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Definitions

• This invention relates to the novel use of cyclic guanylate monophosphate (cGMP)-specific phosphodiesterase type 9 inhibitors (hereinafter referred to as PDE9 inhibitors) for treating a variety of diseases, particularly cardiovascular diseases.
• PDE9 inhibitors cyclic guanylate monophosphate (cGMP)-specific phosphodiesterase type 9 inhibitors
• the invention relates to novel PDE9 inhibitors, to processes for preparing them, intermediates used in their preparation, and compositions containing them.
• cGMP and cAMP hydrolyses cyclic nucleotides cGMP and cyclic adenosine monophosphate (cAMP).
• cGMP and cAMP are central to the control and regulation of a multitude of cellular events, both physiological and pathophysiological.
• PDE9 enzyme as a novel member of the PDE enzyme family that selectively hydrolyses cGMP over cAMP.
• PDE9 was found to be present in a variety of human tissues, namely the testes, brain, small intestine, skeletal muscle, heart, lung, thymus and spleen. Smooth muscle cells in the human vasculature were not analysed for the presence of PDE9.
• the invention provides the use of a PDE9 inhibitor in the manufacture of a medicament for treating or preventing a cardiovascular disorder, disease or condition.
• the cardiovascular disorder, disease or condition is selected from: systemic (or essential) hypertension, pulmonary hypertension (e.g. pulmonary arterial hypertension, pulmonary hypertension of the neonate), congestive heart failure, coronary artery disease, atherosclerosis, stroke, thrombosis, conditions of reduced blood vessel patency (for example post percutaneous transluminal coronary angioplasty), peripheral vascular disease, renal disease (especially that occurring with diabetes), angina (including stable, unstable and variant (Prinzmetal) angina), myocardial ischaemia and any condition where improved blood flow leads to improved end organ function. More preferably the cardiovascular disease is systemic hypertension.
• pulmonary hypertension e.g. pulmonary arterial hypertension, pulmonary hypertension of the neonate
• congestive heart failure e.g. pulmonary arterial hypertension, pulmonary hypertension of the neonate
• coronary artery disease e.g. pulmonary arterial hypertension, pulmonary hypertension of the neonate
• atherosclerosis e.g. pulmonary coronary artery disease
• cardiovascular disease may be associated with other conditions, particularly hypertension associated with diabetes.
• a PDE9 inhibitor in the manufacture of a medicament for treating a condition selected from: male sexual dysfunction (particularly male erectile dysfunction otherwise known as impotence); female sexual dysfunction (FSD) (particularly female hypoactive sexual desire disorder, female sexual arousal disorder, female sexual pain disorder, female orgasmic dysfunction, clitoral dysfunction, dysfunction caused by spinal cord injury and selective serotonin re-uptake inhibitor induced sexual dysfunction), premature labour, dysmenorrhoea, benign prostatic hyperplasia, bladder outlet obstruction, incontinence, nitrate induced tolerance, bronchitis, allergic asthma, chronic asthma, allergic rhinitis, diseases and conditions of the eye (for example glaucoma and optic neuropathy, macular degeneration, elevated intra-ocular pressure, retinal or arterial occlusion), diseases characterised by disorders of gut motility (for example irritable bowel syndrome), pre-eclampsia, Kawasaki's syndrome, nitrate tolerance, multiple s
• the PDE9 inhibitor has a greater than 40% inhibition against PDE9 at a concentration of 1 ⁇ M. More preferably the PDE9 inhibitor has an IC 50 of less than 500 nM, most preferably an IC 50 of less than 50nM. Preferably the PDE9 inhibitor has a selectivity for PDE9 over PDE1 of greater than 10, preferably greater than 50, most preferably greater than 100.
• the PDE9 inhibitors of the invention are bioavailable when taken orally.
• Oral bioavailablity refers to the proportion of an orally administered drug that reaches the systemic circulation.
• the factors that determine oral bioavailability of a drug are dissolution, membrane permeability and metabolic stability.
• a screening cascade of firstly in vitro and then in vivo techniques is used to determine oral bioavailablity.
• the solubilisation of the drug by the aqueous contents of the gastrointestinal tract can be predicted from in vitro solubility experiments conducted at appropriate pH to mimic the GIT.
• the PDE9 inhibitors Preferably have a minimum solubility of 50 ⁇ g/ml. Solubility can be determined by standard procedures known in the art such as described in Lipinski CA et al.; Adv. Drug Deliv. Rev. 23(1-3), 3-25, 1997.
