JP2008522967A - New diazabicyclononene derivatives - Google Patents

Abstract

  The present invention relates to novel 3,9-diazabicyclo [3.3.1] nonene derivatives and their enantiomers and their use as active ingredients in the manufacture of pharmaceutical compositions. The present invention also relates to a low molecular weight renin inhibitor with non-peptidic properties. Has long-acting effects and activates the tissue renin-chymase system to cause pathophysiologically altered local functions such as renal, heart, and vascular remodeling, atherosclerosis, and possibly restenosis Described are orally active renin inhibitors of formula (I) and (I ′) that are active in indications beyond possible blood pressure regulation.

Description

  The present invention relates to novel compounds of formula (I) and their enantiomers of formula (I '). The present invention also relates to a process for the preparation of compounds, pharmaceutical compositions comprising at least one compound of formula (I) or (I ′), and these as renin inhibitors, especially in cardiovascular events and renal failure. Regarding related aspects including use.

In the renin-angiotensin system (RAS), biologically active angiotensin II (Ang II) is produced by a two-step mechanism. The highly specific enzyme renin cleaves angiotensinogen to angiotensin I (Ang I), which is then further processed into Ang II by the less specific angiotensin converting enzyme (ACE). AngII is known to act on at least two receptor subtypes called AT ₁ and AT _2. AT ₁ appears to transmit most of the known functions of Ang II, but the role of AT ₂ is still unclear.

The regulation of RAS represents a major advance in the treatment of cardiovascular disease. ACE inhibitors and AT ₁ blockers have been approved for the treatment of hypertension (Non-patent document 1; Non-patent document 2). In addition, ACE inhibitors prevent kidney disease (Non-patent document 5; Non-patent document 6) and myocardial infarction (Non-patent document 7) for renal protection (Non-patent document 3; Non-patent document 4). Is used.

The rationale for developing a renin inhibitor is the specificity of renin (Non-patent Document 8). The only known substrate for renin is angiotensinogen, which can only be processed by renin (under physiological conditions). In contrast, ACE can cleave bradykinin in addition to Ang I and can be bypassed by the serine protease chymase (9). Therefore, inhibiting ACE in patients causes bradykinin accumulation, resulting in cough (5-20%) and potentially fatal angioedema (0.1-0.2%) (Non-Patent Literature) 10). Chymase is not inhibited by ACE inhibitors. Thus, Ang II formation can still occur in patients treated with ACE inhibitors. On the other hand, blockade of AT ₁ receptor (eg, by losartan) is associated with other AT receptor subtypes (eg, AT ₂ ) versus Ang II whose concentration is significantly increased by blockade of AT ₁ receptor. Overexpose. In summary, renin inhibitors are expected to exhibit a different pharmaceutical profile than ACE inhibitors and AT ₁ blockers in terms of efficacy in blocking RAS and in safety aspects.

  Because renin inhibitors have insufficient oral activity due to their peptidomimetic properties, only limited clinical experience (Non-Patent Document 11) has been obtained (Non-Patent Document 12). The clinical development of some compounds has been discontinued due to the high price of the product along with this problem. Only one compound containing four chiral centers has entered clinical trials (Non-Patent Document 13; Non-Patent Document 14). Therefore, renin inhibitors with excellent oral bioavailability and long duration of action are needed. Recently, the first non-peptide renin inhibitors that exhibit high in vitro activity have been described (Non-Patent Document 15; Patent Document 1; Non-Patent Document 16). However, the development status of these compounds is unknown.

WO97 / 09311 pamphlet GWaeber 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 Rosenberg M. E. et al., Kidney International, 1994, 45, 403 Breyer J. A. et al., Kidney International, 1994, 45, S156 Vaughan D. E. et al., Cardiovasc. Res., 1994, 28, 159 Fouad-Tarazi F. et al., Am. J. Med., 1988, 84 (Suppl. 3A), 83 Pfeffer M. A. et al., N. Engl. J. Med., 1992, 327, 669 Kleinert H. D., Cardiovasc. Drugs, 1995, 9, 645 Husain A., J. Hypertens., 1993, 11, 1155 Israili Z. H. et al., Annals of Internal Medicine, 1992, 117, 234 Azizi M. et al., J. Hypertens., 1994, 12, 419; Neutel J. M. et al., Am. Heart, 1991, 122, 1094 Kleinert H. D., Cardiovasc. Drugs, 1995, 9, 645 Rahuel J. et al., Chem. Biol., 2000, 7, 493 Mealy N. E., Drugs of the Future, 2001, 26, 1139 Oefner C. et al., Chem. Biol., 1999, 6, 127 Marki H. P. et al., Il Farmaco, 2001, 56, 21

  The present invention relates to a low molecular weight renin inhibitor with non-peptidic properties. Has long-acting effects and activates the tissue renin-chymase system to cause pathophysiologically altered local functions such as renal, heart, and vascular remodeling, atherosclerosis, and possibly restenosis Described are orally active renin inhibitors of formula (I) and (I ′) that are active in indications beyond possible blood pressure regulation.

