U.S. patent application number 13/196148 was filed with the patent office on 2011-11-24 for novel compounds.
This patent application is currently assigned to Glaxo Group Limited. Invention is credited to Giuseppe ALVARO, David Amantini, Sandro Belvedere.
Application Number | 20110288098 13/196148 |
Document ID | / |
Family ID | 42267024 |
Filed Date | 2011-11-24 |
United States Patent
Application |
20110288098 |
Kind Code |
A1 |
ALVARO; Giuseppe ; et
al. |
November 24, 2011 |
NOVEL COMPOUNDS
Abstract
This invention relates to imidazopyrazine substituted piperidine
derivatives and their use as pharmaceuticals.
Inventors: |
ALVARO; Giuseppe; (Verona,
IT) ; Amantini; David; (Verona, IT) ;
Belvedere; Sandro; (Verona, IT) |
Assignee: |
Glaxo Group Limited
|
Family ID: |
42267024 |
Appl. No.: |
13/196148 |
Filed: |
August 2, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12576372 |
Oct 9, 2009 |
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13196148 |
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61103967 |
Oct 9, 2008 |
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Current U.S.
Class: |
514/249 |
Current CPC
Class: |
A61P 9/00 20180101; C07D
487/04 20130101; A61P 11/00 20180101; A61P 25/00 20180101 |
Class at
Publication: |
514/249 |
International
Class: |
A61K 31/4985 20060101
A61K031/4985; A61P 9/00 20060101 A61P009/00; A61P 11/00 20060101
A61P011/00; A61P 25/00 20060101 A61P025/00 |
Claims
1. A method of treating a disease or disorder where an antagonist
of a human orexin receptor is required, which comprises
administering to a subject in need thereof an effective amount of a
compound of formula (I): ##STR00007## where Ar is selected from the
group consisting of formula: ##STR00008## R.sub.1 is
(C.sub.1-4)alkyl, halo, halo(C.sub.1-4)alkyl, (C.sub.1-4)alkoxy,
halo(C.sub.1-4)alkoxy, (C.sub.1-4)alkyl-O--(C.sub.1-4)alkyl, CN,
NR.sup.5R.sup.6 wherein R.sup.5 is H or (C.sub.1-4)alkyl and
R.sup.6 is H or (C.sub.1-4)alkyl; R.sub.2 is (C.sub.1-4)alkyl,
halo, halo(C.sub.1-4)alkyl, (C.sub.1-4)alkoxy,
halo(C.sub.1-4)alkoxy, (C.sub.1-4)alkyl-O--(C.sub.1-4)alkyl, CN,
NR.sup.7R.sup.8 wherein R.sup.7 is H or (C.sub.1-4-alkyl and
R.sup.8 is H or (C.sub.1-4)-alkyl; R.sub.3 is (C.sub.1-4)alkyl,
halo, halo(C.sub.1-4)alkyl, (C.sub.1-4)alkoxy,
halo(C.sub.1-4)alkoxy, (C.sub.1-4)alkyl-O--(C.sub.1-4)alkyl, CN,
NR.sup.9R.sup.10 wherein R.sup.9 is H or (C.sub.1-4-alkyl and
R.sup.10 is H or (C.sub.1-4)-alkyl; n is 0 or 1; p is 0 or 1; and q
is 0 or 1; with the proviso that p and q are not both 0; or a
pharmaceutically acceptable salt thereof, wherein the disease or
disorder is a sleep disorder, a depression or mood disorder, an
anxiety disorder, a substance-related disorder or a feeding
disorder.
2. The method according to claim 1, where the disease or disorder
is a sleep disorder.
3. The method according to claim 2, where the sleep disorder is
selected from the group consisting of Primary Insomnia (307.42),
Breathing-Related Sleep Disorders (780.59), Circadian Rhythm Sleep
Disorder (307.45), Dyssomnia Not Otherwise Specified (307.47);
Nightmare Disorder (307.47), Sleep Terror Disorder (307.46),
Sleepwalking Disorder (307.46), Parasomnia Not Otherwise Specified
(307.47); Insomnia Related to Another Mental Disorder (307.42),
Sleep Disorder Due to a General Medical Condition, selected from
sleep disturbances associated with neurological disorders,
neuropathic pain, restless leg syndrome, heart or lung diseases;
and a Substance-Induced Sleep Disorder selected from Insomnia Type,
Parasomnia Type and Mixed Type; Sleep Apnea and Jet-Lag
Syndrome.
4. The method according to claim 1, where, in the compound of
Formula (I), or pharmaceutically acceptable salt thereof, Ar is a
group of formula (II) and n is 0.
5. The method according to claim 1, where, in the compound of
Formula (I), or pharmaceutically acceptable salt thereof, Ar is a
group of formula (II), n is 0, p is 1, q is 0 and R.sub.2 is
(C.sub.1-4)alkyl.
6. The method according to claim 1, where, in the compound of
Formula (I), or pharmaceutically acceptable salt thereof, Ar is a
group of formula (II), n is 0, p is 1, q is 0 and R.sub.2 is
methyl.
7. The method according to claim 1, where, in the compound of
Formula (I), or pharmaceutically acceptable salt thereof, Ar is a
group of formula (II), n is 0, p is 0, q is 1 and R.sub.3 is
(C.sub.1-4)alkyl.
8. The method according to claim 1, where, in the compound of
Formula (I), or pharmaceutically acceptable salt thereof, Ar is a
group of formula (II), n is 0, p is 0, q is 1 and R.sub.3 is
methyl.
9. The method according to claim 1, where, in the compound of
Formula (I), or pharmaceutically acceptable salt thereof, Ar is a
group of formula (II), n is 0, p is 1, q is 1, R.sub.2 is
(C.sub.1-4)alkyl and R.sub.3 is (C.sub.1-4)alkyl.
10. The method according to claim 1, where, in the compound of
Formula (I), or pharmaceutically acceptable salt thereof, Ar is a
group of formula (II), n is 0, p is 1, q is 1, R.sub.2 is methyl
and R.sub.3 is methyl.
11. The method according to claim 1, where, in the compound of
Formula (I), or pharmaceutically acceptable salt thereof, Ar is a
group of formula (III) and n is 0.
12. The method according to claim 1, where, in the compound of
Formula (I), or pharmaceutically acceptable salt thereof, Ar is a
group of formula (III), n is 0, p is 1, q is 0 and R.sub.2 is
(C.sub.1-4)alkyl.
13. The method according to claim 1, where, in the compound of
Formula (I), or pharmaceutically acceptable salt thereof, Ar is a
group of formula (III), n is 0, p is 1, q is 0 and R.sub.2 is
methyl.
14. The method according to claim 1, where, in the compound of
Formula (I), or pharmaceutically acceptable salt thereof, Ar is a
group of formula (III), n is 0, p is 0, q is 1 and R.sub.3 is
(C.sub.1-4)alkyl.