• Membrane permeability refers to the passage of a compound through the cells of the GIT. Lipophilicity is a key property in predicting this and is defined by in vitro Log D 7 . 4 measurements using organic solvents and buffer.
• the PDE9 inhibitors Preferably have a Log D . of -2 to +4, more preferably -1 to +3.
• the Log D can be determined by standard procedures known in the art such as described in Stopher, D and McClean, S; J. Pharm. Pharmacol. 42(2), 144, 1990.
• Cell monolayer assays such as Caco2 add substantially to prediction of favourable membrane permeability in the presence of efflux transporters such as P-glycoprotein, so-called Caco2 flux.
• the PDE9 inhibitors have a Caco2 flux of greater than 2x10 "6 cms "1 , more preferably greater than 5x10 "6 cms "1 .
• the Caco2 flux value can be determined by standard procedures known in the art such as described in Artursson, P and Magnusson, C; J. Pharm. Sci, 79(7), 595-600, 1990.
• Metabolic stability addresses the ability of the GIT or the liver to metabolise compounds during the absorption process: the first pass effect.
• Assay systems such as microsomes, hepatocytes etc are predictive of metabolic lability.
• the PDE9 inhibitors show metabolic stability in the assay system that is commensurate with an hepatic extraction of less then 0.5. Examples of assay systems and data manipulation are described in Obach, RS; Curr. Opin. Drug Disc. Devel. 4(1), 36-44, 2001 and Shibata, Y etal.; Drug Met. Disp. 28(12), 1518-1523, 2000.
• a preferred PDE9 inhibitor is a compound of formula I, a pharmaceutically acceptable salt, solvate or prodrug thereof
• R 1 is H or C ⁇ - 6 alkyl, wherein R 1 is attached to either N 1 or N 2 ;
• R 2 is Ci -6 alkyl optionally substituted by hydroxy or alkoxy; C 3 - 7 cycloalkyl optionally substituted by alkyl, hydroxy or alkoxy; a saturated 5-6- membered heterocycle (preferably tetrahydrofuran, tetrahydrothiophene, pyrrolidine or piperidine) optionally substituted by alkyl, hydroxy or alkoxy; het 1 or Ar 1 ;
• R 3 is C 1 -6 alkyl optionally substituted by 1 or 2 groups independently selected from: Ar 2 ; C 3-7 cycloalkyl optionally substituted by Ci- ⁇ alkyl; OAr 2 ; SAr 2 ;
• R 4 , R 5 and R 6 are independently selected from: hydrogen, halo, phenoxy, phenyl, CF 3 , OCF 3 , R 7 , SR 7 and OR 7 , wherein R 7 is C 1-6 alkyl optionally substituted by het 3 or by a phenyl group optionally substituted by
• R 1 ,2 or 3 groups independently selected from halo, CF 3 , OCF 3 , C ⁇ - 6 alkyl and C 1 -6 alkoxy; or wherein R 4 and R 5 combine to form a 3 or 4 atom link, wherein said link may incorporate one or two heteroatoms independently selected from O, S and N; and wherein het 1 , het 2 and het 3 , which may be the same or different, are aromatic 5-6 membered heterocycles containing 1 , 2 or 3 heteroatoms, independently selected from O, S and N, said heterocycle optionally substituted by 1 , 2 or 3 substituents, independently selected from C- ⁇ - 6 alkyl, C ⁇ -6 alkoxy, halo and phenyl optionally substituted by 1 , 2 or 3 groups independently selected from halo and C ⁇ -6 alkyl; with the provisos that when a) R 1 is attached to N 1 , R 1 is C ⁇ -3 alkyl and R 2 is propyl then R 3
• a more preferred PDE9 inhibitor is a compound of formula la, a pharmaceutically acceptable salt, solvate or prodrug thereof
• R 1 is H or C ⁇ -e alkyl
• R 2 is Ci -6 alkyl optionally substituted by hydroxy or alkoxy; C 3-7 cycloalkyl optionally substituted by alkyl, hydroxy or alkoxy; a saturated 5-6- membered heterocycle (preferably tetrahydrofuran, tetrahydrothiophene, pyrrolidine or piperidine) optionally substituted by alkyl, hydroxy or alkoxy; het 1 or Ar 1 ;