In particular, the invention relates to novel compounds of structural formula (I) and mixtures of enantiomers such as optically pure enantiomers or racemates; and their pharmaceutically acceptable salts, solvent complexes and morphological forms: 1R *, 5S *)-7- {4- [3- (2-Chloro-3,6-difluorophenoxy) -propyl] phenyl} -3,9-diazabicyclo [3.3.1] non-6-ene-6 -Carboxylic acid cyclopropyl- (2,3-dichlorobenzyl) amide

Preferred enantiomers are those represented by formula (I ′): (1R, 5S) -7- {4- [3- (2-chloro-3,6-difluorophenoxy) propyl] phenyl} -3, 9-Diazabicyclo [3.3.1] non-6-ene-6-carboxylic acid cyclopropyl- (2,3-dichlorobenzyl) amide

  The expression pharmaceutically acceptable salt includes hydrochloric acid or hydrobromic acid, sulfuric acid, phosphoric acid, citric acid, formic acid, acetic acid, maleic acid, tartaric acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid, etc. And salts that are not toxic to living organisms with either inorganic or organic acids.

  The compounds of formula (I) contain two interdependent asymmetric carbon atoms with relative stereochemistry (1R *, 5S *) and are optically pure enantiomers of (1R, 5S) -7- {4- [3- (2-Chloro-3,6-difluorophenoxy) -propyl] phenyl} -3,9-diazabicyclo [3.3.1] non-6-ene-6-carboxylic acid cyclopropyl- (2,3-dichloro (Benzyl) amide (ie, a preferred compound of formula (I ′)) and (1S, 5R) -7- {4- [3- (2-chloro-3,6-difluorophenoxy) -propyl] phenyl} -3, May be prepared in the form of 9-diazabicyclo [3.3.1] non-6-ene-6-carboxylic acid cyclopropyl- (2,3-dichlorobenzyl) amide or as a mixture of two enantiomers such as racemates . The present invention encompasses all of these forms. Mixtures may be separated in a manner known per se, ie by column chromatography, thin layer chromatography, HPLC, crystallization or resolution.

  In particular, tartaric acid can be used to resolve compound 3 (see below). Use of D-(-)-tartaric acid will lead to the desired enantiomer.

  Compounds of formula (I) and (I ′) are compounds associated with dysregulation of the renin-angiotensin system, in particular hypertension, congestive heart failure, pulmonary hypertension, renal failure, renal ischemia, renal failure, renal fibrosis, Complications caused by diabetes such as heart failure, cardiac hypertrophy, cardiac fibrosis, myocardial ischemia, cardiomyopathy, glomerulonephritis, renal colic, nephropathy, vascular disorder and neuropathy, glaucoma, elevated intraocular pressure, atherosclerotic artery Sclerosis, restenosis after angioplasty, complications after vascular or cardiac surgery, erectile dysfunction, hyperaldosteronism, pulmonary fibrosis, scleroderma, anxiety, cognitive impairment, complications treated with immunosuppressants As well as other diseases known to be related to or related to the renin-angiotensin system.

  The compounds of formula (I) and (I ′) are notably hypertensive, congestive heart failure, pulmonary hypertension, renal failure, renal ischemia, renal failure, renal fibrosis, heart failure, cardiac hypertrophy, cardiac fibrosis, myocardial ischemia It is useful for the treatment and / or prevention of complications caused by diabetes such as diabetes, cardiomyopathy, nephropathy, vascular disorders and neurological disorders.

  In one embodiment, the present invention relates to a method for the treatment or prevention of diseases associated with dysregulation of the renin-angiotensin system, in particular to a method for the treatment or prevention of the aforementioned diseases, said method comprising Administering to a patient a pharmaceutically active amount of a compound of I) or (I ′).

  A further aspect of the invention relates to a pharmaceutical composition comprising a compound of formula (I) or (I ') and a pharmaceutically acceptable carrier material. These pharmaceutical compositions may be used for the treatment and / or prevention of the diseases mentioned above. The pharmaceutical composition can be used for enteral, parenteral or topical administration. For example, they can be administered orally, for example, in the form of tablets, coated tablets, dragees, hard and soft gelatin capsules, solutions, emulsions or suspensions, e.g. rectally in the form of suppositories, e.g. in the form of injection solutions or infusions. It can be administered orally or topically, for example in the form of an ointment, cream or oil.

  The invention also relates to the use of a compound of formula (I) or (I ') for the manufacture of a pharmaceutical composition for the treatment and / or prevention of the diseases mentioned above.

  The manufacture of the pharmaceutical composition is in any manner familiar to those skilled in the art (eg, Mark Gibson, Editor, Pharmaceutical Preformulation and Formulation, IHS Health Group, Englewood, CO, USA, 2001; Remington, The Science and Practice of Pharmacy, 20th Edition, see Philadelphia College of Pharmacy and Science), described compounds of formula (I) or (I ') or pharmaceutically acceptable salts thereof, optionally other treatments This can be done in combination with a suitable non-toxic, inert, therapeutically compatible solid or liquid carrier material and, if necessary, a conventional pharmaceutical adjuvant into a galenos dosage form.

