NOVEL PIPERAZINES
FIELD OF THE INVENTION
The present invention relates to novel piperazines, to the use of these compounds in pharmaceutical
compositions, to pharmaceutical compositions comprising the compounds, and to
methods of treatment employing these compounds or compositions. The present compounds
show a high and selective binding affinity for the histamine H3 receptor, indicating histamine
H3 receptor antagonistic, inverse agonistic or agonistic activity. As a result, the compounds
are useful for the treatment of diseases or disorders related to the histamine H3 receptor.
BACKGROUND OF THE INVENTION
The existence of the histamine H3 receptor has been known for several years and the receptor
is of current interest for the development of new medicaments. Recently, the human histamine
H3 receptor has been cloned. The histamine H3 receptor is a presynaptic autoreceptor
located both in the central and the peripheral nervous system, the skin and in organs
such as the lung, the intestine, probably the spleen and the gastrointestinal tract. Recent evidence
suggests that the H3 receptor shows intrinsic, constitutive activity, in vitro as well as in
vivo (ie it is active in the absence of an agonist). Compounds acting as inverse agonists can
inhibit this activity. The histamine H3 receptor has been demonstrated to regulate the release
of histamine and also of other neurotransmitters such as serotonin and acetylcholine. A histamine
H3 receptor antagonist or inverse agonist would therefore be expected to increase
the release of these neurotransmitters in the "brain. A histamine H3 receptor agonist, on the
contrary, leads to an inhibition of the biosynthesis of histamine and an inhibition of the release
of histamine and also of other neurotransmitters such as serotonin and acetylcholine.
These findings suggest that histamine H3 receptor agonists, inverse agonists and antagonists
could be important mediators of neuronal activity. Accordingly, the histamine H3 receptor
is an important target for new therapeutics.
Compounds similar to the compounds of the present invention have previously been disclosed,
cf. J. Med. Chem. 1999, 42, 336, J. Med. Chem. 1992, 35, 2369, DE 2804096, J.
Org. Chem. 1996. 61, 3849, Bull. Soc. Chim. Fr. 1969, 319, WO 00/66578, WO 99/21845,
and J. Med. Chem. 1968, 11(6), 1144-1150. However, these references neither disclose nor
suggest that these compounds may have a histamine H3 receptor antagonistic or agonistic
activity.
Several publications disclose the preparation and use of histamine H3 agonists and antagonists.
Most of these are imidazole derivatives. However, recently some imidazole-free ligands
of the histamine H3 receptor have been described (see e.g. Linney et al., J. Med. Chem.
2000, 43, 2362-2370; US 6,316,475, WO 01/66534 and WO 01/74810). However, these
compounds differ structurally from the present compounds.
In view of the art's interest in histamine H3 receptor agonists, inverse agonists and antagonists,
novel compounds which interact with the histamine H3 receptor would be a highly desirable
contribution to the art. The present invention provides such a contribution to the art
being based on the finding that a novel class of piperazines has a high and specific affinity to
the histamine H3 receptor.
Due to their interaction with the histamine H3 receptor, the present compounds are useful in
the treatment of a wide range of conditions and disorders in which an interaction with the histamine
H3 receptor is beneficial. Thus, the compounds may find use, e.g., in the treatment of
diseases of the central nervous system, the peripheral nervous system, the cardiovascular
system, the pulmonary system, the gastrointestinal system and the endocrinological system.
SUMMARY OF THE INVENTION
The invention relates to compounds according to formula I
wherein R1 is selected from fluoro, bromo, iodo, hydroxy, trifluoromethoxy, C2.6-alkoxy,
c1-6-alkyl, amino, C2.6-alkylsulfanyl (-SH-C2.6-alkyl), C2-6-alkylsulfinyl (-SO-C2.6-alkyl), C2.6-
alkylsulfonyl (-S(=O)2-C2.6-alkyl), alkylamino, -alkylamino, cyano, nitro, aryl. heteroaryl
and C3.8-cycloalkyl;
and pharmaceutically acceptable salts, solvates and prodrugs thereof.
The invention also relates to the use of said compounds in therapy, and in particular to
pharmaceutical compositions comprising said compounds.
In another embodiment, the invention relates to methods of treatment, the method comprising
administering to a subject in need thereof an effective amount of one or more compounds
according to formula I.
In a still further embodiment, the invention relates to the use of compounds according to formula
I in the manufacture of medicaments.
DEFINITIONS
In the structural formulae given herein and throughout the present specification, the following
terms have the indicated meaning:
The term "alkyl" as used herein represents a saturated, branched or straight hydrocarbon
group having the indicated number of carbon atoms. Thus, the terms "d-3-alkyl", "d-e-alkyl"
and "C2-6-alkyl" as used herein represent saturated, branched or straight hydrocarbon groups
having from 1 to 3 carbon atoms, 1 to 8 carbon atoms and 2 to 6 carbon atoms, respectively.
Typical alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl,
isobutyl, sec-butyl, ferf-butyl, pentyl, hexyl and the like.
The term "C2.6-alkoxy" as used herein refers to the radical -O-C2-6-alkyl, wherein C2-6-alkyl is
as defined above. Representative examples are ethoxy, n-propoxy, isopropoxy, butoxy, secbutoxy,
terf-butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy and the like.
The term "d-e-alkylamino" as used herein refers to the radical -NH-d-e-alkyl, wherein d-ealkyl
is as defined above. Representative examples are methylamino, ethylamino, isopropylamino,
n-propylamino, butylamino, pentylamino, hexylamino and the like.
The term "di-d.6-alkylamino" as used herein refers to the radical -N(C1.6-alkyl)2, wherein d-ealkyl
is as defined above. It should be understood that the d-e-alkyl groups may be the same
or different. Representative examples are dimethylamino, methylethylamino, diethylamino,
diisopropylamino, di-n-propylamino, dibutylamino, dipentylamino, dihexylamino and the like.
The term "C3.8-cycloalkyl" as used herein represents a monocyclic, carbocyclic group having
from from 3 to 8 carbon atoms. Representative examples are cyclopropyl, cyclobutyl,
cyclopentyl and the like.
The term "C2-6-alkylsulfonyl" as used herein refers to the radical -S(=O)2-C2-alkyl, wherein
C2-6-alkyl is as defined above. Representative examples are ethylsulfonyl, isopropylsulfonyl,
n-propylsulfonyl, butylsulfonyl, pentylsulfonyl and the like.
The term "aryl" as used herein is intended to include carbocyclic aromatic ring systems such
as phenyl, biphenylyl, naphthyl, anthracenyl, phenanthrenyl, fluorenyl, indenyl, pentalenyl,
azulenyl and the like. Aryl is also intended to include the partially hydrogenated derivatives of
the carbocyclic systems enumerated above. Non-limiting examples of such partially hydrogenated
derivatives are 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl and the like.
The term "heteroaryl" as used herein is intended to include heterocyclic aromatic ring systems
containing one or more heteroatoms selected from nitrogen, oxygen and sulfur such as
furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, 1,2,3-triazolyl,
1,2,4-triazolyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl,
1,3,5-triazinyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,
1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, tetrazolyl,
thiadiazinyl, indolyl, isoindolyl, benzofuryl, benzothiehyl, indazolyl, benzimidazolyl, benzothiazolyl,
benzoisothiazolyl, benzoxazolyl, benzisoxazolyl, purinyl, quinazolinyl, quinolizinyl,
quinolinyl, isoquinolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, azepinyl, diazepinyl,
acridinyl and the like. Heteroaryl is also intended to include the partially hydrogenated
derivatives of the heterocyclic systems enumerated above. Non-limiting examples of
such partially hydrogenated derivatives are 2,3-dihydrobenzofuranyl, pyrrolinyl, pyrazolinyl,
indanyl, indolinyl, oxazolidinyl, oxazolinyl, oxazepinyl and the like.
