WO2010024772A1 - Aspartyl protease inhibitors - Google Patents

Abstract

The invention provides compounds of the formula (I wherein Q is trifluoromethyl, C₃-C₆cycloalkyl, phenyl, p-fluorophenyl, pyridyl, thiazolyl, furyl, thienyl or C₃-C₆cycloalkylethynyl; R³ is C₁-C₆alkyl; R⁴ is C₁-C₆alkyl; W is cyano or fluoro; or a pharmaceutically acceptable salt, hydrate or N-oxide thereof. The compounds of the invention are inhibitors of aspartyl proteases such as renin and are among other things useful for the treatment of conditions associated with activities of the RAS, such as hypertension, heart failure and renal insufficiency.

Description

Aspartyl Protease Inhibitors

Technical field

This invention relates to novel compounds having inhibitory activity on aspartyl proteases such as renin. It further concerns pharmaceutical compositions comprising these compounds as active ingredients as well as processes for preparing these compounds and compositions and their use in the preparation of a medicament or their use in therapy.

Background to the invention

The renin-angiotensin system (RAS) is critical for the control of blood pressure and salt balance in mammals. Renin is an aspartyl protease with a high substrate specificity, its only known substrate is angiotensinogen. Renin cleaves the N terminus of circulating angiotensinogento angiotensin I (Ang I), which thereafter is further processed to the active peptide hormone angiotensin II (Ang II) by the less specific angiotensin-converting enzyme (ACE). Ang II increases blood pressure both directly by arterial vascoconstriction and indirectly by liberating the sodium-ion-retaining hormone aldosterone. Ang II is known to work on at least two receptor subtypes called ATI and AT2. ATI seems to transmit most of the known functions of Ang II, while the role of AT2 is still unknown.

Modulation of the RAS represents a major advance in the treatment of cardiovascular diseases. Inhibition of the enzymatic activity of renin leads to a reduction in the formation of Ang I, and as a consequence, a smaller amount of Ang II is produced. The reduced concentration of that active peptide hormone is a direct cause of the hypotensive effect of renin inhibitors.

ACE inhibitors and ATI blockers have been accepted to treat hypertension and ACE inhibitors are used for renal protection in the prevention of congestive heart failure and myocardial infarction. The rationale to develop renin inhibitors is the specificity of renin. 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 has been created with renin inhibitors because of their insufficient oral activity. The clinical development of several compounds has been stopped because of this problem together with the high cost of goods. Only one compound 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. The present invention concerns inhibitors of renin which exhibit beneficial potency, selectivity and/or pharmacokinetic properties. Notably, the compounds of the present invention exhibits advantageous metabolic stability as determined in human liver microsomes (HLM).

Compound 38 on page 4547 of Hanessian et al J. Med. Chem., 2006, 4544-4567, describe the boc-protected peptido mimetic derivative l-benzylsulfanylmethyl-4-(l-butylcarbamoyl-2-methyl- propylcarbamoyl)-2-hydroxy-pentyl]carbamic acid tert-butyi ester, used as an intermediate in the preparation of macrocyclic peptidomimetic BACE inhibitors. Compound 38 is outside the scope of the claims presented below and there is no suggestion that such intermediates could find utility as renin inhibitors.

Compound II on page 246 of Chen et al Bioorg. Med. Chem. Lett. 14 (2004) 245-250 discloses the compound 4-hydroxy-2,5-dimethyl-6-phenyl-hexanoic acid {2-methyl-l-[(pyridin-4- ylmethyl)-carbamoyl]-propyl}-amide used as an intermediate in the synthesis of peptidomimetic BACE inhibitors. The benzyl group attached direct to the backbone is outside the scope of the claims presented below and no suggestion is provided as to alternative utilities in the field of renin inhibition

Brief description of the Invention

In accordance with the present invention, there is provided renin inhibitors which can be represented by the formula (I):

wherein

Q is trifluoromethyl, Cs-Cβcycloalkyl, phenyl, p-fluorophenyl, pyridyl, thiazolyl, furyl, thienyl or

Cs-Cβcycloalkylethynyl;

R³ is Ci-Cealkyl;

R⁴ is Ci-Cealkyl;

W is cyano or fluoro; or a pharmaceutically acceptable salt, hydrate or N-oxide thereof.

The compounds of general formula (I) have several centres of chirality, conveniently the compounds display at least 75%, preferably at least 90%, such as in excess of 95%, enantiomeric purity at each of the chiral centres. In typical embodiments of the invention, the chiral centre whereto the group R is attached has the stereochemistry shown in structure (Ia):

The chiral centre to which the group R⁴ is attached typically has the configuration shown in structure (Ib) below:

i.e. the configuration corresponds typically to that of an L-amino acid.

The chiral centre to which the substituted benzyloxymethyl moiety is attached typically has the configuration shown in structure (Ic) below:

The chiral centre to which the hydroxy group is attached typically has the configuration as shown in structure (Id) below:

Preferred compounds of formula (I) are those having the stereochemistry indicated in structure (Ie):

R³ is Ci-Cβalkyl, preferably ethyl or isopropyl.

R⁴ is Ci-Cβalkyl, preferably isopropyl or more preferably sec. butyl.

Q is as stated above. Preferably Q is p-fluorophenyl, 2-pyridyl, 5-thiazolyl or cyclopropyl, more preferably Q is p-fluorophenyl.

W is as stated above. Preferably, W is fluoro.

In one embodiment of the invention, the compound is not any of:

5-Amino-6-[4'-fluoro-2-(3-methoxy-propoxy)-biphenyl-4-ylmethoxy]-4-hydroxy-2-isopropyl- hexanoic acid [l-(4-cyano-benzylcarbamoyl)-2-methyl-butyl]-amide,

5-Amino-6-[4'-fluoro-2-(3-methoxy-propoxy)-biphenyl-4-ylmethoxy]-4-hydroxy-2-isopropyl- hexanoic acid [l-(4-fluoro-benzylcarbamoyl)-2-methyl-butyl]-amide,

5 - Amino-6- [4-furan-3 -yl-3 -(3 -methoxy-propoxy)-benzyloxy] -4-hydroxy-2-isopropyl-hexano ic acid [ 1 -(4-fluoro-benzylcarbamoyl)-2-methyl-butyl]-amide,

5-Amino-4-hydroxy-2-isopropyl-6-[3-(3-methoxy-propoxy)-4-pyridin-4-yl-benzyloxy]-hexanoic acid [ 1 -(4-fluoro-benzylcarbamoyl)-2-methyl-butyl] -amide, 5-Amino-4-hydroxy-2-isopropyl-6-[3-(3-methoxy-propoxy)-4-thiophen-2-yl-benzyloxy]- hexanoic acid [l-(4-fluoro-benzylcarbamoyl)-2-methyl-butyl]-amide.

It is to be understood that the above defined subgroups, compounds of formulae (Ia), (Ib), (Ic), (Id) and (Ie), as well as any other subgroup defined herein, are meant to also comprise any prodrugs, TV-oxides, addition salts, quaternary amines, metal complexes and stereochemically isomeric forms of such compounds.

As used in the foregoing and hereinafter, the scientific and technological terms and nomenclature have the same meaning as commonly understand by a person of ordinary skill in the art, in addition, the following definitions apply unless otherwise noted.

As used herein 'Ci-Coalkyl' defines a straight or branched chain saturated hydrocarbon radical having from 1 to 6 carbon atoms such as for example methyl, ethyl, 1 -propyl, 2-propyl, 1 -butyl, 2-butyl, 2-methyl-l -propyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 1-hexyl, 2-hexyl, 2- methyl-1 -butyl, 2-methyl- 1-pentyl, 2-ethyl-l -butyl, 3-methyl-2-pentyl, and the like.

As used herein, ' Cs-C₆Cy cloalkyl' defines a saturated carbocyclic ring having 3, 4, 5 or 6 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. Of special interest amongst C₃- CδCycloalkyl is cyclopropyl.

It should be noted that the radical positions on any molecular moiety used in the definitions may be anywhere on such a moiety as long as it is chemically stable.

Radicals used in the definitions of the variables include all possible isomers unless otherwise indicated. For instance pyridyl includes 2-pyridyl, 3-pyridyl and 4-pyridyl; pentyl includes 1- pentyl, 2-pentyl and 3-pentyl.

When any variable occurs more than one time in any constituent, each definition is independent.

Whenever used hereinafter, the term 'compounds of formula (I)', or 'the present compounds' or similar terms, it is meant to include the compounds of formula (I), their prodrugs, TV-oxides, addition salts, quaternary amines, metal complexes, and stereochemically isomeric forms.

