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  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems
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  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
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  • A61P35/00—Antineoplastic agents
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid

Definitions

• the present invention relates to substituted imidazopyrimidine derivatives, substituted imidazopyrazine derivatives and substituted imidazopyridazine derivatives as melanocortin-4 receptor modulators.
• melanocortin-4 receptor modulators are either agonists or antagonists.
• the compounds of the invention are selective antagonists of the human melanocortin-4 receptor (MC-4R).
• the antagonists are useful for the treatment of disorders and diseases such as cachexia induced by e.g. cancer, chronic kidney disease (CKD) or chronic heart failure (CHF), muscle wasting, anorexia induced by e.g. chemotherapy or radiotherapy, anorexia nervosa, amyotrophic lateral sclerosis (ALS), pain, neuropathic pain, anxiety and depression, nausea and emesis.
• MCs Melanocortins stem from pro-opiomelanocortin (POMC) via proteolytic cleavage. These peptides, adrenocorticotropic hormone (ACTH), ⁇ -melanocyte-stimulating hormone ( ⁇ -MSH), ⁇ -MSH and ⁇ -MSH, range in size from 12 to 39 amino acids. The most important endogenous agonist for central MC-4R activation appears to be the tridecapeptide ⁇ - MSH. Among MCs, it was reported that ⁇ -MSH acts as a neurotransmitter or neuromodulator in the brain.
• MC peptides particularly ⁇ -MSH
• ⁇ -MSH have a wide range of effects on biological functions including feeding behavior, pigmentation and exocrine function.
• the biological effects of ⁇ -MSH are mediated by a sub-family of 7- transmembrane G-protein-coupled receptors, termed melanocortin receptors (MC-Rs). Activation of any of these MC-Rs results in stimulation of cAMP formation.
• MC-Rs melanocortin receptors
• MC-1 R was first found in melanocytes.
• Naturally occurring inactive variants of MC-1 R in animals were shown to lead to alterations in pigmentation and a subsequent lighter coat color by controlling the conversion of phaeomelanin to eumelanin through the control of tyrosinase. From these and other studies, it is evident that MC-1R is an important regulator of melanin production and coat color in animals and skin color in humans.
• the MC-2R is expressed in the adrenal gland representing the ACTH receptor.
• the MC- 2R is not a receptor for ⁇ -MSH but is the receptor for the adrenocorticotropic hormone I (ACTH I).
• the MC-3R is expressed in the brain (predominately located in the hypothalamus) and peripheral tissues like gut and placenta, and knock-out studies have revealed that the MC-3R may be responsible for alterations in feeding behavior, body weight and thermogenesis.
• the MC-4R is primarily expressed in the brain. Overwhelming data support the role of MC-4R in energy homeostasis. Genetic knock-outs and pharmacologic manipulation of MC-4R in animals have shown that agonizing the MC-4R causes weight loss and antagonizing the MC-4R produces weight gain (A. Kask, et al., "Selective antagonist for the melanocortin-4 receptor (HS014) increases food intake in free-feeding rats," Biochem. Biophys. Res. Commun., 245: 90-93 (1998)).
• MC-5R is ubiquitously expressed in many peripheral tissues including white fat, placenta and a low level of expression is also observed in the brain. However its expression is greatest in exocrine glands. Genetic knock-out of this receptor in mice results in altered regulation of exocrine gland function, leading to changes in water repulsion and thermoregulation. MC-5R knockout mice also reveal reduced sebaceous gland lipid production (Chen et al., Cell, 91 : 789-798 (1997)).
• MC-3R and MC-4R modulators have potent physiological effects besides their role in regulating pigmentation, feeding behavior and exocrine function.
• ⁇ -MSH recently has been shown to induce a potent anti-inflammatory effect in both acute and chronic models of inflammation including inflammatory bowel-disease, renal ischemia/reperfusion injury and endotoxin-induced hepatitis.
• Administration of ⁇ -MSH in these models results in substantial reduction of inflammation-mediated tissue damage, a significant decrease in leukocyte infiltration and a dramatic reduction in elevated levels of cytokines and other mediators to near baseline levels.
• ⁇ -MSH anti-inflammatory actions of ⁇ -MSH are mediated by MC-1 R.
• the mechanism by which agonism of MC-1 R results in an anti-inflammatory response is likely through inhibition of the pro-inflammatory transcription activator, NF- ⁇ B.
• NF- ⁇ B is a pivotal component of the pro-inflammatory cascade, and its activation is a central event in initiating many inflammatory diseases.
• anti-inflammatory actions of ⁇ -MSH may be, in part, mediated by agonism of MC-3R and/or MC-5R.
• MC-4R signaling is important in mediating feeding behavior (S. Q. Giraudo et al., "Feeding effects of hypothalamic injection of melanocortin-4 receptor ligands," Brain Research, 80: 302-306 (1998)).
• Further evidence for the involvement of MC-Rs in obesity includes: 1) the agouti (A ⁇ ) mouse which ectopically expresses an antagonist of the MC-1 R, MC-3R and MC-4R is obese, indicating that blocking the action of these three MC-R's can lead to hyperphagia and metabolic disorders; 2) MC-4R knockout mice (D.
• MC-4R appears to play a role in other physiological functions as well, namely controlling grooming behavior, erection and blood pressure.
• Erectile dysfunction denotes the medical condition of inability to achieve penile erection sufficient for successful intercourse.
• the term "impotence" is often employed to describe this prevalent condition.
• Synthetic melanocortin receptor agonists have been found to initiate erections in men with psychogenic erectile dysfunction (H. Wessells et al., "Synthetic Melanotropic Peptide Initiates Erections in Men With Psychogenic Erectile Dysfunction: Double-Blind, Placebo Controlled Crossover Study," J. Urol., 160: 389-393, 1998).
• Activation of melanocortin receptors of the brain appears to cause normal stimulation of sexual arousal.
• Evidence for the involvement of MC-R in male and/or female sexual dysfunction is detailed in WO 00/74679.
• Diabetes is a disease in which a mammal's ability to regulate glucose levels in the blood is impaired because the mammal has a reduced ability to convert glucose to glycogen for storage in muscle and liver cells. In Type I diabetes, this reduced ability to store glucose is caused by reduced insulin production.
• Type Il diabetes or “Non-Insulin Dependent Diabetes Mellitus” (NIDDM) is the form of diabetes which is due to a profound resistance to insulin stimulating or regulatory effect on glucose and lipid metabolism in the main insulin-sensitive tissues, muscle, liver and adipose tissue. This resistance to insulin responsiveness results in insufficient insulin activation of glucose uptake, oxidation and storage in muscle, and inadequate insulin repression of lipolysis in adipose tissue and of glucose production and secretion in liver.