The term "treatment" as used herein means the management and care of a patient for the
purpose of combating a disease, disorder or condition. The term is intended to include the
delaying of the progression of the disease, disorder or condition, the alleviation or relief of
symptoms and complications, and/or the cure or elimination of the disease, disorder or condition.
The patient to be treated is preferably a mammal, in particular a human being.
The term "effective amount" as used herein is intended to indicate an amount of a compound
(in casu a compound according to formula I) which when administered a patient gives rise to
a therapeutically relevant response. Said amount may vary depending on e.g. the sex, age,
in the weight and condition of the patient. It lies within the skills of an ordinary trained physician
to determine what an effective amount in any particular treatment is.
DETAILED DESCRIPTION OF THE INVENTION
In one embodiment, R1 represents bromo or cyano.
The compounds of the present invention interact with the histamine H3 receptor and are accordingly
believed to be particularly useful in the treatment of a variety of diseases or conditions
in which histamine H3 interactions are beneficial.
In one aspect, the invention provides the use of a compound according to formula (I) in a
pharmaceutical composition. The pharmaceutical composition may in another aspect of the
invention comprise, as an active ingredient, at least one compound according to formula (I)
together with one or more pharmaceutically acceptable carriers or excipients. In another aspect,
the invention provides such a pharmaceutical composition in unit dosage form, comprising
from about 0.05 mg to about 1000 mg, e.g. from about 0.1 mg to about 500 mg, such
as from about 0.5 mg to about 200 mg of the compound according to formula (I).
In another aspect, the invention provides the use of a compound of formula (I) as defined
above for the preparation of a pharmaceutical composition for the treatment of diseases and
disorders in which an inhibition of the H3 histamine receptor has a beneficial effect.
In another aspect, the invention provides the use of a compound of formula (I) for the preparation
of a pharmaceutical composition having histamine H3 antagonistic activity or histamine
H3 inverse agonistic activity.
In another aspect the invention provides the use of a compound of formula (I) for the preparation
of a pharmaceutical composition for the reduction of weight.
In another aspect, the invention provides the use of a compound of formula (I) for the preparation
of a pharmaceutical composition for the treatment of overweight or obesity.
In another aspect, the invention provides the use of a compound of formula (I) for the preparation
of a pharmaceutical composition for the suppression of appetite or for satiety induction.
In another aspect, the invention provides the use of a compound of formula (I) for the preparation
of a pharmaceutical composition for the prevention and/or treatment of disorders and
diseases related to overweight or obesity, such as dyslipidaemia, coronary heart disease,
gallbladder disease, osteoarthritis and various types of cancer such as endometrial, breast,
prostate and colon cancers.
In another aspect, the invention provides the use of a compound of formula (I) for the preparation
of a pharmaceutical composition for the prevention and/or treatment of eating disorders,
such as bulimia or binge eating.
In another aspect, the invention provides the use of a compound of formula (I) for the preparation
of a pharmaceutical composition for the treatment of IGT (Impaired glucose tolerance).
In another aspect, the invention provides the use of a compound of formula (I) for the preparation
of a pharmaceutical composition for the treatment of type 2 diabetes.
In another aspect, the invention provides the use of a compound of formula (I) for the preparation
of a pharmaceutical composition for the delaying or prevention of the progression from
IGT to type 2 diabetes.
In another aspect, the invention provides the use of a compound of formula (I) for the preparation
of a pharmaceutical composition for the delaying or prevention of the progression from
non-insulin requiring type 2 diabetes to insulin requiring type 2 diabetes.
In another aspect, the invention provides the use of a compound of formula (I) for the preparation
of a pharmaceutical composition for the treatment of diseases and disorders in which a
stimulation of the H3 histamine receptor has a beneficial effect.
In another aspect, the invention provides the use of a compound of formula (I) for the preparation
of a pharmaceutical composition having histamine H3 agonistic activity.
In another aspect, the invention provides the use of a compound of formula (I) for the preparation
of a pharmaceutical composition for the treatment of allergic rhinitis, ulcer or anorexia.
In another aspect, the invention provides the use of a compound of formula (I) for the preparation
of a pharmaceutical composition for the treatment of Alzheimer's disease, narcolepsy,
attention deficit disorders or reduced wakefulness, or for the regulation of sleep.
In another aspect, the invention relates to the use of a compound of formula (I) for the preparation
of a pharmaceutical preparation for the treatment of airway disorders, such as asthma,
for regulation of gastric acid secretion, or for treatment of diarrhoea.
n another aspect, the invention provides a method for the treatment of disorders or diseases
•elated to the H3 histamine receptor, the method comprising administering to a subject in
need thereof an effective amount of a compound of the genera! formula (I) as defined above,
or of a pharmaceutical composition comprising such a compound.
In another aspect, the invention provides a method as described above, wherein the effective
amount of the compound of the genera! formula (I) as defined above is in the range of from
about 0.05 mg to about 2000 mg, preferably from about 0.1 mg to about 1000 mg, and more
preferably from about 0.5 mg to about 500 mg per day.
In one aspect, the invention relates to compounds which exhibit histamine H3 receptor antagonistic
activity or inverse agonistic activity and which may accordingly be useful in the
treatment of a wide range of conditions and disorders in which histamine H3 receptor blockade
is beneficial.
In another aspect, the invention provides a method for reduction of weight, the method comprising
administering to a subject in need thereof an effective amount of a compound of formula
(I) as defined above.
In another aspect, the invention provides a method for treatment of overweight or obesity, the
method comprising administering to a subject in need thereof an effective amount of a compound
of formula (I).
In another aspect, the invention provides a method for suppression of appetite or for satiety
induction, the method comprising administering to a subject in need thereof an effective
amount of a compound of formula (I).
In another aspect, the invention provides a method for prevention and/or treatment of disorders
or diseases related to overweight or obesity, such as dyslipidaemia, coronary heart disease,
gallbladder disease, osteoarthritis and various types of cancer, e.g. endometrial,
breast, prostate or colon cancer, the method comprising administering to a subject in need
thereof an effective amount of a compound of formula (I).
In another aspect, the invention provides a method for prevention and/or treatment of eating
disorders, such as bulimia and binge eating, the method comprising administering to a subject
in need thereof an effective amount of a compound of formula (I).
In another aspect, the invention provides a method for the treatment of IGT (Impaired glucose
tolerance), the method comprising administering to a subject in need thereof an effective
amount of a compound of formula (I).
In another aspect, the invention provides a method for the treatment of type 2 diabetes, the
method comprising administering to a subject in need thereof an effective amount of a compound
of formula (I).
In another aspect, the invention provides a method for the delaying or prevention of the progression
from IGT to type 2 diabetes, the method comprising administering to a subject in
need thereof an effective amount of a compound of formula (I).
In another aspect, the invention provides a method for the delaying or prevention of the progression
from non-insulin requiring type 2 diabetes to insulin requiring type 2 diabetes, the
method comprising administering to a subject in need thereof an effective amount of a compound
of formula (I).
In another aspect, the invention relates to compounds which exhibit histamine H3 receptor
agonistic activity and which may accordingly be useful in the treatment of a wide range of
conditions and disorders in which histamine H3 receptor activation is beneficial.
