WO2007034406A1 - Pyrrolidine-3-carboxylic acid amide derivatives and their use as inhibitors of renin - Google Patents

Abstract

The invention relates to novel pyrrolidine-3-carboxylic acid amide derivatives and their use as active ingredients in the preparation of pharmaceutical compositions. The invention also concerns related aspects including processes for the preparation of these novel compounds, pharmaceutical compositions comprising such compounds and especially the use of such compounds as inhibitors of renin.

Description

PYRROLIDINE-S-CARBOXYLIC ACID AMIDE DERIVATIVES AND THEIR USE AS INHIBITORS

OF RENIN

5 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.

10 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 comprising a compound of formula (I) and especially their use as renin inhibitors in cardiovascular events and renal insufficiency.

15

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

20 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

25 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,

30 403; 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). ACE inhibitors do not inhibit Chymase. 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-pep tide 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)

wherein

W is a phenyl ring (preferred) or a six-membered, non benzofused aromatic ring with one or two nitrogen ring atoms, wherein said rings are substituted by V in para position;

V represents a bond, -(CH₂V, -(CH₂)_S-A-, -CH₂-A- (CH₂)_r, -(CH₂)₂-A-(CH₂)_U-, -A-(CH₂)_V-B-, -CH₂-CH₂-CH₂-A-CH₂-, -A-CH₂-CH₂-B-CH₂-, -CH₂-CH₂-CH₂-A-

CH₂-CH₂-, -CH₂-CH₂-CH₂-CH₂-A-CH₂-, -A-CH₂-CH₂-B-CH₂-CH₂-, -CH₂-A-

CH₂-CH₂-B-CH₂-, -CH₂-A-CH₂-CH₂-CH₂-B-, -O-CH₂-CH(OCH₃)-CH₂-O-, -O-

CH₂-CH(CH₃)-CH₂-O-, -O-CH₂-CH(CF₃)-CH₂-O-, -O-CH₂-C(CH₃)₂-CH₂-O-, -O-

CH₂-C(CHg)₂-O-, -O-CH₂-CH(CH₃)-O-, -O-CH₂-C(CH₂CH₂)-O-, or -0-CH₂-L-, or V represents the heterocyclic group

, wherein this heterocyclic group is bound to the group W of formula (I) via its nitrogen ring atom;

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

L 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;

U represents unsubstituted aryl; mono-, di-, tri- or tetra- substituted aryl wherein the substituents are independently selected from the group consisting of halogen, alkyl, alkoxy, and -CF₃; or mono-, di-, or tri- substituted heteroaryl wherein the substituents are independently selected from the group consisting of halogen, alkyl, alkoxy, and -CF₃;

Q represents methylene or ethylene, preferably methylene;

M represents an aryl, quinolinyl, isoquinolinyl, dihydroquinolinyl or tetrahydroquinolinyl group wherein said groups can optionally be mono- or di- substituted with substituents independently selected from the group consisting of alkyl; alkoxy; -OCF₃; -CF₃; hydroxy- alkyl; halogen; alkyl-O-(CH₂)₀-₄-CH₂-; alkyl-O-(CH₂)₂_₄-O-; and R'₂N-(CH₂)₀-₄-CH₂-, wherein R' is independently selected from the group consisting of hydrogen, alkyl, cyclopropyl, and -C(=O)- R" wherein R" is C₁-C₄-alkyl, -CF₃, -CH₂-CF₃ or cyclopropyl;

R¹ represents alkyl or cycloalkyl, preferably cycloalkyl such as especially cyclopropyl;

r is the integer 3, 4, 5, or 6; s is the integer 2, 3, 4, or 5; t is the integer 1, 2, 3, or 4; u is the integer 1, 2, or 3; and v is the integer 2, 3, or 4;

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

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, relates to a phenyl, naphthyl or indanyl group, preferably a phenyl group.

The term heteroaryl, alone or in combination, 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). In a preferred embodiment of the invention the beginning part of an asymmetric group V is linked to the group W of a compound of formula (I) (that means that for example the -(CH₂)_S part of -(CH₂)_S-A- is linked to the group W of a compound of formula (I)).

