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Definitions

• the present invention relates to novel amido methyl-substituted 1 -(carboxyalkyl)- cyclopentylcarbonylamino-benzazepine-N-acetic acid derivatives which are useful e.g. for the prophylaxis and/or treatment of cardiovascular conditions or diseases, especially cardiac insufficiency, in particular congestive heart failure; hypertension, including secondary forms of hypertension such as essential hypertension, renal hypertension and/or pulmonary hypertension and/or for the prophylaxis and/or treatment of sexual dysfunction and/or for the prophylaxis and/or treatment of adverse conditions associated with apoptosis, and also to medicaments containing these compounds. Furthermore, the invention relates to a process for the preparation of the novel amidomethyl-substituted benzazepine-N-acetic acid derivatives and intermediate products of this process.
• SD sexual dysfunction
• vascular diseases such as those associated with hypertension or diabetes mellitus
• psychiatric disease such as depression.
• Physiological factors include fear, performance anxiety and interpersonal conflict. SD impairs sexual performance, diminishes selfesteem and disrupts personal relationships thereby inducing personal distress.
• Apoptosis is closely involved in morphogenesis and histogenesis in the development process, maintenance of homeostasis, and bio-defense, and it is cell death having an important role in maintaining individual lives.
• apoptosis is excessively induced or inhibited to cause functional disorders in various organs, and thus diseases.
• Drugs showing an apoptosis inhibitory activity can be used as agents for the prophylaxis and treatment of diseases which are thought to be mediated by promotion of apoptosis.
• compositions are known from specification WO 02/094176 A2 which contain compounds having an advantageous combinatory action which inhibits the metalloprotease enzymes NEP and IGS5 and have, inter alia, cardiovascular-active properties. Suitable compounds for such combination preparations are also compounds which fall within the scope of specifications EP 0 733 642 A1 and EP 0 916 679 A1.
• the enzyme IGS5 as it is to be understood in the context of this invention, and its physiological role in connection with cardiovascular diseases, is known per se from the specification WO 01/36610 A1.
• matrix-metalloprotease inhibitors are useful for the treatment of neurodegenerative diseases. Problems associated with that invention are first that matrix-metalloprotease inhibitors comprise a broad group of protease inhibitors, and second that according to the said application the metalloproteases must be used in a pharmaceutical composition also containing a N- NOS inhibitor.
• a group according to the invention of novel amidomethyl-substituted 1 -(carboxyalkyl)-cyclopentylcarbonylamino-benzazepine-N- acetic acid derivatives is distinguished by an action profile which inhibits the enzymes NEP and hSEP, and to a certain extent also ECE, and therefore appears suitable for the prophylaxis and/or treatment of cardiovascular conditions or diseases, especially cardiac insufficiency, in particular congestive heart failure; hypertension, including secondary forms of hypertension such as essential hypertension, renal hypertension and/or pulmonary hypertension; and/or or for the prophylaxis and/or treatment of sexual dysfunction and/or for the prophylaxis and/or treatment of adverse conditions associated with apoptosis.
• the subject of the invention is novel amidomethyl-substituted 1 -(carboxyalkyl)- cyclopentylcarbonylamino-benzazepine-N-acetic acid derivatives of the general formula I,
• R 1 is hydrogen or a group forming a biolabile ester
• R 2 is hydrogen, C ⁇ -alkyl or d- 4 -hydroxyalkyl, the hydroxyl group of which is optionally esterified with C ⁇ -alkanoyl or an amino acid residue
• R 3 is C 1-4 -alkyl; C 1-4 -alkoxy-C ⁇ - 4 -alkyl; C ⁇ - 4 -hydroxyalkyl, which is optionally substituted by a second hydroxyl group and the hydroxyl groups of which are each optionally esterified with C 2 _ 4 -alkanoyl or an amino acid residue; the phenyl group of which is optionally substituted 1-2 times by C ⁇ -4-alkyl, and/or halogen; naphthyl-d- 4 -alkyl; C 3 - 6 -oxoalkyl; phenylcarbonylmethyl, the phenyl group of which is optionally substituted 1-2 times by and/or halogen, or 2- oxoazepanyl, or
• R 2 and R 3 together are C ⁇ -alkylene, the methylene groups of which are optionally replaced 1-2 times by carbonyl, nitrogen, oxygen and/or sulphur and which are optionally substituted once by hydroxy, which is optionally esterified with d ⁇ -alkanoyl or an amino acid residue; C ⁇ -4 -alkyl; C 1-4 -hydroxyalkyl, the hydroxyl group of which is optionally esterified with C 2 - 4 -alkanoyl or an amino acid residue; phenyl or benzyl, and
• R 4 is hydrogen or a group forming a biolabile ester, and physiologically compatible salts of acids of Formula I and/or physiologically compatible acid addition salts of compounds of Formula I.
• a subject of the invention is medicaments containing the compounds of Formula I. Even further, a subject of the invention is a process for the preparation of the compounds of Formula I and intermediate products of this process.
• substituents are or contain d-4-alkyl, these may each be straight-chain or branched.
• substituents in compounds of Formula I stand for halogen, fluorine, chlorine or bromine are suitable. Chlorine is preferred.
• substituents contain C 2-4 -alkanoyl this may be straight-chain or branched. Acetyl is preferred as C 2-4 -alkanoyl.
• amino acid residues may be derived from natural or non-natural, ⁇ - or ⁇ -amino acids.
• amino acid residues which are derived from alanine, asparagine, glutamine, glycine, isoleucine, leucine, lysine, ornithine, phenylalanine, proline and valine.
• the compounds of Formula I represent dicarboxylic acid derivatives optionally esterified with groups forming biolabile esters.
• Groups which can be cleaved under physiological conditions in vivo, releasing bioavailable derivatives of the compounds of Formula I, are suitable as groups forming biolabile esters R 1 and R 4 .
