U.S. patent application number 15/366428 was filed with the patent office on 2017-03-23 for novel compounds.
The applicant listed for this patent is Glaxo Group Limited. Invention is credited to Veronique BIRAULT, Amanda Jennifer Campbell, Stephen Harrison, Joelle Le.
Application Number | 20170081278 15/366428 |
Document ID | / |
Family ID | 44994047 |
Filed Date | 2017-03-23 |
United States Patent
Application |
20170081278 |
Kind Code |
A1 |
BIRAULT; Veronique ; et
al. |
March 23, 2017 |
Novel compounds
Abstract
The present invention is directed to novel retinoid-related
orphan receptor gamma (ROR.gamma.) modulators, processes for their
preparation, pharmaceutical compositions containing these
modulators, and their use in the treatment of inflammatory,
metabolic and autoimmune diseases mediated by ROR.gamma..
Inventors: |
BIRAULT; Veronique;
(Stevenage, GB) ; Campbell; Amanda Jennifer;
(Stevenage, GB) ; Harrison; Stephen; (Stevenage,
GB) ; Le; Joelle; (Stevenage, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Glaxo Group Limited |
Brentford |
|
GB |
|
|
Family ID: |
44994047 |
Appl. No.: |
15/366428 |
Filed: |
December 1, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14819836 |
Aug 6, 2015 |
9540318 |
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15366428 |
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14347018 |
Mar 25, 2014 |
9150508 |
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PCT/EP2012/068845 |
Sep 25, 2012 |
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14819836 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 249/08 20130101;
C07D 213/38 20130101; C07D 239/28 20130101; C07D 213/65 20130101;
C07C 311/44 20130101; C07D 239/26 20130101; C07D 213/30 20130101;
C07D 213/75 20130101; C07D 239/34 20130101; C07C 2601/16 20170501;
C07D 233/22 20130101; C07D 257/04 20130101; C07D 213/42 20130101;
C07D 231/12 20130101; C07D 213/61 20130101; C07D 213/68 20130101;
C07D 233/64 20130101; C07D 213/73 20130101; C07C 311/29 20130101;
C07D 261/08 20130101; C07C 311/21 20130101 |
International
Class: |
C07C 311/44 20060101
C07C311/44; C07D 261/08 20060101 C07D261/08; C07D 233/64 20060101
C07D233/64; C07D 231/12 20060101 C07D231/12; C07D 249/08 20060101
C07D249/08; C07D 239/26 20060101 C07D239/26; C07D 257/04 20060101
C07D257/04; C07C 311/29 20060101 C07C311/29; C07C 311/21 20060101
C07C311/21; C07D 213/68 20060101 C07D213/68; C07D 239/34 20060101
C07D239/34 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 27, 2011 |
GB |
1116641.0 |
Claims
1. A compound of formula (I), or a pharmaceutically acceptable salt
thereof: ##STR00315## wherein R.sub.1 is CH.sub.3 or halo; R.sub.2,
R.sub.3 and R.sub.4 are H; R.sub.5 is CH.sub.3 or halo; R.sub.6 is
selected from the group consisting of C.sub.3-5 alkyl and
--CH.sub.2--C.sub.3-4 cycloalkyl; R.sub.7 is selected from the
group consisting of ##STR00316## R.sub.8 is selected from the group
consisting of C.sub.1-3 alkyl, C.sub.1-3 alkoxy, CH.sub.2CN,
CH.sub.2OH, OH, CN and halo; R.sub.9 is the group
--(CHR.sub.10).sub.s--(X).sub.t--(CHR.sub.10).sub.u--R.sub.11; each
R.sub.10 is independently selected from the group consisting of H,
OH or CH.sub.2OH; R.sub.11 is --C(O)OH, or a 5 or 6-membered
heteroaryl group wherein the 5- or -6 membered heteroaryl group is
optionally substituted with one or two substituents selected from
halo, NH.sub.2 or CH.sub.3 and the 6 membered heteroayl group
contains two, three or four nitrogen atoms as member atoms of the
ring; X is CH.sub.2, NH, O; r is 0, 1 or 2; s is 0, 1 or 2; t is 0
or 1; u is 0, 1 or 2; with the proviso that no more than two
R.sub.10 groups represent OH or CH.sub.2OH.
2. The compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein R.sub.1 and R.sub.5 are
CH.sub.3.
3. The compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein R.sub.6 is isobutyl.
4. The compound according to claim 2, or a pharmaceutically
acceptable salt thereof, wherein R.sub.6 is isobutyl.
5. The compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein R.sub.7 is: ##STR00317##
6. The compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein r is 1 and R.sub.8 is
CH.sub.2OH.
7. The compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein R.sub.1 and R5 are CH.sub.3,
R.sub.6 is isobutyl, r is 1 and R.sub.8 is CH.sub.2OH.
8. The compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein s is 0.
9. The compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein t is 1 and X is O.
10. The compound according to claim 2, or a pharmaceutically
acceptable salt thereof, wherein each R.sub.10 is H.
16. The compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein R.sub.1 and R.sub.5 are CH.sub.3,
R.sub.6 is isobutyl, r is 1, R.sub.8 is CH.sub.2OH, wherein t is 1,
and X is O.
17. The compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein R.sub.11 is ##STR00318##
18. The compound according to claim 1, or a pharmaceutically
acceptable salt thereof, wherein R.sub.11 is selected from the
group consisting of ##STR00319##
19. The compound according to claim 1, or a pharmaceutically
acceptable salt thereof, is selected from the group consisting of:
4-((1H-imidazol-2-yl)methoxy)-N-(2,4-dimethylphenyl)-N-isobutylbenzenesul-
fonamide;
4-((1,3-dimethyl-1H-pyrazol-5-yl)methoxy)-N-(2,4-dimethylphenyl)-
-N-isobutylbenzenesulfonamide;
N-(2,4-dimethylphenyl)-N-isobutyl-4-((pyrimidin-4-yloxy)methyl)benzenesul-
fonamide;
N-(2,4-dimethylphenyl)-N-isobutyl-4-((4-methyl-1H-imidazol-5-yl)-
methoxy)benzenesulfonamide;
N-(2,4-dimethylphenyl)-N-isobutyl-4-((1-methyl-1H-1,2,4-triazol-3-yl)meth-
oxy)benzenesulfonamide;
N-(2,4-dimethylphenyl)-N-isobutyl-4-((1-methyl-1H-imidazol-2-yl)methoxy)b-
enzenesulfonamide;
4-((1H-imidazol-4-yl)methoxy)-N-(2,4-dimethylphenyl)-N-isobutylbenzenesul-
fonamide;
N-(2,4-dimethylphenyl)-N-isobutyl-3-((pyrimidin-4-ylmethoxy)meth-
yl)benzenesulfonamide;
N-(2,4-dimethylphenyl)-N-isobutyl-4-(pyrimidin-4-ylmethoxy)benzenesulfona-
mide;
N-(2,4-dimethylphenyl)-N-isobutyl-4-((1-methyl-1H-imidazol-5-yl)meth-
oxy)benzenesulfonamide;
N-(2,4-dimethylphenyl)-N-isobutyl-4-((1-methyl-1H-1,2,4-triazol-5-yl)meth-
oxy)benzenesulfonamide;
[(4-{[(2,4-dimethylphenyl)(2-methylpropyl)amino]sulfonyl}-2,3-dimethylphe-
nyl)oxy]acetic acid;
3-[(4-{[(4-butyl-2-methylphenyl)(2-methylpropyl)amino]sulfonyl}-2-methylp-
henyl)oxy]propanoic acid;
4-(4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)butanoic
acid;
(R)-3-((4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)-2-methylphenyl)ami-
no)-4-hydroxybutanoic acid;
(S)-2-((4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)amino)-3-hyd-
roxypropanoic acid;
(S)-3-((4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)amino)-4-hyd-
roxybutanoic acid;
N-(2,4-dimethylphenyl)-N-isobutyl-4-(2H-tetrazol-5-yl)benzenesulfonamide;
(R)-3-((4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)amino)-4-hyd-
roxybutanoic acid;
3-(4-(N-isobutyl-N-(2-methyl-5-(trifluoromethyl)phenyl)sulfamoyl)phenyl)p-
ropanoic acid;
3-(4-(N-isobutyl-N-(2-methyl-5-(trifluoromethyl)phenyl)sulfamoyl)phenyl)--
2,2-dimethylpropanoic acid;
5-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)-2-methoxybenzoic
acid;
2-((4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)amino)-3-hydroxy-
propanoic acid;
2-bromo-5-(N-(4-ethylphenyl)-N-isobutylsulfamoyl)benzoic acid;
2-(4-(N-(4-butyl-2-methylphenyl)-N-isobutylsulfamoyl)-2-methylpheno-
xy)acetic acid;
4-((2-(dideuterioamino)pyridin-4-yl)methoxy)-N-(2,4-dimethylphenyl)-N-iso-
butylbenzenesulfonamide;
N-(2,4-dimethylphenyl)-4-(1-hydroxy-3-(2H-tetrazol-5-yl)propyl)-N-isobuty-
lbenzenesulfonamide;
N-(2,4-dimethylphenyl)-4-(1-hydroxy-2-(2H-tetrazol-5-yl)ethyl)-N-isobutyl-
benzenesulfonamide;
N-(2,4-dimethylphenyl)-4-(2-hydroxy-1-(2H-tetrazol-5-yl)propan-2-yl)-N-is-
obutylbenzenesulfonamide;
2-(4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)acetic
acid; 4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)benzoic acid;
4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)benzoic acid; and
3-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)benzoic acid.
19. A pharmaceutical composition comprising a) a compound according
to claim 1, or a pharmaceutically acceptable salt thereof, and b)
one or more pharmaceutically acceptable excipients.
20. A pharmaceutical composition comprising a) a compound according
to claim 18, or a pharmaceutically acceptable salt thereof, and b)
one or more pharmaceutically acceptable excipients.
21. A method of treatment of an inflammatory, metabolic or
autoimmune disease mediated by ROR.gamma. comprising administering
a safe and therapeutically effective amount of a compound as
defined in claim 1, or a pharmaceutically acceptable salt
thereof.
22. The method of treatment according to claim 21, wherein the
disease is asthma, chronic obstructive pulmonary disease (COPD),
bronchitis, allergic diseases, such as allergic rhinitis and atopic
dermatitis, cystic fibrosis, lung allograph rejection, multiple
sclerosis, rheumatoid arthritis, juvenile Rheumatoid arthritis,
Osteoarthritis, ankylosing spondylitis, systemic lupus
erythematosus, psoriasis, Hashimoto's disease, pancreatisis,
autoimmune diabetes, autoimmune ocular disease, ulcerative colitis,
Crohn's disease, inflammatory bowel disease (IBS), inflammatory
bowel syndrome (IBD), Sjorgen's syndrome, optic neuritis, type I
diabetes, neuromyelitis optica, Myastehnia Gravis, uveitis,
Guillain-Barre syndrome, psoriatic arthritis, Graves' disease or
scleritis.
23. The method of treatment according to claim 21, wherein the
disease is selected from the group consisting of multiple
sclerosis, ankylosing spondylitis, and psoriasis.
24. The method of treatment according to claim 21, wherein the
disease is psoriasis.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to novel retinoid-related
orphan receptor gamma (ROR.gamma.) modulators, processes for their
preparation, pharmaceutical compositions containing these
modulators, and their use in the treatment of inflammatory,
metabolic and autoimmune diseases mediated by ROR.gamma..
BACKGROUND OF THE INVENTION
[0002] Retinoid-related orphan receptors (RORs) are transcription
factors that form a subgroup of the nuclear receptor superfamily
(Adv. Dev. Biol. 2006, 16, 313-355). This subgroup consists of
three members: ROR alpha (ROR.alpha.), ROR beta (ROR.beta.) and ROR
gamma (ROR.gamma.). ROR.alpha. and ROR.beta. have approximately 55%
homology in the ligand binding domains to ROR.gamma.. RORs contain
four principal domains shared by the majority of nuclear receptors:
an N-terminal A/B domain, a DNA-binding domain, a hinge domain and
a ligand binding domain.
[0003] The ROR.alpha., ROR.beta. and ROR.gamma. genes have been
mapped to human chromosomes 15q22.2, 9q21.13 and 1q21.3,
respectively. Each ROR gene generates several isoforms, which
differ only in their N-terminal A/B domain. To date, five splice
variants have been recorded for ROR.gamma. and two isoforms of this
member of the ROR family have been identified: ROR.gamma.1 and
ROR.gamma.2 (also known as ROR.gamma.t). ROR.gamma. is a term used
to describe ROR.gamma.1 and/or ROR.gamma.t.
[0004] While ROR.gamma.1 is expressed in a variety of tissues
including thymus, muscle, kidney and liver, ROR.gamma.t is
exclusively expressed in the cells of the immune system and has a
critical role in thymopoiesis, development of several secondary
lymphoid tissues and Th17 lineage specification.
[0005] ROR.gamma.t has been identified as a key regulator of Th17
cell differentiation (A. Jetten, Nuclear Receptor Signalling 2009,
7, 1-32). Th17 cells are a recently discovered subset of T helper
cells which preferentially produce cytokines IL-17A, IL-17F, IL-21
and IL-22. ROR.gamma.t also induces transcription of the gene
encoding IL-17A and IL-17F in naive CD4.sup.+ T helper cells, iNKT
and NKT (Mucosal Immunol. 2009, 2(5), 383-392; J. Immunol. 2008,
180, 5167-5171), .gamma..delta.T cells (Am. J. Respir. Crit. Care
Med. 2010, 182, 464-476), CD8.sup.+ T cells (J. Leukocyte Biol.
2007, 82, 354-360) and finally CD4.sup.-CD8TCR.alpha..beta..sup.+ T
cells (J. Immunol. 2008, 181, 8761-8766). Additional immune cells
such as eosinophils, neutrophils and macrophages can also be a
source of IL-17A in allergic inflammation related to asthma (J.
Allergy Clin. Immunol. 2001, 108, 430-438; J. Immunol. 2008, 181,
6117-6124; Immunity 2004, 21, 467-476).
[0006] Th17 cells and their products have been shown to be
associated with the pathology of a number of human inflammatory and
autoimmune disorders. IL-17A and IL-17F are implicated in numerous
immune and inflammatory responses primarily as pro-inflammatory
regulators inducing the expression of cytokines, chemokines,
adhesion molecules, mucin genes and growth factors. There is
emerging evidence that an increase in IL-17A level is closely
associated with a range of chronic inflammatory diseases such as
rheumatoid arthritis (Curr. Opin. Investig. Drugs 2009, 10,
452-462), multiple sclerosis (Allergol. Int. 2008, 57(2), 115-120),
inflammatory bowel diseases (J. Inflamm. Res. 2010, 3, 33-44),
uveitis, psoriasis (Sci. Transl. Med. 2010, 2(52)) and lung
diseases (Prog. Respir. Res. Base/2010, 39, 141-149; Resp. Research
2010, 11 (78), 1-11).
[0007] There is considerable evidence suggesting that Th17
cells/IL-17 play a key role in the pathogenesis of asthma. In
asthmatic patients, both ROR.gamma.t and IL-17A expression levels
have been shown to be increased in sputum (Chin. Med. J. 2005, 118,
953-956; Resp. Res. 2006, 7(135), 1-9), lung (J. Allergy Clin.
Immunol. 2003, 111(6), 1293-1298), bronchoalveolar lavage (BAL)
fluids and peripheral blood (Immunol. Invest. 2009, 38, 652-664;
Int. Arch. Allergy Immunol. 2005, 137(suppl. 1), 51-54) and levels
directly correlate with disease severity (Int. Arch. Allergy
Immunol. 2010, 151, 297-307). In addition to IL-17A, a recent study
has shown that a further cytokine of the IL-17 family, IL-17F, may
have a crucial role in allergic airway inflammation and hence have
key implications in airway diseases, such as asthma. The
overexpression of the IL-17F gene in mice airways was associated
with airway neutrophilia, cytokine induction, an increase in airway
hyperreactivity and mucus hypersecretion (Inflamm. Allergy Drug
Targets 2009, 8, 383-389). Evidence of role of Th17 cells in
allergens has been discussed in Int. Immunopharmacol. 2010, 10,
226-229.
[0008] The pathogenesis of chronic autoimmune diseases including
multiple sclerosis and rheumatoid arthritis arises from the break
in tolerance towards self-antigens and the development of
auto-aggressive effector T cells infiltrating the target tissues.
Studies have shown that Th17 cells are one of the important drivers
of the inflammatory process in tissue-specific autoimmunity (J.
Exp. Med. 2008, 205, 1517-1522; Cell. Mol. Immunol. 2010, 7,
182-189). There is also evidence that Th17 cells are activated
during the disease process and are responsible for recruiting other
inflammatory cells types, especially neutrophils, to mediate
pathology in the target tissues (Annu. Rev. Immunol. 2009, 27,
485-517). ROR.gamma.t plays a critical role in the pathogenic
responses of Th17 cells (Cell 2006, 126, 1121-1133). ROR.gamma.t
deficient mice show very few Th17 cells. Further support for the
role of ROR.gamma.t in the pathogensis of autoimmune or
inflammatory diseases can be found in the following references:
Immunity 2007, 26, 643-654; Nat. Rev. Immunol. 2006, 6, 205-217; J.
Immunol. 2009, 183, 7169-7177; Brain Pathol. 2004, 14, 164-174;
Brain 2007, 130, 1089-1104; Nat. Rev. Immunol. 2008, 8,
183-192.
[0009] In light of the role ROR.gamma. plays in the pathogenesis of
diseases, it is desirable to prepare compounds that modulate
ROR.gamma. activity and hence have utility in the treatment of
inflammatory, metabolic and autoimmune diseases mediated by
ROR.gamma., such as the respiratory diseases asthma, chronic
obstructive pulmonary disease (COPD) and bronchitis, allergic
diseases including allergic rhinitis and atopic dermatitis, cystic
fibrosis and lung allograph rejection.
SUMMARY OF THE INVENTION
[0010] According to the invention, there is provided novel
retinoid-related orphan receptor gamma (ROR.gamma.) modulators,
processes for their preparation, pharmaceutical compositions
comprising these modulators, and their use in the treatment of
inflammatory, metabolic and autoimmune diseases mediated by
ROR.gamma.. More specifically, the present invention is directed to
compounds of formula (I), and to pharmaceutically acceptable salts
thereof:
##STR00001##
[0011] wherein
[0012] R.sub.1 and R.sub.2 are independently selected from the
group consisting of H, CF.sub.3, halo, C.sub.1-3alkyl and
C.sub.1-3-alkoxy;
[0013] R.sub.3 and R.sub.4 are independently selected from the
group consisting of H, CF.sub.3, halo, CH.sub.3 and OCH.sub.3;
[0014] R.sub.5 is selected from the group consisting of H,
CF.sub.3, halo, OH, CH.sub.2OH, C.sub.1-4alkyl,
CH.sub.2-cyclopropyl and C.sub.1-4alkoxy, wherein C.sub.1-4alkyl
and C.sub.1-4alkoxy, may be optionally substituted with one or two
OH substituents;
[0015] R.sub.6 is selected from the group consisting of
C.sub.3-5alkyl and --CH.sub.2--C.sub.3-4cycloalkyl;
[0016] R.sub.7 is selected from the group consisting of
##STR00002##
[0017] R.sub.8 is selected from the group consisting of
C.sub.1-3alkyl, C.sub.1-3alkoxy, CH.sub.2CN, CH.sub.2OH, OH, CN and
halo;
[0018] R.sub.9 is the group
--(CHR.sub.10).sub.s--(X).sub.t--(CHR.sub.10).sub.u--R.sub.11;
[0019] each R.sub.10 is independently selected from the group
consisting of H, OH or CH.sub.2OH;
[0020] R.sub.11 is an isoxazole, --C(O)OH, or a 5- or 6-membered
heteroaryl group, wherein the isoxazole or the 5- or 6-membered
heteroaryl group may be optionally substituted with one or two
halo, NH.sub.2 or CH.sub.3 substituents;
[0021] X is CH.sub.2, NH, O;
[0022] r is 0, 1 or 2;
[0023] s is 0, 1 or 2;
[0024] t is 0 or 1;
[0025] u is 0, 1 or 2;
[0026] with the proviso that no more than two R.sub.10 groups
represent OH or CH.sub.2OH.
[0027] In one aspect, the present invention provides a
pharmaceutical composition comprising a) a compound of formula (I),
or a pharmaceutically acceptable salt thereof, and b) one or more
pharmaceutically acceptable excipients.
[0028] In a further aspect, the present invention provides a
compound of formula (I), or a pharmaceutically acceptable salt
thereof, for use in therapy.
[0029] Compounds of formula (I), and pharmaceutically acceptable
salts thereof, are modulators of ROR.gamma. and can be useful in
the treatment of inflammatory, metabolic and autoimmune diseases
mediated by ROR.gamma., such as asthma, chronic obstructive
pulmonary disease (COPD) and bronchitis, allergic diseases
including allergic rhinitis and atopic dermatitis, cystic fibrosis,
lung allograph rejection, multiple sclerosis, rheumatoid arthritis,
juvenile rheumatoid arthritis, Osteoarthritis, ankylosing
spondylitis, systemic lupus erythematosus, psoriasis, Hashimoto's
disease, pancreatisis, autoimmune diabetes, autoimmune ocular
disease, ulcerative colitis, Crohn's disease, inflammatory bowel
disease (IBS), inflammatory bowel syndrome (IBD), Sjorgen's
syndrome, optic neuritis, type I diabetes, neuromyelitis optica,
Myastehnia Gravis, uveitis, Guillain-Barre syndrome, psoriatic
arthritis, Graves' disease, scleritis.
[0030] In a further aspect, the present invention provides a
compound of formula (I), or a pharmaceutically acceptable salt
thereof, for use in the treatment of asthma or chronic obstructive
pulmonary disease.
[0031] In a further aspect, the present invention is directed to a
method of treatment of an inflammatory, metabolic or autoimmune
disease mediated by ROR.gamma., which comprises administering to a
subject in need thereof, a safe and therapeutically effective
amount of a compound of formula (I), or a pharmaceutically
acceptable salt thereof.
[0032] In yet a further aspect, the present invention is directed
to a method of treating chronic obstructive pulmonary disease or
asthma, which comprises administering to a subject in need thereof,
a safe and therapeutically effective amount of a compound of
formula (I), or a pharmaceutically acceptable salt thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0033] In a first aspect, the present invention is directed to a
compound of formula (I), or a pharmaceutically acceptable salt
thereof:
##STR00003##
[0034] wherein
[0035] R.sub.1 and R.sub.2 are independently selected from the
group consisting of H, CF.sub.3, halo, C.sub.1-3alkyl and
C.sub.1-3-alkoxy;
[0036] R.sub.3 and R.sub.4 are independently selected from the
group consisting of H, CF.sub.3, halo, CH.sub.3 and OCH.sub.3;
[0037] R.sub.5 is selected from the group consisting of H,
CF.sub.3, halo, OH, CH.sub.2OH, C.sub.1-4alkyl,
CH.sub.2-cyclopropyl and C.sub.1-4alkoxy, wherein C.sub.1-4alkyl
and C.sub.1-4alkoxy, may be optionally substituted with one or two
OH substituents;
[0038] R.sub.6 is selected from the group consisting of
C.sub.3-5alkyl and --CH.sub.2--C.sub.3-4cycloalkyl;
[0039] R.sub.7 is selected from the group consisting of
##STR00004##
[0040] R.sub.8 is selected from the group consisting of
C.sub.1-3alkyl, C.sub.1-3alkoxy, CH.sub.2CN, CH.sub.2OH, OH, CN and
halo;
[0041] R.sub.9 is the group
--(CHR.sub.10).sub.s--(X).sub.t--(CHR.sub.10).sub.u--R.sub.11;
[0042] each R.sub.10 is independently selected from the group
consisting of H, OH or CH.sub.2OH;
[0043] R.sub.11 is an isoxazole, --C(O)OH, or a 5- or 6-membered
heteroaryl group, wherein the isoxazole or the 5- or 6-membered
heteroaryl group may be optionally substituted with one or two
halo, NH.sub.2 or CH.sub.3 substituents;
[0044] X is CH.sub.2, NH, O;
[0045] r is 0, 1 or 2;
[0046] s is 0, 1 or 2;
[0047] t is 0 or 1;
[0048] u is 0, 1 or 2;
[0049] with the proviso that no more than two R.sub.10 groups
represent OH or CH.sub.2OH.
[0050] In a further aspect, the present invention provides subsets
of the compounds of formula (I), of formula (Ia-Ig), or a
pharmaceutically acceptable salt thereof:
##STR00005## ##STR00006##
[0051] In a further aspect, the present invention provides subsets
of the compounds of formula (I), of formula (Ia-Ic), or a
pharmaceutically acceptable salt thereof:
##STR00007##
[0052] In a further aspect, the present invention provides a subset
of compounds of formula (I), of formula (Ia), or a pharmaceutically
acceptable salt thereof:
##STR00008##
[0053] In a further aspect, the present invention provides a subset
of compounds of formula (I), of formula (Ia) above, or a
pharmaceutically acceptable salt thereof, wherein R.sub.1 and
R.sub.5 are each independently CH.sub.3 or halo.
[0054] In a further aspect, the present invention provides a subset
of compounds of formula (I), of formula (Ia) above, or a
pharmaceutically acceptable salt thereof, wherein R.sub.1 and
R.sub.5 are CH.sub.3.
[0055] In yet a further aspect of the present invention, R.sub.6 is
selected from the group consisting of propyl, isobutyl and
--CH.sub.2cyclopropyl.
[0056] In yet a further aspect of the present invention, R.sub.6 is
isobutyl.
[0057] In a further aspect of the present invention, R.sub.7
is:
##STR00009##
[0058] In a further aspect of the present invention, r is 0.
[0059] In a further aspect of the present invention, r is 1 and
R.sub.8 is C.sub.1-3alkyl or C.sub.1-3alkoxy.
[0060] In a further aspect of the present invention, r is 1 and
R.sub.8 is CH.sub.3 or --OCH.sub.3.
[0061] In a further aspect of the present invention, r is 1 and
R.sub.8 is CH.sub.2OH.
[0062] In a further aspect of the present invention, s is 0.
[0063] In a further aspect of the present invention, s is 1.
[0064] In a further aspect of the present invention, u is 1.
[0065] In a further aspect of the present invention, u is 0.
[0066] In a further aspect of the present invention, t is 1 and X
is O.
[0067] In a further aspect of the present invention, each R.sub.10
is H.
[0068] In a further aspect of the present invention, R.sub.11 is
3,5-dimethylisoxazole, of formula:
##STR00010##
[0069] In a further aspect of the present invention, R.sub.11 is
selected from the group consisting of:
##STR00011##
[0070] In a further aspect of the present invention, R.sub.11 is
selected from the group consisting of:
##STR00012##
[0071] In a further aspect of the present invention, the R.sub.11
group is unsubstituted.
[0072] It is understood that the present invention covers all
combinations of substituent groups referred to herein above.
