Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • 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
• • A—HUMAN NECESSITIES
  • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
  • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
  • C07D495/04—Ortho-condensed systems

Definitions

• WO2005/047268 relates to substituted pyrimidine compositions that are capable of modulating the activity of receptors of the NGFI-B family.
• WO2005/116010 relates to phenyl or pyridyl derivatives having EP4 agonistic activity.
• US2006/128729 describes bicyclic pyrazole derivatives for the treatment of diseases associated with cellular proliferation, diseases related to glycosidase expression or inflammatory conditions.
• EP675124 relates to purine derivatives as anti- inflammatory agents.
• US2006084650 discloses (pyrazolyl)(imidazopyrimidinyl)amines as kinase inhibitors.
• the compounds of the invention differ from the prior art compounds in structure, in their pharmacological activity and/or pharmacological potency.
• One aspect of the present invention relates to a compound of formula
• Y represents N or CH
• R 1 represents hydrogen or fluoro
• R represents hydrogen, halo, Ci_6alkyl, Ci_6alkyloxy, cyano, nitro, amino or mono-or di(C i _ 6 alkyl)amino ;
• n represents an integer of value 1, 2 or 3; provided that R 3 may only be other than hydrogen if at least one of R 1 or R 2 is other than hydrogen; and provided the compound is other than 4-(4-methylphenoxy)-2-phenyl-thieno-
• the present invention also relates to the use of a compound of formula (I) for the manufacture of a medicament for preventing or treating a disease by activating the EP4 receptor, in particular for treating a disease by activating the EP4 receptor, in particular for preventing or treating a disease, in particular treating a disease, associated with loss of bone mass, wherein the compound is a compound of formula (I)
• R 2 represents hydrogen, halo, cyano, Ci_ 6 alkyl, Ci_ 6 alkyloxy, Ci_ 6 alkylcarbonyl or
• R represents hydrogen, halo, Ci_6alkyl, Ci_6alkyloxy, cyano, nitro, amino or mono-or di(Ci_6alkyl)amino; n represents an integer of value 1, 2 or 3; provided that R 3 may only be other than hydrogen if at least one of R 1 or R 2 is other than hydrogen; a JV-oxide thereof, a pharmaceutically acceptable salt thereof or a solvate thereof.
• Ci_4alkyl as a group or part of a group defines straight or branched chain saturated hydrocarbon radicals having from 1 to 4 carbon atoms such as methyl, ethyl, propyl, 1-methylethyl, butyl;
• Ci_ 6 alkyl as a group or part of a group defines straight or branched chain saturated hydrocarbon radicals having from 1 to 6 carbon atoms such as the group defined for Ci_4alkyl and pentyl, hexyl,
• Ci_4alkanediyl defines straight or branched chain saturated bivalent hydrocarbon radicals having from 1 to 4 carbon atoms such as methylene,
• halo is generic to fluoro, chloro, bromo and iodo.
• each definition is independent.
• salts of the compounds of formula (I) are those wherein the counterion is pharmaceutically acceptable.
• salts of acids and bases which are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound. All salts, whether pharmaceutically acceptable or not are included within the ambit of the present invention.
• the pharmaceutically acceptable salts as mentioned hereinbefore or hereinafter are meant to comprise the therapeutically active non-toxic acid addition salt forms which the compounds of formula (I) are able to form.
• the latter can conveniently be obtained by treating the base form with such appropriate acids as inorganic acids, for example, hydrohalic acids, e.g.
• primary, secondary and tertiary aliphatic and aromatic amines such as methylamine, ethylamine, propylamine, isopropylamine, the four butylamine isomers, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, quinuclidine, pyridine, quinoline and isoquinoline, the benzathine, JV-methyl-D-glucamine, 2-amino-2-(hydroxymethyl)-l,3- propanediol, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like.
• the salt form can be converted by treatment with acid into the free acid form.
• salt also comprises the quaternary ammonium salts (quaternary amines) which the compounds of formula (I) are able to form by reaction between a basic nitrogen of a compound of formula (I) and an appropriate quaternizing agent, such as, for example, an optionally substituted Ci_6alkylhalide, arylhalide,
• Ci_6alkylcarbonylhalide, arylcarbonylhalide, or arylCi- ⁇ alkylhalide e.g. methyliodide or benzyliodide, wherein aryl represents unsubstituted or substituted phenyl.
