 .mu.l wash buffer. A 1 .mu.g/ml rabbit anti-bovine
TNF.alpha.-biotin conjugated antibody (BioRad, AHP2383B) in
blocking buffer was added to the plates (20 .mu.l/well) which were
incubated for 60 min at 37.degree. C. After removal of the
biotinylated antibody and rinsing of the wells with 50 .mu.l wash
buffer for three times, 20 .mu.l/well of ExtrAvidin.TM.-alkaline
phosphatase (Sigma, E2636), 1:10.000 diluted in blocking buffer,
was added for 1 h at 37.degree. C. After removal of
ExtrAvidin.TM.-alkaline phosphatase and rinsing of the wells with
50 .mu.l wash buffer for three times, the enzymatic reaction/colour
development was initiated by adding 50 .mu.l/well of development
buffer (5 mM para-nitrophenyl phosphate (pNPP) in 50 mM
Na.sub.2CO.sub.3/NaHCO.sub.3 pH 9.6, 2 mM MgCl.sub.2). Optical
density was recorded at 405 nm wavelength. For kinetic measurements
data points were recorded every 5 minutes for 1 hour, endpoint
measurements were taken after 2 hours. By way of example, Example
Compound 12 inhibited the production of TNF.alpha. by bovine PBMCs
stimulated with LPS. This is illustrated by FIGS. 8 and 9.
In Vitro Lipopolysaccharide (LPS)-Induced Cytokine Production by
Porcine Peripheral Blood Mononuclear Cells (PBMCs)
[0503] As a further example, the effect of the present compounds of
the general formula (I) on induced cytokine production in porcine
PBMCs was examined. Cytokine production was induced here by LPS, a
TLR4 ligand, which leads to activation of the IRAK4-mediated signal
path.
[0504] The porcine PBMCs were obtained from anti-coagulated porcine
whole blood. For this purpose, porcine leucocyte rich plasma was
prepared from 36 ml pig blood by centrifugation at 1000 g for 20
min at room temperature (RT), followed by harvest and then
suspension of the porcine PBMC buffy coat in equal volume of PBS/5
mM EDTA (RT). Thirty (30) ml Ficoll-Paque Plus (Fischer Scientific,
Cat. No. 11778538) were pipetted in a Leucosep tube and 30 ml
bovine PBMC buffy coat/PBS/EDTA mixture then layered on top of the
Ficoll-Paque Plus. After centrifugation of the tube at 800 g for 25
min at RT, porcine PBMCs were harvested from the interface of the
porcine plasma and Ficoll-Paque Plus. PBMCs were transferred into a
fresh centrifugation tube and made up with cold PBS/5 mM EDTA. The
cell suspension was centrifuged at 350 g for 10 min at 4.degree. C.
and the supernatant was discarded. The cell pellet was then
re-suspended in 0.2% hypotonic saline to lyse any remaining red
blood cells. After 30 seconds the cell suspension was made isotonic
and centrifuged at 500 g for 5 min at 4.degree. C. The PBMC cell
pellet was then resuspended in complete medium (DMEM with GlutaMAX
(ThermoFisher, Cat. No. 32430100), 10% horse serum (ATCC.RTM.
30-2040.TM.), 20 .mu.M 1-mercaptoethanol (ThermoFisher Cat. No.
31350010 [stock solution: 50 mM]).
[0505] The assay was also carried out in complete medium. The PBMCs
were seeded in 24-well plates at a cell density of 1.times.10.sup.6
cells/well. The present compounds were dissolved in DMSO and
subjected to serial dilution in complete medium. The compound
examples were used in the assay at 8 different concentrations in
the range from 0.003 .mu.M to 10 .mu.M such that the final DMSO
concentration was 0.5%. To induce cytokine secretion, the cells
were stimulated with LPS (LPS from E. coli K12; Invivogen #
tlrl-eklps) in a concentration range of 0.01 to 1 ng/ml for 24
hours. Cell viability was determined using Turk solution (Merck
Millipore #1092770100).