The term 'prodrug' as used throughout this text means the pharmacologically acceptable derivatives such as esters, amides and phosphates, such that the resulting in vivo biotransformation product of the derivative is the active drug as defined in the compounds of formula (I). The reference by Goodman and Gilman (The Pharmacological Basis of Therapeutics, 8^th ed, McGraw-Hill, Int. Ed. 1992, "Biotransformation of Drugs", p 13-15) describing prodrugs generally is hereby incorporated. Prodrugs preferably have excellent aqueous solubility, increased bioavailability and are readily metabolized into the active inhibitors in vivo. Prodrugs of a compound of the present invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either by routine manipulation or in vivo, to the parent compound.

Preferred are pharmaceutically acceptable ester prodrugs that are hydrolysable in vivo and are derived from those compounds of formula (I) having a hydroxy and/or a carboxyl group. An in vivo hydrolysable ester is an ester, which is hydrolysed in the human or animal body to produce the parent acid or alcohol. Suitable pharmaceutically acceptable esters for carboxy include Ci-Cβalkoxymethyl esters for example methoxymethyl, Ci-Cβalkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidyl esters, C₃-CsCyC loalkoxycarbonyloxyCi-Cβalkyl esters for example 1-cyclohexylcarbonyloxyethyl; l,3-dioxolen-2-onylmethyl esters for example 5-methyl-l,3-dioxolen-2-onylmethyl; and Ci -Cβalkoxycarbonyloxy ethyl esters for example 1-methoxycarbonyloxy ethyl which may be formed at any carboxy group in the compounds of this invention.

An in vivo hydrolysable ester of a compound of the formula (I) containing a hydroxy group includes inorganic esters such as phosphate esters and α-acyloxyalkyl ethers and related compounds which as a result of the in vivo hydrolysis of the ester breakdown will give the parent hydroxy group. Examples of α-acyloxyalkyl ethers include acetoxymethoxy and 2,2- dimethylpropionyloxy-methoxy. A selection of in vivo hydrolysable ester forming groups for hydroxy include alkanoyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl, alkoxycarbonyl (to give alkyl carbonate esters), dialkylcarbamoyl and N-(dialkylaminoethyl)-N- alkylcarbamoyl (to give carbamates), dialkylamino acetyl and carboxyacetyl. Examples of substituents on benzoyl include morpholino and piperazino linked from a ring nitrogen atom via a methylene group to the 3- or 4-position of the benzoyl ring.

For therapeutic use, salts of the compounds of formula (I) or any subgroup of compounds of formula (I) are those wherein the counter-ion is pharmaceutically acceptable. However, salts of acids and bases which are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound. All salts, whether pharmaceutically acceptable or not are included within the ambit of the present invention. The pharmaceutically acceptable acid and base addition salts as mentioned hereinabove are meant to comprise the therapeutically active non-toxic acid and base addition salt forms which the compounds of formula (I) are able to form. The pharmaceutically acceptable acid addition salts can conveniently be obtained by treating the base form with such appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric acids and the like; or organic acids such as, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, />toluenesulfonic, cyclamic, salicylic, /^-aminosalicylic, pamoic acids and the like.

Acid addition salt forms can be converted to the free base form by treatment with an appropriate base.

The compounds of formula (I) containing an acidic proton may also be converted into their nontoxic metal or amine addition salt forms by treatment with an appropriate organic or inorganic base. Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g. the lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g. the benzathine, jV-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like.

Base addition salt forms can be converted to the free acid form by treatment with an appropriate acid.

The term addition salt as used hereinabove also comprises the solvates which the compounds of formula (I) or any of the subgroups of compounds of formula (I), as well as the salts thereof, are able to form. Such solvates are for example hydrates, alcoholates and the like.

The term 'quaternary amine' as used above and hereinafter defines the quaternary ammonium salts which the compounds of formula (I) or any of the subgroups of compounds of formula (I), are able to form by reaction between a basic nitrogen of a compound of formula (I) or any of the subgroups of compounds of formula (I), and an appropriate quaternizing agent, such as, for example, an optionally substituted alkylhalide, arylhalide or arylalkylhalide, e.g. methyliodide or benzyliodide. Other reactants with good leaving groups may also be used, such as alkyl trifluoromethanesulfonates, alkyl methanesulfonates, and alkyl p-toluenesulfonates. A quaternary amine has a positively charged nitrogen. Pharmaceutically acceptable counterions include chloro, bromo, iodo, trifluoroacetate and acetate. The counterion of choice can be introduced using ion exchange resins.

The TV-oxide forms of the present compounds are meant to comprise the compounds of formula (I) wherein one or several nitrogen atoms are oxidized to the so-called TV-oxide.

The compounds according to the invention may contain one or more asymmetrically substituted carbon atoms, asymmetric or chiral centre. The presence of one or more of these asymmetric centres in compounds according to the invention can give rise to stereochemically isomeric forms, stereoisomers, and in each case the invention is to be understood to extend to all such stereoisomers, both in pure form and mixed with each others, including enantiomers and diastereomers, and mixtures including racemic mixtures thereof.

Pure stereoisomeric forms of the compounds and intermediates as mentioned herein are defined as isomers substantially free of other enantiomeric or diastereomeric forms of the same basic molecular structure of said compounds or intermediates. In particular, the term 'stereoisomerically pure' concerns compounds or intermediates having a stereoisomeric excess of at least 80% (i.e. minimum 90% of one isomer and maximum 10% of the other possible isomers) up to a stereoisomeric excess of 100% (i.e. 100% of one isomer and none of the other), more in particular, compounds or intermediates having a stereoisomeric excess of 90% up to 100%, even more in particular having a stereoisomeric excess of 94% up to 100% and most in particular having a stereoisomeric excess of 97% up to 100%. The terms 'enantiomerically pure' and 'diastereomerically pure' should be understood in a similar way, but then having regard to the enantiomeric excess, and the diastereomeric excess, respectively, of the mixture in question.

Pure stereoisomeric forms of the compounds and intermediates of this invention may be obtained by application of art-known procedures (cf. Advanced Organic Chemistry: 3rd Edition: author J March, pp 104-107). For instance, enantiomers may be separated from each other using known procedures including, for example, formation of diastereomeric mixtures by reaction with a convenient optically active auxiliary species followed by separation of the diastereomers, using for instance selective crystallisation, and finally cleavage of the auxiliary species. Examples of optically active auxiliary species are optically active acids and bases such as tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid and camphorsulfonic acid. Alternatively, enantiomers may be separated by chromatographic techniques using chiral stationary phases. Pure stereochemically isomeric forms may also be derived from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction occurs stereospecifϊcally. When a specific stereoisomer of a compound is desired, the compound will preferably be synthesized by stereospecifϊc methods of preparation. These methods will advantageously employ enantiomerically pure starting materials.

With reference to the instances where (R) or (S) is used to designate the absolute configuration of a chiral centre within a substituent, the designation is done taking into consideration the whole compound and not the substituent in isolation.

Where tautomers exist in the compounds of the invention, we disclose all individual tautomeric forms and combinations of these as individual specific embodiments of the invention.

It will be appreciated that the compounds of formula (I) may have metal binding, chelating or complex forming properties and therefore may exist as metal complexes or metal chelates. Such metalated derivatives of the compounds of formula (I) are intended to be included within the scope of the present invention.

The invention relates to the compounds of formula (I) or any subgroup of compounds of formula (T) per se, the prodrugs, N-oxides, addition salts, quaternary amines, metal complexes, and stereochemically isomeric forms thereof. One embodiment comprises the compounds of formula (I) or any subgroup of compounds of formula (I) specified herein, as well as the N-oxides, salts, as the possible stereoisomeric forms thereof.

The present invention also includes isotope- labelled compounds of formula I or any subgroup of formula I, wherein one or more of the atoms is replaced by an isotope of that atom, i.e. an atom having the same atomic number as, but an atomic mass different from, the one(s) typically found in nature. Examples of isotopes that may be incorporated into the compounds of formula I or any subgroup of formula I, include but are not limited to isotopes of hydrogen, such as ²H and ³H (also denoted D for deuterium and T for tritium respectively), carbon, such as ¹¹C, ¹³C and ¹⁴C, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³¹P and ³²P, sulphur, such as ³⁵S, fluorine, such as ¹⁸F, chlorine, such as ³⁶Cl, bromine such as ⁷⁵Br, ⁷⁶Br, ⁷⁷Br and ⁸²Br, and iodine, such as ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I. The choice of isotope included in an isotope-labelled compound will depend on the specific application of that compound. For example, for drug or substrate tissue distribution assays, compounds wherein a radioactive isotope such as ³H or ¹⁴C is incorporated will generally be most useful. For radio-imaging applications, for example positron emission tomography (PET) a positron emitting isotope such as ¹¹C, ¹⁸F, ¹³N or ¹⁵O will be useful. The incorporation of a heavier isotope, such as deuterium, i.e. ²H, may provide greater metabolic stability to a compound of formula I or any subgroup of formula I, which may result in, for example, an increased in vivo half life of the compound or reduced dosage requirements.

Isotopically labelled compounds of formula I or any subgroup of formula I can be prepared by processes analogous to those described in the Schemes and/or Examples herein below by using the appropriate isotopically labelled reagent or starting material instead of the corresponding non-isotopically labelled reagent or starting material, or by conventional techniques known to those skilled in the art.