Compounds of the present invention may also be used for the treatment of airway disorders
(such as asthma), as anti-diarrhoeals, and for the modulation of gastric acid secretion.
Furthermore, compounds of the present invention may be used for the treatment of diseases
associated with the regulation of sleep and wakefulness, and for the treatment of narcolepsy
and attention deficit disorders.
Moreover, compounds of the invention may be used as CMS stimulants or as sedatives.
The present compounds may also be used for the treatment of conditions associated with
epilepsy. Additionally, compounds of the invention may be used for the treatment of motion
sickness and vertigo. Furthermore, they may be useful as regulators of hypothalamohypophyseal
secretion, as antidepressants, as modulators of cerebral circulation, and in the
treatment of irritable bowel syndrome.
Further, compounds of the present invention may be used for the treatment of dementia and
Alzheimer's disease.
Compounds of the present invention may also be useful for the treatment of allergic rhinitis,
ulcer or anorexia.
Compounds of the present invention may furthermore be useful for the treatment of migraine
[see, e.g., McLeod et al., The Journal of Pharmacology and Experimental Therapeutics
(1998), 43-50] and for the treatment of myocardial infarction [see Mackins et al., Expert Opinion
on Investigational Drugs 9 (2000), 2537-2542].
In a further aspect of the invention, treatment of a patient with a compound of the present
invention is combined with diet and/or exercise.
In a further aspect of the invention, one of more compounds of the present invention is/are
administered in combination with one or more further active substances in any suitable ratio(
s). Such further active agents may, for example, be selected from antiobesity agents,
antidiabetics, antidyslipidemic agents, antihypertensive agents, agents for the treatment of
complications resulting from or associated with diabetes, and agents for the treatment of
complications arid disorders resulting from or associated with obesity.
Thus, in a further aspect of the invention one or more compounds of the present invention
may be administered in combination with one or more antiobesity agents or appetite regulating
agents. Such agents may, for example, be selected from the group consisting of CART
(cocaine amphetamine regulated transcript) agonists, NPY (neuropeptide Y) antagonists,
MC4 (melanocortin 4) agonists, MC3 (melanocortin 3) agonists, orexin antagonists, TNF
(tumor necrosis factor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP (corticotropin
releasing factor binding protein) antagonists, urocortin agonists, p3 adrenergic
agonists such as CL-316243, AJ-9677, GW-0604, LY362884, LY377267 or AZ-40140, MSH
(melanocyte-stimulating hormone) agonists, MCH (melanocyte-concentrating hormone) antagonists,
CCK (cholecystokinin) agonists, serotonin re-uptake inhibitors such as fluoxetine,
seroxat orcitalopram, serotonin and noradrenaline re-uptake inhibitors, mixed serotonin and
noradrenergic compounds, 5HT (serotonin) agonists, bombesin agonists, galanin antagonists,
growth hormone, growth factors such as prolactin or placental lactogen, growth hormone
releasing compounds, TRH (thyreotropin releasing hormone) agonists, UCP 2 or 3
(uncoupling protein 2 or 3) modulators, leptin agonists, DA agonists (bromocriptin, doprexin),
lipase/amylase inhibitors, PPAR (peroxisome proliferator-activated receptor) modulators,
RXR (retinoid X receptor) modulators, TR p agonists, AGRP (Agouti related protein) inhibi10
tors, opioid antagonists (such as naltrexone), exendin-4, GLP-1 and ciliary neurotrophic factor.
In one embodiment of the invention, an antiobesity agent administered in combination with
one or more compounds of the invention is leptin.
In another embodiment, such an antiobesity agent is dexamphetamine or amphetamine.
In another embodiment, such an antiobesity agent is fenfluramine or dexfenfluramine.
In still another embodiment, such an antiobesity agent is sibutramine.
In a further embodiment, such an antiobesity agent is orlistat.
In another embodiment, such an antiobesity agent is mazindol or phentermine.
In still another embodiment, such an antiobesity agent is phendimetrazine, diethylpropion,
fluoxetine, bupropion, topiramate or ecopipam.
In yet a further aspect of the invention, one or more compounds of the present invention may
be administered in combination with one or more antidiabetic agents. Relevant antidiabetic
agents include insulin, insulin analogues and derivatives such as those disclosed in EP 0 792
290 (Novo Nordisk A/S), e.g. NEB29-tetradecanoyl des (B30) human insulin, EP 0 214 826 and
EP 0 705 275 (Novo Nordisk A/S), e.g. AspB2B human insulin, US 5,504,188 (Eli Lilly), e.g.
Lys628 Pro629 human insulin, EP 0 368 187 (Aventis), e.g. Lantus®, all of which are incorporated
herein by reference, GLP-1 derivatives, such as those disclosed in WO 98/08871
(Novo Nordisk A/S), incorporated herein by reference, as well as orally active hypoglycaemic
agents.
The orally active hypoglycaemic agents preferably comprise imidazolines, sulfonylureas,
biguanides, meglitinides, oxadiazolidinediones, thiazolidinediones, insulin sensitizers. aglucosidase
inhibitors, agents acting on the ATP-dependent potassium channel of the pcells,
e.g. potassium channel openers such as those disclosed in WO 97/26265,
WO 99/03861 and WO 00/37474 (Novo Nordisk A/S) which are incorporated herein by reference,
or mitiglinide, or a potassium channel blocker, such as BTS-67582, nateglinide. glucagon
antagonists, such as one of those disclosed in WO 99/01423 and WO 00/39088 (Novo
Nordisk A/S and Agouron Pharmaceuticals, Inc.), both of which are incorporated herein by
reference, GLP-1 agonists, such as those disclosed in WO 00/42026 (Novo Nordisk A/S and
Agouron Pharmaceuticals, Inc.), incorporated herein by reference, DPP-IV (dipeptidyl peptidase-
IV) inhibitors, PTPase (protein tyrosine phosphatase) inhibitors, inhibitors of hepatic
enzymes involved in stimulation of gluconeogenesis and/or glycogenolysis, glucose uptake
modulators, GSK-3 (glycogen synthase kinase-3) inhibitors, compounds modifying the lipid
metabolism such as antilipidemic agents, compounds lowering food intake, PPAR (perox
isome proliferator-activated receptor) and RXR (retinoid X receptor) agonists, such as ALRT-
268, LG-1268orl_G-1069.
In one embodiment of the invention, one or more compounds of the present invention may
be administered in combination with insulin or an insulin analogue or derivative, such as
NcB29-tetradecanoyl des (B30) human insulin, Asp628 human insulin, Lys628 Pro629 human insulin,
Lantus®, or a mix-preparation comprising one or more of these.
In a further embodiment of the invention, one or compounds of the present invention may be
administered in combination with a sulfonylurea, e.g. tolbutamide, chlorpropamide, tolazamide,
glibenclamide, glipizide, glimepiride, glicazide or glyburide.
In another embodiment of the invention, one or more compounds of the present invention
may be administered in combination with a biguanide, e.g. metformin.
In yet another embodiment of the invention, one or more compounds of the present invention
may be administered in combination with a meglitinide, e.g. repaglinide or nateglinide.
In still another embodiment of the invention, one or more compounds of the present invention
may be administered in combination with a thiazolidinedione insulin sensitizer, e.g. troglitazone,
ciglitazone, pioglitazone, rosiglitazone, isaglitazone, darglitazone, englitazone, CS-
011/CI-1037 or T 174, or a compound disclosed in WO 97/41097, WO 97/41119, WO
97/41120, WO 00/41121 and WO 98/45292 (Dr. Reddy's Research Foundation), all of which
are incorporated herein by reference.