• Other reactants with good leaving groups may also be used, such as for example Ci_6alkyl trifluoromethanesulfonates, Ci_6alkyl methanesulfonates, and Ci_6alkyl /?-toluenesulfonates.
• a quaternary amine has a positively charged nitrogen.
• Counterions include chloro, bromo, iodo, trifluoroacetate, acetate, triflate, sulfate, sulfonate.
• the counterion of choice can be introduced using ion exchange resins.
• iV-oxide forms of the present compounds are meant to comprise the compounds of formula (I) wherein one or several tertiary nitrogen atoms are oxidized to the so-called iV-oxide.
• solvate comprises the hydrates and solvent addition forms which the compounds of formula (I) are able to form, as well as the salts thereof. Examples of such forms are e.g. hydrates, alcoholates and the like.
• stereochemically isomeric forms as used hereinbefore or hereinafter defines all the possible stereoisomeric forms which the compounds of formula (I), and their TV-oxides, salts or solvates may possess.
• chemical designation of compounds denotes the mixture of all possible stereochemically isomeric forms, as well as each of the individual isomeric forms of formula (I) and their TV-oxides, salts or solvates, substantially free, i.e. associated with less than 10%, preferably less than 5%, in particular less than 2% and most preferably less than 1% of the other isomers.
• a compound of formula (I) is for instance specified as (R)
• this means that the compound is substantially free of the (S) isomer.
• an R or S descriptor is assigned (based on Cahn-Ingold-Prelog sequence rule) to the lowest-numbered chiral center, the reference center.
• the configuration of the second stereogenic center is indicated using relative descriptors [R*,R* ] or [i?*,S*], where the first R* is always specified as the reference center and [R*,R*] indicates centers with the same chirality and [i?*,S*] indicates centers of unlike chirality. For example, if the lowest-numbered chiral center in the molecule has an S configuration and the second center is R, the stereo descriptor would be specified as S-[R*, S*].
• the position of the highest priority substituent on the asymmetric carbon atom in the ring system having the lowest ring number is arbitrarily always in the " ⁇ " position of the mean plane determined by the ring system.
• the position of the highest priority substituent on the other asymmetric carbon atom in the ring system relative to the position of the highest priority substituent on the reference atom is denominated " ⁇ ", if it is on the same side of the mean plane determined by the ring system, or " ⁇ ", if it is on the other side of the mean plane determined by the ring system.
• the compounds of (I) may be synthesized in the form of racemic mixtures of enantiomers which can be separated from one another following art-known resolution procedures.
• the racemic compounds of formula (I) may be converted into the corresponding diastereomeric salt forms by reaction with a suitable chiral acid. Said diastereomeric salt forms are subsequently separated, for example, by selective or fractional crystallization and the enantiomers are liberated therefrom by alkali.
• An alternative manner of separating the enantiomeric forms of the compounds of formula (I) involves liquid chromatography using a chiral stationary phase.
• Said pure stereochemically isomeric forms may also be derived from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction occurs stereospecifically.
• Preferably if a specific stereoisomer is desired, said compound will be synthesized by stereospecific methods of preparation. These methods will advantageously employ enantiomerically pure starting materials.
• a first interesting embodiment of the present invention are those compounds of formula (I) having the following formula
• A represents phenyl or cyclohexyl
• ring E represents a partially saturated or aromatic 5-membered heterocycle wherein the dotted lines represent an optional double bond and wherein B, C and D each independently represent CH 2 , CH, N, NH, S or O provided that the 5-membered ring contains 1 or 2 heteroatoms;
• R 2 represents hydrogen, halo, cyano, Ci_ 6 alkyl, Ci_ 6 alkyloxy, Ci_ 6 alkylcarbonyl or
• a second interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein A represents phenyl.
• a third interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein A represents cyclohexyl.
• a sixth interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein R 1 represents hydrogen or R 1 represents fluoro.
• a seventh interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein R 2 represents hydrogen, halo, cyano, C ⁇ alkyl, Ci_6alkyloxy or Ci_6alkylcarbonylamino; in particular hydrogen, halo, cyano, Ci_6alkyl or Ci_6alkyloxy.
• An eighth interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein R 3 represents hydrogen.
• a tenth interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein R 1 is fluoro and R 2 is hydrogen.