• NIDDM Non-Insulin Dependent Diabetes Mellitus
• Hyperinsulemia is associated with hypertension and elevated body weight. Since insulin is involved in promoting the cellular uptake of glucose, amino acids and triglycerides from the blood by insulin sensitive cells, insulin insensitivity can result in elevated levels of triglycerides and LDL which are risk factors in cardiovascular diseases.
• MC-4R agonists might be useful in the treatment of NIDDM and Syndrome X.
• the MC4 receptor is also of interest in terms of the relationship to stress and the regulation of emotional behavior, as based on the following findings. Stress initiates a complex cascade of responses that include endocrine, biochemical and behavioral events. Many of these responses are initiated by release of corticotropin-releasing factor (CRF) (Owen MJ and Nemeroff CB (1991) Physiology and pharmacology of corticotrophin releasing factor. Pharmacol Rev 43: 425-473).
• CCF corticotropin-releasing factor
• MCL0129 (1-[(S)-2-(4-Fluorophenyl)-2-(4-isopropylpiperadin-1-yl)ethyl]-4- [4-(2-methoxynaphthalen-1-yl)butyl]piperazine), a Novel and Potent Nonpeptide Antagonist of the Melanocortin-4 Receptor; Shigeyuki Chaki et al, J. Pharm. Exp. Ther. (2003) 304(2), 818-26).
• cytokines e.g. TNF- ⁇
• MC4 receptors in the hypothalamus by ⁇ -MSH reduces appetite and increases energy expenditure.
• cytokines e.g. leptin
• CKD chronic kidney disease
• AgRP agouti-related peptide
• melanocortin-4 receptor inverse agonist an endogenous melanocortin-4 receptor inverse agonist
• Rats with chronic heart failure show an impaired ability to accumulate and maintain fat mass and lean body mass.
• Treatment of aortic banding induced CHF in rats with AgRP resulted in significantly increases in weight gain, lean body mass, fat accumulation, kidney weights and liver weights.
• Batra A.K. et a/. Central melanocortin blockage attenuates cardiac cachexia in a rat model of chronic heart failure. American Federation for Medical Research, 2008 Western Regional Meeting, abstract 379).
• MC-4R inhibitors could be used to treat emesis, especially in patients undergoing chemotherapy.
• ALS Amytrophic Lateral Sclerosis
• body weight e.g. Ludolph AC, Neuromuscul. Disord. (2006) 16 (8):530-8.
• MC-4R inhibitors could be used to treat ALS patients.
• CKD chronic heart failure
• CHF chronic heart failure
• muscle wasting anorexia induced by e.g. chemotherapy or radiotherapy, anorexia nervosa, amyotrophic lateral sclerosis (ALS), pain, neuropathic pain, anxiety and depression, nausea and emesis and other diseases with MC-4R involvement.
• ALS amyotrophic lateral sclerosis
• the present invention relates to substituted imidazopyrimidine derivatives, substituted imidazopyrazine derivatives and substituted imidazopyridazine derivatives of structural
• the imidazopyrimidine, imidazopyrazine and imidazopyridazine derivatives of structural formula (I) are effective as melanocortin receptor modulators and are particularly effective as selective melanocortin-4 receptor (MC-4R) antagonists. They are therefore useful for the treatment of disorders where the inactivation of the MC-4R is involved.
• the antagonists are useful for the treatment of disorders and diseases such as cachexia induced by e.g. cancer, chronic kidney disease (CKD) or chronic heart failure (CHF), muscle wasting, anorexia induced by e.g. chemotherapy or radiotherapy, anorexia nervosa, amyotrophic lateral sclerosis (ALS), pain, neuropathic pain, anxiety and depression, nausea and emesis.
• the present invention also relates to pharmaceutical compositions comprising the compounds of the present invention and a pharmaceutically acceptable carrier.
• the present invention relates to substituted imidazopyrimidine derivatives, substituted imidazopyrazine derivatives and substituted imidazopyridazine derivatives useful as melanocortin receptor modulators, in particular, as selective MC-4R antagonists.
• WO2008/027812 discloses imidazopyridine and imidazopyrimidine derivatives that act as cannabinoid receptor ligands, e.g., CB2 ligands.
• the compounds are claimed to be useful for treating patients with conditions like an immune disorder, pain, an inflammatory disorder, rheumatoid arthritis, multiple sclerosis, osteoperosis or osteoarthrithis.
• the present invention relates to novel imidazopyrimidines, imidazopyrazines and imidazopyridazines which are used as antagonists of MC-4R.
• the compounds of the present invention are represented by structural formula (I)
• R 1 and R 2 are independently from each other selected from
• Ci -6 alkylene-C 3 - 7 cycloalkyl or
• R 1 and R 2 together with the nitrogen atom to which they are attached to, form a 5 to 6- membered ring which may additionally contain one oxygen atom in the ring and which is unsubstituted or substituted by one or more substituents selected from OH, Ci- 6 alkyl, O- C r6 alkyl, Co-salkylene-Ca-scycloalkyl, Ci-ealkylene-O-d-ealkyl and (CH 2 )o- 3 -phenyl;
• B, D and E are independently from each other selected form CH and N with the proviso that one of B, D and E represents N;
• A is -NH-,
• R 7 is independently selected from
• NR 15a R 15b halogen, phenyl and heteroaryl, wherein phenyl and heteroaryl are unsubstituted or substituted by 1 to 3 R 4 * 1 ;
• X is H 1 CN,
• C 3 - 8 cycloalkyl unsubstituted or substituted with one or more halogen atoms, phenyl, phenyl which is fused with a saturated heterocyclic 6-membered ring, wherein the heterocyclic ring contains 1 or 2 heteroatoms independently selected from O and N,
• NR 15a R 15b wherein each phenyl, heterocyclyl and heteroaryl is unsubstituted or substituted by 1 to 3 R 43 and/or 1 R 4b and/or 1 R 5 ;
• R 48 ishalogen
• Ci- 6 alkyl unsubstituted or substituted with one or more substituents independently selected from halogen, OH and O-Ci. 6 alkyl, O-Ci -6 alkyl, wherein alkyl is unsubstituted or substituted with one or more halogen atoms, or
• R 4b is C(O)NH 2 ,
• R 5 is 5 to 6-membered saturated or unsaturated heterocyclyl containing 1 to 3 heteroatoms independently selected from N, O and S or
• heteroaryl containing 1 to 3 heteroatoms independently selected from N, O and S wherein each heterocyclyl and heteroaryl is unsubstituted or substituted by 1 or 2
• R 6 is H
• R 8 and R 9 are independently from each other selected from H,
• R 10 is H, NH 2 ,
• alkyl is unsubstituted or substituted by 1 to 3 halogens Ci -6 alkyl, halogen,
• R 12 and R 13 are independently from each other selected from H,
• Ci-ealkylene-O-Ci-ealkyl wherein alkyl, alkylene and cycloalkyl are unsubstituted or substituted by 1 to 3 R 48 ;
• R 14 isH
• R 15a and R 15b are independently from each other selected from
• Ci. 6 alkyl unsubstituted or substituted with one or more substituents selected from halogen, OH, O(C 1-6 alkyl), NH 2 , NH(Ci -6 alkyl) and N(C 1-6 alkyl) 2 , phenyl and heteroaryl, wherein phenyl and heteroaryl are unsubstituted or substituted by 1 to 3 R 48 , and
• R 16 , R 16a and R 16b are independently from each other selected from
• Ci -6 alkyl unsubstituted or substituted with one or more substituents selected from halogen, OH, OCd-ealkyl), NH 2 , NH(C 1-6 alkyl) and N(Ci -6 alkyl) 2 ,
• the variant A represents -NH- or a bond. More preferably, A represents a bond.