[0506] The amount of secreted TNF.alpha. in the cell culture
supernatant of LPS-exposed porcine PBMCs was determined using a
rabbit anti-porcine TNF.alpha. antibody based ELISA read-out. The
ELISA assay was performed in 384 well ELISA plates, which were
coated with 3 .mu.g/ml rabbit anti-porcine TNF.alpha. antibody
(BioRad, AHP2397) in 50 mM Na.sub.2CO.sub.3/NaHCO.sub.3 pH 9.6
buffer in 10 .mu.l/well for 48 h at 4.degree. C. After removal of
antibody and rinsing of wells for three times with 50 .mu.l of wash
buffer (PBS, 0.05% (v/v) Tween 20), the wells were incubated for 60
min at 37.degree. C. with 50 .mu.l blocking buffer (PBS, 0.05%
(v/v) Tween 20, 1% (w/v) bovine serum albumin). Thereafter,
blocking buffer was removed and culture supernatant samples were
added (20 .mu.l/well). After an incubation for 90 min at 37.degree.
C., the samples were removed and the wells were rinsed for three
times with 50 .mu.l wash buffer. A 0.25 .mu.g/ml rabbit
anti-porcine TNF.alpha.-biotin conjugated antibody (BioRad,
AHP2397B) in blocking buffer was added to the plates (20
.mu.l/well) which were incubated for 60 min at 37.degree. C. After
removal of the biotinylated antibody and rinsing of the wells with
50 .mu.l wash buffer for three times, 20 .mu.l/well of
ExtrAvidin.TM.-alkaline phosphatase (Sigma, E2636), 1:10.000
diluted in blocking buffer, was added for 1 h at 37.degree. C.
After removal of ExtrAvidin.TM.-alkaline phosphatase and rinsing of
the wells with 50 .mu.l wash buffer for three times, the enzymatic
reaction/colour development was initiated by adding 90 .mu.l/well
of development buffer (5 mM para-nitrophenyl phosphate (pNPP) in 50
mM Na.sub.2CO.sub.3/NaHCO.sub.3 pH 9.6, 2 mM MgCl.sub.2). Optical
density was recorded at 405 nm wavelength. For kinetic measurements
data points were recorded every 5 minutes for 1 hour, endpoint
measurements were taken after 2 hours. By way of example, at 10
.mu.M Example Compound 12 inhibited the production of TNF.alpha. by
bovine PBMCs stimulated with 0.1 ng/ml LPS. This is illustrated by
FIG. 10.
In Vivo Model of House Dust Mite Induced Canine Allergic
Dermatitis
[0507] To evaluate the potential anti-allergic/anti-inflammatory
efficacy of the present compounds of the general formula (I) a
model of house dust mites (HDM)-sensitized Beagle dogs was used.
Therein, HDM-sensitization consisted of a series of subcutaneous
injections of HDM antigen (10 .mu.g, Greer Laboratories, Lenoir,
N.C., USA) and Alhydrogel.RTM. (0.2 mL, InvivoGen, San Diego,
Calif. 921221, USA) as adjuvant in time intervals of approximately
two weeks. The sensitization process was monitored and confirmed by
intradermal skin testing. Once the dogs were positive to HDM skin
intradermal testing, one month apart from the last sensitization,
HDM antigen (135 .mu.g) was topically applied and pricked into the
skin (with 2 mm long micro needles) of the adult beagle dogs in the
inner part of the posterior legs and the effect of the present
compounds on signs of allergic dermatitis, e.g. erythema and edema,
was examined. There were 2 groups of 4 animals each: 1 placebo
control group and 1 group treated with Example Compound 12. The
control group was orally treated with gelatin capsules containing
micro cellulose while the group treated with Example Compound 12
was orally treated with gelatin capsules containing Example
Compound 12 and micro cellulose. The administration of Example
Compound 12 or the placebo started 5 days before the challenge with
HDM antigen and continued until 2 days after the challenge. The
treatment frequency was once daily, with a dose of 10 mg/kg body
weight in the case of Example Compound 12. Starting 30 min after
challenge and at different time points for 48 h, erythema and edema
were evaluated using VAS (Visual Analogue Scale) in the 2 groups.