• Suitable examples of this are C ⁇ -4-alkyl groups, in particular methyl, ethyl, n-propyl and isopropyl; C ⁇ -alkyloxy-C ⁇ -alkyloxy-C ⁇ -4-alkyl groups, in particular methoxyethoxy methyl; C ⁇ -cycloalkyl groups, in particular cyclohexyl; C 3 - 7 - alkyl groups; phenyl or groups optionally substituted in the phenyl ring once or twice by halogen, C ⁇ -4 -alkyl or C ⁇ .
• biolabile ester represents an optionally substituted phenyl-C ⁇ - -alkyl group
• this may contain an alkylene chain with 1 to 3, preferably 1, carbon atoms and preferably stands for optionally substituted benzyl, in particular for 2- chlorobenzyl or 4-chlorobenzyl.
• the group forming a biolabile ester represents an optionally substituted phenyl group
• the phenyl ring of which is substituted by a lower alkylene chain this may contain 3 to 4, preferably 3, carbon atoms and in particular be indanyl.
• the group forming a biolabile ester represents an optionally substituted C 2-6 -alkanoyloxy-C ⁇ - 4 -alkyl group
• the C 2- 6-alkanoyl group may be straight-chain or branched.
• R 1 preferably has the meanings hydrogen, ethyl, methoxyethoxymethyl, (RS)-1- [[(isopropyl)carbonyl]oxy]ethyl, (RS)-1-[[(ethyl)carbonyl]oxy]-2-methylpropyl, (RS)-1- [[(cyclohexyloxy)carbonyl]oxy]ethyl, 5-methyl-2-oxo-1 ,3-dioxolen-4-yl-methyl, 2-oxo-1 ,3- dioxolan-4-yl-methyl or (RS)-1-[[(ethoxy)carbonyl]oxy]ethyl.
• R 2 preferably has the meanings hydrogen, methyl, ethyl, 2-hydroxyethyl or 3- hydroxypropyl, each hydroxyl group optionally being esterified with C 2 -*-alkanoyl or an amino acid residue.
• R 3 has the meaning (Co ⁇ -alkyl ⁇ amino-d-e-alkyl, one or two Cr -alkyl groups can independently of each other be present. More specifically, "(Co- 4 -alkyl) 2 amino-C ⁇ . 6 - alkyf expressly comprises the meanings "(C 0 ) 2 -alkylamino-C ⁇ - 6 -alky , "(C 0 )(C ⁇ - )-alkyl- amino-C L g-alkyl” and "(C 1 - ) 2 -alkylamino-C ⁇ - 6 -alkyr.
• R 3 preferably has the meanings isopropyl; methoxyethyl; 2-hydroxyethyl or 3-hydroxypropyl, each hydroxyl group optionally being esterified with C 2 - 4 -alkanoyl or an amino acid residue; 3-acetyloxy-n-propyl; cyclopropylmethyl; 2-methoxybenzyl, 4- methoxy benzyl; 4-methoxyphenylethyl; 2,4 -dimethoxybenzyl; 1-naphthyl methyl; 3-oxo- 1,1-dimethylbutyl; phenyl-2-oxoethyl; 2-(4-methoxyphenyl)-2-oxoethyl; 3-(2-oxoaze- panyl); (Co- -alkyl) 2 amino-C ⁇ . 6 -alkyl, in particular dimethylamino-n-propyl, (methyl)amino- ethyl, amino-n-prop
• R 2 and R 3 together are C 4 . 7 -alkylene, the methylene groups of which are optionally replaced or optionally substituted, optionally in each case morpholine; piperidine; 4-ketopiperidine; 4-hydroxypiperidine, optionally being esterified with C 2-4 -alkanoyl or an amino acid residue at the hydroxyl group; piperazine or pyrrolidine is preferred.
• R* preferably has the meanings hydrogen, C ⁇ -4-alkyl, p-methoxybenzyl, N,N-di-(Co- 4 - alkyl)amino-C ⁇ . 6 -alkyl, (RS)-1-[[(isopropyl)carbonyl]oxy]ethyl, (RS)-1-[[(ethyl)carbonyl]- oxy]-2-methylpropyl, (RS)-1-[[(cyclohexyloxy)carbonyl]oxy]ethyl, 5-methyl-2-oxo-1 ,3- dioxolen-4-yl-methyl, 2-oxo-1,3-dioxolan-4-yl-methyl or (RS)-1-[[(ethoxy)carbonyl]oxy]- ethyl.
• Particularly preferred compounds of Formula I are selected from the group consisting of
• novel compounds of Formula I and their salts are obtained by reacting a compound of the general formula II,
• R 01 and R 401 independently of each other, are each an acid-protecting group, with a compound of the general formula III, R 3 R 2 — NH III
• R 2 and R 3 have the above meanings, wwhheerree RR 22 aanndd//oorr RR 33 ccoonnttaaiinn ffrreeee I hydroxyl groups, if desired these are reacted with a com- pound of the general formula IV, C 1-3 -C(0)-X iv wherein X stands for a leaving group, or with an amino acid derivative protected by a suitable protective group, where R 01 and/or R 401 do not represent desired groups forming a biolabile ester and/or where R 2 and/or R 3 comprise protective groups in any present amino acid residue, these are cleaved off in succession in the resulting compounds simultaneously or individually in any desired sequence and if desired the acid functions released in each case are converted into biolabile ester groups, and if desired resulting acids of Formula I are converted into their physiologically compatible salts, or salts of the acids of Formula I are converted into the free acids and/or bases of Formula I are converted into their
• Suitable physiologically compatible salts of acids of Formula I are in each case alkali metal, alkaline-earth metal or ammonium salts thereof, for example sodium, potassium or calcium salts thereof, physiologically compatible, pharmacologically neutral organic salts thereof with amines such as for example ammonia, diethylamine, tert. bu- tylamine, N-methylglucamine, choline, or with amino acids such as for example arginine.