[0073] Specific examples of compounds of formula (I) are: [0074]
N-(4-butyl-2-methylphenyl)-N-isobutyl-4-(pyridin-4-yloxy)benzenesulfonami-
de; [0075]
N-(4-butyl-2-methylphenyl)-4-{[(3,5-dimethyl-4-isoxazolyl)methy-
l]oxy}-N-(2-methylpropyl)benzenesulfonamide; [0076]
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(2,4-dimethylphenyl)-N-isobutylb-
enzenesulfonamide; [0077]
4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(4-fluoro-2-methylphenyl)-N--
(2-methylpropyl)benzenesulfonamide; [0078]
N-(2,4-difluorophenyl)-4-{[(3,5-di
methyl-4-isoxazolyl)methyl]oxy}-N-(2-methylpropyl)benzenesulfonamide;
[0079]
N-(4-butylphenyl)-4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(2--
methylpropyl)benzenesulfonamide; [0080]
4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-[4-(1-methylethyl)phenyl]-N--
(2-methylpropyl)benzenesulfonamide; [0081]
4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(2-fluoro-4-methylphenyl)-N--
(2-methylpropyl)benzenesulfonamide; [0082]
N-(4-chlorophenyl)-4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-isobutylbenze-
nesulfonamide; [0083] 4-((3,5-dimethyl
isoxazol-4-yl)methoxy)-N-isobutyl-N-(4-propylphenyl)benzenesulfonamide;
[0084]
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-isobutyl-N-(3-(trifluorom-
ethyl)phenyl)benzenesulfonamide; [0085]
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-isobutyl-N-(2-methyl-5-(trifluor-
omethyl)phenyl)benzenesulfonamide; [0086]
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-isobutyl-N-phenyl
benzenesulfonamide; [0087]
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(2-ethyl
phenyl)-N-propylbenzenesulfonamide; [0088]
4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(3-methyl
phenyl)-N-(2-methylpropyl)benzenesulfonamide; [0089]
4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(2,5-dimethyl
phenyl)-N-(2-methylpropyl)benzenesulfonamide; [0090]
4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(3-ethyl
phenyl)-N-(2-methylpropyl)benzenesulfonamide; [0091]
N-(5-chloro-2-fluorophenyl)-4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N--
(2-methylpropyl)benzenesulfonamide; [0092]
N-(2-chloro-5-fluorophenyl)-4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N--
(2-methylpropyl)benzenesulfonamide; [0093]
4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(4-methyl
phenyl)-N-(2-methylpropyl)benzenesulfonamide; [0094]
N-(cyclopropylmethyl)-4-{[(3, 5-di
methyl-4-isoxazolyl)methyl]oxy}-N-(2,4-dimethylphenyl)benzenesulfonamide;
[0095]
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(4-hydroxy-2-methylphenyl-
)-N-isobutylbenzenesulfonamide; [0096]
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-phenyl-N-propyl
benzenesulfonamide; [0097]
N-(4-butyl-2-methylphenyl)-N-isobutyl-4-(pyridin-3-yloxy)benzenesulfonami-
de; [0098]
4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-[3-(methyloxy)phen-
yl]-N-(2-methylpropyl)benzenesulfonamide; [0099]
N-(2,4-dimethylphenyl)-4-hydroxy-N-isobutylbenzenesulfonamide;
[0100]
N-(2,4-dimethylphenyl)-N-(2-methylpropyl)-4-[(4-pyridinylmethyl)oxy]benze-
nesulfonamide; [0101]
N-(5-chloro-2-fluorophenyl)-N-isobutyl-4-(pyridin-4-ylmethoxy)benzenesulf-
onamide; [0102]
N-(3-fluoro-5-methylphenyl)-N-isobutyl-4-(pyridin-4-ylmethoxy)benzenesulf-
onamide; [0103]
N-(3,5-dimethylphenyl)-N-isobutyl-4-(pyridin-4-ylmethoxy)benzenesulfonami-
de; [0104]
N-isobutyl-N-(5-methoxy-2-methylphenyl)-4-(pyridin-4-ylmethoxy)-
benzenesulfonamide; [0105]
N-(2,5-difluorophenyl)-N-isobutyl-4-(pyridin-4-ylmethoxy)benzenesulfonami-
de; [0106]
N-(5-fluoro-2-methylphenyl)-N-isobutyl-4-(pyridin-4-ylmethoxy)b-
enzenesulfonamide; [0107]
N-(2,5-dimethylphenyl)-N-isobutyl-4-(pyridin-4-ylmethoxy)benzenesulfonami-
de; [0108]
N-(3-ethylphenyl)-N-isobutyl-4-(pyridin-4-ylmethoxy)benzenesulf-
onamide; [0109]
N-isobutyl-N-(3-methoxyphenyl)-4-(pyridin-4-ylmethoxy)benzenesulfonamide;
[0110]
N-isobutyl-4-(pyridin-4-ylmethoxy)-N-(m-tolyl)benzenesulfonamide;
[0111] N-[4-(cyclopropylmethyl)-2,6-di methyl
phenyl]-4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(2-methylpropyl)benz-
enesulfonamide; [0112]
N-(3,5-difluorophenyl)-N-(2-methylpropyl)-4-[(phenylmethyl)oxy]benzenesul-
fonamide; [0113] N-(2,5-difluorophenyl)-4-{[(3,5-di
methyl-4-isoxazolyl)methyl]oxy}-N-(2-methylpropyl)benzenesulfonamide;
[0114]
N-(3,5-dichlorophenyl)-4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}b-
enzenesulfonamide; [0115]
N-(2,5-dichlorophenyl)-4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(2-me-
thylpropyl)benzenesulfonamide; [0116]
N-(3,4-dichlorophenyl)-4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(2-me-
thylpropyl)benzenesulfonamide; [0117]
N-(3,5-difluorophenyl)-4-{[(3,5-di
methyl-4-isoxazolyl)methyl]oxy}-N-(2-methylpropyl)benzenesulfonamide;
[0118] 4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(3,4-dimethyl
phenyl)-N-(2-methylpropyl)benzenesulfonamide; [0119]
4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(3-fluoro-5-methylphenyl)-N--
(2-methylpropyl)benzenesulfonamide; [0120]
4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(3,5-dimethyl
phenyl)-N-(2-methylpropyl)benzenesulfonamide; [0121]
4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-[2-methyl-5-(methyloxy)pheny-
l]-N-(2-methylpropyl)benzenesulfonamide; [0122]
4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(5-fluoro-2-methylphenyl)-N--
(2-methylpropyl)benzenesulfonamide; [0123]
N-(3-chlorophenyl)-4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(2-methyl-
propyl)benzenesulfonamide; [0124]
4-((1H-imidazol-2-yl)methoxy)-N-(2,4-dimethylphenyl)-N-isobutylbenzenesul-
fonamide; [0125]
N-(2,4-dimethylphenyl)-N-isobutyl-4-(pyridin-4-ylmethoxy)benzenesulfonami-
de; [0126]
4-((1,3-dimethyl-1H-pyrazol-5-yl)methoxy)-N-(2,4-dimethyl
phenyl)-N-isobutylbenzenesulfonamide; [0127]
N-(2,4-dimethylphenyl)-N-isobutyl-4-((5-methylisoxazol-3-yl)methoxy)benze-
nesulfonamide; [0128]
N-(2,4-dimethylphenyl)-N-isobutyl-4-((pyridin-4-yloxy)methyl)benzenesulfo-
namide; [0129]
N-(2,4-dimethylphenyl)-N-isobutyl-4-((pyrimidin-4-yloxy)methyl)benzenesul-
fonamide; [0130] N-(2,4-dimethyl
phenyl)-N-isobutyl-4-((pyridin-4-ylmethoxy)methyl)benzenesulfonamide;
[0131]
3-(((3,5-dimethylisoxazol-4-yl)methoxy)methyl)-N-(2,4-dimethylphen-
yl)-N-isobutylbenzenesulfonamide; [0132] N-(2,4-dimethyl
phenyl)-N-isobutyl-3-((pyridin-4-ylmethoxy)methyl)benzenesulfonamide;
[0133]
N-(2,4-dimethylphenyl)-N-isobutyl-4-((4-methyl-1H-imidazol-5-yl)me-
thoxy)benzenesulfonamide; [0134]
N-(2,4-dimethylphenyl)-N-isobutyl-4-((1-methyl-1H-1,2,4-triazol-3-yl)meth-
oxy)benzenesulfonamide; [0135]
N-(2,4-dimethylphenyl)-N-isobutyl-4-((1-methyl-1H-imidazol-2-yl)methoxy)b-
enzenesulfonamide; [0136]
4-((1H-imidazol-4-yl)methoxy)-N-(2,4-dimethylphenyl)-N-isobutylbenzenesul-
fonamide; [0137]
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-isobutyl-N-(5-isopropyl-2-methyl-
phenyl)benzenesulfonamide; [0138]
3-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(2,4-di methyl
phenyl)-N-isobutylbenzenesulfonamide; [0139] 4-((3,5-dimethyl
isoxazol-4-yl)methoxy)-N-isobutyl-N-(o-tolyl)benzenesulfonamide;
[0140]
N-(2,4-dimethylphenyl)-N-isobutyl-3-(pyridin-4-ylmethoxy)benzenesulfonami-
de; [0141]
N-(2,4-dimethylphenyl)-N-isobutyl-3-((pyrimidin-4-ylmethoxy)met-
hyl)benzenesulfonamide; [0142]
N-(2,4-dimethylphenyl)-N-isobutyl-4-(pyridin-4-ylmethoxy)benzenesulfonami-
de; [0143] N-(2,4-dimethyl
phenyl)-N-isobutyl-4-(2-(pyridin-4-yl)ethoxy)benzenesulfonamide;
[0144]
4-(2-(3,5-dimethylisoxazol-4-yl)ethoxy)-N-(2,4-dimethylphenyl)-N-isobutyl-
benzenesulfonamide; [0145]
N-(2,4-dimethylphenyl)-N-isobutyl-4-(pyrimidin-4-ylmethoxy)benzenesulfona-
mide; [0146]
N-(4-bromo-2-methylphenyl)-4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-isobu-
tylbenzenesulfonamide; [0147]
N-(2,4-dimethylphenyl)-4-((2-fluoropyridin-4-yl)methoxy)-N-isobutylbenzen-
esulfonamide; [0148]
N-(2,4-dimethylphenyl)-N-isobutyl-4-((1-methyl-1H-imidazol-5-yl)methoxy)b-
enzenesulfonamide; [0149]
N-(2,4-dimethylphenyl)-N-isobutyl-4-((1-methyl-1H-1,2,4-triazol-5-yl)meth-
oxy)benzenesulfonamide; [0150]
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(2,4-dimethylphenyl)-N-isobutyl--
3-methoxybenzenesulfonamide; [0151]
N-(2,4-dimethylphenyl)-4-(2-hydroxy-2-(pyridin-4-yl)ethoxy)-N-isobutylben-
zenesulfonamide; [0152]
4-((2-aminopyridin-4-yl)methoxy)-N-(2,4-dimethyl phenyl)-N-isobutyl
benzenesulfonamide; [0153]
[(4-{[(2,4-dimethylphenyl)(2-methylpropyl)amino]sulfonyl}-2,3-dimethyl
phenyl)oxy]acetic acid; [0154]
N-(2,4-dimethylphenyl)-4-(1-hydroxy-2-((pyridin-3-ylmethyl)amino)ethyl)-N-
-isobutylbenzenesulfonamide; [0155]
3-[(4-{[(4-butyl-2-methylphenyl)(2-methylpropyl)amino]sulfonyl}-2-methy
phenyl)oxy]propanoic acid; [0156]
N-(2,4-dimethylphenyl)-4-(1-hydroxy-2-((pyridin-4-ylmethyl)amino)ethyl)-N-
-isobutylbenzenesulfonamide; [0157]
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(4-(2-hydroxypropan-2-yl)-2-meth-
ylphenyl)-N-isobutylbenzenesulfonamide; [0158]
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(4-(2-hydroxyethoxy)-2-methylphe-
nyl)-N-isobutylbenzenesulfonamide; [0159]
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(4-(hydroxymethyl)-2-methyl
phenyl)-N-isobutylbenzenesulfonamide; [0160]
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(4-(3-hydroxypropyl)phenyl)-N-is-
obutylbenzenesulfonamide; [0161]
4-(4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)butanoic
acid; [0162] 4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(4-ethyl
phenyl)-N-isobutylbenzenesulfonamide; [0163]
(R)-3-((4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)-2-methylphenyl)ami-
no)-4-hydroxybutanoic acid; [0164]
(S)-2-((4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)amino)-3-hyd-
roxypropanoic acid; [0165]
(S)-3-((4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)amino)-4-hyd-
roxybutanoic acid; [0166]
N-(2,4-dimethylphenyl)-N-isobutyl-4-(2H-tetrazol-5-yl)benzenesulfonamide;
[0167]
(R)-3-((4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)amino-
)-4-hydroxybutanoic acid; [0168]
3-(4-(N-isobutyl-N-(2-methyl-5-(trifluoromethyl)phenyl)sulfamoyl)phenyl)p-
ropanoic acid; [0169]
3-(4-(N-isobutyl-N-(2-methyl-5-(trifluoromethyl)phenyl)sulfamoyl)phenyl)--
2,2-dimethylpropanoic acid; [0170]
5-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)-2-methoxybenzoic
acid; [0171]
2-((4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)amino)-3--
hydroxypropanoic acid; [0172]
N-(2,6-difluorophenyl)-4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(2-me-
thylpropyl)benzenesulfonamide; [0173]
2-bromo-5-(N-(4-ethylphenyl)-N-isobutylsulfamoyl)benzoic acid;
[0174]
2-(4-(N-(4-butyl-2-methylphenyl)-N-isobutylsulfamoyl)-2-methylphenoxy)ace-
tic acid; [0175]
4-((2-(dideuterioamino)pyridin-4-yl)methoxy)-N-(2,4-dimethylphenyl)-N-iso-
butylbenzenesulfonamide; [0176]
N-(2,4-dimethylphenyl)-4-(1-hydroxy-3-(2H-tetrazol-5-yl)propyl)-N-isobuty-
lbenzenesulfonamide; [0177]
N-(2,4-dimethylphenyl)-4-(1-hydroxy-2-(2H-tetrazol-5-yl)ethyl)-N-isobutyl-
benzenesulfonamide; [0178]
N-(2,4-dimethylphenyl)-4-(2-hydroxy-1-(2H-tetrazol-5-yl)propan-2-yl)-N-is-
obutylbenzenesulfonamide; [0179]
N-(4-(1,3-dihydroxypropan-2-yl)phenyl)-4-(((3,5-dimethylisoxazol-4-yl)oxy-
)methyl)-N-isobutylbenzenesulfonamide; [0180]
2-(4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)acetic
acid; [0181] 4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)benzoic
acid; [0182] 4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)benzoic
acid; and [0183]
3-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)benzoic acid.
[0184] As used herein, the term "alkyl" refers to a saturated
hydrocarbon chain having the specified number of member atoms. For
example, C.sub.1-4 alkyl refers to an alkyl group having from 1 to
4 member atoms. Unless otherwise stated, alkyl groups are
unsubstituted. Alkyl groups may be straight chain or branched. The
term "alkyl" includes, but is not limited to, methyl, ethyl, propyl
(n-propyl and isopropyl), butyl (n-butyl, sec-butyl, isobutyl and
tert-butyl).
[0185] As used herein, the term "alkoxy" refers to an --O-alkyl
group wherein "alkyl" is defined above.
[0186] As used herein, the term "halo" refers to the halogen
radicals fluoro, chloro, bromo and iodo.
[0187] As used herein, the phrase "5- or 6-membered heteroaryl"
indicates an aromatic ring containing 5 or 6 member atoms, wherein
the member atoms are either carbon or nitrogen. The 5- or
6-membered heteroaryl group may contain one, two, three or four
nitrogen atoms as member atoms of the ring.
[0188] As used herein, the phrase "optionally substituted"
indicates that a group may be unsubstituted or substituted with one
or two substituents as defined herein. "Substituted" in reference
to a group indicates that a hydrogen atom attached to a member atom
with a group is replaced.
[0189] As used herein, the term "ROR.gamma." refers to all isoforms
of this member of the ROR family, including ROR.gamma.1 and
ROR.gamma.t.
[0190] As used herein, the term "ROR.gamma. modulator" refers to a
chemical compound of formula (I) that inhibits, either directly or
indirectly, the activity of ROR.gamma.. ROR.gamma. modulators
include antagonists and inverse agonists of ROR.gamma..
[0191] The compounds of formula (I) and pharmaceutically acceptable
salts thereof may contain one or more asymmetric center (also
referred to as a chiral center) and may, therefore, exist as
individual enantiomers, diastereomers, or other stereoisomeric
forms, or as mixtures thereof. Chiral centers, such as chiral
carbon atoms, may also be present in a substituent such as an alkyl
group. Where the stereochemistry of a chiral center present in a
compound of formula (I), or in any chemical structure illustrated
herein, is not specified the structure is intended to encompass all
individual stereoisomers and all mixtures thereof. Thus, compounds
of formula (I) and pharmaceutically acceptable salts thereof
containing one or more chiral center may be used as racemic
mixtures, enantiomerically enriched mixtures, or as
enantiomerically pure individual stereoisomers.
[0192] Individual stereoisomers of a compound of formula (I), or a
pharmaceutically acceptable salt thereof, which contain one or more
asymmetric center may be resolved by methods known to those skilled
in the art. For example, such resolution may be carried out (1) by
formation of diastereoisomeric salts, complexes or other
derivatives; (2) by selective reaction with a stereoisomer-specific
reagent, for example by enzymatic oxidation or reduction; or (3) by
gas-liquid or liquid chromatography in a chiral environment, for
example, on a chiral support such as silica with a bound chiral
ligand or in the presence of a chiral solvent. The skilled artisan
will appreciate that where the desired stereoisomer is converted
into another chemical entity by one of the separation procedures
described above, a further step is required to liberate the desired
form. Alternatively, specific stereoisomers may be synthesized by
asymmetric synthesis using optically active reagents, substrates,
catalysts or solvents, or by converting one enantiomer to the other
by asymmetric transformation.
[0193] In certain aspects, compounds of formula (I) may contain an
acidic functional group. In certain other embodiments, compounds of
formula (I) may contain a basic functional group. Thus, the skilled
artisan will appreciate that pharmaceutically-acceptable salts of
the compounds of formula (I) may be prepared. Indeed, in certain
embodiments of the invention, pharmaceutically-acceptable salts of
the compounds of formula (I) may be preferred over the respective
free base or free acid because such salts may impart greater
stability or solubility to the molecule thereby facilitating
formulation into a dosage form.
[0194] In certain embodiments, compounds according to formula (I)
may contain a basic functional group and are therefore capable of
forming pharmaceutically acceptable acid addition salts by
treatment with a suitable acid. Suitable acids include
pharmaceutically acceptable inorganic acids and pharmaceutically
acceptable organic acids. Representative pharmaceutically
acceptable acid addition salts include hydrochloride, hydrobromide,
nitrate, methylnitrate, sulfate, bisulfate, sulfamate, phosphate,
acetate, hydroxyacetate, phenylacetate, propionate, butyrate,
isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate,
malate, tartrate, citrate, salicylate, p-aminosalicyclate,
glycollate, lactate, heptanoate, phthalate, oxalate, succinate,
benzoate, o-acetoxybenzoate, chlorobenzoate, methylbenzoate,
dinitrobenzoate, hydroxybenzoate, methoxybenzoate, naphthoate,
hydroxynaphthoate, mandelate, tannate, formate, stearate,
ascorbate, palmitate, oleate, pyruvate, pamoate, malonate, laurate,
glutarate, glutamate, estolate, methanesulfonate (mesylate),
ethanesulfonate (esylate), 2-hydroxyethanesulfonate,
benzenesulfonate (besylate), p-aminobenzenesulfonate,
p-toluenesulfonate (tosylate), and napthalene-2-sulfonate.
[0195] In certain embodiments, compounds according to formula (I)
may contain an acidic functional group. Suitable
pharmaceutically-acceptable salts include salts of such acidic
functional groups. Representative salts include pharmaceutically
acceptable metal salts such as sodium, potassium, lithium, calcium,
magnesium, aluminum, and zinc salts; carbonates and bicarbonates of
a pharmaceutically acceptable metal cation such as sodium,
potassium, lithium, calcium, magnesium, aluminum, and zinc;
pharmaceutically acceptable organic primary, secondary, and
tertiary amines including aliphatic amines, aromatic amines,
aliphatic diamines, and hydroxy alkylamines such as methylamine,
ethylamine, 2-hydroxyethylamine, diethylamine, TEA,
ethylenediamine, ethanolamine, diethanolamine, and
cyclohexylamine.
[0196] For reviews on suitable pharmaceutical salts see Berge et
al, J. Pharm, Sci., 66, 1-19, 1977; P L Gould, International
Journal of Pharmaceutics, 33 (1986), 201-217; and Bighley et al,
Encyclopaedia of Pharmaceutical Technology, Marcel Dekker Inc, New
York 1996, Volume 13, page 453-497. Other salts that are not deemed
pharmaceutically acceptable may be useful in the preparation of
compounds of formula (I) and are included within the scope of the
invention, such as ammonia and trifluoroacetic acid. The present
invention encompasses all possible stoichiometric and
non-stoichiometric forms of the salts of the compounds of formula
(I).
[0197] As used herein, the term "pharmaceutically-acceptable salts"
refers to salts that retain the desired biological activity of the
subject compound and exhibit minimal undesired toxicological
effects. These pharmaceutically-acceptable salts may be prepared in
situ during the final isolation and purification of the compound,
or by separately reacting the purified compound in its free acid or
free base form with a suitable base or acid, respectively.
[0198] The invention also includes all suitable isotopic variations
of a compound of formula (I) or a pharmaceutically acceptable salt
thereof. An isotopic variation of a compound of formula (I), or a
pharmaceutically acceptable salt thereof, is defined as one in
which at least one atom is replaced by an atom having the same
atomic number but an atomic mass different from the atomic mass
usually found in nature. Examples of isotopes that can be
incorporated into compounds of the invention include isotopes of
hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine such as
.sup.2H, .sup.3H, .sup.13C, .sup.14C, .sup.15N, .sup.17O, .sup.18O,
.sup.18F and .sup.36Cl, respectively. Certain isotopic variations
of a compound of formula (I) or a salt or solvate thereof, for
example, those in which a radioactive isotope such as .sup.3H or
.sup.14C is incorporated, are useful in drug and/or substrate
tissue distribution studies. Tritiated, i.e., .sup.3H, and
carbon-14, i.e., .sup.14C, isotopes are particularly preferred for
their ease of preparation and detectability. Further, substitution
with isotopes such as deuterium, i.e., .sup.2H, may afford certain
therapeutic advantages resulting from greater metabolic stability,
for example, increased in vivo half-life or reduced dosage
requirements and hence may be preferred in some circumstances.
Isotopic variations of a compound of formula (I), or a
pharmaceutically salt thereof, can generally be prepared by
conventional procedures such as by the illustrative methods or by
the preparations described in the Examples hereafter using
appropriate isotopic variations of suitable reagents.
[0199] A compound of formula (I), or a pharmaceutically acceptable
salt thereof, may be in amorphous or crystalline form. Moreover, a
compound of formula (I), or a pharmaceutically acceptable salt
thereof, may exist in one or more crystalline forms. Consequently,
the present invention includes within its scope all forms of a
compound of formula (I), or a pharmaceutically acceptable salt
thereof.
[0200] The person skilled in the art will appreciate that many
organic compounds can form complexes with solvents in which they
are reacted or from which they are precipitated or crystallised.
These complexes are known as "solvates". Where the solvent is water
the complex is known as a "hydrate". The present invention
encompasses all solvates of the compounds of formula (I). In
addition, prodrugs are also included within the context of this
invention. As used herein, the term "prodrug" means a compound
which is converted within the body, e.g. by hydrolysis in the
blood, into its active form that has medical effects.
Pharmaceutically acceptable prodrugs are described in T. Higuchi
and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the
A.C.S. Symposium Series, Edward B. Roche, ed., Bioreversible
Carriers in Drug Design, American Pharmaceutical Association and
Pergamon Press, 1987, and in D. Fleisher, S. Ramon and H. Barbra
"Improved oral drug delivery: solubility limitations overcome by
the use of prodrugs", Advanced Drug Delivery Reviews (1996) 19(2)
115-130, each of which are incorporated herein by reference.
[0201] Prodrugs are any covalently bonded carriers that release a
compound of formula (I) in vivo when such prodrug is administered
to a patient. Prodrugs are generally prepared by modifying
functional groups in a way such that the modification is cleaved,
either by routine manipulation or in vivo, yielding the parent
compound. Prodrugs include, for example, compounds of this
invention wherein hydroxy, amine or sulfhydryl groups are bonded to
any group that, when administered to a patient, cleaves to form the
hydroxy, amine or sulfhydryl groups. Thus, representative examples
of prodrugs include (but are not limited to) acetate, formate and
benzoate derivatives of alcohol, sulfhydryl and amine functional
groups of the compounds of formula (I). Further, in the case of a
carboxylic acid (--COOH), esters may be employed, such as methyl
esters, ethyl esters, and the like. Esters may be active in their
own right and/or be hydrolysable under in vivo conditions in the
human body. Suitable pharmaceutically acceptable in vivo
hydrolysable ester groups include those which break down readily in
the human body to leave the parent acid or its salt.
EXPERIMENTAL
[0202] Compounds of the invention may be prepared by methods known
in the art of organic synthesis as set forth in part by the
following synthesis schemes. In the following reaction schemes and
hereafter, unless otherwise stated, all the groups are defined in
the first aspect. It is also recognised that in all of the schemes
described below, it is well understood that protecting groups for
sensitive or reactive groups are employed where necessary in
accordance with general principles of organic synthesis (T. W.
Greene and P. G. M. Wuts (1991) Protecting Groups in Organic
Synthesis, John Wiley & Sons). These groups are removed at a
convenient stage of the compound synthesis using methods that are
readily apparent to those skilled in the art. The selection of
processes as well as the reaction conditions and order of their
execution shall be consistent with the preparation of compounds of
the invention.
General Reaction Schemes
Scheme 1a and 1b
##STR00013##
##STR00014##
[0204] Compounds of formula (A)a and (A)b may be prepared from
sulfonyl chlorides of formula (C)a and (C)b respectively, by
reaction with an aniline of formula (B), according to Scheme 1a and
1b. Typical reaction conditions comprise mixing together a sulfonyl
chloride of formula (C)a or (C)b with the appropriate aniline (B),
in a basic solvent such as pyridine, for a suitable time, such as
16 hours, at a suitable temperature, such as ambient.
Scheme 2a and 2b
##STR00015##
##STR00016##
[0206] Compounds of formula (A)c and (A)d may be prepared from
intermediate compounds of formula (D)a and (D)b respectively, by
coupling with an appropriately substituted alcohol according to
Scheme 2a and 2b. Typical coupling conditions would include the
`Mitsunobu reaction` and comprise mixing the alcohol together with
an intermediate compound of formula (D)a or (D)b and
triphenylphosphine, in a suitable solvent, such as tetrahydrofuran.
The mixture is then treated with a suitable coupling reagent, such
as diisopropyl azodicarboxylate, and the reaction stirred for a
suitable time, such as 16 hours, at a suitable temperature, such as
ambient.
Scheme 3a and 3b
##STR00017##
##STR00018##
[0208] Compounds of formula (A)e and (A)f may be prepared from
intermediate compounds of formula (D)c and (D)d respectively, by
reaction with an appropriately substituted alkyl or heteroaromatic
alcohol or amine according to Scheme 3a and 3b. Typical reaction
conditions comprise mixing the alcohol or amine together with an
intermediate compound of formula (D)c or (D)d, with a strong base
such as sodium hydride, in a suitable solvent such as
2-methyltetrahydrofuran, under nitrogen at a suitable temperature,
such as ambient for a suitable time, such as 3 hours.
Scheme 4a and 4b
##STR00019##
##STR00020##
[0210] Compounds of formula (A)g and (A)h may be prepared from
intermediate compounds of formula (D)e and (D)f respectively, by
reaction with an appropriate alkylating agent according to Scheme
4a and 4b. Typical reaction conditions comprise mixing an
intermediate compound of formula (D)e or (D)f, with a strong base
such as sodium hydride in a suitable solvent, such as
dimethylsulfoxide, for a suitable time, such as 5 minutes, under
nitrogen. The mixture is then treated with the alkylating agent and
stirred at a suitable temperature such as ambient for a suitable
time, such as 18 hours.
Scheme 5a and 5b
##STR00021##
##STR00022##
[0212] Key intermediate compounds of formula (D), where R.sub.12 is
a suitable functional group for later transformation into R.sub.9;
may be prepared from sulfonyl chlorides of formula (E)a and (E)b,
by reaction with an aniline of formula (B) according to Scheme 5a
and 5b. Typical reaction conditions comprise mixing together a
sulfonyl chloride of formula (E)a or (E)b with the appropriate
aniline (B) in a basic solvent such as pyridine, for a suitable
time, such as 16 hours, at a suitable temperature such as
ambient.
[0213] R.sub.12 may include functionality (which can be
protected/masked) that is inert to reaction under the above
conditions and may then be converted to R.sub.9 in subsequent
step(s). Suitable examples for R.sub.12 can include -halo,
--(CHR.sub.10).sub.s-halo, --OMe/--OBn, --CO.sub.2Me/--CO.sub.2Et,
--CN, --NHAc (the latter four which may then be deprotected or
transformed to --OH, --CH.sub.2OH, --CH.sub.2NH.sub.2 and
--NH.sub.2 using methods known to those skilled in the art of
organic synthesis).
Scheme 6a and 6b
##STR00023##
##STR00024##
[0215] Compounds of formula (D) where R.dbd.R.sub.6, may be
prepared from intermediate compounds of formula (D), where R.dbd.H,
by reaction with a suitable alkylating agent according to Scheme 6a
and 6b. Typical reaction conditions comprise mixing together a
compound of formula (D), where R.dbd.H, with a strong base, such as
Barton's base, in a suitable solvent such as acetonitrile, for a
suitable time, such as 1 hour, at a suitable temperature such as
ambient. The mixture is then treated with the appropriate
alkylating agent and heated in a sealed vessel to a suitable
temperature, for example 150.degree. C., by microwaves, for a
suitable time, such as 25 minutes.
Scheme 7a and 7b
##STR00025##
##STR00026##
[0217] Secondary sulfonamide compounds of formula (F)a and (F)b may
be prepared from sulfonyl chlorides of formula (C)a and (C)b
respectively, by reaction with a primary aniline of formula (B),
R.dbd.H, according to Scheme 7a and 7b. Typical reaction conditions
comprise mixing together a sulfonyl chloride of formula (C)a or
(C)b with the appropriate aniline (B), R.dbd.H, in a basic solvent
such as pyridine, for a suitable time, such as 16 hours, at a
suitable temperature such as ambient.
Scheme 8a and 8b
##STR00027##
##STR00028##
[0219] Compounds of formula (A)i and (A)j, may be prepared from
intermediate compounds of formula (F)a and (F)b, by reaction with a
suitable alkylating agent according to Scheme 8a and 8b. Typical
reaction conditions comprise mixing together a compound of formula
(F)a or (F)b, with a strong base, such as Barton's base, in a
suitable solvent such as acetonitrile, for a suitable time, such as
1 hour, at a suitable temperature such as ambient. The mixture is
then treated with the appropriate alkylating agent and heated in a
sealed vessel to a suitable temperature, for example 150.degree.
C., by microwaves, for a suitable time, such as 25 minutes.
##STR00029##
[0220] Secondary anilines of formula (B), R.dbd.R.sub.6 may be
prepared from primary anilines of formula (B), R.dbd.H, by reaction
with an appropriate primary alcohol according to Scheme 9. Typical
reaction conditions comprise mixing together a primary aniline (B),
R.dbd.H, with the appropriate alcohol, a suitable base such as
potassium iodide and a suitable catalyst such as
pentamethylcyclopentadienyliridium(III) chloride in a suitable
solvent, such as water. The mixture is then heated in a sealed
vessel to a suitable temperature, for example 170.degree. C., by
microwaves, for a suitable time, such as 1 hour.
##STR00030##
[0221] Secondary anilines of formula (B), R.dbd.R.sub.6 may be
prepared from aryl chlorides of formula (G), by reaction with an
appropriate primary alkylamine according to Scheme 10. Typical
reaction conditions comprise mixing together an aryl chloride (G),
with with the appropriate primary alkyl amine, a suitable catalyst
such as
{1,3-bis[2,6-bis(1-methylethyl)phenyl]-2-imidazolidinyl}(chloro)(2-methyl-
-2-propen-1-yl)palladium and a suitable base such as lithium
hexamethyldisilizide, in a suitable solvent, such as
tetrahydrofuran. The mixture is then heated in a sealed vessel to a
suitable temperature, for example 70.degree. C., by microwaves, for
a suitable time, such as 45 minutes.