• the variant A represents -Ci -6 alkylene, -C 2 6 alkenylene or -C 2-6 alkinylene wherein alkylene, alkenylene and alkinylene are unsubstituted or substituted with one ore more R 7 such as 1 , 2 or 3 R 7 .
• R 7 such as 1 , 2 or 3 R 7 .
• A represents C 1-3 alkylene, such as methylene, ethylene, propylene or isopropylene, C 2 . 3 alkenylene, such as ethenylene or prop-1-enylene, or C 2-3 alkinylene, such as ethinylene or prop-2-inylene. It is further preferred that alkylene, alkenylene and alkinylene are unsubstituted or substituted by 1 R 7 . More preferably, alkylene, alkenylene and alkinylene are unsubstituted.
• R 7 is as defined above.
• R 7 represents d-ealkyl, OR 14 , NR 15a R 15b or halogen, wherein R 14 , R 15a and R 15b are defined as above. More preferably, R 7 represents C 1-6 alkyl, OH, NH 2 or fluorine.
• R 1 and R 2 independently from each other represent C 3 - 6 alkyl or that R 1 and R 2 , together with the nitrogen atom to which they are attached to, form a 5 to 6-membered ring which may additionally contain one oxygen atom in the ring and which is unsubstituted or substituted by one or more substituents, preferably 1 , 2 or 3 substituents, independently selected from OH, Ci -6 alkyl, Co-salkylene-Cs-scycloalkyl, O-Ci -6 alkyl, Ci -6 alkylene-O-Ci -6 alkyl and (CH 2 ) 0 - 3 -phenyl.
• R 1 and R 2 independently from each other represent C 3-6 alkyl.
• R 3 is -(CR 8 R 9 ) n -rCHR 9 -T.
• B represents a nitrogen atom whereas D and E mean a CH group or, alternatively, D represents a nitrogen atom whereas B and E mean a CH group.
• the variant T is NR 12 R 13 .
• the variants R 12 and R 13 are preferably independently from each other selected from H, d ⁇ alkyl and (CH 2 ) 0-2 -C 3-6 cycloalkyl, wherein alkyl and cycloalkyl are unsubstituted or substituted by 1 to 3 R 48 such as 1 , 2 or 3 substituents R 48 and wherein R 48 is defined as above
• variable bond in variant T indicated by the dash crossed with a wiggly line shows a bond to a carbon atom or to a nitrogen atom of the respective heterocycle.
• variable bond in variant T indicates a bond to a nitrogen atom.
• the variant T is selected from
• variant Y is CH 2 or NR 11 wherein R 11 is defined as above.
• R 11 is hydrogen.
• R 10 is selected from H, NH 2 , C 1-6 alkyl, NH(C 1-6 alkyl) and N(C 1-6 alkyl) 2 . More preferably, R 10 is H, NH 2 or d ⁇ alkyl.
• said variant preferably represents a 4 to 8-membered saturated or unsaturated heterocyclyl containing 1 , 2, 3 or 4 heteroatoms independently selected from N, O and S, or a 5- to 6-membered heteroaryl containing 1 , 2, 3 or 4 heteroatoms independently selected from N, O and S, wherein each heterocyclyl or heteroaryl is unsubstituted or substituted by 1 to 3 R 4a such as 1 , 2 or 3 R 4a and/or 1 R 4b and/or 1 R 5 wherein R 48 , R 4b and R 5 are defined as above. More preferably, X represents a 4 to 8-membered saturated or unsaturated heterocyclyl containing 1 , 2, 3 or 4 heteroatoms independently selected from N, O and S, or a 5- to 6-membered heteroaryl containing 1 , 2, 3 or 4 heteroatoms independently selected from N, O and S, wherein each heterocyclyl or heteroaryl is unsubstituted or substituted by 1 to 3 R 4a such
• each heterocyclyl or heteroaryl is unsubsubstituted or substituted by 1 to 3 R 48 such as 1 , 2 or 3 R 43 and/or 1 R 4b and/or 1 R 5 wherein R 43 , R 4b and R 5 are defined as above.
• R 48 is selected from Ci -6 alkyl, unsubstituted or substituted with one or more substituents selected from halogen, OH and O-Ci- 6 alkyl.
• the variant X represents phenyl which is unsubstituted or substituted by 1 to 3 R 43 such as 1 , 2 or 3 R 43 and/or 1 R 4b and/or 1 R 5 wherein R 43 , R 4b and R 5 are defined as above.
• the variant X represents -C(O)-R 6 , OR 14 , or NR 15a R 15b wherein R 6 , R 14 , R 15a and R 15b are defined as above. More preferably, X is -C(O)-R 6 or NR 153 R 15 ", most preferably X is -C(O)-R 6 .
• R 6 is preferably O-Ci -6 alkyl, wherein alkyl is unsubstituted or substituted with one or more R 16 .
• the variant R 6 is NR 16a R 16b , more preferably NH-Ci -6 alkyl.
• the index n represents an integer from 1 to 6, such as the integers 1 , 2, 3, 4, 5 or 6.
• n represents the integers 1 , 2, 3, or 4.
• the index q represents the integers 1 or 2. Preferably, q is 1.