• the substituents R 2 and/or R 3 contain basic groups, in particular nitrogen, the compounds of Formula I may also occur in the form of acid addition salts.
• Physiologically compatible acid addition salts of compounds of Formula I are their conventional salts with inorganic acids, for example sulphuric acid, phosphoric acid or hydrohalic acids, preferably hydrochloric acid, or with organic acids, for example lower aliphatic monocarboxylic, dicarboxylic or tricarboxylic acids such as maleic acid, fumaric acid, tartaric acid, citric acid, or with sulphonic acids, for example lower alkanesulphonic acids such as methanesulphonic acid.
• inorganic acids for example sulphuric acid, phosphoric acid or hydrohalic acids, preferably hydrochloric acid
• organic acids for example lower aliphatic monocarboxylic, dicarboxylic or tricarboxylic acids such as maleic acid, fumaric acid, tartaric acid, citric acid, or with sulphonic acids, for example lower alkanesulphonic acids such as methanesulphonic acid.
• Conventional protective groups for protecting carboxylic acid functions may be selected as acid-protecting groups R 101 and R 401 , which can then be cleaved off again using known methods.
• Suitable protective groups for carboxylic acids are known, for example, from McOmie, "Protective Groups in Organic Chemistry", Plenum Press (cited as “McOmie” hereinafter), and Greene, Wuts, "Protective Groups in Organic Synthesis", Wiley Interscience Publication (cited as “Greene” hereinafter), each in the most recent edition. Groups forming a biolabile ester may also be used as acid-protecting groups.
• the compounds obtained upon reaction of compounds of Formula II with compounds of Formula III in these cases already represent esters of Formula I according to the invention.
• Suitable acid-protecting groups R 101 and R 401 are in particular those groups which can be selectively cleaved or selectively introduced independently of each other.
• acid-protecting groups which are cleavable under different conditions which may also represent groups forming biolabile esters, are: unbranched lower alkyl groups such as ethyl, which can be cleaved off relatively easily under basic conditions; branched lower alkyl groups such as tert.
• the person skilled in the art is familiar with selecting suitable protective groups to obtain a desired substitution pattern.
• the compounds can thus be present in a total of four stereoisomeric forms.
• the present invention comprises both the mixtures of stereoisomers and enantiomers, and also the isomerically pure compounds of Formula I.
• Isomerically pure compounds of Formula I are preferred. Particularly preferred are compounds of Formula I wherein the carbon atom bearing the amide side chain in position 3 of the benzazepine skeleton is in the "S" configuration.
• the reaction of the acids of Formula II with the amines of Formula III can be earned out according to conventional methods for the formation of amide groups by aminoacyla- tion.
• the carboxylic acids of Formula II or their reactive derivatives may be used as acy- lation agents.
• mixed acid anhydrides and acid halides are suitable reactive derivatives.
• acid chlorides or acid bromides of the acids of Formula II or mixed esters of the acids of Formula II with organic sulphonic acids for example with lower-alkanesul phonic acids optionally substituted by halogen, such as methanesul- phonic acid or trifluoromethanesulphonic acid, or with aromatic sulphonic acids such as benzenesulphonic acids or with benzenesulphonic acids substituted by lower alkyl or halogen, e.g. toluenesulphonic acids or bromobenzenesul phonic acids, can be used.
• the acylation can expediently, in particular if a mixed anhydride of the acids of Formula II with a sulphonic acid is used as acylation agent, be carried out in the presence of an acid-binding reagent.
• Suitable acid-binding agents are for example organic bases which are soluble in the reaction mixture such as tertiary nitrogen bases, for example tert.-lower alkylamines and pyridines such as triethylamine, tripropylamine, N-methylmorpholine, pyridine, 4-dimethylaminopyridine, 4-diethylaminopyridine or 4-pyrrolidinopyridine.
• Organic bases used in excess can also serve as solvents at the same time.
• the reaction of the amino compounds of Formula III with the carboxylic acids of Formula II can expediently also be carried out in the presence of a coupling reagent known e.g. from peptide chemistry as being suitable for amide formation.
• a coupling reagent known e.g. from peptide chemistry as being suitable for amide formation.
• Examples of coupling reagents which promote amide formation with the free acids by reacting with the acid in situ, forming a reactive acid derivative are in particular: ethyl chloroformate, alkylcarbodiimides, e.g.
• the reaction in the presence of a coupling reagent can be carried out expediently at temperatures of -30" to +50°C in solvents such as halogenated hydrocarbons and/or aromatic solvents and optionally in the presence of an acid-binding amine described above.
• the protective groups R 0 a nd R 401 provided that they do not represent any desired groups forming a biolabile ester, and/or the protective groups which may be present in any present amino acid moiety in R 2 and/or R 3 , can be cleaved in known manner and if desired selectively from the compounds obtained by reacting the compounds of Formula II with the compounds of Formula III.
• the starting compounds of Formula II are novel compounds which are suitable as intermediate products for the preparation of novel active substances, for example for the preparation of the compounds of Formula I.
• the compounds of Formula II can be prepared by reacting compounds of the general formula V,
• R 5 is an acid-protecting group and R 01 has the above meaning, with compounds of the general formula VI,
• R 401 has the above meaning, and subsequently cleaving off the acid -protecting groups R 5 again in known manner.
• the reaction can be carried out in a manner known for aminoacylations, for example corresponding to the manner indicated above for the reaction of compounds of Formula II with compounds of Formula III.
• the amines of Formula III are known per se or can be prepared in known manner from known compounds.
• the reactive acid derivatives of Formula IV are known per se or can be prepared in known manner from known compounds. These are straight-chain or branched C- - carboxylic acid derivatives.
• R 101 and R 5 have the above meanings, with cyclopentanecarboxylic acid.
• the reaction can take place in known manner under the conditions of a Michael condensation in an organic solvent which is inert under the reaction conditions by reaction of the cyclopentanecarboxylic acid with a strong base capable of forming the dianion of the cyclopentanecarboxylic acid and subsequent reaction with the acrylic ester derivative of Formula VII.
