WO2007034445A2

WO2007034445A2 - Novel piperazine derivatives as renin inhibitors for cardiovascular events and renal insufficiency

Info

Publication number: WO2007034445A2
Application number: PCT/IB2006/053445
Authority: WO
Inventors: Olivier Bezencon; Christoph Boss; Daniel Bur; Olivier Corminboeuf; Corinna Grisostomi; Lubos Remen; Sylvia Richard-Bildstein; Thomas Weller
Original assignee: Actelion Pharmaceuticals Ltd
Current assignee: Actelion Pharmaceuticals Ltd
Priority date: 2005-09-26
Filing date: 2006-09-22
Publication date: 2007-03-29
Anticipated expiration: 2008-03-26
Also published as: WO2007034445A3

Abstract

The invention relates to novel piperazine derivatives and related compounds and their use as active ingredients in the preparation of pharmaceutical compositions. The invention also concerns related aspects including processes for the preparation of the compounds, pharmaceutical compositions containing one or more of those compounds and especially their use as inhibitors of renin. In particular, the present invention relates to novel compounds of the formula (I),

Description

Novel Piper azine Derivatives

The present claimed invention was made as a result of activities undertaken within the scope of a research collaboration agreement between Merck & Co., Inc., Actelion Pharmaceuticals Ltd, and Actelion Ltd. The agreement was executed on December 4, 2003.

The invention relates to novel compounds of the formula (I). The invention also concerns related aspects including processes for the preparation of the compounds, pharmaceutical compositions containing one or more compounds of formula (I) and especially their use as renin inhibitors in cardiovascular events and renal insufficiency.

In the renin-angiotensin system (RAS) the biologically active angiotensin II (Ang II) is generated by a two-step mechanism. The highly specific enzyme renin cleaves angiotensinogen to angiotensin I (Ang I), which is then further processed to Ang II by the less specific angiotensin-converting enzyme (ACE). Ang II is known to work on at least two receptor subtypes called ATi and AT2. Whereas ATi seems to transmit most of the known functions of Ang II, the role of AT2 is still unknown.

Modulation of the RAS represents a major advance in the treatment of cardiovascular diseases. ACE inhibitors and ATi blockers have been accepted to treat hypertension (Waeber B. et al, "The renin-angiotensin system: role in experimental and human hypertension", in Birkenhager W. H., Reid J. L. (eds): Hypertension, Amsterdam, Elsevier Science Publishing Co, 1986, 489-519; Weber M. A., Am. J. Hypertens., 1992, 5, 247S). In addition, ACE inhibitors are used for renal protection (Rosenberg M. E. et al, Kidney International, 1994, 45, 403; Breyer J. A. et al., Kidney International, 1994, 45, S156), in the prevention of congestive heart failure (Vaughan D. E. et al, Cardiovasc. Res., 1994, 28, 159; Fouad-Tarazi F. et al, Am. J. Med., 1988, 84 (Suppl. 3A), 83) and myocardial infarction (Pfeffer M. A. et al, N. Engl. J. Med., 1992, 327, 669).

The rationale to develop renin inhibitors is the specificity of renin (Kleinert H. D., Cardiovasc. Drugs, 1995, 9, 645). The only substrate known for renin is angiotensinogen, which can only be processed (under physiological conditions) by renin. In contrast, ACE can also cleave bradykinin besides Ang I and can be bypassed by chymase, a serine protease (Husain A., J. Hypertens., 1993, 11, 1155). In patients inhibition of ACE thus leads to bradykinin accumulation causing cough (5-20%) and potentially life-threatening angioneurotic edema (0.1-0.2%) (Israili Z. H. et al, Annals of Internal Medicine, 1992, 117, 234). Chymase is not inhibited by ACE inhibitors. Therefore, the formation of Ang II is still possible in patients treated with ACE inhibitors. Blockade of the ATi receptor (e.g. by losartan) on the other hand overexposes other AT-receptor subtypes (e.g. AT₂) to Ang II, whose concentration is significantly increased by the blockade of ATi receptors. In summary, renin inhibitors are expected to demonstrate a different pharmaceutical profile than ACE inhibitors and ATi blockers with regard to efficacy in blocking the RAS and in safety aspects.

Only limited clinical experience (Azizi M. et al., J. Hypertens., 1994, 12, 419; Neutel J. M. et al., Am. Heart, 1991, 122, 1094) has been created with renin inhibitors because of their insufficient oral activity due to their peptidomimetic character (Kleinert H. D., Cardiovasc. Drugs, 1995, 9, 645). The clinical development of several compounds has been stopped because of this problem together with the high cost of goods. Only one compound containing four chiral centers has entered clinical trials (Rahuel J. et al., Chem. Biol., 2000, 7, 493; Mealy N. E., Drugs of the Future, 2001, 26, 1139). Thus, renin inhibitors with good oral bioavailability and long duration of action are required. Recently, the first non-peptide renin inhibitors were described which show high in vitro activity (Oefner C. et al., Chem. Biol., 1999, 6, 127; Patent Application WO 97/09311; Marki H. P. et al., Il Farmaco, 2001, 56, 21). However, the development status of these compounds is not known. The present invention relates to renin inhibitors of a non-peptidic nature and of low molecular weight. Described are orally active renin inhibitors of formula (I) which have a long duration of action and which are active in indications beyond blood pressure regulation where the tissular renin-chymase system may be activated leading to pathophysiological^ altered local functions such as renal, cardiac and vascular remodeling, atherosclerosis, and possibly restenosis. So, the present invention describes these non-peptidic renin inhibitors of formula (I).

In particular, the present invention relates to novel compounds of the formula (I),

_|__|

Formula (I)

wherein

W represents a phenyl substituted by V in para position; a five-membered heteroaryl with two ring heteroatoms independently selected from N, O, and S; or a six-membered heteroaryl with one or two nitrogen ring atoms;

V represents -NH-R³-, -N(CH₃)-R³-, -NH-R³-O-, -N(CH₃)-R³-O-, -0-CH₂-Q-, -CH₂-CH₂-CH₂-, -CH₂-A-CH₂-, -CH₂-CH₂-A-, -CH₂-CH₂-CH₂-CH₂-, -CH₂-CH₂- A-CH₂-, -CH₂-CH₂-CH₂-A-, -A-CH₂-CH₂-B-, -CH₂-CH₂-CH₂-CH₂-CH₂-, -CH₂- CH₂-A-CH₂-CH₂-, -CH₂-CH₂-CH₂-A-CH₂-, -CH₂-CH₂-CH₂-CH₂-A-, -A-CH₂- CH₂-CH₂-B-, -CH₂-A-CH₂-CH₂-B-, -CH₂-N(R²)-CH₂-, or -CH₂-CH₂-N(R²)-CH₂-, preferably -0-CH₂-Q-, -CH₂-CH₂-A-, -CH₂-CH₂-CH₂-A- or -A-CH₂-CH₂-B-; or V represents a heterocyclic group selected from

W , W W ;

U represents aryl that is optionally mono-, di-, tri-, or tetra-substituted with substituents independently selected from the group consisting of halogen, alkyl, -CF₃, -OCF₃, methoxy, and hydroxy-alkyl; or heteroaryl that is optionally mono-, di-, or tri- substituted with substituents independently selected from the group consisting of halogen, alkyl, -CF₃, -OCF₃, methoxy, and hydroxy-alkyl;

M represents aryl or pyridinyl that is optionally mono-, di-, or tri- substituted, wherein the substituents are independently selected from the group consisting of halogen, alkyl, -CF₃, -OCF₃, alkoxy, alkyl-O-(CH₂)₀-₄-CH₂-, alkyl-O-(CH₂)₂_₄-O-, and R⁴ ₂N-(CH₂)o-₄-CH₂-;

Q represents a five-membered heteroaryl with two or three, preferably two, ring heteroatoms independently selected from nitrogen and oxygen, preferably an oxadiazolyl or isoxazolyl, most preferably an isoxazolyl;

A and B independently represent -O- or -S-, preferably -0-;

R¹ represents cycloalkyl, preferably cyclopropyl;

R² represents alkyl, cycloalkyl, or CF₃CH₂-, preferably methyl;

R³ represents alkylene, preferably ethylene or propylene;

R⁴ represents hydrogen, alkyl, cyclopropyl, or -C(=O)-R' wherein R' is C₁-C₄- alkyl, -CF₃, -CH₂-CF₃, or cyclopropyl;

m is the integer O or 1 ; and n represents the integer 1 or 2, preferably 1 ;

with the exception of l-{4-[2-(2,5-difluoro-phenoxy)-ethoxy]-phenyl}-piperazine- 2-carboxylic acid benzyl-cyclopropyl-amide and l-{4-[2-(2,5-difluoro-phenoxy)- ethoxy] -phenyl }-piperazine-2-carboxy lie acid (2-chloro-benzyl)-cyclopropyl- amide;

and optically pure enantiomers, mixtures of enantiomers such as racemates, diastereomers, mixtures of diastereomers, diastereomeric racemates, mixtures of diastereomeric racemates, and meso-forms, as well as salts and solvates of such compounds, and morphological forms.