• Alkyl is a straight chain or branched alkyl having 1 , 2, 3, 4, 5 or 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, or hexyl.
• Alkenyl is a straight chain or branched alkyl having 1 , 2, 3, 4, 5, or 6 carbon atoms and one to three double bonds, preferably one or two double bonds, most preferably one double bound.
• Preferred examples of a C 2-6 alkenyl group are ethenyl, prop-1-enyl, prop-2- enyl, isoprop-1-enyl, n-but-1-enyl, n-but-2-enyl, n-but-3-enyl, isobut-1-enyl, isobut-2-enyl, n-pent-1-enyl, n-pent-2-enyl, n-pent-3-enyl, n-pent-4-enyl, n-pent-1 ,3-enyl, isopent-1-enyl, isopent-2-enyl, neopent-1-enyl, n-hex-1-enyl, n-hex-2-en
• Alkinyl is a straight chain or branched alkyl having 1 , 2, 3, 4, 5, or 6 carbon atoms and one to three triple bonds, preferably one or two triple bonds, most preferably one triple bond.
• Preferred examples of a C 2 - 6 alkinyl group are ethinyl, prop-1-inyl, prop-2-inyl, n-but-1-inyl, n-but-2-inyl, n-but-3-inyl, n-pent-1 -inyl, n-pent-2-inyl, n-pent-3-inyl, n-pent-4-inyl, n-pent- 1 ,3-inyl, isopent-1-inyl, neopent-1-inyl, n-hex-1 -inyl, n-hex-2-inyl, n-hex-3-inyl, n-hex-4-inyl, n-hex-5-
• Cycloalkyl is an alkyl ring having preferably 3, 4, 5, 6, 7 or 8 carbon atoms at the most, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl, more preferably 3, 4, 5 or 6 carbon atoms.
• Heteroaryl is an aromatic moiety having 1 , 2, 3, 4 or 5 carbon atoms and at least one heteroatom independently selected from O, N and/or S.
• Heteroaryl is preferably selected from thienyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, isothiazolyl, isoxazyl, furanyl, and indazolyl, more preferably from thienyl, furanyl, imidazolyl, pyridyl, and pyrimidinyl.
• Heterocyclyl is a saturated or unsaturated ring containing at least one heteroatom independently selected from O, N and/or S and 1 , 2, 3, 4, 5, 6 or 7 carbon atoms.
• heterocyclyl is a 4, 5, 6, 7 or 8-membered ring and is preferably selected from tetrahydrofuranyl, azetidinyl, pyrrolidinyl, piperidinyl, pyranyl, morpholinyl, thiomorpholinyl, more preferably from piperidinyl and pyrrolidinyl.
• Halogen is a halogen atom selected from F, Cl, Br and I, preferably from F, Cl and Br.
• the compounds of structural formula (I) are effective as melanocortin receptor modulators and are particularly effective as selective modulators of MC-4R. They are useful for the treatment and/or prevention of disorders responsive to the inactivation of MC-4R, such as cachexia induced by e.g. cancer, chronic kidney disease (CKD) or chronic heart failure
• CKD chronic kidney disease
• CHF amyotrophic lateral sclerosis
• Compounds of structural formula (I) contain one or more asymmetric centers and can occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers.
• the present invention is meant to comprehend all such isomeric forms of the compounds of structural formula (I).
• Compounds of structural formula (I) may be separated into their individual diastereoisomers by, for example, fractional crystallization from a suitable solvent, for example methanol or ethyl acetate or a mixture thereof, or via chiral chromatography using an optically active stationary phase.
• Absolute stereochemistry may be determined by X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing an asymmetric center of known absolute configuration.
• any stereoisomer of a compound of the general formula (I) may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known absolute configuration.
• salts refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids.
• Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc and the like. Particularly preferred are the ammonium, calcium, lithium, magnesium, potassium and sodium salts.
• Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylamino- ethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
• basic ion exchange resins such as arg
• salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids.
• acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, formic, furnaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, malonic, mucic, nitric, parnoic, pantothenic, phosphoric, propionic, succinic, sulfuric, tartaric, p-toluenesulfonic, trifluoroacetic acid and the like.
• Particularly preferred are citric, fumaric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric and tartaric acids.
• Compounds of formula (I) are melanocortin receptor antagonists and as such are useful in the treatment, control or prevention of diseases, disorders or conditions responsive to the inactivation of one or more of the melanocortin receptors including, but not limited to, MC- 1 R, MC-2R, MC-3R, MC-4R or MC-5R.
• diseases, disorders or conditions include, but are not limited to, cachexia induced by e.g. cancer, chronic kidney disease (CKD) or chronic heart failure (CHF), muscle wasting, anorexia induced by e.g. chemotherapy or radiotherapy, anorexia nervosa, amyotrophic lateral sclerosis (ALS), pain, neuropathic pain, anxiety and depression, nausea and emesis.
• the compounds of formula (I) can be further used in the treatment, control or prevention of hypertension, hyperlipidemia, osteoarthritis, cancer, gall bladder disease, sleep apnea, compulsion, neuroses, insomnia/sleep disorder, substance abuse, pain, fever, inflammation, immune-modulation, rheumatoid arthritis, skin tanning, acne and other skin disorders, neuroprotective and cognitive and memory enhancement including the treatment of Alzheimer's disease.
• the compound of formula (I) can be used as a melanocortin-4 receptor antagonist.
• the compound of formula (I) can be used for the prophylaxis or treatment of disorders, diseases or conditions responsive to the inactivation of the melanocortin-4 receptor in a mammal.
• the compound of formula (I) can be used for the prophylaxis or treatment of a disorder, disease or condition selected from cachexia, muscle wasting, anorexia, anxiety, depression, amyotrophic lateral sclerosis (ALS), pain, nausea and emesis.
• a disorder, disease or condition selected from cachexia, muscle wasting, anorexia, anxiety, depression, amyotrophic lateral sclerosis (ALS), pain, nausea and emesis.
• the compound of formula (I) can be used for the prophylaxis or treatment of cachexia selected from cancer cachexia, cachexia induced by chronic kidney disease (CKD) or cachexia induced by chronic heart failure (CHF).
• cachexia selected from cancer cachexia, cachexia induced by chronic kidney disease (CKD) or cachexia induced by chronic heart failure (CHF).
• the compound of formula (I) can be used for the prophylaxis or treatment of anorexia selected from anorexia nervosa or anorexia induced by radiotherapy or chemotherapy.
• the compound of formula (I) can be used for the prophylaxis or treatment of pain, in particular neuropathic pain.