• Suitable solvents are ethers, in particular cyclic ethers such as THF.
• Suitable strong bases are non-nucleophilic organic alkali metal amides or alkali metal lower alkyls such as lithium diisopropylamide or n -butyl lithium.
• the cyclopentanecarboxylic acid is reacted in THF with two equivalents of n -butyl lithium and the reaction mixture is then reacted further with the compound of Formula VII.
• the reaction temperature may be between -80° and 0°C.
• Compounds of Formula VI are known, for example from the specification EP 0 733 642 A1 , and can be prepared in the form of their racemates or alternatively in isomerically pure form according to the methods described therein or methods analogous thereto.
• Compounds of Formula VII can be prepared by esterifying compounds of the general formula VIM,
• R 5 represents an acid-protecting group, in known manner with a desired alcohol.
• Compounds of Formula VIII can for example obtained by reacting itaconic acid anhydride under known conditions which open the anhydride group with a reagent capable of formation of the acid -protecting group R 5 such as a correspondingly substituted alcohol.
• the compounds of Formula V have a chiral centre at the carbon atom bearing the radical "-COOR 101 " and are obtained upon synthesis from acrylic ester derivatives of Formula VII in the form of their racemates.
• the optically active compounds can in principle be obtained from the racemic mixtures in a manner known per se, e.g. by chroma- tographic separation on chiral separating materials or by reaction with suitable optically active bases, e.g. ⁇ -methylbenzylamine, cinchonidine or pseudoephedrine, and subsequent separation into their optical antipodes by fractional crystallisation of the salts obtained.
• suitable optically active bases e.g. ⁇ -methylbenzylamine, cinchonidine or pseudoephedrine
• the compounds of Formula I and their pharmacologically compatible salts are distinguished by advantageous pharmacological properties. In particular, the substances inhibit the enzyme NEP.
• ANP atrial natriuretic peptide
• the compounds according to the invention reduce the production of endothelin by inhibiting the ECE activity and additionally inhibiting the hSEP activity and thus counteract an increase in the peripheral resistance, which consequently results in relieving myo- cardial strain.
• Results hitherto furthermore suggest that the substances according to the invention by inhibiting the NEP activity result in higher ANP levels and an extended dura- tion of action of ANP. This should result in intensification of the ANP-mediated endogenous mechanism of cardioprotective action and impart to the substances of Formula I high effectiveness with respect to intensification of the diuretic/natriuretic ANP-induced activities.
• NEP is involved not only in the breakdown of ANP but also in the breakdown of endothelin. It follows from this that pure NEP inhibition in addition to the desired increase in the ANP levels would also lead to an unfavourable increase in the endothelin levels. For this reason, a mixed profile of NEP, hSEP and a certain proportion of ECE inhibition should be regarded as particularly beneficial, since it prevents both the breakdown of the natriuretic diuretie ANP (by NEP blockade), and simultaneously inhibits the formation of endothelin (by hSEP and ECE inhibition). As a result, a positive influence can be brought to bear on the adverse attendant effect of pure NEP inhibitors (namely undesirable increase in the endothelin levels).
• pathological conditions like conditions or
• the compounds of Formula I may also be used beneficially in the prophylaxis or treatment of damage to the heart, in particular to the myocardium, induced by cardiotoxic doses of medicaments, in particular of cytostatic agents, preferably of cytostatic antibiotics or chemicals; angina abdominal is, cerebral ischaemias, peripheral vascular disease, subarachnoid haemorrhage, chronic obstructive pulmonary disease (COPD), asthma, renal disease (renal failure), atherosclerosis, and pain in cases of colorectal or prostatic carcinoma in larger mammals, particularly humans.
• cytostatic agents preferably of cytostatic antibiotics or chemicals
• COPD chronic obstructive pulmonary disease
• COPD chronic obstructive pulmonary disease
• asthma asthma
• renal disease renal failure
• atherosclerosis and pain in cases of colorectal or prostatic carcinoma in larger mammals, particularly humans.
• Test buffer 100 mM Tris pH 7.0, 250 mM NaCI Enzyme: soluble, human recombinant NEP Prof. Crine, University of Montreal, Canada stock solution: 100 ⁇ g/ml in 20 mM Tris pH 7.0, Working solution: Stock solution with test buffer diluted to 2 ⁇ g/ml
• Substrate Mca*-Asp-lle-Ala-T ⁇ -Phe-Dpa**-Thr-Pro-Glu-His-Val-Val-Pro-Tyr- Gly-Leu-Gly-COOH; a fluorescence-quenched Big-ET-1 analogon, i.e. a substrate of metalloproteases which is detectable via the fluorescence signal, in particular of NEP and ECE-1.
• the fluorescence of the MCA fluorophore is initially quenched by the presence of the "quencher" Dpa.
• Test substances All the substances were dissolved in DMSO (10 mM) and diluted to the concentration to be tested with test buffer.
• the substrate was separated from cleavage products by means of reversed-phase HPLC (CC 125/4 Nucleosil 300/5 C i ⁇ RP column with CC 8/4 Nucleosil 100/5 C18 pre- colu n, from Macherey-Nagel, D ⁇ ren, Germany).
• HPLC reversed-phase HPLC
• 60 ⁇ l of the test mixture was injected into the HPLC sample injection point and the column was then eluted at a flow rate of 1 ml/min with the following gradient:
• All the peptides were detected by absorption at 214 nm and by fluorescence with an excitation wavelength of 328 nm and an emission wavelength of 393 nm.
• the increasing fluorescence signal (corresponds to the surface, A) of the HPLC peak of the peptide with the non-quenched Mca fluorophore is used for the further calculations.
• test substances of Formula I listed in Table 1 below had the IC 50 values given below: Table 1 : NEP-inhibiting action of the test substances in vitro
• COOH occurring as a result of the enzymatic activity of the hSEP was investigated in a standard test in vitro.