  Suitable carrier materials include not only inorganic carrier materials, but also organic carrier materials. Thus, for example, lactose, corn starch or derivatives thereof, talc, stearic acid or its salts can be used as carrier materials for tablets, coated tablets, dragees and hard gelatine capsules. Suitable carrier materials for soft gelatin capsules are, for example, vegetable oils, waxes, fats, and semi-solid and liquid polyols (but depending on the nature of the active ingredient, in the case of soft gelatin capsules the carrier is Not needed). Suitable carrier materials for the production of solutions and syrups are, for example, water, polyols, sucrose, invert sugar and the like. Suitable carrier materials for injection are, for example, water, alcohols, polyols, glycerol and vegetable oils. Suitable carrier materials for suppositories are, for example, natural or hardened oils, waxes, fats and semi-liquid or liquid polyols. Suitable carrier materials for topical formulations are glycerides, semi-synthetic and synthetic glycerides, hydrogenated oils, liquid waxes, liquid paraffins, liquid fatty alcohols, sterols, polyethylene glycols and cellulose derivatives.

  Conventional stabilizers, preservatives, wetting and emulsifying agents, consistency-improving agents, flavor-improving agents, salts for changing osmotic pressure, buffer substances, solubilizers, coloring agents, and masking and antioxidant agents Are also considered as pharmaceutical adjuvants.

  The dosage of the compounds of formulas (I) and (I ′) can vary within wide limits depending on the disease to be controlled, the age and personal condition of the patient, and the mode of administration, of course, each specific Tailored to the individual requirements of the specific case.

  In a preferred embodiment, this amount is comprised between 2 mg and 1000 / mg per day.

  In certain preferred embodiments, this amount is comprised between 1 mg to 500 / mg per day.

  In a particularly more preferred embodiment, this amount is comprised between 5 mg and 200 / mg per day.

  Another aspect of the present invention relates to a process for the manufacture of a pharmaceutical composition comprising a compound of formula (I) or (I '). According to said method, one or more active ingredients of the formula (I) or (I ') are mixed with inert excipients in a manner known per se.

  Also, the compounds of formula (I) and (I ′) or the above-mentioned pharmaceutical composition are ACE-inhibitor, neutral endopeptidase inhibitor, angiotensin II receptor antagonist, endothelin receptor antagonist, vasodilator, calcium antagonist Drugs, potassium activators, diuretics, sympathomimetic drugs, β-adrenergic antagonists, α-adrenergic antagonists and / or aldosterone antagonists, 11β-hydroxysteroid dehydrogenase type 1 inhibitors and soluble guanylate cyclase activation It is useful in combination with other pharmacologically active compounds such as other agents useful for the prevention or treatment of the aforementioned diseases such as factors.

  The present invention also relates to prodrugs of compounds of formula (I) or (I ') that are converted in vivo to compounds of formula (I) or (I') per se. Thus, it is understood that any reference to a compound of formula (I) or (I ′) also refers to the corresponding prodrug of the compound of formula (I) or (I ′), as appropriate and expedient. .

  Compounds of formula (I) and (I ') can be prepared by the methods outlined below, by the methods described in the examples or by analogous methods.

Preparation of compounds of formula (I) and (I ′):
The synthesis of the compounds of formula (I) and (I ′) described herein is one possible synthesis of many other variations. Other synthetic routes and methodologies will be apparent to those skilled in the art.

  Bicyclononane derivative 3 (Scheme 1) can be prepared as a racemate as previously described (WO 2003/093267). Production of vinyl triflate 6 proceeds using sodium hydride and N-phenyl-bis (trifluoromethanesulfonimide). Negishi-coupling between bicyclo 6 and bromophenyl derivative 10 leads to diazabicyclone 7. The bromophenyl derivative 10 is a 1,4-dibromobenzene that undergoes Grignard reaction with allyl bromide (→ compound 8), then hydroboration reaction (→ compound 9), and finally protects the hydroxy substituent as a silyl ether. After that, it is manufactured in 3 steps. Bicyclononane 7 then transprotects another place to bicyclononane 11. Derivative 12 is derived by Mitsunobu type coupling with 2-chloro-3,6-difluorophenol. Saponification of the ethyl ester under strongly basic conditions leads to a mixture of carboxylic acid derivatives 13 and 14.

Scheme 1

The mixture of compounds 13 and 14 is not separated and is further used directly in the amide coupling step with amine derivative 16, which is prepared in one step using reductive amination (Scheme 2). The amide coupling product 15 is obtained. Cleavage of both Boc-protecting groups leads to compounds of formula (I).

Scheme 2

Enantioselective synthesis:
The compounds of the present invention contain two chiral centers, which are not independent of one another. The racemic compounds were derived by the synthetic methods shown so far. Racemates can be separated into compounds of formula (I ′) and its enantiomers using a chiral HPLC-column. Both enantiomers also used enantioselective acylation (Majewski M., Lasny R., J. Org. Chem., 1995, 60, 5825) as described in WO 2003/093267. Thus, it will be selectively produced from a meso-bicyclononane derivative such as compound 17 (Scheme 3). Another variation is the resolution of racemates using chiral organic acid derivatives.

Scheme 3

The following examples serve to illustrate the invention in more detail. However, they are not intended to limit the scope to any manner.