• the compound of formula (I) in another embodiment in connection with any of the above or below embodiments can be used for the prophylaxis or treatment of cancer cachexia.
• the compound of formula (I) can be used for the prophylaxis or treatment of muscle wasting.
• the compound of formula (I) in another embodiment in connection with any of the above or below embodiments can be used for the prophylaxis or treatment of anorexia.
• the compound of formula (I) can be used for the prophylaxis or treatment of anxiety and/or depression. In another embodiment in connection with any of the above or below embodiments the compound of formula (I) can be used for the prophylaxis or treatment of emesis.
• the compound of formula (I) can be used for the prophylaxis or treatment of amytrophic lateral sclerosis (ALS).
• ALS amytrophic lateral sclerosis
• Any suitable route of administration may be employed for providing a mammal, especially a human with an effective dosage of a compound of the present invention.
• oral, rectal, topical, parenteral, ocular, pulmonary, nasal and the like may be employed.
• Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols and the like.
• compounds of formula (I) are administered orally or topically.
• the effective dosage of active ingredient employed may vary depending on the particular compound employed, the mode of administration, the condition being treated and the severity of the condition being treated. Such dosage may be ascertained readily by a person skilled in the art.
• CKD chronic kidney disease
• CHF chronic heart failure
• ALS amyotrophic lateral sclerosis
• ALS amyotrophic lateral sclerosis
• nausea and emesis generally satisfactory results are obtained when the compounds of the present invention are administered at a daily dosage of from about 0.001 milligram to about 100 milligrams per kilogram of body weight, preferably given in a single dose or in divided doses two to six times a day, or in sustained release form.
• the total daily dose will generally be from about 0.07 milligrams to about 3500 milligrams. This dosage regimen may be adjusted to provide the optimal therapeutic response.
• the compounds of formula (I) are preferably formulated into a dosage form prior to administration. Accordingly the present invention also includes a pharmaceutical composition comprising a compound of formula (I) and a suitable pharmaceutical carrier.
• the active ingredient (a compound of formula (I)) is usually mixed with a carrier, or diluted by a carrier, or enclosed within a carrier, which may be in the form of a capsule, sachet, paper or other container.
• a carrier which may be in the form of a capsule, sachet, paper or other container.
• the carrier serves as a diluent, it may be a solid, semisolid or liquid material which acts as a vehicle, excipient or medium for the active ingredient.
• compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosol (as a solid or in a liquid medium), soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.
• Suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, methyl cellulose, methyl and propylhydroxybenzoates, talc, magnesium stearate and mineral oil.
• the formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.
• the compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient.
• the compounds of formula (I) when existing as a diastereomeric mixture may be separated into diastereomeric pairs of enantiomers by fractional crystallization from a suitable solvent such as methanol, ethyl acetate or a mixture thereof.
• the pair of enantiomers thus obtained may be separated into individual stereoisomers by conventional means by using an optically active acid as a resolving agent.
• any enantiomer of a compound of the formula (I) may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known configuration.
• the compounds of formula (I) of the present invention can be prepared according to the procedures of the following Schemes and Examples, using appropriate materials and are further exemplified by the following specific examples. Moreover, by utilizing the procedures described herein, in conjunction with ordinary skills in the art, additional compounds of the present invention claimed herein can be readily prepared. The compounds illustrated in the examples are not, however, to be construed as forming the only genus that is considered as the invention. The Examples further illustrate details for the preparation of the compounds of the present invention. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds. The instant compounds are generally isolated in the form of their pharmaceutically acceptable salts, such as those described previously.
• the free amine bases corresponding to the isolated salts can be generated by neutralization with a suitable base, such as aqueous sodium hydrogencarbonate, sodium carbonate, sodium hydroxide and potassium hydroxide, and extraction of the liberated amine free base into an organic solvent followed by evaporation.
• a suitable base such as aqueous sodium hydrogencarbonate, sodium carbonate, sodium hydroxide and potassium hydroxide
• the amine free base isolated in this manner can be further converted into another pharmaceutically acceptable salt by dissolution in an organic solvent followed by addition of the appropriate acid and subsequent evaporation, precipitation or crystallization. All temperatures are degrees Celsius.
• optionally substituted amine and 2-amino-pyhmidine-5- carboxylic acid or 5-amino-pyrazine-2-carboxylic acid are reacted in an amide coupling reaction in the presence of a coupling reagent such as EDC in an organic solvent such as DMF or DCM at a suitable temperature.
• a coupling reagent such as EDC in an organic solvent such as DMF or DCM at a suitable temperature.
• the resulting amide can then be reacted with a sulfonylchloride in a solvent such as pyridine or any other appropriate solvent and an organic base such as triethylamine to yield the corresponding sulfonylamino-amides.
• 6-oxo-1. ⁇ -dihydro-pyridazine-S-carboxylic acid can be reacted with a reagent such as oxalyl chloride in a suitable solvent like 1,2-dichloroethane in the presence of DMF to form the corresponding acid chloride which can subsequently be converted to the corresponding amide using an optionally substituted amine in the presence of a base such as DIEA in a suitable solvent such like DCM.
• a base such as DIEA
• suitable solvent such like DCM
• Optionally substituted 6-chloro-pyridazine-3-carboxylic acid amide can be converted to the corresponding sulfonylamino-amide using p-toluenesulfonamide in the presence of a base such as cesium carbonate in a suitable solvent such as DMF under microwave irradiation.
• optionally substituted ⁇ -alkoxycarbonyl- ⁇ -bromoketones can be obtained from the corresponding ketone by reacting it for example with copper(ll) bromide in a solvent such as mixture of ethyl acetate and chloroform at an appropriate temperature for a given time.
• the resulting ⁇ -bromoketones can then be reacted with sulfonylamino-amides in a solvent such as MeCN in the presence of an appropriate base, for example DIEA, to yield the N-alkylated sulfonylamino-amides.
• the resulting acid can be activated with a reagent such as isobutyl chloroformate in the presence of a suitable base such as N-methylmorpholine in an appropriate solvent such as THF and subsequently be reduced to the corresponding alcohol with a reducing agent such as sodium borohydride in an appropriate solvent such as a mixture of THF and water.
• a suitable base such as N-methylmorpholine
• a reducing agent such as sodium borohydride in an appropriate solvent such as a mixture of THF and water.
• the alcohol function can be converted to a leaving group with a reagent such as mesyl chloride or tosyl chloride in an appropriate solvent such as mixture of DCM and THF in the presence of a suitable base like TEA.
• Product of this reaction can be treated with an amine T-H in an appropriate solvent like MeCN to yield the target molecule.