• the measure of the inhibitory activity of the substances which was determined was their IC 5 0 value.
• the ⁇ C W value of a test substance having enzyme-inhibitory activity is that concentration of the test substance at which 50% of the enzymatic activity of the hSEP is blocked.
• Test buffer 100 mM Tris pH 7.0, 250 mM NaCI
• Enzyme His6-tagged hSEP ectodomain from Innogenetics, Ghent, Belgium
• Stock solution 53 mg/ml in 20 mM HEPES pH 7.2, 5% glycerol, 0.005% Tween20, 100 mM NaCI, purity >99%
• Working solution stock solution with test buffer diluted to 10 mg/ml
• Substrate Mca-Asp-lle-Ala-T ⁇ -Phe-Dpa-Thr-Pro-Glu-His-Val-Val-Pro-Tyr-Gly- Leu-Gly-COOH; fluorescence-quenched Big-ET-1 analogon.
• test buffer 100 ⁇ M in test buffer from Polypeptide Laboratories, Wolfenb ⁇ ttel, Germany
• Test substances All the substances were dissolved in DMSO (10 mM) and diluted to the concentration to be tested with test buffer.
• the test and the HPLC procedure were carried out analogously to the manner set forth above for determining the in vitro inhibitory action of the test substances on NEP. 10 nM phosphoramidon served as standard inhibitor in the HPLC procedure.
• a pressure transducer (Statham) was inserted into the carotid artery to measure blood pressure.
• One jugular vein was cannulated for administering the substance, and the other for administering Big-ET-1.
• the rats were administered the corresponding test substance of Formula I in a concentration as a rule of 10 ⁇ mol/kg, or a vehicle. Five minutes later, 0.5 nmol/kg Big-ET-1 was infused over a period of one minute.
• the maximum Big-ET-induced increase in blood pressure and the maximum lowering of heart rate were calculated from the measured values as the difference between the value measured at the moment of maximum development of the Big-ET action (typically after 5 min.) and the value measured before Big-ET infusion.
• AUC area under the curve).
• the AUC value provides information about the entire extent and duration of the Big-ET action or the reduction thereof by substances; the AUC value can therefore - in addition to the maximum Big-ET action - provide additional information about the effect of the substances, for example in the event that the substances e.g. do not, or only slightly, influence the maximum Big-ET action, but considerably accelerate the subsiding of this action.
• the compounds of Formula I also do exhibit ECE-inhibitory properties to a certain extent.
• the ECE-inhibitory properties of the substances of Formula I can be demonstrated in a standard test in vitro.
• the compounds of Formula I are dually acting compounds which are capable of inhibiting NEP and hSEP and are also suited for prophylaxis and/or treatment of SD.
• MED is defined as: “... the inability to achieve and/or maintain a penile erection for satisfactory sexual performance (see NIH Consensus Development Panel on Impotence (1993). NIH Consensus Conference Impotence. JA. M. A. 270: 83) .".
• FSD is best defined as the difficulty or inability of a woman to find satisfaction in sexual expression.
• FSD is a collective term for several diverse female sexual disorders (Leiblum, S. R. (1998). Definition and classification of female sexual disorders. Int. J. Impotence Res., 10, S104-S106 ; Berman, J. R., Berman, L.& Goldstein, I. (1999).
• Female sexual dysfunction Incidence, pathophysiology, evaluations and treatment options. Urology, 54,385-391.). The woman may have lack of desire, difficulty with arousal or orgasm, pain with intercourse or a combination of these problems.
• Several types of disease, medications, injuries or psychological problems can cause FSD.
• FSD FSD
• desire arousal
• orgasm arousal and orgasm
• Desire or libido is the drive for sexual expression. Its manifestations often include sexual thoughts either when in the company of an interested partner or when exposed to other erotic stimuli.
• Arousal is the vascular response to sexual stimulation, an important component of which is genital engorgement and includes increased vaginal lubrication, elongation of the vagina and increased genital sensation/sensitivity.
• Orgasm is the release of sexual tension that has culminated during arousal, hence, FSD occurs when a woman has an inadequate or unsatisfactory response in any of these phases, usually desire, arousal or orgasm.
• FSD categories include hypoactive sexual desire disorder, sexual arousal disorder, orgasmic disorders and sexual pain disorders.
• the compounds of the invention will improve the genital response to sexual stimulation (as in female sexual arousal disorder), in doing so they may also improve the associated pain, distress and discomfort associated with intercourse and so treat other female sexual disorders.
• a compound of the invention in the preparation of a medicament for the treatment or prophylaxis of hypoactive sexual desire disorder, sexual arousal disorder, orgasmic disorder and sexual pain disorder, more preferably for the treatment or prophylaxis of sexual arousal disorder, orgasmic disorder, and sexual pain disorder, and preferably in the treatment or prophylaxis of sexual arousal disorder.
• Hypoactive sexual desire disorder is present if a woman has no or little desire to be sexual, and has no or few sexual thoughts or fantasies.
• This type of FSD can be caused by low testosterone levels, due either to natural menopause or to surgical menopause. Other causes include illness, medications, fatigue, depression and anxiety.
• FSAD is characterised by inadequate genital response to sexual stimulation.
• the genitalia do not undergo the engorgement that characterises normal sexual arousal.
• the vaginal walls are poorly lubricated, so that intercourse is painful. Orgasms may be impeded.
• Arousal disorder can be caused by reduced oestrogen at menopause or after childbirth and during lactation, as well as by illnesses, with vascular components such as diabetes and atherosclerosis.
• SSRIs selective serotonin re-uptake inhibitors
• Sexual pain disorders (includes dyspareunia and vaginismus) is characterised by pain resulting from penetration and may be caused by medications which reduce lubrication, endometriosis, pelvic inflammatory disease, inflammatory bowel disease or urinary tract problems.