• An eleventh interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein R 1 is hydrogen and R 2 is halo, cyano, Ci_ 6 alkyl, Ci_ 6 alkyloxy, Ci_6alkylcarbonyl or Ci_6alkylcarbonylamino.
• a twelfth interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein R 1 is fluoro and R 2 is halo, cyano, Ci_6alkyl, Ci_6alkyloxy, Ci_6alkylcarbonyl or Ci_6alkylcarbonylamino.
• a thirteenth interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein R 1 and R 2 are both hydrogen.
• a fourteenth interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein Y represents N.
• a fifteenth interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein Y represents CH.
• a nineteenth interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein the compound of formula (I) is having the following formula including any stereochemically isomeric form thereof, a JV-oxide thereof, a pharmaceutically acceptable salt thereof or a solvate thereof.
• a twentieth interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein the compound of formula (I) is a compound having the following formula
• a twenty first interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein the compound of formula (I) is a compound having the following formula
• a twenty second interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein the compound of formula (I) is a compound having the following formula
• a twenty third interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein the compound of formula (I) is a compound having the following formula
• a twenty fourth interesting embodiment of the present invention are those compounds of formula (I) or any subgroup thereof as mentioned hereinbefore as interesting embodiment wherein the compound of formula (I) is a compound having the following formula
• a twenty fifth interesting embodiment of the present invention are those compounds of formula (1-1) or (I-l-a) wherein one or more, preferably all, of the following restrictions apply : a) A represents phenyl or cyclohexyl; b) R 1 represents hydrogen or fluoro, in particular hydrogen; c) R 2 represents hydrogen, cyano, halo, Ci_6alkyl or Ci_6alkyloxy; d) R 3 represents hydrogen; e) X represents a direct bond.
• a twenty seventh interesting embodiment of the present invention are those compounds of formula (I) selected from the group consisting of 4-(2-phenyl-thieno[3,2- d]pyrimidin-4-yloxy)-benzonitrile; 4-(2-cyclohexyl-thieno[3,2-d]pyrimidin-4-yloxy)-3- fluoro-benzonitrile; a JV-oxide thereof, a pharmaceutically acceptable salt thereof or a solvate thereof.
• compounds of formula (I-l-c) can be prepared by reacting in a first step (a), an intermediate of formula (II-a) with an intermediate of formula P-W 2 wherein P represents a suitable protective group, such as for example benzyl or Ci_4alkyloxy- Ci_4alkyl and wherein W 2 represents a suitable leaving group, such as for example halo, e.g.
• step (a) is then reacted in a next step (b) with an intermediate of formula (III) in the presence of a suitable base, such as for example K2CO3 or sodium hydride, and a suitable solvent, such as for example acetonitrile or dimethylsulfoxide.
• a suitable base such as for example K2CO3 or sodium hydride
• a suitable solvent such as for example acetonitrile or dimethylsulfoxide.
• step (b) is then deprotected in a next step (c) by reaction with a suitable acid, such as for example hydrochloric acid, in the presence of a suitable solvent, such as for example an alcohol, e.g. methanol, or acetonitrile.
• Compounds of formula (I) wherein A represents phenyl, said compounds being represented by formula (I-b), can also be prepared by dehalogenating an intermediate of formula (XII) in the presence OfH 2 , a suitable catalyst, such as for example palladium on charcoal, a catalyst poison, such as for example a thiophene solution, a suitable base, such as for example KOAc, and a suitable solvent, such as for example an alcohol, e.g. methanol.
• a suitable catalyst such as for example palladium on charcoal
• a catalyst poison such as for example a thiophene solution
• a suitable base such as for example KOAc
• a suitable solvent such as for example an alcohol, e.g. methanol.
• the compounds of formula (I) may further be prepared by converting compounds of formula (I) into each other according to art-known group transformation reactions.
• the compounds of formula (I) may be converted to the corresponding JV-oxide forms following art-known procedures for converting a trivalent nitrogen into its iV-oxide form.
• Said JV-oxidation reaction may generally be carried out by reacting the starting material of formula (I) with an appropriate organic or inorganic peroxide.
• Appropriate inorganic peroxides comprise, for example, hydrogen peroxide, alkali metal or earth alkaline metal peroxides, e.g. sodium peroxide, potassium peroxide;
• appropriate organic peroxides may comprise peroxy acids such as, for example, benzenecarboper- oxoic acid or halo substituted benzenecarboperoxoic acid, e.g.