##STR00031##
[0222] A secondary aniline of formula (B), R.dbd.R.sub.6 may be
prepared from a primary aniline of formula (B), R.dbd.H, by
reaction with an appropriate aldehyde according to Scheme 11.
Typical reaction conditions comprise mixing together a primary
aniline (B), R.dbd.H, with the appropriate aldehyde, in a suitable
solvent such as tetrahydrofuran, for a suitable time, such as 20
minutes. The mixture is then treated with a suitable reducing
agent, such as sodium triacetoxyborohydride and stirred for a
suitable time, such as 18 hours, at a suitable temperature, such as
ambient.
##STR00032##
[0223] A secondary aniline of formula (B), R.dbd.R.sub.6 may be
prepared from a primary aniline (B), in a two step process, via an
intermediate primary amide of formula (H), according to Scheme 12.
Intermediate (H) may be prepared from a primary aniline (B) by
reaction with a suitable anhydride. Typical reaction conditions
comprise mixing together a primary aniline of formula (B) with the
appropriate anhydride in a suitable solvent, such as
dichloromethane, with a suitable base such as triethylamine, under
nitrogen, for a suitable time, such as 20 hours, at a suitable
temperature, such as ambient.
[0224] Secondary aniline (B), R.dbd.R.sub.6 may then be prepared
from intermediate (H), by reduction of the amide. Typical reaction
conditions comprise mixing together an intermediate primary amide
(H), with a suitable reducing agent, such as a solution of
borane-tetrahydrofuran complex in tetrahydrofuran, in a suitable
solvent such as tetrahydrofuran, under nitrogen. The mixture is
then warmed to a suitable temperature, such as 60.degree. C., and
stirred for a suitable time, such as 2 hours.
##STR00033##
[0225] Secondary anilines of formula (B), R.dbd.R.sub.6 may be
prepared from primary anilines of formula (B), R.dbd.H, by reaction
with an appropriate dialkylamine according to Scheme 13. Typical
reaction conditions comprise mixing together a primary aniline (B),
R.dbd.H, with the appropriate dialkylamine, a suitable base such as
potassium iodide and a suitable catalyst such as
pentamethylcyclopentadienyliridium(III) chloride in a suitable
solvent such as xylene. The mixture is then heated in a sealed
vessel to a suitable temperature, for example 190.degree. C., by
microwaves, for a suitable time, such as 2 hours.
Scheme 14
##STR00034##
##STR00035##
[0227] Compounds of formula (L)a/(M)a and (L)b/(M)b may be prepared
from epoxide-containing intermediate compounds of formula (K)a and
(K)b respectively, by reaction with an appropriate amine, according
to Scheme 14a and 14b. Typical reaction conditions comprise mixing
together epoxide-containing intermediate compound (K)a or (K)b with
an appropriate amine, in a suitable solvent, such as ethanol, with
a suitable base, such as triethylamine, at a suitable temperature,
such as 50.degree. C., for a suitable time, such as overnight. The
ratio of products (L) to (M) may vary with selection of amine, and
where a mixture of products results, separation may be achieved
using a suitable purification system, such as preparative HPLC.
Scheme 15
##STR00036##
##STR00037##
[0229] Epoxide-containing intermediate compounds of formula (K)a
and (K)b, may be prepared from intermediate compounds of formula
(D)g and (D)h respectively, by reaction with a vinyl organometal
reagent, then oxidation of the vinyl group, according to Scheme 15.
Typical reaction conditions for step (i) comprise mixing together
intermediate compound (D)g or (D)h with a suitable vinyl
organometal reagent, such as potassium trifluoro(vinyl)borate, an
appropriate base such as cesium carbonate and a suitable catalyst
such as palladium(II)chloride with a suitable ligand, such as
triphenylphosphine. A suitable solvent, such as
tetrahydrofuran/water mixture is then added and the reaction heated
in a sealed vessel to a suitable temperature, for example
140.degree. C., by microwaves, for a suitable time, such as 1
hour.
[0230] The resulting vinyl intermediate can then be oxidised to the
epoxide-containing intermediate (K)a or (K)b by treating with a
suitable oxidising agent, such as m-chloroperbenzoic acid in an
appropriate solvent such as dichloromethane, for a suitable time,
such as 16 hours, at a suitable temperature, such as 0.degree. C.
to ambient.
Example ROR.gamma. Modulators
[0231] The present invention is further illustrated by the
following non-limiting examples of ROR.gamma. modulators, which
have been prepared by a number of different methods.
Preparation of Products P1a to P21
[0232] The secondary anilines (P1a to P21--see Table 1) were
prepared by one of six different routes (R1 to R6 below). The
particular route used for the preparation of each secondary aniline
product is outlined in Table 1
Route 1 (R1)
##STR00038##
[0233] Generic Route
[0234] An aqueous solution of ammonium formate (5 equivalents, 0.2
mL/mmol) was diluted with isopropanol (1.6 mL) and added to
palladium on carbon 10% wet (1 equivalent) under nitrogen. A
solution of an aniline (1 equivalent, see Table 1 for specific
aniline used) and isobutyraldehyde (1.1 equivalents) in isopropanol
(0.3 mL/mmol) was added to the suspension and the mixture was
stirred for 1 h. The mixture was filtered through celite, the
celite cake washed with isopropanol. The combined liquid phases
were concentrated in vacuo. The residue was purified using a
pre-conditioned aminopropyl solid phase extraction cartridge
(NH.sub.2 SPE) (20 g) using methanol as an eluent. The methanolic
organic phase was concentrated in vacuo to give a second residue
which was further purified on a Biotage Flashmaster II using silica
(Si) 10 g/mmol using a 0-100% dichloromethane-cyclohexane gradient.
The fractions containing the expected product were combined and
concentrated in vacuo to give the desired product.
Specific Example of Route 1 (R1)
Preparation of (2,4-dimethylphenyl)(2-methylpropyl)amine
[0235] An aqueous solution of ammonium formate (3.15 g, 50.0 mmol,
10 mL) was diluted with isopropanol (80 mL) and added to palladium
on carbon 10% wet (1.064 g, 10.00 mmol) under nitrogen. A solution
of 2,4-dimethylaniline (1.212 g, 10 mmol) and isobutyraldehyde
(1.004 mL, 11.00 mmol) in isopropanol (3 mL) was added and the
mixture was stirred for 1 h. The mixture was filtered through
celite, the celite cake washed with isopropanol and combined liquid
phases concentrated in vacuo. The residue (1.8 g) was purified
using a pre-conditioned aminopropyl solid phase extraction
cartridge (NH.sub.2 SPE) (20 g) using methanol as an eluent. The
methanolic organic phase was concentrated under vacuo to give a
second residue which was further purified on a Biotage Flashmaster
II using silica (Si) 10 g/mmol using a 0-100%
dichloromethane-cyclohexane gradient over 40 mins. The fractions
containing the expected product were combined and concentrated in
vacuo to give the desired product 1.5 g as a yellow oil.
Route 2 (R2)
##STR00039##
[0236] Specific Example of Route 2 (R2)
Preparation of (2,4-dimethylphenyl)(2-methylpropyl)amine
[0237] Isobutyraldehyde (5 mL, 55.1 mmol) was added to
(2,4-dimethylphenyl)amine (7.01 g, 57.8 mmol) in Tetrahydrofuran
(50 mL) to give a brown solution. The solution was stirred for 20
mins at room temperature before sodium triacetoxy borohydride
(16.34 g, 77 mmol) was added. The reaction mixture was stirred at
room temperature for 18 hours and the reaction was analysed by LCMS
to confirm conversion to the desired product. The solution was
diluted with ethyl acetate (100 mL) and the organic phase washed
with water (100 mL). The organic phase was separated from the
aqueous phase using a separating funnel. The organic phase was
concentrated in vacuo to give the product as a brown oil.
Route 3 (R3)
##STR00040##
[0238] Generic Route
[0239] A mixture of an aryl chloride (1 equivalent, see Table 1 for
specific aryl chloride used), isobutylamine (2 equivalents), and
{1,3-bis[2,6-bis(1-methylethyl)phenyl]-2-imidazolidinyl}(chloro)(2-methyl-
-2-propen-1-yl)palladium `Caddick catalyst` (prepared according to
reference: Org. Biomol. Chem., 2008, 6, 2820-2825) (0.02
equivalents) was treated with lithium hexamethyl disilizide in
tetrahydrofuran (1M in THF) (1.25 equivalents, 1.25 mL/mmol) under
nitrogen. The reaction mixture was heated to 70.degree. C. for 45
min in a septum-sealed vessel. The cooled mixture was concentrated
in vacuo and the residue was partitioned between a solution of
citric acid in water (4 mL/mmol) and tert butyl methyl ether
(2.times.4 mL/mmol). The organic phase was treated with dried
MgSO.sub.4, the solid removed by filtration and the organic phase
was concentrated in vacuo to give the product.
Specific Example of Route 3 (R3)
Preparation of (2,4-dimethylphenyl)(2-methylpropyl)amine
[0240] A mixture of 1-chloro-2,4-dimethylbenzene (1687 mg, 12
mmol), isobutylamine (1755 mg, 24.00 mmol), and Caddick catalyst
(140 mg, 0.238 mmol) was treated with lithium hexamethyl disilizide
in tetrahydrofuran (1M LHMDS in THF, 15 mL, 15.00 mmol) under
nitrogen. The reaction mixture was heated to 70.degree. C. for 45
min in a septum-sealed vessel. The cooled mixture was concentrated
in vacuo and the residue was partitioned between aqueous citric
acid (50 mL) and tert butyl methyl ether (TBME) (2.times.50 mL).
The organic phase was treated with dried MgSO.sub.4, the solid
removed by filtration and the organic phase was concentrated in
vacuo to give (2,4-dimethylphenyl)(2-methylpropyl)amine (2.05 g,
11.56 mmol, 96% yield) as an orange oil.
Route 4 (R4)
##STR00041##
[0241] Generic Route
[0242] 2-methyl-1-propanol (2 equivalents), an aniline (1
equivalent, see Table 1 for specific aniline used), potassium
iodide (2 equivalents) and pentamethylcyclopentadienyliridium (III)
chloride (obtainable from, for example, Sigma Aldrich),
[Cp*IrCl.sub.2].sub.2 (0.01 equivalents) were dissolved in water
(0.6 mL/mmol). The resulting mixture was irradiated under
microwaves (CEM microwave) at 170.degree. C. for 1 hour. To the
reaction mixture was added dichloromethane (1.5 mL/mmol) and the
crude material organic was purified by mass directed autoprep using
a formic acid modifier. Relevant fractions were combined and
concentrated in vacuo to give the product.
Specific Example of Route 4 (R4)
Preparation of
2-methyl-N-(2-methylpropyl)-5-(trifluoromethyl)aniline
[0243] 2-methyl-1-propanol (0.556 mL, 6.00 mmol),
2-methyl-5-(trifluoromethyl)aniline (525 mg, 3 mmol), potassium
iodide (996 mg, 6.00 mmol) and [Cp*IrCl.sub.2].sub.2 (33.1 mg,
0.030 mmol) were dissolved in water (2 mL). The resulting mixture
was irradiated under microwaves (CEM microwave) at 170.degree. C.
for 1 hour. To the reaction mixture was added dichloromethane (5
mL) and the crude material was purified by mass directed autoprep
(250 mg scale.times.3, Method C, formic acid modifier). Relevant
fractions were combined and evaporated to give
2-methyl-N-(2-methylpropyl)-5-(trifluoromethyl)aniline (260 mg,
1.102 mmol, 36.7% yield) as a dark yellow oil.
Route 5 (R5)
##STR00042##
[0244] Specific Example of Route 5 (R5)
Preparation of 4-butyl-N-(2-methylpropyl)aniline
[0245] Isobutyric anhydride (2.034 mL, 12.25 mmol) was added
dropwise to a stirred solution of 4-butyl-2-methylaniline (2 g,
12.25 mmol) and triethylamine (3.41 mL, 24.50 mmol) in
dichloromethane (DCM) (20 mL) under nitrogen. The solution was
stirred for 20 hours and then treated with 2M HCl (75 mL) and
stirred for 5 minutes. The reaction mixture was extracted with DCM
and the combined organic layers were dried over magnesium sulfate.
The magnesium sulfate was filtered and the DCM phase concentrated
in vacuo to gave N-(4-butyl-2-methylphenyl)-2-methylpropanamide
(2.402 g, 10.29 mmol, 84% yield). A stirred solution of
N-(4-butyl-2-methylphenyl)-2-methylpropanamide (2.402 g, 10.29
mmol) in tetrahydrofuran (30 mL) under nitrogen was treated
dropwise with 1M borane in tetrahydrofuran (BH.sub.3 in THF, 30.9
mL, 30.9 mmol; obtainable from, for example, Aldrich). The solution
was heated at 60.degree. C. for 2 hours before being quenched with
2M HCl (18 mL) and stirred for a further 15 minutes. It was then
basified with aqueous Na.sub.2CO.sub.3 and extracted with DCM
(2.times.150 mL). The combined organic phases were then dried over
magnesium sulfate and concentrated in vacuo. The sample was loaded
in dichloromethane and purified on Biotage Flashmaster II on silica
(Si) 100 g using a 0-100% dichloromethane-cyclohexane over 60 mins.
The appropriate fractions were combined and evaporated in vacuo to
give 1.66 g of the title compound as a colourless liquid.
Route 6 (R6)
##STR00043##
[0246] Generic Route
[0247] Pentamethylcyclopentadienyliridium(III) chloride,
[Cp*IrCl.sub.2].sub.2 (0.01 equivalents), an aniline (1 equivalent,
see Table 1 for specific aniline used), potassium iodide (2
equivalents) and diisobutylamine (3 equivalents) were dissolved in
xylene (1 mL/mmol). The mixture was irradiated under microwaves
(CEM microwave) for 2 hours at 190.degree. C. To the mixture was
added dichloromethane (1.5 mL/mmol) and water (1.5 mL/mmol). The
organic fraction were separated and concentrated in vacuo. The
crude material was purified on a Biotage Flashmaster II using
silica (7 g/mmol, eluting with 0-100% DCM in cyclohexane). The
relevant fractions were combined and concentrated to give the
desired product.
Specific Example of Route 6 (R6)
Preparation of 4-(1-methylethyl)-N-(2-methylpropyl)aniline
[0248] [Cp*IrCl.sub.2].sub.2 (33.1 mg, 0.030 mmol),
4-isopropylaniline (406 mg, 3 mmol), potassium iodide (996 mg, 6.00
mmol) and diisobutylamine (1.572 mL, 9.00 mmol) were dissolved in
xylene (3 mL). The mixture was irradiated under microwaves (CEM
microwave) for 2 hours at 190.degree. C. To the mixture was added
Dichloromethane (DCM) (1.5 mL/mmol) and water (1.5 mL/mmol). The
organic fraction were separated and concentrated in vacuo. The
crude material was purified by silica chromatography (20 g, eluting
with 0-100% DCM in cyclohexane). The relevant fractions were
combined and concentrated to leave the title compound (201 mg,
1.040 mmol, 34.7% yield) as a yellow oil.
TABLE-US-00001 TABLE 1 Preparation Details for Products P1a to P21
by Routes 1 to 6 Product SM1 SM2 Scale Yield Work- Base ion ID
Route ID Source ID Source (mmol) (%) up Purification RT (M +1) P1a
R1 A1 Aldrich B1 Aldrich 10 88 S D2 1.04 178 P1b R2 A1 Aldrich B1
Aldrich 55.1 100 A None 1.34 178 P1c R3 A2 Avocado B2 Aldrich 12 96
A None 1.03 178 Research Chemicals P1d R4 A1 Aldrich B3 Aldrich
16.92 63 A D3 1.02 178 P2a R1 A3 ABCR B1 Aldrich 4 57 A D1 1.49 220
Gelest (UK) Ltd P2b R4 A3 ABCR B3 Aldrich 6 85 A D3 1.5 220 Gelest
(UK) Ltd P2c R5 A3 ABCR B4 Aldrich 2.45 47 A D2 3.8 220 Gelest (UK)
Ltd P3 R4 A4 Aldrich B3 Aldrich 6 62 A D2 1.18 182 P4 R4 A5 Aldrich
B3 Aldrich 6 70 A D2 1.25 184 P5 R4 A6 Apollo B3 Aldrich 3 49 A D2
1.3 182 Scientific Ltd P6 R4 A7 Aldrich B3 Aldrich 3 53 A D2 1.27
186 P7 R6 A8 Aldrich B5 Sigma 6 45 A D2 1.26 206 Aldrich P8 R6 A9
Aldrich B5 Sigma 6 34 A D2 1.1 192 Aldrich P9 R6 A10 Aldrich B5
Sigma 6 34 A D1 1.31 218 Aldrich P10 R6 A11 Aldrich B5 Sigma 6 26 A
N3 1.13 192 Aldrich P11 R4 A12 Avocado B3 Aldrich 3 37 A F 1.42 232
Research Chemicals P12 R4 A13 Aldrich B3 Aldrich 3 80 A D2 0.98 164
P13 R4 A14 Fluorochem B3 Aldrich 3 10 A D5 1.37 202 P14 R4 A15
Avocado B3 Aldrich 3 27 A D2 1.35 202 Research Chemicals P15 R4 A16
Acros Organics B3 Aldrich 6 46 A D3 1.02 180 P16 R4 A17 Aldrich B3
Aldrich 3 74 A D3 1.12 178 P17 R4 A1 Aldrich B3 Aldrich 3 56 A D3
1.24 178 P18 R4 A18 Aldrich B3 Aldrich 3 85 A None 0.77 164 P19 R4
A1 Aldrich B6 Sigma 12.38 33 A N2 1.32 176 Aldrich P20 R3 A19
Aldrich B2 Aldrich 2 34 A None 1.03 180 P21 R4 A20 Refer to
Additional B3 Aldrich 0.802 63 A D2 1.07 322 Experimental
Section
Preparation of Products P22 to P82
[0249] The secondary and tertiary sulfonamide products P21 to P82
(see Table 2) were prepared by either Route 7 or Route 8 described
below. The particular route used for the preparation of each
secondary of tertiary sulfonamide product is outlined in Table
2.
Route 7 (R7)
##STR00044##
[0250] Generic Route
[0251] To a solution of an aniline (1 equivalent, See Table 2 for
specific aniline used) in pyridine (4 mL/mmol) was added a sulfonyl
chloride (1 equivalent, See Table 2 for specific sulfonyl chloride
used) in one charge at room temperature. In some cases
4-dimethylaminopyridine (0.02 equivalents) was also added to
reaction mixture (see Table 2 for further details). The reaction
mixture was left to stand at 20.degree. C. for 16 hr. In some
cases, the reaction solution was heated (80 or 95.degree. C.) for 1
h before being left at room temperature overnight. The solvent was
evaporated in vacuo (Biotage V10 evaporator) to give a crude
material. The crude material was treated in one of the following
ways: 1. Purification on SPE cartridges; 2. aqueous work up,
evaporation of the organic phase followed by purification by mass
directed autoprep.
Specific Example of Route 7 (R7)
Preparation of
N-(4-chlorophenyl)-4-((3,5-dimethylisoxazol-4-yl)methyl)-N-isobutylbenzen-
esulfonamide
[0252]
N-(4-chlorophenyl)-4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-isobuty-
lbenzenesulfonamide was prepared in an array format.
4-chloro-N-isobutylaniline (1.493 mg, 0.099 mmol) was pre-weighed
into a 2 mL vial, and
4-((3,5-dimethylisoxazol-4-yl)methoxy)benzene-1-sulfonyl chloride
(30 mg, 0.099 mmol) then added, followed by 4-DMAP (approx 2 mg).
Pyridine (0.3 mL) was added last. The reaction solution was capped
and stood at RT for 3 days. After this time the reaction was not
complete and the reaction were warmed to 80.degree. C. for 2 hours.
The pyridine was removed to almost dryness. DMSO (1 mL) was added
to the residues which were then purified by mass directed autoPrep
on a Sunfire C18 column using Acetonitrile Water with a Formic acid
modifier. The solvent was dried under a stream of nitrogen in the
Radleys blowdown apparatus to give the required products.
Route 8 (R8)
##STR00045##
[0253] Generic Route
[0254] To a solution of an amine (1 equivalent, see Table 2 for
specific amine used) and sulfonyl chloride (1.1 equivalents, see
Table 2 for specific sulfonyl chloride used) in pyridine (2
mL/mmol) was added neat Barton's base
(2-(tert-butyl)-1,1,3,3-tetramethylguanidine) (1 equivalent). The
reaction vessel was sealed and heated in Emrys Optimiser using
initial high absorbance to 100.degree. C. for 30 min. After cooling
the reaction was analysed and product presence confirmed. The
solvent was evaporated in vacuo using the Vaportec V10 to give the
crude product. The sample was loaded in methanol and purified by
SPE on aminopropyl (NH.sub.2) 10 g/mmol using methanol. The
appropriate fractions were concentrated and further purified on SPE
on sulfonic acid (SCX) 4 g/mmol using methanol. The appropriate
fractions were combined and dried under a stream of nitrogen in the
Radley's blowdown apparatus to give the crude product. The samples
were dissolved in 1:1 MeOH:DMSO 1 mL and purified by mass directed
autoPrep on Sunfire C18 column using Acetonitrile Water with a
Formic acid modifier. The fractions containing the product were
combined and solvent removed under a stream of nitrogen in the
Radleys blowdown apparatus to give the required products.
Specific Example of Route 8 (R8)
Preparation of
N-[4-(cyclopropylmethyl)-2,6-dimethylphenyl]-4-{[(3,5-dimethyl-4-isoxazol-
yl)methyl]oxy}benzenesulfonamide
[0255] To a solution of
[4-(cyclopropylmethyl)-2,6-dimethylphenyl]amine hydrochloride (100
mg, 0.472 mmol) and
4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}benzenesulfonyl chloride
(157 mg, 0.520 mmol) in Pyridine (1 mL) was added neat Barton's
base (2-(tert-butyl)-1,1,3,3-tetramethylguanidine) (0.095 mL, 0.472
mmol). The reaction vessel was sealed and heated in Emrys Optimiser
using initial high absorbance to 100.degree. C. for 30 min. After
cooling the reaction was analysed and product presence confirmed.
The solvent was evaporated in vacuo using the Vaportec V10 to give
the crude product. The sample was loaded in methanol and purified
by SPE on aminopropyl (NH.sub.2) 5 g using methanol. The
appropriate fractions were concentrated and the crude material
further purified by SPE on sulfonic acid (SCX) 2 g using methanol.
The appropriate fractions were combined and dried under a stream of
nitrogen in the Radleys blowdown apparatus to give the crude
product. The samples were dissolved in 1:1 MeOH:DMSO 1 mL and
purified by mass directed autoPrep on Sunfire C18 column using
Acetonitrile Water with a Formic acid modifier. The solvent was
dried under a stream of nitrogen in the Radleys blowdown apparatus
to give the required products (7.4 mg, 0.017 mmol, 3.56% yield)
TABLE-US-00002 TABLE 2 Preparation of Products P22 to P82 by Route
7 or 8 Product SM1 SM2 Scale Yield Work Base ion ID Route ID Source
ID Source (mmol) DMAP .degree. C. (%) up Purification RT (M + 1)
P22 R7 P2c See Table 1 B7 Apollo 0.1 yes 95 3 E F 1.25 453
Scientific Ltd P23 R7 P2c See Table 1 B8 Manchester 0.1 yes 95 15 E
F 1.55 485 Organics P24 R7 P1a See Table 1 B8 ACB 0.2 no 20 19 E F
1.4 443 Blocks Ltd P25 R7 P3 See Table 1 B8 Manchester 0.3 no 20 16
S F 1.36 447 Organics P26 R7 P6 See Table 1 B8 ACB 0.27 no 20 18 S
F 1.3 451 Blocks Ltd P27 R7 P7 See Table 1 B8 Manchester 0.24 no 20
13 S N1 1.51 471 Organics P28 R7 P8 See Table 1 B8 Manchester 0.26
no 20 23 S F & ED1 1.44 457 Organics P29 R7 P5 See Table 1 B8
ACB 0.276 no 20 5 S F 1.34 447 Blocks Ltd P30 R7 P4 See Table 1 B8
Manchester 0.1 yes 80 13 E F 1.45 449 Organics P31 R7 P10 See Table
1 B8 Manchester 0.1 yes 80 47 E F 1.53 457 Organics P32 R7 P9 See
Table 1 B8 Manchester 0.1 yes 80 43 E F 1.43 483 Organics P33 R7
P11 See Table 1 B8 Manchester 0.1 yes 80 3 E F 1.47 497 Organics
P34 R7 A21 Otava B8 Manchester 0.099 yes 20 26 E F 1.27 415
Building Organics Blocks P35 R7 A22 UkrOrgSynthesis B8 Manchester
0.1 yes 20 15 E A 1.3 429 Ltd Organics P36 R7 P12 See Table 1 B8
ACB 0.184 no 20 66 S F 1.36 429 Blocks Ltd P37 R7 P17 See Table 1
B8 ACB 0.169 no 20 48 E F 1.41 443 Blocks Ltd P38 R7 P16 See Table
1 B8 ACB 0.186 no 20 61 E F 1.41 443 Blocks Ltd P39 R7 P14 See
Table 1 B8 ACB 0.149 no 20 27 E F 1.33 467 Blocks Ltd P40 R8 P13
See Table 1 B8 ACB 0.297 no 150 4 E F 1.32 467 Blocks Ltd P41 R7
P15 See Table 1 B9 Enamine 1 no 20 50 A & S none 1.41 426 Ltd
P42 R7 P18 See Table 1 B8 Manchester 0.306 no 20 52 S F 1.35 429
Organics P43 R7 P19 See Table 1 B8 Manchester 0.1 no 20 40 E F 1.36
441 Organics P44 R7 P20 See Table 1 B8 Manchester 0.575 no 20 64 A
none 1.16 445 Organics P45 R7 A23 ABCR B8 Manchester 0.370 no 20 50
S F 3.22 401 Organics P46 R7 P1c See Table 1 B9 Enamine 5.6 no 20
41 A R 1.54 424 Ltd P47 R7 P1d See Table 1 B11 Aldrich 5.64 no 20
92 A none 1.42 336 P48 R7 A24 ABCR B9 Enamine 1 no 20 54 A E3 1.23
375 Ltd P49 R7 A25 Aldrich B8 Manchester 0.3 no 20 51 S none 1.04
395 Organics P50 R7 A26 SigmaAldrich B8 Manchester 0.9 no 20 91 S
none 1.21 427 Organics P51 R7 A27 Aldrich B8 Manchester 0.9 no 20
36 S none 1.17 427 Organics P52 R7 A28 Aldrich B8 Manchester 0.321
no 20 25 S none 1.18 427 Organics P53 R7 A24 ABCR B8 Manchester
0.194 no 20 81 E F 1.09 395 Organics P54 R7 A29 Aldrich B8
Manchester 0.3 no 20 98 S none 1.12 387 Organics P55 R7 A30 Apollo
B8 Manchester 0.3 no 20 96 S none 1.1 391 Scientific Ltd Organics
P56 R7 A25 Aldrich B8 Manchester 0.3 no 20 100 S none 1.14 387
Organics P57 R7 A31 Sigma B8 Manchester 0.3 no 20 44 S none 1.05
403 Aldrich Organics P58 R7 A32 Aldrich B8 Manchester 0.3 no 20 98
S none 1.08 391 Organics P59 R7 A33 Aldrich B8 Manchester 0.3 no 20
75 S none 1.11 393 Organics P60 R7 P12 See Table 1 B9 Enamine 1 no
20 78 A none 1.5 410 Ltd P61 R7 P16 See Table 1 B9 Enamine 1 no 20
62 A none 1.54 424 Ltd P62 R7 P17 See Table 1 B9 Enamine 1 no 20 57
A none 1.51 424 Ltd P41 R7 P15 See Table 1 B9 Enamine 1 no 20 50 A
& S none 1.41 426 Ltd P63 R7 A31 Sigma B11 Aldrich 1 no 20 63 S
none 1.02 296 Aldrich P64 R7 A34 Acros Organics B11 Aldrich 1 no 20
91 S none 1.1 280 P65 R7 A30 Apollo Scientific B11 Aldrich 1 no 20
76 S none 1.06 284 Ltd P66 R7 A25 Aldrich B11 Aldrich 1 no 40 31 E
E2 1.01 288 P67 R7 A32 Aldrich B9 Enamine 1 no 40 71 A E3 1.23 372
Ltd P68 R7 A15 Avocado B11 Aldrich 0.687 no 40 96 A none 1.09 303
P69 R7 A35 TCI UK Ltd B8 Manchester 0.67 no 20 90 S none 1.23 415
Organics P70 R7 P1d See Table 1 B12 Alfa 2.096 no 20 59 A E3 1.44
348 Aesar P71 R7 P1d See Table 1 B13 Apollo 1.918 no 20 61 A D3
1.41 179 Scientific Ltd P72 R7 A36 Fluka B8 Manchester 0.467 no 20
42 A none 1.07 373 Organics P73 R7 P1d See Table 1 B14 Sigma 0.564
no 20 58 A E1 1.47 366 Aldrich P74 R7 A37 Sigma B8 Manchester 1.29
no 20 98 A none 1.17 451 Aldrich Organics P75 R7 P1d See Table 1
B15 ChemCollect 0.6 no 20 49 E F 1.26 364 GmbH P76 R8 A38 WO 2009
B8 Manchester 0.472 no 100 4 S T 1.28 441 045830 Organics P77 R7
P2a See Table 1 B16 ABCR 0.1 yes 95 38 E F 1.52 352 P78 R7 P1d See
Table 1 B17 Aldrich 2.82 no 20 67 S none 1.51 336 P79 R7 P1d See
Table1 B18 Apollo 2.8 no 20 98 A E3 1.56 340 international P80 R7
P21 See Table 1 B8 Manchester 0.506 no 20 93 A none 1.69 344
Organics P81 R7 P15 See Table 1 B8 Manchester 0.167 no 20 70 E F
1.28 338 Organics P82 R7 A39 Fluorochem B8 Manchester 1 no 20 94 A
none 1.11 428 Organics
Preparation of Products P83 to P97
[0256] The tertiary sulfonamide products P83 to P97 were prepared
by a number of routes as described below. The particular route used
for the preparation of each tertiary sulfonamide product is
outlined in Table 3.