• the prevalence of FSD is difficult to gauge because the term covers several types of problem, some of which are difficult to measure, and because the interest in treating FSD is relatively recent.
• FSD FSD consists of several subtypes that express symptoms in separate phases of the sexual response cycle, there is not a single therapy.
• FSD FSD pharmacologically
• therapy consists of the following: psychological counseling, over-the-counter sexual lubricants, and investigational candidates, including drugs approved for other conditions.
• These medications consist of hormonal agents, either testosterone or combinations of oestrogen and testosterone and more recently vascular drugs, that have proved effective in MED. None of these agents has yet been demonstrated to be effective in treating FSD.
• DSM Diagnostic and Statistical Manual
• FSAD The Diagnostic and Statistical Manual
• the arousal response consists of vasocongestion in the pelvis, vaginal lubrication and expansion and swelling of the external genitalia.
• the disturbance causes marked distress and/or interpersonal difficulty.
• Studies investigating sexual dysfunction in couples reveals that up to 76% of women have complaints of sexual dysfunction and that 30-50% of women in the USA experience FSD (Berman, J. R., Berman, L. A., Werbin, T. J. et al. (1999).
• FSAD hormone replacement therapy
• the present invention is advantageous as it helps provide a means for restoring a normal sexual arousal response-namely increased genital blood flow leading to vaginal, clitoral and labial engorgement. This will result in increased vaginal lubrication via plasma transudation, increased vaginal compliance and increased genital sensitivity.
• the present invention provides a means to restore, or potentiate, the normal sexual arousal response.
• the genital organs consist of an internal and external group.
• the internal organs are situated within the pelvis and consist of ovaries, the uterine tubes, uterus and the vagina.
• the external organs are superficial to the urogenital diaphragm and below the pelvic arch. They comprise the mons pubis, the labia majora and minora pudendi, the clitoris, the vestibule, the bulb of the vestibule, and the greater vestibular glands" (Gray's Anatomy, C. D. Clemente, 13th American Edition).
• R. J. Levin teaches that, because "...
• penile erection is a haemodynamic event which is dependent upon the balance of contraction and relaxation of the corpus caver- nosal smooth muscle and vasculature of the penis (see Lerner, S. E. et al (1993). A review of erectile dysfunction: new insights and more questions. J. Urology 149: 1246- 1255).
• Corpus cavernosal smooth muscle is also referred to herein as corporal smooth muscle or in the plural sense corpus cavernosa. Relaxation of the corpus cavernosal smooth muscle leads to an increased blood flow into the trabecular spaces of the corpus cavernosa, causing them to expand against the surrounding tunica and compress the draining veins.
• NANC non-adrenergic, non-cholinergic
• NO nitric oxide
• CGRP calcitonin gene related peptide
• VIP VIP
• NOS nitric oxide synthase
• reducing corporal smooth muscle tone may aid NO to induce relaxation of the corpus cavernosum.
• NO is released from neurones and the endothelium and binds to and activates soluble guanylate cyclase (sGC) located in the smooth muscle cells and endothelium, leading to an elevation in intracellular cyclic guanosine 3', 5'-monophosphate (cGMP) levels.
• sGC soluble guanylate cyclase
• PDE5-inhibitors e.g. sildenafil increase intracellular cGMP in corpus cavernosum tissue by inhibiting its breakdown.
• PDE5-inhibitors are inactive in the absence of a stimulator of cGMP formation, e.g. in the absence of NO.
• VIP positive nerve fibres have been found in the trabecular meshwork of the corpus cavernosum, suggesting a role of VIP release in penile erection. Effects of VIP are thought to be mediated via increases in cAMP and are thus complementary to those of cGMP-elevating agents. In patients with ED an intracavemosal injection of VIP (combined with the ⁇ -adrenoceptor antagonist phentolamine) was found to be a safe and effective treatment, with a response rate of 67% (erections sufficient for sexual intercourse).
• NEP and hSEP both degrade CNP and VIP and thereby limit the effects of CNP and VIP on cavernosal smooth muscle. Inhibition of CNP and VIP breakdown will lead to increased availability of these vasorelaxing factors thereby increasing blood flow to the corpus cavernosum which finally should result in improved erectile function. Support can be found for this from experimental data in rabbits, showing a significant increase in intracavemosal pressure and female genital blood flow after application of an NEP-inhibitor (see document WO 02/079143).
• SMR1 a gene encoding a pro-peptide of the endogenous NEP-inhibitor sialorphine was found (see User H.M., Zelner D.J., McKenna K.E., McVary K.T. (2003). Microarray analysis and description of SMR1 gene in rat penis in a post-radical prostatectomy model of erectile dysfunction. J Urol.;170(1 ):298-301 ) to be markedly downregulated (> 80-fold) in a rat model of neurogenic erectile dysfunction suggesting that in this disease NEP activity may be enhanced and contribute to the development of erectile dysfunction.
• Enzymes a) hSEP (sol hu)(his)6; or: His6-tagged hSEP ectodomai ⁇ .
• stock solution 53 ⁇ g/ml in 20 mM HEPES pH 7.2, 5% glycerol, 0.005% Tween20, 100 mM NaCI, purity >99% working solution: stock solution diluted with assay buffer to 5 ⁇ g/ml Supplier: Innogenetics, Ghent, Belgium. Preparation and purification of the protein were performed as described in WO 02/094176.
• NEP prepared from pig kidney cortex
• stock solution 120 ⁇ g/ml in 20 mM bisTris, purity >95% working solution: stock solution diluted with assay buffer to 5 ⁇ g/ml Supplier: Dr. Philippe Crine, Univ. of Montreal, Canada
• Substrates a) VIP b) CNP (32-53) stock solution: 100 ⁇ M in assay buffer Supplier: Bachem, Weil am Rhein, Germany
• Assay buffer 100 mM Tris pH 7.0, 250 mM NaCI
• test compounds were dissolved in DMSO at 10 mM and further diluted with assay buffer.