The general terms used hereinbefore and hereinafter preferably have, within this disclosure, the following meanings, unless otherwise indicated:

Where the plural form is used for compounds, salts, pharmaceutical compositions, diseases and the like, this is intended to mean also a single compound, salt, or the like.

Any reference to a compound of formula (I) is to be understood as referring also to optically pure enantiomers, mixtures of enantiomers such as racemates, diastereomers, mixtures of diastereomers, diastereomeric racemates, mixtures of diastereomeric racemates, and meso-forms, as well as salts (especially pharmaceutically acceptable salts) and solvates (including hydrates) of such compounds, and morphological forms, as appropriate and expedient.

In the definitions of formula (I) - if not otherwise stated - the term alkyl, alone or in combination with other groups, means saturated, straight or branched chain groups with one to seven carbon atoms, preferably one to four carbon atoms, i.e. Ci-C4-alkyl. Examples of alkyl groups are methyl, ethyl, n-propyl, iso-propyl, n- butyl, iso-butyl, sec -butyl, tert-butyl, 2,2-dimethylpropyl, pentyl, hexyl and heptyl. The methyl, ethyl and isopropyl groups are preferred. The term hydroxy-alkyl, alone or in combination with other groups, refers to an HO-R group, wherein R is alkyl. Examples are hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, and CH₃CH(OH)-.

The term alkoxy, alone or in combination with other groups, refers to an R-O- group, wherein R is an alkyl. Examples of alkoxy groups are methoxy, ethoxy, propoxy, iso-propoxy, iso-butoxy, sec-butoxy and tert-butoxy.

The term alkylene, alone or in combination with other groups, means straight or branched divalent chain groups with one to seven carbon atoms, preferably one to four carbon atoms. Examples of alkylene are methylene, ethylene, propylene and butylene.

The term halogen means fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.

The term cycloalkyl, alone or in combination with other groups, means a saturated cyclic hydrocarbon ring system with 3 to 7 carbon atoms, e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. The cyclopropyl group is a preferred group.

The term aryl, alone or in combination with other groups, relates to a phenyl, naphthyl or indanyl group, preferably a phenyl group.

The term heteroaryl, alone or in combination with other groups, means six- membered aromatic rings with one to four nitrogen ring atoms; six-membered aromatic rings with one to three nitrogen ring atoms, wherein said rings are fused to a benzene ring; five-membered aromatic rings with one to three ring heteroatoms independently selected from oxygen, nitrogen and sulfur, wherein said rings are optionally fused to a benzene ring; a tetrazolyl ring; a thiazinyl ring; or a coumarinyl. Examples of such ring systems are furanyl, thienyl, pyrrolyl, pyridinyl, pyrimidinyl, indolyl, quinolinyl, isoquinolinyl, imidazolyl, triazinyl, thiazolyl, isothiazolyl, pyridazinyl, pyrazolyl, oxazolyl, isoxazolyl, benzothienyl, quinazolinyl and quinoxalinyl.

If the term V within the present invention represents an asymmetric bivalent group, such a group may be connected in both possible ways to the group W and U of a compound of formula (I) unless indicated otherwise. In a preferred embodiment of the invention the beginning part of the V groups -0-CH₂-Q-, -CH₂-CH₂-A-, and -CH₂-CH₂-CH₂-A- is linked to the group W of a compound of formula (I) (that means that for example the -CH₂ part of -CH₂-CH₂-A- is linked to the group W of a compound of formula (I)).

The group U preferably represents tri- or tetra- substituted phenyl wherein the substituents are independently selected from halogen, methyl and hydroxy-alkyl. Most preferably U represents 2,6-dichloro-4-methyl-phenyl, 2-chloro-3,6- difluoro-phenyl, 2,6-dichloro-4-hydroxymethyl-phenyl, 2,6-dichloro-3-methyl-4- hydroxymethyl-phenyl, 2,6-dichloro-4-(l-hydroxyethyl)-phenyl or 2,6-dichloro-3- methyl-4-(l-hydroxyethyl)-phenyl, especially 2,6-dichloro-4-methyl-phenyl or 2- chloro-3,6-difluoro-phenyl.

The group M preferably represents the radical

wherein t is 2 or 3, X is CH or N, R⁵ is chloro or methyl and R⁶ is hydrogen, chloro, methyl, methoxy or -CF₃. Most preferably t is 2, X is CH or N, R⁵ is chloro and R⁶ is hydrogen. The expression pharmaceutically acceptable salts encompasses either salts with inorganic acids or organic acids like hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, phosphorous acid, nitrous acid, citric acid, formic acid, acetic acid, oxalic acid, maleic acid, lactic acid, tartaric acid, fumaric acid, benzoic acid, mandelic acid, cinnamic acid, palmoic acid, stearic acid, glutamic acid, aspartic acid, methanesulfonic acid, ethanesulfonic acid, ethanedisulfonic acid, p-toluenesulfonic acid, salicylic acid, succinic acid, trifluoroacetic acid, and the like that are non toxic to living organisms or, in case the compound of formula (I) is acidic in nature, with an inorganic base like an alkali or earth alkali base, e.g. sodium hydroxide, potassium hydroxide, calcium hydroxide and the like. For other examples of pharmaceutically acceptable salts, reference can be made to "Salt selection for basic drugs", Int. J. Pharm. (1986), 33, 201-217.

The compounds of the formula (I) contain one or more asymmetric carbon atoms and can be prepared in form of optically pure enantiomers, mixtures of enantiomers such as racemates, diastereomers, mixtures of diastereomers, diastereomeric racemates, mixtures of diastereomeric racemates, or meso-forms.

The present invention encompasses all these forms. Mixtures can be separated in a manner known per se, e.g. by column chromatography, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), or crystallization.

Compounds of the invention also include nitrosated compounds of formula (I) that have been nitrosated through one or more sites such as oxygen (hydroxyl condensation), sulfur (sulfydryl condensation) and/or nitrogen.

The nitrosated compounds of the present invention can be prepared using conventional methods known to one skilled in the art. For example, known methods for nitrosating compounds are described in U.S. Pat. Nos. 5,380,758, 5,703,073, 5,994,294, 6,242,432 and 6,218,417; WO 98/19672; and Oae et al., Org. Prep. Proc. Int., 15(3): 165-198 (1983). A group of preferred compounds of formula (I) is that wherein W is a phenyl, or represents

Another group of preferred compounds of formula (I) is that wherein W is a phenyl.

Another group of also more preferred compounds of formula (I) is that wherein R² represents methyl.

Another group of also more preferred compounds of formula (I) is that wherein R³ represents ethylene or propylene.

A group of preferred compounds of formula (I) is that wherein V is -OCH₂CH₂O-, -CH₂CH₂CH₂O-, or -CH₂CH₂O-.

A group of preferred compounds of formula (I) is that wherein V is -OCH₂Q-.

Another group of also more preferred compounds of formula (I) is that wherein V is -OCH₂Q- and Q is an isoxazolyl or an oxadiazolyl, preferably an isoxazolyl.

A group of preferred compounds of formula (I) is that wherein V represents

W Another group of preferred compounds of formula (I) is that wherein R¹ is cyclopropyl.

Another group of also more preferred compounds of formula (I) is that wherein M represents the radical

wherein t is 2 or 3, X is CH or N, R⁵ is chloro or methyl and R⁶ is hydrogen, chloro, methyl, methoxy or -CF₃.

Another group of also more preferred compounds of formula (I) is that wherein U is 2,6-dichloro-4-methyl-phenyl or 2-chloro-3,6-difluoro-phenyl.

Another group of also more preferred compounds of formula (I) is that wherein m represents the integer 1.

Another group of also more preferred compounds of formula (I) is that wherein n represents the integer 1.

Another group of also more preferred compounds of formula (I) is that wherein the absolute configuration of the chiral center at the piperazine core moiety of formula (I) is R.