Salts are preferably the pharmaceutically acceptable salts of the compounds of formula (I).

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, ethane sulfonic 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 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 M represents aryl (preferably phenyl), which is especially optionally mono- or di- substituted with substituents independently selected from the group consisting of methyl, methoxy, -CF₃, -(CH₂)₂_₃OCH₃, and halogen.

A further group of preferred compounds of formula (I) is that wherein Q is methylene.

Another group of preferred compounds of formula (I) is that wherein V is -OCH₂CH₂O- or especially -CH₂CH₂CH₂O- [preferably wherein the -CH₂- part of -CH₂CH₂CH₂O- is linked to the group W of formula (I)].

An additional group of preferred compounds of formula (I) is that wherein V represents -0-CH₂-L-, wherein L represents isoxazolyl.

A further group of preferred compounds of formula (I) is that wherein U is a mono-, di-, tri- or tetra- substituted aryl (preferably a mono-, di-, or tri- substituted phenyl), wherein the substituents are independently selected from the group consisting of halogen and methyl.

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

An additional group of preferred compounds of formula (I) is that wherein R¹ is cyclopropyl.

A further group of preferred compounds of formula (I) is that with an (i?)-absolute configuration at the 3-position of the pyrrolidine core structure of formula (I), and an (^-absolute configuration at the 4-position of the pyrrolidine core structure of formula (I).

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.

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

W is a phenyl ring, substituted by V in para position;

V represents -A-(CH₂)_V-B-;

A and B are both -O- ; U represents tri- substituted phenyl, wherein the substituents are independently selected from alkyl (preferably methyl) and halogen;

Q represents methylene;

M represents di- substituted phenyl, wherein the substituents are independently selected from alkyl, preferably from methyl; R¹ is cycloalkyl, preferably cyclopropyl; and v is the integer 2.

An especially preferred compound of formula (I) is:

(3R, 45')-4-{4-[2-(2,6-dichloro-4-methyl-phenoxy)-ethoxy]-phenyl}-pyrrolidine-3- 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.

A compound of formula (I) can be prepared as described in Scheme 1. Starting from a dihydropyrrolyl derivative protected with a suitable protecting group PG (that may be changed along the synthesis) a compound of type A is prepared typically using any kind of Michael addition, often catalyzed by a transition metal. A subsequent amide coupling leads to a compound of type B, then removal of the protecting group to a compound of formula (I). Scheme 1

B

Example

Abbreviations (as used herein):

ACE Angiotensin Converting Enzyme

Ang Angiotensin aq. aqueous

BINAP 2,2'-Bis-diphenylphosphanyl-5,6,7,8,5',6',7',8'-octahydro-

[1,1 ']binaphthalenyl

BSA Bovine serum albumine

BuLi n-Butyllithium cod 1 ,5-cyclooctadiene

DIPEA Diisopropylethylamine

DMAP 4-N,N-Dimethylaminopyridine

DMSO Dimethylsulfoxide

EDC HC] [ Ethyl-NN-dimethylaminopropylcarbodiimide hydrochloride

EDTA Ethylenediamine tetraacetate

EIA Enzyme immunoassay

ELSD Evaporative Light-Scattering Detection

ES Electro spray

Et Ethyl

EtOAc Ethyl acetate

EtOH Ethanol

FC Flash Chromatography h hour(s)

HOBt Hydroxybenzotriazol

HPLC High Performance Liquid Chromatography

LC-MS Liquid Chromatography - Mass Spectroscopy

MeOH Methanol min minute(s)

MS Mass Spectroscopy

OD Optical density org. organic

PBS Phosphate Buffered Saline

PG protecting group

Ph Phenyl

Rf Retention value (on TLC) rt room temperature sol. Solution

TFA Trifluoroacetic acid

THF Tetrahydrofuran

TLC Thin Layer Chromatography

Rt 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.