• 3-chlorobenzenecarbo- peroxoic acid peroxoalkanoic acids, e.g. peroxoacetic acid, alkylhydroperoxides, e.g. tert.butyl hydro-peroxide.
• Suitable solvents are, for example, water, lower alcohols, e.g. ethanol and the like, hydrocarbons, e.g. toluene, ketones, e.g. 2-butanone, halogenated hydrocarbons, e.g. dichloromethane, and mixtures of such solvents.
• the compounds of formula (I) and some of the intermediates in the present invention may contain an asymmetric carbon atom.
• Pure stereochemically isomeric forms of said compounds and said intermediates can be obtained by the application of art-known procedures.
• diastereoisomers can be separated by physical methods such as selective crystallization or chromatographic techniques, e.g. counter current distribution, chiral liquid chromatography and the like methods.
• Enantiomers can be obtained from racemic mixtures by first converting said racemic mixtures with suitable resolving agents such as, for example, chiral acids, to mixtures of diastereomeric salts or compounds; then physically separating said mixtures of diastereomeric salts or compounds by, for example, selective crystallization or chromatographic techniques, e.g. liquid chromatography and the like methods; and finally converting said separated diastereomeric salts or compounds into the corresponding enantiomers.
• suitable resolving agents such as, for example, chiral acids
• intermediates of formula (II) wherein Wi represents chloro can be prepared by reacting an intermediate of formula (IV) with SOCl 2 or phosphoric trichloride optionally in the presence of a suitable solvent, such as for example chloroform, N,N- dimethylformamide or N, ⁇ /-dimethylbenzeneamine.
• a suitable solvent such as for example chloroform, N,N- dimethylformamide or N, ⁇ /-dimethylbenzeneamine.
• a suitable base such as for example ⁇ /,iV-diisopropylethanamine
• Intermediates of formula (IV) wherein Y represents N, said intermediates being represented by formula (IV-a), can be prepared by cyclizing an intermediate of formula (V) in the presence of an aqueous ammoniak solution or NH 4 OH or NH 3 in acetonitrile or an alcohol, e.g. methanol.
• Intermediates of formula (IV-a) can also be prepared by reacting an intermediate of formula (VI) in the presence of a suitable base, such as for example K2CO3, NaOCH3 or l,8-diazabicyclo[5.4.0]undecene-7 (DBU), and a suitable solvent, such as for example water, alcohol, e.g. ethanol or isopropanol, or chloroform.
• a suitable base such as for example K2CO3, NaOCH3 or l,8-diazabicyclo[5.4.0]undecene-7 (DBU)
• a suitable solvent such as for example water, alcohol, e.g. ethanol or isopropanol, or chloroform.
• Intermediates of formula (IV-a) can also be prepared by reacting an intermediate of formula (XIII) with an intermediate of formula (XIV) in the presence of a base, such as for example potassium t-butoxide, and a suitable solvent, such as for example tetrahydrofuran.
• a base such as for example potassium t-butoxide
• a suitable solvent such as for example tetrahydrofuran.
• Intermediates of formula (IV-b) can be prepared by reacting an intermediate of formula (VII) with an intermediate of formula (VIII) in the presence of a suitable base, such as for example K2CO3, and a suitable solvent, such as for example water.
• a suitable base such as for example K2CO3
• a suitable solvent such as for example water.
• Intermediates of formula (IV-c) can be prepared by reacting an intermediate of formula (XV) with an intermediate of formula (XIV) in the presence of lithium diisopropylamide (LDA) and a suitable solvent, such as for example tetrahydrofuran.
• LDA lithium diisopropylamide
• intermediates of formula (V) can be prepared by reacting an intermediate of formula (IX) with an intermediate of formula (X) wherein W 3 represents a suitable leaving group, such as for example halo, e.g. chloro and the like, in the presence of a suitable solvent, such as for example acetone or pyridine, and optionally a suitable base, such as for example pyridine or 4- ⁇ /, ⁇ /-dimethylaminopyridine
• a suitable solvent such as for example acetone or pyridine
• a suitable base such as for example pyridine or 4- ⁇ /, ⁇ /-dimethylaminopyridine
• Intermediates of formula (VI) can be prepared by reacting an intermediate of formula (XI) with an intermediate of formula (X) in the presence of a suitable base, such as for example JV,jV-dimethyl-4-pyridine amine, and a suitable solvent, such as for example pyridine.