In a preferred embodiment the present invention relates to a compound of formula (I), wherein

W represents a phenyl substituted by V in para position;

V represents -0-CH₂-CH₂-O- or -0-CH₂-Q-, wherein Q is an isoxazolyl; U respresents di- or tri- substituted phenyl, wherein the substituents are independently selected from the group consisting of alkyl (especially methyl), halogen and -CF₃;

M represents phenyl which is di- substituted with alkyl, preferably with methyl, and most preferably represents 2,3-dimethyl-phenyl; R¹ represents cyclopropyl; and m and n both represent the integer 1.

The present invention also relates to compounds of formula (I) wherein the meanings of one or more of the substituents and symbols as defined for formula (I), or a preferred embodiment of formula (I), are replaced by their preferred meanings as defined herein, such as those defined for the above-given preferred compounds.

Especially preferred compounds of formula (I) are selected from the group consisting of:

(i?)-l-{4-[3-(2,6-dichloro-phenyl)-isoxazol-5-ylmethoxy]-phenyl}-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(i?)-l-{4-[3-(2-chloro-6-fluoro-phenyl)-isoxazol-5-ylmethoxy]-phenyl}-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(i?)-l-{4-[3-(2-chloro-4-fluoro-phenyl)-isoxazol-5-ylmethoxy]-phenyl}-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(i?)-l-{4-[3-(2,4-dichloro-phenyl)-isoxazol-5-ylmethoxy]-phenyl}-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(i?)-l-{4-[3-(4-fluoro-2-trifluoromethyl-phenyl)-isoxazol-5-ylmethoxy]-phenyl}- 6-oxo-piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide, (^-1-(4-[3-(2-ChIOrO-S, 6-difluoro-phenyl)-isoxazol-5-ylmethoxy] -phenyl }-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(5>l-{4-[3-(2,6-dichloro-phenyl)-isoxazol-5-ylmethoxy]-phenyl}-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(i?)-l-{4-[2-(2,6-dichloro-4-methyl-phenoxy)-ethoxy]-phenyl}-6-oxo-piperazine- 2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(i?)-l-{4-[2-(2-chloro-3,6-difluoro-phenoxy)-ethoxy]-phenyl}-6-oxo-piperazine- 2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(i?)-l-{4-[2-(2,6-dichloro-4-fluoro-phenoxy)-ethoxy]-phenyl}-6-oxo-piperazine- 2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(i?)-l-{4-[2-(2-chloro-6-fluoro-3-methyl-phenoxy)-ethoxy]-phenyl}-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(i?)-6-oxo- 1 - { 4- [2- (2,4,6-trimethyl-phenoxy)-ethoxy] -phenyl } -piperazine-2- carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(R)- 1 - { 4- [2-(2-chloro-5-fluoro-phenoxy)-ethoxy]-phenyl } -6-oxo-piperazine-2- carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(i?)-l-{4-[2-(2-chloro-4,5-dimethyl-phenoxy)-ethoxy]-phenyl}-6-oxo-piperazine- 2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(R)-6-oxo-l-{4-[2-(2,3,6-trifluoro-phenoxy)-ethoxy]-phenyl}-piperazine-2- carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(i?)-l-{4-[2-(2,6-dichloro-phenoxy)-ethoxy]-phenyl}-6-oxo-piperazine-2- carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide, and (R)- 1 - { 4- [2-(2-chloro-4-methyl-phenoxy)-ethoxy] -phenyl } -6-oxo-piperazine-2- carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide.

The compounds of formula (I) are useful for the treatment and/or prophylaxis of diseases such as or related to hypertension, congestive heart failure, pulmonary hypertension, renal insufficiency, renal ischemia, renal failure, renal fibrosis, cardiac insufficiency, cardiac hypertrophy, cardiac fibrosis, myocardial ischemia, cardiomyopathy, glomerulonephritis, renal colic, complications resulting from diabetes such as nephropathy, vasculopathy and neuropathy, glaucoma, elevated intra-ocular pressure, atherosclerosis, restenosis post angioplasty, complications following vascular or cardiac surgery, erectile dysfunction, hyperaldosteronism, lung fibrosis, scleroderma, anxiety, cognitive disorders, complications of treatments with immunosuppressive agents, and other diseases known to be related to the renin-angiotensin system.

The compounds of formula (I) are especially useful for the treatment and/or prophylaxis of hypertension, congestive heart failure, pulmonary hypertension, renal insufficiency, renal ischemia, renal failure, renal fibrosis, cardiac insufficiency, cardiac hypertrophy, cardiac fibrosis, myocardial ischemia, cardiomyopathy, complications resulting from diabetes such as nephropathy, vasculopathy and neuropathy.

In one embodiment, the invention relates to a method for the treatment and/or prophylaxis of diseases, which are associated with a dysregulation of the renin- angiotensin system, in particular to a method for the treatment or prophylaxis of the above-mentioned diseases, said methods comprising administering to a patient a pharmaceutically active amount of a compound of formula (I).

A further aspect of the present invention relates to pharmaceutical compositions comprising a compound of formula (I) and a pharmaceutically acceptable carrier material. These pharmaceutical compositions may be used for the treatment and/or prophylaxis of the above-mentioned diseases. The pharmaceutical compositions can be used for enteral, parenteral, or topical administration. They can be administered, for example, perorally, e.g. in the form of tablets, coated tablets, dragees, hard and soft gelatine capsules, solutions, emulsions or suspensions, rectally, e.g. in the form of suppositories, parenterally, e.g. in the form of injection solutions or infusion solutions, or topically, e.g. in the form of ointments, creams or oils.

The invention also relates to the use of a compound of formula (I) for the preparation of pharmaceutical compositions for the treatment and/or prophylaxis of the above-mentioned diseases.

The production of the pharmaceutical compositions can be effected in a manner which will be familiar to any person skilled in the art (see for example Mark Gibson, Editor, Pharmaceutical Preformulation and Formulation, IHS Health Group, Englewood, CO, USA, 2001; Remington, The Science and Practice of Pharmacy, 20th Edition, Philadelphia College of Pharmacy and Science) by bringing the described compounds of formula (I) or their pharmaceutically acceptable salts, optionally in combination with other therapeutically valuable substances, into a galenical administration form together with suitable, non-toxic, inert, therapeutically compatible solid or liquid carrier materials and, if desired, usual pharmaceutical adjuvants.

Compounds of formula (I) or the above-mentioned pharmaceutical compositions are also of use in combination with other pharmacologically active compounds such as ACE-inhibitors, neutral endopeptidase inhibitors, aldosterone antagonists, angiotensin II receptor antagonists, endothelin receptors antagonists, vasodilators, calcium antagonists, potassium activators, diuretics, sympatholitics, beta- adrenergic antagonists, alpha- adrenergic antagonists and/or other drugs beneficial for the prevention or the treatment of the above-mentioned diseases such as 1 lbeta-hydroxysteroid dehydrogenase type 1 inhibitors and soluble guanylate cyclase activators. The present invention also relates to pro-drugs of a compound of formula (I) that convert in vivo to the compound of formula (I) as such. Any reference to a compound of formula (I) is therefore to be understood as referring also to the corresponding pro-drugs of the compound of formula (I), as appropriate and expedient.

The preparation of compounds of formula (I) can begin with a 2-chloromalonate derivative as described in Scheme 1, wherein R^a stands for a suitable substituent such as methyl or ethyl. An alkylation with a compound of type A, wherein R^b stands for a modifiable precursor of the V-linker as defined for formula (I), and PG for a suitable protecting group (typically a carbamate), and subsequent removal of the protecting group PG, leads to a diketopiperazine of type B. Reduction and protection with BoC₂O leads to a compound of type C. Oxidation leads to a compound of type D. A compound of type F can be obtained from a compound of type D. Achievement of the U-V group, as defined for formula (I) leads to a piperazine of type G. Deprotection leads to a desired compound of formula (T).

Scheme 1

Cleavage of PG

An enantiomerically pure ketopiperazine H (F. Rϋbsam et al, Tetrahedron, 2000, 56, 8481; Daniel D. Holsworth et al, Bioorg. Med. Chem., 2005, 13, 2657; Powell, N. A.; Ciske, F. L.; Clay, E. C; Cody, W. L.; Downing, D. M.; Blazecka, P. G.; Holsworth, D. D.; Edmunds, J. J., Org. Lett., 2004, 6, 4069) can be used as starting point to prepare a ketopiperazine, as described in Scheme 2. A coupling with an iodoaryl derivative, catalyzed by a transition metal, leads to a compound of type J. Oxidation leads to a compound of type K. A compound of type M can be obtained from a compound of type K. Achievement of the U-V group leads to a compound of type N, then deprotection to a desired compound of formula (I").