Route 9 (R9)
##STR00046##
[0257] Generic Route
[0258] To a round bottomed flask was added a sulfonamide (1
equivalent, see Table 3 for specific sulfonamide used), ammonium
formate (5 equivalents), palladium(II) hydroxide (20% on carbon)
(0.1 equivalents) and ethanol (28 mL/mmol). The reaction mixture
was stirred and left to reflux under nitrogen overnight at
90.degree. C. An LCMS was taken, and showed full conversion into
the product, and then reaction mixture was left to cool. To remove
the palladium (II) hydroxide, the reaction mixture was then passed
through a celite column (10 g/mmol). The residue remaining on the
celite was thoroughly dampened with water, and then deactivated
with a sodium metabisulfite solution. The crude mixture was
concentrated in vacuo, diluted with ethyl acetate and washed with
water, then brine. An LCMS confirmed the presence of the product in
the organic phase, which was then dried down under a stream of
nitrogen.
Specific Example of Route 9
N-(2,4-dimethylphenyl)-4-hydroxy-N-(2-methylpropyl)benzenesulfonamide
[0259] To a round bottomed flask was added a stirrer bar,
N-(2,4-dimethylphenyl)-N-(2-methylpropyl)-4-[(phenylmethyl)oxy]benzenesul-
fonamide (974 mg, 2.300 mmol), ammonium formate (725 mg, 11.50
mmol), palladium(II) hydroxide (20% on carbon) (164 mg, 0.230 mmol)
and Ethanol (65 mL). A reflux condenser was placed on the top of
the flask and the reaction mixture was heated to 90.degree. C.
under reflux with stirring overnight. A product peak was observed
by LCMS analysis, but only partial conversion had been seen
overnight. White crystals thought to be ammonium formate were
observed on the side of the reflux condenser and consequently a
further 5 equivalents of ammonium formate (725 mg, 11.50 mmol) were
added to the reaction mixture. The temperature of the heater was
raised to 95.degree. C. after which the reaction mixture had
reached boiling point. After a further 30 minutes of heating,
another LCMS sample was taken, showing no change in the ratio of
starting material:product. The reaction mixture was cooled for 5
minutes before the addition of another measure of palladium(II)
hydroxide (20% on carbon) (164 mg, 0.230 mmol). The reaction
mixture was then reheated to 95.degree. C. for another 30 minutes
after which full conversion to the product was observed. The
reaction mixture was filtered through a 10 g Celite cartridge
before concentrating in vacuo to give 727 mg of crude product. The
crude mixture was diluted with ethyl acetate and washed with water,
then brine. The organic phase was then concentrated in vacuo to
give 663 mg of the title compound.
Route 10 (R10)
##STR00047##
[0260] Generic Route
[0261] To a solution of a sulfonamide (1 equivalent, see Table 4
for specific sulfonamide used) in Acetonitrile (8 mL/mmol) was
added neat Barton's base
(2-(tert-butyl)-1,1,3,3-tetramethylguanidine)(1 equivalent). The
reaction mixture was stirred at room temperature for 1 hour.
1-bromo-2-methylpropane (2 equivalents) was then added. The
reaction vessel was sealed and heated in Biotage Initiator
microwave using initial high absorbance power setting to
150.degree. C. for 25 min. The solvent was dried under a stream of
nitrogen in the Radleys blowdown apparatus. The sample was loaded
in dichloromethane and purified on automated normal chromatograph
on silica (Si) 80 g/mmol using a 0-50% ethyl acetate-cyclohexane.
The appropriate fractions were combined and evaporated to give the
required product.
Specific Example of Route 10 (R10)
4-fluoro-N-[2-methyl-5-(methyloxy)phenyl]-N-(2-methylpropyl)benzenesulfona-
mide
[0262] To a solution of
4-fluoro-N-[2-methyl-5-(methyloxy)phenyl]benzenesulfonamide (185.8
mg, 0.629 mmol) in Acetonitrile (5 mL) was added neat Barton's base
(2-(tert-butyl)-1,1,3,3-tetramethylguanidine) (0.131 mL, 0.629
mmol). The reaction mixture was stirred at room temperature for 1
hour. 1-bromo-2-methylpropane (0.137 mL, 1.258 mmol) was then
added. The reaction vessel was sealed and heated in Biotage
Initiator microwave using initial high absorbance power setting to
150.degree. C. for 25 min. The solvent was dried under a stream of
nitrogen in the Radleys blowdown apparatus. The sample was loaded
in dichloromethane and purified on automated normal chromatograph
on silica (Si) 50 g using a 0-50% ethyl acetate-cyclohexane over 40
mins. The appropriate fractions were combined and dried under a
stream of nitrogen in the Radleys blowdown apparatus to give the
required product 171 mg as a white solid.
Route 11 (R11)
##STR00048##
[0263] Specific Example of Route 11 (R11)
[0264]
N-(2,4-dimethylphenyl)-N-isobutyl-3-methoxybenzenesulfonamide
(431.4 mg, 1.242 mmol) was dissolved in dichloromethane (DCM) (10
mL) and cooled to -78.degree. C. BBr.sub.3 in DCM (1M) (6.21 mL,
6.21 mmol) was added dropwise and the reaction put under nitrogen.
The reaction was allowed to reach room temperature and left to stir
overnight. Water (20 mL) was added dropwise into the reaction
mixture, until no more fumes were released. The crude product was
then extracted to the organic phase of an aqueous work up between
DCM (30 mL) and water (20 mL). The aqueous phase was washed twice
with DCM (20 mL.times.2). The organic phase was then dried and
concentrated in vacuo. The crude product was then dissolved in a
minimum of DCM, and loaded onto a silica column before being
purified by silica column chromatography (20 mins, 20 g, 0-25%
EtOAc in cyclohexane). The relevant fractions were then combined
and condensed, to give the product,
N-(2,4-dimethylphenyl)-3-hydroxy-N-isobutylbenzenesulfonamide,
384.4 mg.
Route 12 (R12)
##STR00049##
[0265] Specific Example of Route 12 (R12)
[0266] Methyl
3-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)benzoate (400 mg,
1.065 mmol) was dissolved in tetrahydrofuran (THF) (10 mL). To this
solution was added lithium borohydride (2M in THF) (0.932 mL, 1.864
mmol). The reaction was left to stir overnight, at room
temperature, overnight. The reaction was quenched by the addition
of 5% citric acid (10 mL), and the reaction allowed to stir, under
nitrogen for 1 hour. The reaction mixture was then diluted with
ethyl acetate (25 mL), and the product extracted to the organic
phase. The organic phase was dried by passing it through a
hydrophobic frit, and then concentrated in vacuo to give the
desired product,
N-(2,4-dimethylphenyl)-3-(hydroxymethyl)-N-isobutylbenzenesulfonamide,
378.7 mg.
TABLE-US-00003 TABLE 3 Preparation of Products P83 to P97 by Routes
9 to 12 Product SM1 SM2 Scale Yield Work- Base ion ID Route ID
Source ID Source (mmol) (%) up Purification RT (M + 1) P83 R9 P46
See Table 1 N/A N/A 2.3 86 A none 1.3 334 P84 R9 P60 See Table 2
N/A N/A 0.779 92 A none 1.2 320 P85 R9 P61 See Table 2 N/A N/A
0.619 79% F none 1.2 334 P86 R9 P62 See Table 2 N/A N/A 0.567 90 A
none 1.2 334 P87 R9 P41 See Table 2 N/A N/A 0.504 79 A none 1.1 336
P88 R10 P63 See Table 2 B19 Aldrich 0.629 77 E E2 1.3 352 P89 R10
P64 See Table 2 B19 Aldrich 0.909 73 E E2 1.4 336 P90 R10 P65 See
Table 2 B19 Aldrich 0.762 63 E E2 1.3 340 P91 R10 P66 See Table 2
B19 Aldrich 0.313 45 E E2 1.3 344 P92 R9 P93 See Table 2 N/A N/A
0.327 85 A none 1.2 338 P93 R10 P67 See Table 2 B19 Aldrich 0.528
65 A E3 1.5 428 P94 R10 P68 See Table 2 B19 Aldrich 0.731 57 A E3
1.4 360 P95 R11 P70 See Table 2 N/A N/A 1.242 93 E E3 1.3 334 P96
R12 P71 See Table 2 N/A N/A 1.065 102 A none 1.2 348 P97 R10 P82
See Table 2 B19 Aldrich 0.945 33 A F 1.4 501
Preparation of Products P98 to P149
[0267] The tertiary sulfonamide products P98 to P149 were prepared
by a number of different routes as described below. The particular
route used for the preparation of each tertiary sulfonamide product
is outlined in Table 4.
Route 13 (R13)
##STR00050##
[0268] Generic Route
[0269] This route was performed in an array format. A mixture of a
phenol (1 equivalent, see Table 4 for specific phenol used), and
potassium fluoride, 40% by weight on alumina (40%
KF.Al.sub.2O.sub.3 3 equivalents) in acetonitrile (2 mL/mmol) was
stirred for 5 mins, before addition of an alkylating reagent (see
Table 4 for specific alkylating agent used for each reaction) (1
equivalent) in acetonitrile (2 mL/mmol). The reaction mixture was
capped and stirred at RT for 18 hours. Additional 40%
KF.Al.sub.2O.sub.3 (3 equivalents) and more alkylating reagent (1
equivalent) were added to the tube, which was capped and stirred
for a further 18 hours. The sample was filtered through cotton
wool, and DMSO added to bring the total volume to 1 mL. The
solution was purified by CAS mass directed autoPrep on Sunfire C18
column using acetonitrile:water with a formic acid modifier. The
solvent was dried under a stream of nitrogen in the Radleys
blowdown apparatus to give the required product.
Specific Example of Route 13
Preparation of
N-(2,4-dimethylphenyl)-N-isobutyl-4-(pyridine-4-ylmethoxy)benzenesulfonam-
ide
[0270] A mixture of
N-(2,4-dimethylphenyl)-4-hydroxy-N-isobutylbenzenesulfonamide (33
mg, 0.099 mmol), and potassium fluoride, 40% by weight on alumina
(40% KF.Al.sub.2O.sub.3 43.1 mg, 0.297 mmol) in acetonitrile (0.2
mL) were stirred for 5 mins, before addition of
4-(bromomethyl)pyridine (0.099 mmol) in acetonitrile (0.2 mL). The
reaction mixture was capped and stirred at RT for 18 hours.
Additional 40% KF.Al.sub.2O.sub.3 (43 mg) and more alkylating
reagent (0.1 mmol) were added to the tube, which was capped and
stirred for a further 18 hours. The reaction had additional
potassium fluoride, 40% by weight on alumina (43 mg) and alkylating
reagent (0.1 mmol), and then recapped and stirred for 2 days. The
sample was filtered through cotton wool, and DMSO added to bring
the total volume to 1 mL. The solutions were purified by mass
directed autoprep on Sunfire C18 column using acetonitrile water
with a formic acid modifier. The solvent was dried under a stream
of nitrogen in the Radleys blowdown apparatus to give the required
product.
Route 14 (R14)
##STR00051##
[0271] Generic Route
[0272] This route was performed in an array format. A phenol (1
equivalent, see Table 4 for specific phenol used), an alcohol (1.5
equivalents, see Table 4 for specific alcohol used in each
reaction), triphenylphosphine (1.5 equivalents mmol) were
pre-weighed into a 4 mL glass vial. Tetrahydrofuran (THF) (4
mL/mmol) was added followed by diisopropyl
diazene-1,2-dicarboxylate (DIAD) (1.5 quivalents). The vial was
capped and stirred at RT for 2 days. The reaction mixture was
filtered to remove any insoluble materials. The sample was purified
by mass directed autoPrep on Xbridge column using
acetonitrile:water with an ammonium carbonate modifier. The solvent
was dried under a stream of nitrogen in the Radleys blowdown
apparatus to give the required products,
Specific Example
Preparation of
N-(2,4-dimethylphenyl)-N-isobutyl-4-(pyridine-4-ylmethoxy)benzenesulfonam-
ide
[0273]
N-(2,4-dimethylphenyl)-4-hydroxy-N-isobutylbenzenesulfonamide (33
mg, 0.099 mmol), pyridine-4ylmethanol (2.52 mg, 0.148 mmol),
triphenylphosphine (39 mg, 0.149 mmol) were pre-weighed into a 4 mL
glass vial. Tetrahydrofuran (THF) (0.6 mL) was added followed by
diisopropyl diazene-1,2-dicarboxylate (DIAD) (0.029 mL, 0.148
mmol). The vial was capped and stirred at RT for 2 days. The
reaction mixture was filtered to remove any insoluble materials.
The sample was purified by mass directed autoprep on an Xbridge
column using acetonitrile:water with an ammonium carbonate
modifier. The solvent was dried under a stream of nitrogen in the
Radleys blowdown apparatus to give the required product.
Route 15 (R15)
##STR00052##
[0274] Generic Route
[0275] A phenol (1 equivalent, see table 5 for specific phenol
used), an alcohol (1.25 equivalents, see Table 4 for specific
alcohol used),
(4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyl)phenyl)diph-
enylphosphine (Fluoroflash, 1.5 equivalents mmol) were added to
Biotage 2-5 mL microwave vessel. Tetrahydrofuran (THF) (14 mL/mmol)
was added followed by diisopropyl diazene-1,2-dicarboxylate (DIAD)
(1.25 quivalents). The vials were capped and stirred at RT
overnight. The reaction mixture was concentrated in vacuo and then
diluted with ethyl acetate (85 mL/mmol) and water (85 mL). The
organic fraction was separated, dried and then concentrated in
vacuo to give the crude product. The crude product was dissolved in
DMF:H.sub.2O (9:1) 3.5 mL/mmol, and loaded onto a flurous column
(pre-conditioned with 1 mL DMF, followed by 6 mL MeOH:H.sub.2O
(5:1). The semi purified fraction was eluted with 20 mL/mmol
MeOH:H.sub.2O (5:1). The fraction was concentrated down, and
dissolved in 1:1 MeOH:DMSO 1 mL and purified by mass directed
autoprep on Sunfire C18 column using acetonitrile:water with a
formic acid modifier. The solvent was dried under a stream of
nitrogen in the Radleys blowdown apparatus to give the required
product.
Specific Example of Route 15
Preparation of
3-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(2,4-dimethylphenyl)-N-isobutylb-
enzenesulfonamide
[0276]
N-(2,4-dimethylphenyl)-3-hydroxy-N-isobutylbenzenesulfonamide (76.9
mg, 0.231 mmol), (3,5-dimethylisoxazol-4-yl)methanol (36.7 mg,
0.288 mmol) and
(4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyl)p-
henyl)diphenylphosphine (204 mg, 0.288 mmol) were added to a 2-5 mL
Biotage microwave vessel. Tetrahydrofuran (THF) (4 mL) was added
followed by diisopropyl diazene-1,2-dicarboxylate (DIAD) (0.056 mL,
0.288 mmol). The reaction vial was sealed and left to stir
overnight at RT. The reaction mixture was concentrated in vacuo and
then diluted with ethyl acetate (25 mL) and water (25 mL). The
organic fraction was separated, dried and then concentrated in
vacuo to give the crude product. The crude product was dissolved in
DMF: H.sub.2O (9:1) 1 mL, and loaded onto a flurous column
(preconditioned with 1 mL DMF, followed by 6 mL MeOH:H.sub.2O
(5:1). The semi purified fraction was eluted with 6 mL
MeOH:H.sub.2O (5:1). The fraction was concentrated down, and
dissolved in 1:1
[0277] MeOH:DMSO 1 mL and purified by mass directed autoPrep on
Sunfire C18 column using Acetonitrile Water with a Formic acid
modifier. The solvent was dried under a stream of nitrogen in the
Radleys blowdown apparatus to give the required product, 37.7
mg.
Route 16 (R16)
##STR00053##
[0278] Generic Route
[0279] This route was performed in an array format.
N-(2,4-dimethylphenyl)-4-fluoro-N-isobutylbenzenesulfonamide (8
equivalents) was dissolved in DMSO or DMF (0.75 mL/mmol.times.8)
and an aliquot (representing 1 equivalent) was added to an alcohol
(1 equivalent, see Table 4 for specific alcohol used for each
reaction). 60% sodium hydride dispersed on oil (1 equivalent) was
added to the reaction. The reaction was capped and sonicated to aid
dispersion. The reaction was left to stand at RT for 18 hours. The
reaction was quenched with methanol (0.5 mL), and sonicated to aid
dispersion. All the samples were purified by mass directed autoprep
on an Xbridge column using acetonitrile:water with an ammonium
carbonate modifier. The solvent was dried under a stream of
nitrogen in the Radleys blowdown apparatus to give the required
product (as an ammonium salt where appropriate).
Specific Example
Preparation of
N-(3-fluoro-5-methylphenyl)-N-isobutyl-4-(61yridine-4-ylmethoxy)benzenesu-
lfonamide
[0280] A solution was prepared of an alcohol (0.15 mmol
pyridine-4-methanol-16 mg) dissolved in DMF (0.5 mL). This solution
was added to the core,
4-fluoro-N-(3-fluoro-5-methylphenyl)-N-(2-methylpropyl)benzenesulfonamide
(0.15 mmol, 50.9 mg). 60% sodium hydride dispersed on oil (0.006 g,
0.150 mmol) was added to the reaction. The reaction was capped and
sonicated to aid dispersion. The reaction was left to stand at room
temperature for 18 hours. Additional 60% sodium hydride dispersed
on oil (0.005 g) and alcohol monomer, pyridine-4-methanol (0.5
equivalent) in DMF (0.2 mL) was added and the reaction capped and
stirred for an additional 18 hours. Solvent removed to almost a
third dry. The sample was dissolved in 1:1 MeOH:DMSO 1 mL (filtered
through cotton wool) and split into two batches and purified by
mass directed autoprep on an Xbridge column using
acetonitrile:water with an ammonium carbonate modifier. The solvent
was dried under a stream of nitrogen in the Radleys blowdown
apparatus to give the title compound, 12.9 mg.
Route 17 (R17)
##STR00054##
[0281] Generic Route
[0282] This generic route was performed in an array format. To a
solution of an alcohol (1 equivalent, see Table 4 for specific
alcohol used) and
4-(bromomethyl)-N-(2,4-dimethylphenyl)-N-isobutylbenzenesulfonamide
(1 equivalent) in 2-methyltetrahydrofuran (2-MeTHF) (13 mL/mmol)
stirred under nitrogen at room temperature was added solid sodium
hydride (60% dispersed in oil, 1 equivalent). The reaction mixture
was stirred at 20.degree. C. for 3 hours. The reaction was
carefully quenched with water (1 mL/mmol). The solvent was dried
under a stream of nitrogen in the Radleys blowdown apparatus to
give the crude products. The samples were dissolved in 1:1
MeOH:DMSO 1 mL and purified by mass directed autoprep on Xbridge
column using acetonitrile:water with an ammonium carbonate
modifier. The solvent was dried under a stream of nitrogen in the
Radleys blowdown apparatus to give the required product.
Specific Example
Preparation of
N-(2,4-dimethylphenyl)-N-isobutyl-4-((pyridin-4-yloxy)methyl)benzenesulfo-
namide
[0283] To a solution of an alcohol (1.276 mg, 0.075 mmol, see Table
4 for specific alcohol used for each reaction) and
4-(bromomethyl)-N-(2,4-dimethylphenyl)-N-isobutylbenzenesulfonamide
(0.031 g, 0.075 mmol) in 2-Methyltetrahydrofuran (2-MeTHF) (1 mL)
stirred under nitrogen at room temperature was added solid sodium
hydride (1.800 mg, 0.075 mmol)(tip of spatula). The reaction
mixture was stirred at 20.degree. C. for 3 hrs. The reaction was
carefully quenched with water (5 drops per reaction). The solvent
was dried under a stream of nitrogen in the Radleys blowdown
apparatus to give the crude product. The sample was dissolved in
1:1 MeOH:DMSO 1 mL and purified by mass directed autoprep on an
Xbridge column using acetonitrile:water with an ammonium carbonate
modifier. The solvent was dried under a stream of nitrogen in the
Radley's blowdown apparatus to give the required product (3.89 mg,
10% yield).
Further Example of Route 17 (not Prepared in an Array Format)
Preparation of
N-(2,4-dimethylphenyl)-N-isobutyl-4-((pyridine-4-ylmethoxy)methyl)benzene-
sulfonamide
[0284] To a solution of crude
4-(bromomethyl)-N-(2,4-dimethylphenyl)-N-isobutylbenzenesulfonamide
(100 mg, 0.122 mmol) and pyridine-4-ylmethanol (13.30 mg, 0.122
mmol) in 2-Methyltetrahydrofuran (2-MeTHF) (1 mL) and dimethyl
sulfoxide (DMSO) (0.5 mL) stirred in air at RT was added solid
sodium hydride (4.87 mg, 0.122 mmol) in one charge, tip of spatula.
The reaction mixture was stirred at 20.degree. C. for 16 hours.
Reaction was carefully quenched with methanol (0.5 mL) and water
(0.5 mL) and evaporated in vacuo to give a residue in DMSO. This
was diluted with dichloromethane (10 mL) and water (10 mL) and
stirred vigorously for 10 min. The layers were separated by
hydrophobic frit and the organic fraction evaporated to give the
crude product. The samples were dissolved in 1:1 MeOH:DMSO 1 mL and
purified by mass directed autoprep on Sunfire C18 column using
acetonitrile:water with a formic acid modifier.
[0285] The solvent was dried under a stream of nitrogen in the
Radleys blowdown apparatus to give the required product
N-(2,4-dimethylphenyl)-N-isobutyl-4-((pyridine-4-ylmethoxy)methyl)benzene-
sulfonamide (4.8 mg, 10.94 .mu.mol, 8.98% yield)
Route 18 (R18)
##STR00055##
[0286] Specific Example
Preparation of
N-(2,4-dimethylphenyl)-N-isobutyl-3-((pyridin-4-ylmethoxy)methyl)benzenes-
ulfonamide
[0287]
N-(2,4-dimethylphenyl)-3-(hydroxymethyl)-N-isobutylbenzenesulfonami-
de (50 mg, 0.144 mmol) and Sodium hydride (60% in Mineral Oil)
(6.33 mg, 0.158 mmol) were dissolved in N,N-Dimethylformamide (DMF)
(3 mL). To this solution was added 4-(bromomethyl)pyridine (24.75
mg, 0.144 mmol), and the reaction heated to 60.degree. C. and left
to stir for 30 min. The reaction solution concentrated on the
Biotage V10 and then extracted the crude product to the organic
phase of an aqueous work up between ethyl acetate (15 mL) and water
(15 mL). The organic phase was separated (hydrophobic frit) and
then concentrated in vacuo. The sample was dissolved in 1:1
MeOH:DMSO 1 mL and purified by mass directed autoprep on Sunfire
C18 column using acetonitrile:water with a formic acid modifier.
The solvent was evaporated in vacuo. The crude product was run
through an NH.sub.2 SPE column (1 g) eluting with methanol, to
remove the formic acid. The product was then dried under a stream
of nitrogen on a Radleys blow down unit, to give the final product,
13.5 mg.
TABLE-US-00004 TABLE 4 Preparation of Products P98 to P149 by
Routes 13 to 18 Product SM1 SM2 Scale Yield Work- Base ion ID Route
ID ID SM2 Source (mmol) (%) up Purification RT (M + 1) P98 R13 P83
B20 Aldrich 0.1 15 F F 1.14 425 P98 R14 P83 B21 Aldrich 0.1 15 F A
1.13 425 P99 R16 P94 B21 Aldrich 0.15 2 E A 1.06 449 P100 R16 P90
B21 Aldrich 0.15 18 E A 1.06 429 P101 R16 P89 B21 Aldrich 0.15 2 E
A 1.1 425 P102 R16 P88 B21 Aldrich 0.15 9 E A 1.03 441 P103 R16 P91
B21 Aldrich 0.15 4 E A 0.99 433 P104 R14 P92 B21 Aldrich 0.121 9 F
A 1.05 429 P105 R14 P86 B21 Aldrich 0.1 3 F A 1.09 425 P106 R14 P85
B21 Aldrich 0.1 11 F A 1.1 425 P107 R14 P87 B21 Aldrich 0.1 3 F A
0.98 427 P108 R14 P84 B21 Aldrich 0.1 22 F A 1.03 411 P109 R10 P76
B19 Aldrich 0.017 71 S F 1.5 497 P110 R10 P48 B19 Aldrich 0.537 6 E
E3 1.44 432 P111 R10 P49 B19 Aldrich 0.152 35 S A 1.28 451 P112 R10
P50 B19 Aldrich 0.819 32 S A 1.43 483 P113 R10 P51 B19 Aldrich
0.328 28 S A 1.38 483 P114 R10 P52 B19 Aldrich 0.082 10 S A 1.42
483 P115 R10 P53 B19 Aldrich 0.157 60 A F 1.32 451 P116 R10 P54 B19
Aldrich 0.295 48 S A 1.37 443 P117 R10 P55 B19 Aldrich 0.289 53 S A
1.34 447 P118 R10 P56 B19 Aldrich 0.342 50 S A 1.38 443 P119 R10
P57 B19 Aldrich 0.131 48 S A 1.32 459 P120 R10 P58 B19 Aldrich
0.295 48 S A 1.33 447 P121 R10 P59 B19 Aldrich 0.224 49 S A 1.34
449 P122 R13 P83 B22 ASDI 0.099 4 F F 0.97 414 P123 R16 P47 B21
Aldrich 0.596 20 S T 1.11 425 P124 R13 P83 B23 Maybridge Chemical
0.099 67 F F 1.36 442 Co Ltd P125 R13 P83 B24 Maybridge Chemical
0.099 74 F F 1.41 429 Co Ltd P126 R17 P73 B25 Sigma 0.075 10 E A
1.04 425 Aldrich P127 R17 P73 B26 Sigma 0.075 5 E A 1.05 426
Aldrich P128 R17 P73 B21 Aldrich 0.122 9 A F 1.09 439 P129 R14 P96
B27 Fluorochem 0.173 10 A F 1.4 457 P130 R18 P96 B28 Aldrich 0.144
21 A F 1.11 439 P131 R16 P47 B29 TCI Ltd 0.149 20 E A 0.96 428 P132
R16 P47 B30 Matrix 0.149 34 E A 1.24 429 Scientific P133 R16 P47
B31 Key Organics Ltd 0.149 52 E A 1.03 428 P134 R16 P47 B32 Aldrich
0.149 5 E A 1 414 P135 R10 P69 B19 Aldrich 0.15 21 S F 1 471 P136
R15 P95 B27 Fluorochem 0.231 37 A & FL F 1.51 443 P137 R10 P72
B19 Aldrich 0.188 13 A F 1.36 429 P138 R15 P95 B21 Aldrich 0.231 27
A & FL F 1.18 425 P139 R14 P96 B33 Cayman 0.144 16 A F 1.31 440
P140 R13 P83 B28 Aldrich 0.099 15 F F 1.14 425 P141 R14 P83 B34
ABCR-Gelest (UK) Ltd 0.099 44 F A 1.04 439 P142 R14 P83 B35
Chembridge 0.099 50 F A 1.41 457 P143 R14 P83 B33 Cayman 0.099 20 F
A 1.28 426 P144 R10 P74 B19 Aldrich 1.059 72 E E1 1.43 507 P145 R14
P83 B36 Activate Scientific Ltd 0.099 34 F A 1.1 433 P146 R16 P47
B37 Activate Scientific Ltd 0.149 63 E A 0.99 428 P147 R16 P47 B38
Activate Scientific Ltd 0.149 59 E A 1.24 429 P148 R13 P75 B39
Aldrich 0.107 64 F F 1.38 473 P149 R16 P47 B40 ABCR 0.745 95 A 1.34
440
Additional Experimental
Preparation of Starting Material A20
4-(3-((tert-butyldimethylsilyl)oxy)propyl)aniline
[0288] To a 50 mL round bottomed flask was added 1H-imidazole (59.4
mg, 0.873 mmol), 3-(4-aminophenyl)propan-1-ol 120 mg, 0.794 mmol)
and Tetrahydrofuran (THF) (5 mL). To this solution was added
tert-butylchlorodimethylsilane (132 mg, 0.873 mmol), and this was
left to stir for 2 days, under nitrogen at room temperature.
Another 0.5eq of tert-butylchlorodimethylsilane was added to the
reaction and the reaction was left to stir for another 2 hours.
After this, reaction was stopped and the suspension concentrated
down on a Biotage V10 evaporator, and then the product extracted to
the organic phase of an aqueous work up between ethyl acetate (10
mL) and water (10 mL). The organic phase was then passed through a
hydrophobic frit, and concentrated in vacuo, leaving the crude
product (212 mg, 100%). LCMS (M+1): 266, RT 1.14 mins.