• methyl esters of optionally substituted imidazo[1 ,2- a]pyrimidines, -pyrazines and imidazo[1 ,2-b]pyridazines can be reduced to the corresponding alcohol with a reagent such as sodium borohydride in an appropriate solvent like methanol.
• a reagent such as sodium borohydride in an appropriate solvent like methanol.
• the alcohol can be further reacted to the target molecules as depicted in Reaction Scheme 4.
• N-protected ⁇ -amino- ⁇ - bromoketones can be reacted with sulfonylamino-amides in a solvent such as MeCN in the presence of an appropriate base, for example DIEA, to yield the N-alkylated sulfonylamino-amides.
• a solvent such as MeCN
• an appropriate base for example DIEA
• These intermediates can then be further cyclised to the corresponding imidazo[1,2-a]pyrimidines, -pyrazines and imidazo[1,2-b]pyridazines by treating them with TFAA in a suitable solvent such as DCM or 1 ,2-dichloroethane at an appropriate temperature for a given time.
• the side chain amine function can be deprotected using a reagent such as TMSI in a suitable solvent such as MeCN in the case of a Z-protecting group.
• Phthalimids can be cleaved with hydrazine hydrate in an appropriate solvent like ethyl acetate.
• Optionally substituted imidazo[1 ,2-a]pyrimidines, -pyrazines and imidazo[1,2-b]pyridazines bearing the primary amino group in the side chain can be directly tested in the biologcial assay or are subjected to further derivatization.
• reaction with 1 ,5-dibromopentane in an appropriate solvent like 1 ,2-dichloroethane in the presence of a suitable base such as DIEA leads to the corresponding piperidine derivatives.
• optionally substituted ⁇ -chloro- ⁇ -bromoketones can be obtained from the corresponding ketone by reacting it for example with copper (II) bromide in a solvent such as mixture of ethyl acetate and chloroform at an appropriate temperature for a given time.
• the resulting cc-bromoketones can then be reacted with sulfonylamino- amides in a solvent such as MeCN in the presence of an appropriate base, for example DIEA 1 to yield the N-alkylated sulfonylamino-amides.
• optionally substituted ⁇ -chloro- ⁇ -bromoketones can also be reacted with a sulfonylamino-ester in a solvent such as MeCN in the presence of an appropriate base, for example DIEA, to yield the N-alkylated sulfonylamino-esters.
• a sulfonylamino-ester in a solvent such as MeCN in the presence of an appropriate base, for example DIEA
• These intermediates can then be further cyclised to the corresponding imidazo[1 ,2-a]pyrimidines, -pyrazines and imidazo[1 ,2-b]pyridazines by treating them with TFAA in a suitable solvent such as DCM or 1 ,2-dichloroethane at an appropriate temperature for a given time.
• the capping group T can be inserted by reacting the chloroalkyl substituted imidazo[1 ,2- a]pyrimidines, -pyrazines and imidazo[1 ,2-b]pyridazines with a capping group T-H in an appropriate solvent such as MeCN.
• a suitable base such as DIEA is used in addition to liberate the free amine T-H.
• Ester function of optionally substituted imidazo[1 ,2-a]pyrimidines, -pyrazines and imidazo[1 ,2- b]pyridazines can be hydrolyzed under basic conditions using a reagent like lithium hydroxide monohydrate in a suitable solvent such as a mixture of water, THF and MeOH.
• the product of the saponification can be isolated as lithium salt or as the corresponding acid.
• the ester function can also be cleaved under acidic conditions for example using a reagent such as aqueous hydrochloric acid.
• the product of the ester cleavage can be introduced into the next step as acid or lithium salt.
• Amide formation can be achieved using standard peptide coupling procedures.
• the acid can be coupled with an amine HNR 1 R 2 in the presence of EDC/HOBt, a base such as diisopropylethylamine and a solvent such as dichloromethane.
• a suitable solvent such as DCM, DMF, THF or a mixture of the above solvents, can be used for the coupling procedure.
• a suitable base includes triethylamine (TEA), diisopropylethylamine (DIEA), N-methylmorpholine (NMM), collidine or 2,6-lutidine.
• TSA triethylamine
• DIEA diisopropylethylamine
• NMM N-methylmorpholine
• collidine or 2,6-lutidine.
• a base may not be needed when EDC/HOBt is used.
• Optionally substituted imidazo[1 ,2-a]pyrimidines, -pyrazines and imidazo[1 ,2-b]pyridazines bearing a chloropyridine or bromopyridine as residue -A-X can be converted to the corresponding pyridones using a reagent such as aqueous hydrochloric acid at a suitable temperature as shown in Reaction Scheme 9. At the same time the ester function is also hydrolyzed.
• the acids can be coupled with amines HNR 1 R 2 as described above.
• optionally substituted aminopyrimidine- and aminopyrazine-amides which can be obtained as shown in Reaction Scheme 1 , can be converted to imidazo[1 ,2-a]pyrimidine and -pyrazine 6-carboxylic acid amides by reaction with ⁇ -bromoketones in a solvent like MeCN.
• This reaction can be carried out either in a flask in refluxing solvent or any other appropriate temperature or in a microwave reaction system.
• the reaction products can be purified by standard procedures or may precipitate directly from the solution upon cooling and may thus be used in subsequent reactions without further purification.
• products from Reaction Scheme 10 optionally substituted imidazo[1 ,2-a]pyrimidine and -pyrazine 6-carboxylic acid amides may be used in a Mannich-reaction to give 3-aminomethyl-imidazo[1 ,2-a] pyrimidine and -pyrazine 6- carboxylic acid amides by reacting the imidazo[1 ,2-a]pyrimidine and -pyrazine 6-carboxylic acid amides with an appropriate amine and aqueous formaldehyde solution in a solvent such as acetic acid.
• Diamines containing one nitrogen-protecting group can be further deprotected by treating the compound with an acid such as for example HCI in dioxane or TFA in DCM. Such compounds can then be purified by standard purification procedures such as flash chromatography or preparative HPLC.
• Reaction Scheme 12 :
• optionally substituted imidazo[1 ,2-a]pyrimidine and -pyrazine 6-carboxylic acid amides can be reacted in a Michael addition reaction with ⁇ , ⁇ - unsaturated aldehydes in a solvent such as a mixture of acetic acid and acetic anhydride at elevated temperature.
• the reaction may also be carried out in a microwave reactor.
• the product of this reaction can be treated with a base such as sodium bicarbonate in a suitable solvent like a mixture of water and methanol to yield the corresponding aldehydes which can be subjected to a reductive amination with an amine T-H in the presence of a reducing agent such as sodium triacetoxyborohydride in an appropriate solvent like DCE.