Scheme 2

Also, a compound of type H can be reduced directly to a piperazine of type O as described in Scheme 3. A coupling with an iodoaryl leads to compound of type C.

Scheme 3

Other combinations of sequences are always possible, as long as the chemistry allows it. The skilled person in the art shall notice such possibilities as obvious variations of the sequences presented hereby.

An enantiomerically pure compound can be prepared by separation of an intermediate or of a final compound by HPLC, using a chiral column. Otherwise, an enantiomerically pure material can be prepared by enantio selective synthesis.

Examples

Abbreviations (as used herein):

ACE Angiotensin Converting Enzyme

AcOH Acetic acid

Ang Angiotensin aq. aqueous

Boc teTt-Butyloxycarbonyl

BSA Bovine serum albumine

Bu Butyl

DIPEA Diisopropylethylamine

DMF N,N-Dimethylformamide

DMSO Dimethylsulfoxide

EIA Enzyme immunoassay

ELSD Evaporative Light-Scattering Detection

ES+ Electro- spray, positive ionization

Et Ethyl

EtOAc Ethyl acetate

EtOH Ethanol

FC Flash Chromatography h hour(s)

HPLC High Performance Liquid Chromatography

LC-MS Liquid Chromatography - Mass Spectroscopy

MeOH Methanol min minute(s) MS Mass Spectroscopy

NCS N-chlorosuccinimide

OD Optical density org. organic PBS Phosphate Buffered Saline

PG protecting group rt room temperature sat. saturated sol. Solution TBAF Tetrafluoroammonium fluoride trihydrate

TFA trifluoroacetic acid

THF Tetrahydrofuran

TLC Thin Layer Chromatography t_R retention time UV ultra violet

Vis visible

HPLC and LC-MS conditions (if not indicated otherwise):

Analytic: Zorbax 59 SB Aqua column, 4.6 x 50 mm from Agilent Technologies. Eluents: A: acetonitrile; B: H₂O + 0.5% TFA. Gradient: 90% B → 5% B over 2 min. Flow: 1 mL/min. Detection: UV/Vis + MS.

Preparative: Zorbax SB Aqua column, 20 x 500 mm from Agilent Technologies.

Eluent: A: Acetonitrile; B: H₂O + 0.05% ammonium hydroxide (25% aq.).

Gradient: 80% B → 10% B over 6 min. Flow: 40 mL/min. Detection: UV + MS, or UV + ELSD.

Chiral analytic: Regis Whelk column, 4.6 x 250 mm, 10 μm. Eluent A: EtOH +

0.05% Et₃N. Eluent B: hexane. Isocratic conditions, 60% B, over 40 min, 1 mL/min. The isocratic mixture may vary, depending on the compounds.

Chiral, preparative: As analytical conditions, but on a Regis Whelk 01 column, 50x250 mm and a flow of 100 mL/min.

All t_R are given in min. 4-Flu or o-2-tr iflu or omethyl-b enzaldehyde oxime

4-Fluoro-2-trifluoromethyl-benzaldehyde (5 g; 26 mmol) was dissolved in CH₃CN (44 mL). NaHCO₃ (6.55 g; 78.1 mmol) was added, and the reaction mixture was stirred for 5 min followed by the addition of water (44 mL), hydroxylamine hydrochloride (3.61 g; 52.1 mmol), and tetra-butyl ammoniumchloride (361 mg, 1.3 mmol). Stirring at rt was continued for 90 min, followed by careful addition of glacial AcOH (5 mL) to reach a pH between 6 and 7. The layers were separated, and the aq. phase was extracted with Et₂O (3x). The combined org. extracts were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude material was purified by FC (CH₂Cl₂) to give the title compound (4.96 g, 46%). LC-MS: t_R = 0.88; ES+: 208.13.

5-(?ert-Butyl-dimethyl-silanyloxymethyl)-3-(4-flu or o-2-tr iflu or omethyl- phenyl)-isoxazole To a sol. of NCS (3.2 g; 23.95 mmol) and pyridine (0.38 mL) in DMF (48 mL) was added dropwise a solution of 4-fluoro-2-trifluoromethyl-benzaldehyde oxime (4.96 g; 23.95 mmol) in DMF (14.4 mL). Stirring was continued at rt for 1 h, followed by the dropwise addition of a solution of teτt-butyl-dimethyl-prop-2- ynyloxy-silane (1.63 g; 9.58 mmol) in DMF (7.6 mL). The reaction mixture was heated to 85 ⁰C, and a sol. of Et₃N (3.33 mL; 23.95 mmol) in DMF (19 mL) was slowly added. Stirring at 85 ⁰C was continued for 15 min. The reaction mixture was cooled to rt, diluted with water and extracted with heptane (2x). The combined org. extracts were washed with water and brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by FC (EtOAc/heptane 1:4) to give the title compound (2.33 g, 26%). LC-MS: t_R = 1.16; ES+: 376.22.

[3-(4-FIu or o-2-tr ifluoromethyl-phenyl)-isoxazol-5-yl]-methanol To a sol. of 5-(rm-butyl-dimethyl-silanyloxymethyl)-3-(4-fluoro-2- trifluoromethyl-phenyl)-isoxazole (2.33 g; 6.21 mmol) in MeOH (20 mL) was added TBAF (1.78 g; 6.82 mmol), and stirring at rt was continued for 3 h followed by the addition of aq. sat. NaHCO₃ (50 mL). The reaction mixture was concentrated under reduced pressure to remove the MeOH, and the remaining aq. layer was extracted with EtOAc (2x). The combined org. layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by FC (EtOAc/heptane 3:7) to give the title compound (1.38 g, 85%). LC-MS: t_R = 0.86; ES+: 262.08.

2-Chloro-4-fluoro-benzaldehyde oxime

Prepared as 4-fluoro-2-trifluoromethyl-benzaldehyde oxime, from 2-chloro-4- fluoro-benzaldehyde. LC-MS: t_R = 0.84; [M+H]⁺ = 174.6.

5-(?ert-Butyl-dimethyl-silanyloxymethyl)-3-(2-chloro-4-fluoro-phenyl)- isoxazole

Prepared as 5-(?ert-butyl-dimethyl-silanyloxymethyl)-3-(4-fluoro-2- trifluoromethyl-phenyl)-isoxazole, from 2-chloro-4-fluoro-benzaldehyde oxime. LC-MS: t_R = 1.18; ES+: 343.12.

[3-(2-Chloro-4-fluoro-phenyl)-isoxazol-5-yl]-methanol

Prepared as [3-(4-fluoro-2-trifluoromethyl-phenyl)-isoxazol-5-yl]-methanol, from 5-(?ert-butyl-dimethyl-silanyloxymethyl)-3-(2-chloro-4-fluoro-phenyl)-isoxazole. LC-MS: t_R = 0.83; ES+: 228.05.

5-(?ert-Butyl-dimethyl-silanyloxymethyl)-3-(2-chloro-6-fluoro-phenyl)- isoxazole

Prepared as 5-(?ert-butyl-dimethyl-silanyloxymethyl)-3-(4-fluoro-2- trifluoromethyl-phenyl)-isoxazole, from 2-chloro-6-fluoro-benzaldehyde oxime. LC-MS: t_R = 1.15; ES+: 342.18.

[3-(2-Chloro-6-fluoro-phenyl)-isoxazol-5-yl]-methanol

Prepared as [3-(4-fluoro-2-trifluoromethyl-phenyl)-isoxazol-5-yl]-methanol, from 5-(?ert-butyl-dimethyl-silanyloxymethyl)-3-(2-chloro-6-fluoro-phenyl)-isoxazole. LC-MS: t_R = 0.81; ES+: 228.06. 5-(tm-Butyl-dimethyl-silanyloxymethyl)-3-(2,6-dichloro-phenyl)-isoxazole Prepared as 5-(rm-butyl-dimethyl-silanyloxymethyl)-3-(4-fluoro-2- trifluoromethyl-phenyl)-isoxazole, from 2,6-dichloro-benzaldehyde oxime. LC- MS: t_R = 1.16; ES+: 358.17.

[3-(2,6-Dichloro-phenyl)-isoxazol-5-yl]-methanol

Prepared as [3-(4-fluoro-2-trifluoromethyl-phenyl)-isoxazol-5-yl]-methanol, from 5-(?ert-butyl-dimethyl-silanyloxymethyl)-3-(2,6-dichloro-phenyl)-isoxazole. LC- MS: t_R = 0.84; ES+: 244.02.