Preparation of Product P150
N-(2,4-dimethylphenyl)-4-(2-hydroxy-2-(pyridin-4-yl)ethoxy)-N-isobutylbenz-
enesulfonamide
a) preparation of 1-(pyridine-4-yl)ethane-1,2-diol
[0289] 4-vinylpyridine (2.523 g, 24 mmol) was dissolved in acetone
(30 mL) and cooled to .about.2.degree. C. (internal temp), with
stirring. A solution of potassium permanganate (2.530 g, 16.01
mmol) and magnesium sulfate (962 mg, 7.99 mmol) in water (50 mL)
was prepared and added dropwise to the acetone solution, over 20
minutes. The mixture was stirred for an additional 10 minutes, then
temperature increased to RT. A small quantity of hydroquinone (26.4
mg, 0.240 mmol) was added to the reaction, then the mixture was
filtered through glass sinter. The filtrate was concentrated to 5
mL and ethanol (20 mL) added to precipitate potassium sulfate. The
solution stored in the fridge over weekend. The resulting brown
precipitate was filtered from the mixture. The mother liquors were
evaporated to give a brown oil, 2.85 g. Product was purified by
silica (Si) column on Companion (0-25% methanol in DCM, over 23
minutes, UV collection). Combination of the clean
product-containing fractions and removal of solvent in vacuo
provided desired product, 954 mg, as a pale yellow oil which
solidified on cooling. LCMS (M+1) 139, RT 0.36 mins.
b) preparation of
N-(2,4-dimethylphenyl)-4-(2-hydroxy-2-(pyridin-4-yl)ethoxy)-N-isobutylben-
zenesulfonamide
[0290] A solution of
N-(2,4-dimethylphenyl)-4-fluoro-N-isobutylbenzenesulfonamide (90
mg, 0.268 mmol) was prepared in N,N-dimethylformamide (2 mL) and
added to a microwave vial containing potassium carbonate (74.2 mg,
0.537 mmol) and 1-(pyridin-4-yl)ethane-1,2-diol (41.1 mg, 0.295
mmol). The vial was sealed and heated to 130.degree. C. for 30 min
by microwaves. After this time, a further portion of potassium
carbonate (74.2 mg, 0.537 mmol) was added to the reaction and the
mixture heated to 140.degree. C. by microwaves for an additional 30
min. 18-crown-6 (70.9 mg, 0.268 mmol) was then added to the
reaction and the mixture heated to 140.degree. C. by microwaves,
for a further 30 min. Water (15 mL) was added and the product
extracted with DCM (3.times.15 mL). The combined organics were
dried with a hydrophobic frit, then the crude material purified by
high-pH MDAP to give the desired product, 18.2 mg. LCMS (M+1) 455,
RT 1.27 mins.
Preparation of Product P151
2-(4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)-2,3-dimethylphenoxy)acet-
ic acid
a) preparation of ethyl
{[4-(chlorosulfonyl)-2,3-dimethylphenyl]oxy}acetate
[0291] Ethyl [(2,3-dimethylphenyl)oxy]acetate (6.1 g, 29.3 mmol)
was added dropwise to chlorosulfonic acid (8.78 mL, 132 mmol) at
0.degree. C., with stirring, causing a blood red solution. Reaction
stirred for 30 minutes, then allowed to warm to RT. Mixture was
stirred for a further 2 hours, then quenched by pouring over
ice/water (200 mL). A dark red gum formed, this was extracted into
dichloromethane (3.times.100 mL). Organics were concentrated in
vacuo, to give a thick red oil, which crystallized on standing.
Product was dried overnight in vacuum oven, to yield 8.179 g as red
crystals. No further purification carried out. RT 1.25 mins.
b) preparation of ethyl
[(4-{[(2,4-dimethylphenyl)(2-methylpropyl)amino]sulfonyl}-2,3-dimethylphe-
nyl)oxy]acetate
[0292] In a 15 mL test tube was placed
N-isobutyl-2,4-dimethylamiline (100 mg, 0.564 mmol, prepared, for
example, according to P1a, Table 1), ethyl
{[4-(chlorosulfonyl)-2,3-dimethylphenyl]oxy}acetate (173 mg, 0.564
mmol,), and di-isopropyl-ethyl-amine (0.197 mL, 1.128 mmol) in
dichloromethane (DCM) (2 mL) to give an orange solution. The
reaction was stirred at 30.degree. C. for 18 hours. The solvent was
removed in vacuo and the residue purified by MDAP in two portions.
The fractions from the MDAP were identified by LCMS, combined and
the solvent removed in vacuo to give the expected compound. The
compound was dissolved in dioxane and freeze dried over night.
Yield 16% (41.7 mg) of a white powder. LCMS (M+1) 448, RT 1.52
mins.
c) preparation of
2-(4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)-2,3-dimethylphenoxy)ace-
tic acid
[0293] In a 25 mL round-bottomed flask was placed ethyl
[(4-{[(2,4-dimethylphenyl)(2-methylpropyl)amino]sulfonyl}-2,3-dimethylphe-
nyl)oxy]acetate (50 mg, 0.112 mmol) and lithium hydroxide (5.35 mg,
0.223 mmol) in tetrahydrofuran (THF) (3 mL) and Water (1 mL) to
give a white suspension. The reaction was stirred overnight at room
temperature. The reaction mixture was diluted with ethyl acetate
and washed with water (dilute HCl). The organic layer was separated
and the solvent removed in vacuo to give the expected compound. The
residue was taken up in dioxane and freeze dried overnight. Yield
43%. LCMS (M-1) 418, RT 1.26 mins.
Preparation of Product P152
3-[(4-{[(4-butyl-2-methylphenyl)(2-methylpropyl)amino]sulfonyl}-2-methylph-
enyl)oxy]propanoic acid
[0294] To (4-butyl-2-methylphenyl)(2-methylpropyl)amine (21.94 mg,
0.100 mmol, prepared, for example, according to P2, Table 1) in
pyridine (1 mL) was added
3-{[4-(chlorosulfonyl)-2-methylphenyl]oxy}propanoic acid (27.9 mg,
0.100 mmol) and left to stand at RT in air for 2 hours. The sample
was dissolved in a 1:1 mixture of MeOH and DMSO and was purified by
method A (see Table 8) using Waters Xbridge Shield RP18 column.
Yield 37%. LCMS (M+1) 462, RT 1.46 mins.
Preparation of Products P153 and P154
N-(2,4-dimethylphenyl)-4-(1-hydroxy-2-((pyridin-3-ylmethyl)amino)ethyl)-N--
isobutylbenzenesulfonamide and
N-(2,4-dimethylphenyl)-4-(1-hydroxy-2-((pyridin-4-ylmethyl)amino)ethyl)-N-
-isobutylbenzenesulfonamide
a) preparation of
N-(2,4-dimethylphenyl)-N-isobutyl-4-vinylbenzenesulfonamide
[0295] A suspension of potassium trifluoro(vinyl)borate (241 mg,
1.800 mmol),
4-bromo-N-(2,4-dimethylphenyl)-N-isobutylbenzenesulfonamide (595
mg, 1.5 mmol), cesium carbonate (1466 mg, 4.50 mmol), palladium(II)
chloride (5.32 mg, 0.030 mmol) and triphenylphosphine (23.61 mg,
0.090 mmol) was prepared in tetrahydrofuran (THF) (2 mL) and Water
(0.2 mL). The mixture was heated to 140.degree. C. by microwaves
for 30 min, to yield a black suspension. Additional 1/2 eq of
potassium trifluoro(vinyl)borate (121 mg, 0.9 mmol) was added,
along with extra water (0.2 mL) and THF (2 mL) and reaction heated
for a further 1 hour at 140.degree. C. The reaction mixture was
diluted with dichloromethane (15 mL) and water (5 mL), then
filtered through a plug of celite to remove palladium catalyst. The
resulting yellow solution was dried using a hydrophobic frit,
concentrated, then purified by flash (Si) using 0-25% ethyl
acetate/cyclohexane over 40 minutes. Fractions were combined to
give a clear oil, 385.3 mg, and the product taken directly to the
next step, without further purification. LCMS (M+1) 344, RT 1.48
mins.
b) preparation of
N-(2,4-dimethylphenyl)-N-isobutyl-4-(oxiran-2-yl)benzenesulfonamide
[0296] A solution of
N-(2,4-dimethylphenyl)-N-isobutyl-4-vinylbenzenesulfonamide (380.2
mg, 1.107 mmol) was prepared in dichloromethane (DCM) (5 mL) and
mCPBA (764 mg, 4.43 mmol) added at 0.degree. C. The reaction was
stirred over the weekend from 0.degree. C. to 25.degree. C. The
reaction was washed with water (15 mL), sodium hydroxide
(2.times.15 mL) and brine (15 mL), then concentrated in vacuo.
Purification by flash (Si) using 0-25% ethyl acetate in cyclohexane
gave the desired product 195.8 mg as a clear oil. Yield 49%. LCMS
(M+1) 360, RT 1.38 mins.
c) preparation of
N-(2,4-dimethylphenyl)-4-(1-hydroxy-2-((pyridin-3-yl)methyl)amino)ethyl)--
N-isobutylbenzenesulfonamide
[0297] A solution of pyridin-3-ylmethanamine (10.38 mg, 0.096 mmol)
in ethanol (0.25 mL) was prepared in a 4 mL reaction vial and
triethylamine (0.027 mL, 0.192 mmol) added. A solution of
N-(2,4-dimethylphenyl)-N-isobutyl-4-(oxiran-2-yl)benzenesulfonamide
(28.8 mg, 0.08 mmol) in ethanol (0.75 mL) was then added and the
reaction heated at 40.degree. C. overnight. After this time,
further pyridin-3-ylmethanamine (10.38 mg, 0.096 mmol) was added
and the reaction heated at 50.degree. C. for an additional 2 hours.
The reaction mixture was then concentrated under a stream of
nitrogen and the crude mixture purified by high-pH MDAP to give the
required product, 10.6 mg. LCMS (M+1) 468, RT 1.24 mins.
d) preparation of
N-(2,4-dimethylphenyl)-4-(1-hydroxy-2-((pyridin-4-ylmethyl)amino)ethyl)-N-
-isobutylbenzenesulfonamide
[0298] A solution of pyridin-4-ylmethanamine (10.38 mg, 0.096 mmol)
in ethanol (0.25 mL) was prepared in a 4 mL reaction vial and
triethylamine (0.027 mL, 0.192 mmol) added. A solution of
N-(2,4-dimethylphenyl)-N-isobutyl-4-(oxiran-2-yl)benzenesulfonamide
(28.8 mg, 0.08 mmol) in ethanol (0.75 mL) was then added and the
reaction heated at 50.degree. C. overnight. The reaction mixture
was then concentrated under a stream of nitrogen and the crude
mixture purified by high-pH MDAP to give the required product, 10.5
mg. LCMS (M+1) 468, RT 1.22 mins.
Preparation of Product P155
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(4-(2-hydroxypropan-2-yl)-2-methy-
lphenyl)-N-isobutylbenzenesulfonamide
[0299] To a solution of ethyl
4-(4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-isobutylphenylsulfonamido)-3--
methylbenzoate (66 mg, 0.132 mmol) in toluene (0.53 mL) and
tetrahydrofuran (THF) (0.530 mL) stirred under nitrogen at
0.degree. C. was added a solution of methylmagnesium bromide in
butyl ether (0.264 mL, 0.264 mmol) dropwise. The reaction mixture
was stirred at 0.degree. C. for 30 min and then allowed to warm up
to RT and stirred for 2 hours. The reaction mixture was diluted
with dichoromethane (5 mL). The organic phase was washed with water
10 mL, dried using a hydrophobic frit and dried under a stream of
nitrogen in the Radleys blowdown apparatus to give the crude
product. The samples was dissolved in 1:1 MeOH:DMSO 1 mL and
purified by mass directed autoprep on a Sunfire C18 column using
acetonitrile:water with a formic acid modifier. The solvent was
dried under a stream of nitrogen in the Radleys blowdown apparatus
to give the required product, 22 mg as a colorless oil. Yield 34%.
LCMS (M-18) 469, RT 1.25 mins.
Preparation of Product P156
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(4-(2-hydroxyethoxy)-2-methylphen-
yl)-N-isobutylbenzenesulfonamide
a) preparation of
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-isobutyl-N-(2-methyl-4-(2-((tetr-
ahydro-2H-pyran-2-yl)oxy)ethoxy)phenyl)benzenesulfonamide
[0300] An aliquot of tetrahydrofuran (THF) (1 mL) containing
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(4-hydroxy-2-methylphenyl)-N-iso-
butylbenzenesulfonamide (50 mg, 0.112 mmol) and DIAD (0.048 mL,
0.247 mmol) was added to triphenylphosphine (64.9 mg, 0.247 mmol).
The reaction mixture was sealed and left to stir for 10 minutes
before addition of 2-((tetrahydro-2H-pyran-2-yl)oxy)ethanol (0.015
mL, 0.112 mmol). The sealed reaction mixture was then heated to
50.degree. C. over night. LCMS showed the formation of the product.
LCMS (M+1) 572, RT 1.4 mins. Used without further purification in
next reaction.
b) preparation of
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(4-(2-hydroxyethoxy)-2-methylphe-
nyl)-N-isobutylbenzenesulfonamide
[0301] To a solution of
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-isobutyl-N-(2-methyl-4-(2-((tetr-
ahydro-2H-pyran-2-yl)oxy)ethoxy)phenyl)benzenesulfonamide (51.2 mg,
0.089 mmol) in tetrahydrofuran (THF) (4 mL) stirred at room
temperature was added a solution of 4M HCl in dioxane (4 mL, 16.00
mmol). The reaction mixture was stirred at room temperature for 1
hour. The solvent was dried under a stream of nitrogen in the
Radleys blowdown apparatus to give the crude product. The sample
was dissolved in 1:1 MeOH:DMSO 1 mL and purified by Open Access
Mass Directed AutoPrep on Sunfire C18 column using Acetonitrile
Water with a Formic acid modifier. The solvent was dried under a
stream of nitrogen in the Radleys blowdown apparatus to give the
required product, 27 mg as colourless oil which solidified. LCMS
(M+1) 489, RT 1.14 mins.
Preparation of Product P157
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(4-(hydromethyl)-2-methylphenyl)--
N-isobutylbenzenesulfonamide
[0302] To a solution of ethyl
4-(4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-isobutylphenylsulfonamido)-3--
methylbenzoate (40 mg, 0.080 mmol) in THF (5 mL) stirred under
nitrogen at 0.degree. C. was added a solution of LiAlH.sub.4 1 M in
diethyl ether (0.160 mL, 0.160 mmol) dropwise. The reaction mixture
was stirred at 0.degree. C. for 30 minutes. The reaction was then
allowed to warm up to room temperature and stirred for 2 hours. The
reaction mixture was diluted with dichloromethane (10 mL). The
organic phase was washed with water 10 mL, and saturated brine 10
mL, dried using a hydrophobic frit and concentrated under a stream
of nitrogen in the Radleys blowdown apparatus to give the crude
product. The crude was dissolved in 1:1 MeOH:DMSO 1 mL and purified
by mass directed autoprep on a Sunfire C18 column using
acetonitrile:water with a formic acid modifier. The solvent was
dried under a stream of nitrogen in the Radleys blowdown apparatus
to give the required product, 23.6 mg colorless oil which
solidified. LCMS (M+1) 459, RT 1.14 mins.
Preparation of Product P158
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(4-(3-hydroxypropyl)phenyl)-N-iso-
butylbenzenesulfonamide
[0303]
N-(4-(3-((tert-butyldimethylsilyl)oxy)propyl)phenyl)-4-((3,5-dimeth-
ylisoxazol-4-yl)methoxy)-N-isobutylbenzenesulfonamide (276.8 mg,
0.472 mmol) was dissolved in Tetrahydrofuran (THF) (10 mL). To this
solution was added TBAF in THF (1M) (0.707 mL, 0.707 mmol), and the
solution was left to stir for 1 hr at room temperature. The
reaction mixture was concentrated on the Biotage V10 evaporator,
extracted to the organic phase of an aqueous workup between ethyl
acetate (10 mL) and water (10 mL) followed by a brine (10 mL) wash.
The organic phase was passed through a hydrophobic frit, and
concentrated in vacuo, before being dissolved in DCM and loaded
onto a silica column and purified by column chromatography on
Biotage II Flashmaster (20 g silica (Si), 40 mins, 0-50% EtOAc in
cyclohexane). The relevant fractions were combined and condensed
and the sample was freeze dried using a minimum of dioxane in
water, to give the final product, 67%. LCMS (M+1) 473.1, RT 1.16
mins.
Preparation of Product P159
4-(4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)butanoic
acid
a) preparation of methyl
4-(4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)-4-nitrobutanoate
[0304] To a solution of methyl 4-nitrobutanoate (0.047 mL, 0.378
mmol), 4-bromo-N-(2,4-dimethylphenyl)-N-isobutylbenzenesulfonamide
(100 mg, 0.252 mmol),
di-tert-butyl(2'-methyl-[1,1'-biphenyl]-2-yl)phosphine (7.88 mg,
0.025 mmol) and cesium carbonate (99 mg, 0.303 mmol) in
1,2-Dimethoxyethane (DME) (1.5 mL) at RT was added solid
Pd(II)(dba)2 (7.25 mg, 0.013 mmol), ensuring all particles were
under the solvent level. The vial was flushed with nitrogen for 2
minutes. The reaction vessel was sealed and heated in Emrys
Optimiser using initial high absorbance to 120.degree. C. for 30
min. After cooling the reaction was analysed. Product was present
as well as starting material. The vessel was resealed and heated at
120.degree. C. for a further 30 min. No change in analysis. The
reaction mixture was passed through a pre-packed silica cartridge
(500 mg) to remove palladium residue; the cartridge was washed with
methanol (5 mL). The resulting filtrate was evaporated in vacuo to
give a residue. The samples were dissolved in 1:1 MeOH:DMSO 1 mL
and purified by Open Access Mass Directed AutoPrep on Sunfire C18
column using Acetonitrile Water with a Formic acid modifier. The
solvent was dried under a stream of nitrogen in the Radleys blow
down apparatus to give the required product 28 mg. (43% yield).
LCMS (M+1) 418; RT 1.45.
b) preparation of methyl
4-(4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)butanoate
[0305] A solution of methyl
4-(4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)-4-nitrobutanoate
(28 mg, 0.061 mmol) in Ethanol (2 mL) was prepared. The reaction
was hydrogenated using the H-cube (settings: 20.degree. C., 1 bar,
1 mL/min flow rate) and 10% Pd/C CatCart 30 as the catalyst.
Analysis showed loss of nitro group. The solvent was evaporated in
vacuo. The samples were dissolved in 1:1 MeOH:DMSO 1 mL and
purified by Open Access Mass Directed AutoPrep on Sunfire C18
column using Acetonitrile Water with a Formic acid modifier. The
solvent was dried under a stream of nitrogen in the Radleys
blowdown apparatus to give the required product 10.9 mg. LCMS (M+1)
418; Rt 1.45 mins.
c) preparation of
4-(4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)butanoic
acid
[0306] To a solution of methyl
4-(4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)butanoate
(10.9 mg, 0.026 mmol) in tetrahydrofuran (THF) (0.25 mL) stirred in
air at RT was added a solution of lithium hydroxide (0.625 mg,
0.026 mmol) in water (0.250 mL) in one charge. The reaction mixture
was stirred at 20.degree. C. for 30 min, to ensure dissolution and
then left to stand overnight. Analysis showed complete conversion.
The reaction mixture was evaporated and the residue partitioned
between ethyl acetate 5 mL and water 5 mL, stirring vigorously for
2 hours before separation with a hydrophilic frit. Analysis showed
product to be in both layers. Both layers were separately acidified
(2N hydrochloric acid, ca. 1 mL), diluted with water (2 mL) and
dichloromethane (2.times.3 mL). The organic fractions were
separated by hydrophobic frit. The appropriate fractions were
combined and dried under a stream of nitrogen in the Radleys
blowdown apparatus to give the two batches of the required product
5.75 mg and 3.05 mg. LCMS (M+1) 404, RT 1.27 mins.
Preparation of Product P160
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(4-ethylphenyl)-N-isobutylbenzene-
sulfonamide
a) preparation of 4-ethyl-N-(2-methylpropyl) aniline
[0307] A mixture of 2-methyl-1-propanol (5.94 mL, 64.40 mmol),
4-ethylaniline (2 mL, 16.09 mmol), potassium iodide (5.342 g, 32.2
mmol) and [Cp*IrCl.sub.2].sub.2 (128 mg, 0.161 mmol) was prepared
in water (10 mL). This mixture was heated by microwaves to
150.degree. C. for 1.5 hours. After cooling, the reaction mixture
was diluted with dichloromethane (20 mL) and water (10 mL) then
stirred vigorously for 3 minutes. The organic phase was separated
(hydrophobic frit) and the aqueous phase was diluted with further
dichloromethane (10 mL) then stirred vigorously for 2 minutes and
separated (hydrophobic frit). The combined organic fractions were
evaporated in vacuo to give the crude product as a brown oil. The
crude was purified by flash silica (Si) chromatography (using a
0-50% dichloromethane-cyclohexane gradient). The appropriate
fractions were combined and evaporated in vacuo to give the title
compound as a pale yellow oil. (1.9285 g, 77%). LCMS (M+1) 178, RT
0.91 mins.
b) preparation of
4-((3,5-dimethylisoxazol-4-yl)methoxy)-N-(4-ethylphenyl)-N-isobutylbenzen-
esulfonamide
[0308] To 4-ethyl-N-isobutylaniline (50 mg, 0.282 mmol) in pyridine
(1 mL) was added
4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}benzenesulfonyl chloride
(94 mg, 0.310 mmol) and the reaction stood at room temperature, in
air, for 16 hours. The crude was then purified by MDAP (Method F),
to provide 103.6 mg of desired product. LCMS (M+1) 443, RT 1.39
mins.
Preparation of Product P161
(R)-3-((4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)-2-methylphenyl)amin-
o)-4-hydroxybutanoic acid
a) preparation of
N-(2,4-dimethylphenyl)-4-fluoro-N-isobutyl-3-methylbenzenesulfonamide
[0309] To (2,4-dimethylphenyl)(2-methylpropyl)amine (300 mg, 1.692
mmol) in pyridine (5 mL) was added
4-fluoro-3-methylbenzene-1-sulfonyl chloride (353 mg, 1.692 mmol)
and the reaction stood at room temperature, in air, for 16 hours.
The crude was then passed through an aminopropyl (NH.sub.2) SPE
(solid phase extraction) cartridge eluting with methanol followed
by a sulfonic acid (SCX) SPE, eluting with methanol. The
appropriate fractions were combined and concentrated under a stream
of nitrogen to give the crude product. The crude purified by flash
silica (Si) chromatography (using a 0-100% ethyl
acetate-cyclohexane gradient). The appropriate fractions were
combined and evaporated in vacuo to give the required product, 527
mg as a yellow oil which solidified on standing. LCMS (M+15) 458,
RT 1.46 mins.
b) preparation of
(R)-3-((4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)-2-methylphenyl)ami-
no)-4-hydroxybutanoic acid
[0310] A solution of (S)-3-amino-4-hydroxybutanoic acid (17.04 mg,
0.143 mmol) was prepared in DMSO (1.0 mL) and
N-(2,4-dimethylphenyl)-4-fluoro-N-isobutyl-3-methylbenzenesulfonamide
(50 mg, 0.143 mmol) added. The solution was then treated with
sodium hydride (60% dispersion in oil, 17.17 mg, 0.429 mmol). The
reaction was stirred under nitrogen for 2 hours to aid dispersion,
then stood at room temperature for 5 days. The reaction was
quenched with isopropanol (0.5 mL) and water (0.5 mL), then
concentrated in vacuo. The crude was dissolved in a 1:1 mixture of
MeOH:DMSO (1 mL), filtered (cotton wool plug) and purified by MDAP
(Method A) to give the title compound, 8.2 mg. LCMS (M+1) 449, RT
0.93 mins.
Preparation of Product P162
(S)-2-((4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)amino)-3-hydr-
oxypropanoic acid
[0311] A solution of (S)-2-amino-3-hydroxypropanoic acid (15.67 mg,
0.149 mmol) was prepared in DMSO (1.0 mL) and
N-(2,4-dimethylphenyl)-4-fluoro-N-isobutylbenzenesulfonamide (50
mg, 0.149 mmol) added. The solution was then treated with sodium
hydride (60% dispersion in oil, 17.89 mg, 0.447 mmol). The reaction
was stirred under nitrogen for 2 hours to aid dispersion, then
quenched with isopropanol (0.5 mL) and water (0.5 mL) and
concentrated in vacuo. The sample was partitioned between water (5
mL) and dichloromethane (5 mL) and the organics separated
(hydrophobic frit). The crude was purified by MDAP (Method A) to
give the required product 1.6 mg. LCMS (M+1) 421, RT 0.93 min.
Preparation of Product P163
(S)-3-((4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)amino)-4-hydr-
oxybutanoic acid
[0312] A solution of (S)-3-amino-4-hydroxybutanoic acid (17.76 mg,
0.149 mmol) was prepared in DMSO (1.0 mL) and
N-(2,4-dimethylphenyl)-4-fluoro-N-isobutylbenzenesulfonamide (50
mg, 0.149 mmol) added. The solution was then treated with sodium
hydride (60% dispersion in oil, 17.89 mg, 0.447 mmol). The reaction
was stirred under nitrogen for 2 hours to aid dispersion, then
stood at room temperature for 5 days. The reaction was quenched
with isopropanol (0.5 mL) and water (0.5 mL), then concentrated in
vacuo. The crude was dissolved in a 1:1 mixture of MeOH:DMSO (1
mL), filtered (cotton wool plug) and purified by MDAP (Method A) to
give the title compound, 7.98 mg. LCMS (M+1) 435, RT 0.91 mins.
Preparation of Product P164
N-(2,4-dimethylphenyl)-N-isobutyl-4-(2H-tetrazol-5-yl)benzenesulfonamide
[0313] To a solution of (2,4-dimethylphenyl)(2-methylpropyl)amine
(100 mg, 0.564 mmol) in pyridine (2 mL) was added
4-(2H-tetrazol-5-yl)benzene-1-sulfonyl chloride (138 mg, 0.564
mmol) and the mixture stirred at 20.degree. C. for 30 minutes, then
stood at room temperature, in air, for 16 hours. The crude was then
purified by flash silica (Si) chromatography (using a 25-100% ethyl
acetate-cyclohexane gradient) to give a colourless gum. This was
further dried under a stream of nitrogen, then under vacuum for 3
hours, to give the final product, 104.87 mg. LCMS (M+1) 386, RT
1.22 mins.
Preparation of Product P165
(R)-3-((4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)amino)-4-hydr-
oxybutanoic acid
[0314] A solution of (R)-3-amino-4-hydroxybutanoic acid
hydrochloride (23.19 mg, 0.149 mmol) was prepared in DMSO (1.0 mL)
and N-(2,4-dimethylphenyl)-4-fluoro-N-isobutylbenzenesulfonamide
(50 mg, 0.149 mmol) added. The solution was then treated with
sodium hydride (60% dispersion in oil, 17.89 mg, 0.447 mmol). The
reaction was stirred under nitrogen for 2 hours to aid dispersion,
then stood at room temperature for 5 days. The reaction was
quenched with isopropanol (0.5 mL) and water (0.5 mL), then
concentrated in vacuo. The crude was dissolved in a 1:1 mixture of
MeOH:DMSO (1 mL), filtered (cotton wool plug) and purified by MDAP
(Method A) to give the title compound, 6.09 mg. LCMS (M+1) 435, RT
0.91 mins.
Preparation of Product P166
3-(4-(N-isobutyl-N-(2-methyl-5-(trifluoromethyl)phenyl)sulfamoyl)phenyl)pr-
opanoic acid
a) preparation of methyl
3-[4-({(2-methylpropyl)[2-methyl-5-(trifluoromethyl)phenyl]amino}sulfonyl-
)phenyl]propanoate
[0315] To a solution of
(2-methylpropyl)[2-methyl-5-(trifluoromethyl)phenyl]amine (50 mg,
0.216 mmol) in pyridine (5 mL) was added methyl
3-[4-(chlorosulfonyl)phenyl]propanoate (56.8 mg, 0.216 mmol). The
mixture was stirred for 30 minutes to ensure dilution, then stood
at room temperature, in air, for 16 hours. The solvent was
evaporated in vacuo to give the crude product which was purified by
MDAP (Method F) to give the required product, 21.5 mg. LCMS (M+1)
458, RT 1.39 mins.
b) preparation of
3-(4-(N-isobutyl-N-(2-methyl-5-(trifluoromethyl)phenyl)sulfamoyl)phenyl)p-
ropanoic acid
[0316] To a stirred solution of methyl
3-(4-(N-isobutyl-N-(2-methyl-5-(trifluoromethyl)phenyl)sulfamoyl)phenyl)p-
ropanoate (15 mg, 0.033 mmol) in tetrahydrofuran (THF) (1.5 mL) at
room temperature, was added a solution of lithium hydroxide
(approximately 0.785 mg, 0.033 mmol--tip of spatula used) in water
(1.5 mL). The reaction mixture was stirred at 20.degree. C. for 4
hours and then stood overnight. The mixture was concentrated in
vacuo then partitioned between water (2 mL) and ethyl acetate
(2.times.5 mL). The aqueous layer was separated (hydrophilic frit)
and acidified, then extracted with ethyl acetate (5 mL). The
organic layer was separated (hydrophobic frit) and concentrated in
vacuo to give required product, 2.4 mg. LCMS (M+1) 444, RT 1.25
mins.
Preparation of Product P167
3-(4-(N-isobutyl-N-(2-methyl-5-(trifluoromethyl)phenyl)sulfamoyl)phenyl)-2-
,2-dimethylpropanoic acid
a) preparation of ethyl
3-(4-(N-isobutyl-N-(2-methyl-5-(trifluoromethyl)phenyl)sulfamoyl)phenyl)--
2,2-dimethylpropanoate
[0317] To a stirred solution of
(2-methylpropyl)[2-methyl-5-(trifluoromethyl)phenyl]amine (50 mg,
0.216 mmol) in pyridine (5 mL) in air, at room temperature, was
added ethyl 3-[4-(chlorosulfonyl)phenyl]-2,2-dimethylpropanoate
(65.9 mg, 0.216 mmol). The reaction mixture was stirred at
20.degree. C. for 30 minutes, then stood overnight. The solvent was
evaporated in vacuo to give the crude material which was then
purified by MDAP (Method F) to give the required product, 20 mg.