• a base such as sodium bicarbonate
• a suitable solvent like a mixture of water and methanol
• a reducing agent such as sodium triacetoxyborohydride in an appropriate solvent like DCE.
• Michael addition of optionally substituted imidazo[1,2- a]pyrimidine and -pyrazine 6-carboxylic acid amides can also be performed with ⁇ , ⁇ - unsaturated ketones using the reaction conditions described in Reaction Scheme 12.
• the product of the Michael addition reaction can be directly subjected to the reductive amination reaction.
• Rink amide resin Novagel, 0.62 mmol/g, 1.45 g was treated with DCM for 15 min.
• the reaction mixture was shaken until testing with bromophenol blue indicated a full conversion.
• the resin was filtered and washed with DCM (3 x).
• the product was cleaved from the resin by treatment with TFA/DCM 1 :1.
• the resin was filtered off and washed three times with DCM. The filtrates were combined and the solvent was distilled off.
• Example 2 A mixture of Example 2 (71 mg, free base), 1 ,5-dibromopentane (59 ⁇ l) and DIEA (75 ⁇ l) in 1 ,2-dichloroethane (1 ml) was stirred at 90 0 C overnight. The reaction mixture was absorbed on silica gel and the product was purified by column chromatography. The pure product was dissolved in 4 N HCI in dioxane (2 ml) and lyophilized to yield a yellow solid.
• intermediate 4a 25 mg
• the reaction mixture was heated to reflux for 3 h.
• the reaction mixture was allowed to cool to room temperature and the solvent was removed under reduced pressure.
• the crude product was purified by flash chromatography to yield a white solid.
• example 8 (40 mg) in tert-butanol (3 ml) was added potassium hydroxide (25 mg) in form of a powder and the resulting mixture was heated to 70 0 C for 4 h. The mixture was partitioned between ethyl acetate and brine. The aqueous layer was extracted twice with brine.
• ⁇ -Chloro-pyrazine ⁇ -carboxylic acid methyl ester (0.86 g) was dissolved in DMF (15 ml) and p-toluensulfonamide (0.94 g) and cesium carbonate (2.44 g) were added. The solution was reacted in the microwave reactor at 160 0 C for 30 min. The reaction mixture was poured in water and the aqueous phase was extracted three times with DCM. The combined organic layer was washed with brine, dried over sodium sulfate and the solvent was removed under reduced pressure. The aqueous layer was saturated with sodium chloride and extracted three times with DCM. The combined organic layer was washed with brine, dried over sodium sulfate and the solvent was removed under reduced pressure to yield a second batch of crude material. The crude product was purified by column chromatography.
• a membrane binding assay is used to identify competitive inhibitors of fluorescence labeled NDP-alpha-MSH binding to HEK293 cell membrane preparations expressing human melanocortin receptors.
• test compound or unlabeled NDP-alpha-MSH is dispensed at varying concentrations to a 384 well microtiter plate. Fluorescence labeled NDP-alpha-MSH is dispensed at a single concentration, followed by addition of membrane preparations. The plate is incubated for 5 h at room temperature.
• the degree of fluorescence polarization is determined with a fluorescence polarization microplate reader.
• Agonistic activity of human melanocortin receptors is determined in a homogeneous membrane based assay. Competition between unlabeled cAMP and a fixed quantity of fluorescence labeled cAMP for a limited number of binding sites on a cAMP specific antibody is revealed by fluorescence polarization.
• test compound or unlabeled NDP-alpha-MSH is dispensed at varying concentrations to a 384 well microtiter plate.
• Membrane preparations from HEK293 cells expressing the human melanocortin receptors are added.
• an appropriate amount of ATP, GTP and the cAMP antibody is added and the plate is further incubated before the fluorescence labeled cAMP conjugate is dispensed.
• the plate is incubated for 2 h at 4°C before it is read on a fluorescence polarization microplate reader.
• the amount of cAMP produced as a response to a test compound is compared to the production of cAMP resulting from stimulation with NDP-alpha-MSH.
• Representative compounds of the present invention were tested and found to bind to the melanocortin-4 receptor. These compounds were generally found to have IC 5O values less than 2 ⁇ M.
• IC 50 values of the hMC-4R binding assay were listed in the table: a ⁇ 0.1 ⁇ M; b > 0.1 ⁇ M and ⁇ 1.0 ⁇ M; c > 1.0 ⁇ M
• Food intake in rats is measured after s.c, i.p. or p.o. administration of the test compound (see e.g. Chen, A.S. et al. Transgenic Res 2000 Apr;9(2): 145-54).
• LPS lipopolysaccharide
• A monoamino-oxidase-A
• NA noradrenaline
• MAO-B inhibitors such as selegiline and tranylcypromine
• SSRI serotonin uptake inhibitors
• Benzodiazepines and other types of psychoactive compounds have been found to be inactive in this test (see e.g. Porsolt R.D., Bertin A., Jaffre M. Behavioral despair in rats and mice: strain differences and the effect of imipramine. Eur. J. Pharmacol. 1978, 51 : 291-294, Borsini F. and MeIi A. Is the forced swimming test a suitable model for revealing antidepressant activity (Psychopharmacol. 1988, 94: 147- 160).
• mice Male Swiss mice (4-5 weeks old). Animals are randomly assigned to different groups (10 mice per group).
• the mouse is considered to be immobile when it passively floats on the water making only small movements to keep its head above the surface.
• the water is replaced with clean water after 3 animals tested.
• Treatment is administered before the test as vehicle or test compound at different doses.
• Compounds are usually administered by p.o. i.p. or s.c. routes.
• the final incubation volume is 25 ⁇ l.
• a control incubation is included for each compound tested where 0.1 M phosphate buffer pH7.4 is added instead of NADPH (minus NADPH). Two control compounds are included with each species. All incubations are performed singularly for each test compound. Each compound is incubated for 0, 5, 15, 30 and 45 min. The control (minus NADPH) is incubated for 45 min only.
• the reactions are stopped by the addition of 50 ⁇ l methanol containing internal standard at the appropriate time points.
• the incubation plates are centrifuged at 2,500 rpm for 20 min at 4°C to precipitate the protein.
• sample supernatants are combined in cassettes of up to 4 compounds and analysed using generic LC-MS/MS conditions.
• cryopreserved hepatocytes Suspensions of cryopreserved hepatocytes are used for human hepatocyte stability assay (pooled from 3 individuals). All cryopreserved hepatocytes are purchased from In Vitro Technologies, Xenotech or TCS.