• R 3 is C- ⁇ - 6 alkyl optionally substituted by 1 or 2 groups independently selected from: Ar 2 ; C 3-7 cycloalkyl optionally substituted by C ⁇ -6 alkyl; OAr 2 ; SAr 2 ; NHC(0)Ci -6 alkyl; het 2 ; xanthene; and naphthalene; wherein Ar 1 and Ar 2 are independently groups of formula
• R 4 , R 5 and R 6 are independently selected from: hydrogen, halo, phenoxy, phenyl, CF 3) OCF 3 , R 7 , SR 7 and OR 7 , wherein R 7 is C ⁇ -6 alkyl optionally substituted by het 3 or by a phenyl group optionally substituted by 1 , 2 or 3 groups independently selected from halo, CF 3 , OCF 3 , C ⁇ -6 alkyl and C ⁇ -6 alkoxy; or wherein R 4 and R 5 combine to form a 3 or 4 atom link, wherein said link may incorporate one or two heteroatoms independently selected from O, S and N; and wherein het 1 , het 2 and het 3 , which may be the same or different, are aromatic 5-6 membered heterocycles containing 1 , 2 or 3 heteroatoms, independently selected from O, S and N, said heterocycle optionally substituted by 1 , 2 or 3 substituents, independently selected from Ci- ⁇ alkyl,
• the invention provides a compound of formula la, wherein R 1 is H or C-i- ⁇ alkyl; R 2 is selected from C-i- ⁇ alkyl, straight chain or branched chain, C 3-7 cycloalkyl and heteroaryl; R 3 is C ⁇ - 6 alkyl, straight chain or branched chain, optionally substituted by 1-2 groups each independently selected from: Ar, C 3-7 cycloalkyl, OAr, SAr, NC(0)C ⁇ - 6 alkyl, heteroaryl, xanthene and naphthalene, wherein: Ar is a group of formula
• R 4 , R 5 and R 6 are each independently selected from: H, halo, OPh, Ph, CF 3 , OCF 3 , SCi-6 alkyl, C ⁇ - 6 alkyl, OC ⁇ - 6 alkyl, said alkyl optionally substituted by a heteroaryl group or by a Ph group, wherein said Ph group is optionally substituted by 1-3 groups selected from halo, CF 3 , OCF 3 and C-i -6 alkyl; or wherein R 4 and R 5 may combine to form a C ⁇ -3 alkyl link, wherein said link may optionally incorporate one or two heteroatoms selected from O, S and N, wherein heteroaryl is aromatic 5-6 membered heterocycle containing 1 -3 heteroatoms, each independently selected from O, S and N, said heterocycle optionally substituted by 1-3 substituents, each independently selected from C ⁇ -6 alkyl, halo and Ph, said Ph optionally substituted by 1-3 groups selected from halo and C ⁇ -6 alkyl
• Halo means fluoro, chloro, bromo or iodo.
• R 1 is hydrogen or CH 3 . More preferably R 1 is hydrogen.
• R 2 is C 3- alkyl, cyclopentyl or pyridyl. More preferably R 2 is 3-pyridyl.
• R 3 is C ⁇ -3 alkyl optionally substituted by 1 or 2 groups independently selected from: Ar 2 , C 3 - 7 cycloalkyl optionally substituted by C 1-6 alkyl and het 2 . More preferably R 3 is C 1-3 alkyl optionally substituted by Ar 2 . Most preferably R 3 is methyl substituted by Ar 2 .
• R 4 , R 5 and R 6 are independently selected from: hydrogen, halo, phenoxy, phenyl, CF 3 , OCF 3 , R 7 , SR 7 , and OR 7 , wherein R 7 is C ⁇ -6 alkyl optionally substituted by a het 3 group or by a phenyl group optionally substituted by 1 ,2 or 3 groups independently selected from halo, CF 3 , OCF 3 , Ci-e alkyl and Ci- ⁇ alkoxy; or wherein R 4 and R 5 combine to form a 3 atom link wherein said link contains an oxygen atom.
• R 4 , R 5 and R 6 are independently selected from hydrogen, halo, CF 3) OCF 3 , phenoxy, and OC ⁇ -6 alkyl optionally substituted by phenyl optionally substituted by halo, CF 3 , OCF 3 or C ⁇ -6 alkyl.
• R 4 , R 5 and R 6 are independently selected from hydrogen, chloro, OCF 3 , CF 3 , phenoxy and OC ⁇ -6 alkyl substituted by phenyl.
• R 4 , R 5 and R 6 are independently selected from hydrogen, chloro, OCF 3 and OC ⁇ - 3 alkyl substituted by phenyl.