In still another embodiment of the invention, one or more compounds of the present invention
may be administered in combination with an insulin sensitizer, e.g. such as Gl 262570, YM-
440, MCC-555, JTT-501, AR-H039242, KRP-297, GW-409544, CRE-16336, AR-H049020,
LY510929, MBX-102, CLX-0940, GW-501516, or a compound disclosed in WO 99/19313,
WO 00/50414, WO 00/63191, WO 00/63192 or WO 00/63193 (Dr. Reddy's Research Foundation)
or in WO 00/23425, WO 00/23415, WO 00/23451, WO 00/23445, WO 00/23417,
WO 00/23416, WO 00/63153, WO 00/63196, WO 00/63209, WO 00/63190 or WO 00/63189
(Novo Nordisk A/S), all of which are incorporated herein by reference.
In a further embodiment of the invention, one or more compounds of the present invention
may be administered in combination with an a-glucosidase inhibitor, e.g. voglibose, emiglitate,
miglitol or acarbose.
In another embodiment of the invention, one or more compounds of the present invention
may be administered in combination with an agent acting on the ATP-dependent potassium
channel of the p-cells, e.g. tolbutamide, glibenclamide, glipizide, glicazide, BTS-67582 or repaglinide.
in yet another embodiment of the invention, one or more compounds of the present invention
may be administered in combination with nateglinide.
In still another embodiment, one or more compounds of the present invention may be administered
in combination with an antihyperlipidemic agent or antilipidemic agent, e.g. cholestyramine,
colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol or dextrothyroxine.
In still another embodiment of the invention, one or more compounds of the present invention
may be administered in combination with an antilipidemic agent, e.g. cholestyramine, colestipol,
clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol or dextrothyroxine.
In another aspect of the invention, one or more compounds of the present invention may be
administered in combination with more than one of the above-mentioned compounds, e.g. in
combination with metformin and a sulfonylurea such as glyburide; a sulfonylurea and acarbose;
nateglinide and metformin; acarbose and metformin; a sulfonylurea, metformin and
troglitazone; insulin and a sulfonylurea; insulin and metformin; insulin, metformin and a sulfonylurea;
insulin and troglitazone; insulin and lovastatin; etc.
Furthermore, one or more compounds of the present invention may be administered in combination
with one or more antihypertensive agents. Examples of antihypertensive agents are
p-blockers such as alprenolol, atenolol, timolol, pindolol, propranolol and metoprolol. ACE
(angiotensin converting enzyme) inhibitors such as benazepril, captopril, enalapril, fosinopril,
lisinopril, quinapril and ramiprii, calcium channel blockers such as nifedipine, felodipine,
nicardipine, isradipine, nimodipine, diltiazem and verapamil, and a-blockers such as doxazosin,
urapidil, prazosin and terazosin. Further reference can be made to Remington: The
Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co.. Easton,
PA, 1995.
It should be understood that any suitable combination of compounds according to the invention
with diet and/or exercise, one or more of the above-mentioned compounds and optionally
one or more other active substances are considered to be within the scope of the present
invention.
Furthermore, some compounds of the present invention may exist in different tautomeric
forms, and it is intended that any tautomeric forms which the compounds are able to form are
included within the scope of the present invention.
The present invention also encompasses pharmaceutically acceptable salts of the present
compounds. Such salts include pharmaceutically acceptable acid addition salts, pharmaceutically
acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition
salts include salts of inorganic acids as well as organic acids. Representative examples of
suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric,
nitric acids and the like. Representative examples of suitable organic acids include formic,
acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic,
lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic,
ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic,
gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic,
benzenesulfonic, p-toluenesulfonic acids and the like. Further examples of pharmaceutically
acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable
salts listed in J. Pharm. Sci. 1977, 66, 2, which is incorporated herein by reference. Examples
of metal salts include lithium, sodium, potassium, magnesium salts and the like. Examples
of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium,
trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium,
butylammonium, tetramethylammonium salts and the like.
Also intended as pharmaceutically acceptable acid addition salts are the hydrates which the
present compounds are able to form.
The acid addition salts may be obtained as the direct products of compound synthesis. Alternatively,
the free base may be dissolved in a suitable solvent containing the appropriate acid,
and the salt isolated by evaporating the solvent or otherwise separating the salt and solvent.
Compounds of the present invention may form solvates with standard low molecular weight
solvents using methods well known to the person skilled in the art. Such solvates are also to
be understood as being within the scope of the present invention.
The invention also encompasses prodrugs of the present compounds which following administration
undergo chemical conversion by metabolic processes before becoming active
pharmacological substances. In general, such prodrugs will be functional derivatives of the
present compounds which are readily convertible in vivo into the required compound of the
formula (I). Conventional procedures for the selection and preparation of suitable prodrug
derivatives are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier,
1985.
The invention also encompasses active metabolites of the present compounds.
PHARMACEUTICAL COMPOSITIONS
The compounds of the invention may be administered alone or in combination with pharmaceutically
acceptable carriers or excipients, in either single or multiple doses. The pharmaceutical
compositions according to the invention may be formulated with pharmaceutically
acceptable carriers or diluents as well as any other known adjuvants and excipients in accor14
dance with conventional techniques, such as those disclosed in Remington: The Science and
Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995.
The pharmaceutical compositions may be specifically formulated for administration by any
suitable route, such as the oral, rectal, nasal, pulmonary, topical (including buccal and sublingual),
transdermal, intracisternal, intraperitoneal, vaginal or parenteral (including subcutaneous,
intramuscular, intrathecal, intravenous and intradermal) route, the oral route being
preferred. It will be appreciated that the preferred route will depend on the general condition
and age of the subject to be treated, the nature of the condition to be treated and the active
ingredient chosen.
Pharmaceutical compositions for oral administration include solid dosage forms such as capsules,
tablets, dragees, pills, lozenges, powders and granules. Where appropriate, they can
be prepared with coatings, such as enteric coatings, or they can be formulated so as to provide
controlled release of the active ingredient, such as sustained or prolonged release according
to methods well known in the art.
Liquid dosage forms for oral administration include solutions, emulsions, suspensions, syrups
and elixirs.
Pharmaceutical compositions for parenteral administration include sterile aqueous and nonaqueous
injectable solutions, dispersions, suspensions or emulsions as well as sterile powders
to be reconstituted in sterile injectable solutions or dispersions prior to use. Depot injectable
formulations are also to be understood as being within the scope of the present invention.
Other suitable administration forms include suppositories, sprays, ointments, cremes, gels,
inhalants, dermal patches, implants etc.
A typical oral dosage is in the range of from about 0.001 to about 100 mg/kg body weight per
day, preferably from about 0.01 to about 50 mg/kg body weight per day, and more preferably
from about 0.05 to about 10 mg/kg body weight per day, administered in one or more doses,
such as from 1 to 3 doses. The exact dosage will depend upon the frequency and mode of
administration, the sex, age, weight and general condition of the subject treated, the nature
and severity of the condition treated and any concomitant diseases to be treated, and other
factors evident to those skilled in the art.
The formulations may conveniently be presented in unit dosage form by methods known to
those skilled in the art. A typical unit dosage form for oral administration one or more times
per day, such as from 1 to 3 times per day, may contain from 0.05 to about 1000 mg, preferably
from about 0.1 to about 500 mg, and more preferably from about 0.5 mg to about 200
mg of a compound (or a salt or other derivative thereof as set forth above), according to the
invention.
For parenteral routes, such as intravenous, intrathecal, intramuscular and similar administration,
typical doses are of the order of about half the dose employed for oral administration.