The invention further relates to methods for the preparation of the compounds of formula (I) or any subgroup of compounds of formula (I), the prodrugs, TV-oxides, addition salts, quaternary amines, metal complexes, and stereochemically isomeric forms thereof, its intermediates, and the use of the intermediates in the preparation of the compounds of formula (I) or any subgroup of compounds of formula (I).

The invention also relates to the use of a compound of formula (I) or any subgroup of compounds of formula (I), or a prodrug, TV-oxide, addition salt, quaternary amine, metal complex, or stereochemically isomeric form thereof, for the manufacture of a medicament. Or the invention relates to the use of a of a compound of formula (I) or any subgroup of compounds of formula (I), or a prodrug, TV-oxide, addition salt, quaternary amine, metal complex, or stereochemically isomeric form thereof in therapy.

In the context of the present specification, the term 'therapy' also includes 'prophylaxis' unless there are specific indications to the contrary. The terms 'therapeutic' and 'therapeutically' should be construed accordingly.

The invention further relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or a compound of any of the subgroups of formula (I) or a pharmaceutically acceptable salt thereof as specified herein, and a pharmaceutically acceptable adjuvant, diluent or carrier for administration to a subject in need thereof. A therapeutically effective amount in this context is an amount sufficient to act in a prophylactic way against, to stabilize or to reduce adverse conditions associated with RAS activity, such as or related to hypertension, heart failure, pulmonary hypertension, renal insufficiency, renal ischemia, in affected subjects or subjects being at risk of being affected.

The invention further relates to a process of preparing a pharmaceutical composition as specified herein, which comprises intimately mixing a pharmaceutically acceptable adjuvant, diluent or carrier with a therapeutically effective amount of a compound of formula (I) or any of the subgroups of formula (I) as specified herein, or a pharmaceutically acceptable salt or a solvate, prodrug, N-oxide, quaternary amine, metal complex or stereochemically isomeric form thereof as specified herein.

The compounds of formula (I) or any of the subgroups of formula (I) have enzyme inhibiting properties and are modulators of the renin-angiotensin system, in particular they are inhibitors of the natural enzyme renin and may be used in 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. In one embodiment, the invention relates to a method for the treatment and/or prophylaxis of diseases or conditions which are associated with a dysregulation of the renin-angiotensin system, in particular to a method for the treatment or profylaxis of the above mentioned diseases, said method comprising administering to a patient a pharmaceutically active amount of a compound of formula (I) or any of the subgroups of formula (I).

The invention further provides a method of treating a disease or condition known to be related to the renin-angiotensin system which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, a solvate, prodrug, TV-oxide, quaternary amine, metal complex, or stereochemically isomeric form thereof, as hereinbefore defined. The invention also provides a method of treating diseases or conditions such as or related to the above mentioned (e.g. hypertension) which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) or any of the subgroups of formula (I) or a pharmaceutically acceptable salt, or solvate thereof as hereinbefore defined.

For the above-mentioned therapeutic uses the dosage administered will, of course, vary with the compound employed, the mode of administration, the treatment desired and the disorder indicated. The daily dosage of the compound of formula I/salt/so lvate (active ingredient) may be in the range from 0.001 mg/kg to 75 mg/kg, in particular from 0.5 mg/kg to 30 mg/kg. This daily dose may be given in divided doses as necessary. Typically unit dosage forms will contain about 1 mg to 500 mg of a compound of this invention.

The compounds of formula (I) and pharmaceutically acceptable salts, solvates, prodrugs, TV-oxides, quaternary amines, metal complexes, or stereochemically isomeric forms thereof may be used on their own but will generally be administered in the form of a pharmaceutical composition in which the compound of formula (I) /salt/solvate (active ingredient) is in association with a pharmaceutically acceptable adjuvant, diluent or carrier. Depending on the mode of administration, the pharmaceutical composition will preferably comprise from 0.05 to 99 %w (per cent by weight), more preferably from 0.10 to 70 %w/w, of active ingredient, and, from 1 to 99.95 %w/w, more preferably from 30 to 99.90 %w/w, of a pharmaceutically acceptable adjuvant, diluent or carrier, all percentages by weight being based on total composition. A representative tablet within the scope of the pharmaceutical composition of the invention could have a mass of 500 - 1500 mg with a loading of active ingredient in the range 35 - 75%, with the balance being excipients, such as binders, disintegrants, antioxidants and the like.

The pharmaceutical compositions of this invention may be administered in standard manner for the disease or condition that it is desired to treat, for example by oral, topical, parenteral, buccal, nasal, vaginal or rectal administration or by inhalation. For these purposes the compounds of this invention may be formulated by means known in the art into the form of, for example, tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely divided powders or aerosols for inhalation, and for parenteral use (including intravenous, intramuscular or infusion) sterile aqueous or oily solutions or suspensions or sterile emulsions. The oral delivery route, particularly capsules or tablets is favoured. In addition to the compounds of the present invention the pharmaceutical composition of this invention may also contain, or be co- administered (simultaneously or sequentially) with, one or more pharmacological agents of value in treating one or more of the diseases or conditions referred to hereinabove. A representative example are 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 ibeta-hydroxy steroid dehydrogenase type 1 inhibitors and soluble guanylate cyclase activators.

General synthetic methodology

The compounds of the present invention and intermediates useful for the synthesis of these compounds are prepared by methods and techniques known to those skilled in the art. The general schemes below illustrate typical synthetic routes to the compounds of the invention and to intermediates thereof. Alternative routes, which will be readily apparent to the ordinary skilled organic chemist, may alternatively be used to synthesize various portions of the molecules as illustrated by the general schemes and the preparative examples below.

Scheme 1 illustrates a synthetic route to a lactone which is a useful intermediate in the preparation of compounds of formula (I).

Scheme 1

The isopropylidene derivative (Ia) achieved for example as described in Tetrahedron Lett., 1987, 28, 1143, can be transferred into the methyl glycoside (Ib) by acidic hydrolysis of the acetal group effected by treatment with a suitable acid such as sulphuric acid, in the presence of methanol. The achieved free secondary hydroxy group can then be reductively removed effected for instance by transformation of the hydroxy group into a thiocarbonyl group by reaction with thiocarbonyl diimidazole (TCDI) followed by reductive removal of the formed thiocarbonyl group using for instance conditions such as tributyltin hydride the presence of a radical initiator like azobis-(2-methylpropyonitrile) (AIBN) or the like to give the 2,3-dideoxy glycoside (Ic). Oxidative cleavage of the methyl ether performed for example by oxidation with m- chloroperbensoesyra or the like in the presence of BF3-etherate, gives the lactone (Id). The ring substituent R³ can then be introduced for example by treatment of the lactone with a base such as LDA or equivalent followed by reaction with a suitable alkylating agent such as an alkyl halide like an alkyl bromide or alkyl iodide or a derivative of sulphonic acid such as a mesylate, triflate or tosylate or the like of the desired alkyl radical, thus providing the α-alkylated lactone (Ie). Removal of the benzyl groups using any suitable conditions well known to the skilled person, such as catalytic hydrogenation, provides the diol (If). The primary hydroxy group of the afforded diol (If) can then be selectively alkylated for example by activation of the hydroxy group with dibutyltinoxide followed by reaction with a desired alkylating agent Q'-(CH₂)_n-Lg wherein Q' is a disubstituted phenyl moiety and Lg is a suitable leaving group such as a halide like bromide or iodide in the presence of tetrabutylammonium bromide or the like thus forming the O-alkylated lactone (Ig). Alternatively, the substituent (2'-(CH₂)- can be introduced by using the Mitsunobu conditions (Mitsunobu, 1981, Synthesis, January, 1-28; Rano et al., Tetrahedron Lett., 1995, 36, 22, 3779-3792; Krchnak et al., Tetrahedron Lett., 1995, 36, 5, 6193-6196; Richter et al., Tetrahedron Lett., 1994, 35, 27, 4705-4706) i.e. reaction of the alcohol (If) with an azodicarboxylate such as DIAD or the like in the presence of triphenylphosphine followed by displacement with a desired alcohol Q'-(CH₂)-OH. Replacement of the hydroxy group of the secondary alcohol (Ig) by azide with concomitant inversion of the stereochemistry may be effected by transforming the hydroxy group to a leaving group, for example a derivative of sulphonic acid like a triflate or tosylate or the like by subjecting the alcohol to sulphonylating conditions such as treatment with the appropriate anhydride or halide optionally in the presence of a base, for instance pyridine, followed by displacement of the leaving group with azide for example sodium azide, thus giving the azide derivative (Ih). Alternatively, the azide moiety can be introduced by treatment of the alcohol with diisopropyl azodicarboxylate or any other suitable azodicarboxylate, in combination with triphenylphosphine, followed by reaction with azide for example DPPA, in a solvent like THF.