15. The method according to claim 1, where, in the compound of
Formula (I), or pharmaceutically acceptable salt thereof, Ar is a
group of formula (III), n is 0, p is 0, q is 1 and R.sub.3 is
methyl.
16. The method according to claim 1, where, in the compound of
Formula (I), or pharmaceutically acceptable salt thereof, Ar is a
group of formula (III), n is 0, p is 1, q is 1, R.sub.2 is
(C.sub.1-4)alkyl and R.sub.3 is (C.sub.1-4)alkyl.
17. The method according to claim 1, where, in the compound of
Formula (I), or pharmaceutically acceptable salt thereof, Ar is a
group of formula (III), n is 0, p is 1, q is 1, R.sub.2 is methyl
and R.sub.3 is methyl.
18. The method according to claim 1, where the compound of Formula
(I) is
6,8-dimethyl-2-({(2S)-1-[(2-methyl-5-phenyl-1,3-thiazol-4-yl)carbonyl]-2--
piperidinyl}methyl)imidazo[1,2-a]pyrazine, or pharmaceutically
acceptable salt thereof.
Description
[0001] This application is a divisional of application Ser. No.
12/576,372, filed 9 Oct. 2009, which claims benefit of U.S.
Provisional Application No. 61/103,967, filed Oct. 9, 2008.
FIELD OF THE INVENTION
[0002] This invention relates to imidazopyrazinylmethyl substituted
piperidine derivatives and their use as pharmaceuticals.
BACKGROUND OF THE INVENTION
[0003] Many medically significant biological processes are mediated
by proteins participating in signal transduction pathways that
involve G-proteins and/or second messengers.
[0004] Polypeptides and polynucleotides encoding the human
7-transmembrane G-protein coupled neuropeptide receptor, orexin-1
(HFGAN72), have been identified and are disclosed in EP-A-875565,
EP-A-875566 and WO 96/34877. Polypeptides and polynucleotides
encoding a second human orexin receptor, orexin-2 (HFGANP), have
been identified and are disclosed in EP-A-893498.
[0005] Polypeptides and polynucleotides encoding polypeptides which
are ligands for the orexin-1 receptor, e.g. orexin-A (Lig72A) are
disclosed in EP-A-849361.
[0006] Orexin receptors are found in the mammalian host and may be
responsible for many biological functions, including pathologies
including, but not limited to, depression; anxiety; addictions;
obsessive compulsive disorder; affective neurosis/disorder;
depressive neurosis/disorder; anxiety neurosis; dysthymic disorder;
behaviour disorder; mood disorder; sexual dysfunction; psychosexual
dysfunction; sex disorder; sexual disorder; schizophrenia; manic
depression; delerium; dementia; severe mental retardation and
dyskinesias such as Huntington's disease and Gilles de la Tourett's
syndrome; disturbed biological and circadian rhythms; feeding
disorders, such as anorexia, bulimia, cachexia, and obesity;
diabetes; appetite/taste disorders; vomiting/nausea; asthma;
cancer; Parkinson's disease; Cushing's syndrome/disease; basophil
adenoma; prolactinoma; hyperprolactinemia; hypopituitarism;
hypophysis tumor/adenoma; hypothalamic diseases; Froehlich's
syndrome; adrenohypophysis disease; hypophysis disease; hypophysis
tumor/adenoma; pituitary growth hormone; adrenohypophysis
hypofunction; adrenohypophysis hyperfunction; hypothalamic
hypogonadism; Kallman's syndrome (anosmia, hyposmia); functional or
psychogenic amenorrhea; hypopituitarism; hypothalamic
hypothyroidism; hypothalamic-adrenal dysfunction; idiopathic
hyperprolactinemia; hypothalamic disorders of growth hormone
deficiency; idiopathic growth hormone deficiency; dwarfism;
gigantism; acromegaly; disturbed biological and circadian rhythms;
and sleep disturbances associated with such diseases as
neurological disorders, neuropathic pain and restless leg syndrome,
heart and lung diseases; acute and congestive heart failure;
hypotension; hypertension; urinary retention; osteoporosis; angina
pectoris; myocardial infarction; ischaemic or haemorrhagic stroke;
subarachnoid haemorrhage; head injury such as sub-arachnoid
haemorrhage associated with traumatic head injury; ulcers;
allergies; benign prostatic hypertrophy; chronic renal failure;
renal disease; impaired glucose tolerance; migraine; hyperalgesia;
pain; enhanced or exaggerated sensitivity to pain, such as
hyperalgesia, causalgia and allodynia; acute pain; burn pain;
atypical facial pain; neuropathic pain; back pain; complex regional
pain syndromes I and II; arthritic pain; sports injury pain; pain
related to infection, e.g. HIV, post-polio syndrome, and
post-herpetic neuralgia; phantom limb pain; labour pain; cancer
pain; post-chemotherapy pain; post-stroke pain; post-operative
pain; neuralgia; nausea and vomiting; conditions associated with
visceral pain including irritable bowel syndrome, migraine and
angina; urinary bladder incontinence e.g. urge incontinence;
tolerance to narcotics or withdrawal from narcotics; sleep
disorders; sleep apnea; narcolepsy; insomnia; parasomnia; jet-lag
syndrome; and neurodegenerative disorders, which includes
nosological entities such as
disinhibition-dementia-parkinsonism-amyotrophy complex;
pallido-ponto-nigral degeneration, epilepsy, and seizure
disorders.
[0007] Experiments have shown that central administration of the
ligand orexin-A (described in more detail below) stimulated food
intake in freely-feeding rats during a 4 hour time period. This
increase was approximately four-fold over control rats receiving
vehicle. These data suggest that orexin-A may be an endogenous
regulator of appetite. Therefore, antagonists of its receptor may
be useful in the treatment of obesity and diabetes, see Cell, 1998,
92, 573-585.
[0008] There is a significant incidence of obesity in westernised
societies. According to WHO definitions a mean of 35% of subjects
in 39 studies were overweight and a further 22% clinically obese.
It has been estimated that 5.7% of all healthcare costs in the USA
are a consequence of obesity. About 85% of Type 2 diabetics are
obese, and diet and exercise are of value in all diabetics. The
incidence of diagnosed diabetes in westernised countries is
typically 5% and there are estimated to be an equal number
undiagnosed. The incidence of both diseases is rising,
demonstrating the inadequacy of current treatments which may be
either ineffective or have toxicity risks including cardiovascular
effects. Treatment of diabetes with sulfonylureas or insulin can
cause hypoglycaemia, whilst metformin causes GI side-effects. No
drug treatment for Type 2 diabetes has been shown to reduce the
long-term complications of the disease. Insulin sensitisers will be
useful for many diabetics, however they do not have an anti-obesity
effect.