LCMS (M+1) 500, RT 1.53 mins.
b) preparation of
3-(4-(N-isobutyl-N-(2-methyl-5-(trifluoromethyl)phenyl)sulfamoyl)phenyl)--
2,2-dimethylpropanoic acid
[0318] To a stirred solution of ethyl
3-(4-(N-isobutyl-N-(2-methyl-5-(trifluoromethyl)phenyl)sulfamoyl)phenyl)--
2,2-dimethylpropanoate (15 mg, 0.030 mmol) in tetrahydrofuran (THF)
(1.5 mL), at room temperature, was added a solution of lithium
hydroxide (approximately 0.719 mg, 0.030 mmol--tip of spatula used)
in water (1.5 mL). The reaction mixture was stirred at 20.degree.
C. for 4 hours and then stood overnight. The mixture was evaporated
in vacuo, then partitioned between water (2 mL) and ethyl acetate
(2.times.5 mL). The organic fraction was separated (hydrophilic
frit) and evaporated in vacuo to give a residue. This was
redissolved in THF (1 mL) and water (1 mL) and new lithium
hydroxide (tip of spatula) was added. The reaction was stood for 3
days, after which time the solvents had evaporated to give a white
residue. The residue was acidified (2N HCl, 3 mL) and extracted
with ethyl acetate (2.times.5 mL). The organics were separated
(hydrophilic frit) and concentrated in vacuo, then purified by MDAP
(Method F) to give the required product, 2.38 mg. LCMS (M+1) 472,
RT 1.37 mins.
Preparation of Product P168
5-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)-2-methoxybenzoic
acid
[0319] To a solution of (2,4-dimethylphenyl)(2-methylpropyl)amine
(200 mg, 1.128 mmol) in pyridine (3 mL) was added
5-(chlorosulfonyl)-2-methoxybenzoic acid (283 mg, 1.128 mmol) and
the reaction stirred at 20.degree. C. for 4 hours. The solvent was
removed in vacuo and the crude purified by flash silica (Si)
chromatography (using a 0-100%, ethyl acetate-dichloromethane
gradient, with extra 0-20% methanol added). The appropriate
fractions were combined and evaporated in vacuoto give the required
product, 108 mg. LCMS (M+1) 392, RT 1.18 mins.
Preparation of Product P169
2-((4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)amino)-3-hydroxyp-
ropanoic acid
[0320] A solution of 2-amino-3-hydroxypropanoic acid (15.67 mg,
0.149 mmol) was prepared in DMSO (1.0 mL) and
N-(2,4-dimethylphenyl)-4-fluoro-N-isobutylbenzenesulfonamide (50
mg, 0.149 mmol) added. The solution was then treated with sodium
hydride (60% dispersion in oil, 5.96 mg, 0.149 mmol) and the
reaction stirred under nitrogen for 2 hours. The mixture was then
quenched with isopropanol (0.5 mL) and water (0.5 mL) and the
solvent removed in vacuo. The sample was partitioned between water
(5 mL) and dichloromethane (5 mL) and the organics separated
(hydrophobic frit). The crude was purified by MDAP (Method A) to
give the required product 1.0 mg. LCMS (M+1) 421, RT 1.17 mins.
Preparation of Product P170
N-(2,6-difluorophenyl)-4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(2-met-
hylpropyl)benzenesulfonamide
a) preparation of 2,6-difluoro-N-(2-methylpropyl)aniline
[0321] 2,6-difluoroaniline (387 mg, 3 mmol), potassium iodide (996
mg, 6.00 mmol) and [Cp*IrCl.sub.2].sub.2 (33.1 mg, 0.030 mmol) were
dissolved in 2-methyl-1-propanol (3 mL, 32.4 mmol). The resulting
mixture was heated by microwaves to 190.degree. C. for 8 hours. To
the reaction mixture was added dichloromethane (10 mL) and water
(10 mL) and the organic phase separated (hydrophobic frit). Diethyl
ether was added and the solution filtered through a silica
cartridge (10 g) eluting with further diethyl ether. The filtrate
was evaporated in vacuo, then purified by flash silica (Si)
chromatography (using a 0-100% dichloromethane-cyclohexane
gradient). The appropriate fractions were combined and concentrated
to give the title product (80 mg) as a colourless oil. LCMS (M+1)
186, RT 1.26 mins.
b) preparation of
N-(2,6-difluorophenyl)-4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}-N-(2-me-
thylpropyl)benzenesulfonamide
[0322] To a solution of (2,6-difluorophenyl)(2-methylpropyl)amine
(80 mg, 0.432 mmol) in pyridine (1 mL) was added
4-{[(3,5-dimethyl-4-isoxazolyl)methyl]oxy}benzenesulfonyl chloride
(156 mg, 0.518 mmol) and the reaction stood at room temperature, in
air, for 16 hours. The solvent was removed in vacuo and the crude
then passed through a sulfonic acid (SCX) SPE (solid phase
extraction) cartridge eluting with methanol followed by 2M
ammonia/methanol solution. The product-containing fractions were
concentrated in vacuo and the crude then passed through an
aminopropyl (NH.sub.2) SPE cartridge eluting with methanol followed
by 2M ammonia/methanol solution. The appropriate fractions were
combined and concentrated in vacuo, then further purified by MDAP
(Method F) to give the required product, 0.6 mg. LCMS (M+18) 468,
RT 1.28 mins.
Preparation of Product P171
2-bromo-5-(N-(4-ethylphenyl)-N-isobutylsulfamoyl)benzoic acid
a) preparation of methyl
2-bromo-5-(N-(4-ethylphenyl)-N-isobutylsulfamoyl)benzoate
[0323] To a solution of 4-ethyl-N-isobutylaniline (400 mg, 2.256
mmol) in pyridine (2 mL) was added methyl
2-bromo-5-(chlorosulfonyl)benzoate (707 mg, 2.256 mmol)
portionwise, over 30 minutes. The reaction mixture was stirred at
20.degree. C. for 30 minutes then stood at room temperature, in
air, for 16 hours. The solvent was evaporated in vacuo to give a
crude yellow solid. The crude was then triturated with methanol,
filtered and dried in vacuo to give the desired product (600 mg) as
a white solid. LCMS (M+1) 454/456, RT 1.41 mins.
b) preparation of
2-bromo-5-(N-(4-ethylphenyl)-N-isobutylsulfamoyl)benzoic acid
[0324] To a suspension of methyl
2-bromo-5-(N-(4-ethylphenyl)-N-isobutylsulfamoyl)benzoate (300 mg,
0.660 mmol), palladium(II) acetate (14.82 mg, 0.066 mmol),
1-(vinyloxy)butane (BVE) (0.427 mL, 3.30 mmol) and
triphenylphosphine (34.6 mg, 0.132 mmol) in acetonitrile (1 mL) in
air, at room temperature, was added triethylamine (0.110 mL, 0.792
mmol). The reaction vessel was sealed and heated by microwaves to
150.degree. C. for 1 hour then the solvent was evaporated in vacuo
and the residue redissolved in tetrahydrofuran (THF) (1.0 mL) and
treated with HCl (2N, 1 mL, 3.29 mmol). The mixture was stirred
vigorously for 1 hour then stood overnight. Analysis showed
hydrolysed starting material was present. The crude was purified by
MDAP (Method F) to isolate the hydrolysed starting material as the
product, 84.8 mg. LCMS (M+1) 440/442, RT 1.25 mins.
Preparation of Product P172
2-(4-(N-(4-butyl-2-methylphenyl)-N-isobutylsulfamoyl)-2-methylphenoxy)acet-
ic acid
[0325] To (4-butyl-2-methylphenyl)(2-methylpropyl)amine (21.94 mg,
0.100 mmol) in pyridine (1 mL) was added
{[4-(chlorosulfonyl)-2-methylphenyl]oxy}acetic acid (26.5 mg, 0.100
mmol) and reaction stood for 2 hours at room temperature. The
sample was then dissolved in a 1:1 mixture of MeOH and DMSO and
purified by MDAP Method A2, to provide the desired product, 11 mg.
LCMS (M+1) 448, RT 1.44 mins.
Preparation of Product P173
4-((2-(dideuterioamino)pyridin-4-yl)methoxy)-N-(2,4-dimethylphenyl)-N-isob-
utylbenzenesulfonamide
[0326] To a solution of (2-aminopyridin-4-yl)methanol (37.0 mg,
0.298 mmol) and
N-(2,4-dimethylphenyl)-4-fluoro-N-isobutylbenzenesulfonamide (100
mg, 0.298 mmol) in d6-DMSO (1 mL) stirred in air at room
temperature, was added sodium hydride (60% dispersion in mineral
oil, 23.85 mg, 0.596 mmol). The reaction mixture was stirred at
20.degree. C. for 2 hours then carefully quenched with methanol (2
mL) and water (2 mL). The reaction mixture was evaporated in vacuo
and purified by MDAP Method A, to give the desired dideuterated
product, 47.5 mg, as a yellow oil. High-pH LCMS (M+1) 441, RT 1.34
mins.
Preparation of Product P174
N-(2,4-dimethylphenyl)-4-(1-hydroxy-3-(2H-tetrazol-5-yl)propyl)-N-isobutyl-
benzenesulfonamide
a) preparation of 5-methyl-2-trityl-2H-tetrazole
[0327] To a solution of 5-methyl-1H-tetrazole (1.513 g, 18.00
mmol), tetrabutylammonium bromide (0.029 g, 0.090 mmol) and sodium
hydroxide (18.90 mL, 18.90 mmol) in dichloromethane (DCM) (25 mL)
stirred under nitrogen at room temperature, was added
(chloromethanetriyl)tribenzene (5.02 g, 18 mmol). The reaction
mixture was stirred at 20.degree. C. for 72 hours. The reaction was
quenched with water, then partitioned between dichloromethane (10
mL) and water (25 mL). The organic phase was separated (hydrophobic
frit) and the aqueous phase re-extracted (stirred for 5 min) with
additional DCM (10 mL), then separated with a hydrophobic frit. The
combined organic fractions were washed with water (2.times.25 mL),
5% aqueous sodium bicarbonate (10 mL) and brine (10 mL). The
organic phase was evaporated in vacuo to give a white solid.
Recrystallisation from hot ethyl acetate (8 mL/g) provided the
product as a white solid (2.2 g). LCMS RT 1.35 mins.
b) preparation of
N-(2,4-dimethylphenyl)-4-(1-hydroxy-3-(2-trityl-2H-tetrazol-5-yl)propyl)--
N-isobutylbenzenesulfonamide
[0328] To a solution of 5-methyl-2-trityl-2H-tetrazole (68.5 mg,
0.21 mmol) in tetrahydrofuran (THF) (1 mL), stirred under nitrogen
at -70.degree. C., was added a solution of n-BuLi (1.6 M in
hexanes, 0.144 mL, 0.231 mmol) in Tetrahydrofuran (THF) (1 mL)
dropwise over 1 minute. The reaction mixture was then stirred at
-78.degree. C. for 45 minutes.
N-(2,4-dimethylphenyl)-N-isobutyl-4-(oxiran-2-yl)benzenesulfonamide
(108 mg, 0.300 mmol) in Tetrahydrofuran (THF) (1 mL) was added
dropwise at -78.degree. C. over 1 minute. The reaction mixture was
then stirred at -78.degree. C. for a further 1 hour and then
allowed to warm to room temperature. The reaction was carefully
quenched using a few drops of water, under nitrogen. Ethyl acetae
(3 mL) and water (2 mL) were added and the reaction stirred
vigorously for 5 minutes. The organic phase was separated using a
hydrophilic frit and concentrated in vacuo to give a residue. LCMS
analysis showed incomplete reaction, so the above reaction
procedure repeated using fresh n-butyl lithium solution and
reintroducing the crude residue instead of new starting material,
this resulted in improved conversion. The residue was then purified
by flash (Si) chromatography (using a 0-25% ethyl
acetate-cyclohexane gradient) to give the required product (20 mg)
as a colourless gum. LCMS RT 1.56 mins.
c) preparation of
N-(2,4-dimethylphenyl)-4-(1-hydroxy-3-(2H-tetrazol-5-yl)propyl)-N-isobuty-
lbenzenesulfonamide
[0329] N-(2,4-di methyl
phenyl)-4-(1-hydroxy-3-(2-trityl-2H-tetrazol-5-yl)propyl)-N-isobutylbenze-
nesulfonamide (20 mg, 0.029 mmol), at room temperature was treated
with a solution of HCl (6N in isopropanol, 2 mL, 12.00 mmol) and
isopropanol (2 mL). The reaction mixture was stirred at 20.degree.
C. for 2 hours, then the solvent evaporated in vacuo and the crude
partitioned between ethyl acetate (5 mL) and water (5 mL). The
organic phase was separated by hydrophilic frit then evaporated in
vacuo to give a residue. LCMS analysis showed the reaction was
incomplete, so the residue was re-dissolved in dichloromethane
(DCM) (2 mL) and treated with formic acid (0.5 mL, 13.04 mmol). The
solution was left to stand for 2 hours. The solvent was evaporated
in vacuo then dissolved in a 1:1 mixture of MeOH and DMSO and
purified by MDAP (Method F) to give the required product, 3.69 mg.
LCMS (M+1) 444, RT 1.09 mins.
Preparation of Product P175
N-(2,4-dimethylphenyl)-4-(1-hydroxy-2-(2H-tetrazol-5-yl)ethyl)-N-isobutylb-
enzenesulfonamide
a) preparation of
N-(2,4-dimethylphenyl)-4-formyl-N-isobutylbenzenesulfonamide
[0330] To a solution of (2,4-dimethylphenyl)(2-methylpropyl)amine
(400 mg, 2.256 mmol) in pyridine (5 mL), stirred in air at room
temperature, was added 4-formylbenzene-1-sulfonyl chloride (760 mg,
3.71 mmol) in one charge. The reaction mixture was stirred at
20.degree. C. for 30 minutes, then left to stand 6 hours. The
solvent was removed in vacuo and the crude material purified by
flash (Si) chromatography (using a 0-25% ethyl acetate-cyclohexane
gradient). The appropriate fractions were combined and evaporated
in vacuo to give the required product (794 mg) as a colourless gum.
LCMS (M+1) 346, RT 1.33 mins.
b) preparation of
N-(2,4-dimethylphenyl)-4-(1-hydroxy-2-(2H-tetrazol-5-yl)ethyl)-N-isobutyl-
benzenesulfonamide
[0331] To a solution of 5-methyl-2-trityl-2H-tetrazole (68.5 mg,
0.21 mmol) in tetrahydrofuran (THF) (1 mL), stirred under nitrogen
at -70.degree. C., was added a solution of n-BuLi (1.6 M in
hexanes, 0.144 mL, 0.231 mmol) and tetrahydrofuran (THF) (1 mL)
dropwise over 1 minute. The reaction mixture was stirred at
-78.degree. C. for 45 minutes.
N-(2,4-dimethylphenyl)-4-formyl-N-isobutylbenzenesulfonamide (222
mg, 0.643 mmol) in tetrahydrofuran (THF) (1 mL) was then added
dropwise at -78.degree. C. over 1 minute. The reaction mixture was
stirred at -78.degree. C. for 2 hours, then allowed to warm to room
temperature. The reaction was carefully quenched using a few drops
of water under nitrogen. Ethyl acetate (3 mL) and water (2 mL) were
added and the reaction stirred vigorously for 5 minutes. The
organic phase was separated (hydrophilic frit) then evaporated in
vacuo to give a residue. The residue was redissolved in
dichloromethane (DCM) (2 mL) and treated with formic acid (0.5 mL,
13.04 mmol). The solution was stood overnight, then the solvent
evaporated in vacuo and the crude purified by MDAP (Method F) to
give the required product, 17.3 mg. LCMS (M+1) 430, RT 1.09
mins.
Preparation of Product P176
N-(2,4-dimethylphenyl)-4-(2-hydroxy-1-(2H-tetrazol-5-yl)propan-2-yl)-N-iso-
butylbenzenesulfonamide
a) preparation of
4-acetyl-N-(2,4-dimethylphenyl)-N-isobutylbenzenesulfonamide
[0332] To a solution of (2,4-dimethylphenyl)(2-methylpropyl)amine
(300 mg, 1.692 mmol) in pyridine (5 mL), stirred in air at room
temperature, was added 4-acetylbenzene-1-sulfonyl chloride (370 mg,
1.692 mmol) in one charge. The reaction mixture was stirred at
20.degree. C. for 30 minutes, then left to stand overnight. The
solvent was evaporated in vacuo then redissolved in methanol and
passed through an aminopropyl (NH2) SPE cartridge, eluting with
methanol, followed by 2M ammonia/methanol. The product containing
fractions were combined and passed through a sulphonic acid (SCX)
SPE cartridge eluting with methanol, followed by 2M
ammonia/methanol. The product containing fractions were combined
and evaporated under a stream of nitrogen to give the crude
product. The crude was purified by flash (Si) chromatography (using
a 0-50% ethyl acetate-cyclohexane gradient) to give the required
product (282 mg) as a yellow gum. LCMS (M+1) 360, RT 1.35 mins.
b) preparation of
N-(2,4-dimethylphenyl)-4-(2-hydroxy-1-(2-trityl-2H-tetrazol-5-yl)propan-2-
-yl)-N-isobutylbenzenesulfonamide
[0333] To a solution of 5-methyl-2-trityl-2H-tetrazole (68.5 mg,
0.21 mmol) in tetrahydrofuran (THF) (1 mL) stirred under nitrogen
at -70.degree. C., was added a solution of n-BuLi (1.6 M in
hexanes, 0.144 mL, 0.231 mmol) and tetrahydrofuran (THF) (1 mL)
dropwise over 1 minute. The reaction mixture was stirred at
-78.degree. C. for 45 minutes, then
4-acetyl-N-(2,4-dimethylphenyl)-N-isobutylbenzenesulfonamide (106
mg, 0.294 mmol) in tetrahydrofuran (THF) (1 mL) was added dropwise
at -78.degree. C. over 1 minute. The reaction mixture was stirred
at -78.degree. C. for 1 hour, then allowed to warm to room
temperature. The reaction was carefully quenched using a few drops
of water under nitrogen. Ethyl acetate (3 mL) and water (2 mL) were
then added and the reaction stirred vigorously for 5 minutes. The
organic phase was separated using a hydrophilic frit and
concentrated in vacuo to give a residue. LCMS analysis showed
incomplete reaction, so the above reaction procedure repeated using
fresh n-butyl lithium solution and reintroducing the crude residue
instead of new starting material, this resulted in improved
conversion. The residue was then purified by flash silica (Si)
chromatography (using a 0-25% ethyl acetate-cyclohexane gradient)
to give the required product (22 mg) as a colourless gum. LCMS
(M+1) 686, RT 1.57 mins.
c) preparation of
N-(2,4-dimethylphenyl)-4-(2-hydroxy-1-(2H-tetrazol-5-yl)propan-2-yl)-N-is-
obutylbenzenesulfonamide
[0334]
N-(2,4-dimethylphenyl)-4-(2-hydroxy-1-(2-trityl-2H-tetrazol-5-yl)pr-
opan-2-yl)-N-isobutylbenzenesulfonamide (22 mg, 0.032 mmol) at room
temperature, was treated with a solution of HCl (6N in isopropanol,
2 mL, 12.00 mmol) and isopropanol (2 mL). The reaction mixture was
stirred at 20.degree. C. for 2 hours then the solvent evaporated in
vacuo and the crude partitioned between ethyl acetate (5 mL) and
water (5 mL). The organic phase was separated by hydrophilic frit
then evaporated in vacuo to give a residue. LCMS analysis showed
the reaction was incomplete, so the residue was redissolved in
dichloromethane (DCM) (2 mL) and treated with formic acid (0.5 mL,
13.04 mmol). The solution was left to stand for 2 hours. The
solvent was evaporated in vacuo then dissolved in a 1:1 mixture of
MeOH and DMSO and purified by MDAP (Method F) to give the required
product, 5.3 mg. LCMS (M+1) 444, RT 1.10 mins.
Preparation of Product P177
N-(4-(1,3-dihydroxypropan-2-yl)phenyl)-4-(((3,5-dimethylisoxazol-4-yl)oxy)-
methyl)-N-isobutylbenzenesulfonamide
a) preparation of
2,2,3,3,9,9,10,10-octamethyl-6-(4-nitrophenyl)-4,8-dioxa-3,9-disilaundeca-
ne
[0335] To a solution of 2-(4-nitrophenyl)propane-1,3-diol (120 mg,
0.609 mmol) and 1H-imidazole (83 mg, 1.217 mmol) in tetrahydrofuran
(THF) (5 mL) was added tert-butylchlorodimethylsilane (220 mg,
1.461 mmol). The solution was stirred overnight at room
temperature, under nitrogen. An additional 1 eq of
tert-butylchlorodimethylsilane was added to the reaction and the
mixture stirred, under nitrogen, over the weekend. The reaction was
concentrated in vacuo and the product extracted into the organic
phase of an aqueous work up between ethyl acetate and water. The
organic phase was then passed through a hydrophobic frit and
concentrated in vacuo to give the desired crude product, 219.6 mg,
which was used directly in the next step without further
purification. LCMS RT 1.84 mins.
b) preparation of
4-(2,2,3,3,9,9,10,10-octamethyl-4,8-dioxa-3,9-disilaundecan-6-yl)aniline
[0336] A solution of
2,2,3,3,9,9,10,10-octamethyl-6-(4-nitrophenyl)-4,8-dioxa-3,9-disilaundeca-
ne (219.6 mg, 0.516 mmol) was prepared in ethanol (10.32 mL). This
was hydrogenated using the H-cube flow hydrogenator (settings:
20.degree. C., 10 bar H.sub.2, 1 mL/min flow rate) and a 10% Pd/C
CatCart 30 catalyst. The collected solvent was evaporated by a
stream nitrogen to give the desired product, 193.1 mg, which was
used in the next step without purification. LCMS (M+1) 396, RT 1.72
mins.
c) preparation of
N-isobutyl-4-(2,2,3,3,9,9,10,10-octamethyl-4,8-dioxa-3,9-disilaundecan-6--
yl)aniline
[0337] A mixture of
4-(2,2,3,3,9,9,10,10-octamethyl-4,8-dioxa-3,9-disilaundecan-6-yl)aniline
(193.1 mg, 0.488 mmol), isobutanol (0.090 mL, 0.976 mmol),
potassium iodide (162 mg, 0.976 mmol), [Cp*IrCl.sub.2].sub.2 (3.89
mg, 4.88 .mu.mol) and water (3 mL) was prepared in a microwave
vial. The reaction vessel was sealed and heated by microwaves to
150.degree. C. for 1.5 hours. After cooling, the solution was
diluted with water and dichloromethane (20 mL), then the organic
phase separated (hydrophobic frit) and concentrated in vacuo. The
crude was purified by flash silica (Si) chromatography (using a
0-100% dichloromethane-cyclohexane gradient) to give the desired
product, 155.9 mg. LCMS (M+1) 452, RT 1.96 mins.
d) preparation of
4-(((3,5-dimethylisoxazol-4-yl)oxy)methyl)-N-isobutyl-N-(4-(2,2,3,3,9,9,1-
0,10-octamethyl-4,8-dioxa-3,9-disilaundecan-6-yl)phenyl)benzenesulfonamide
[0338] A solution of
4-((3,5-dimethylisoxazol-4-yl)methoxy)benzene-1-sulfonyl chloride
(125 mg, 0.414 mmol) and
N-isobutyl-4-(2,2,3,3,9,9,10,10-octamethyl-4,8-dioxa-3,9-disilaundecan-6--
yl)aniline (155.9 mg, 0.345 mmol) was prepared in pyridine (3 mL)
and stood overnight. The solution was concentrated in vacuo and the
product extracted to the organic phase of an acidic work up between
ethyl acetate and 5% citric acid. The organic phase was separated
(hydrophobic frit) and concentrated in vacuo to give the crude
product, 214.7 mg, which was used directly in the next step with no
further purification. LCMS (M+1) 717, RT 1.87 mins.
e) preparation of
N-(4-(1,3-dihydroxypropan-2-yl)phenyl)-4-(((3,5-dimethylisoxazol-4-yl)oxy-
)methyl)-N-isobutylbenzenesulfonamide
[0339] To a solution of
4-(((3,5-dimethylisoxazol-4-yl)oxy)methyl)-N-isobutyl-N-(4-(2,2,3,3,9,9,1-
0,10-octamethyl-4,8-dioxa-3,9-disilaundecan-6-yl)phenyl)benzenesulfonamide
(214.7 mg, 0.299 mmol) in tetrahydrofuran (THF) (10 mL) was added
TBAF (1 M in THF, 0.748 mL, 0.748 mmol), and this was stirred at
room temperature for 1 hour. The reaction mixture was concentrated
in vacuo and extracted to the organic phase of an aqueous workup
between ethyl acetate and water/brine. The organic phase was
separated (hydrophobic frit), then concentrated in vacuo and
purified by flash silica (Si) chromatography (using a 0-100% ethyl
acetate-cyclohexane gradient). The product containing fractions
were combined and condensed, then freeze-dried to provide 76.4 mg
of desired product. LCMS (M+1) 489, RT 1.02 mins.
Preparation of Product P178
2-(4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)acetic
acid
a) preparation of methyl
2-(4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)acetate
[0340] A solution of N-isobutyl-2,4-dimethylaniline (740 mg, 4.17
mmol) and methyl 2-(4-(chlorosulfonyl)phenyl)acetate (1246 mg, 5.01
mmol) was prepared in pyridine (4 mL) and stood for 2 hours. The
reaction was then concentrated in vacuo and the product extracted
to the organic phase of an acidic work up between ethyl acetate and
5% citric acid. The organic phase was separated (hydrophobic frit)
then concentrated in vacuo and purified by flash silica (Si)
chromatography (using a 0-100% dichloromethane-cyclohexane
gradient) to give the desired product, 1.4199 g. LCMS (M+1) 390, RT
1.37 mins.
b) preparation of
2-(4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)acetic
acid
[0341] A solution of methyl
2-(4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)phenyl)acetate
(1.3799 g, 3.54 mmol) in tetrahydrofuran (THF) (8 mL) and water (2
mL), was prepared and treated with sodium hydroxide (1.063 mL,
10.63 mmol). The reaction was then heated by microwaves to
110.degree. C., for 30 minutes. After cooling, crude material from
a test of the above reaction run on a smaller scale (.about.40 mg)
was combined with this mixture. The combined solution was then
neutralised with 2M HCl and diluted with further water and ethyl
acetate. The product was extracted into the organic phase and the
organics separated (hydrophobic frit) then concentrated in vacuo to
provide 1.3033 g of desired material. LCMS (M+1) 376, RT 1.24
mins.
Preparation of Product P179
4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)benzoic acid
a) preparation of methyl
4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)benzoate
[0342] A solution of N-isobutyl-2,4-dimethylaniline (0.63 g, 3.55
mmol) and methyl 4-(chlorosulfonyl)benzoate (1.001 g, 4.26 mmol)
was prepared in pyridine (4 mL) and stood for 2 hours. The reaction
was then concentrated in vacuo and the product extracted to the
organic phase of an acidic work up between ethyl acetate and 5%
citric acid. The organic phase was separated (hydrophobic frit)
then concentrated in vacuo and purified by flash silica (Si)
chromatography (using a 0-25% ethyl acetate-cyclohexane gradient)
to give the desired product, 1.2698 g. LCMS (M+1) 376, RT 1.44
mins.
b) preparation of
4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)benzoic acid
[0343] A solution of methyl
4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)benzoate (1.2698 g,
3.38 mmol) in tetrahydrofuran (THF) (8 mL) and water (2 mL), was
prepared and treated with sodium hydroxide (1.015 mL, 10.15 mmol).
The reaction was then heated by microwaves to 110.degree. C., for
30 minutes. After cooling, the solution was neutralised with 2M HCl
and concentrated in vacuo. The product was extracted into the
organic phase of an aqueous work up between water and ethyl
acetate. The organic phase was then separated (hydrophobic frit)
and concentrated in vacuo to give the desired product, 1.063 g.
LCMS (M+1) 362, RT 1.27 mins.
Preparation of Product P180
4-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)benzoic acid, ammonia
salt
[0344] N-(2,4-dimethylphenyl)-N-isobutyl-4-vinylbenzenesulfonamide
(0.06 g, 0.175 mmol) was dissolved in acetone (0.240 mL) and cooled
to .about.2.degree. C., with stirring. A solution of potassium
permanganate (0.018 g, 0.117 mmol) and magnesium sulfate (7.00 mg,
0.058 mmol) in water (0.4 mL) was prepared and added dropwise to
the acetone solution, over 20 minutes. The mixture was stirred for
an additional 10 minutes, then the temperature increased to room
temperature and the mixture filtered and concentrated under a
stream of nitrogen. LCMS analysis confirmed presence of some acid
product as well as dihydroxylated product, but showed mainly
unreacted starting material. So above procedure was repeated again
using this crude material, with the post-addition stirring
increased to 2.75 hours at 0.degree. C. then 15 minutes warming to
rt. Analysis confirmed improved conversion, but still remaining
starting material. So the procedure was repeated again using this
crude material, but with the post-addition stirring increased to
overnight and temperature allowed to warm from 0.degree. C. to room
temperature over this time. Analysis now confirmed all starting
material had been consumed with an increased conversion to the acid
product. The crude was dissolved in methanol (5 mL) and passed
through an aminopropyl (NH.sub.2) cartridge (washed through with
methanol), however the initial flush failed to adequately separate
the acid form the dihydroxylated product. A second flush of the
cartridge with methanol-ammonia solution successfully eluted the
desired ammonia salt of the acid product as a white solid, 9 mg.