Examples General observations The compounds have been characterized by at least LC-MS and ¹ H-NMR. Only LC-MS data is shown here.
HPLC- or LC-MS conditions (unless otherwise indicated):
Analytical: Zorbax 59 SB Aqua column from Agilent Technologies, 4.6 x 50 mm.
Eluent: A: acetonitrile; B: H ₂ O + 0.5% TFA. Gradient: 90% B to 5% B over 2 minutes. Flow rate: 1 mL / min. Detection: UV / Vis + MS.
For preparation: Zorbax SB Aqua column from Agilent Technologies, 20 x 500 mm.
Eluent: A: acetonitrile; B: H ₂ O + 0.05% ammonium hydroxide (25% aqueous solution).
Gradient: 80% B to 10% B over 6 minutes. Flow rate: 40 mL / min. Detection: UV + MS or UV + ELSD.
Chiral analytical: Regis Whelk column, 4.6 × 250 mm, 10 μm. Eluent A: EtOH + 0.05% Et ₃ N. Eluent B: hexane. Uniform solvent conditions, 60% B over 40 min, 1 mL / min. The homogeneous solvent mixture may vary depending on the compound.
For chiral preparation: Regis Whelk 01 column, 50 x 250 mm and flow rate of 100 mL / min, but the same analytical conditions.
All t _R are given in minutes.

Abbreviations (as used in this specification)
ACE angiotensin converting enzyme
Ang angiotensin
aq. Aqueous
Boc tert-butyloxycarbonyl
BSA bovine serum albumin
Bu Butyl
BuLi n-Butyllithium
DIPEA Diisopropylethylamine
DMAP 4-N, N-dimethylaminopyridine
DMF N, N-dimethylformamide
DMSO Dimethyl sulfoxide
EDC.HCl Ethyl-N, N-dimethylaminopropylcarbodiimide hydrochloride
EIA enzyme immunoassay
Light scattering detection of ELSD vaporization
ES + electrospray, positive ionization
Et ethyl
EtOAc ethyl acetate
EtOH ethanol
FC flash chromatography
h hours
HOBt hydroxybenzotriazole
HPLC high performance liquid chromatography
LC-MS liquid chromatography-mass spectrometry
MeOH methanol
min minutes
MS mass spectrometry
org. organic
Ph phenyl
rt room temperature
sat. saturated
sol. solution
TBDMS tert-butyldimethylsilyl
Tf trifluoromethylsulfonyl
TFA trifluoroacetic acid
THF tetrahydrofuran
t _R retention time
UV UV
Vis Visible.

Compound 6
Compound 3 (WO 2003/093267, 99.58 g, 305 mmol) was dissolved in dry THF (1450 mL) under a nitrogen atmosphere and the mixture was cooled to 0 ° C. NaH (16.64 g; 55% dispersion in mineral oil (381 mmol)) was added in 2 g portions over a period of 35 minutes while maintaining the temperature between 0-4 ° C. H ₂ gas was generated. After the addition, the mixture was yellowish green and slightly suspended. The reaction mixture was stirred at 0-4 ° C. for 75 minutes. Tf ₂ NPh (128.6 g, 360 mmol) was then added as a solid within 5 minutes. The reaction mixture turned brown. The reaction was stirred over the weekend at room temperature, with the cooling bath removed. The reaction mixture was poured onto 1 L ice / water and THF was removed under reduced pressure. The remaining aqueous phase (3 × 500 mL) was extracted with EtOAc. The combined organic layers were washed with water (500 mL) and brine (500 mL). The organic phase was then dried over MgSO ₄ , filtered and evaporated under reduced pressure. To the crude brown residue (174 g), 50 mL of pentane was added and the mixture was stirred at 4 ° C. overnight. The crystals were filtered and washed with a cold mixture of cold hexane (70 mL) and hexane / diethyl ether (4: 1, 100 mL). This yielded 84 g of product containing some TfNHPh. This material was filtered through silica gel (75 g). TfNHPh was washed away with CH ₂ Cl ₂ . The product was then rinsed with EtOAc (3 × 1 L) to give the title compound after evaporation at reduced pressure. The title compound was obtained in 3 fractions: a) 44.45 g of off-white crystals, b) 27.98 g of slightly brown crystals and c) 15 g of yellow oil containing the product and TfNHPh. Two days later, TfNHPh contained in fraction c) crystallized. This was filtered to give 9.43 g of product as a brown oil.

Mother liquor treatment:
The combined mother liquor obtained above was concentrated under vacuum. The brown oil residue (75 g) was purified by FC (1500 g silica gel) using a gradient of (EtOAc / heptane 1: 9 → EtOAc). The column was then washed with EtOAc / MeOH 9: 1. The title compound was isolated as a pure product as 25.44 g of an off-white solid. LC-MS: t _R = 0.87 min; ES +: 459.24.