The compounds of this invention are generally utilized as the free substance or as a pharmaceutically
acceptable salt thereof. One example is an acid addition salt of a compound having
a free base functionality. When a compound of the formula (I) contains a free base functionality,
such salts are prepared in a conventional manner by treating a solution or suspension
of the free base form of the compound of formula (I) with a chemical equivalent (acid-base
equivalent) of a pharmaceutically acceptable acid. Representative examples of relevant inorganic
and organic acids.are mentioned above. Physiologically acceptable salts of a compound
of the invention having an hydroxy group include the anion of said compound in combination
with a suitable cation, such as sodium or ammonium ion.
For parenteral administration, solutions of the novel compounds of the formula (I) in sterile
aqueous solution, aqueous propylene glycol or sesame or peanut oil may be employed. Such
aqueous solutions should be suitably buffered if necessary, and the liquid diluent first rendered
isotonic with sufficient saline or glucose. The aqueous solutions are particularly suitable
for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The
sterile aqueous media employed are all readily available by standard techniques known to
those skilled in the art.
Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solution
and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose,
cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid or lower
alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids,
fatty acids, fatty acid amines, polyoxyethylenes or water. Similarly, the carrier or diluent
may include any sustained release material known in the art, such as glyceryl monostearate
or glyceryl distearate, alone or mixed with a wax. The pharmaceutical compositions formed
by combining the novel compounds of the formula (I) and the pharmaceutically acceptable
carriers are then readily administered in a variety of dosage forms suitable for the disclosed
routes of administration. The formulations may conveniently be presented in unit dosage
form by methods known in the art of pharmacy.
Formulations of the present invention suitable for oral administration may be presented as
discrete units such as capsules or tablets, each containing a predetermined amount of the
active ingredient, and which may include a suitable excipient. These formulations may be in
the form of powder or granules, as a solution or suspension in an aqueous or non-aqueous
liquid, or as an oil-in-water or water-in-oil liquid emulsion.
If a solid carrier is used for oral administration, the preparation may be tabletted, placed in a
hard gelatine capsule in powder or pellet form or it can be in the form of a troche or lozenge.
The amount of solid carrier may vary widely, but will usually be from about 25 mg to about 1
g. If a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft
gelatine capsule or sterile injectable liquid, such as an aqueous or non-aqueous liquid suspension
or solution.
A typical tablet, which may be prepared by conventional tabletting techniques, may contain:
Core:
Compound of the invention, e.g. compound of any of
Examples 1-8 (as free compound or salt thereof) 5.0 mg
Lactosum Ph. Eur. 67.8 mg
Cellulose, microcryst. (Avicel) 31.4 mg
Amberlite® IRP88* 1.0 mg
Magnesii stearas Ph. Eur. q.s.
Coating:
Hydroxypropyl methylcellulose approx. 9 mg
Mywacett 9-40 T approx. 0.9 mg
Polacrillin potassium NF, tablet disintegrant, Rohm and Haas.
Acylated monoglyceride used as plasticizer for film coating.
If desired, the pharmaceutical composition of the invention may comprise the compound of
the formula (I) in combination with one or more further pharmacologically active substances,
e.g. substances chosen among those described in the foregoing.
EXAMPLES
In the examples the following terms are intended to have the following, general meanings:
DIPEA: diisopropylethylamine
DMSO: dimethylsulphoxide
NMR spectra were recorded on Bruker 300 MHz and 400 MHz instruments.
HPLC-MS was performed on a Perkin Elmer instrument (AP1100). The column used was XTerra
C18, 5 ^m, 50 X 3 mm, and elution was done at 1.5 ml/min at room temperature with a
gradient of 5-90% acetonitrile in water with 0.01% trifluoroacetic acid over a period of 7.5
min.
The RP-analyses was performed using an Alliance Waters 2695 system fitted with a Waters
2487 dual-band detector. UV detections were collected using a Symmetry C18 , 3.5 urn, 3.0
mm x 100 mm column. The elution is done with a linear gradient consisting of 5-90% acetonitrile,
90-0% water and 5% of 1% aqueous trifluoroacetic acid over a period of 8 minutes at a
flow-rate of 1.0 min/min.
General procedure (A)
Compounds of the general formula (I) may be prepared by the general procedure (A):
R1 (I)
N—N
(Hal = halogen, notably Cl or Br)
A mixture of isopropylpiperazine (2.00 mmol), DMSO (1.0 ml), a suitable halopyridazine (2.00
mmol), and a base such as DIPEA(0.20 ml) is stirred for one hour at 100 °C and then for 18
hours at 120 °C. Water and potassium carbonate are added and the mixture is extracted with
a solvent such as ethyl acetate (3 x 20 ml). The combined extracts are washed with brine,
dried over magnesium sulphate, and concentrated under reduced pressure. The residue may
be converted into an appropriate salt, such as the hydrochloride salt, by co-evaporation with
an acid, such as 1 molar aqueous hydrochloric acid, ethanol and toluene, and the residue is
then purified by recrystallization
General procedure (B)
Compounds of the general formula (I) may be prepared by the general procedure (B):
A compound of formula I may be prepared from a suitable monosubstituted piperazine and a
suitable bromopyridazine in the presence of a suitable catalyst, such as, e. g.,
tris(dibenzylideneacetone)dipalladium, in a suitable solvent, such as toluene, at a suitable
temperature between 0°C and 150°C.
General procedure (C)
Chloropyridazines of the general formula (lla), (lib) or (lie) may be prepared by the general
procedure (C):
Example 1
4-[6-(4-lsopropylpiperazin-1-yl)-pyridazin-3-yl]benzonitrile
19
A suspension of 4-(6-ch!oro-pyridazin-3-yl)-benzonitrile (1 g, 4.64 mmol; prepared as described
in US patent No. 4,112,095), isopropylpiperazine (0.654 g, 5.1 mmol), DIPEA (1.199
g, 9.27 mmol) and 4-(dimethylamino)pyridine (0.057g, 0.464 mmol) in DMSO (4 ml) was
stirred and heated to 100 °C for 20h. After cooling to room temperature, the mixture was diluted
with dichloromethane (25 ml) and water (35 ml) and stirred for 5 min. The organic
phase was separated, washed with water (50 ml) and brine (50 ml), and acidified to pH 2 by
addition of 1N hydrochloric acid. The mixture was extracted with water (30 ml), and the aqueous
phase was washed with dichloromethane (10 ml) and concentrated in vacuo to give a
solid, which was collected and stripped with ethanol to afford the title compound as a crystalline
hydrochloride (1.33 g, 76%).
1H NMR (D2O) £1.30 (d, 6H), 3.26 (broad t, 2H), 3.45-3.68 (m, 5H), 4.52 (broad d, 2H), 7.75
(d, 1H), 7.77 (d, 2H), 7.87 (d, 2H), 8.12 (d, 1H);
HPLC-MS: m/z 308.2 (MH+); R 1.76 min.
Example 2
3-(4-Bromophenyl)-6-(4-isopropylpiperazin-1-yl)pyridazine
This compound was prepared according to General Procedure (A), starting from 1-
isopropylpiperazine and 3-chloro-6-(4-bromophenyl)pyridazine, prepared as described in US
patent No. 4,112,095. The compound was isolated as its hydrochloride salt.
1H NMR (D20): S 1.30 (d, 6H), 3.23 (broad t, 2H), 3.47 (broad t, 2H), 3.52-3.67 (m, 3H), 4.52
(broad d, 2H), 7.64 (d, 2H), 7.67 (d, 2H), 7.82 (d, 1H), 8.15 (d, 1H);
HPLC: R, = 3.49 min.