Compounds of formula (I) can then be achieved from the intermediate lactone (Ih) as shown in scheme 2.

2c

Scheme 2 The linear amino compound (2b) can be achieved by opening of the lactone (Ih) with a desired amino derivative (2a) in the presence of a coupling agent for example 2-hydroxypyridine and a base like isopropyl diethylamine. Reduction of the azide using conditions compatible with the group (CH₂)-Q\ for example hydrogenation at atmospheric pressure in the presence of Lindlar Catalyst or equivalent then provides the amino derivative (2c).

Amino derivatives (2a) to be used for the opening of the lactone in the scheme 2 above are commercially available or they can be prepared by the skilled person according to literature procedures. An example of their preparation is illustrated in scheme 3.

Scheme 3

A suitably protected amino acid (3a), carrying the desired side chain R , can be coupled to the amine W-Ph-CH₂-NH₂ using any convenient method for peptide coupling known in the art. For example, a coupling agent like HATU or isobutylchloro formate in the presence of a tertiary amine such as ethyldiisopropylamine (DIEA) or N-methylmorpholine in a solvent like dimethyl formamide can be used thus providing the amide. Subsequent removal of the N-protecting group using the appropriate conditions according to the protecting group used, such as acidic treatment in the case of a Boc-group, provides the free amine (3b).

An alternative route to compounds of formula (I) is shown in scheme 4.

Y is a leaving group or OH

Pg¹ and Pg² are orthogonal protecting groups

Scheme 4

The azide derivative (4a), prepared for example as outlined in scheme 2, wherein Pg¹ is a hydroxy protecting group for example a benzyl group can be transformed to the corresponding amine by reduction of the azide group using any convenient reduction method such as hydrogenation in the presence of a suitable catalyst, such as Lindlars catalyst or the like in the presence Of BoC₂O to provide the boc protected derivative (4b). Protection of the secondary hydroxy group, using a protecting group (Pg²) which is orthogonal to the one used for the primary hydroxy group (Pg¹), followed by removal of the primary hydroxy protecting group using the appropriate conditions according to the group used, such as for example catalytic hydrogenation in the case of a benzyl group provides the primary alcohol (4c). Suitable protecting groups for the above route will be recognized by the skilled person and a numerous of useful protecting groups are described in Greene "Protective Groups in Organic Synthesis", John Wiley and sons, New York (1981). For example benzyl can be used as Pg¹ and acetyl as Pg². The disubstituted benzyl group can then be introduced as described above. For example, reaction of the primary alcohol (4c) with an alkylating agent Q '-CH₂-Lg wherein Q' is the disubstituted phenyl moiety and Lg is a leaving group such as halide like a bromide, chloride or iodide or a derivative of sulphonic acid such as a triflate or mesylate or the like in the presence of a base like NaH or the like, or alternatively, a trichloroacetimidate of a desired group, Q'-CH₂-O- C(=NH)Cl3 may be reacted with the primary alcohol (4c) in the in the presence of a Lewis acid such as BFsOEt₂. Trichloroacetimidates are conveniently prepared by reaction of the corresponding alcohol with trichloroacetonitrile in the presence of a base like NaH.

Alternatively, the disubstituted benzyl moiety may be introduced by a Mitsunobu reaction of the primary alcohol (4c) with a desired alcohol, QJ-CH₂-OH, as described above.

The disubstituted benzyl derivatives Q '-CH₂- used in the schemes above can be prepared according to literature procedures or as described herein. A general example is illustrated in scheme 6.

6c

6d 6e

Scheme 6

Alkylation of the phenolic hydroxy group of ester (6a) with 3-methoxypropanol using for example the Mitsunobu conditions, such as in the presence OfPh₃P, an azodicarboxylate like DIAD provides the methoxypropoxy substituted benzoic ester derivative 6b. The desired substituent Q can then be introduced by using for instance a Pd-catalyzed cross coupling reaction, whereof many are extensively described in the literature. For example the Suzuki conditions may be used, i.e. reaction of the bromo derivative (6b) with the boronic acid of a desired substituent Q in the presence of a palladium catalyst such as Pd(PPh₃)₄ or Pd(OAc)₂ or the like and a suitable base such as potassium carbonate or potassium fluoride or the like, thus providing the Q-substituted compound (6c). Other suitable reactions that can be used for the introduction of the substituent Q are for instance the Stille reaction, wherein a tin derivative, such as a trialkyltin derivative, of the desired group Q is reacted with the bromo derivative (6b) in the presence of Pd(O), or the Heck coupling reaction wherein the bromo derivative (6b) is reacted with a double bond of the desired group Q in the presence of a Pd catalyst such as Pd(PPli3)4 PdCl₂ or Pd(OAc)₂ and a base such as triethylamine, potassium carbonate or the like. Reduction of the ester function of compound 6c using any convenient reduction method known in the art, such as treatment with DIBAL-H, provides benzylic alcohol (6d). The afforded alcohol can then either be used directly in the coupling to the primary hydroxy group of the lactone (If) or the linear compound (4c) employing the Mitsunobu conditions, or the hydroxy group can be transferred to a leaving group, such as a halide like bromide by treatment with for instance bromine or tetrabromomethane in the presence of triphenylphosphine, and subsequently coupled to the primary hydroxy group of the lactone (If) or the linear compound (4c) as described above.

Alternatively, the substituent Q of the benzene ring may be introduced after coupling of the benzyl moiety to the primary hydroxy group of the lactone (If), as illustrated in scheme 7.

Scheme 7

Transformation of the bromo substituted benzoic acid (7a), into the corresponding benzyl bromide derivative (7b) by reduction of the ester function followed by replacement of the hydroxy group to a bromide as described in Scheme 6. Subsequent coupling to the lactone (If) as outlined in scheme 1, then provides the alkylated lactone derivative 7c. The substituent Q of the benzene ring can then be introduced using any convenient coupling method, such as the Stille, Heck or Suzuki conditions as described above in scheme 6 to afford the Q-substituted compound 7d. Replacement of the secondary hydroxy group with an azide followed by opening of the lactone with a desired amine and reduction of the azide function, as described above, then yields the compound of formula (I).

Scheme 8 shows an alternative route to compounds of the invention, starting from Garner's aldehyde.

Lg is a leaving group Pg is a protecting group

Scheme 8

α-Alkylation of the aldehyde (8a) by reaction with a suitable propiolic acid ester, for example the methyl ester, in the presence of a base like buthyllithium followed by reduction of the triple bond for example by catalytic hydrogenation using a catalyst like palladium on carbon provides the alcohol (8b). Heating of the afforded hydroxy ester in the presence of acetic acid effects the ring closing and thus affords lactone (8c). The afforded lactone can then be alkylated at the α-carbon with a desired group R³ as described above, i.e. by treatment of the lactone with a base such as LDA optionally followed by addition of tripyrrolidine phosphorus oxide and finally addition of the alkylating agent or by any other suitable alkylation method which provides the alkylated lactone (8d). Cleavage of the cyclic aminal by treatment with acid such as TFA followed by reaction with BoC₂O in order to reprotect the amino function affords the primary alcohol (8e). The group Q-CH₂ can then be introduced using any suitable method such as any of those described above. For example, a trichloroimidate of the desired group Q-CH₂ in the presence of TMS triflate can be used. The lactone may then be opened either directly with a desired amine as described above to give the amide (8h), or alternatively, the lactone may be opened by treatment with hydroxide such as lithium hydroxide, thus affording the acid (8g). Protection of the hydroxy group, using any conventional protecting group for example a silyl group such as a tert-butyl dimethylsilyl group, followed by coupling of the acid to a suitable amine using standard peptide coupling conditions such as using a coupling agent like EDAC in the presence of HOBt and a tertiary amine like triethylamine, and finally removal of the hydroxy protecting group provide the amide (8h).

Any functional groups present on any of the constituent compounds used in the preparation of the compounds of the invention are appropriately protected where necessary. For example functionalities on the natural or non-natural amino acids are typically protected as is appropriate in peptide synthesis. Those skilled in the art will appreciate that the selection and use of appropriate protecting groups depend upon the reaction conditions. Suitable protecting groups are described in Greene, "Protective Groups in Organic Synthesis", John Wiley & Sons, New York (1981) and "The Peptides: Analysis, Synthesis, Biology", Vol. 3, Academic Press, New York (1981), the disclosure of which are hereby incorporated by reference.

Detailed Description

Various embodiments of the compounds of the invention and key intermediates towards such compounds will now be described by way of illustration only with reference to the following non-limiting examples.