2-(4-Bromophenoxy)ethanol

To a sol. of 4-bromophenol (40 g, 231 mmol) in EtOH (140 mL) was added NaOH (10.2 g, 254 mmol). The resulting mixture was stirred at 70 ⁰C for 30 min until the whole amount of NaOH had dissolved. A sol. of 2-bromoethanol (17.3 mL, 231 mmol) in EtOH (40 mL) was added dropwise at 7O⁰C. The sol. rapidly turned milky. The mixture was heated to reflux overnight. The solvents were removed under reduced pressure, and the residue was dissolved in EtOAc. The mixture was washed with water and brine, dried over MgSO₄, filtered, and the solvents were removed under reduced pressure. Purification of the crude by FC (EtO Ac/heptane 1:5 → 1:4 → 1:3 → 1:2 → EtOAc) yielded the title compound (39.2 g, 78%) as a pale brown oil that crystallized when placed at -18 ⁰C. R_f = 0.3 in (EtO Ac/heptane 1:1).

l-Bromo-4-(2-iodoethoxy)benzene

To a sol. of compound 2-(4-bromophenoxy)ethanol (39.2 g, 181 mmol) in dry toluene (500 mL) was added imidazole (61.5 g, 903 mmol), PPh₃ (90 g, 343 mmol), and iodine (87.1 g, 343 mmol). This mixture was stirred at 60 ⁰C for 2 h. The mixture was filtered over Celite, and the filtrate was concentrated under reduced pressure. Purification of the residue by FC (EtO Ac/heptane 1:5 → 1:4 → 1:3 → 1:2 → 1:1) yielded the title compound (39.9 g, 67%).

l-Bromo-4-[2-(2,6-dichloro-4-methylphenoxy)ethoxy]benzene In a 500-mL 3-neecked flask equipped with a mechanical stirrer and a reflux condenser were mixed compound l-bromo-4-(2-iodoethoxy)benzene (39.9 g, 122 mmol) and 2,6-dichloro-/?-cresol (21.6 g, 122 mmol) in dry acetone (1200 mL). To this mixture was added K₂CO₃ (16.86 g, 122 mmol), and the resulting suspension was heated to reflux for 20 h. The reaction mixture was concentrated under reduced pressure, the residue was dissolved in EtOAc and this org. phase was washed with water (2x), and with brine (Ix). The org. layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. Purification of the residue by a short FC (EtO Ac/heptane 1:9) yielded the title compound (44.4 g, 96%).

4-[2-(2,6-Dichloro-4-methylphenoxy)ethoxy]phenylboronic acid To a sol. of l-bromo-4-[2-(2,6-dichloro-4-methylphenoxy)ethoxy]benzene (2.25 g, 6.0 mmol) in THF (15 mL) at -78 °C and under Ar, was added BuLi (1.5M in hexane, 4.40 mL, 6.6 mmol). After being stirred for 20 min at -78 ⁰C, the sol. was transferred to a solution of triisopropylborate (6.90 mL, 30 mmol) in THF (20 mL) at -78⁰C. After 15 min at -78 ⁰C, the sol. was poured on cold aq. 4M HCl (45 mL, 180 mmol), and stirred for 10 min. The solution was extracted with EtOAc (3x). The combined org. layers were washed with brine, dried over Na₂SO₄, filtered, and the solvents were removed under reduced pressure. The solid was recrystallized in EtOAc to give the title compound (1.6 g, 78%). LC- MS: R₁ = 0.99 min, ES+ = not seen.

(3R, 45')-l-Benzyl-4-{4-[2-(2,6-dichloro-4-methylphenoxy)ethoxy]phenyl} pyrrolidine-3-carboxylic acid tert-butyl ester (Al) Prepared according to the procedure described by K. M. Belik et al. {Tetrahedron Letters, 2004, 45, 3265) from l-benzyl-2,5-dihydro-lH-pyrrole-3-carboxylic acid tert-butyl ester (Martin, C. et al. J. Org. Chem., 2001, 66, 3797; 0.96 g, 3.70 mmol), 4-[2-(2,6-dichloro-4-methylphenoxy)ethoxy]phenylboronic acid (3.0 g, 8.80 mmol), (R)-BINAP (0.23 g, 0.37 mmol) and [Rh(OH) (cod)] ₂ (Uson, R., et al., Inorg. Synth., 1985, 23, 129; 0.049 mg, 0.09 mmol) in THF (25 mL) and water (0.48 mL). The brown oil was not purified but used directly in the next step. LC- MS: R_t = 1.02 min, ES+=556.42.