Compound 7
A solution of compound 10 (102.1 g, 310 mmol) in THF (1.50 L) was cooled to −78 ° C. under nitrogen. BuLi (1.5M hexane solution, 212 mL, 318 mmol) was added. After 30 minutes, ZnCl ₂ (1M THF solution, 465 mL, 465 mmol) was added. The mixture was warmed to room temperature. A solution of compound 6 (83.6 g, 155 mmol) in THF (100 mL) was added followed by Pd (PPh ₃ ) ₄ (4.48 g, 3.87 mmol). The mixture was heated to reflux for 30 minutes and allowed to cool to room temperature. The mixture was diluted with EtOAc and washed with 1M aqueous NaOH (1 ×). The organic extract was dried over MgSO ₄ , filtered and the solvent removed under reduced pressure. Purification of the residue by FC (MeOH / CH ₂ Cl ₂ 1: 49 → 1: 24 → 3: 47 → 2: 23) gave the title product (88.6 g, substantially quantitative). LC-MS: t _R = 0.98 min; ES +: 559.24.

Compound 8
Mg (8.76 g, 360 mmol) was suspended in THF (90 mL) under nitrogen in a three neck flask equipped with a concentrator and addition funnel. The addition funnel was filled with a solution of 1,4-dibromobenzene (77.3 g, 327 mmol) in THF (40 mL). About 5% 1,4-dibromobenzene solution was carefully added to the Mg suspension to initiate the reaction with the help of a heat gun. At the start of the reaction, the 1,4-dibromobenzene solution was added at such a rate that the reaction mixture was gently refluxed (about 20 minutes). The mixture was stirred for an additional 30 minutes and cooled to 0 ° C. THF (100 mL) was added and the addition funnel was filled with a solution of allyl bromide (30.5 mL, 360 mmol) in THF (50 mL). The allyl bromide solution was added slowly while maintaining the reaction temperature below 20 ° C. When the addition was complete, the mixture was stirred for an additional 30 minutes while being cooled to 0 ° C. 1M aqueous HCl was added. The mixture was diluted with Et ₂ O and washed with 1M aqueous HCl and brine. The combined aqueous extracts were extracted back with Et ₂ O. The combined organic extracts were dried over MgSO ₄ , filtered and the solvent removed under reduced pressure. Distillation of the residue (11 mbar, 88-92 ° C.) gave the title compound (39.3 g, ca. 61%) with another unidentified impurity.

Compound 9
BH ₃ (1M THF solution, 412 mL, 412 mmol) was added to a THF (1.00 L) solution of compound 8 (204 g, 1.03 mmol) at 0 ° C. under nitrogen. The mixture was stirred overnight while warming to room temperature. Aqueous NaOH (2.5 M, 1.65 L, 4.12 mol) was added and the mixture was cooled to 0 ° C. H ₂ O ₂ (35%, 480 mL, 5.15 mol) was carefully added dropwise and the mixture was stirred for 3 hours. Et ₂ O was added and the phases were separated. The organic layer was washed with water (1 ×) and brine (1 ×). The organic layer was dried over MgSO ₄ , filtered and the solvent removed under reduced pressure. Purification by FC (Et ₂ O / petroleum ether 1: 1 → Et ₂ O) gave the title compound (97.4 g, 44%).

Compound 10
A solution of compound 9 (49.4 g, 230 mmol) in DMF (500 mL) was cooled to 0 ° C. and imidazole (23.77 g, 349 mmol) and TBDMS-Cl (52.6 g, 349 mmol) were added. The mixture was stirred overnight while warming to room temperature. The mixture was diluted with heptane (1.0 L) and saturated aqueous NH ₄ Cl (800 mL) and the mixture was shaken. The layers were separated. The aqueous layer was extracted with heptane and the combined organic extracts were washed with brine. The organic extract was dried over MgSO ₄ , filtered and the solvent removed under reduced pressure. Purification by FC (Et ₂ O / heptane 1: 99 → 1: 19) gave the title compound (68.1 g, 90%). LC-MS: t _R = 1.24 min; ES +: not visible.

Compound 11
Compound 7 (32.0 g, 57.3 mmol) was dissolved in dry 1,2-dichloroethane (590 mL). NaHCO ₃ (48.2 g, 573 mmol) and 1-chloroethyl chloroformate (62.5 mL, 573 mmol) were added and the suspension was heated to 80 ° C. After 3 hours, the reaction mixture was allowed to cool to room temperature. The mixture was filtered and the filtrate was evaporated under reduced pressure. The residue was dried under high vacuum for 15 minutes. The product was then diluted in MeOH (400 mL) and the mixture was heated to 50 ° C. for 20 minutes. The reaction mixture was allowed to cool to room temperature and the solvent was removed under reduced pressure. The yellow solid was dried under high vacuum for 1 hour. The solid was dissolved in CH ₂ Cl ₂ (190 mL) and the solution was cooled to 0 ° C. DIPEA (49.1 mL, 287 mmol) and Boc ₂ O (37.5 g, 172 mmol) were added. The reaction mixture was stirred overnight while warming to room temperature. The reaction mixture was diluted with CH ₂ Cl ₂ (110 mL). The organic layer was washed with 1M aqueous HCl (2 × 300 mL) and saturated aqueous NaHCO ₃ (300 mL). The organic layer was dried over MgSO ₄ , filtered and the solvent was evaporated under reduced pressure. Purification of the residue by FC (CH ₂ Cl ₂ / MeOH 100: 0 → 2: 98 → 5: 95) gave the title compound (26.2 g, 86%). LC-MS: t _R = 1.05 min; ES +: 531.33.