[0009] Rat sleep/EEG studies have also shown that central
administration of orexin-A, an agonist of the orexin receptors,
causes a dose-related increase in arousal, largely at the expense
of a reduction in paradoxical sleep and slow wave sleep 2, when
administered at the onset of the normal sleep period. Therefore
antagonists of its receptor may be useful in the treatment of sleep
disorders including insomnia.
SUMMARY OF THE INVENTION
[0010] WO03/002561 discloses N-aroyl cyclic amine derivatives as
orexin antagonists. Compounds disclosed in WO03/002561 include
piperidine derivatives substituted at the 2-position with bicyclic
heteroarylmethyl groups. We have now unexpectedly found that some
piperidine derivatives substituted at the 2-position with an
imidazopyrazinylmethyl group have surprisingly beneficial
properties including, for example, increased oral bioavailability
and significantly increase solubility in physiologically relevant
media compared to the prior art compounds. Such properties make
these imidazopyrazinylmethyl substituted piperidine derivatives
very attractive as potential pharmaceutical agents which may be
useful in the prevention or treatment of obesity, including obesity
observed in Type 2 (non-insulin-dependent) diabetes patients, sleep
disorders, anxiety, depression, schizophrenia, drug dependency or
compulsive behaviour. Additionally these compounds may be useful in
the treatment of stroke, particularly ischemic or haemorrhagic
stroke, and/or blocking the emetic response, i.e. useful in the
treatment of nausea and vomiting.
[0011] Accordingly the present invention provides a compound of
formula (I)
##STR00001## [0012] where Ar is selected from the group consisting
of formula:
##STR00002##
[0013] R.sub.1 is (C.sub.1-4)alkyl, halo, halo(C.sub.1-4)alkyl,
(C.sub.1-4)alkoxy, halo(C.sub.1-4)alkoxy,
(C.sub.1-4)alkyl-O--(C.sub.1-4)alkyl, CN, NR.sup.5R.sup.6 wherein
R.sup.5 is H or (C.sub.1-4)alkyl and R.sup.6 is H or
(C.sub.1-4)alkyl;
[0014] R.sub.2 is (C.sub.1-4)alkyl, halo, halo(C.sub.1-4)alkyl,
(C.sub.1-4)alkoxy, halo(C.sub.1-4)alkoxy,
(C.sub.1-4)alkyl-O--(C.sub.1-4)alkyl, CN, NR.sup.7R.sup.8 wherein
R.sup.7 is H or (C.sub.1-4-alkyl and R.sup.8 is H or
(C.sub.1-4)-alkyl;
[0015] R.sub.3 is (C.sub.1-4)alkyl, halo, halo(C.sub.1-4)alkyl,
(C.sub.1-4)alkoxy, halo(C.sub.1-4)alkoxy,
(C.sub.1-4)alkyl-O--(C.sub.1-4)alkyl, CN, NR.sup.9R.sup.10 wherein
R.sup.9 is H or (C.sub.1-4-alkyl and R.sup.10 is H or
(C.sub.1-4)-alkyl;
[0016] n is 0 or 1;
[0017] p is 0 or 1; and
[0018] q is 0 or 1;
[0019] with the proviso that p and q are not both 0;
[0020] or a pharmaceutically acceptable salt thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0021] In one embodiment Ar is a group of formula (II).
[0022] In another embodiment Ar is a group of formula (III).
[0023] In one embodiment n is 0.
[0024] In one embodiment Ar is a group of formula (II), n is 0, p
is 1, q is 0 and R.sub.2 is (C.sub.1-4)alkyl.
[0025] In another embodiment Ar is a group of formula (II), n is 0,
p is 1, q is 0 and R.sub.2 is methyl.
[0026] In one embodiment Ar is a group of formula (II), n is 0, p
is 0, q is 1 and R.sub.3 is (C.sub.1-4)alkyl.
[0027] In another embodiment Ar is a group of formula (II), n is 0,
p is 0, q is 1 and R.sub.3 is methyl.
[0028] In one embodiment Ar is a group of formula (II), n is 0, p
is 1, q is 1, R.sub.2 is (C.sub.1-4)alkyl and R.sub.3 is
(C.sub.1-4)alkyl.
[0029] In another embodiment Ar is a group of formula (II), n is 0,
p is 1, q is 1, R.sub.2 is methyl and R.sub.3 is methyl.
[0030] In one embodiment n is 0.
[0031] In one embodiment Ar is a group of formula (III), n is 0, p
is 1, q is 0 and R.sub.2 is (C.sub.1-4)alkyl.
[0032] In another embodiment Ar is a group of formula (III), n is
0, p is 1, q is 0 and R.sub.2 is methyl.
[0033] In one embodiment Ar is a group of formula (III), n is 0, p
is 0, q is 1 and R.sub.3 is (C.sub.1-4)alkyl.
[0034] In another embodiment Ar is a group of formula (III), n is
0, p is 0, q is 1 and R.sub.3 is methyl.
[0035] In one embodiment Ar is a group of formula (III), n is 0, p
is 1, q is 1, R.sub.2 is (C.sub.1-4)alkyl and R.sub.3 is
(C.sub.1-4)alkyl.
[0036] In another embodiment Ar is a group of formula (III), n is
0, p is 1, q is 1, R.sub.2 is methyl and R.sub.3 is methyl.
[0037] Examples of the compounds of the invention include
6,8-dimethyl-2-({(2S)-1-[(2-methyl-5-phenyl-1,3-thiazol-4-yl)carbonyl]-2--
piperidinyl}methyl)imidazo[1,2-a]pyrazine.
[0038] When the compound contains a (C.sub.1-4)alkyl group, whether
alone or forming part of a larger group, e.g. (C.sub.1-4)alkoxy,
the alkyl group may be straight chain, branched or cyclic, or
combinations thereof. Examples of (C.sub.1-4)alkyl are methyl or
ethyl. An example of (C.sub.1-4)alkoxy is methyloxy.
[0039] Examples of halo(C.sub.1-4)alkyl include trifluoromethyl
(i.e. --CF.sub.3).
[0040] Examples of (C.sub.1-4)alkoxy include methyloxy and
ethyloxy.
[0041] Examples of halo(C.sub.1-4)alkoxy include trifluoromethyloxy
(i.e. --OCF.sub.3).
[0042] Halogen or "halo" (when used, for example, in
halo(C.sub.1-4)alkyl) means fluoro, chloro, bromo or iodo.
[0043] It is to be understood that the present invention covers all
combinations of particularised groups and substituents described
herein above.
[0044] The compounds of formula (I) are S enantiomers. Where
additional chiral centres are present in compounds of formula (I),
the present invention includes within its scope all possible
enantiomers and diastereoisomers, including mixtures thereof. The
different isomeric forms may be separated or resolved one from the
other by conventional methods, or any given isomer may be obtained
by conventional synthetic methods or by stereospecific or
asymmetric syntheses. The invention also extends to any tautomeric
forms or mixtures thereof.