• a suitable base such as for example JV,jV-dimethyl-4-pyridine amine
• a suitable solvent such as for example pyridine.
• Intermediates of formula (VI) can also be prepared from the corresponding carboxylic acid derivative of formula (VF) by reaction with ethyl chloro formate and NH3 in the presence of a suitable base, such as for example triethylamine, in a suitable solvent, such as for example acetonitrile.
• a suitable base such as for example triethylamine
• Intermediates of formula (VF) can be prepared by hydrolysis of an intermediate of formula (V) in the presence of a suitable base, such as for example KOH, and a suitable solvent, such as for example an alcohol, e.g. ethanol.
• a suitable base such as for example KOH
• a suitable solvent such as for example an alcohol, e.g. ethanol.
• a suitable base such as for example K2CO3 or sodium hydride
• a suitable solvent such as for example acetonitrile or dimethylsulfoxide.
• EP4 is one of the four subtype receptors (EPl, EP2, EP3 EP4) of prostaglandine E2.
• Prostaglandins are arachidonic acid metabolites that are synthetized by the cyclo- oxygenase pathway.
• a major cyclo-oxygenase product is prostaglandin E 2 (PGE 2 ), which participates in a broad range of biological activities, such as smooth muscle relaxation, vasodilation, fever, inflammatory pain, enteric mucus secretion, renal regulation and bone formation.
• PGE 2 can exert agonistic activities on four G-protein-coupled receptor subtypes, which are termed EPl, EP2, EP3 and EP4. Each of these receptor subtypes has a distinct pharmacological signature based on their ligand preference and coupling to intracellular signalling pathways.
• EPl and EP3 receptors are coupled to calcium metabolism and inhibition of cyclic adenosine 5 -monophosphate (cAMP) via GqZG 1 G-proteins.
• cAMP cyclic adenosine 5 -monophosphate
• EP2 and EP4 are linked to the stimulation of adenylyl cyclase and increased cAMP synthesis via G s G-proteins.
• PGE 2 effects are linked to its activation of the EP4 receptor, as evidenced by experiments using EP4-deficient mice and specific EP4 agonists or antagonists.
• EP4-deficient mice produce a reduced vasodepressor response following PGE 2 infusion, exhibit decreased contact hypersensitivity and show reduced incidence and intensity of disease in the collagen antibody- induced arthritis model, while they have increased colitis induced by dextran sulphate treatment. Furthermore, the absence of EP4 decreases bone mass and impaired fracture healing in aged male mice, whilst PGE 2 administration fails to induce bone formation in EP4-deficient mice.
• EP4 selective agonists suppresses dextran sulphate colitis, restores bone mass and strength in both normal and aged, ovariectomized animals, reduces indomethacin-induced small intestinal ulceration, attenuates endotoxin/galactosamine-induced liver injury, reduces mercury chloride-evoked acute kidney failure and attenuated pain responses in Freund's complete adjuvant-induced joint inflammation.
• topical application of an EP4 agonist reduces the increased intraocular pressure.
• EP4 antagonists block the bone anabolic effects Of PGE 2 in rats.
• the use of PGE 2 in human therapy is compromised by its inability to discern EP receptor subtypes, which leads to a number of untoward effects, including nausea, emesis, diarrhea and hypotension.
• the compounds of formula (I), their JV-oxides, pharmaceutically acceptable salts, or solvates are useful for the treatment or prevention, in particular for the treatment, of a disease by activating the EP4 receptor.
• the compounds of formula (I), their JV-oxides, pharmaceutically acceptable salts or solvates may be used as a medicine, in particular for use as a medicine for the treatment of a disease by activating the EP4 receptor.
• the present compounds can be used for the manufacture of a medicament for treating or preventing a disease by activating the EP4 receptor, in particular for treating a disease by activating the EP4 receptor.
• a method of treating a warm-blooded mammal, including a human, suffering from or a method of preventing a warm-blooded mammal, including a human, to suffer from a disease by activating the EP4 receptor in particular a method of treating a warm-blooded mammal, including a human, suffering from a disease by activating the EP4 receptor.
• Said methods comprise the administration of an effective amount of a compound of formula (I), a JV-oxide form thereof, a pharmaceutically acceptable salt thereof or a solvate thereof, to a warm-blooded mammal, including a human.