5-(?ert-Butyl-dimethyl-silanyloxymethyl)-3-(2,4-dichloro-phenyl)-isoxazole Prepared as 5-(rm-butyl-dimethyl-silanyloxymethyl)-3-(4-fTuoro-2- trifluoromethyl-phenyl)-isoxazole, from 2,6-dichloro-benzaldehyde oxime. LC- MS: t_R = 1.20; ES+: 358.14.

[3-(2,4-Dichloro-phenyl)-isoxazol-5-yl]-methanol

Prepared as [3-(4-fluoro-2-trifluoromethyl-phenyl)-isoxazol-5-yl]-methanol, from 5-(?er?-butyl-dimethyl-silanyloxymethyl)-3-(2,4-dichloro-phenyl)-isoxazole. LC- MS: t_R = 0.88; ES+: 243.99.

(5')-4-(4-Benzyloxy-phenyl)-3-hydr oxymethyl-5-oxo-piper azine- 1 -car b oxylic acid tert-butyl ester (J l) l-Benzyloxy-4-iodo-benzene (3.68 g; 11.65 mmol) and (5)-3-hydroxymethyl-5- oxo-piperazine-1-carboxylic acid tert-butyl ester (Tetrahedron, 2000, 56, 8481; 3.22 g; 13.98 mmol) were dissolved in degassed DMF (75 mL) under inert atmosphere, followed by addition of K₃PO₄ (4.95 g; 23.3 mmol), copper(I)-iodide (111 mg; 0.58 mmol) and (ii?,2i?)-(-)-l,2-diaminocyclohexane (133 mg; 1.16 mmol). The reaction mixture was heated to 90 ⁰C for 20 h, concentrated under reduced pressure and EtOAc (250 mL) was added to the residue. The sol. was washed with aq. 12.5% ammonia (250 mL), dried over MgSO₄, filtered, and the solvents were removed under reduced pressure. Purification of the crude by FC (EtOAc) yielded the title compound (1.96 g, 40%) as a slightly brown powder. LC-MS: t_R = 0.93; ES+: 413.31.

(i?)-4-(4-Benzyloxy-phenyl)-3-hydroxymethyl-5-oxo-piperazine-l-carboxylic acid tert-butyl ester (J 2)

(i?)-3-Hydroxymethyl-5-oxo-piperazine-l-carboxylic acid tert-butyl ester (Org. Lett, 2004, 6, 4069; 10 g, 43.4 mmol) and l-benzyloxy-4-iodobenzene (11.43 g, 36.1 mmol) were dissolved in DMF (250 mL), followed by the addition of K₃PO₄ (15.3 g, 72.3 mmol) and CuI (344 mg, 1.8 mmol). The mixture was degassed with N₂, and (ii?,2i?)-(-)-l,2-diaminocyclohexane (413 mg; 3.6 mmol) was added. The mixture was heated to 90 ⁰C in an inert atmosphere for 72 h, cooled to rt, filtered, and concentrated under reduced pressure. The residue was dissolved in EtOAc, washed with aq. 12.5% ammonia (2x), dried over MgSO₄, filtered, and the solvents were removed under reduced pressure. The residue was dissolved in as little hot propan-2-ol as possible, and Et₂O was added. The sol. was slowly cooled to 4 ⁰C. The crystallized product was filtered off and dried under high vacuum to give the title compound (9.55 g, 64%). LC-MS: t_R = 0.93; ES+: 413.08.

(5')-4-(4-Benzyloxy-phenyl)-5-oxo-piperazine-l,3-dicarboxylic acid 1-tert- butyl ester (Kl)

Compound J l (1.95 g; 4.73 mmol) was dissolved in DMF (25 mL), followed by the addition of pyridinium dichromate (6.08 g, 16.17 mmol). Stirring at rt was continued for 20 h. Water (500 mL) was added to the reaction mixture. The product was extracted with EtOAc (3x). The combined org. extracts were dried over MgSO₄, filtered, and the solvents were removed under reduced pressure.

Purification of the crude product by FC (EtOAc / hexane 2:1) yielded the title compound (1.51 g, 75%). LC-MS: t_R = 0.92; ES+: 427.08.

(i?)-4-(4-Benzyloxy-phenyl)-5-oxo-piperazine-l,3-dicarboxylic acid 1-tert- butyl ester (K2) In an inert atmosphere compound J 2 (538 mg, 1.3 mmol) was dissolved in CH₂Cl₂ (20 mL), and the sol. was cooled to 0 ⁰C. 2,2,6, 6-Tetramethylpiperidine 1-oxyl radical (2 mg, 0.013 mmol), aq. sat. NaHCO₃ (5.28 mL), KBr (15.5 mg, 0.13 mmol), and tetra-butylammonium chloride (18 mg, 0.065 mmol) were added, followed by the dropwise addition of a mixture of aq. NaOCl (10%, 3 mL), aq. sat. NaHCO₃ (1.4 mL), and brine (3 mL) within 5 min. Stirring at 0 ⁰C was continued for 45 min, followed by the addition of a sol. of sodium thiosulfate (0.6 g) in water (5 mL). The pH of the reaction mixture was adjusted to 11-12 by the addition of aq. 2M NaOH (ca. 1 mL), and the layers were separated. The aq. layer was acidified by the addition of aq. IM HCl (ca. 2.5 mL), upon which a white precipitate formed. The mixture was extracted with Et₂O (4x 35 mL). The combined org. extracts were dried over MgSO₄. Activated charcoal (0.3 g) was added and filtered off over Celite, and the solvents were removed under reduced pressure to give the title compound (371 mg, 67%). LC-MS: t_R = 0.93; ES+: 427.31.

(,S')-4-(4-Benzyloxy-phenyl)-3-[cyclopropyl-(2,3-dimethyl-benzyl)-carbamoyl]-

5-oxo-piperazine-l-carboxylic acid tert-butyl ester (Ml)

Compound Kl (1.2 g, 2.81 mmol) was suspended in CH₂Cl₂ (15 mL) and 1- chloro-N,N,2-trimethylpropenylamine (395 mg, 2.955 mmol) was added. Stirring was continued for 20 min at rt, followed by the addition of a sol. of cyclopropyl- (2,3-dimethyl-benzyl)-amine hydrochloride (prepared by reductive amination of 2,3-dimethylbenzaldehyde and cyclopropylamine; 596 mg, 2.81 mmol) and DIPEA (727 mg, 5.63 mmol) in CH₂Cl₂ (15 mL) within 20 min at rt. Stirring was continued for 1 h. The reaction mixture was diluted with CH₂Cl₂, subsequently washed with aq. 10% citric acid (250 mL), and aq. sat. NaHCO₃. The org. layer was dried over MgSO₄, filtered, and the solvents were removed under reduced pressure. Purification of the residue by FC (hexane/EtOAc 2:1) yielded the title compound (763 mg, 46 %). LC-MS: t_R = 1.11; ES+: 584.47.

(S)-3-[Cyclopropyl-(2,3-dimethyl-benzyl)-carbamoyl]-4-(4-hydroxy-phenyl)- 5-oxo-piperazine-l-carboxylic acid tert-butyl ester (M2) In an autoclave purged with N₂, Pd(OH)₂ (250 mg) was suspended in EtOH (10 mL), followed by the addition of compound Ml (910 mg, 1.559 mmol) dissolved in EtOH (10 mL). AcOH (5 mL) was added, and the mixture was hydrogenated overnight at rt (5 bar H₂-pressure). The reaction mixture was filtered over Celite, and the solvents were removed under reduced pressure. Purification of the residue by FC (EtOAc) yielded the title compound (528 mg, 68 %). LC-MS: t_R = 0.95; ES+: 494.18.

(i?)-4-(4-Benzyloxy-phenyl)-3-[cyclopropyl-(2,3-dimethyl-benzyl)- carbamoyl]-5-oxo-piperazine-l-carboxylic acid tert-butyl ester (M3)

Compound K2 (3 g, 7.035 mmol) was dissolved in CH₂Cl₂ (45 mL) in an inert atmosphere at rt, and l-chloro-N,N,2-trimethylpropenylamine (1.05 g, 7.84 mmol) was added. Stirring was continued for 20 min, followed by the slow addition (20 min) of a sol. of cyclopropyl-(2,3-dimethyl-benzyl)-amine (1.49 g, 7.035 mmol) and DIPEA (1.83 g, 14.22 mmol) in CH₂Cl₂ (45 mL). Stirring was continued for 10 min. The reaction mixture was washed with water (3x), aq. 10% citric acid (2x) and water again (2x). The org. layer was dried over MgSO₄, filtered, and the solvents were removed under reduced pressure. Purification of the residue by FC (EtOAc/hexane 1:1) yielded the title compound (3.75 g, 91 %). LC-MS: t_R = 1.11; ES+: 584.47.