Plasma samples were analyzed to determine exposure to the compound
in relationship to the clinical evaluations. Edema and erythema
were significantly reduced after treatment with Example Compound
12. This is illustrated by Tables 4 and 5, and by FIGS. 11 and
12.
TABLE-US-00005 TABLE 4 Erythema (in VAS units) after treatment with
Example Compound 12 versus placebo Time post challenge Placebo
Control Example Compound 12 [hours] [VAS units] [VAS units] 0 0.0
0.0 0.5 5.7 2.2 1 5.8 1.2 4 5.1 0.7 6 4.7 0.8 24 3.9 0.5 48 1.4
0.2
TABLE-US-00006 TABLE 5 Edema (in VAS units) after treatment with
Example Compound 12 versus placebo Time post challenge Placebo
Control Example Compound 12 [hours] [VAS units] [VAS units] 0 0.0
0.0 0.5 6.7 3.8 1 5.0 1.9 4 0.8 0.3 6 0.9 0.1 24 0.6 0.2 48 0.1
0.0
In Vivo Pruritic Model of Flea Allergy Dermatitis
[0508] To evaluate the potential anti-pruritic effect of the
present compounds of the general formula (I) a model of Flea
Allergy Dermatitis (FAD) was used. Only adult dogs with a history
of FAD were enrolled in the study. The in-life phase of the study
consisted of two phases: a Pruritus Induction Phase (2 weeks)
followed by a Treatment Phase (2 weeks). Dogs were infested with
Ctenocephalides fleas (first challenge with 100 fleas/dog, all
subsequent challenges with 30 fleas/dog) twice weekly during both
study phases. There were 2 groups of 12 animals each: 1 placebo
control group and 1 group treated with Example Compound 12. The
control group was orally treated with gelatin capsules containing
micro cellulose while the group treated with Example Compound 12
was orally treated with gelatin capsules containing Example
Compound 12 and micro cellulose. The treatment frequency was once
daily, with a dose of 20 mg/kg body weight in the case of Example
Compound 12. Starting 1 day after treatment and at every third day,
dogs were recorded for 4 hours and time spent in pruritic behavior
was determined as seconds spent in scratching, licking, biting.
Plasma samples were analyzed to determine exposure to the compound
in relationship to the clinical evaluations. Pruritus was
substantially reduced after 10 days of treatment with Example
Compound 12. This is illustrated by Table 6 and FIG. 13.
TABLE-US-00007 TABLE 6 Reduction of puritic behavior compared to
baseline after treatment with Example Compound 12 versus placebo
(shown as percent-change from baseline at the listed day after
treatment) Day 4 Day 7 Day 10 Day 13 Placebo Control -12.0% -9.9%
1.7% -20.0% Example Compound 12 -26.7% 5.7% -57.7% -48.0%
[0509] FIG. 1: Inhibition of IL-23 in human monocyte-generated DCs
for Example Compound 12. Data are shown as mean values with
standard deviations.
[0510] FIG. 2: Inhibition of INF-.alpha. in (A) imiquimod (R837)-
or (B) CpG-A-stimulated human plasmacytoid DCs for Example Compound
12. Data are shown as mean values with standard deviations.
[0511] FIG. 3: Treatment of an LPS-induced inflammation with
Example Compound 11 leads to a reduced amount of secreted
TNF-.alpha.. Data are shown as mean values with standard
deviations.
[0512] FIG. 4: Treatment of an IL-1.beta.-induced inflammation with
Example Compounds 11 (left) and 12 (right) leads to a
dose-dependent reduction in the amount of secreted TNF-.alpha..
Data are shown as mean values with standard deviations.
[0513] FIG. 5: Anti-inflammatory effects of Example Compound 11 in
an animal model of rheumatoid arthritis (adjuvant-induced rat
model). Significant and dose-dependent inhibition of rheumatic
joint inflammation measured on the basis of the disease activity
score. The data corresponds to the mean values+standard deviations.
Single-factor ANOVA variance analysis with subsequent multiple
comparative analysis with the CFA control group by means of
Dunnett's test; *p<0.05; **p<0.01;***p&