Intermediate 1

Step a) 5-(l,2-Bis-benzyloxyethyl)-2-methoxytetrahvdrofuran-3-ol (I-la) Sulfuric acid (13 mL, 240 mmol) was added dropwise to a solution of 5-(l,2-bis-benzyloxy- ethyl)-2,2-dimethyl-tetrahydro-furo[2,3-d][l,3]dioxole (prepared as described by Hanessian et al in Tetrahedron Lett., 1987, 28, 1142) (30.2 g, 78.6 mmol) in MeOH (180 mL) at 0 ⁰C. The reaction mixture was then stirred at room temperature for 2 h, cooled down to 0 ⁰C and carefully neutralized with sat. aq. NaHCOs. Methanol was distilled off under reduced pressure and the residue was taken into EtOAcZH₂O. The phases were separated and the aqueous phase was extracted with EtOAc. The combined organic extracts were dried (Na₂SO₄) and the solvent evaporated to give the crude product as a yellow oil. Purification by flash chromatography on silica gel (Hex:EtOAc 10:1 to 2.5:1) afforded 18.7 g (67%) of the title compound as a colourless oil (mixture of anomers).

Step b) Imidazole- 1-carbothioic acid 0^5-0.2-bis-benzyloxyethyl)-2-methoxytetrahydrofuran- 3-yli ester Q- lfr)

A solution of the alcohol I-la (14.2 g, 39.7 mmol) and l,l '-thiocarbonyldiimidazole (7.1 g, 39.8 mmol) in THF (120 mL) was refluxed under N₂ for 2 h. The reaction mixture was concentrated under vacuum and the residue was purified by flash chromatography on silica gel (Hex:EtOAc 2.5:1) which gave the title compound (15.8 g, 85%) (mixture of anomers).

Step c) 2-(l,2-Bis-benzyloxyethyl)-5-methoxytetrahvdrofuran (I- Ic)

A solution of compound I-lb (15.8 g, 33.8 mmol) and AIBN (0.55 g, 3.3 mmol) in toluene was heated to 100 ⁰C under N₂. Tributyltinhydride (13.6 mL, 50.6 mmol) was then added carefully dropwise (gas evolution). The reaction mixture was stirred further 20 min at 100 ⁰C after the addition was completed. Evaporation of the solvent afforded a syrup that was purified by flash chromatography on silica gel (Hex:EtOAc 20:1 to 2.5:1) which gave the title compound (8.2 g, 71%) (mixture of anomers). Step d) 5-π.2-Bis-benzyloxyethyiydihydrofuran-2-one (I- Id)

A solution of compound I-lc (10.7 g, 31.29 mmol) and m-CPBA (19.6 g, 62.5 mmol) in dichloromethane (10 niL) was treated with BFsOEt₂ (2 mL, 15.8 mmol) at 0 ⁰C. The reaction mixture was then allowed to warm up to room temperature and stirring was continued for 17 h. Evaporation of the solvent afforded the crude product that was purified by flash chromatography on silica gel (Hex:EtOAc 20:1 to 2.5:1) which gave the title compound (6.1 g, 60%) as a colourless oil.

Step e) 5-π,2-Bis-benzyloxyethyl)-3-isopropyldihvdrofuran-2-one (I-le)

A solution of compound I-ld (3.1 g, 9.51 mmol) in THF (30 mL) was added to a 1 M solution of LDA in THF (20 mL, 20 mmol) at -60 ⁰C under N₂. The reaction mixture was then treated with tris(pyrrolidinophosphine) oxide (15 mL) followed by 2-iodopropane (3.8 mL, 38.1 mmol). Stirring at -60 ⁰C was continued for 1 h and the reaction mixture was quenched with aq. NH₄Cl. The phases were separated and the aqueous phase was extracted with ¹BuOMe. The combined organic extracts were dried (Na₂SO₄) and the solvent evaporated. The residue was purified by flash chromatography on silica gel (Hex:EtOAc 10:1) which gave the title compound (1.2 g, 34%) as a colourless oil.

Step f) 5-(1.2-Dihydroxyethyl)-3-isopropyldihydrofuran-2-one (I-lf)

A solution of compound I-le (2.58 g, 7.01 mmol) in MeOH (50 mL) was hydrogenated in the presence of 10% Pd/C at 3 bar H₂ pressure. Filtration of the catalyst and evaporation of the solvent afforded the title compound (1.2 g, 92%) as a white solid.

Example 1

I i₁ R = NH₂

Step a) 4-Bromo-3-(3-methoxy-propoxy)-benzoic acid methyl ester (Ia) 4-Bromo-3-hydroxy-benzoic acid methyl ester [4.2 g, 18 mmol (prepared according J. Med. Chem. 2005, pp. 1596-1609)] and 3-methoxy-l-propanol (9.5 g, 36.4 mmol) were dissolved in dichloromethane [DCM (200 ml)]. Triphenylphosphine (9.5 g, 36.4 mmol) and 1, 1 '- (azodicarbonyl) dipiperidine (9.2 g, 36.4 mmol) were added and the reaction mixture was stirred at room temperature over night. The reaction mixture was filtered, the solvent evaporated and the afforded residue was purified on several silica gel columns eluted with hexanes/ethyl acetate 9:1 which gave 4.15 g (75%) of the title compound with a purity of 80% according to GC.

Step b) 4-Cyclopropyl-3-(3-methoxy-propoxy)-benzoic acid methyl ester (Ib) Cyclopropylboronic acid pinacol ester (0.75 g, 4.46 mmol) was stirred in H₂O (0.8 mL) in the presence Of KsPO₄ (2.36 g, 11.1 mmol) for 15 min. The mixture was diluted with toluene (18 mL) and bromo derivative Ia was added. The mixture was degassed, and palladium(II)acetate (116 mg, 0.51 mmol) and tricyclohexylphosphine was added. The mixture was heated to 100 ⁰C, and stirred for 24 h. Evaporation and purification by column chromatography (gradient elution with hexanes/THF 30:1 - 4:1) gave the title compound (0.88 g, 88%). GC/MS:[M+ ion at 264.

Step c) [4-Cyclopropyl-3 -(3 -methoxy-propoxy)-phenyl1 -methanol (Ic)

Methyl ester Ia was dissolved in heptane, the mixture was cooled to 0 ⁰C and stirred. DIBAL-H

(3 eq.) was added dropwise. The reaction was quenched after 30 min by careful dropwise addition of 3M HCL. The mixture was diluted with Et₂O and the combined organic layers were washed with 1 M HCl and brine, dried (MgSO4), filtered and evaporated, which gave the title compound (93%).

Step d) 4-Bromomethyl-l-cvclopropyl-2-(3-methoxy-propoxy)-benzene (Id) The benzyl alcohol Ic and triphenylphosphine (1.05 eq.) was dissolved in DCM and the solution was stirred and cooled to 0 ⁰C on an ice bath. A solution of tetrabromo methane (1.1 eq.) in DCM was added dropwise after which the cooling bath was removed and stirring was continued for 2 h. Additional triphenylphosphine (0.13 eq.) was added and stirring was continued for 2 h. of SiO2 was added and the solvent was evaporated. The product was purified by column chromatography (gradient elution with hexanes/ethyl acetate 50:1 - 20:1) which gave the title compound (73%).

Step e) 5- (2-[4-Cyclopropyl-3-(3-methoxy-propoxy)-benzyloxyl- 1 -hydroxy-ethyll -3-isopropyl- dihydro-furan-2-one (Ie)

A solution of the dihydroxy compound I-lf and Bu₂SnO (1 eq.) in toluene was refluxed with a Dean-Stark trap for 4 h. The reaction mixture was cooled down to 90 ⁰C and Id (leq.) and Bu₄NBr (1 eq.) were added. The mixture was stirred for further 22 h at 90 ⁰C, concentrated under vacuum and the residue purified by flash chromatography on silica gel (Hex:EtOAc 4:1 to 1 :1 with 1% MeOH throughout) which gave the title compound in 84% yield. LC/MS [M+NH₄]⁺ 424, [M+Na]⁺ 429.

Step f) 5- ( 1 -Azido-2-[4-cyclopropyl-3-(3-methoxy-propoxy)-benzyloxyl-ethyl| -3-isopropyl- dihydro-furan-2-one (If)

A solution of compound Ie (0.85 g, 2.09 mmol) and pyridine (1.7 eq.) in CH₂Cl₂ was treated with Tf₂O (1.3 eq.) at room temperature. After stirring for 30 min at room temperature, the reaction was quenched with H₂O. The product was extracted into CH₂Cl₂ and the combined extracts were dried (Na₂SO₄) and the solvent evaporated. The afforded residue was dissolved in DMF, sodium azide (5 eq.) was added and the resulting suspension was stirred at 60 ⁰C for 1 h. The reaction mixture was concentrated under vacuum and the residue was purified by flash chromatography on silica gel (Hex:EtOAc 7:1- 3:1) which gave the title compound (475 mg, 53%) along with some minor impurities. Step g) 2-Amino-3-methyl-pentanoic acid 4-fluoro-benzylamide (Ig)

A solution of Boc- VaI-OH (2.5 g, 12.5 mmol), EtN¹Pr (2.3 eq) and HATU (0.9 eq.) in DMF was stirred for 5 min at 0 ⁰C whereafter 4-fluorobenzylamine (1 eq.) was added. The reaction mixture was stirred for 20 min and then concentrated under vacuum. The residue was taken into EtOAcZH₂O and the organic phase was washed with H₂O, dried (Na₂SO₄) and evaporated. The crude product was purified by flash chromatography on silica gel (Hex: EtOAc 2.5:1) which gave the boc-protected title compound. The afforded residue was dissolved in TFA and the resulting solution was stirred at room, temperature. After Ih. the solvent was evaporated and the residue was taken into CH₂Cl₂ and washed with 1 M NaOH. The organic phase was dried (Na₂SO₄) and concentrated which gave the title compound (78%).