[0045] It will be understood that the invention includes
pharmaceutically acceptable derivatives of compounds of formula (I)
and that these are included within the scope of the invention.
[0046] Particular compounds according to the invention include
those mentioned in the examples and their pharmaceutically
acceptable derivatives.
[0047] As used herein "pharmaceutically acceptable derivative"
includes any pharmaceutically acceptable salt, ester or salt of
such ester of a compound of formula (I) which, upon administration
to the recipient is capable of providing (directly or indirectly) a
compound of formula (I) or an active metabolite or residue
thereof.
[0048] It will be appreciated that for use in medicine the salts of
the compounds of formula (I) should be pharmaceutically acceptable.
Suitable pharmaceutically acceptable salts will be apparent to
those skilled in the art. Pharmaceutically acceptable salts include
those described by Berge, Bighley and Monkhouse J. Pharm. Sci
(1977) 66, pp 1-19. Such pharmaceutically acceptable salts include
acid addition salts formed with inorganic acids e.g. hydrochloric,
hydrobromic, sulphuric, nitric or phosphoric acid; and organic
acids e.g. succinic, maleic, acetic, fumaric, citric, tartaric,
benzoic, p-toluenesulfonic, methanesulfonic or naphthalenesulfonic
acid. Other salts e.g. oxalates or formates, may be used, for
example in the isolation of compounds of formula (I) and are
included within the scope of this invention. Also included within
the scope of the invention are solvates and hydrates of compounds
of formula (I).
[0049] Certain of the compounds of formula (I) may form acid
addition salts with one or more equivalents of the acid. The
present invention includes within its scope all possible
stoichiometric and non-stoichiometric forms.
[0050] The compounds of formula (I) may be prepared in crystalline
or non-crystalline form and, if crystalline, may optionally be
solvated, eg. as the hydrate. This invention includes within its
scope stoichiometric solvates (eg. hydrates) as well as compounds
containing variable amounts of solvent (eg. water).
[0051] The subject invention also includes isotopically-labeled
compounds which are identical to those recited in formula (I) and
following, but for the fact that one or more atoms are replaced by
an atom having an atomic mass or mass number different from the
atomic mass or mass number most commonly found in nature. Examples
if isotopes that can be incorporated into compounds of the
invention include isotopes of hydrogen, carbon, nitrogen, oxygen,
fluorine, iodine and chlorine such as .sup.3H, .sup.11C, .sup.14C,
.sup.18F, .sup.123I or .sup.125I.
[0052] Compounds of the present invention and pharmaceutically
acceptable salts of said compounds that contain the aforementioned
isotopes and/or other isotopes of other atoms are within the scope
of the present invention. Isotopically labeled compounds of the
present invention, for example those into which radioactive
isotopes such as .sup.3H or .sup.14C have been incorporated, are
useful in drug and/or substrate tissue distribution assays.
Tritiated, ie. .sup.3H, and carbon-14, ie. .sup.14C, isotopes are
particularly preferred for their ease of preparation and
detectability. .sup.11C and .sup.18F isotopes are particularly
useful in PET (positron emission tomography).
[0053] Since the compounds of formula (I) are intended for use in
pharmaceutical compositions it will readily be understood that they
are each preferably provided in substantially pure form, for
example at least 60% pure, more suitably at least 75% pure and
preferably at least 85%, especially at least 98% pure (% are on a
weight for weight basis). Impure preparations of the compounds may
be used for preparing the more pure forms used in the
pharmaceutical compositions.
[0054] According to a further aspect of the present invention there
is provided a process for the preparation of compounds of formula
(I) and derivatives thereof. The following scheme details an
example of a synthetic route to compounds of the invention. In the
following scheme reactive groups can be protected with protecting
groups and deprotected according to well established
techniques.
Scheme
[0055] According to a further feature of the invention there is
provided a process for the preparation of compounds of formula (I)
and derivatives thereof. The following is an example of a synthetic
scheme that may be used to synthesise the compounds of the
invention.
##STR00003##
[0056] It will be understood by those skilled in the art that
certain compounds of the invention can be converted into other
compounds of the invention according to standard chemical
methods.
[0057] The starting materials for use in the scheme are
commercially available, known in the literature or can be prepared
by known methods.
[0058] The compounds of formula (I) may be prepared singly or as
compound libraries comprising at least 2, e.g. 5 to 1000,
preferably 10 to 100 compounds of formula (I). Compound libraries
may be prepared by a combinatorial `split and mix` approach or by
multiple parallel synthesis using either solution phase or solid
phase chemistry, by procedures known to those skilled in the
art.
[0059] Thus according to a further aspect of the invention there is
provided a compound library comprising at least 2 compounds of
formula (I), or pharmaceutically acceptable derivatives
thereof.
[0060] Pharmaceutically acceptable salts may be prepared
conventionally by reaction with the appropriate acid or acid
derivative.
[0061] The present invention provides compounds of formula (I) and
their pharmaceutically acceptable derivatives for use in human or
veterinary medicine.
[0062] The compounds of formula (I) and their pharmaceutically
acceptable derivatives are useful for the treatment of diseases or
disorders where an antagonist of a human Orexin receptor is
required such as obesity, including obesity observed in Type 2
(non-insulin-dependent) diabetes patients, schizophrenia, anxiety,
depression, obsessive compulsive disorder, drug dependency and/or
sleep disorders selected from the group consisting of dyssomnias
such as primary insomnia (307.42), primary hypersomnia (307.44),
narcolepsy (347), breathing-related sleep disorders (780.59),
circadian rhythm sleep disorder (307.45) and dyssomnia not
otherwise specified (307.47); parasomnias such as nightmare
disorder (307.47), sleep terror disorder (307.46), sleepwalking
disorder (307.46) and parasomnia not otherwise specified (307.47);
sleep disorders related to another mental disorder such as insomnia
related to another mental disorder (307.42) and hypersomnia related
to another mental disorder (307.44); sleep disorder due to a
general medical condition; and substance-induced sleep disorder
including the subtypes insomnia type, hypersomnia type, parasomnia
type and mixed type, sleep apnea and jet-lag syndrome (numbers in
brackets after the listed diseases refer to the classification code
in DSM-IV: Diagnostic and Statistical Manual of Mental Disorders,
4th Edition, published by the American Psychiatric Association. The
various subtypes of the disorders mentioned herein are contemplated
as part of the present invention).
[0063] Additionally the compounds of formula (I) and
pharmaceutically acceptable derivatives are useful for the
treatment of stroke, particularly ischemic or haemorrhagic and/or
in blocking an emetic response i.e. nausea and vomiting.
[0064] The invention also provides a method of treating or
preventing diseases or disorders where an antagonist of a human
orexin receptor is required, for example those diseases and
disorders mentioned hereinabove, which comprises administering to a
subject in need thereof an effective amount of a compound of
formula (I), or a pharmaceutically acceptable derivative
thereof.