LCMS (M+1) 362, RT 1.24 mins.
Preparation of Product P181
3-(N-(2,4-dimethylphenyl)-N-isobutylsulfamoyl)benzoic acid
[0345] A solution of 3-(chlorosulfonyl)benzoic acid (439 mg, 1.992
mmol) and N-isobutyl-2,4-dimethylaniline (294 mg, 1.660 mmol) was
prepared in pyridine (5 mL) and stood overnight at room
temperature. An additional 0.5 eq of 3-(chlorosulfonyl)benzoic acid
was then added and the reaction stood for a second night at room
temperature. The solvents were then removed in vacuo and the
product extracted to the organic phase of an acidic work up between
ethyl acetate and 5% citric acid. The organic phase was separated
(hydrophobic frit) then concentrated in vacuo and purified by flash
silica (Si) chromatography (using a 0-25% ethyl acetate-cyclohexane
gradient). The product containing fractions were combined and
condensed to provide 99.1 mg of desired material. LCMS (M+1) 362,
RT 1.24 mins.
TABLE-US-00005 TABLE 5 Chemical Structures of A References ID
Structure A1 ##STR00056## A2 ##STR00057## A3 ##STR00058## A4
##STR00059## A5 ##STR00060## A6 ##STR00061## A7 ##STR00062## A8
##STR00063## A9 ##STR00064## A10 ##STR00065## A11 ##STR00066## A12
##STR00067## A13 ##STR00068## A14 ##STR00069## A15 ##STR00070## A16
##STR00071## A17 ##STR00072## A18 ##STR00073## A19 ##STR00074## A20
##STR00075## A21 ##STR00076## A22 ##STR00077## A23 ##STR00078## A24
##STR00079## A25 ##STR00080## A26 ##STR00081## A27 ##STR00082## A28
##STR00083## A29 ##STR00084## A30 ##STR00085## A31 ##STR00086## A32
##STR00087## A33 ##STR00088## A34 ##STR00089## A35 ##STR00090## A36
##STR00091## A37 ##STR00092## A38 ##STR00093## A39 ##STR00094##
TABLE-US-00006 TABLE 6 Chemical Structures of B References ID
Structure B1 ##STR00095## B2 ##STR00096## B3 ##STR00097## B4
##STR00098## B5 ##STR00099## B6 ##STR00100## B7 ##STR00101## B8
##STR00102## B9 ##STR00103## B11 ##STR00104## B12 ##STR00105## B13
##STR00106## B14 ##STR00107## B15 ##STR00108## B16 ##STR00109## B17
##STR00110## B18 ##STR00111## B19 ##STR00112## B20 ##STR00113## B21
##STR00114## B22 ##STR00115## B23 ##STR00116## B24 ##STR00117## B25
##STR00118## B26 ##STR00119## B27 ##STR00120## B28 ##STR00121## B29
##STR00122## B30 ##STR00123## B31 ##STR00124## B32 ##STR00125## B33
##STR00126## B34 ##STR00127## B35 ##STR00128## B36 ##STR00129## B37
##STR00130## B38 ##STR00131## B39 ##STR00132## B40 ##STR00133##
TABLE-US-00007 TABLE 7 Chemical Structures of Products (P) ID
Structure P1a-d ##STR00134## P2a-c ##STR00135## P3 ##STR00136## P4
##STR00137## P5 ##STR00138## P6 ##STR00139## P7 ##STR00140## P8
##STR00141## P9 ##STR00142## P10 ##STR00143## P11 ##STR00144## P12
##STR00145## P13 ##STR00146## P14 ##STR00147## P15 ##STR00148## P16
##STR00149## P17 ##STR00150## P18 ##STR00151## P19 ##STR00152## P20
##STR00153## P21 ##STR00154## P22 ##STR00155## P23 ##STR00156## P24
##STR00157## P25 ##STR00158## P26 ##STR00159## P27 ##STR00160## P28
##STR00161## P29 ##STR00162## P30 ##STR00163## P31 ##STR00164## P32
##STR00165## P33 ##STR00166## P34 ##STR00167## P35 ##STR00168## P36
##STR00169## P37 ##STR00170## P38 ##STR00171## P39 ##STR00172## P40
##STR00173## P41 ##STR00174## P42 ##STR00175## P43 ##STR00176## P44
##STR00177## P45 ##STR00178## P46 ##STR00179## P47 ##STR00180## P48
##STR00181## P49 ##STR00182## P50 ##STR00183## P51 ##STR00184## P52
##STR00185## P53 ##STR00186## P54 ##STR00187## P55 ##STR00188## P56
##STR00189## P57 ##STR00190## P58 ##STR00191## P59 ##STR00192## P60
##STR00193## P61 ##STR00194## P62 ##STR00195## P63 ##STR00196## P64
##STR00197## P65 ##STR00198## P66 ##STR00199## P67 ##STR00200## P68
##STR00201## P69 ##STR00202## P70 ##STR00203## P71 ##STR00204## P72
##STR00205## P73 ##STR00206## P74 ##STR00207## P75 ##STR00208## P76
##STR00209## P77 ##STR00210## P78 ##STR00211## P79 ##STR00212## P80
##STR00213## P81 ##STR00214## P82 ##STR00215## P83 ##STR00216## P84
##STR00217## P85 ##STR00218## P86 ##STR00219## P87 ##STR00220## P88
##STR00221## P89 ##STR00222## P90 ##STR00223## P91 ##STR00224## P92
##STR00225## P93 ##STR00226## P94 ##STR00227## P95 ##STR00228## P96
##STR00229## P97 ##STR00230## P98 ##STR00231## P99 ##STR00232##
P100 ##STR00233## P101 ##STR00234## P102 ##STR00235## P103
##STR00236## P104 ##STR00237## P105 ##STR00238## P106 ##STR00239##
P107 ##STR00240## P108 ##STR00241## P109 ##STR00242## P110
##STR00243## P111 ##STR00244## P112 ##STR00245## P113 ##STR00246##
P114 ##STR00247## P115 ##STR00248## P116 ##STR00249## P117
##STR00250## P118 ##STR00251## P119 ##STR00252## P120 ##STR00253##
P121 ##STR00254## P122 ##STR00255## P123 ##STR00256##
P124 ##STR00257## P125 ##STR00258## P126 ##STR00259## P127
##STR00260## P128 ##STR00261## P129 ##STR00262## P130 ##STR00263##
P131 ##STR00264## P132 ##STR00265## P133 ##STR00266## P134
##STR00267## P135 ##STR00268## P136 ##STR00269## P137 ##STR00270##
P138 ##STR00271## P139 ##STR00272## P140 ##STR00273## P141
##STR00274## P142 ##STR00275## P143 ##STR00276## P144 ##STR00277##
P145 ##STR00278## P146 ##STR00279## P147 ##STR00280## P148
##STR00281## P149 ##STR00282## P150 ##STR00283## P151 ##STR00284##
P152 ##STR00285## P153 ##STR00286## P154 ##STR00287## P155
##STR00288## P156 ##STR00289## P157 ##STR00290## P158 ##STR00291##
P159 ##STR00292## P160 ##STR00293## P161 ##STR00294## P162
##STR00295## P163 ##STR00296## P164 ##STR00297## P165 ##STR00298##
P166 ##STR00299## P167 ##STR00300## P168 ##STR00301## P169
##STR00302## P170 ##STR00303## P171 ##STR00304## P172 ##STR00305##
P173 ##STR00306## P174 ##STR00307## P175 ##STR00308## P176
##STR00309## P177 ##STR00310## P178 ##STR00311## P179 ##STR00312##
P180 ##STR00313## P181 ##STR00314##
[0346] Analytical Methodology
[0347] Outlined below are general methods for work-up and
purification.
[0348] Work-Up
[0349] Reactions were worked up in a number of ways which may be
combined for example by solid-phase extraction (SPE) using either
sulfonic acid (SCX) or aminopropyl (NH.sub.2) cartridges eluting
with methanol and then 2M methanolic ammonia (Method S);
solid-phase extraction using fluorous cartridges eluting with
methanol:water (Method FL); evaporation either in vacuo or by
blowing nitrogen across sample (Method E); and aqueous work up
where the sample is diluted with water or dilute acid or dilute
base and then extracted with a suitable solvent, for example ethyl
acetate or dichloromethane (Method A) or filtration of sample
through a filter tube (Method F).
[0350] Evaporation
[0351] Samples were concentrated using Radley's nitrogen blow down
unit, rotary evaporator or Biotage V10 evaporator to give crude
residue.
[0352] Purification
[0353] Purification was by a range of methods including:
mass-directed autoprep (MDAP) using either low or high pH modifiers
see below for column details; automated normal phase chromatography
on for example a Biotage Flashmaster II or a ISCO companion, using
silica or aminopropyl column and a range of solvents, which
included, for example, ethyl acetate/cyclohexane/dichloromethane
and methanol; or recrystallisation from suitable solvent.
TABLE-US-00008 TABLE 8 Purification Methods Key Purification (as
used in Tables 1 to 4) MDAP (formic modifier) F MDAP (high pH) A
MDAP (high pH) A2 MDAP (TFA modifier) T Normal Phase
Chromatography: Silica: E1 EtOAc-cyclohexane 0-100% Normal Phase
Chromatography: Silica E2 0-50% ethyl acetate-cyclohexane Normal
Phase Chromatography: Silica E3 EtOAc-cyclohexane 0-25% Normal
Phase Chromatography: Silica D1 DCM Normal Phase Chromatography:
Silica D2 0-100% DCM in cycohexane Normal Phase Chromatography
Silica D3 0-50% DCM in cyclohexane Normal Phase Chromatography
Silica D4 0-30% DCM in Cyclohexane Normal Phase Chromatography:
Silica D5 0-25% DCM in Cyclohexane Normal phase chromatography:
Amino Propyl N1 Silica 0-100% EtOAc in cyclohexane Normal Phase
Chromatography: Amino Propyl N2 Silica DCM-cyclohexane 0-50% Normal
Phase Chromatography: Amino Propyl N3 Silica DCM-cyclohexane 0-30%
Normal Phase Chromatography: Silica ED1 EtOAc-DCM 0-100%
Re-crystallisation from Methanol R
[0354] MDAP Purification
[0355] MDAP (Formic Modifier): Method F
[0356] The HPLC analysis was conducted on a Sunfire C18 column (150
mm.times.30 mm i.d. 5 .mu.m packing diameter) at ambient
temperature.
[0357] The solvents employed were:
[0358] A=0.1% v/v solution of Formic Acid in Water.
[0359] B=0.1% v/v solution of Formic Acid in Acetonitrile.
[0360] The gradient was selected according to the analytical
retention time.
[0361] The UV detection was an averaged signal from wavelength of
210 nm to 350 nm and mass spectra were recorded on a mass
spectrometer using alternate-scan positive and negative mode
electrospray ionization.
[0362] MDAP (High pH): Method A
[0363] The HPLC analysis was conducted on an XBridge C18 column
(150 mm.times.30 mm i.d. 5 .mu.m packing diameter) at ambient
temperature.
[0364] The solvents employed were:
[0365] A=10 mM Ammonium Bicarbonate in water adjusted to pH 10 with
Ammonia solution.
[0366] B=Acetonitrile.
[0367] The gradient was selected according to the analytical
retention time.
[0368] The UV detection was an averaged signal from wavelength of
210 nm to 350 nm and mass spectra were recorded on a mass
spectrometer using alternate-scan positive and negative mode
electrospray ionization.
[0369] MDAP (High pH): Method A2
[0370] The HPLC analysis was conducted on a Waters Xbridge Shield
RP18 C18 column (100 mm.times.19 mm i.d. 5 .mu.m packing diameter)
at ambient temperature.
[0371] The solvents employed were:
[0372] A=10 mM Ammonium Bicarbonate in water adjusted to pH 10 with
Ammonia solution.
[0373] B=Methanol.
[0374] The gradient was selected according to the analytical
retention time.
[0375] The UV detection was an averaged signal from wavelength of
210 nm to 400 nm and mass spectra were recorded on a mass
spectrometer using alternate-scan positive and negative mode
electrospray ionization.
[0376] MDAP (TFA Modifier): Method T
[0377] The HPLC analysis was conducted on a Sunfire C18 column (150
mm.times.30 mm i.d. 5 .mu.m packing diameter) at ambient
temperature.
[0378] The solvents employed were:
[0379] A=0.1% v/v solution of Trifluoroacetic Acid in Water.
[0380] B=0.1% v/v solution of Trifluoroacetic Acid in
Acetonitrile.
[0381] The gradient was selected according to the analytical
retention time.
[0382] The UV detection was an averaged signal from wavelength of
210 nm to 350 nm and mass spectra were recorded on a mass
spectrometer using positive electrospray ionisation.
[0383] LCMS Analysis Conditions
[0384] The following conditions are representative of those used
for the generation of analytical LCMS data. Unless otherwise
stated, all LCMS data were generated using the formic acid LCMS
method described below.
[0385] Formic Add Generic Analytical UPLC Open Access LC/MS (2
Minute Method)
[0386] The UPLC analysis was conducted on an Acquity UPLC BEH C18
column (2.1 mm.times.50 mm i.d. 1.7 .mu.m packing diameter) at 40
degrees centigrade.
[0387] The solvents employed were:
[0388] A=0.1% v/v solution of Formic Acid in Water.
[0389] B=0.1% v/v solution of Formic Acid in Acetonitrile.
[0390] The gradient employed was:
TABLE-US-00009 Time (min) Flow Rate (mL/min) % A % B 0 1 97 3 1.5 1
0 100 1.9 1 0 100 2.0 1 97 3
[0391] The UV detection was an averaged signal from wavelength of
210 nm to 350 nm and mass spectra were recorded on a mass
spectrometer using alternate-scan positive and negative mode
electrospray ionization.
[0392] High pH LC/MS (2 Minute Method)
[0393] UPLC analysis was conducted on an Acquity UPLC BEH C18
column (50 mm.times.2.1 mm i.d. 1.7 .mu.m packing diameter) at
40.degree. C.
[0394] The solvents employed were:
[0395] A=10 mM ammonium bicarbonate in water adjusted to pH 10 with
ammonia solution.
[0396] B=Acetonitrile.
[0397] The gradient employed was:
TABLE-US-00010 Time (min) Flow Rate (mL/min) % A % B 0 1 99 1 1.5 1
3 97 1.9 1 3 97 2.0 1 99 1
[0398] The UV detection was a summed signal from wavelength of 210
nm to 350 nm and mass spectra were recorded on a mass spectrometer,
such as a Waters ZQ, using alternate-scan positive and negative
mode electrospray ionization.
[0399] .sup.1H NMR Analysis
[0400] .sup.1H NMR spectra were recorded on a Bruker DRX 400 (400
MHz) instrument. The following abbreviations have been used: s,
singlet; d, doublet; t, triplet; Hz, Hertz.
TABLE-US-00011 TABLE 9 .sup.1H NMR Data for a Selection of Example
ROR.gamma. Modulators Product No. .sup.1H NMR Data P34 .sup.1H NMR
(600 MHz, DMSO-d6) .delta. = 7.47 (2H, d), 7.33-7.38 (2H, m),
7.29-7.33 (1H, m), 7.15 (2H, d), 7.07 (2H, d), 5.01 (2H, s), 3.30
(2H, d), 2.41 (3H, s), 2.21 (3H, s), 1.41 (1H, m), 0.84 (6H, d) P35
.sup.1H NMR (600 MHz, DMSO-d6) .delta. = 7.58 (2H, d), 7.38 (1H,
d), 7.30 (1H, t), 7.20 (2H, d), 7.11 (1H, t), 6.61 (1H, d),
5.01-5.10 (2H, m), 3.54-3.62 (1H, m), 3.15 (1H, ddd), 2.64-2.78
(2H, m), 2.40-2.44 (3H, m, H-15), 2.23 (3H, s, H-6), 1.20-1.39 (2H,
m), 1.17 (3H, t), 0.80 (3H, t) P36 .sup.1H NMR (400 MHz, DMSO-d6)
.delta. = 7.53-7.44 (m, 2 H), 7.22 (t, 1 H), 7.18-7.08 (m, 3 H),
6.90 (s, 1 H), 6.81 (d, 1 H), 5.02 (s, 2 H), 3.28 (d, 2 H), 2.42
(s, 3 H), 2.26 (s, 3 H), 2.22 (s, 3 H), 1.50-1.36 (m, 1 H), 0.84
(d, 6 H) P37 .sup.1H NMR (400 MHz, DMSO-d6) .delta. = 7.61-7.34 (m,
2 H), 7.26-7.10 (m, 3 H), 7.04 (d, 1 H), 6.40 (s, 1 H), 5.05 (s, 2
H), 3.44-3.34 (m, 1 H), 3.05 (dd, 1 H), 2.43 (s, 3 H), 2.22 (s, 6
H), 2.12 (s, 3 H), 1.58-1.26 (m, 1 H), 0.94 (d, 3 H), 0.76 (d, 3 H)
P38 .sup.1H NMR (400 MHz, DMSO-d6) .delta. = 7.52-7.43 (m, 2 H),
7.25 (t, 1 H), 7.18-7.08 (m, 3 H), 6.88 (d, 1 H), 6.81 (br. s., 1
H), 5.01 (s, 2 H), 3.29 (d, 2 H), 2.59-2.52 (m, 2 H), 2.42 (s, 3
H), 2.21 (s, 3 H), 1.52-1.32 (m, 1 H), 1.08 (t, 3 H), 0.84 (d, 6 H)
P39 .sup.1H NMR (400 MHz, DMSO-d6) .delta. = 7.65-7.56 (m, 2 H),
7.53-7.45 (m, 1 H), 7.40-7.30 (m, 1 H), 7.25-7.16 (m, 3 H), 5.05
(s, 2 H), 3.27 (d, 2 H), 2.42 (s, 3 H), 2.22 (s, 3 H), 1.52-1.35
(m, 1 H), 0.84 (d, 6 H) P40 .sup.1H NMR (400 MHz, DMSO-d6) .delta.
= 7.71-7.55 (m, 3 H), 7.31 (ddd, 1 H), 7.25-7.18 (m, 2 H), 7.00
(dd, 1 H), 5.05 (s, 2 H), 3.38-3.32 (m, 1 H), 3.27-3.18 (m, 1 H),
2.43 (s, 3 H), 2.22 (s, 3 H), 1.49-1.39 (m, 1 H), 0.91 (d, 3 H),
0.79 (d, 3 H) P41 .sup.1H NMR (400 MHz, DMSO-d6) .delta. =
7.52-7.45 (m, 4 H), 7.44-7.38 (m, 2 H), 7.38-7.32 (m, 1 H), 7.25
(t, 1 H), 7.20-7.13 (m, 2 H), 6.89 (dd, 1 H), 6.63 (dd, 1 H), 6.56
(t, 1 H), 5.19 (s, 2 H), 3.67 (s, 3 H), 3.29 (d, 2 H), 1.51-1.36
(m, 1 H), 0.84 (d, 6 H) P42 .sup.1H NMR (400 MHz, METHANOL-d4)
.delta. = 7.55-7.40 (m, 2 H), 7.19-7.01 (m, 4 H), 6.90 (d, 2 H),
4.95 (s, 2 H), 3.29 (s, 2 H), 2.40 (s, 3 H), 2.29 (s, 3 H), 2.25
(s, 3 H), 1.59-1.41 (m, 1 H), 0.87 (d, 6 H) P43 .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta. = 7.74-7.56 (m, 2 H), 7.11 (s, 1 H),
7.05-6.93 (m, 2 H), 6.86 (d, 1 H), 6.51 (d, 1 H), 4.86 (s, 2 H),
3.52 (dd,, 1 H), 3.14 (dd, 1 H), 2.48-2.43 (m, 3 H), 2.42-2.38 (m,
3 H), 2.31 (s, 6 H), 0.94-0.75 (m, 1 H), 0.51-0.30 (m, 2 H),
0.25-0.10 (m, 1 H), 0.01--0.10 (m, 1 H) P44 .sup.1H NMR (400 MHz,
DMSO-d6) .delta. = 7.65-7.41 (m, 2 H), 7.24-7.09 (m, 2 H), 6.64 (d,
1 H), 6.49-6.43 (m, 1 H), 6.42-6.37 (m, 1 H), 5.04 (s, 2 H),
3.38-3.33 (m, 1 H), 2.99 (dd, 1 H), 2.42 (s, 3 H), 2.22 (s, 3 H),
2.18 (s, 3 H), 1.53-1.34 (m, 1 H), 0.94 (d, 3 H), 0.76 (d, 3 H)
P109 .sup.1H NMR (400 MHz, METHANOL-d4) d ppm 7.60-7.74 (m, 2 H)
7.06-7.23 (m, 2 H) 6.94 (br. s., 2 H) 5.00 (s, 2 H) 2.37-2.51 (m, 5
H) 2.27 (s, 3 H) 2.01 (br. s., 6 H) 1.53-1.75 (m, 1 H) 0.93-1.00
(m, 1 H) 0.89 (d, 6 H) 0.38-0.65 (m, 2 H) 0.06-0.28 (m, 2 H) P111
.sup.1H NMR (400 MHz, METHANOL-d4) d ppm 7.56-7.67 (m, 2 H),
7.08-7.21 (m, 4 H), 6.91-7.02 (m, 1 H), 5.00 (s, 2 H), 3.33 (d, 2
H), 2.43 (s, 3 H), 2.27 (s, 3 H), 1.48-1.59 (m, 1 H), 0.90 (d, 6 H)
P112 .sup.1H NMR (400 MHz, CHLOROFORM-d) d ppm 7.54 (d, 2 H), 7.31
(t, 1 H), 6.97-7.03 (m, 4 H), 4.86 (s, 2 H), 3.26 (d, 2 H), 2.44
(s, 3 H), 2.31 (s, 3 H), 1.57-1.65 (m, 1 H), 0.92 (d, 6 H) P113
.sup.1H NMR (400 MHz, CHLOROFORM-d) d ppm 7.60-7.75 (m, 2 H),
7.31-7.41 (m, 1 H), 7.24-7.30 (m, 1 H), 7.18 (d, 1 H), 6.98-7.03
(m, 2 H), 4.86 (s, 2 H), 3.27-3.44 (m, 2 H), 2.44 (s, 3 H), 2.31
(s, 3 H), 1.57-1.68 (m, 1 H), 0.96 (d, 3 H), 0.92 (d, 3 H) P114
.sup.1H NMR (400 MHz, CHLOROFORM-d) d ppm 7.53 (d, 2 H), 7.40 (d, 1
H), 7.16 (d, 1 H), 6.93-7.04 (m, 3 H), 4.85 (s, 2 H), 3.27 (d, 2
H), 2.44 (s, 3 H), 2.31 (s, 3 H), 1.58-1.64 (m, 1 H), 0.92 (d, 6 H)
P116 .sup.1H NMR (400 MHz, METHANOL-d4) d ppm 7.45-7.54 (m, 2 H),
7.07-7.13 (m, 2 H), 7.04 (d, 1 H), 6.82 (d, 1 H), 6.70 (dd, 1 H),
4.98 (s, 2 H), 3.27-3.29 (m, 2 H), 2.42 (s, 3 H), 2.26 (s, 3 H),
2.23 (s, 3 H), 2.18 (s, 3 H), 1.51 (m, 1 H), 0.88 (d, 6 H) P117
.sup.1H NMR (400 MHz, METHANOL-d4) d ppm 7.47-7.55 (m, 2 H),
7.08-7.16 (m, 2 H), 6.89 (d, 1 H), 6.73 (s, 1 H), 6.62 (d, 1 H),
4.98 (s, 2 H), 3.29-3.34 (m, 2 H), 2.42 (s, 3 H), 2.28 (s, 3 H),
2.26 (s, 3 H), 1.54 (m, 1 H), 0.90 (d, 6 H) P118 .sup.1H NMR (400
MHz, METHANOL-d4) d ppm 7.46-7.53 (m, 2 H), 7.06-7.14 (m, 2 H),
6.94 (s, 1 H), 6.63 (s, 2 H), 4.99 (s, 2 H), 3.28-3.30 (m, 2 H),
2.42 (s, 3 H), 2.27 (s, 3 H), 2.22 (br. s, 6 H), 1.52 (m, 1 H),
0.89 (d, 6 H) P123 .sup.1H NMR (400 MHz, METHANOL-d4) d ppm 8.89
(d, 2 H) 8.22 (d, 2 H) 7.50-7.66 (m, 2 H) 7.18-7.32 (m, 2 H) 7.09
(s, 1 H) 6.80-6.96 (m, 1 H) 6.49 (d, 1 H) 5.58 (s, 2 H) 3.39-3.56
(m, 1 H) 2.96-3.16 (m, 1 H) 2.29 (d, 6 H) 1.42-1.63 (m, 1 H) 1.00
(d, 3 H) 0.80 (d, 3 H) P126 .sup.1H NMR (400 MHz, METHANOL-d4) d
ppm 7.79-7.95 (m, 2 H) 7.59-7.71 (m, 2 H) 7.41-7.54 (m, 2 H) 7.07
(s, 2 H) 6.76-6.93 (m, 2 H) 6.38-6.57 (m, 6 H) 5.29 (s, 2 H)
3.37-3.53 (m, 1 H) 3.04-3.18 (m, 1 H) 2.26 (d, 6 H) 1.42-1.60 (m, 1
H) 0.98 (d, 3 H) 0.79 (d, 3 H) P127 .sup.1H NMR (400 MHz,
METHANOL-d4) d ppm 8.54 (d, 1 H) 7.81 (dd, 1 H) 7.66 (d, 2 H) 7.52
(d, 2 H) 7.08 (br. s., 1 H) 6.77-6.91 (m, 1 H) 6.38-6.55 (m, 1 H)
6.16-6.38 (m, 1 H) 5.26 (s, 2 H) 3.37-3.53 (m, 1 H) 2.99-3.20 (m, 1
H) 2.26 (d, 6 H) 1.40-1.63 (m, 1 H) 0.93-1.04 (m, 3 H) 0.72-0.84
(m, 3 H) P128 .sup.1H NMR (400 MHz, METHANOL-d4) d ppm 8.42-8.59
(m, 2 H) 7.50-7.64 (m, 5 H) 7.46 (d, 2 H) 7.08 (s, 1 H) 6.85 (d, 1
H) 6.48 (d, 1 H) 4.65-4.71 (m, 2 H) 4.71-4.74 (m, 2 H) 3.38-3.55
(m, 1 H) 3.05-3.14 (m, 1 H) 2.27 (br. s., 6 H) 1.46-1.58 (m, 1 H)
0.97-1.02 (m, 3 H) 0.77-0.82 (m, 3 H) P135 .sup.1H NMR (400 MHz,
METHANOL-d4) d ppm 7.54 (d, 2 H) 6.93-7.23 (m, 4 H) 6.20-6.47 (m, 1
H) 4.90-5.12 (m, 2 H) 3.41-3.59 (m, 1 H) 3.00-3.19 (m, 1 H)
2.57-2.76 (m, 1 H) 2.42 (s, 3 H) 2.33 (s, 3 H) 2.27 (s, 3 H)
1.41-1.60 (m, 1 H) 0.91-1.10 (m, 9 H) 0.80 (d, 3 H)
[0401] Biological Evaluation
[0402] The compounds of formula (I) and pharmaceutically acceptable
salts thereof are ROR.gamma. modulators, and hence have utility in
the treatment of inflammatory, metabolic and autoimmune diseases
mediated by ROR.gamma.. The biological activities of exemplified
compounds of formula (I) were assessed in the following disclosed
assays.
[0403] Dual Fluorescence Energy Transfer (FRET) Assay
[0404] This assay is based on the knowledge that nuclear receptors
interact with cofactors (transcription factors) in a ligand
dependent manner. ROR.gamma. is a typical nuclear receptor in that
it has an AF2 domain in the ligand binding domain (LBD) which
interacts with co-activators. The sites of interaction have been
mapped to the LXXLL motifs in the co-activator SRC1(2) sequences.
Short peptide sequences containing the LXXLL motif mimic the
behavior of full-length co-activator.
[0405] This assay measures ligand-mediated interaction of the
co-activator peptide with the purified bacterial-expressed
ROR.gamma. ligand binding domain (ROR.gamma.-LBD) to indirectly
assess ligand binding. ROR.gamma. has a basal level of interaction
with the co-activator SRC1(2) in the absence of ligand, Thus, it is
possible to find ligands that inhibit or enhance the
ROR.gamma./SRC1(2) interaction.
[0406] Materials
[0407] Generation of ROR.gamma.-LBD Bacterial Expression
Plasmid
[0408] Human ROR.gamma. Ligand Binding Domain (ROR.gamma.-LBD) was
expressed in E. coli strain BL21(DE3) as an amino-terminal
polyhistidine tagged fusion protein. DNA encoding this recombinant
protein was sub-cloned into a modified pET21a expression vector
(Novagen). A modified polyhistidine tag (MKKHHHHHHLVPRGS) was fused
in frame to residues 263-518 of the human ROR.gamma. sequence.
[0409] Protein Purification
[0410] Approximately 50 g E. coli cell pellet was resuspended in
300 mL of lysis buffer (30 mM imidazole pH 7.0 and 150 mM NaCl).
Cells were lysed by sonication and cell debris was removed by
centrifugation for 30 minutes at 20,000 g at 4.degree. C. The
cleared supernatant was filtered through a 0.45 uM cellulose
acetate membrane filter. The clarified lysate was loaded onto a
column (XK-26) packed with ProBond Nickel Chelating resin
(InVitrogen), pre-equilibrated with 30 mM imidazole pH 7.0 and 150
mM NaCl. After washing to baseline absorbance with the
equilibration buffer, the column was developed with a gradient from
30 to 500 mM imidazole pH 7.0. Column fractions containing the
ROR.gamma.-LBD protein were pooled and concentrated to a volume of
5 mLs. The concentrated protein was loaded onto a Superdex 200
column pre-equilibrated with 20 mM Tris-Cl pH 7.2 and 200 mM NaCl.