(i?)-3-[Cyclopropyl-(2,3-dimethyl-benzyl)-carbamoyl]-4-(4-hydroxy-phenyl)-

5-oxo-piperazine-l-carboxylic acid tert-butyl ester (M4)

Pd(OH)₂ (20% on carbon, ca. 50% water, 688 mg) was suspended in EtOH (40 mL) and AcOH (100%, 20 mL). Compound M3 (3.75 g, 6.42 mmol) was added, and the mixture was stirred at rt under hydrogen pressure (5 bar) for 18 h. The mixture was filtered off over Celite, and the filtrate was concentrated under reduced pressure. The residue was dissolved in EtOAc, washed with aq. sat.

NaHCO₃ and water (2x). The org. layer was dried over MgSO₄, filtered, and the solvents were removed under reduced pressure to give the title compound (2.62 g,

82%). LC-MS: t_R = 0.95; ES+: 494.18. (i?)-4-{4-[2-(tert-Butyl-dimethyl-silanyloxy)-ethoxy]-phenyl}-3-[cyclopropyl- (2,3-dimethyl-benzyl)-car b amoyl] -5-oxo-piper azine- 1 -car b oxylic acid tert- butyl ester (M5)

Compound M4 (1.33 g, 2.69 mmol) and 2-(teτt-butyl-dimethyl-silanyloxy)- ethanol (475 mg, 2.69 mmol) were dissolved in a mixture of toluene (13 mL) and

DIPEA (0.7 mL). Azodicarboxylic dipiperidide (906 mg 3.59 mmol) and PBu₃

(85%, 1.27 g, 6.3 mmol) were added. The reaction mixture was heated to 80 ⁰C for 30 min, cooled to rt, and hexane (40 mL) was added. The precipitate was filtered off. The filtrate was concentrated under reduced pressure. Purification of the residue by FC (CH₂Cl₂) yielded the title compound (1.14 g, 64%). LC-MS: t_R

= 1.20; ES+: 652.53.

(i?)-3-[Cyclopropyl-(2,3-dimethyl-benzyl)-carbamoyl]-4-[4-(2-hydroxy- ethoxy)-phenyl]-5-oxo-piperazine-l-carboxylic acid tert-hλxty\ ester (M6) Compound M5 (1.14 g, 1.75 mmol) was dissolved in THF (20 mL) and TBAF (541 mg 1.75 mmol) was added. Stirring at rt was continued for 20 min. The mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (80 mL) and washed with brine (5x). The org. layer was dried over MgSO₄, filtered, and the solvents were removed under reduced pressure to give the title compound (913 mg, 97%). LC-MS: t_R = 0.95; ES+: 538.47.

Examples

Example 1 (i?)-l-{4-[3-(2,6-Dichloro-phenyl)-isoxazol-5-ylmethoxy]-phenyl}-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide Compound M4 (70 mg, 0.142 mmol) and [3-(2,6-dichloro-phenyl)-isoxazol-5-yl]- methanol (52 mg, 0.213 mmol) were dissolved in toluene (1.5 mL). Azodicarboxylic dipiperidide (73 mg, 0.284 mmol) and PBu₃ (85%, 84 mg, 0.355 mmol) were added. The reaction mixture was heated to 110 ⁰C for 2 h, cooled to rt, and EtOAc (2 mL) was added. The precipitate was filtered off. The filtrate was concentrated under reduced pressure, and the residue was purified by preparative HPLC. The purified intermediate was dissolved in dioxane (0.5 mL), and HCl (4M in dioxane, 0.5 mL) was added. Stirring was continued for 2 h. The solvents were evaporated under reduced pressure to give the title compound (32 mg, 34%). LC-MS: t_R = 0.91; ES+: 619.20.

Example 2

(i?)-l-{4-[3-(2-Chloro-6-fluoro-phenyl)-isoxazol-5-ylmethoxy]-phenyl}-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide Compound M4 (70 mg, 0.142 mmol) and [3-(2-chloro-6-fluoro-phenyl)-isoxazol- 5-yl]-methanol (48 mg, 0.213 mmol) were dissolved in toluene (1.5 mL). Azodicarboxylic dipiperidide (73 mg, 0.284 mmol) and PBu₃ (85%, 84 mg, 0.355 mmol) were added. The reaction mixture was heated to 110 ⁰C for 2 h, cooled to rt, and EtOAc (2 mL) was added. The precipitate was filtered off. The filtrate was concentrated under reduced pressure and the residue was purified by preparative HPLC. The purified intermediate was dissolved in dioxane (0.5 mL), and HCl (4M in dioxane, 0.5 mL) was added. Stirring was continued for 2 h. The solvents were evaporated under reduced pressure to give the title compound (41 mg, 45%). LC-MS: t_R = 0.90; ES+: 603.31.

Example 3

(i?)-l-{4-[3-(2-Chloro-4-fluoro-phenyl)-isoxazol-5-ylmethoxy]-phenyl}-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide Compound M4 (70 mg, 0.142 mmol) and [3-(2-chloro-4-fluoro-phenyl)-isoxazol- 5-yl]-methanol (48 mg, 0.213 mmol) were dissolved in toluene (1.5 mL). Azodicarboxylic dipiperidide (73 mg, 0.284 mmol) and PBu₃ (85%, 84 mg, 0.355 mmol) were added. The reaction mixture was heated to 110 ⁰C for 2 h, cooled to rt, and EtOAc (2 mL) was added. The precipitate was filtered off. The filtrate was concentrated under reduced pressure, and the residue was purified by preparative HPLC. The purified intermediate was dissolved in dioxane (0.5 mL), and HCl in dioxane (4M in dioxane, 0.5 mL) was added. Stirring was continued for 2 h. The solvents were evaporated under reduced pressure to give the title compound (24 mg, 26%). LC-MS: t_R = 0.91; ES+: 603.28. Example 4

(R)-I- {4-[3-(2,4-Dichloro-phenyl)-isoxazol-5-ylmethoxy]-phenyl}-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide Compound M4 (70 mg, 0.142 mmol) and [3-(2,4-dichloro-phenyl)-isoxazol-5-yl]- methanol (52 mg, 0.213 mmol) were dissolved in toluene (1.5 mL). Azodicarboxylic dipiperidide (73 mg, 0.284 mmol) and PBu₃ (85%, 84 mg, 0.355 mmol) were added. The reaction mixture was heated to 110 ⁰C for 2 h, cooled to rt, and EtOAc (2 mL) was added. The precipitate was filtered off. The filtrate was concentrated under reduced pressure, and the residue was purified by preparative HPLC. The purified intermediate was dissolved in dioxane (0.5 mL), and HCl (4M in dioxane, 0.5 mL) was added. Stirring was continued for 2 h. The solvents were evaporated under reduced pressure to give the title compound (29 mg, 23 %). LC-MS: t_R = 0.93; ES+: 619.30.

Example 5

(R)-I- {4-[3-(4-Flu or o-2-triflu or omethyl-phenyl)-isoxazol-5-ylmethoxy]- phenyl}-6-oxo-piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl- benzyl)-amide Compound M4 (70 mg, 0.142 mmol) and [3-(4-fluoro-2-trifluoromethyl-phenyl)- isoxazol-5-yl] -methanol (56 mg, 0.213 mmol) were dissolved in toluene (1.5 mL). Azodicarboxylic dipiperidide (73 mg, 0.284 mmol) and PBu₃ (85%, 84 mg, 0.355 mmol) were added. The reaction mixture was heated to 110 ⁰C for 2 h, cooled to rt, and EtOAc (2 mL) was added. The precipitate was filtered off. The filtrate was concentrated under reduced pressure, and the residue was purified by preparative HPLC. The purified intermediate was dissolved in dioxane (0.5 mL), and HCl (4M in dioxane, 0.5 mL) was added. Stirring was continued for 2 h. The solvents were evaporated under reduced pressure to give the title compound (34 mg, 36%). LC-MS: t_R = 0.92; ES+: 637.37. Example 6

(5>l-{4-[3-(2-Chloro-3,6-difluoro-phenyl)-isoxazol-5-ylmethoxy]-phenyl}-6- oxo-piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide Compound M2 (50 mg, 0.101 mmol), [3-(2-chloro-3,6-difluoro-phenyl)-isoxazol- 5-yl]-methanol (WO 2006/064484; 37 mg, 0.15 mmol), and azodicarboxylic dipiperidide (51 mg, 0.202 mmol) were dissolved in toluene (15 mL), followed by the addition of PBu₃ (85%, 60.2 mg, 0.25 mmol). The reaction mixture was heated to 100 ⁰C for 90 min, cooled to rt and Et₂O (3 mL) was added. The precipitate was filtered off and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC. The intermediate compound was dissolved in dioxane (0.5 mL), followed by the addition of HCl (4M in dioxane, 0.5 mL). Stirring was continued for 60 min. The reaction mixture was concentrated under reduced pressure to yield the title compound (34.6 mg, 52%). LC-MS: t_R = 0.90; ES+: 621.31.