• het 2 is an aromatic 5-6 membered heterocycle containing 1 or 2 nitrogen atoms optionally containing a further heteroatom, said heterocycle being optionally substituted by 1 , 2 or 3 substituents, each independently selected from C- ⁇ - 6 alkyl, halo and phenyl optionally substituted by 1 , 2 or 3 groups independently selected from halo and C ⁇ -6 alkyl.
• het 2 is an aromatic 5-membered heterocycle containing 1 or 2 nitrogen atoms optionally containing a further heteroatom, said heterocycle being optionally substituted by 1 substituent selected from C ⁇ -6 alkyl, halo and phenyl optionally substituted by 1 , 2 or 3 groups independently selected from halo and Ci-e alkyl.
• het 2 is an aromatic 5 membered heterocycle containing 1 or 2 nitrogen atoms optionally containing a further heteroatom and optionally substituted by phenyl optionally substituted by halo.
• het 2 is imidazolyl or oxadiazolyl.
• Preferred compounds are:
• substituted means substituted by one or more defined groups.
• the term independently means that where more than one substituent is selected from a number of possible substituents, those substituents may be the same or different.
• the pharmaceutically acceptable salts of the compounds of formula I which contain a basic centre are, for example, non-toxic acid addition salts formed with inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid, with carboxylic acids or with organo-sulfonic acids.
• Examples include the HCI, HBr, HI, sulfate or bisulfate, nitrate, phosphate or hydrogen phosphate, acetate, benzoate, succinate, saccharate, fumarate, maleate, lactate, citrate, tartrate, gluconate, camsylate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate salts.
• Compounds of formula I which contain an acidic centre can provide pharmaceutically acceptable metal salts, in particular non-toxic alkali and alkaline earth metal salts, with bases. Examples include the sodium, potassium, aluminium, calcium, magnesium and zinc salts.
• organic salts can be made, for example the diethanolamine salt.
• suitable pharmaceutical salts see Berge et al, J. Pharm, Sci., 66, 1-19, 1977; P L Gould, International Journal of Pharmaceutics, 33 (1986), 201-217; and Bighley etal, Encyclopaedia of Pharmaceutical Technology, Marcel Dekker Inc, New York 1996, Volume 13, page 453-497.
• the pharmaceutically acceptable solvates of the compounds of formula I include hydrates thereof.
• the compounds of the invention may possess one or more chiral centres and so exist in a number of stereoisomeric forms. All stereoisomers and mixtures thereof are included in the scope of the present invention. Racemic compounds may either be separated using preparative HPLC and a column with a chiral stationary phase or resolved to yield individual enantiomers utilising methods known to those skilled in the art. In addition, chiral intermediate compounds may be resolved and used to prepare chiral compounds of the invention.
• the compounds of the invention may exist in one or more tautomeric forms. All tautomers and mixtures thereof are included in the scope of the present invention. For example, a claim to 2-hydroxypyridinyl would also cover its tautomeric form, ⁇ -pyridonyl.
• pro-drugs examples include Drugs of Today, Volume 19, Number 9, 1983, pp 499 - 538 and in Topics in Chemistry, Chapter 31 , pp 306 - 316 and in "Design of Prodrugs" by H. Bundgaard, Elsevier, 1985, Chapter 1 (the disclosures in which documents are incorporated herein by reference). It will further be appreciated by those skilled in the art, that certain moieties, known to those skilled in the art as “pro-moieties”, for example as described by H. Bundgaard in "Design of Prodrugs” (the disclosure in which document is incorporated herein by reference) may be placed on appropriate functionalities when such functionalities are present within compounds of the invention.
• Preferred prodrugs for compounds of the invention include: esters, carbonate esters, hemi-esters, phosphate esters, nitro esters, sulfate esters, sulfoxides, amides, carbamates, azo-compounds, phosphamides, glycosides, ethers, acetals and ketals.
• the invention also includes all suitable isotopic variations of a compound of the invention.
• An isotopic variation of a compound of the invention is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature.
• isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2 H, 3 H, 13 C, 14 C, 15 N, 17 0, 18 0, 31 P, 32 P, 35 S, 18 F and 36 CI, respectively.
• isotopic variations of the invention are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3 H, and carbon-14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2 H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances.
• Isotopic variations of the compounds of the invention can generally be prepared by conventional procedures such as by the illustrative methods or by the preparations described in the Examples and Preparations hereafter using appropriate isotopic variations of suitable reagents.