• a reversed phase HPLC technique with a CC 125/4 Nucleosil 300/5 d 8 RP column and a CC 8/4 Nucleosil 100/5 C18 precolumn (Macherey-Nagel, D ⁇ ren, Germany) was used. 60 ⁇ l of the reac- tion samples were injected into the HPLC and the column was eluted at a flow rate of 1 ml/min with the following gradient:
• Solution A 100% H20 + 0.5M H3P04 pH 2.0
• CNP and VIP were cleaved by NEP and hSEP in vitro. Breakdown of both peptides was faster with hSEP than with NEP, as is shown in table 4 below:
• test compounds according to the invention were able to prevent degradation of CNP and VIP by both NEP and SEP.
• test substances of Formula I listed in Table 5 below had the IC50 values given below: Table 5: Prevention of degradation of CNP and VIP by the test compounds breakdown of CNP breakdown of VIP inhibition of hSEP NEP hSEP NEP breakdown by example ICso (nM) ICso (nM) ICso (nM) ICso (nM) no. 1.0 10.1 3.1 3.1
• the compounds of Formula I are also suited for the prophylaxis and/or treatment of adverse conditions associated with apoptosis.
• neuro-degenerative disorders such as e.g. ischemic stroke, cerebral ischemia, traumatic brain injury, acute disseminated encepha- lomyelitis, amyotrophic lateral sclerosis (ALS), retinitis pigmentosa, mild cognitive impairment, Alzheimer's disease, Pick's disease, senile dementia, progressive supranu- clear palsy, subcortical dementias, Wilson disease, multiple infarct disease, arterioscle- rotic dementia, AIDS associated dementia, cerebellar degeneration, spinocerebellar degeneration syndromes, Friedreichs ataxia, ataxia telangiectasia, epilepsy related brain damage, spinal cord injury, restless legs syndrome, Huntington's disease and Parkinson's disease, striatonigral degeneration, cerebral vasculitis, mitochondrial encephalo- myopathies, neuronal ceroid lipofuscinosis, spinal muscular atrophies, lysosomal storage disorders with central nervous system involvement, le
• alcoholic hepatitis viral hepatitis, metabolic hepatitis, autoimmune hepatitis, radiation-induced hepatitis, liver cirrhosis, hemolytic uremic syndrome, glomerulonephritis, lupus nephritis, viral diseases such as fulminant hepatitis: joint-diseases such as trauma and osteoarthritis; immuno- suppression or immunodeficiency, in particular autoimmune diseases like idiopathic inflammatory myopathy, chronic neutropenia, thrombotic thrombocytopenic purpura, rheumatoid arthritis, idiopathic thrombocytopenic purpura, autoimmune haemolytic syn- dromes, antiphospholipid antibody syndromes, myocarditis, multiple sclerosis and its diagnostic sub-classifications relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, progressive relapsing multiple sclerosis, acute multiple
• acoustic trauma- induced auditory hair cell death and hearing loss aminoglycoside induced auditory hair cell death and hearing loss, ototoxic drug-induced hearing loss, perilymphatic fistula, cholesteatoma, cochlear or vestibular ischemia, Meniere's disease, radiation-induced hearing loss, hearing loss induced by bacterial or viral infections and idiopathic hearing loss; transplantation: graft-versus-host disease, acute and chronic rejection of heart-, lung-, kidney-, skin-, corneal-, bone marrow- or liver-transplants; wound healing and tissue rejection.
• Traumatic brain injury Delayed apoototic neuronal death
• the contusing device consisted of a stainless steel tube, 40 cm in length, perforated at 1 cm intervals to prevent air compression in the tube.
• the center of the footplate was stereotaxically positioned 1.5 mm posterior and 2.5 mm lateral to the bregma.
• the rats underwent per- fusion fixation 3 days after brain injury with a solution containing 4% paraformaldehyde in phosphate buffer.
• An unbiased counting frame (0.05 mm x 0.05 mm; disector height 0.01 mm) and a high-aperture objective (x40) were used for sampling.
• Normal neurons were identified by the presence of the typical nuclei with clear nucleoplasm and distinct nucleolus surrounded by cytoplasm containing Nissl substance.
• the border between CA2 and CA3 subfields was considered as the point where the looser arrangement of large pyramidal cells goes into densely packed pyramidal cells of the subfield CA3.
• An arbitrary line connecting the lateral ends of the dentate granule cell layers was considered a junction between subfields CA3 and CA4.
• Example 3 elicited a dose-dependent neuroprotective effect.
• a neuroprotective effect was still evident when the test substance of Example 3 was administered i.c.v. up to 8 hrs after trauma: Dose response of the neuroprotective effect of the test substance of Example 3 when administered i.c.v. 15 min after trauma to adult Wistar rats was measured. Neuronal densities were determined in the CA3 hippocampal subfield as described in the methods.
• n indicate the number of rats per group, where applicable.
• the time window of the neuroprotective effect of test substance of Example 3 when administered i.c.v. 2, 4 or 8 hrs after trauma to adult Wistar rats was measured.
• Neuronal densities were determined in the CA3 hippocampal subfield as described in the methods. Densities of CA3 neurons ⁇ SEM in 6 stereotactic levels in the traumatized right side of rats treated with either vehicle or the test compound of Example 3 were measured and the results listed in table 7 below.
• Table 7 Neuronal densities CA3 hippocampus, cells x 10 3 /mm 3
• Adriamycin toxicity Determination of anti-apoptotic activity
• the pumps had been filled with either vehicle or solution containing compounds of the invention at the appropriate concentration and primed prior to implantation.
• Animals subsequently received adriamycin at three equal daily doses of 5 mg/kg i.p. on days 1, 2 and 3. Rats were euthanized 5 days after the first injection of adriamycin and transcardially perfused with a solution containing 4% paraformaldehyde in phosphate buffer. The heart, liver and kidneys were subsequently removed and embedded in para- fin.