• Incubations are performed at a test or control compound concentration of 3 ⁇ M at a cell density of 0.5x10 6 viable cells/mlL The final DMSO concentration in the incubation is 0.25%. Control incubations are also performed in the absence of cells to reveal any non- enzymatic degradation.
• Duplicate samples (50 ⁇ l) are removed from the incubation mixture at 0, 5, 10, 20, 40 and 60 min (control sample at 60 min only) and added to methanol, containing internal standard (100 ⁇ l), to stop the reaction. Tolbutamide, 7-hydroxycoumarin, and testosterone are used as control compounds.
• the samples are centrifuged (2500 rpm at 4°C for 20 min) and the supernatants at each time point are pooled for cassette analysis by LC-MS/MS using generic methods.
• Caco-2 cells obtained from the ATCC at passage number 27 are used. Cells (passage number 40-60) are seeded on to Millipore Multiscreen Caco-2 plates at 1 x 105 cells/cm 2 . They are cultured for 20 days in DMEM and media is changed every two or three days. On day 20 the permeability study is performed.
• HBSS Hanks Balanced Salt Solution
• HEPES HEPES
• glucose at 37°C is used as the medium in permeability studies. Incubations are carried out in an atmosphere of 5% CO 2 with a relative humidity of 95%.
• the monolayers are prepared by rinsing both basolateral and apical surfaces twice with HBSS at 37°C. Cells are then incubated with HBSS in both apical and basolateral compartments for 40 min to stabilize physiological parameters.
• HBSS is then removed from the apical compartment and replaced with test compound dosing solutions.
• the solutions are made by diluting 10 mM test compound in DMSO with HBSS to give a final test compound concentration of 10 ⁇ M (final DMSO concentration 1%).
• the fluorescent integrity marker lucifer yellow is also included in the dosing solution.
• Analytical standards are made from dosing solutions. Test compound permeability is assessed in duplicate. On each plate compounds of known permeability characteristics are run as controls.
• the apical compartment inserts are then placed into 'companion' plates containing fresh HBSS.
• Basolateral to apical (B-A) permeability determination the experiment is initiated by replacing buffer in the inserts then placing them in companion plates containing dosing solutions. At 120 min the companion plate is removed and apical and basolateral samples diluted for analysis by LC-MS/MS. The starting concentration (C 0 ) and experimental recovery is calculated from both apical and basolateral compartment concentrations. The integrity of the monolayers throughout the experiment is checked by monitoring lucifer yellow permeation using fluorimetric analysis. Lucifer yellow permeation is low if monolayers have not been damaged. Test and control compounds are quantified by LC- MS/MS cassette analysis using a 5-point calibration with appropriate dilution of the samples. Generic analytical conditions are used.
• the permeability coefficient for each compound (P app ) is calculated from the following equation:
• C 0 is the donor compartment concentration at time zero and A is the area of the cell monolayer.
• C 0 is obtained from analysis of donor and receiver compartments at the end of the incubation period. It is assumed that all of the test compound measured after 120 min incubation was initially present in the donor compartment at 0 min.
• An asymmetry index (Al) is derived as follows:
• An asymmetry index above unity shows efflux from the Caco-2 cells, which indicates that the compound may have potential absorption problems in vivo.
• the apparent permeability (P app (A-B)) values of test compounds are compared to those of control compounds, atenolol and propranolol, that have human absorption of approximately 50 and 90% respectively (Zhao, Y.H., et al., (2001). Evaluation of Human Intestinal Absorption Data and Subsequent Derivation of a Quantitative Structure-Activity Relationship (QSAR) with the Abraham Descriptors. Journal of Pharmaceutical Sciences. 90 (6), 749-784).
• Talinolol (a known P-gp substrate (Deferme, S., MoIs, R., Van Driessche, W., Augustijns, P. (2002). Apricot Extract Inhibits the P-gp-Mediated Efflux of Talinolol. Journal of Pharmaceutical Sciences. 91(12), 2539-48)) is also included as a control compound to assess whether functional P-gp is present in the Caco-2 cell monolayer.
• the selective CYP1A inhibitor, alpha-naphthoflavone is screened alongside the test compounds as a positive control.
• the selective CYP2C9 inhibitor, sulphaphenazole is screened alongside the test compounds as a positive control.
• the selective CYP2C19 inhibitor, tranylcypromine is screened alongside the test compounds as a positive control.
• the selective CYP2D6 inhibitor, quinidine is screened alongside the test compounds as a positive control.
• test compound concentrations (0.05, 0.25, 0.5, 2.5, 5, 25 ⁇ M in DMSO; final DMSO concentration 0.26%) are incubated with human liver microsomes (0.25 mg/ml) and NADPH (1 mM) in the presence of the probe substrate midazolam (2.5 ⁇ M) for 5 min at 37°C.
• the selective CYP3A4 inhibitor, ketoconazole is screened alongside the test compounds as a positive control.
• emission wavelength 595 nm.
• the reactions are terminated by the addition of methanol containing internal standard. The samples are then centrifuged, and the supernatants are combined, for the simultaneous analysis of 4-hydroxytolbutamide, 4- hydroxymephenytoin, dextrorphan, and 1-hydroxymidazolam plus internal standard by LC- MS/MS.
• test compound 5 ⁇ M, 0.5% final DMSO concentration
• buffer pH 7.4
• 10% plasma v/v in buffer
• the experiment is performed using equilibrium dialysis with the two compartments separated by a semi-permeable membrane.
• the buffer solution is added to one side of the membrane and the plasma solution to the other side.
• Standards are prepared in plasma and buffer and are incubated at 37°C.
• Corresponding solutions for each compound are analyzed in cassettes by LC-MS/MS.
• samples are taken from both sides of the membrane.
• the solutions for each batch of compounds are combined into two groups (plasma-free and plasma- containing) then cassette analyzed by LC-MS/MS using two sets of calibration standards for plasma-free (7 points) and plasma-containing solutions (6 points).
• Generic LC-MS/MS conditions are used. Samples are quantified using standard curves prepared in the equivalent matrix. The compounds are tested in duplicate. A control compound is included in each experiment.
• PC sample concentration in protein containing side
• PF sample concentration in protein free side fu at 10% plasma is converted to fu 100% plasma using the following equation:
• Example 5 As a specific embodiment of an oral composition of a compound of the present invention, 34 mg of Example 5 is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.
• Example 7 As another specific embodiment of an oral composition of a compound of the present invention, 36 mg of Example 7 is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.

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