• Compounds of general formula I may be prepared from compounds of general formula II according to reaction scheme 1.
• Suitable conditions are well known to a man skilled in the art and include base catalysed cyclisation using reagents such as potassium tert-butoxide, sodium hydroxide and potassium carbonate in an alcoholic solvent such as ethanol or isopropanol or an alcohol/water mixture.
• the reaction may be carried out at a temperature between room temperature and the reflux temperature of the solvent, optionally in the presence of hydrogen peroxide.
• Compounds of general formula II may be prepared according to reaction scheme 2, by reacting compounds of formula IV with compounds of formula III. Such amide bond forming reactions may be carried out under a wide variety of conditions well known to the skilled man.
• compounds of formula IV may be activated by treatment with an agent such as 1 ,1-carbonyldiimidazole (GDI) or fluoro-N.N.N'.N'-tetramethylformamidinium hexafluorophosphate (TFFH), or a combination of agents such as azabenzotriazol-l-yloxytris(pyrrolidino)- phosphonium hexafluorophosphate (PyAOP) and 1-hydroxy-7-azabenzotriazole (HOAt), followed by addition of the compound of formula III.
• an agent such as 1 ,1-carbonyldiimidazole (GDI) or fluoro-N.N.N'.N'-tetramethylformamidinium hexafluor
• reaction shown in reaction scheme 2 may be carried out by addition of a peptide coupling agent such as 0-(7-azabenzotriazol-1-yl) ⁇ N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) to a mixture of the compounds of formula III and IV.
• a peptide coupling agent such as 0-(7-azabenzotriazol-1-yl) ⁇ N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU)
• HATU hydroxybenzotriazol-1-yl
• This reaction is carried out in a suitable solvent such as dichloromethane, pyridine, N,N-dimethylformamide (DMF), N,N- dimethylacetamide (DMA) or 1 -methyl-2-pyrrolidinone at a temperature between 0°C and the reflux temperature of the solvent.
• the reaction is preferentially carried out by activation of the compound of
• Compounds of general formula III may be prepared from easily obtained starting materials using processes described in the preparative examples hereinafter and described in European Patent EP0463756.
• a pharmaceutically acceptable salt of a compound of the formula I may be readily prepared by mixing together solutions of a compound of the formula I and the desired acid or base, as appropriate.
• the salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.
• the compounds of the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
• the compounds of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, multi-particulates, gels, films, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.
• the compounds of the invention may also be administered as fast-dispersing or fast-dissolving dosage forms or in the form of a high energy dispersion or as coated particles. Suitable formulations of the compounds of the invention may be in coated or uncoated form, as desired.
• Such solid pharmaceutical compositions may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine and starch (preferably corn, potato or tapioca starch), disintegrants such as sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
• excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine and starch (preferably corn, potato or tapioca starch), disintegrants such as sodium starch glycollate, croscarmellose sodium and certain complex silicates, and
• a formulation of the tablet could typically contain between about 0.01 mg and 500 mg of active compound whilst tablet fill weights may range from 50 mg to 1000 mg.
• An example of a formulation for a 10mg tablet is illustrated below:
• the tablets are manufactured by a standard process, for example, direct compression or a wet or dry granulation process.
• the tablet cores may be coated with appropriate overcoats.
• Modified release and pulsatile release dosage forms may contain excipients such as those detailed for immediate release dosage forms together with additional excipients that act as release rate modifiers, these being coated on and/or included in the body of the device.
• Release rate modifiers include, but are not exclusively limited to, hydroxypropylmethyl cellulose, methyl cellulose, sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate, polyethylene oxide, Xanthan gum, Carbomer, ammonio methacrylate copolymer, hydrogenated castor oil, carnauba wax, paraffin wax, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, methacrylic acid copolymer and mixtures thereof.
• Modified release and pulsatile release dosage forms may contain one or a combination of release rate modifying excipients.
• Release rate modifying excipients may be present both within the dosage form i.e. within the matrix, and/or on the dosage form, i.e. upon the surface or coating.
• Fast dispersing or dissolving dosage formulations may contain the following ingredients: aspartame, acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, diascorbic acid, ethyl acrylate, ethyl cellulose, gelatin, hydroxypropylmethyl cellulose, magnesium stearate, mannitol, methyl methacrylate, mint flavouring, polyethylene glycol, fumed silica, silicon dioxide, sodium starch glycolate, sodium stearyl fumarate, sorbitol, xylitol.