• TUNEL staining For terminal deoxynucleotide transferase-mediated dUTP nick end-label (TUNEL) based histological analysis, organs were post-fixed for 5 days at 4 °C and paraffin-embedded. TUNEL staining was performed on 10 ⁇ m thick paraffin sections using the ApopTag Peroxidase kit (S 7100, Oncor Appligene, Heidelberg, Germany) according to the manufacturer's instructions.
• Example 4 conferred significant protection against adriamycin toxicity in the heart, liver and kidney in that it significantly reduced the densities of TUNEL positive cells in the three organs. This effect was dose-dependent with the dose of 100 mg/kg and day being the most effective:
• Wistar rats were administered adriamycin at the cumulative dose of 15 mg/kg i.p.
• the test substance of Example 4 was administered s.c. at the doses of 20, 50 or 100 mg/kg and day by means of Alzet osmotic minipumps over 5 days. Animals were euthanized and transcardially perfused 5 days after the first injection of adriamycin and the heart, kidney and liver were processed for TUNEL staining. Densities of TUNEL positive cells were determined as described in the methods. Results for each organ (heart, liver, kidney) were measured as mean densities of TUNEL positive cells ⁇ SEM for the control groups and the different test groups (20, 50 or 100 mg/kg and day of test compound of Example 4) and listed in table 8 below.
• the test substance of Example 4 dose-dependently decreased the cytotoxic effect of adriamycin in all three organs. Comparisons between groups were performed by means of Student's t test ( ** P ⁇ 0.01; ***P ⁇ 0.001 compared to vehicle treated rats).
• the present invention also provides a method of treating or preventing cardiovascular disorders or diseases and/or treatment of adverse conditions associated with apoptosis in mammals and humans comprising administering to a subject in need thereof an effective amount of a compound of Formula I.
• the present invention further provides a method of treating or preventing sexual dysfunction in mammals and humans comprising administering to a subject in need thereof an effective amount of a dually acting compound capable of inhibiting NEP and hSEP, in particular of a compound of Formula I, according to the invention.
• the compounds of Formula I may be administered in conventional pharmaceutical compositions.
• the doses to be used may vary individually and will naturally vary according to the type of condition to be treated and the substance used. In general, however, medicinal forms with an active substance content of 0.2 to 500 mg, in particular 10 to 200 mg, active substance per individual dose are suitable for administration to humans and larger mammals.
• the agents of the present invention may also be administered by intravenous infusion, at a dose which is likely to range from 0.001-10 mg/kg/hr. The above dosages are exemplary of the average case.
• the compounds may be contained according to the invention, together with conventional pharmaceutical auxiliaries and/or excipients, in solid or liquid pharmaceutical compositions.
• solid pharmaceutical compositions a re compositions which can be administered orally, such as tablets, coated tablets, capsules, powders or granules, or alternatively suppositories.
• These pharmaceutical compositions may contain conventional pharmaceutical inorganic and/or organic excipients, such as talcum, lactose or starch, in addition to conventional pharmaceutical auxiliaries, for example lubricants or tablet disintegrating agents.
• Liquid pharmaceutical compositions such as suspensions or emulsions of the active substances may contain the usual diluents such as water, oils and/or suspension agents such as polyethylene glycols and the like.
• auxiliaries may additionally be added, such as preservatives, taste correctives and the like.
• the active substances may be mixed and formulated with the pharmaceutical auxiliaries and/or excipients in known manner.
• the active substances may for example be mixed with the auxiliaries and/or excipients in conventional manner and may be wet or dry granulated.
• the granules or powder may be poured directly into capsules or be pressed into tablet cores in conventional manner. These may be coated in known manner if desired.
• the following examples are intended to explain the invention further, without limiting its scope.
• HPLC-MS API100 Quadrupol mass spectrometer (PE Applied Biosystems) coupled to a LC200 pump (PE). Electrospray ionisation, positive mode. Scan range m/z 100 to 1000. Software MassChrom 1.2. Xterra ® column (4.6 mm x 50 mm, 2.5 ⁇ m).
• Solvent system Water (10 mM ammonium acetate, pH 5) and acetonitrile, linear gradient from 5% acetonitrile to 95% in 10 min.
• the filter cake was subsequently washed with 1.5 I EA and the combined organic phases were very largely evaporated at reduced pressure.
• the residue was taken up in 500 ml EA/cyclohexane (1 :1, v/v) and extracted twice with 200 ml semi- saturated Na 2 C0 3 s olution each time.
• the aqueous phase was acidulated with cone.
• KHS0 4 solution and extracted 3 times with 200 ml EA each time. After drying over sodium sulphate, it was evaporated under reduced pressure. Drying of the remaining residue in an oil pump vacuum yielded 71 g 3- ⁇ [1-( ⁇ [(3S)-1 -(2-tert.
• the aqueous phase was neutralised with saturated aqueous KHS0 solution and extracted three times with EA.
• the combined organic phases were washed with 100 ml saturated aqueous common salt solution and dried over sodium sulphate. Evaporation of the solvent at reduced pressure and drying of the remaining residue in an oil pump vacuum yielded 5.59 g of the title compound.
• Stationary phase Nucleosil 100-10; column: 250 mm long, 20 mm diameter; flow rate: 8 ml/min.; mobile phase: n-heptane (800 ml), 2-propanol (200 ml), TFA (1 ml).
• stationary phase EC 250/4 Nucleosil 100-10; column 250 ml long, 4 mm diameter, flow rate: 1.5 ml/min.; mobile phase: n-heptane (800 ml), 2-propanol (200 ml), TFA (1 ml).
• the active substance, the corn starch and the lactose were processed into a homogeneous pasty mixture using EA.
• the paste was ground and the resulting granules were placed on a suitable tray and dried at 45 C C in order to remove the solvent.
• the dried granules were passed through a crusher and mixed in a mixer with the further following auxiliaries:

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• 2004
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• 2007
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