Compound 12
Of a mixture of compound 11 (56.0 g, 106 mmol), 2-chloro-3,6-difluorophenol (34.8 g, 211 mmol), azadicarboxylic acid dipiperidine (53.4 g, 211 mmol) and PBu ₃ (85%, 83 mL, 317 mmol) A toluene (1.20 L) solution was heated to reflux under nitrogen for 1 hour. The mixture was allowed to cool to room temperature. The mixture was diluted with EtOAc (2.00 L) and the mixture was washed with 1M aqueous NaOH (2 × 900 mL). The organic extract was dried over MgSO ₄ , filtered and the solvent removed under reduced pressure. Purification of the residue by FC (EtOAc / heptane 1: 19 → 1: 1) gave the title compound (67.5 g, 94%). LC-MS: t _R = 1.24 min; ES +: 617.25.

Compounds 13 and 14
A mixture of 1M NaOH aqueous solution (700 mL) of Compound 12 (67.5 g, 99.6 mmol) and EtOH (1.40 L) was stirred at 80 ° C. overnight. The mixture was partially evaporated under reduced pressure and EtOAc (500 mL) was added. The aqueous phase was acidified with 3M aqueous HCl and the mixture was extracted. The organic layer was separated, dried over MgSO ₄ , filtered and the solvent removed under reduced pressure. The residue was dried under high vacuum to give a 1: 1 mixture of compounds 13 and 14, which was used without further purification (61.8 g, 95%). LC-MS: t _R = 1.12 and 1.14 min; ES +: 649.37.

Compound 15
Compounds 13 and 14 (20.7 g, 32.0 mmol), Compound 16 (17.3 g, 80 mmol), EDC.HCl (18.4 g, 96 mmol), HOBt (5.40 g, 40.0 mmol), DMAP (0.98 g, 8.00 mmol) and DIPEA A mixture of (22.0 mL, 128 mmol) in CH ₂ Cl ₂ (540 mL) was stirred at room temperature. The reaction was checked daily by LC-MS and if the reaction was not complete, EDC.HCl (6.13 g, 32 mmol) and DIPEA (5.5 mL, 32 mmol) were added. When the reaction was complete, the mixture was diluted with more CH ₂ Cl ₂ and washed with 1M aqueous HCl (3 ×) and saturated aqueous NaHCO ₃ (2 ×). The organic layer was dried over MgSO ₄ , filtered and the solvent removed under reduced pressure. Purification by FC gave the title compound (16.5 g, 60%). LC-MS: t _R = 1.29 min; ES +: 848.35.

Compound 16
A mixture of 2,3-dichlorobenzaldehyde (90.0 g, 514 mmol) and cyclopropylamine (72 mL, 1.02 mol) in dry MeOH (1300 mL) was stirred at room temperature overnight. The reaction mixture was cooled to 0 ° C. and NaBH ₄ (25.3 g, 668 mmol) was added. The reaction mixture was again stirred overnight at room temperature. Ice was added to the reaction mixture and the solvent was evaporated under reduced pressure. The residue was dissolved in EtOAc and washed with 1M aqueous NaOH. The aqueous layer was extracted with EtOAc (3x). The combined organic extracts were dried over MgSO ₄ , filtered and the solvent was removed under reduced pressure. Purification of the residue by FC (EtOAc / heptane 9: 1 → 1: 1) gave the title compound (97.4 g, 87%). LC-MS: t _R = 0.64 min; ES +: 216.15.

Compound (I): (rac.)-(1R *, 5S *)-7- {4- [3- (2-Chloro-3,6-difluorophenoxy) -propyl] phenyl} -3,9-diazabicyclo [ 3.3.1] Non-6-ene-6-carboxylic acid cyclo-propyl- (2,3-dichlorobenzyl) amide A solution of compound 15 (16.5 g, 19.4 mmol) in CH ₂ Cl ₂ (100 mL) cooled to 0 ° C. did. HCl (4M dioxane solution, 100 mL) was added. The mixture was stirred at 0 ° C. for 1 hour and then at room temperature for 1 hour. The solvent was removed under reduced pressure and the residue was dried under high vacuum. The residue was diluted with CH ₂ Cl ₂ and washed with 1M aqueous NaOH until the aqueous layer settled basic. The organic extract was dried over MgSO ₄ , filtered and the solvent removed under reduced pressure. Purification of the residue by FC (MeOH / CH ₂ Cl ₂ 5: 95 → 10: 90 → 20: 80) gave the title compound (12.0 g, 95%). LC-MS: t _R = 0.88 min; ES +: 648.36.