[0065] The invention also provides a compound of formula (I), or a
pharmaceutically acceptable derivative thereof, for use in the
treatment or prophylaxis of diseases or disorders where an
antagonist of a human orexin receptor is required, for example
those diseases and disorders mentioned hereinabove.
[0066] The invention also provides the use of a compound of formula
(I), or a pharmaceutically acceptable derivative thereof, in the
manufacture of a medicament for the treatment or prophylaxis of
diseases or disorders where an antagonist of a human Orexin
receptor is required, for example those diseases and disorders
mentioned hereinabove.
[0067] The invention also provides the use of a compound of formula
(I), or a pharmaceutically acceptable derivative thereof, in the
manufacture of a medicament for the treatment or prophylaxis of
diseases or disorders where an antagonist of a human Orexin
receptor is required such as obesity, including obesity observed in
Type 2 (non-insulin-dependent) diabetes patients, schizophrenia,
anxiety, depression, obsessive compulsive disorder, drug dependency
and/or sleep disorders selected from the group consisting of
dyssomnias such as primary insomnia (307.42), primary hypersomnia
(307.44), narcolepsy (347), breathing-related sleep disorders
(780.59), circadian rhythm sleep disorder (307.45) and dyssomnia
not otherwise specified (307.47); parasomnias such as nightmare
disorder (307.47), sleep terror disorder (307.46), sleepwalking
disorder (307.46) and parasomnia not otherwise specified (307.47);
sleep disorders related to another mental disorder such as insomnia
related to another mental disorder (307.42) and hypersomnia related
to another mental disorder (307.44); sleep disorder due to a
general medical condition; and substance-induced sleep disorder
including the subtypes insomnia type, hypersomnia type, parasomnia
type and mixed type, sleep apnea and jet-lag syndrome (numbers in
brackets after the listed diseases refer to the classification code
in DSM-IV: Diagnostic and Statistical Manual of Mental Disorders,
4th Edition, published by the American Psychiatric Association. The
various subtypes of the disorders mentioned herein are contemplated
as part of the present invention).
[0068] For use in therapy the compounds of the invention are
usually administered as a pharmaceutical composition. The invention
also provides a pharmaceutical composition comprising a compound of
formula (I), or a pharmaceutically acceptable derivative thereof,
and a pharmaceutically acceptable carrier.
[0069] The compounds of formula (I) and their pharmaceutically
acceptable derivatives may be administered by any convenient
method, e.g. by oral, parenteral, buccal, sublingual, nasal, rectal
or transdermal administration, and the pharmaceutical compositions
adapted accordingly.
[0070] The compounds of formula (I) and their pharmaceutically
acceptable derivatives which are active when given orally can be
formulated as liquids or solids, e.g. as syrups, suspensions,
emulsions, tablets, capsules or lozenges.
[0071] A liquid formulation will generally consist of a suspension
or solution of the active ingredient in a suitable liquid
carrier(s) e.g. an aqueous solvent such as water, ethanol or
glycerine, or a non-aqueous solvent, such as polyethylene glycol or
an oil. The formulation may also contain a suspending agent,
preservative, flavouring and/or colouring agent.
[0072] A composition in the form of a tablet can be prepared using
any suitable pharmaceutical carrier(s) routinely used for preparing
solid formulations, such as magnesium stearate, starch, lactose,
sucrose and cellulose.
[0073] A composition in the form of a capsule can be prepared using
routine encapsulation procedures, e.g. pellets containing the
active ingredient can be prepared using standard carriers and then
filled into a hard gelatin capsule; alternatively a dispersion or
suspension can be prepared using any suitable pharmaceutical
carrier(s), e.g. aqueous gums, celluloses, silicates or oils and
the dispersion or suspension then filled into a soft gelatin
capsule.
[0074] Typical parenteral compositions consist of a solution or
suspension of the active ingredient in a sterile aqueous carrier or
parenterally acceptable oil, e.g. polyethylene glycol, polyvinyl
pyrrolidone, lecithin, arachis oil or sesame oil. Alternatively,
the solution can be lyophilised and then reconstituted with a
suitable solvent just prior to administration.
[0075] Compositions for nasal administration may conveniently be
formulated as aerosols, drops, gels and powders. Aerosol
formulations typically comprise a solution or fine suspension of
the active ingredient in a pharmaceutically acceptable aqueous or
non-aqueous solvent and are usually presented in single or
multidose quantities in sterile form in a sealed container which
can take the form of a cartridge or refill for use with an
atomising device. Alternatively the sealed container may be a
disposable dispensing device such as a single dose nasal inhaler or
an aerosol dispenser fitted with a metering valve. Where the dosage
form comprises an aerosol dispenser, it will contain a propellant
which can be a compressed gas e.g. air, or an organic propellant
such as a fluorochlorohydrocarbon or hydrofluorocarbon. Aerosol
dosage forms can also take the form of pump-atomisers.
[0076] Compositions suitable for buccal or sublingual
administration include tablets, lozenges and pastilles where the
active ingredient is formulated with a carrier such as sugar and
acacia, tragacanth, or gelatin and glycerin.
[0077] Compositions for rectal administration are conveniently in
the form of suppositories containing a conventional suppository
base such as cocoa butter.
[0078] Compositions suitable for transdermal administration include
ointments, gels and patches.
[0079] Preferably the composition is in unit dose form such as a
tablet, capsule or ampoule.
[0080] The dose of the compound of formula (I), or a
pharmaceutically acceptable derivative thereof, used in the
treatment or prophylaxis of the abovementioned disorders or
diseases will vary in the usual way with the particular disorder or
disease being treated, the weight of the subject and other similar
factors. However, as a general rule, suitable unit doses may be
0.05 to 1000 mg, more suitably 0.05 to 500 mg. Unit doses may be
administered more than once a day for example two or three times a
day, so that the total daily dosage is in the range of about 0.01
to 100 mg/kg; and such therapy may extend for a number of weeks or
months. In the case of pharmaceutically acceptable derivatives the
above figures are calculated as the parent compound of formula
(I).
[0081] No toxicological effects are indicated/expected when a
compound of formula (I) is administered in the above mentioned
dosage range.
[0082] Human Orexin-A has the amino acid sequence:
TABLE-US-00001 (SEQ ID NO: 1) pyroGlu Pro Leu Pro Asp Cys Cys Arg
Gln Lys Thr 1 5 10 Cys Ser Cys Arg Leu Tyr Glu Leu Leu His Gly Ala
15 20 Gly Asn His Ala Ala Gly Ile Leu Thr Leu-NH.sub.2 25 30
[0083] Orexin-A can be employed in screening procedures for
compounds which inhibit the ligand's activation of the orexin-1
receptor.