• compositions for preventing or treating a disease by activating the EP4 receptor in particular for treating a disease by activating the EP4 receptor.
• Said compositions comprise a therapeutically effective amount of a compound of formula (I), a JV-oxide form thereof, a pharmaceutically acceptable salt thereof or a solvate thereof, and a pharmaceutically acceptable carrier or diluent.
• compositions of the present invention may be formulated into various pharmaceutical forms for administration purposes.
• compositions there may be cited all compositions usually employed for systemically administering drugs.
• an effective amount of the particular compound, optionally in salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration.
• a pharmaceutically acceptable carrier which carrier may take a wide variety of forms depending on the form of preparation desired for administration.
• These pharmaceutical compositions are desirable in unitary dosage form suitable, particularly, for administration orally, rectally, percutaneously, or by parenteral injection.
• any of the usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs, emulsions and solutions; or solid carriers such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules, and tablets. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid pharmaceutical carriers are obviously employed.
• the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included.
• injectable solutions for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution.
• injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed.
• solid form preparations which are intended to be converted, shortly before use, to liquid form preparations.
• the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin.
• the compounds of the present invention may also be administered via inhalation or insufflation by means of methods and formulations employed in the art for administration via this way.
• the compounds of the present invention may be administered to the lungs in the form of a solution, a suspension or a dry powder.
• Any system developed for the delivery of solutions, suspensions or dry powders via oral or nasal inhalation or insufflation are suitable for the administration of the present compounds.
• the compounds of the present invention may also be topically administered in the form of drops, in particular eye drops. Said eye drops may be in the form of a solution or a suspension. Any system developed for the delivery of solutions or suspensions as eye drops are suitable for the administration of the present compounds.
• Unit dosage form refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, suppositories, injectable solutions or suspensions and the like, and segregated multiples thereof.
• the exact dosage and frequency of administration depends on the particular compound of formula (I) used, the particular condition being treated, the severity of the condition being treated, the age, weight, sex, extent of disorder and general physical condition of the particular patient as well as other medication the individual may be taking, as is well known to those skilled in the art. Furthermore, it is evident that said effective daily amount may be lowered or increased depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compounds of the instant invention.
• the pharmaceutical composition will preferably comprise from 0.05 to 99 % by weight, more preferably from 0.1 to 70 % by weight, even more preferably from 0.1 to 50 % by weight of the active ingredient, and, from 1 to 99.95 % by weight, more preferably from 30 to 99.9 % by weight, even more preferably from 50 to 99.9 % by weight of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.
• DMF N, ⁇ /-dimethylformamide
• the LC measurement was performed using an Acquity UPLC (Ultra Performance Liquid Chromatography) system (Waters) comprising a binary pump, a sample organizer, a column heater (set at 55 0 C), a diode-array detector (DAD) and a column as specified in the respective methods below.
• Flow from the column was split to a MS spectrometer.
• the MS detector was configured with an electrospray ionization source. Mass spectra were acquired by scanning from 100 to 1000 in 0.18 seconds using a dwell time of 0.02 seconds.
• the capillary needle voltage was 3.5 kV and the source temperature was maintained at 140 0 C. Nitrogen was used as the nebulizer gas.
• Data acquisition was performed with a Waters-Micromass MassLynx-Openlynx data system.
• the HPLC measurement was performed using an Alliance HT 2790 (Waters) system comprising a quaternary pump with degasser, an autosampler, a column oven (set at 40 0 C), a diode-array detector (DAD) and a column as specified in the respective methods below.
• Flow from the column was split to a MS spectrometer.
• the MS detector was configured with an electrospray ionization source. Mass spectra were acquired by scanning from 100 to 1000 in 1 second using a dwell time of 0.1 second.
• the capillary needle voltage was 3 kV and the source temperature was maintained at 140 0 C. Nitrogen was used as the nebulizer gas.
• Data acquisition was performed with a Waters- Micromass MassLynx-Openlynx data system.
• Reversed phase UPLC was carried out on a bridged ethylsiloxane/silica hybrid (BEH) Cl 8 column (1.7 ⁇ m, 2.1 x 50 mm; Waters Acquity) with a flow rate of 0.8 ml/min.