• dispersing or dissolving as used herein to describe FDDFs are dependent upon the solubility of the drug substance used i.e. where the drug substance is insoluble a fast dispersing dosage form can be prepared and where the drug substance is soluble a fast dissolving dosage form can be prepared.
• the compounds of the invention can also be administered parenterally, for example, intracavemouslly, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion or needleless injection techniques.
• parenteral administration they are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
• the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
• the preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
• dosage levels and other dosage levels herein are for the average human subject having a weight range of about 65 to 70 kg.
• the skilled person will readily be able to determine the dosage levels required for a subject whose weight falls outside this range, such as children and the elderly.
• the daily dosage level of the compounds of the invention will usually be from 0.01 mg to 500 mg (in single or divided doses).
• tablets or capsules of the compound of the invention may contain from 0.01 mg to 500 mg (for example 10 mg to 250 mg) of active compound for administration singly or two or more at a time, as appropriate.
• the physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient.
• the above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.
• the skilled person will appreciate that the compounds of the invention may be taken as a single dose as needed or desired.
• the compounds of the invention can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray, atomiser or nebuliser, with or without the use of a suitable propellant, e.g.
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• the pressurised container, pump, spray, atomiser or nebuliser may contain a solution or suspension of the active compound, e.g.
• Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.
• Aerosol or dry powder formulations are preferably arranged so that each metered dose or "puff" contains from 1 ⁇ g to 50 mg of a compound of the invention for delivery to the patient.
• the overall daily dose with an aerosol will be in the range of from 1 ⁇ g to 50 mg which may be administered in a single dose or, more usually, in divided doses throughout the day.
• the compounds of the invention can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder.
• the compounds of the invention may also be dermally or transdermally administered, for example, by the use of a skin patch. They may also be administered by the pulmonary or rectal routes.
• the compounds may also be administered by the ocular route.
• the compounds can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride.
• they may be formulated in an ointment such as petrolatum.
• the compounds of the invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
• they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
• the compounds of the invention may also be used in combination with a cyclodextrin.
• Cyclodextrins are known to form inclusion and non-inclusion complexes with drug molecules. Formation of a drug-cyclodextrin complex may modify the solubility, dissolution rate, bioavailability and/or stability property of a drug molecule. Drug-cyclodextrin complexes are generally useful for most dosage forms and administration routes.
• the cyclodextrin may be used as an auxiliary additive, e.g. as a carrier, diluent or solubiliser.
• Alpha-, beta- and gamma-cyclodextrins are most commonly used and suitable examples are described in WO-A-91/11172, WO-A- 94/02518 and WO-A-98/55148.
• the present invention additionally comprises the combination of a PDE9 inhibitor, (particularly a compound of formula I as defined in the various embodiments of the first aspect) and one or more additional active agent selected from:
• a PGI2 prostaglandin such as prostacyclin or iloprost
• an ⁇ - adrenergic receptor antagonist compound also known as ⁇ - adrenoceptor antagonists, -receptor antagonists or -blockers
• suitable compounds for use herein include: the -adrenergic receptor antagonists as described in PCT application WO99/30697 published on 14th June 1998, the disclosures of which relating to ⁇ -adrenergic receptor antagonists are incorporated herein by reference and include, selective ⁇ i -adrenoceptor antagonists or ⁇ 2 -adrenoceptor antagonists and non-selective adrenoceptor antagonists, suitable oi -adrenoceptor antagonists include: phentolamine, phentolamine mesylate, trazodone, alfuzosin, indoramin, naftopidil, tamsulosin, d
• an angiotensin II receptor blocker such as candesartan, eprosartan, irbesartan, losartan, olmesartan, olmesartan medoxomil, saralasin, telmisartan and valsartan.
• a substrate for NO-synthase such as L-arginine
• a calcium channel blocker such as amlodipine, verapamil, pranidipine, azelnidipine and vatanidipine
• k) a cholesterol lowering agent such as statins.
• statins are atorvastatin calcium (Lipitor), cerivastatin sodium (Baycol), fluvastatin sodium
• an antiplatelet or antithrombotic agent e.g. tPA, uPA, warfarin, hirudin and other thrombin inhibitors, aspirin, plavix, cilastozol, heparin, thromboplastin activating factor inhibitors;
• a PDE5 inhibitor such as 5-[2-ethoxy-5-(4-methyl-1 - piperazinylsulphonyl)phenyl]-1-methyl-3-n-propyl-1 ,6-dihydro-7H-pyrazolo[4,3- ]pyrimidin-7-one (sildenafil); (6 : ?,12a/ : ?)-2,3,6,7,12,12a-hexahydro-2-methyl-6- (3,4-methylenedioxyphenyl)pyrazino[2', 1 ':6, 1 ]pyrido[3,4-b]indole-1 ,4-dione (tadalafil, IC
• a combination of active agents are administered, then they may be administered simultaneously, separately or sequentially.