Compound (I ′): (1R, 5S) -7- {4- [3- (2-chloro-3,6-difluorophenoxy) propyl] -phenyl} -3,9-diazabicyclo [3.3.1] non- 6-ene-6-carboxylic acid cyclopropyl- (2,3-dichlorobenzyl) amide Compound (I) (1.00 g, 1.55 mmol) in homogeneous solvent conditions (25% EtOH, 0.1% diethylamine, 75% Hexane) and a flow rate of 100 mL / min, purified on a Regis Whelk 01 column, 50 × 250 mm. The desired compound came out as the first enantiomer (t _R = 20.00 min). After evaporating the solvent under reduced pressure, the residue was dried under high vacuum. The residue was dissolved in CH ₂ Cl ₂ and washed with 10% aqueous K ₂ CO ₃ solution. The organic extract was dried over MgSO ₄ , filtered and the solvent removed under reduced pressure. The residue was dried under high vacuum to give the title compound (350 mg).

Biological assay
1. Enzyme immunoassay (EIA) to estimate AngI accumulation and renin inhibition
1.1 Manufacture of AngI-BSA conjugate
1.3 mg (1 μmol) AngI [1-10 (Bachem, H-1680)] and 17 mg (0.26 μmol) BSA (Fluka, 05475) were dissolved in 4 mL 0.1 M phosphate buffer (pH 7.4), 2 mL of a 1: 100 dilution of glutaraldehyde in H ₂ O (Sigma G-5882) was added dropwise. The mixture was incubated overnight at 4 ° C., then dialyzed twice against 2 liters of 0.9% NaCl for 4 hours at room temperature followed by overnight dialysis against 2 liters of PBS 1X at room temperature. . The solution was then filtered through a syringe filter, 0.45 μm (Nalgene, Cat # 194-2545). Conjugates can be stored in 0.05% sodium azide in polypropylene tubes at 4 ° C. for at least 12 months.

1.2 Manufacture of BSA-AngI coated MTP Dilute microtiter plate (MPT384, MaxiSorp ™, Nunc) 1: 100,000 in 1x PBS in Teflon beaker (exact dilution is conjugate Incubate overnight at 4 ° C. with 80 μl of AngI (1-10) / BSA conjugate (depending on the batch of) and emptied into 90 μl blocking solution [0.5% BSA (Sigma A-2153) in PBS Filled with 1 × solution, 0.02% NaN ₃ ] and incubated at room temperature for at least 2 hours or overnight at 4 ° C. 96 well MTP (MaxiSorp ™, Nunc) was coated with 200 μl conjugate and blocked with 250 μl blocking solution as described above, except that the blocking solution contained 3% BSA. . Plates can be stored in blocking solution for 1 month at 4 ° C.

1.3 AngI-EIA in 384-well MTP
AngI (1-10) / BSA coated MTP was washed 3 times with wash buffer (PBS 1 ×, 0.01% Tween 20) and 75 μl primary antibody solution (anti-AngI antiserum, 1: 1 in horse serum). 10 pre-diluted) and diluted to a final concentration of 1: 100,000 in assay buffer (PBS 1X, 1 mM EDTA, 0.1% BSA, pH 7.4). 5 μl renin reaction (or standard in assay buffer) (see below) was added to the primary antibody solution and the plate was incubated overnight at 4 ° C. After incubation, the plate was washed 3 times with wash buffer and at room temperature with secondary antibody [horseradish peroxidase conjugated anti-rabbit IgG (Amersham Bioscience, NA 934V) diluted 1: 2,000 in wash buffer]. Incubated for 2 hours. The plate was washed 3 times with wash buffer, then the substrate solution [1.89 mM ABTS (2.2′-azino-di- (3-ethyl-benzthiazoline sulfonate)] (Roche Diagnostics, 102 946) and 2.36 mM H ₂ O ₂ [30% (Fluka, 95300] substrate buffer (0.1 M sodium acetate, 0.05 M sodium dihydrogen phosphate, pH 4.2) was incubated for 1 hour at room temperature. FLUOStar Optima) at 405 nm The production of AngI during the renin reaction was quantified by comparing the OD of the standard curve of AngI (1-10) measured in parallel with the OD of the sample.

2. Primary renin inhibition assay: IC _{50 in} buffer (384 well MTP)
The renin assay is adapted from a previously described assay (Fischli W. et al., Hypertension, 1991, 18: 22-31) and consists of two steps: in the first step, recombinant human Incubate renin with its substrate (commercial human tetradecapeptide renin substrate) to produce the product angiotensin I (AngI). In the second step, the accumulated AngI is measured by an immunological assay (enzyme immunoassay, EIA). A detailed description of this assay is found below. EIA is very sensitive and is very suitable for the measurement of renin activation in buffer or plasma. Because the renin used in this assay is low (2 fmol tubes or 10 pM per assay), the measurement of inhibitor affinity in this primary assay can be reduced to low pM concentrations.