• Two mobile phases (mobile phase A: 0.1 % formic acid in H2 ⁇ /methanol 95/5; mobile phase B: methanol) were used to run a gradient condition from 95 % A and 5 % B to 5 % A and 95 % B in 1.3 minutes and hold for 0.2 minutes.
• An injection volume of 0.5 ⁇ l was used.
• Cone voltage was 10 V for positive ionization mode and 20 V for negative ionization mode.
• LCMS analytical data (R(t) means retention time in minutes; MH(+) means the protonated mass of the compound; LCMS method refers to the method used for LCMS described above).
• HEK293 cells stably transfected with hEP4 were grown up to 80-90% confluence in T175 Falcon flasks in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 100 IU/ml penicillin G, 100 ⁇ g/ml streptomycin sulphate, 1 mM sodium pyruvate, 300 ⁇ g/ml L-glutamine and 10% heat inactivated foetal calf serum (Biochrom AG) in a humidified atmosphere of 5%CO 2 at 37°C.
• DMEM Dulbecco's modified Eagle's medium
• Biochrom AG heat inactivated foetal calf serum
• the experiments were performed with the cAMP Dynamic HTRF kit (CIS bio international, France), used according to the supplier's instructions. Specifically, cells were thawed rapidly by warming up the vials in a warm water bath at 37°C. The thawed cell suspension (2 ml; 10 7 cells/ml) was transferred to a 50 ml Falcon tube and for each vial, 10 ml prewarmed culture medium was added. The falcon tube was centrifuged at 1,500 RPM for 5 minutes. The pellet was washed by adding 15 ml HBSS (per vial) and centrifuged at 1,500 RPM for another 5 minutes.
• the final pellet was resuspended in stimulation buffer (HBSS Ix, IBMX ImM, Hepes 5mM, MgCl 2 1OmM, BSA 0.1%, pH 7.4).
• the suspension was counted in a nucleocounter and further diluted in stimulation buffer at a concentration of 500,000 cells/ml.
• the cells were seeded out in a MW384 COSTAR 3710 with the compounds using a Multidrop 384 at a density of 10,000 cells/well in 20 ⁇ l.
• the cells were incubated for 30 minutes at room temperature in the dark in the presence of different concentrations of the compounds diluted in stimulation buffer in a final volume of 30 ⁇ l/well.
• the final concentration of DMSO whenever needed to dissolve the compounds) did not exceed 1% (v/v) and was also included in the corresponding control samples. Reaction was stopped by adding 10 ⁇ l cAMP-d2 conjugate and subsequently 10 ⁇ l of anti-cAMP with the Multidrop.
• Selectivity of the compounds for EP4 can also be demonstrated by determining whether the compounds have activity on the EPl receptor, for instance by [Ca 2+ J 1 measurements in response to activation or inhibition of the monkey EPl receptor as follows : The antagonistic and agonistic effect of the test compounds on intracellular Ca 2+ concentrations ([Ca 2+ J 1 ) was measured in a fluorescent based assay, using the calcium assay kit (Molecular Devices, Crawley, England).
• HEK293 cells stably transfected with monkey EPl receptor were cultured in T 175 Falcon flasks in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 100 IU/ml penicillin G, 100 ⁇ g/ml streptomycin sulphate, 1 mM sodium pyruvate, 300 ⁇ g/ml L-glutamine and 10% heat inactivated foetal calf serum (Biochrom AG) in a humidified atmosphere of 5%CO 2 at 37°C. Before the experiments, the cells were grown on 384-well (black wall/transparent bottom) plates from Greiner for 1 day until they reached confluency.
• DMEM Dulbecco's modified Eagle's medium
• the cells were loaded with loading buffer supplied by the kit supplemented with 10 niM probenecid and 0.1% fatty acid free bovine serum albumine, adjusted to pH 7.4 with 1 M Hepes-acid, for 90 minutes at 37°C in a CO 2 incubator. Ca 2+ signals were measured in a Fluorometric Imaging Plate Reader (FLIPR, from Molecular Devices).
• FLIPR Fluorometric Imaging Plate Reader
• PGE2 prostaglandin E2
• To test agonistic activity the compounds were added to the loaded cells during the measurement in the FLIPR where 1000 nM PGE2 was used as the reference agonist.
• PEC50 (agonism) and pICso (antagonism) values for the tested compounds were ⁇ 5.

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  • 2008-01-29 WO PCT/EP2008/051041 patent/WO2008092860A1/en active Application Filing

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