• a method of treating a cardiovascular disorder in a mammal wherein the mammal is treated with an effective amount of a PDE9 inhibitor.
• the preferred embodiments specified hereinabove for the first aspect extend to this aspect.
• a series of carboxylic acids of general formula IV (where R 3 is defined in Table 3) (80 ⁇ mol) were each dissolved in a 3.75% solution of triethylamine in dimethylacetamide (400 ⁇ l) and added to a 96 well plate. Carbonyldiimidazole (13mg, 80 ⁇ mol) dissolved in pyridine (212 ⁇ l) was then added into each well, and the plates were left to stand at room temperature for 2 hours. A solution of the product from Preparation 6 (13.5mg, 80 ⁇ mol) dissolved in dimethylacetamide (100 ⁇ l) was then added. The plates were sealed and heated to 70°C in an oven under nitrogen for 18 hours. The plates were removed and allowed to cool to room temperature over 2 hours.
• the solvent was removed using a GENEVAC (45°C, 0.15mbar) over 5.5 hours.
• a solution of potassium t- butoxide (268 mg, 240 ⁇ mol) in isopropyl alcohol (0.5ml) was added to each well, and the plates were sealed and transferred to an oven at 110°C under nitrogen for 15 hours. The plates were removed and allowed to cool to room temperature over 2 hours.
• the solvent was removed using the GENEVAC (45°C, 0.15mbar) over 5.5 hours, and a solution of p-toluenesulfonic acid (30 mg, 160 ⁇ l) in isopropyl alcohol (0.5ml) was added to each well.
• Palladium tetrakis triphenylphosphine (22mg, 5 mole %) was added to a nitrogen- purged solution of the product from preparation 36 (164 mg, 0.376 mmol), phenyl boronic acid (69 mg, 0.56 mmol), sodium carbonate (119 mg, 1.13 mmol as a solution in 0.8 ml water) in ethylene glycol dimethyl ether (3 ml). The mixture was heated at 83°C for 18 h. On cooling, the mixture was diluted with ethyl acetate/tetrahydrofuran and washed with brine, dried (MgS0 4 ), filtered and concentrated in vacuo.
• the product from preparation 3 (5g, 32.5mmol) was added portionwise to concentrated sulfuric acid (25ml) at room temperature with stirring.
• the reaction mixture was then heated to 60°C and concentrated nitric acid (70%, 6ml, 90mmol) was added dropwise whilst keeping the temperature at 60°C.
• the reaction was stirred at 60°C for 3h, cooled to room temperature and then poured onto 50ml of ice with stirring.
• Oxalyl chloride (6.8ml, 77.6mmol) was added dropwise to a suspension of the product from preparation 4 (5.15g, 25.9mmol) in dichloromethane (80ml) containing dimethylformamide (0.1ml) under nitrogen at 0°C.
• the reaction was stirred at 0°C for 1 h, allowed to warm to room temperature and stirred for a further 2h.
• the solvent was removed under reduced pressure, the residue was dissolved in toluene (100ml) and ammonia gas was bubbled into the solution for 2h.
• the reaction was stirred under nitrogen at room temperature for 18h, concentrated under reduced pressure and the residue was dissolved in hot methanol (300ml).
• 2,4-Dimethylbenzylcyanide 70g, 0.48mol was mixed with water (134ml) and concentrated sulfuric acid (106ml, 1.98mol) was added slowly. The reaction was heated to reflux for 3h, then cooled to room temperature over 18h. The mixture was poured onto crushed ice (500ml), stirred for 1 h and the resulting precipitate was isolated by filtration. After washing with water the solid was dissolved in 1.2M sodium hydroxide solution (500ml), extracted with dichloromethane (2x250ml) and the aqueous phase was treated with decolourising carbon (2g) at reflux for 10min and filtered hot through hyflo supercel.
• Carbonyldiimidazole (84mg, 0.515mmol) was added to a solution of 2- trifluoromethyoxyphenyl acetic acid (113mg, 0.515mmol) in tetrahydrofuran (4ml) under nitrogen at room temperature, and the mixture was stirred for 3h.