[0084] In general, such screening procedures involve providing
appropriate cells which express the orexin-1 receptor on their
surface. Such cells include cells from mammals, yeast, Drosophila
or E. coli. In particular, a polynucleotide encoding the orexin-1
receptor is used to transfect cells to express the receptor. The
expressed receptor is then contacted with a test compound and an
orexin-1 receptor ligand to observe inhibition of a functional
response. One such screening procedure involves the use of
melanophores which are transfected to express the orexin-1
receptor, as described in WO 92/01810.
[0085] Another screening procedure involves introducing RNA
encoding the orexin-1 receptor into Xenopus oocytes to transiently
express the receptor. The receptor oocytes are then contacted with
a receptor ligand and a test compound, followed by detection of
inhibition of a signal in the case of screening for compounds which
are thought to inhibit activation of the receptor by the
ligand.
[0086] Another method involves screening for compounds which
inhibit activation of the receptor by determining inhibition of
binding of a labelled orexin-1 receptor ligand to cells which have
the receptor on their surface. This method involves transfecting a
eukaryotic cell with DNA encoding the orexin-1 receptor such that
the cell expresses the receptor on its surface and contacting the
cell or cell membrane preparation with a compound in the presence
of a labelled form of an orexin-1 receptor ligand. The ligand may
contain a radioactive label. The amount of labelled ligand bound to
the receptors is measured, e.g. by measuring radioactivity.
[0087] Yet another screening technique involves the use of FLIPR
equipment for high throughput screening of test compounds that
inhibit mobilisation of intracellular calcium ions, or other ions,
by affecting the interaction of an orexin-1 receptor ligand with
the orexin-1 receptor.
[0088] Throughout the specification and claims which follow, unless
the context requires otherwise, the word `comprise`, and variations
such as `comprises` and `comprising` will be understood to imply
the inclusion of a stated integer or step or group of integers but
not to the exclusion of any other integer or step or group of
integers or steps.
[0089] All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
[0090] The following Examples illustrate the preparation of
pharmacologically active compounds of the invention. The
Descriptions 1 and 2 illustrate the preparation of intermediates to
compounds of the invention.
EXAMPLES
[0091] In the procedures that follow, after each starting material,
reference to a description is typically provided. This is provided
merely for assistance to the skilled chemist. The starting material
may not necessarily have been prepared from the batch referred
to.
[0092] The compounds described in the Examples described
hereinafter have all been prepared as a first step from
stereochemically pure methyl 5-oxo-L-prolinate or ethyl
5-oxo-D-prolinate. The stereochemistry of the compounds of the
Descriptions and Examples have been assigned on the assumption that
the pure configuration of 5-oxo-prolinate is maintained.
[0093] Compounds are named using ACD/Name PRO 6.02 chemical naming
software (Advanced Chemistry Development Inc., Toronto, Ontario,
M5H2L3, Canada). Proton Magnetic Resonance (NMR) spectra were
recorded either on Varian instruments at 300, 400 or 500 MHz, or on
a Bruker instrument at 300 and 400 MHz. Chemical shifts are
reported in ppm (.delta.) using the residual solvent line as
internal standard. Splitting patterns are designed as s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; b, broad.
[0094] The NMR spectra were recorded at a temperature ranging from
25 to 90.degree. C. When more than one conformer was detected the
chemical shifts for the most abundant one is reported.
[0095] HPLC analysis indicated by R.sub.t(HPLC): x min, was
performed on an Agilent 1100 series instrument using a Luna 3u
C18(2) 100A (50.times.2.0 mm) column (mobile phase: 100%
[water+0.05% TFA] to 95% [acetonitrile+0.05% TFA] in 8 min, flux=1
ml/min, detection wavelength 220 nm.
[0096] Mass spectra (MS) were taken on a 4 II triple quadrupole
Mass Spectrometer (Micromass UK) or on a Agilent MSD 1100 Mass
Spectrometer, operating in ES (+) and ES (-) ionization mode or on
an Agilent LC/MSD 1100 Mass Spectrometer, operating in ES (+) and
ES (-) ionization mode coupled with HPLC instrument Agilent 1100
Series [LC/MS--ES (+):analysis performed on a Supelcosil ABZ +Plus
(33.times.4.6 mm, 3 .mu.m) (mobile phase: 100% [water +0.1%
HCO.sub.2H] for 1 min, then from 100% [water+0.1% HCO.sub.2H] to 5%
[water +0.1% HCO.sub.2H] and 95% [CH.sub.3CN] in 5 min, finally
under these conditions for 2 min; T=40.degree. C.; flux=1 mL/min;
LC/MS--ES (-):analysis performed on a Supelcosil ABZ +Plus
(33.times.4.6 mm, 3 .mu.m) (mobile phase: 100% [water +0.05%
NH.sub.3] for 1 min, then from 100% [water +0.05% NH.sub.3 to 5%
[water +0.05% NH.sub.3] and 95% [CH.sub.3CN] in 5 min, finally
under these conditions for 2 min; T=40.degree. C.; flux=1 mL/min].
In the mass spectra only one peak in the molecular ion cluster is
reported.
[0097] For reactions involving microwave irradiation, a Personal
Chemistry Emrys.TM. Optimizer was used.
[0098] Flash silica gel chromatography was carried out on silica
gel 230-400 mesh (supplied by Merck AG Darmstadt, Germany) or over
Varian Mega Be--Si pre-packed cartridges or over pre-packed Biotage
silica cartridges.
[0099] SPE-SCX cartridges are ion exchange solid phase extraction
columns supplied by Varian. The eluent used with SPE-SCX cartridges
is methanol followed by 2N ammonia solution in methanol.
[0100] In a number of preparations, purification was performed
using either Biotage manual flash chromatography (Flash+) or
automatic flash chromatography (Horizon) systems. All these
instruments work with Biotage Silica cartridges.
[0101] SPE-Si cartridges are silica solid phase extraction columns
supplied by Varian.