(JR , 4S)- 1 -Benzyl-4- {4- [2-(2,6-dichlor o-4-methylphenoxy)ethoxy]phenyl }- pyrrolidine-3-carboxylic acid (A2)

TFA (2.27 mL, 29.6 mmol) was added to a sol. of compound Al (2.06 g, 3.7 mmol) in 1,2-dichloroethane (8 mL) and heated under reflux for 2 h. Aq. IM NaOH was added at 20 ⁰C until pH=7. The layers were separated, and the aq. layer was extracted with CH₂Cl₂. The combined org. extracts were washed with brine, dried over MgSO₄, filtered, and the solvents were removed under reduced pressure. The gummy solid was triturated and washed with EtOAc/heptane 1/1 to give the title compound (1.3 g, 70%). LC-MS: R₁ = 0.91 min, ES+ = 502.16.

(3R , 4S)- 1 -Benzyl-4- {4- [2-(2,6-dichlor o-4-methylphenoxy)ethoxy]phenyl } pyrrolidine-3-carboxylic acid cyclopropyl-(2,3-dimethylbenzyl)amide (B) A sol. of compound A2 (0.113 g, 0.23 mmol), (2,3- dimethylbenzyl)cyclopropylamine (prepared by reductive amination of 2,3- dimethylbenzaldehyde and cyclopropylamine, 0.119 g, 0.68 mmol), DMAP (0.007 g, 0.06 mmol), DIPEA (0.157 mL, 0.90 mmol), HOBt (0.043 g, 0.28 mmol) and

EDC-HCl (0.065 g, 0.34 mmol) in CH₂Cl₂ (4 mL) was stirred for 5 h. The mixture was diluted with EtOAc, washed with water (2x) and brine. The org. phase was dried over MgSO₄, filtered, and the solvents were removed under reduced pressure. Purification of the residue by FC (CH₂Cl₂/Me0H 95/5) yielded the title compound (0.044 g, 30%). LC-MS: R₁ = 1.05 min, ES+=657.37.

Example 1

(3R, 45>4-{4-[2-(2,6-Dichloro-4-methyl-phenoxy)-ethoxy]-phenyl}- pyrrolidine-3-carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide

To a sol. of compound B (0.044 g, 0.067 mmol) in 1,2-dichloroethane (1 mL) in a vial under Ar was added NaHCO₃ (0.056 mg, 0.067 mmol) and 1- chloroethylchloroformate (0.074 mL, 0.067 mmol). The suspension was shaken for 5 min at rt and then 3 h at 80 ⁰C. The reaction mixture was filtered, washed with CH₂Cl₂ and the solvents were removed under reduced pressure. MeOH (1 mL) was added and the solution was heated at 60 ⁰C for 1 h. The solvents were removed under reduced pressure, and the residue was purified by HPLC (H₂O, MeOH, NH₄OH) to yield the title product (0.016g, 42%). LC-MS: R_t = 0.97 min, ES+=567.34; chiral HPLC: R₁ = 25.8 min (only one peak seen).

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 Microtiter plates (MPT384, MaxiSorpTM, Niαnc) 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 l:100'000 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, ImM 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) [30 mM 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). The compounds of formula (I) exhibit IC50 values between 0.1 nM to 300 nM, especially between 1 nM to 30 nM.

Examples of inhibition:

Compound of Example 1: IC50 = 3.9 nM

Claims

Claims

1. A compound selected from the group consisting of pyrrolidine-3-carboxylic acid amides of the formula (I)

M

wherein

W is a phenyl ring or a six-membered, non benzofused aromatic ring with one or two nitrogen ring atoms, wherein said rings are substituted by V in para position;