• the product from preparation 10 (100mg, 0.515mmol) was then added and the reaction was stirred for 18h.
• the reaction mixture was diluted with water (20ml), acidified to pH2 with 2N HCI and extracted with ethyl acetate (2x20ml).
• N-lodosuccinimide (326 mg, 1.45 mmol) was added to a solution of the product from preparation 37 (300 mg, 0.967 mmol) in dry dimethylformamide (5 ml) at room temperature. The mixture was heated at 55°C for 18 h, cooled and concentrated in vacuo. The residue was dissolved in ethyl acetate/tetrahydrofuran and washed with water and brine, dried (MgS0 4 ), filtered and concentrated.
• the ability of the compounds of the invention to inhibit the PDE9 enzyme was determined using the following in vitro assay.
• the assay used purified recombinant human PDE9.
• the assay uses [ 3 H]cGMP which is hydrolysed by the PDE9 enzyme to the 5'- nucleotide [ 3 H]GMP.
• the [ 3 H]GMP binds to yttrium silicate scintillation proximity assay (SPA) beads, and detected by scintillation counting. Inhibition of activity is determined relative to the activity of uninhibited controls.
• SPA yttrium silicate scintillation proximity assay
• Recombinant human PDE-9 enzyme (Flag-tagged) was obtained by expression in a baculovirus/S/9 cell system and purified by anti-FLAG monoclonal antibody affinity chromatography (D A Fisher, J F Smith, J S Pillar, S H Denis, J B Cheng (1998), J. Biol. Chem., 273: 15559-15564).
• SPA beads Yttrium Silicate
• Buffer B was prepared containing Bovine Serum Albumin (2 mg/ml) (BSA) in Buffer A. It was prepared fresh and filter sterilised.
• BSA Bovine Serum Albumin (2 mg/ml)
• PDE9 enzyme solution was prepared in Buffer B (dilution factor determined such that no more than 30% breakdown of substrate occurred, but typically 1:35,000).
• cGMP substrate was prepared from a 50nM stock of guanosine 3':5'-cyclic monophosphate (cGMP) prepared to give final assay concentration of
• SPA beads were prepared by creating a suspension of beads in water (20 mg/ml) (28ml per pack) containing 3mM cold cGMP to effectively quench the reaction.
• the compounds of the invention were diluted by a factor of 50 (i.e. 2 ⁇ l in 10O ⁇ l) when constituted in the final assay mix.
• Compound stock was prepared at 4mM in DMSO. Dilute 1/8 with DMSO to give 500 ⁇ M solutions.
• a 4mM stock of standard inhibitor was prepared in DMSO.
• the standard inhibitor chosen was 5-(3-bromobenzyl)-3-n-propyl-1 ,6-dihydro-7H-pyrazolo[4,3- d]pyrimidin-7-one.
• the solution was further diluted with DMSO to give a 500 ⁇ M solution.
• the plates were then analysed using a scintillation counter (such as the NXT TopCount)TM.
• a scintillation counter such as the NXT TopCount
• IC50 values were determined from sigmoid dose response curves of enzyme activity vs. compound concentration. The IC 5 o of the standard was expected to fall in the range 30 - 50nM.
• the ability of the compounds to inhibit the PDE1 enzyme was determined by an assay procedure broadly analogous to that described above for PDE9. The differences are as follows: i) human PDE1 enzyme was isolated from human ventricle homogenate by chromatographic separation of a high speed centrifugation supernatant fraction; ii) the cGMP substrate (1 ⁇ M) was prepared by combining 0.436 ml of a 10 ⁇ M stock of unlabelled guanosine 3':5'-cyclic monophosphate (cGMP) with 10 ⁇ M [3H]cGMP (15.6Ci/mmol) and 4.554 ml Buffer A; the final assay concentration of cGMP in the assay being 0.5 ⁇ M, iii) the PDE1 inhibitor used as a standard was 5-[4-(N,N-diethylamino)benzy ⁇ ]-1-methyl- 3-n-propyl-1 ,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin
• the compounds of the invention were tested using the above assays and found to inhibit the PDE9 enzyme.
• compound 52 was found to have an IC50 against PDE9 of 126 nM; compound 204 was found to have an IC50 against PDE9 of 143 nM; and compound 258 was found to have an IC50 against PDE9 of 141 nM.
• Compounds 52, 204 and 258 were all greater than 10 fold selective for PDE9 over PDE1.

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