Example 3
3-(4-Ethanesulfonylphenyl)-6-(4-isopropylpiperazin-1-yl)pyridazine
This compound was prepared according to General Procedure (A), starting from 1-
isopropylpiperazine and 3-chloro-6-(4-ethanesulfonylphenyl)pyridazine, prepared according
to General Procedure (C). The compound was isolated as its hydrochloride salt.
nH NMR (de-DMSO): 8 1.15 (t, 3H), 1.33 (d, 6H), 3.17 (m, 2H), 3.36 (q, 2H), 3.45-3.60 (m,
3H), 3.67 (t, 2H), 4.66 (d, 2H), 7.71 (d, 1H), 8.00 (d, 2H), 8.29 (d. 1H), 8.33 (d, 2H);
HPLC: R, = 4.73min.
Example 4
3-(4-Ethanesulfinylphenyl)-6-(4-isopropylpiperazin-1-yl)pyridazine
This compound was prepared according to General Procedure (A), starting from 1-
isopropylpiperazine and 3-chloro-6-(4-ethanesulfinylphenyl)pyridazine, prepared according to
General Procedure (C). The compound was isolated as its hydrochloride salt.
1H NMR (de-DMSO): 5 1.06 (t, 3H), 1.32 (d, 6H), 2.83 (m, 1H), 3.05-3.24 (m, 3H), 3.46-3.59
(m, 3H), 3.68 (broad t. 2H), 4.64 (broad d, 2H), 7.75-7.83 (m, 3H), 8.26 (d, 2H), 8.32 (d, 1H),
11.47 (broads, 1H);
HPLC: R, = 4.10min.
Example 5
3-[4-(Butane-1-sulfonyl)phenyl]-6-(4-isopropylpiperazin-1-yl)pyridazine
ChL
This compound was prepared according to General Procedure (A), starting from 1-
isopropylpiperazine and 3-[4-(butane-1-sulfonyl)phenyl]-6-chloropyridazine, prepared according
to General Procedure (C). The compound was isolated as its hydrochloride salt.
1H NMR (de-DMSO): <> 0.84 (t, 3H), 1.33 (d,6H), 1.3-1.4 (m, 2H), 1.54 (m, 2H), 3.17 (m, 2H),
3.37 (m, 2H), 3.45-3.60 (m, 3H), 3.67 (broad t, 2H), 4.66 (broad d, 2H), 7.73 (d, 1H), 8.03 (d,
2H), 8.30 (d, 1H), 8.34 (d, 2H), 11.41 (broad s, 1H);
HPLC: R,=6.46min.
Example 6
3-[4-(Butane-1-sulfinyl)phenyl]-6-(4-isopropylpiperazin-1-yl)pyridazine
CH,
This compound was prepared according to General Procedure (A), starting from 1-
isopropylpiperazine and 3-[4-(butane-1-sulfinyl)phenyl]-6-chloropyridazine, prepared according
to General Procedure (C). The compound was isolated as its hydrochloride salt.
1H NMR (de-DMSO): S 0.87 (t, 3H), 1.26-1.50 (m, 9H), 1.65 (m, 1H), 2.83 (m, 1H), 3.02 (m,
1H), 3.18 (m, 2H), 3.45-3.60 (m, 3H), 3.68 (broad t, 2H), 4.64 (broad d, 2H), 7.74-7.86 (m,
3H), 8.26 (d, 2H), 8.32 (d. 1H), 11.41 (broad s, 1H);
HPLC: R, = 5.52min.
Example 7
3-(4-lsopropylpiperazin-1-yl)-6-[4-(propane-1-sulfonyl)phenyl]pyridazine
This compound was prepared according to General Procedure (A), starting from 1-
isopropylpiperazine and 3-chloro-6-[4-(propane-1-sulfonyl)phenyl]pyridazine, prepared according
to General Procedure (C). The compound was isolated as its hydrochloride salt.
1H NMR (de-DMSO): S 0.94 (t, 3H), 1.33 (d, 6H), 1.59 (m, 2H), 3.17 (m, 2H), 3.35 (m. 2H),
3.45-3.60 (m, 3H), 3.68 (broad t, 2H), 4.67 (broad d, 2H), 7.74 (d, 1H), 8.02 (d, 2H), 8.81 (d,
1H), 8.84 (d, 2H), 11.44 (broad s, 1H);
HPLC: R, = 5.60min.
Example 8
3-(4-lsopropylpiperazin-1-yl)-6-[4-(propane-1-sulfinyl)phenyl]pyridazine
This compound was prepared according to General Procedure (A), starting from 1-
isopropylpiperazine and 3-chloro-6-[4-(propane-1 -sulfinyl)phenyl]pyridazine, prepared according
to General Procedure (C). The compound was isolated as its hydrochloride salt.
1H NMR (de-DMSO): S 0.98 (t, 3H), 1.33 (d, 6H), 1.50 (m, 1H), 1.70 (m, 1H), 2.82 (m, 1H),
2.99 (m, 1H), 3.18 (m, 2H), 3.45-3.60 (m, 3H), 3.69 (broad t, 2H), 4.65 (broad d, 2H), 7.75-
7.85 (m, 3H), 8.26 (d, 2H), 8.32 (d, 1H), 11.47 (broad s, 1H);
HPLC: R, = 4.76min.
PHARMACOLOGICAL METHODS
The ability of the compounds to interact with the histamine H3 receptor can be determined by
the following in vitro binding assays.
Binding assay I
Rat cerebral cortex is homogenized in ice cold K-Hepes, 5 mM MgCI2 pH 7.1 buffer. After two
differential centrifugations the last pellet is resuspended in fresh Hepes buffer containing
mg/ml bacitracin. Ali,quots of the membrane suspension (400 ug/ml) are incubated for 60 min
at 25°C with 30 pM [125l]-iodoproxifan (a known histamine H3 receptor antagonist) and the
test compound at various concentrations. The incubation is stopped by dilution with ice-cold
medium, followed by rapid filtration through Whatman GF/B filters pretreated for 1 hour with
0.5% polyethyleneimine. The radioactivity retained on the filters is counted using a Cobra II
auto gamma counter. The radioactivity of the filters is indirectly proportional to the binding
affinity of the tested compound. The results are analyzed by non-linear regression analysis.
Binding assay II
The H3-receptor agonist ligand R-a-methyl[3H]histamine (RAMHA) is incubated with isolated
rat cortex cell-membranes at 25 °C for 1 hour, followed by a filtration of the incubate through
Whatman GF/B filters. Radioactivity retained on the filters is measured using a beta counter.
Male Wistar rats (150-200 g) are decapitated, and cerebral cortex is quickly dissected out
and frozen immediately on dry ice. Tissue is kept at -80 °C until membrane preparation. During
the membrane preparation the tissue is kept on ice all the time. Rat cerebral cortex is
homogenized in 10 volumes (w/w) ice-cold Hepes buffer (20 mM Hepes, 5 mM MgCI2 pH 7.1
(KOH) + 1 mg/ml bacitracin) using an Ultra-Turrax homogenizer for 30 seconds. The homogenate
is centrifuged at 140 g in 10 min. The supernatant is transferred to a new test tube
and centrifuged for 30 min at 23 000 g. Pellet is resuspended in 5-10 ml Hepes buffer, homogenized
and centrifuged for 10 min at 23 000 g. This short centrifugation step is repeated
twice. After the last centrifugation the pellet is resuspended in 2-4 ml Hepes buffer and the
protein concentration is determined. The membranes are diluted to a protein concentration of
5 mg/ml using Hepes buffer, aliquoted and stored at -80 °C until use.