V represents a bond, -(CH₂V, -(CH₂VA-, -CH₂-A-(CH₂V, -(CH₂)₂-A-(CH₂)_U-, -A-(CH₂)_V-B-, -CH₂-CH₂-CH₂-A-CH₂-, -A-CH₂-CH₂-B-CH₂-, -CH₂-CH₂-CH₂-A- CH₂-CH₂-, -CH₂-CH₂-CH₂-CH₂-A-CH₂-, -A-CH₂-CH₂-B-CH₂-CH₂-, -CH₂-A- CH₂-CH₂-B-CH₂-, -CH₂-A-CH₂-CH₂-CH₂-B-, -O-CH₂-CH(OCH₃)-CH₂-O-, -O- CH₂-CH(CH₃)-CH₂-O-, -O-CH₂-CH(CF₃)-CH₂-O-, -O-CH₂-C(CH₃)₂-CH₂-O-, -O- CH₂-C(CHg)₂-O-, -O-CH₂-CH(CH₃)-O-, -O-CH₂-C(CH₂CH₂)-O-, or -0-CH₂-L-, or V represents the heterocyclic group

₅ wherein this heterocyclic group is bound to the group W of formula (I) via its nitrogen ring atom; A and B independently represent -O- or -S-;

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

U represents unsubstituted aryl; mono-, di-, tri- or tetra- substituted aryl wherein the substituents are independently selected from the group consisting of halogen, alkyl, alkoxy, and -CF₃; or mono-, di-, or tri- substituted heteroaryl wherein the substituents are independently selected from the group consisting of halogen, alkyl, alkoxy, and -CF₃;

Q represents methylene or ethylene;

M represents an aryl, quinolinyl, isoquinolinyl, dihydroquinolinyl or tetrahydroquinolinyl group wherein said groups can optionally be mono- or di- substituted with substituents independently selected from the group consisting of alkyl; alkoxy; -OCF₃; -CF₃; hydroxy- alkyl; halogen; alkyl-O-(CH₂)₀-₄-CH₂-; alkyl-O-(CH₂)₂_₄-O-; and R'₂N-(CH₂)₀-₄-CH₂-, wherein R' is independently selected from the group consisting of hydrogen, alkyl, cyclopropyl, and -C(=0)- R' ' wherein R' ' is C₁-C₄-alkyl, -CF₃, -CH₂-CF₃ or cyclopropyl;

R¹ represents alkyl or cycloalkyl;

r is the integer 3, 4, 5, or 6; s is the integer 2, 3, 4, or 5; t is the integer 1, 2, 3, or 4; u is the integer 1, 2, or 3; and v is the integer 2, 3, or 4;

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 M represents aryl which is optionally mono- or di- substituted with substituents independently selected from the group consisting of methyl, methoxy, -CF₃, -(CH₂)₂_₃OCH₃, and halogen.

3. A compound according to claim 1 or 2, wherein Q is methylene.

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

5. A compound according to any one of claims 1 to 3, wherein V is -0-CH₂-L-, wherein L is isoxazolyl.

6. A compound according to any one of claims 1 to 5, wherein U is a mono-, di-, or tri-substituted phenyl, wherein the substituents are independently selected from the group consisting of halogen and methyl.

7. A compound according to any one of claims 1 to 6, wherein W is phenyl.

8. A compound according to any one of claims 1 to 7, wherein R¹ is cyclopropyl.

9. A compound according to any one of claims 1 to 8 with an (i?)-absolute configuration at the 3-position of the pyrrolidine core structure of formula (I), and an (^-absolute configuration at the 4-position of the pyrrolidine core structure of formula (I).

10. A compound according to claim 1, wherein

W is a phenyl ring, substituted by V in para position; V represents -A-(CH₂)_V-B-; A and B are both -O- ;

U represents tri-substituted phenyl, wherein the substituents are independently selected from alkyl and halogen; Q represents methylene; M represents di- substituted phenyl, wherein the substituents are independently selected from alkyl; R¹ is cycloalkyl; and v is the integer 2.

11. A compound according to claim 1, which is:

(3R, 45')-4-{4-[2-(2,6-dichloro-4-methyl-phenoxy)-ethoxy]-phenyl}-pyrrolidine-3- carboxylic acid cyclopropyl-(2,3-dimethyl-benzyl)-amide.

12. A pharmaceutical composition comprising a compound according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier material.

13. A compound according to any one of claims 1 to 11, or composition according to claim 12, for use as a medicament.

14. Use of a compound according to any one of claims 1 to 11 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.