Example 7

(S)-I- {4- [3-(2,6-Dichlor o-phenyl)-isoxazol-5-ylmethoxy] -phenyl }-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide

As described in Example 6, compound M2 (50 mg; 0.101 mmol) was reacted with [3-(2,6-dichloro-phenyl)-isoxazol-5-yl]-methanol (37 mg; 0.15 mmol) to give, after Boc-deprotection, the title compound (41.9 mg, 63%). LC-MS: t_R = 0.91; ES+: 619.29.

Example 8 (i?)-l-{4-[2-(2,6-Dichloro-4-methyl-phenoxy)-ethoxy]-phenyl}-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide Compound M6 (76 mg, 0.142 mmol) was dissolved in toluene (1.5 mL), followed by the addition of 2,6-dichloro-4-methyl-phenol (37.3 mg, 0.213 mmol), azodicarboxylic dipiperidide (73 mg; 0.284 mmol) and PBu₃ (85%, 85 mg, 0.355 mmol). The reaction mixture was heated to 110 ⁰C for 2 h, cooled to rt, and EtOAc (3 mL) was added. The precipitate was filtered off, the filtrate concentrated under reduced pressure, and the residue was purified by preparative HPLC. The intermediate compound was dissolved in dioxane (0.5 mL), and HCl (4M in dioxane, 0.5 mL) was added. The reaction mixture was stirred for 16 h and concentrated in vacuo to give the title compound (19 mg, 21%). LC-MS: tR = 0.94; ES+: 596.33.

Example 9

(i?)-l-{4-[2-(2-Chloro-3,6-difluoro-phenoxy)-ethoxy]-phenyl}-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide According to example 8, from compound M6 (76 mg, 0.142 mmol) and 2-chloro- 3,6-difluoro-phenol (35.4 mg; 0.213 mmol), the title compound (47 mg, 53%) was obtained. LC-MS: t_R = 0.91; ES+: 584.37.

Example 10

(i?)-l-{4-[2-(2,6-Dichloro-4-fluoro-phenoxy)-ethoxy]-phenyl}-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide

According to example 8, from compound M6 (76 mg, 0.142 mmol) and 2,6- dichloro-4-fluoro-phenol (39 mg, 0.213 mmol), the title compound (23 mg, 25 %) was obtained. LC-MS: t_R = 0.93; ES+: 599.88.

Example 11

(i?)-l-{4-[2-(2-Chloro-6-fluoro-3-methyl-phenoxy)-ethoxy]-phenyl}-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide According to example 8, from compound M6 (76 mg, 0.142 mmol) and 2-chloro- 6-fluoro-3-methyl-phenol (34.5 mg, 0.213 mmol), the title compound (28 mg, 32%) was obtained. LC-MS: t_R = 0.92; ES+: 580.36.

Example 12

(i?)-6-Oxo-l-{4-[2-(2,4,6-trimethyl-phenoxy)-ethoxy]-phenyl}-piperazine-2- carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide According to example 8, from compound M6 (76 mg, 0.142 mmol) and 2,4,6- trimethyl-phenol (29 mg; 0.213 mmol), the title compound (39 mg, 46%) was obtained. LC-MS: t_R = 0.94; ES+: 556.45. Example 13

(R)-I- {4-[2-(2-Chloro-5-flu or o-phenoxy)-ethoxy]-phenyl}-6-oxo-piperazine-2- carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide According to example 8, from compound M6 (76 mg, 0.142 mmol) and 2-chloro- 5-fluoro-phenol (31.5 mg, 0.213 mmol), the title compound (34 mg, 39%) was obtained. LC-MS: t_R = 0.91; ES+: 566.39.

Example 14 (i?)-l-{4-[2-(2-Chloro-4,5-dimethyl-phenoxy)-ethoxy]-phenyl}-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide

According to example 8, from compound M6 (76 mg, 0.142 mmol) and 2-chloro-

4,5-dimethyl-phenol (34 mg, 0.213 mmol), the title compound (44 mg, 50%) was obtained. LC-MS: t_R = 0.94; ES+: 576.36.

Example 15

(i?)-6-Oxo-l-{4-[2-(2,3,6-trifluoro-phenoxy)-ethoxy]-phenyl}-piperazine-2- carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide

According to example 8, from compound M6 (76 mg, 0.142 mmol) and 2,3,6- trifluoro-phenol (31.5 mg, 0.213 mmol), the title compound (34 mg, 39%) was obtained. LC-MS: t_R = 0.90; ES+: 568.42.

Example 16

(i?)-l-{4-[2-(2,6-Dichloro-phenoxy)-ethoxy]-phenyl}-6-oxo-piperazine-2- carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide

According to example 8, from compound M6 (76 mg, 0.142 mmol) and 2,6- dichloro-phenol (35 mg, 0.213 mmol), the title compound (26 mg, 29 %) was obtained. LC-MS: t_R = 0.92; ES+: 582.23.

Example 17

(i?)-l-{4-[2-(2-Chloro-4-methyl-phenoxy)-ethoxy]-phenyl}-6-oxo-piperazine- 2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide According to example 8, from compound M6 (76 mg, 0.142 mmol) and 2-chloro- 4-methyl-phenol (31 mg, 0.213 mmol), the title compound was obtained (23 mg, 27%). LC-MS: t_R = 0.92; ES+: 562.28.

Biological Assays

1. Enzyme immuno assay (EIA) to estimate Angl accumulation and renin inhibition

1.1 Preparation of Angl-BSA conjugate

1.3 mg (1 μmol) of Angl [1-10 (Bachem, H-1680)] and 17 mg (0.26 μmol) of BSA (Fluka, 05475) were dissolved in 4 mL of 0.1M phosphate buffer, pH 7.4, after which 2 mL of a 1:100 dilution of glutaraldehyde in H₂O (Sigma G-5882) was added dropwise. The mixture was incubated overnight at 4 ⁰C, then dialyzed against 2 liters of 0.9% NaCl, twice for 4 h at rt, followed by dialysis against 2 liters of PBS IX overnight at rt. The solution was then filtered with a Syringe filter, 0.45 μm (Nalgene, Cat. No. 194-2545). The conjugate can be stored in polypropylene tubes in 0.05% sodium azide at 4 ⁰C for at least 12 months.

1.2 Preparation of BSA- Angl coated MTP

Micro titer plates (MPT384, MaxiSorpTM_; N_unc) were incubated overnight at 4 ⁰C with 80 μl of Angl (l-10)/BSA conjugate, diluted l:100'000 in PBS IX in a teflon beaker (exact dilution dependent on batch of conjugate), emptied, filled with 90 μl of blocking solution [0.5% BSA (Sigma A-2153) in PBS IX, 0.02% NaN₃], and incubated for at least 2 h at rt, or overnight at 4 ⁰C. 96 well MTP (MaxiSorp™, Nunc) were coated with 200 μl conjugate and blocked with 250 μl blocking solution as above, except that the blocking solution contained 3% BSA. The plates can be stored in blocking solution at 4 ⁰C for 1 month.

1.3 Angl-EIA in 384 well MTP

The Angl (l-10)/BSA coated MTP were washed 3 times with wash buffer (PBS IX, 0.01% Tween 20) and filled with 75 μl of primary antibody solution (anti- Angl antiserum, pre-diluted 1:10 in horse serum), diluted to a final concentration of lilOO'OOO in assay buffer (PBS IX, ImM EDTA, 0.1% BSA, pH 7.4). 5 μl of the renin reaction (or standards in assay buffer) (see below) were added to the primary antibody solution and the plates were incubated overnight at 4 ⁰C. After the incubation the plates were washed 3 times with wash buffer and incubated with secondary antibody [anti-rabbit IgG, linked to horseradish peroxidase (Amersham Bioscience, NA 934V), diluted l:2'000 in wash buffer] for 2 h at rt. The plates were washed 3 times with wash buffer and then incubated for 1 h at rt with substrate solution [1.89mM ABTS (2.2'-azino-di-(3-ethyl- benzthiazolinsulfonate)] (Roche Diagnostics, 102 946) and 2.36mM H₂O₂ [30%, (Fluka, 95300] in substrate buffer (0.1M sodium acetate, 0.05M sodium dihydrogen phosphate, pH 4.2). The OD of the plate was read at 405 nm in a microplate reader (FLUOStar Optima from BMG). The production of Angl during the renin reaction was quantified by comparing the OD of the sample with the OD of a standard curve of Angl(l-lO), measured in parallel.