50 1 test-compound, 100 l membrane (200 ng/ml), 300 nl Hepes buffer and 50 ^l R-amethyl[
3H]histamine (1 nM) are mixed in a test tube. The compounds to be tested are dissolved
in DMSO and further diluted in HaO to the desired concentrations. Radioligand and
membranes are diluted in Hepes buffer + 1 mg/ml bacitracin. The mixture is incubated for 60
min at 25 "C. Incubation is terminated by addition of 5 ml ice-cold 0.9% NaCI, followed by
rapid filtration through Whatman GF/B filters pre-treated for 1 hour with 0.5% polyethyleneimine.
The filters are washed with 2 x 5 ml ice-cold NaCI. To each filter is added a 3 ml
scintillation cocktail, and the retained radioactivity is measured with a Packard Tri-Carb beta
counter.
IC50 values are calculated by non-linear regression analysis of binding curves (6 points minimum)
using the windows program GraphPad Prism, GraphPad software, USA.
Binding assay III
The human H3 receptor is cloned by PCR and subcloned into the pcDNAS expression vector.
Cells stably expressing the H3 receptor are generated by transfecting the H3-expression
vectors into HEK 293 cells and using G418 to select for H3 clones. The human H3-HEK 293
clones are cultured in DMEM (GIBCO-BRL) with glutamax, 10% foetal calf serum, 1%
peniciliin/streptavidin and 1 mg/ml G 418 at 37 °C and 5% C02. Before harvesting, the
confluent cells are rinsed with PBS and incubated with Versene (proteinase, GIBCO-BRL) for
approximately 5 min. The cells are flushed with PBS and DMEM and the cell suspension
collected in a tube and centrifuged for 5-10 min at 1500 rpm in a Heraeus Sepatech
Megafuge 1.0. The pellet is resuspended in 10-20 vol. Hepes buffer [20 mM Hepes, 5 mM
MgCI2, pH 7.1 (KOH)] and homogenized for 10-20 seconds using an Ultra-Turrax
homogenizer. The homogenate is centrifuged for 30 min at 23 000 g. The pellet is
resuspended in 5-10 ml Hepes buffer, homogenized 5-10 seconds with the Ultra-Turrax and
centrifuged for 10 min at 23 000 g. Following this centrifugation step, the membrane pellet is
resuspended in 2-4 ml Hepes buffer, homogenized with a syringe or Teflon homogenizer,
and the protein concentration determined. The membranes are diluted to a protein
concentration of 1-5 mg/ml in Hepes buffer, aliquoted and kept at -80 °C until use.
Aliquots of the membrane suspension are incubated for 60 min at 25 °C with 30 pM [125I]-
iodoproxifan (a known compound with high affinity for the H3 receptor) and the test compound
at various concentrations. The incubation is stopped by dilution with ice-cold medium,
followed by rapid filtration through Whatman GF/B filters pretreated for 1 hour with 0.5%
polyethyleneimine. The radioactivity retained on the filters is counted using a Cobra II auto
gamma counter. The radioactivity of the filters is indirectly proportional to the binding affinity
of the tested compound. The results are analysed by non-linear regression analysis.
When tested, the present compounds of the formula (I) generally show a high binding affinity
to the histamine H3 receptor.
Preferably, the compounds according to the invention have an !C5o value as determined by
one or more of the assays of less than 10 uM, more preferably less than 1 nM, and still more
preferably less than 500 nM, such as less than 100 nM.
Functional assay I
The ability of the compounds to interact with the histamine H3 receptor as agonists, inverse
agonists and/or antagonists, is determined by an in vitro functional assay utilizing membranes
from HEK 293 cell expressing the human H3 receptors.
The H3 receptor is cloned by PCR and subcloned into the pcDNAS expression vector. Cells
stably expressing the H3 receptor are generated by transfecting the HS-expression vectors
into HEK 293 cells and using G418 to select for H3 clones. The human H3-HEK 293 clones
are cultured in DMEM with glutamax, 10% foetal calf serum, 1% penicillin/streptavidin and 1
mg/ml G 418 at 37°C and 5% C02.
The H3 receptor expressing cells are washed once with phosphate buffered saline (PBS)
and harvested using versene (GIBCO-BRL). PBS is added and the cells are centrifuged for 5
min at 188 g. The cell pellet is resuspended in stimulation buffer to a concentration of 1 x 106
cells/ml. cAMP accumulation is measured using the Flash Plate® cAMP assay (NEN™ Life
Science Products). The assay is generally performed as described by the manufacturer.
Briefly, 50 |il cell suspension is added to each well of the Flashplate which also contained 25
ul 40 |^M isoprenaline, to stimulate cAMP generation, and 25 |J of test compound (either
agonists or inverse agonists alone, or agonist and antagonist in combination). The assay can
be run in "agonist-mode" in which the test compound is added, in increasing concentration,
on its own, to the cells, and cAMP is measured. If cAMP goes up, the compound in question
is an inverse agonist; if cAMP does not change, it is a neutral antagonist, and if camp goes
down, it is an agonist. The assay can also be run in the "antagonist-mode" in which a test
compound is added, in increasing concentrations, together with increasing concentrations of
a known H3 agonist (e.g. RAMHA). If the test compound is an antagonist, increasing concentrations
of it cause a right-ward shift in the H3-agonist's dose-response curves. The final volume
in each well is 100 nl. Test compounds are dissolved in DMSO and diluted in H2O. The
fixture is shaken for 5 min, and allowed to stand for 25 min at room temperature. The reaction
is stopped with 100 (J "Detection Mix" per well. The plates are then sealed with plastic,
shaken for 30 min, allowed to stand overnight, and finally the radioactivity is counted in the
Cobra II auto gamma topcounter. EC50 values are calculated by non-linear regression analysis
of dose response curves (6 points minimum) using Graph Pad Prism. Kb values are calculated
by Schild plot analysis.
Functional assay II
The ability of the compounds to bind and interact with the human H3 receptor as agonists,
inverse agonists and/or antagonists, is determined by a functional assay, named [35S]
GTPyS assay. The assay measures the activation of G proteins by catalyzing the exchange
of guanosine 5'-diphosphate (GDP) by guanosine 5'-triphosphate (GTP) at the a-subunit.
The GTP-bounded G proteins dissociate into two subunits, GO.GTP and Gpy, which in turn
regulate intracellular enzymes and ion channels. GTP is rapidly hydrolysed by the Ga-subunit
(GTPases) and the G protein is deactivated and ready for a new GTP exchange cycle.
To study the function of ligand-induced G protein coupled receptor (GPCR) activation by an
increase in guanine nucleotide exchange at the G proteins, the binding of [35S]-guanosine-5'-
O-(S-thio) triphosphate [S5S] GTPyS, a non-hydrolysed analogue of GTP, is determined. This
process can be monitored in vitro by incubating cell membranes containing the G protein
coupled receptor H3 with GDP and [35S] GTPyS. Cell membranes are obtained from CHO
cells stably expressing the human H3 receptor. The cells are washed twice in PBS, harvested
with PBS+1 mM EDTA, pH 7.4 and centrifuged at 1000 rpm for 5 min. The cell pellet
is homogenized in 10 ml ice-cold Hepes buffer (20 mM Hepes, 10 mM EDTA pH 7.4 (NaOH))
using an Ultra-Turrax homogenizer for 30 seconds and centrifuged for 15 min at 20.000 rpm.