2.1 Methodology Recombinant human renin (3 pg / μl) assay buffer (PBS 1 ×, 1 mM EDTA, 0.1% BSA, pH 7.4) solution, human tetradecapeptide (1-14) substrate (Bachem, M-1120) [5 μM 10 mM HCl solution), hydroxyquinoline sulfate (Fluka, 55100) [30 mM H ₂ O solution] and assay buffer were premixed at 4 ° C. in a ratio of 100: 30: 10: 145. 47.5 μl of this premix per well was transferred to a polypropylene plate (MTP384, Nunc). Test compounds were dissolved and diluted in 100% DMSO, 2.5 μl was added to the premix and then incubated at 37 ° C. for 3 hours. At the end of the incubation period, 5 μl of the renin reaction (or standard in assay buffer) was transferred to the EIA assay (as described above) to quantify the AngI produced by renin. The percentage of renin inhibition (AngI reduction) was calculated for each concentration of compound, and the concentration of renin inhibition that inhibited enzyme activity by 50% (IC ₅₀ ) was determined. IC ₅₀ values for all compounds tested are below 100 nM. However, the selected compounds exhibit very good bioavailability and are more metabolically stable than the prior art compounds.

The compound of formula (I ′) exhibits an IC ₅₀ value of 0.5 nM.
Hemodynamic measurement (telemetry method)
Animals-Female double transgenic rats with human renin and human angiotensinogen were purchased from RCC Ltd, Fullingsdorf, Switzerland. All animals were maintained under the same conditions and had free access to normal pelleted rat chow and water. Rats were initially treated with enalapril (1 mg / kg / day) for 2 months. Approximately 2 weeks after cessation of enalapril treatment, double transgenic rats become hypertensive and reach mean arterial blood pressure in the range of 160-170 mmHg.

  Transmitter implantation-Rats were anesthetized intraperitoneally with a mixture of 90 mg / kg ketamine-HCl (Ketavet, Parke-Davis, Berlin FRG) and 10 mg / kg xylazine (Rompun, Bayer, Leverkusen, FRG). The pressure transmitter was implanted intraperitoneally under aseptic conditions with a sensing catheter placed in the descending aorta below the renal artery showing upstream. The transmitter was sutured to the abdominal musculature and the skin was closed.

  The telemetering-system-telemetry unit was obtained from Data Sciences (St. Paul, MN). The implanted sensor is a liquid-filled catheter (0.7mm diameter, connected to a highly stable low conductance strain gauge pressure transducer (which measures absolute arterial pressure compared to vacuum) and a high frequency transmitter. Length 8cm; model TA11PA-C40). The tip of the catheter was filled with a viscous gel that prevented blood reflux and coated with an antithrombogenic film to inhibit thrombus formation. The implant (length = 2.5 cm, diameter = 1.2 cm) weighs 9 g and has a typical battery life of 6 months. The receiver platform (RPC-1, Data Sciences) connected the radio signal to a digitized input and sent it to a dedicated personal computer (Compaq, deskpro). Arterial pressure was calibrated by using input from an atmospheric pressure reference (APR-1, Data Sciences). Systolic, mean and diastolic blood pressures were expressed in millimeters of mercury (mmHg).

  Hemodynamic measurements-Double transgenic rats implanted with pressure transmitter were dosed with vehicle or 10 mg / kg test substance by oral gavage (n = 6 per group) and the mean arterial blood pressure was continuously monitored . The effect of the test substance was expressed as the maximum reduction in mean arterial pressure (MAP) in the treatment group versus the control group.

  The compound of formula (I ') is active in this animal model. A single oral dose of 10 mg / kg caused a 26 mm Hg blood pressure decrease and a single 3 mg / kg oral dose caused a 13 mm Hg blood pressure decrease.

Claims (5)

(1R *, 5S *)-7- {4- [3- (2-Chloro-3,6-difluorophenoxy) propyl] phenyl} -3,9-diazabicyclo [3.3.1] non-6-ene-6 A compound of formula (I), designated as -carboxylic acid cyclopropyl- (2,3-dichlorobenzyl) amide, and mixtures of enantiomers such as optically pure enantiomers or racemates; and their pharmaceutically acceptable Salts, solvent complexes and morphological types:
Represented by the formula (I ′) and (1R, 5S) -7- {4- [3- (2-chloro-3,6-difluorophenoxy) propyl] phenyl} -3,9-diazabicyclo- [3.3. 1] Enantiomers of the compound of formula (I) according to claim 1, named as non-6-ene-6-carboxylic acid cyclopropyl- (2,3-dichlorobenzyl) amide, and their pharmaceutically acceptable Salts, solvent complexes and form types
  A pharmaceutical composition comprising the compound according to claim 1 or 2 and a pharmaceutically acceptable carrier substance.   4. A compound according to claim 1 or 2 or a composition according to claim 3 for use as a medicament.   Hypertension, congestive heart failure, pulmonary hypertension, renal failure, renal ischemia, renal failure, renal fibrosis, heart failure, cardiac hypertrophy, cardiac fibrosis, myocardial ischemia, cardiomyopathy, glomerulonephritis, renal colic, nephropathy Complications caused by diabetes such as vascular and neurological disorders, glaucoma, elevated intraocular pressure, atherosclerosis, restenosis after angioplasty, complications after vascular or cardiac surgery, erectile dysfunction, high aldosterone Treatment and / or treatment of diseases selected from symptom, pulmonary fibrosis, scleroderma, anxiety, cognitive impairment, complications of treatment with immunosuppressants, and other diseases known to be related to the renin-angiotensin system Or use of a compound according to claim 1 or 2 for the manufacture of a pharmaceutical composition for prevention.