2. Primary renin inhibition assay: IC₅₀ in buffer, 384 well MTP

The renin assay was adapted from an assay described before (Fischli W. et ah,

Hypertension, 1991, 18:22-31) and consists of two steps: in the first step, recombinant human renin is incubated with its substrate (commercial human tetradecapeptide renin substrate) to create the product Angiotensin I (Angl). In the second step, the accumulated Angl is measured by an immunological assay (enzyme immuno assay, EIA). The detailed description of this assay is found below. The EIA is very sensitive and well suited for renin activity measurements in buffer or in plasma. Due to the low concentration of renin used in this assay (2 fmol per assay tube or 10 pM) it is possible to measure inhibitor affinities in this primary assay down to low pM concentration.

2.1 Methodology Recombinant human renin (3 pg/μl) in assay buffer (PBS IX, 1 mM EDTA, 0.1% BSA, pH 7.4), human tetradecapeptide (1-14) substrate (Bachem, M-1120) [5 μM in 10 mM HCl], hydroxyquinoline sulfate (Fluka, 55100) [3OmM in H₂O] and assay buffer were premixed at 4 ⁰C at a ratio of 100:30:10:145. 47.5 μl per well of this premix was transferred into polypropylene plates (MTP384, Nunc). Test compounds were dissolved and diluted in 100% DMSO and 2.5 μl added to the premix, then incubated at 37 ⁰C for 3 h. At the end of the incubation period, 5 μl of the renin reaction (or standards in assay buffer) were transferred into EIA assays (as described above) and Angl produced by renin was quantified. The percentage of renin inhibition (Angl decrease) was calculated for each concentration of compound and the concentration of renin inhibition was determined that inhibited the enzyme activity by 50% (IC50).

Examples of inhibition:

Claims

1. A compound selected from the group consisting of piperazine compounds of the formula (I)

wherein

V represents -NH-R³-, -N(CH₃)-R³-, -NH-R³-O-, -N(CH₃)-R³-O-, -0-CH₂-Q-, -CH₂-CH₂-CH₂-, -CH₂-A-CH₂-, -CH₂-CH₂-A-, -CH₂-CH₂-CH₂-CH₂-, -CH₂-CH₂- A-CH₂-, -CH₂-CH₂-CH₂-A-, -A-CH₂-CH₂-B-, -CH₂-CH₂-CH₂-CH₂-CH₂-, -CH₂- CH₂-A-CH₂-CH₂-, -CH₂-CH₂-CH₂-A-CH₂-, -CH₂-CH₂-CH₂-CH₂-A-, -A-CH₂- CH₂-CH₂-B-, -CH₂-A-CH₂-CH₂-B-, -CH₂-N(R²)-CH₂-, or -CH₂-CH₂-N(R²)-CH₂-; or V represents a heterocyclic group selected from

U represents aryl that is optionally mono-, di-, tri-, or tetra- substituted with substituents independently selected from the group consisting of halogen, alkyl, -CF₃, -OCF₃, methoxy, and hydroxy-alkyl; or heteroaryl that is optionally mono-, di-, or tri- substituted with substituents independently selected from the group consisting of halogen, alkyl, -CF₃, -OCF₃, methoxy, and hydroxy-alkyl;

M represents aryl or pyridinyl that is optionally mono-, di-, or tri-substituted, wherein the substituents are independently selected from the group consisting of halogen, alkyl, -CF₃, -OCF₃, alkoxy, alkyl-O-(CH₂)₀-₄-CH₂-, alkyl-O-(CH₂)₂_₄-O-, and R⁴ ₂N-(CH₂)o-₄-CH₂-;

Q represents a five-membered heteroaryl with two or three ring heteroatoms independently selected from nitrogen and oxygen;

A and B independently represent -O- or -S-;

R¹ represents cycloalkyl;

R² represents alkyl, cycloalkyl, or CF₃CH₂-;

R³ represents alkylene;

m is the integer O or 1 ; and n represents the integer 1 or 2;

with the exception of l-{4-[2-(2,5-difluoro-phenoxy)-ethoxy]-phenyl}-piperazine- 2-carboxylic acid benzyl-cyclopropyl-amide and l-{4-[2-(2,5-difluoro-phenoxy)- ethoxy] -phenyl } -piperazine-2-carboxylic acid (2-chloro-benzyl)-cyclopropyl- amide; and optically pure enantiomers, mixtures of enantiomers such as racemates, diastereomers, mixtures of diastereomers, diastereomeric racemates, mixtures of diastereomeric racemates, and meso-forms, as well as salts and solvates of such compounds, and morphological forms.

2. A compound according to claim 1, wherein W is a phenyl, or represents

3. A compound according to claim 2, wherein W is a phenyl.

4. A compound according to any one of claims 1 to 3, wherein R² represents methyl.

5. A compound according to any one of claims 1 to 3, wherein R³ represents ethylene or propylene.

6. A compound according to any one of claims 1 to 3, wherein V is -OCH₂CH₂O- , -CH₂CH₂CH₂O-, or -CH₂CH₂O-.

7. A compound according to any one of claims 1 to 3, wherein V is -OCH₂Q-.

8. A compound according to claim 7, wherein Q is an isoxazolyl or an oxadiazolyl.

9. A compound according to any one of claims 1 to 3, wherein V represents

W

10. A compound according to any one of claims 1 to 9, wherein R¹ is cyclopropyl.

11. A compound according to any one of claims 1 to 10, wherein M represents the radical

12. A compound according to any one of claims 1 to 11, wherein U is 2,6- dichloro-4-methyl-phenyl or 2-chloro-3,6-difluoro-phenyl.

13. A compound according to any one of claims 1 to 12, wherein m represents the integer 1.

14. A compound according to any one of claims 1 to 13, wherein n represents the integer 1.

15. A compound according to any one of claims 1 to 14, wherein the absolute configuration of the chiral center at the piperazine core moiety of formula (I) is R.

16. A compound according to claim 1, selected from the group consisting of:

(i?)-l-{4-[3-(4-fluoro-2-trifluoromethyl-phenyl)-isoxazol-5-ylmethoxy]-phenyl}- 6-oxo-piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(^-1-(4-[3-(2-ChIOrO-S, 6-difluoro-phenyl)-isoxazol-5-ylmethoxy] -phenyl }-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(i?)-l-{4-[2-(2,6-dichloro-4-fluoro-phenoxy)-ethoxy]-phenyl}-6-oxo-piperazine- 2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide, (i?)-l-{4-[2-(2-chloro-6-fluoro-3-methyl-phenoxy)-ethoxy]-phenyl}-6-oxo- piperazine-2-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide,

(i?)-l-{4-[2-(2,6-dichloro-phenoxy)-ethoxy]-phenyl}-6-oxo-piperazine-2- carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide, and

(R)- 1 - { 4- [2-(2-chloro-4-methyl-phenoxy)-ethoxy] -phenyl } -6-oxo-piperazine-2- carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide.

17. A pharmaceutical composition comprising a compound according to any one of claims 1 to 16 and a pharmaceutically acceptable carrier material.

18. A compound according to any one of claims 1 to 16, or composition according to claim 17, for use as a medicament.

19. Use of a compound according to any one of claims 1 to 16 for the preparation of a pharmaceutical composition for the treatment and/or prophylaxis of diseases selected from hypertension, congestive heart failure, pulmonary hypertension, renal insufficiency, renal ischemia, renal failure, renal fibrosis, cardiac insufficiency, cardiac hypertrophy, cardiac fibrosis, myocardial ischemia, cardiomyopathy, glomerulonephritis, renal colic, complications resulting from diabetes such as nephropathy, vasculopathy and neuropathy, glaucoma, elevated intra-ocular pressure, atherosclerosis, restenosis post angioplasty, complications following vascular or cardiac surgery, erectile dysfunction, hyperaldosteronism, lung fibrosis, scleroderma, anxiety, cognitive disorders, complications of treatments with immunosuppressive agents, and other diseases known to be related to the renin-angiotensin system.