Following this centrifugation step, the membrane pellet is resuspended in 10 ml ice-cold
Hepes buffer (20 mM Hepes, 0.1 mM EDTA pH 7.4 (NaOH)) and homogenized as described
above. This procedure is repeated twice except for the last homogenization step, the protein
concentration is determined, and membranes are diluted to a protein concentration of 2
mg/ml, aliquoted and kept at -80 °C until use.
In order to study the presence and the potency of an inverse agonist/antagonist, the H3-
receptor agonist ligand R-a-methyl histamine (RAMHA) is added. The ability of the test compound
to counteract the effect of RAMHA is measured. When studying the effect of an agonist,
RAMHA is not added to the assay medium. The test compound is diluted in the assay
buffer (20 mM HEPES, 120 mM NaCI, 10 mM MgCI2 pH 7.4 (NaOH)) at various concentrations
followed by addition of 10"8 nM RAMHA (only in the case where an inverse ago26
ftst/antagonist is examined), 3 μM GDP, 2.5μg membranes, 0.5 mg SPA beads and 0.1 nM
[35S] GTPyS, and incubation for 2 hours with gentle shaking at room temperature. The plates
are centrifuged at 1500 rpm for 10 min and the radioactivity is measured using a Topcounter.
The results are analyzed by non-linear regression and the IC5o value is determined.
RAMHA and other H3 agonists stimulate the binding of [35S] GTPyS to membranes expressing
the H3 receptor. In the antagonist/inverse agonist test, the ability of increasing amounts
of test compound to inhibit the increased [35S] GTPyS binding by 10"8 M RAMHA is measured
as a decrease in radioactivity signal. The IC50 value determined for an antagonist is the ability
of this compound to inhibit the effect of lO-8M RAMHA by 50%. In the agonist test, the ability
of increasing amounts of test compound is measured as an increase in radioactivity signal.
The EC50 value determined for an agonist is the ability of this compound to increase the signal
by 50% of the maximal signal that is obtained by 10"5 M RAMHA.
Preferably, the antagonists and agonists according to the invention have an IC5o/EC5o value
(as determined by one or more of the assays described above) of less than 10 μM, more
preferably less than 1 µM, and still more preferably less than 500 µM, such as less than 100
The open cage Schedule-fed rat model
The ability of the present compounds to reduce weight is determined using the in vivo open
cage Schedule-fed rat model.
Sprague-Dawley (SD) male rats of an age of about 1/4 to 2 months and a weight of about
200-250 g are purchased from Mollegard Breeding and Research Centre A/S (Denmark). On
arrival they are allowed some days of acclimatisation before being placed in individual open
plastic cages. They are habituated to the presence of food (Altromin pelleted rat chow) in
their home cage for only 7 hours each day (from 07.30 to14.30, seven days a week). Water
is present ad libitum. Once the consumption of food has stabilised after 7 to 9 days, the animals
are ready for use.
Each animal is used only once to avoid carry-over effects between treatments. During the
test sessions, the test compound is administered intraperitoneally or orally 30 min before the
start of the sessions. One group of animals is administered the test compound at different
doses, and a control group of animals receives vehicle. Food and water intake are monitored
at 1, 2 and 3 hours post administration.
Any side effects (manifested as barrel-rolling, bushy fur etc.) may rapidly be detected, since
the animals are kept in transparent plastic cages to enable continuous monitoring.

CLAIMS
1. A compound according to formula I
(Figure Removed)
wherein R1 is independently selected from fluoro, bromo, iodo, hydroxy, trifluoromethoxy, C2-6-alkoxy, C1-6-alkyl, amino, C2-6-alkylsulfanyl,C2-6-alkylsulfinyl, C2-6-alkylsulfonyl, C1-6-alkylamino, di-C1-6-alkylamino, cyano, nitro, aryl, heteroaryl and C3-6 -cycloalkyl; and pharmaceutically acceptable salts, solvates and prodrugs thereof.
2. A compound according to claim 1, wherein R1 represents bromo or cyano.
3. A compound according to claim 1 or 2 for use in therapy.
4. A pharmaceutical composition comprising, as an active ingredient, at least one compound
according to claim 1 or 2 together with one or more pharmaceutically acceptable carriers or
excipients.
5. A pharmaceutical composition according to claim 4 in unit dosage form, comprising from
about 0.05 mg to about 1000 mg, preferably from about 0.1 mg to about 500 mg, more pref
erably from about 0.5 mg to about 200 mg of a compound according to claim 1 or 2.
6. Use of a compound according to claim 1 or 2 for the preparation of a pharmaceutical com
position for the treatment of disorders and diseases related to the histamine H3 receptor.
7. The use according to claim 6 for the preparation of a pharmaceutical composition for the
reduction of weight.
8. The use according to claim 6 for the preparation of a pharmaceutical composition for the
treatment of overweight or obesity.
9. The use according to claim 6 for the preparation of a pharmaceutical composition for the
suppression of appetite or for satiety induction.
10. The use according to claim 6 for the preparation of a pharmaceutical composition for the
prevention and/or treatment of disorders and diseases related to overweight or obesity.
11. The use according to claim 6 for the preparation of a pharmaceutical composition for the
prevention and/or treatment of eating disorders, such as bulimia or binge eating.
12. The use according to claim 6 for the preparation of a pharmaceutical composition for the
treatment of IGT.
13. The use according to claim 6 for the preparation of a pharmaceutical composition for the
treatment of type 2 diabetes.
14. The use according to claim 6 for the preparation of a pharmaceutical composition for the
delaying or prevention of the progression from IGT to type 2 diabetes.
15. The use according to claim 6 for the preparation of a pharmaceutical composition for the
delaying or prevention of the progression from non-insulin requiring type 2 diabetes to insulin
requiring type 2 diabetes.
16. The use according to claim 6 for the preparation of a pharmaceutical composition for the
treatment of allergic rhinitis, ulcer or anorexia.
17. The use according to claim 6 for the preparation of a pharmaceutical composition for the
treatment of Alzheimer's disease, narcolepsy or attention deficit disorders.
18. A method for the treatment of disorders or diseases related to the H3 histamine receptor,
the method comprising administering to a subject in need thereof an effective amount of a
compound according to claim 1 or 2.
19. A method according to claim 18, wherein the effective amount of the compound is in the
range of from about 0.05 mg to about 2000 mg, preferably from about 0.1 mg to about 1000
mg, more preferably from about 0.5 mg to about 500 mg per day.
20. A method according to claim 18 or 19 for the reduction of weight.
21. A method according to claim 18 or 19 for the treatment of overweight or obesity.
22. A method according to claim 18 or 19 for the suppression of appetite or for satiety induc
tion.
23. A method according to claim 18 or 19 for the prevention and/or treatment of disorders
and diseases related to overweight or obesity.
24. A method according to claim 18 or 19 for the prevention and/or treatment of eating disor
ders, such as bulimia or binge eating.
25. A method according to claim 18 or 19 for the treatment of IGT.
26. A method according to claim 18 or 19 for the treatment of type 2 diabetes.
27. A method according to claim 18 or 19 for the delaying or prevention of the progression
from IGT to type 2 diabetes.
28. A method according to claim 18 or 19 for the delaying or prevention of the progression
from non-insulin requiring type 2 diabetes to insulin requiring type 2 diabetes.
29. A method according to claim 18 or 19 for the treatment of allergic rhinitis, ulcer or ano
rexia.
30. A method according to claim 18 or 19 for the treatment of Alzheimer's disease, narco
lepsy or attention deficit disorders.