[0102] The following table lists the abbreviations used in the
text:
TABLE-US-00002 AcCl Acetyl chloride BINAP
2,2'-Bis(diphenylphosphino)-1,1'-binaphthyl Boc t-Butoxycarbonyl
n-BuLi N Butyl Lithium s-BuLi Sec Butyl Lithium Cy Cyclohexanes DCM
Dichloromethane DIPA N,N-diisopropylamine DIPEA
N,N-diisopropyl-N-ethylamine DME 1,2-Dimethoxyethane DMF
Dimethylformamide Et.sub.2O Diethylether EtOAc Ethylacetate LAH
Lithium Aluminum Hydride LDA Lithiumdiisopropylamide MsCl
Mesylchloride NBS N-Bromosuccinimide NCS N-Chlorosuccinimide rt
Room Temperature TBTU
O-(benzotriazol-1-yl)-N,N,N'N'-tetramethyluronium tetrafluoroborate
TEA Triethylamine TFA Trifluoroacetic acid THF Tetrahydrofuran
Descriptions
Description 1:
1,1-dimethylethyl(2S)-2-[2-(methyloxy)-2-oxoethyl]-1-piperidinecarboxylat-
e (D1)
##STR00004##
[0104] Into a 250 ml round bottom flask
((2S)-1-{[(1,1-dimethylethyl)oxy]carbonyl}-2-piperidinyl)acetic
acid (1.00 g, 4.11 mmol), DMF (25 ml), DIPEA (2.15 ml, 12.33 mmol)
and TBTU (1.98 g, 6.17 mmol) were added. The mixture was stirred at
rt for 20 min and a brown colour was formed. After this time MeOH
(0.25 ml, 6.17 mmol) was added and the resulting solution stirred
at rt for 30 min. Then it was transferred into e separatory funnel
containing brine (20 ml) and extracted with EtOAc (20 ml.times.2),
the combined organic layers were washed with water/ice (5.times.20
ml). The organic layer was dried (Na.sub.2SO.sub.4), filtered and
concentrated. The crude obtained was purified by column
chromatography (Biotage SP1, Cy-EtOAc from 100/0 to 85/15).
Collected fractions gave the title compound (1.01 g, 3.92 mmol, 95%
yield) as a colorless oil. .sup.1H-NMR (500 MHz, CDCl.sub.3)
.delta.(ppm): 4.67-4.75 (m, 1H), 3.96-4.05 (m, 1H), 3.67 (s, 3H),
2.79 (t, 1H), 2.61 (dd, 1H), 2.53 (dd, 1H), 1.60-1.70 (m, 6H), 1.46
(s, 9H).
Description 2:
1,1-dimethylethyl(2S)-2-(3-bromo-2-oxopropyl)-1-piperidinecarboxylate
(D2)
##STR00005##
[0106] In a 500 ml round-bottomed flask under nitrogen at rt, D1
(11.1 g, 43.1 mmol) was dissolved in THF (100 ml) to give a pale
yellow solution. This solution was cooled to -78.degree. C. and the
Tebbe reagent (104 ml of a 0.5 M solution in toluene, 51.8 mmol)
was added dropwise. The thick mixture was diluted with further 70
ml of dry toluene. The resulting brown-orange mixture was stirred
at this temperature for 30 min and then slowly warmed up to rt and
left under stirring for 2 h. The reaction mixture was charged into
a dropping funnel and then added dropwise to a 2 L round-bottomed
flask containing .about.400 ml of NaOH 1 M aqueous solution cooled
at 0.degree. C. At the end of the quench, the resulting grey
suspension was diluted with EtOAc (250 ml) and allowed to stir
overnight (mechanical stirring). The resulting yellow suspension
was then filtered over a Gooch funnel (using Sterimat): salts were
washed with EtOAc (.about.500 ml). Phases were then separated and
the organic layer was washed with brine (2.times.500 ml). The
combined organic phases were dried (Na.sub.2SO.sub.4), filtered and
concentrated to give a deep orange oil. This material was diluted
with .about.500 ml of Et.sub.2O: some salts precipitated, so the
resulting suspension was filtered over a Gooch funnel (using
Sterimat). The filtrate was concentrated under vacuum to give 12.4
g of crude
1,1-dimethylethyl(2S)-2-[2-(methyloxy)-2-propen-1-yl]-1-piperidinecarboxy-
late as an orange-brown oil. The material contained some residual
salts (as the overall recovered amount was higher than the
theoretical amount). The material was used without further
purification in the next reaction and supposed to be pure at 89 wt
%. In a 1 L round-bottomed flask under nitrogen at rt
1,1-dimethylethyl(2S)-2-[2-(methyloxy)-2-propen-1-yl]-1-piperidinecarboxy-
late (12.4 g, 43.1 mmol) was dissolved in THF (125 ml) and water
(35 ml) to give a pale yellow solution. NBS (7.67 g, 43.1 mmol) was
then added dissolved in .about.100 ml of THF. The resulting grey
mixture was stirred at rt for 1 h. Then additional NBS (0.2 eq, 1.5
g) dissolved in 50 ml of THF was added and the reaction mixture
stirred at rt for 1 h. The mixture was concentrated under vacuum to
remove THF, then was diluted with EtOAc (.about.500 ml) and water
(200 ml). Phases were separated and the aqueous layer was
back-extracted with EtOAc (250 ml). The combined organic layers
were dried (Na.sub.2SO.sub.4) filtered and concentrated to give:
17.8 g of a brown oil. This material was purified by
flash-chromatography (Biotage 75L, Cy-EtOAc from 100-0 to 90-10) to
give the title compound (6.0 g, 18.7 mmol, 43% yield from D1, two
steps) as a slightly yellow oil that solidified upon standing.
UPLC: rt=0.79, peaks observed: 344 [M+Na, 100%], 342 [M+Na, 100%],
266 [M-tBu,100%] and 264 [M-tBu,100%]. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta.(ppm): 4.72-4.79 (m, 1H), 3.91-4.10 (m, 3H),
2.77-2.97 (m, 3 H), 1.49-1.75 (m, 6H), 1.46 (s, 9H).
EXAMPLES
Example 1
6,8-dimethyl-2-({(2S)-1-[(2-methyl-5-phenyl-1,3-thiazol-4-yl)carbonyl]-2-p-
iperidinyl}methyl)imidazo[1,2-a]pyrazine (HCl salt) (E1)
##STR00006##
[0108] To a solution of
1,1-dimethylethyl(2S)-2-(3-bromo-2-oxopropyl)-1-piperidinecarboxylate
D2 (0.140 g, 0.44 mmol) in DMF (2 ml) was added
3,5-dimethyl-2-pyrazinamine (0.054 g, 0.44 mmol) and the mixture
was stirred at 150.degree. C. for 30 min. The reaction mixture was
charged into a SCX column and was eluted with methanol and ammonia
2 M in methanol. Collected fractions gave 0.115 g of a crude
containing the desired
6,8-dimethyl-2-[(2S)-2-piperidinylmethyl]imidazo[1,2-a]pyrazine.
UPLC: rt=0.34, peak observed: 245 (M+1). C.sub.14H.sub.20N.sub.4
requires 244. Into a 7 ml screw capped vial
2-methyl-5-phenyl-1,3-thiazole-4-carboxylic acid (0.114 g, 0.52
mmol) was dissolved in DCM (1 ml), Oxalyl chloride (0.100 ml, 1.14
mmol) then DMF (0.036 ml, 0.47 mmol) were added and the resulting
mixture was stirred for 30 min at rt. The solvent was removed under
reduced pressure and the resulting yellow solid dissolved in DCM (1
ml) and added dropwise to the solution containing the crude (0.115
g) 6,8-dimethyl-2-[(2S)-2-piperidinylmethyl]imidazo[1,2-a]pyrazine
and TEA (0.197 ml, 0.47 mmol) in DCM (1 ml) cooled at 0.degree. C.