Step h) 5-Azido-6-r4-cvclopropyl-3-(3-methoxy-propoxy)-benzyloxyl-4-hvdroxy-2-isopropyl- hexanoic acid ri-(4-fluoro-benzylcarbamoyl)-2-methyl-butyl^"|-amide (Ih) Lactone If (I eq.), the amine Ig (4 eq.), 2-hydroxypyridine (4 eq.) and DIPEA (1.1 eq.) were stirred and heated to 70 ⁰C for 3 days. The mixture was cooled to room temperature and t- BuOMe was added and the afforded suspension was heated to 50 ⁰C and sonicated for 5 min, then centrifuged at 3500 rpm at 7 ⁰C for 12 min after which the supernatant was siphoned off. This process was repeated three times which gave the title compound in 28% yield. LC/MS [M+H]⁺ 670.

Step i) 5-Amino-6-[4-cyclopropyl-3-(3-methoxy-propoxy)-benzyloxyl-4-hydroxy-2-isopropyl- hexanoic acid [l-(4-fluoro-benzylcarbamoyl)-2-methyl-butyl^"|-amide (Ii) A solution of the azide Ih (22 mg, 0.033 mmol) in MeOH was hydrogenated at atmospheric pressure in the presence of Lindlar's catalyst. The mixture was filtered and the solvent evaporated, the residue was dissolved in 1,4-dioxane and freeze dried which gave the title compound (21 mg, 97%). LC/MS [M+H]⁺ 644.3. Purity according to HPLC was >95%.

Example 2

2f, R = N₃ 2g, R = NH₂

Step a) [4-Bromo-3-(3-methoxy-propoxy)-phenyl]-methanol (2a)

The methyl ester Ia (3.00 g, 9.90 mmol) was reduced according to the procedure described in

Example 1 step c, which gave the title compound (2.60 g, 95%).

Step b) l-Bromo-4-bromomethyl-2-(3-methoxy-propoxy)-benzene (2b)

The benzyl alcohol 2a (2.60 g, 9.45 mmol) was reacted according to the procedure described in Example 1 step d, which gave the title compound (2.62 g, 82%) after purification by column chromatography (gradient elution with hexanes/ethyl acetate 30:1 - 1 :1).

Step c) 5- (2-[4-Bromo-3-(3-methoxy-propoxy)-benzyloxy^"|- 1 -hydroxy-ethyll -3-isopropyl- dihydro-furan-2-one (2c)

The benzyl bromide derivative 2b (2.62 g, 7.75 mmol) was coupled to the lactone I- If according to the procedure described in Example 1 step e, which, after column chromatography (gradient elution with hexanes/ethyl acetate 7:1 - 1 :1 with 1% MeOH throughout), gave the title compound (3.21 g, 85%). LC/MS [M+H]⁺ 445/447 , [M+Na]⁺ 467/469.

Step d) 5- { 1 -Azido-2-r4-bromo-3-(3-methoxy-propoxy)-benzyloxy^"|-ethvU -3-isopropyl-dihvdro- furan-2-one (2d)

Alcohol 2c (153 mg, 0.336 mmol) was reacted according to the procedure described in Example 1 step f which gave the title compound (121 mg, 77%). Step e) 5- ( 1 -Azido-2-[3-(3-methoxy-propoxy)-4-pyridin-3-yl-benzyloxyl-ethyl| -3-isopropyl- dihydro-furan-2-one (2e)

A microwave reactor tube was charged with bromide 2d (50 mg, 0.106 mmol), pyridine-3- boronic acid (17 mg, 0.138 mmol), l,l '-bis(diphenylphosphino)ferrocene- palladium(II)dichloride dichloromethane complex ((dppf)2PdCl2) (4.3 mg, 5.3 μmol), 3.8 mL of a 5:2:1 mixture of 1,4-dioxane, ethanol and 2 M K3PO4 solution. The mixture was degassed by 5 cycles of vacuum/^, and subsequently heated to 140 ⁰C for 15 min in the microwave reactor. The mixture was diluted with Et₂O, EtOAc, and sat aq NaHCO₃ (7 mL of each). The phases were separated and the aqueous layer was extracted with Et₂O (2 x 10 mL). The combined organic layers were washed with 2 x 7 mL sat aq NaHCO₃, dried (MgSO₄), filtered and evaporated. The product was purified by column chromatography (gradient elution with hexanes/ethyl acetate 3:2 - 100% EtOAc) which gave the title compound (21 mg, 42%)

Step f) 5-Azido-4-hydroxy-2-isopropyl-6-[3-(3-methoxy-propoxyV4-pyridin-3-yl-benzyloxy^"|- hexanoic acid [l-(4-fluoro-benzylcarbamoylV2-methyl-butyl^"|-amide (2f) The lactone 2e (31 mg, 0.066 mmol) was reacted with the amine Ig according to the method described in Example 1 step h. The product was purified by diluting the reaction mixture with DCM (10 mL) and saturated aqueous NaHCO₃ solution (10 mL). The phases were separated and the organic phase was evaporated and redispersed in MeCN (1 mL), sonicated, centrifuged, and the supernatant siphoned off (process repeated 4 times) which gave title compound (19 mg, 41%) . LC/MS [M+H]⁺ 707.3.

Step g) 5-Amino-4-hydroxy-2-isopropyl-6-[3-(3-methoxy-propoxyV4-pyridin-3-yl-benzyloxy^"|- hexanoic acid [l-(4-fluoro-benzylcarbamoylV2-methyl-butyl^"|-amide (2g) The azide 2f (19 mg, 0.033 mmol) was reacted as described in Example 1 step i, which gave the title compound (18 mg, 99%). LC/MS [M+H]⁺ 681.4. Purity according to HPLC was >95%.

Example 3

3f, R = N₃ 3g, R = NH₂

Step a) 3-(3-Methoxy-propoxy)-4-trifluoromethyl-benzoic acid methyl ester (3a) Bromide Ia (0.80 g, 2.64 mmol), DMF (5.3 ml), methyl 2-chloro-2,2-difluoroacetate (0.76 g, 5.28 mmol), KF (0.16 g, 2.77 mmol), and CuI (0.53 g, 2.77 mmol) were added in sequence to a screw-cap tube under N₂ atmosphere. The tube was sealed and heated to 120 ⁰C for 18 h after which the tube was allowed to cool and opened to release pressure and more methyl 2-chloro- 2,2-difluoroacetate (3 mL), KF (80 mg), and CuI (250 mg) were added, the mixture was degassed and heated again for 18 h. Three further additions of methyl 2-chloro-2,2- difluoroacetate were made during the following 48 h. The mixture was diluted with Et₂O (25 mL) and filtered through Celite which was washed with Et₂O (25 mL). The filtrate was washed with sat aq NaHCO₃ (2 x 20 mL) and IM HCl (2 x 20 mL), brine (20 mL) and dried (MgSO₄), filtered and evaporated which gave a crude brown oil. Purification by column chromatography (gradient elution with hexanes/ethyl acetate 20:1 - 5:1) gave the title compound along with some minor impurities (0.61 g), which was used as such in the next step. GC/MS (EI) m/z 292, 260, 189, 160, 141, 73, 45.

Step b) [3 -(3 -Methoxy-propoxy)-4-trifluoromethyl-phenvH -methanol (3b) The methyl ester 3a (0.60 g, 2.05 mmol) was reduced according to the procedure described in Example 1 step c, which gave the title compound along with some minor impurities (0.52 g). The afforded compound was used as such in the next step. Step c) 4-Bromomethyl-2-(3-methoxy-propoxy)-l-trifluoromethyl-benzene (3 c) The benzyl alcohol 3b was reacted according to the procedure described in Example 1 step d, the product was purified by column chromatography (gradient elution with hexanes/ethyl acetate 100:1 - 5:1) which gave the title compound (199 mg, 23% over two steps).

Step d) 5- { 1 -Hvdroxy-2-r3-(3-methoxy-propoxy)-4-trifluoromethyl-benzyloxyl-ethvU -3- isopropyl-dihydro-furan-2-one (3d)

The bromide derivative 3c was reacted with the diol If according to the procedure described in Example 1 step e, which gave the title compound in 64% yield after column chromatography (gradient elution with hexanes/ethyl acetate 10:1 - 2:1 with 1% MeOH throughout). LC/MS [M+H]⁺ 434, [M+ NH₄J⁺ 452.