The fractions containing the desired ROR.gamma.-LBD protein were
pooled together.
[0411] Protein Biotinylation
[0412] Purified ROR.gamma.-LBD was buffer exchanged by exhaustive
dialysis [3 changes of at least 20 volumes (>8000.times.)]
against PBS [100 mM NaPhosphate, pH 8 and 150 mM NaCl]. The
concentration of ROR.gamma.-LBD was approximately 30 uM in PBS.
Five-fold molar excess of NHS-LC-Biotin (Pierce) was added in a
minimal volume of PBS. This solution was incubated with occasional
gentle mixing for 60 minutes at ambient room temperature. The
modified ROR.gamma.-LBD was dialyzed against 2 buffer changes--TBS
pH 8.0 containing 5 mM DTT, 2 mM EDTA and 2% sucrose--each at least
20 times of the volume. The modified protein was distributed into
aliquots, frozen on dry ice and stored at -80.degree. C. The
biotinylated ROR.gamma.-LBD was subjected to mass spectrometric
analysis to reveal the extent of modification by the biotinylation
reagent. In general, approximately 95% of the protein had at least
a single site of biotinylation and the overall extent of
biotinylation followed a normal distribution of multiple sites
ranged from one to five.
[0413] A biotinylated peptide corresponding to amino acid 676 to
700 (CPSSHSSLTERHKILHRLLQEGSPS) of the co-activator steroid
receptor coactivator SRC1(2) was generated using similar
method.
[0414] Assay
[0415] Protocol Step 1: Preparation of Europium Labeled SRC1(2)
Peptide
[0416] Biotinylated SRC1(2) solution was prepared by adding an
appropriate amount of biotinylated SRC1(2) from the 100 uM stock
solution to a buffer containing 10 mM of freshly added DTT from
solid to give a final concentration of 40 nM. An appropriate amount
of Europium labeled Streptavidin was then added to the biotinylated
SRC1(2) solution in a tube to give a final concentration of 10 nM.
The tube was inverted gently and incubated for 15 minutes at room
temperature. Twenty-fold excess biotin from the 10 mM stock
solution was added and the tube was inverted gently and incubated
for 10 minutes at room temperature.
[0417] Protocol Step 2: Preparation of APC Labeled
ROR.gamma.-LBD
[0418] Biotinylated ROR.gamma.-LBD solution was prepared by adding
an appropriate amount of biotinylated ROR.gamma.-LBD from the stock
solution to a buffer containing 10 mM of freshly added DTT from
solid to give a final concentration of 40 nM. An appropriate amount
of APC labeled Streptavidin was then added to the biotinylated
ROR.gamma.-LBD solution in a tube to give a final concentration of
20 nM. The tube was inverted gently and incubated for 15 minutes at
room temperature. Twenty-fold excess biotin from the 10 mM stock
solution was then added and the tube was inverted gently and
incubated for 10 minutes at room temperature.
[0419] Protocol Step 3: Testing
[0420] Equal volumes of the above-described Europium labeled
SRC1(2) peptide and the APC labeled ROR.gamma.-LBD were gently
mixed together to give 20 nM ROR.gamma.-LBD, 10 nM APC-Strepavidin,
20 nM SRC1(2) and 5 nM Europium-Streptavidin. The reaction mixtures
were incubated for 5 minutes. Using a Thermo Combi Multidrop 384
stacker unit, 25 ul of the reaction mixtures per well was added to
the 384-well assay plates containing 1 ul of test compound per well
in 100% DMSO. The plates were incubated for 1 hour and then read on
ViewLux in Lance mode for EU/APC.
[0421] Results
[0422] The exemplified compounds of formula (I) were tested in the
dual FRET assay described above. All exemplified compounds of
formula (I), with the exception of P77, 169-171 and 181 that were
not tested, were found to have a mean pIC50 between 5.0 and 8.0.
The exemplified compounds of formula (I) P24, P28, P30, P31, P33,
P98, P104, P114, P123, P140, P141, P142, P150 and P160 were found
to have a mean pIC50 value of 7.8.
[0423] Peripheral Blood Mononucleocyte Cell Assay (PBMC
Assay--IL-17)
[0424] RORs (Retinoic Acid Related Orphan Receptors) are members of
the class 1 nuclear receptor family. RORs regulate gene
transcription by binding to specific DNA response element (RORE) as
a monomer and have critical roles of in development, immunity,
circadian rhythm, and cellular metabolism (recently reviewed by A
Jetten, Nuclear Receptor Signaling, 2009, 7, 1-32) One member of
this nuclear receptor family, ROR.gamma.t, has been identified as a
regulator of differentiation and development of IL-17-expressing
human and mouse CD4+ T cells, so called Th17 cells which play a
role in both host defence and inflammatory disorders. ROR.gamma.t
is also required for transcription of the genes encoding IL-17A and
IL-17F in iNKT, NKT (Immuno) 2009, 2(5), 383-392, J. Immunol, 2008,
180, 51(67), 5171-5171), .gamma..delta.T cells (J. Resp and Crit
Care Med 2010, 182(4), 464-476), CD8.sup.+ Tcell (J Leukocyte Biol,
2007, 82(2), 354-360) and finally
CD4.sup.-CD8TCR.alpha..beta..sup.+ T cells (J. Immunol. 2008, 181
8761-8766). Additional immune cells such as eosinophils,
neutrophils and macrophages can also be source of IL17A in some
allergic inflammation related to asthma (J Allergy Clin Immunol
2001, 108, 430-438; J. Immunol, 2008, 181, 6117-6124; Immunity,
2004, 21, 467-476), however, the link with ROR.gamma.t has not yet
been confirmed in the literature.
[0425] This assay is designed to measure levels of IL-17A secreted
from antiCD3/CD28 stimulated frozen Peripheral Blood Mononuclear
cells (PBMC) isolated from human blood with the aim of identifying
inhibitors of IL-17A release.
[0426] Assay Solutions
[0427] Assay Media Components:
[0428] RPMI 1640 (as supplied, for example, by Gibco) -90%
[0429] FCS (as supplied, for example, by Invitrogen) (endotoxin
tested) -10%
[0430] Penicillyn/Streptomycin solution .times.1
[0431] Preparation: 50 mL Heat Inactivated Australian FBS, 5 mL
Glutamax and 5 mL
[0432] Penicillin/Streptomycin are aseptically added to 500 mL RPMI
in a biosafety cabinet. The Penicllin/Streptomycin 100.times. stock
is supplied by, for example, Gibco (10,000 Units/mL Penicillin,
10,000 ug/mL Streptomycin). Stock L-glutamine 100.times. (as
supplied, for example, by Invitrogen) Note: To be kept in a fridge
(4.degree. C.) for 4 weeks. Warm up in a water bath set at
37.degree. C. prior to use.
[0433] Anti-Human IL-17 Detection Antibody Components:
[0434] IL-17 detection antibody and Blocking buffer B (supplied,
for example, by Mesoscale Discovery) Dulbecco's PBS without
Ca.sup.2+ and Mg.sup.2+ (supplied, for example, by Gibco) Note:
Prepare detection anti body at final concentration of 1 ug/mL.
Solution to be kept refrigerated.
[0435] MSD Read Buffer T.times.2 Components:
[0436] Water and MSD Read Buffer T.times.4 (as supplied, for
example, by MSD)
[0437] Note: Dilute MSD Read Buffer T.times.4 in half with water.
To be kept at room temperature.
[0438] Assay Capacity: 384
[0439] Equipment and Materials
[0440] MSD Sector Imager 6000 supplied by MesoScale Discovery
(MSD)
[0441] Multidrop 384 supplied by Thermo Scientific
[0442] CyBi-Well, model 7518-00 supplied by CyBio AG
[0443] Microplates 384 clear supplied by Greiner
[0444] Assay
[0445] Protocol Step 1: Assay Plates Preparation Before Adding Cell
Suspension [0446] 1. Ensure no external endotoxin is present in
media and reagents used in the assay. [0447] 2. The compounds for
screening are dispensed into a master plate at 10 mM top
concentration which are serially diluted 1:3 across 11 points in
DMSO, then 500 nl is transferred into a 384-well flat-bottomed
Greiner plate to which 50 ul of cell suspension is added: for the
single shot screening the highest concentration of compound is
10.sup.-5M; for 11 point full curve dose response studies the
highest concentration is 10.sup.-4 M. [0448] CONTROLS: [0449] As a
low control, DMSO (as supplied, for example, by VWR) (final
concentration 1%) in column 6 (16 points). [0450] As a high
control, 5-(4-fluorophenyl)-2-ureidothiophene-3-carboxamide
(obtainable from, for example, Sigma) at a final concentration of
10.sup.-4 M in DMSO should be used in column 18 (16 points). [0451]
If the compounds dispensed earlier than the day of the assay, they
should be kept at-20.degree. C.
[0452] Protocol Step 2: Day 1: Thawing and Handing of PBMC [0453]
1. Thaw PBMC in the vial using water bath (37.degree. C.). Ensure
that water does not cover the vial (the level should be lower than
the screw cup of the vial) [0454] 2. Transfer the contents of the
vial into 50 mL Falcon tube. [0455] 3. Add 10 mL of Assay Media
drop by drop to decrease the concentration of DMSO (as supplied,
for example, by VWR) in the freezing media gradually. [0456] 4.
Spin down the cells in a centrifuge (1000 rpm-5 mins). [0457] 5.
Decant the supernatant. [0458] 6. Re-suspend the cells in 10 mL of
Assay Media. [0459] 7. Transfer of 0.1 mL of suspension into Cedex
counting tube. [0460] 8. Add 0.9 mL of media to achieve volume of
the suspension for counting up to 1 mL. Count the cells on Cedex
using 1:10 dilution factor settings. [0461] 9. Make the cell
suspension at the concentration 8.times.10.sup.5 cells/mL. to give
a final number of 40,000 cells/well.
[0462] Protocol Step 3: Day 1: Stimulation of PBMC with CD3/CD28
Beads [0463] 1. Add well mixed CD3/CD28 Dynabeads (as supplied, for
example, by Dynal) to achieve ratio bead:cell=2:1 (i.e a dilution
of 1 in 20). Mix thoroughly. [0464] 2. Dispense the suspension into
the 384 Assay Plates using Multidrop (50 ul per well). If the
volume of cell suspension is large, mix the suspension after
dispensing into every other plate. [0465] 3. Cover the plates with
the lids and place them to the humidified incubator (37.degree. C.,
5% CO.sub.2) for 48 hours.
[0466] Protocol Step 4: Day 2: MSD Plates Preparation [0467] 1.
Block cytokine capture Mesoscale Discovery MSD plates with 0.1%
Block buffer B (provided by Mesoscale Dsicovery) in D-PBS solution
using 40 ul per well. [0468] 2. Leave the plates covered with lids
in the fridge over night. [0469] 3 Plates are washed manually using
PBS and a multidrop combi. Blocker B buffer is flicked out into a
waste pot and 40 ul of PBS is dispensed into the plate using a
combi. This is then flicked out manually and the plates tapped on
to blue roll to remove as much residual liquid as possible before
transferring the cell supernatant. [0470] 4 Tap the plates over a
paper towel.
[0471] Protocol Step 5: Day 3: IL-17 Detection on MSD Plates [0472]
1. Transfer 10 ul of supernatants from assay plates to the MSD
plates using Cybiwell. Ensure that all wells are covered with the
solution. Tap the plate gently, if some of the wells are not
covered with the supernatant. [0473] 2. Cover the plates with
adhesive foil (brown stickers) and leave them for 1 hour of
incubation on shaker at room temperature (RT). [0474] 3. Add 10 ul
of MSD IL-17 detecting antibody using multidrop (1 ug/mL in D-PBS
without Ca.sup.2+ and Mg.sup.2+ (supplied, for example, by Gibco)).
[0475] 4. Cover the plates with adhesive foil and incubated with
shaking for 3 hours at room temperature [0476] 5. Plates are washed
manually twice using PBS and a multidrop combi as before. [0477] 6.
Tap the plates over a paper towel. [0478] 7. Add 35 ul of MSD Read
Buffer T.times.2 using multidrop. [0479] 8. Read plates on MSD
MA6000 reader using the 384 well plate protocol as per
manufacturer's instructions.
[0480] Results
[0481] The exemplified compounds of formula (I) were tested in the
PBMC assay described above. All exemplified compounds of formula
(I), with the exception of P99, P105, P107, P153, P154, P159,
P161-165, P168-171, P174-176, P178, P180 and P181 that were not
tested and P179 which had a value of <4.0, were found to have a
mean pIC50 between 4.0 and 7.0. The exemplified compounds of
formula (I) P23, P24, P28, P30, P31, P33, P37, P118, P123, P128,
P132, P137, P140, P143, P144, P147, P155 and P160 were found to
have a mean pIC50 value of 6.0.
[0482] Utility
[0483] Compounds of formula (I), and pharmaceutically acceptable
salts thereof, are modulators of ROR.gamma. and can be useful in
the treatment of inflammatory, metabolic and autoimmune diseases
mediated by ROR.gamma. such as asthma, chronic obstructive
pulmonary disease (COPD) and bronchitis, allergic diseases, such as
allergic rhinitis and atopic dermatitis, cystic fibrosis, lung
allograph rejection, multiple sclerosis, rheumatoid arthritis,
juvenile Rheumatoid arthritis, Osteoarthritis, ankylosing
spondylitis, systemic lupus erythematosus, psoriasis, Hashimoto's
disease, pancreatisis, autoimmune diabetes, autoimmune ocular
disease, ulcerative colitis, Crohn's disease, inflammatory bowel
disease (IBS), inflammatory bowel syndrome (IBD), Sjorgen's
syndrome, optic neuritis, type I diabetes, neuromyelitis optica,
Myastehnia Gravis, uveitis, Guillain-Barre syndrome, psoriatic
arthritis, Graves' disease and scleritis. The use of ROR.gamma.
modulators for the treatment of the respiratory diseases listed
above, such as asthma and COPD is of particular interest.
[0484] In a further aspect, the present invention also provides a
compound of formula (I), or a pharmaceutically acceptable salt or
solvate thereof, for use in therapy.
[0485] In a further aspect, the present invention also provides a
compound of formula (I), or a pharmaceutically acceptable salt or
solvate thereof, for use in the treatment of inflammatory,
metabolic and autoimmune diseases mediated by ROR.gamma..
[0486] In a further aspect, the present invention provides a
compound of formula (I), or a pharmaceutically acceptable salt
thereof, for use in the treatment of asthma or chronic obstructive
pulmonary disease.
[0487] In a further aspect, the present invention is directed to a
method of treatment of an inflammatory, metabolic or autoimmune
disease mediated by ROR.gamma., which comprises administering to a
subject in need thereof, a safe and therapeutically effective
amount of a compound of formula (I), or a pharmaceutically
acceptable salt thereof.
[0488] In yet a further aspect, the present invention is directed
to a method of treating chronic obstructive pulmonary disease or
asthma, which comprises administering to a subject in need thereof,
a safe and therapeutically effective amount of a compound of
formula (I), or a pharmaceutically acceptable salt thereof.
[0489] As used herein, the term "treatment" refers to prophylaxis
of the condition, ameliorating or stabilising the specified
condition, reducing or eliminating the symptoms of the condition,
slowing or eliminating the progression of the condition, and
preventing or delaying reoccurrence of the condition in a
previously afflicted patient or subject.
[0490] As used herein, the term "therapeutically effective amount"
refers to the quantity of a compound of formula (I), or a
pharmaceutically acceptable salt thereof, which will elicit the
desired biological response in an animal or human body.
[0491] As used herein, the term "subject" refers to an animal or
human body
[0492] Pharmaceutical Development
[0493] A compound of formula (I), or a pharmaceutically acceptable
salt thereof, will normally, but not necessarily, be formulated
into pharmaceutical compositions prior to administration to a
patient.
[0494] Accordingly, in another aspect the invention is directed to
pharmaceutical compositions comprising a compound of formula (I),
or a pharmaceutically acceptable salt thereof, and one or more
pharmaceutically-acceptable excipients.
[0495] Pharmaceutical compositions comprising a compound of formula
(I), or a pharmaceutically acceptable salt thereof, may be prepared
using techniques and methods known to those skilled in the art.
Some of the methods commonly used in the art are described in
Remington's Pharmaceutical Sciences (Mack Publishing Company).
[0496] A pharmaceutical composition of a compound of formula (I),
or a pharmaceutically acceptable salt thereof, may be formulated
for administration by any appropriate route, for example by the
inhaled, nasal, oral (including buccal or sublingual), topical
(including buccal, sublingual, transdermal, epicutaneous) or
parenteral (subcutaneous, intramuscular, intravenous, intradermal)
route. Thus, a pharmaceutical composition of a compound of formula
(I), or a pharmaceutically acceptable salt thereof, may be
formulated as, for example, a solution or suspension (aqueous or
non-aqueous), tablet, capsule, powder, granule, lozenge, lotion,
cream, ointment, gel, foam or reconstitutable powder depending on
the particular route of administration. Such pharmaceutical
compositions may be prepared by any method known in the art of
pharmacy, for example by bringing into association the active
ingredient with the excipient(s).
[0497] Tablets and capsules for oral administration may be in unit
dose presentation form, and may contain conventional excipients
such as binding agents, for example syrup, acacia, gelatine,
sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example
lactose, sugar, maize-starch, calcium phosphate, sorbitol or
glycine; tabletting lubricants, for example magnesium stearate,
talc, polyethylene glycol or silica; disintegrants, for example
potato starch; or acceptable wetting agents such as sodium lauryl
sulfate. The tablets may be coated according to methods well known
in normal pharmaceutical practice.
[0498] Oral liquid preparations may be in the form of, for example,
aqueous or oily suspensions, solutions, emulsions, syrups or
elixirs, or may be presented as a dry product for reconstitution
with water or other suitable vehicle before use. Such liquid
preparations may contain conventional additives, such as suspending
agents, for example sorbitol, methyl cellulose, glucose syrup,
gelatine, hydroxyethyl cellulose, carboxymethyl cellulose,
aluminium stearate gel or hydrogenated edible fats, emulsifying
agents, for example lecithin, sorbitan monooleate, or acacia;
non-aqueous vehicles (which may include edible oils), for example
almond oil, oily esters such as glycerine, propylene glycol, or
ethyl alcohol; preservatives, for example methyl or propyl
p-hydroxybenzoate or sorbic acid, and, if desired, conventional
flavouring or colouring agents.
[0499] Pharmaceutical compositions of a compound of formula (I) or
a pharmaceutically acceptable salt thereof, for topical
administration, may be presented as, for instance, ointments,
creams or lotions, eye ointments and eye or ear drops, impregnated
dressings and aerosols, and may contain appropriate conventional
additives such as preservatives, solvents to assist drug
penetration and emollients in ointments and creams. The
compositions may also contain compatible conventional carriers,
such as cream or ointment bases and ethanol or oleyl alcohol for
lotions. Such carriers may be present as from about 1% up to about
98% of the composition. More usually they will form up to about 80%
of the composition.
[0500] Pharmaceutical compositions adapted for parenteral
administration include aqueous and non-aqueous sterile injection
solutions which may contain anti-oxidants, buffers, bacteriostats
and solutes which render the composition isotonic with the blood of
the intended recipient; and aqueous and non-aqueous sterile
suspensions which may include suspending agents and thickening
agents. The compositions may be presented in unit-dose or
multi-dose containers, for example sealed ampoules and vials, and
may be stored in a freeze-dried (lyophilized) condition requiring
only the addition of the sterile liquid carrier, for example water
for injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets.
[0501] Pharmaceutical compositions for topical administration to
the lung may include aerosol compositions and dry powder
compositions.
[0502] Dry powder compositions for topical delivery to the lungs or
nose generally contain a powder mix of a compound of formula (I) or
a pharmaceutically acceptable salt thereof and a suitable carrier,
such as lactose or starch. Dry powder compositions for topical
delivery to the lung or nose may, for example, be presented in
capsules and cartridges for use in an inhaler or insufflator of,
for example, gelatine. Each capsule or cartridge may generally
contain between 20 .mu.g-10 mg of the compound of formula (I), or a
pharmaceutically acceptable salt thereof. Alternatively, the
compounds of formula (I), or pharmaceutically acceptable salts
thereof, may be presented without excipients. Packaging of the
pharmaceutical composition may be suitable for unit dose or
multi-dose delivery. In the case of multi-dose delivery, the
composition can be pre-metered (eg as in Diskus, see GB 2242134 or
Diskhaler, see GB 2178965, 2129691 and 2169265) or metered in use
(eg as in Turbuhaler, see EP 69715). An example of a unit-dose
device is Rotahaler (see GB 2064336). The Diskus inhalation device
comprises an elongate strip formed from a base sheet having a
plurality of recesses spaced along its length and a lid sheet
hermetically but peelably sealed thereto to define a plurality of
containers, each container having therein an inhalable composition
comprising a compound of formula (I), or a pharmaceutically
acceptable salt thereof, preferably combined with a carrier, such
as lactose. Preferably, the strip is sufficiently flexible to be
wound into a roll. The lid sheet and base sheet will preferably
have leading end portions which are not sealed to one another and
at least one of the said leading end portions is constructed to be
attached to a winding means. Also, preferably the hermetic seal
between the base and lid sheets extends over their whole width. The
lid sheet may preferably be peeled from the base sheet in a
longitudinal direction from a first end of the said base sheet.
[0503] Medicaments for administration by inhalation desirably have
a controlled particle size. The optimum particle size for
inhalation into the bronchial system is usually 1-10 .mu.m,
preferably 2-5 .mu.m. Particles having a size above 20 .mu.m are
generally too large when inhaled to reach the small airways. To
achieve these particle sizes the particles of compound of formula
(I), or a pharmaceutically acceptable salt thereof, may be size
reduced by conventional means eg by micronisation. The desired
fraction may be separated out by air classification or sieving.
Preferably, the particles will be crystalline, prepared for example
by a process which comprises mixing in a continuous flow cell in
the presence of ultrasonic radiation a flowing solution of compound
of formula (I), or a pharmaceutically acceptable salt thereof, as
medicament in a liquid solvent with a flowing liquid antisolvent
for said medicament (eg as described in International Patent
Application PCT/GB99/04368). Alternatively, the particles may be
prepared by a process which comprises admitting a stream of
solution of the substance in a liquid solvent and a stream of
liquid antisolvent for said substance tangentially into a
cylindrical mixing chamber having an axial outlet port such that
said streams are thereby intimately mixed through formation of a
vortex and precipitation of crystalline particles of the substance
is thereby caused (eg as described in International Patent
Application PCT/GB00/04237). When an excipient such as lactose is
employed, generally, the particle size of the excipient will be
much greater than the inhaled medicament within the present
invention. When the excipient is lactose it will typically be
present as milled lactose, wherein not more than 85% of lactose
particles will have a MMD of 60-90 .mu.m and not less than 15% will
have a MMD of less than 15 .mu.m.
[0504] Aerosol compositions may be developed, with the use of a
suitable liquefied propellant, for delivery from pressurised packs,
such as a metered dose inhaler. Aerosol compositions can be either
a suspension or a solution and generally contain the compound of
formula (I), or a pharmaceutically acceptable salt thereof, and a
suitable propellant such as a fluorocarbon or hydrogen-containing
chlorofluorocarbon or mixtures thereof, particularly
hydrofluoroalkanes, especially 1,1,1,2-tetrafluoroethane,
1,1,1,2,3,3,3-heptafluoro-n-propane or a mixture thereof. The
aerosol composition may optionally contain additional formulation
excipients well known in the art such as surfactants eg oleic acid
or lecithin and cosolvents eg ethanol. Aerosol compositions will
generally be retained in a pressurised canister (eg an aluminium
canister) closed with a valve (eg a metering valve) and fitted into
an actuator with a mouthpiece. Aerosol compositions may also
include aqueous solutions or suspensions that are delivered to the
nose or lungs by nebulisation.
[0505] Pharmaceutical compositions for topical administration to
the nose may also be developed for delivery by nasal spray or as
nasal droplets. Pharmaceutical compositions for nasal
administration may be developed in such a way to allow the
medicament(s) to be delivered to all appropriate areas of the nasal
cavities (the target tissue). Moreover, a pharmaceutical
composition may be developed for nasal administration, which
permits the medicament(s) to remain in contact with the target
tissue for an increased period of time.
[0506] A suitable dosing regimen for a pharmaceutical composition
administered topically to the nose by use of a nasal spray may be
for the patient to inhale slowly through the nose subsequent to the
nasal cavity being cleared. During inhalation, the composition may
be administered to one nostril while the other is manually
compressed. This procedure may then be repeated for the other
nostril. Generally, one or two sprays per nostril may be
administered by the above procedure up to two or three times each
day. Typically, each spray to the nostril may deliver from about 25
to about 100 .mu.L of the pharmaceutical composition.
[0507] Pharmaceutical compositions for topical administration to
the nose by nasal spray or as nasal drops may be prepared as a
solution or suspension. The solution or suspension may be aqueous
or non-aqueous based, and may contain one or more pharmaceutically
acceptable excipients, such as suspending agents, for example
carboxymethylcellulose, methylcellulose, veegum, tragacanth,
bentonite and polyethylene glycols; preservatives, for example
chelating agents (e.g EDTA), quaternary ammonium compounds (e.e
benzalkonium chloride, benzethonium chloride, cetrimide and
cetylpyridinium chloride), mercurial agents (e.g. phenylmercuric
nitrate, phenylmercuric acetate and thimerosal), alcoholic agents
(e.g. chlorobutanol, phenylethyl alcohol and benzyl alcohol),
antibacterial esters (e.g. esters of para-hydroxybenzoic acid) and
other anti-microbial agents such as chlorhexidine, chlorocresol,
sorbic acid and its salts (e.g. potassium sorbate), and polymyxin;
isotonicity adjusting agents, for example sodium chloride,
dextrose, xylitol and calcium chloride; buffering agents, wetting
agents, for example fatty alcohols, esters and ethers, such as
polyoxyethylene (20) sorbitan monooleate (polysorbate 80);
anti-oxidants, sweetening agents and taste-masking agents.
[0508] It should be understood that in addition to the ingredients
particularly mentioned above, the pharmaceutical compositions may
include other agents conventional in the art having regard to the
type of formulation in question.
[0509] A compound of formula (I), or a pharmaceutically acceptable
salt thereof, may also be used in combination with one or more
other therapeutic agents, selected from the group consisting of
R.sub.2-adrenoreceptor agonists, anti-inflammatory agents (e.g.
corticosteroids and NSAID's) and anticholinergic agents.
[0510] .beta..sub.2-adrenoreceptor agonists that may be used in
combination with a compound of formula (I), or a pharmaceutically
acceptable salt thereof, include, for example, salmeterol,
salbutamol, formoterol, and salts thereof, for example the
xinafoate salt of salmeterol, the sulfate salt of salbutamol or the
fumarate salt of formoterol). Further .beta..sub.2-adrenoreceptor
agonists include those described in WO03/024439, such as
4-{(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)amino]-1-hydroxyet-
hyl}-2-(hydroxymethyl)phenol and its pharmaceutically acceptable
salts, such as triphenylacetate.
[0511] Corticosteroids that may be used in combination with a
compound of formula (I), or a pharmaceutically acceptable salt
thereof, include, for example, fluticasone propionate and
6.alpha.,9.alpha.-difluoro-17a-[(2-furanylcarbonyl)oxy]-11.beta.-hydroxy--
16.alpha.-methyl-3-oxo-androsta-1,4-diene-17.beta.-carbothioic acid
S-fluoromethyl ester (fluticasone furoate).
[0512] Anticholinergic agents may also be used in combination with
a compound of formula (I), or a pharmaceutically acceptable salt
thereof. Examples of anticholinergic agents are those compounds
that act as antagonists at the muscarinic receptors, in particular
those compounds which are antagonists of the M.sub.1 or M.sub.3
receptors, dual antagonists of the M.sub.1/M.sub.3 or
M.sub.2/M.sub.3, receptors or pan-antagonists of the
M.sub.1/M.sub.2/M.sub.3 receptors. Antimuscarinic compounds for
administration via inhalation include, for example, ipratropium
(for example, as the bromide, CAS 22254-24-6, sold under the name
Atrovent), tiotropium (for example, as the bromide, CAS
136310-93-5, sold under the name Spiriva),
(3-endo)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1-
]octane bromide, and
4-[hydroxy(diphenyl)methyl]-1-{2-[(phenylmethyl)oxy]ethyl}-1-azoniabicycl-
o[2.2.2]octane bromide.
[0513] It will be clear to a person skilled in the art that, where
appropriate, the other therapeutic agent(s) may be used in the form
of pharmaceutically acceptable salts, or prodrugs, or as esters
(e.g lower alkyl esters), or as solvates (e.g. hydrates) to
optimise the activity and/or stability and/or physical
characteristics (e.g. solubility) of the therapeutic agent. It will
be clear also that, where appropriate, the therapeutic agent(s) may
be used in optically pure form.
[0514] The invention thus provides in a further aspect a
combination comprising a compound of formula (I), or a
pharmaceutically acceptable salt thereof, and one or more other
therapeutic agents.
Sequence CWU 1
1
315PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 1Leu Xaa Xaa Leu Leu1 5
215PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 2Met Lys Lys His His His His His His
Leu Val Pro Arg Gly Ser1 5 10 15 325PRTArtificial SequenceAmino
acid sequence identified using molecular biology techniques. 3Cys
Pro Ser Ser His Ser Ser Leu Thr Glu Arg His Lys Ile Leu His1 5 10
15 Arg Leu Leu Gln Glu Gly Ser Pro Ser 20 25
* * * * *