The ice-bath was removed and the reaction mixture left under
stirring at rt for 1 h. DCM was added and the mixture washed with a
saturated NaHCO.sub.3 aqueous solution, the organic phase was
separated, dried (Na.sub.2SO.sub.4), filtered and concentrated. The
residue was purified by column chromatography on silica gel
(Biotage 25 M, DCM/MeOH, 90/01) and by fraction lynx (basic
method). The free base of the title compound was obtained (0.039 g,
0.079 mmol, 20% yield from D2, three steps) as a yellow solid.
UPLC: rt=0.58, peak observed: 446 (M+1). C.sub.25H.sub.27N.sub.5OS
requires 445. .sup.1H NMR [the product is present as a mixture of
conformers (ratio c.ca 60/40)] (500 MHz, DMSO-d.sup.6). The free
base (0.037 g, 0.08 mmol) was transferred into a 7 ml screw capped
vial with anhydrous DCM (1 ml) and the solution cooled to 0.degree.
C. HCl (0.125 ml of a 1 M solution in Et.sub.2O, 0.13 mmol) was
added dropwise and the mixture stirred for 15 min. The solvent was
removed under reduced pressure and the resulting solid triturated
with anhydrous Et.sub.2O. The title compound was obtained (0.041 g,
0.08 mmol, 98% yield) as a white solid. UPLC: rt=0.58, peak
observed: 446 (M+1-HCl). C.sub.25H.sub.28ClN.sub.5OS requires
482.
Example 2
Determination of Antagonist Affinity at Human Orexin-1 and 2
Receptors Using FLIPR
Cell Culture
[0109] Adherent Chinese Hamster Ovary (CHO) cells, stably
expressing the recombinant human Orexin-1 (hOX1) or human Orexin-2
receptors (hOX2), were maintained in culture in Alpha Minimum
Essential Medium (Gibco/Invitrogen, cat. no.; 22571-020),
supplemented with 10% decomplemented foetal bovine serum (Life
Technologies, cat. no. 10106-078) and 400 ug/mL Geneticin G418
(Calbiochem, cat. no. 345810). Cells were grown as monolayers under
95%:5% air:CO.sub.2 at 37.degree. C. and passaged every 3-4 days.
The highest passage used was 25.
Measurement of [Ca.sup.2+].sub.i Using the FLIPR.TM.
[0110] CHO-hOX1 or CHO-hOX2 cells were seeded into black
clear-bottom 384-well plates at a density of 20,000 cells per well
in culture medium as described above and maintained overnight
(95%:5% air:CO.sub.2 at 37.degree. C.).
[0111] On the day of the experiment, culture medium were discarded
and the cells washed three times with standard buffer (NaCl, 145
mM; KCl, 5 mM; HEPES, 20 mM; Glucose, 5.5 mM; MgCl.sub.2, 1 mM;
CaCl.sub.2, 2 mM) added with Probenecid 2.5 mM.
[0112] The plates were then incubated at room temperature for 60
minutes in the dark with 1 .mu.M FLUO-4AM dye to allow cell uptake
of the FLUO-4AM, which is then converted by intracellular esterases
to FLUO-4, which is unable to leave the cells.
[0113] After incubation, cells were washed three times with
standard buffer to remove extracellular dye and 30 .mu.L of buffer
were left in each well after washing. Compounds of the invention
were tested in a final assay concentration range from 1.66E-05M to
1.58E-11M.
[0114] Compounds of the invention were dissolved in
dimethylsulfoxide (DMSO) at a stock concentration of 10 mM. These
solutions are serially diluted with DMSO in a 384 compound plate
and 1 .mu.L of each dilution is transferred to the test compound
plate. Just prior compounds addition to the cells, buffer (50
.mu.l/well) was added to the 1 .mu.L compound copy plate.
[0115] An agonist stimulus 384-well plate containing 50 .mu.L/well
of human orexin A (hOrexinA) was prepared just before using by
diluting with buffer a stock plate: final concentration is
equivalent to the calculated EC80 for hOrexinA. This value was
obtained by testing hOrexinA in concentration response curve (at
least 16 replicates) the same day of the experiment.
[0116] The loaded cells were then incubated for 10 min at
37.degree. C. with test compound. The plates were then placed into
a FLIPR.TM. (Molecular Devices, UK) to monitor cell fluorescence
(.lamda..sub.ex=488 nm, .lamda..sub.EM=540 nm) (Sullivan E, Tucker
E M, Dale I L. Measurement of [Ca.sup.2+].sub.i using the
fluometric imaging plate reader (FLIPR). In: Lambert D G (ed.),
Calcium Signaling Protocols. New Jersey: Humana Press, 1999,
125-136). A baseline fluorescence reading was taken over a 5 to 10
second period, and then 10 .mu.L of EC80 hOrexinA solution was
added. The fluorescence was then read over a 4-5 minute period.
Data Analysis
[0117] Functional responses using FLIPR were measured as peak
fluorescence intensity minus basal fluorescence and expressed as a
percentage of a non-inhibited Orexin-A-induced response on the same
plate. Iterative curve-fitting and parameter estimations were
carried out using a four parameter logistic model and Microsoft
Excel (Bowen W P, Jerman J C. Nonlinear regression using
spreadsheets. Trends Pharmacol. Sci. 1995; 16: 413-417). Antagonist
affinity values (IC.sub.50) were converted to functional pK.sub.i
values using a modified Cheng-Prusoff correction (Cheng Y C,
Prusoff W H. Relationship between the inhibition constant (K.sub.i)
and the concentration of inhibitor which causes 50 percent
inhibition (IC.sub.50) of an enzymatic reaction. Biochem.
Pharmacol. 1973, 22: 3099-3108).
fpKi = - log ( IC 50 ) ( 2 + ( [ agonist ] ( EC 50 ) ) n ) 1 / n -
1 ##EQU00001##
[0118] Where [agonist] is the agonist concentration, EC.sub.50 is
the concentration of agonist giving 50% activity derived from the
agonist dose response curve and n=slope of the dose response curve.
When n=1 the equation collapses to the more familiar Cheng-Prusoff
equation.
[0119] The compound of Example 1 tested according to this method
had an fpKi value of 8.3 at the human cloned orexin-1 receptor and
8.2 at the human cloned orexin-2 receptor.
Sequence CWU 1
1
1133PRTHomo Sapien 1Glu Pro Leu Pro Asp Cys Cys Arg Gln Lys Thr Cys
Ser Cys Arg Leu1 5 10 15Tyr Glu Leu Leu His Gly Ala Gly Asn His Ala
Ala Gly Ile Leu Thr 20 25 30Leu
* * * * *