Step e) 5- { 1 -Azido-2-r3-(3-methoxy-propoxy)-4-trifluoromethyl-benzyloxyl-ethyl| -3-isopropyl- dihydro-furan-2-one (3e)

Alcohol 3d (169 mg, 0.388 mmol) and PPh₃ (133 mg, 0.505 mol) were dissolved in dry THF (15 mL), cooled to -15 ⁰C, and stirred. DIAD (107 μL, 0.544 mmol) was added, and the stirring was continued. After 10 min at -15 ⁰C the reaction was allowed to warm to 0 ⁰C and DPPA (117 μL, 0.544 mmol) was added. The mixture was stirred for 18 h, whereafter sat aq Na₂CO₃ (15 mL), and Et₂O (10 mL) was added. The layers were separated, the aqueous layer was extracted with 2 x 15 mL EtOAc, and the combined organic layers were washed with aq sat NaCO₃, IM HCl, brine (15 mL of each), dried (MgSO₄), filtered and evaporated. The afforded crude product was purified by column chromatography (gradient elution with hexanes/ethyl acetate 9:1 - 2:1) which gave the title compound (44 mg, 25%).

Step f) 5-Azido-4-hydroxy-2-isopropyl-6-[3-(3-methoxy-propoxy)-4-trifluoromethyl- benzyloxyl-hexanoic acid ri-(4-fluoro-benzylcarbamoyl)-2-methyl-butvH -amide (3f) The lactone 3e (44 mg, 0.096 mmol) was opened with the amine Ig according to the procedure described in Example 1 step h. The reaction mixture was diluted with DCM (10 mL) and IM HCl (10 mL). The phases were separated and the organic phase was evaporated and redispersed in MeCN (0.8 ml), sonicated, centrifuged, and the supernatant siphoned off (process repeated 3 times) which gave the title compound (34 mg, 51%) of. LC/MS [M+H]⁺ 698. Step g) 5-Amino-4-hydroxy-2-isopropyl-6-[3-(3-methoxy-propoxy)-4-trifluoromethyl- benzyloxyl-hexanoic acid [l-(4-fluoro-benzylcarbamoyl)-2-methyl-butyl^"| -amide (3g) Azide 3g (22 mg, 0.033 mmol) was reacted as described in Example 1 step i, which gave the title compound (32 mg, 96%). LC/MS [M+H]⁺ 672.4. Purity according to HPLC was >95%.

Example 4

4c 4d

Step a) 5- ( 1 -Hydroxy-2-[3-(3-methoxy-propoxy)-4-thiazol-2-yl-benzyloxyl-ethyl| -3-isopropyl- dihydro-furan-2-one (4a)

The bromide 2c (100 mg, 0.225 mmol), 2-tributylstannylthiazol (141 μL, 0.45 mmol), dichloro- bis(tricyclohexylphosphine)palladium(II) (25 mg, 0,033 mmol), CuI (9 mg, 0.045 mmol), LiCl (38mg, 0.898 mmol), 1,4-dioxane (1.5 mL), and DMF (0.2 mL) were mixed in a microwave reactor tube, which was sealed, thoroughly degassed, and heated in a microwave reactor for 1 h at 165 ⁰C. The mixture was diluted with MeCN (25 mL) and filtered through a syringe filter. The filtrate was extracted with isohexane (5 x 5 mL) and the isohexane extracts were discarded. The MeCN phase was evaporated and the residue redissolved in Et₂O (5 mL), EtOAc (5mL), and H₂O (20 mL). The phases were separated and the aqueous phase was extracted with a 1 : 1 Et₂O/EtOAc mixture (3 x 7 mL). The combined organic phases were washed with H₂O (2 x 7 mL) and brine (7 mL) and evaporated. The product was purified by column chromatography (gradient elution with hexanes/ethyl acetate 3:1 - 1 :1 with 1% MeOH throughout) which gave the title compound as a mixture of diastereomers (31 mg, 30%). Step b) 5- ( 1 -Azido-2-[3-(3-methoxy-propoxy)-4-thiazol-2-yl-benzyloxyl-ethyl| -3-isopropyl- dihydro-furan-2-one (4b)

Alcohol 4a (40 mg, 0.089 mmol) was dissolved in DCM (1 niL) and pyridine (14 μL, 0.178 mmol) was added, and the solution cooled to -20 ⁰C. Trifluoromethanesulfonic anhydride (19 μL, 0.11 mmol) was added, and the reaction was stirred for 10 min. Cooling was maintained while reaction was concentrated in vacuo. The residue was cooled to -78 ⁰C and DMF (1 mL) was added dropwise. NaN₃ (0.178 mmol, 12mg) was added (one portion), and the reaction was allowed to warm to -5 ⁰C for 1 h. The reaction was diluted with H₂O (20 mL) and extracted with a 1 :1 Et₂OZEtOAc mixture (3 x 10 mL). The organic extracts were washed with H₂O (2 x 10 mL), brine (10 mL), dried (MgSO₄), filtered and evaporated. The reaction was repeated to give 64 mg of crude title compound in total, which was used as such in the next step.

Step c) 5-Azido-4-hydroxy-2-isopropyl-6-[3-(3-methoxy-propoxy)-4-thiazol-2-yl-benzyloxy^"|- hexanoic acid [l-(4-fluoro-benzylcarbamoyl)-2-methyl-butyl^"|-amide (4c) The lactone 4b (28 mg, 0.059 mmol) was reacted with the amine Ig according to the procedure described in Example 1 step h. The reaction mixture containing the title compound as a mixture of diastereomers was taken to the next step without prior purification.

Step d) 5-Amino-4-hydroxy-2-isopropyl-6-[3-(3-methoxy-propoxy)-4-thiazol-2-yl-benzyloxy^"|- hexanoic acid [l-(4-fluoro-benzylcarbamoyl)-2-methyl-butyl^"|-amide (4d) The crude reaction mixture from step 4c was diluted with MeOH (30 mL) and was reacted as described in Example 1 step i which gave a diastereomeric mixture containing the title compound in 47% yield total yield from 4a. Separation of the diastereomeric mixture by preparative HPLC (NH₄OAc - MeCN gradient) gave the title compound (10.3 mg). Purity according to HPLC was > 95%. LC/MS [M+H]⁺ 687.4.

Example 5

5-Amino-4-hvdroxy-2-isopropyl-6-r3-(3-methoxy-propoxy)-4-thiazol-2-yl-benzyloxyl-hexanoic acid ri-(4-fluoro-benzylcarbamovi)-2-methyl-butyl1 -amide (5 a)

Separation of the diastereomeric mixture produced in Example 4 step d by preparative HPLC (NH₄OAc - MeCN gradient) yielded 8.8 mg of the title compound. Purity according to HPLC was > 95%. LC/MS [M+H]⁺ 687.3.

Example 6

Step a) 5- { 1 -Hvdroxy-243-(3-methoxy-propoxy)-4-pyridin-2-yl-benzyloxy1-ethyU -3-isopropyl- dihydro-furan-2-one (6a)

The bromide 2c was coupled to 2-tributylstannylpyridine (145 μL, 0.45 mmol) according to the procedure described in Example 4 step a. The reaction was worked up by dilution with EtOAc (5 mL), Et₂O (5 mL), and sat aq NaHCO₃ (15 mL), followed by filtration, and the phases were separated. The aqueous phase was extracted with a 1 :1 Et₂OZEtOAc mixture (3 x 10 mL), and the combined organic extracts were washed with aq sat NaHCO₃ (2 x 5 mL), dried (MgSO₄), filtered and evaporated. The product was purified by column chromatography (gradient elution with hexanes/ethyl acetate 3:1 - 100% EtOAc). The step was repeated to give 36 mg of title compound as a mixture of diastereomers (31 mg).

Step b) 5- { 1 -Azido-2-r3-(3-methoxy-propoxy)-4-pyridin-2-yl-benzyloxyl-ethvU -3-isopropyl- dihydro-furan-2-one (6b)

The alcohol 6a (31 mg, 0.070 mmol) was reacted according to the procedure described in Example 4 step b, which gave the title compound as a mixture of diastereomers (32 mg, 97% total yield).

Step c) 5-Azido-4-hvdroxy-2-isopropyl-6-r3-(3-methoxy-propoxy)-4-pyridin-2-yl-benzyloxyl- hexanoic acid ri-(4-fluoro-benzylcarbamoyl)-2-methyl-butvH-amide (6c) The lactone 6b (32 mg, 0.068 mmol) was reacted with he amine Ig according to the procedure described in Example 1 step h. The product was isolated from the reaction mixture by preparative HPLC (NH₄OAc - MeCN gradient) as a mixture of diastereomers along with some minor impurities and was used as such in the next step.

Step d) 5-Amino-4-hydroxy-2-isopropyl-6-[3-(3-methoxy-propoxy)-4-pyridin-2-yl-benzyloxy|- hexanoic acid [l-(4-fluoro-benzylcarbamoyl)-2-methyl-butyl^"|-amide (6d) The crude reaction mixture from Example 6 step c was reacted as described in Example 1 step i which gave the title compound as a diastereomeric mixture (43% total yield from 6b). The title compound was isolated by preparative HPLC (NH₄OAc - MeCN gradient) which yielded 9.9 mg. Purity according to HPLC was > 95%. LC/MS [M+H]⁺ 681.4.

Example 7

5-Amino-4-hvdroxy-2-isopropyl-6-r3-(3-methoxy-propoxy)-4-pyridin-2-yl-benzyloxyl-hexanoic acid ri-(4-fluoro-benzylcarbamoyl)-2-methyl-butvH -amide (7) Separation of the diastereomeric mixture produced in Example 6 step d by preparative HPLC (NH₄OAc - MeCN gradient) gave the title compound (7.8 mg). Purity according to HPLC was > 95%. LC/MS [M+H]⁺ 681.4.

Example 8

8d, R = NH₂

Step a) 5- { 1 -Hvdroxy-2-r3-(3-methoxy-propoxy)-4-thiazol-5-yl-benzyloxyl-ethvU -3-isopropyl- dihydro-furan-2-one (8a)

Bromide 2c (100 mg, 0.225 mmol), KOAc (33 mg, 0.33 mol), Pd(PPh₃)₄ (13 mg, 0.011 mmol), thiazole (78 μL, 1.12 mmol), and N,N-dimethylacetamide (1.5 mL) were mixed in a vial fitted with a septum. The mixture was degassed and heated to 145 ⁰C for 3 h. The mixture was filtered through a syringe filter, and the filter was washed with Et₂O (7 mL), EtOAc (7 mL), and H₂O (15 mL). The phases were separated and the aqueous layer was extracted with an Et₂O/EtOAc 1 : 1 mixture (15 mL). The combined organic phases were washed with H₂O (10 mL) and brine (10 mL), and evaporated. The crude product was redissolved in 1,4-dioxane and freeze-dried. The procedure was repeated, and the combined material was purified by column chromatography (gradient elution with hexanes/ethyl acetate 4:1 - 100% EtOAc with 1% MeOH throughout) which gave the title compound (74 mg, 37%).

Step b) 5- { 1 -Azido-2-r3-(3-methoxy-propoxy)-4-thiazol-5-yl-benzyloxyl-ethvU -3-isopropyl- dihydro-furan-2-one (8b)

The alcohol 8a (39 mg, 0.070 mmol) was reacted according to the procedure described in Example 4 step b, which gave the title compound along with some minor impurities, and the product was used as such in the next step (average yield 74% from two experiments, 64 mg).

Step c) 5-Azido-4-hvdroxy-2-isopropyl-6-r3-(3-methoxy-propoxy)-4-thiazol-5-yl-benzyloxyl- hexanoic acid ri-(4-fluoro-benzylcarbamoyl)-2-methyl-butyl^"|-amide (8c) The lactone 8b (64 mg, 0.13 mmol) was reacted with the amine Ig according to the procedure described in Example 1 step h. The reaction mixture was diluted with DCM (10 mL) and H₂O (10 mL). The phases were separated and the organic phase was evaporated and redispersed in MeCN (1 ml), sonicated, centrifuged, and the supernatant siphoned off (process repeated 3 times), which gave the title compound along with some minor impurities. The product was used as such in the next step.

Step d) 5-Amino-4-hvdroxy-2-isopropyl-6-r3-(3-methoxy-propoxy)-4-thiazol-5-yl-benzyloxy^"|- 5 -methyl- hexanoic acid ri-(4-fluoro-benzylcarbamoyl)-2-methyl-butyl1 -amide (8d) The crude azide 8c was reacted as described in Example 1 step i which, after purification by preparative HPLC (NH₄OAc - MeCN gradient), gave the title compound (42.6 mg, 48% yield from 8b). Purity according to HPLC was > 95%. LC/MS [M+H]⁺ 687.4.

Example 9

9b, R = N₃ 9c, R = NH₂

Step a) 2-Amino-3-methyl-pentanoic acid 4-cyano-benzylamide (9a)

4-Cyanobenzylamine was reacted with Boc- VaI-OH according to the procedure described in

Example 1 step g, which gave the title compound (43%).

Step b) 5-Azido-6-r4-cvclopropyl-3-(3-methoxy-propoxy)-benzyloxyl-4-hvdroxy-2-isopropyl- hexanoic acid ri-(4-cvano-benzylcarbamoyl)-2-methyl-butyl^"|-amide (9b)

Lactone If (30 mg, 0.070 mmol) was opened with the amine 9a (68 mg, 0.28 mmol) according to the procedure described in Example 1 step h, which gave the title compound (17 mg, 36%).

Step c) 5-Amino-6-r4-cvclopropyl-3-(3-methoxy-propoxy)-benzyloxyl-4-hydroxy-2-isopropyl- hexanoic acid ri-(4-cvano-benzylcarbamoyl)-2-methyl-butyl^"|-amide (9c) Azide 9b was reacted as described in Example 1 step i. Purification by preparative HPLC (NH₄OAc - MeCN gradient) gave the title compound (8.0 mg, 50%). Purity according to HPLC was > 95%. LC/MS [M+H]⁺ 651.4.

Example 10

10c 10d, R = N₃ 10e, R = NH₂

Step a) 5-π.2-Dihydroxy-ethy0-3-methyl-dihydro-furan-2-one (IQa) The procedure described for the preparation of intermediate I- Ie was followed but using iodomethane instead of iodopropane, subsequent removal of the benzyl groups as described in intermediate 1 , step f, gave the title compound.

Step b) 5- (2-[4-cyclopropyl-3-(3-methoxy-propoxy)-benzyloxyl- 1 -hydroxy-ethyll -3-methyl- dihydro-furan-2-one (IQb)

The diol 10a was alkylated with the benzyl bromide derivative Id according to the procedure described in Example 1 step e. Silica gel column chromatography (gradient Q - ¹A - I - 2%EtOH/DCM) gave the title compound (81 mg, 74%). LC/MS [M+NH₄]⁺ 396.

Step c) 5- { 1 -azido-2-r4-cvclopropyl-3-(3-methoxy-propoxy)-benzyloxyl-ethvU -3-methyl- dihydro-furan-2-one (IQc)

The alcohol 10b (81 mg, 0.21 mmol) and Ph₃P (75 mg, 0.28 mmol) were dissolved in dry THF (1.6 ml). The mixture was cooled to -15⁰C and then DIAD (60 mg, 0.3 mmol) was added and the mixture was stirred for 15 min. The temperature was settled at 0 ⁰C and then DPPA (82 mg, 0.3 mmol) was added and the mixture was stirred for 30 min and then stirred at room temperature over night. The mixture was evaporated on rotavapor. Silica gel column chromatography (gradient 0 - Y₂ - l%EtOH/DCM) gave the title compound (51 mg, 59%). LC/MS [M+NH₄]⁺ 421.

Step d) 5-Azido-6-r4-cvclopropyl-3-(3-methoxy-propoxy)-benzyloxyl-4-hvdroxy-2-methyl- hexanoic acid ri-(4-fluorobenzylcarbamoyl)-2-methyl-butvH -amide (IQd) The lactone 10c (51 mg, 0.126 mmol) was reacted with the amine Ig according to the procedure described in 1 step h which gave the title compound (40 mg, 49%). LC/MS [M+H]⁺ 642.

Step e) 5-Amino-6-r4-cvclopropyl-3-(3-methoxy-propoxy)-benzyloxyl-4-hvdroxy-2-methyl- hexanoic acid ri-(4-fluorobenzylcarbamoyl)-2-methyl-butvH -amide (IQe) The azide 1Od (40 mg, 0.062 mmol) was dissolved in MeOH (40 ml) and the solution was ran through H-Cube with a cassette containing 5% Pd on calcium carbonate and the resulting reaction solution was evaporated on rotavapor. Freeze drying from water gave the title compound as a solid (29.3 mg, 77%). LC/MS [M+H]⁺ 616. Purity >95%.

Example 11

11c 11 d, R = N₃ 11e, R = NH₂

Step a) 5-(l,2-Dihvdroxy-ethyl)-3-ethyl-dihvdro-furan-2-one (Ha) The procedure described for the preparation of intermediate I- Ie was followed but using iodoethane instead of iodopropane, subsequent removal of the benzyl groups as described in intermediate 1 , step f, gave the title compound.

Step b) 5- {2-r4-cvclopropyl-3-(3-methoxy-propoxy)-benzyloxyl- 1 -hydroxy-ethyll -3-ethyl- dihydro-furan-2-one (1 Ib)

The diol 1 Ia (50.5 mg, 0.29 mmol) was alkylated with the benzyl bromide derivative Id according to the procedure described in Example 1 step e. Silica gel column chromatography

(gradient 0 - ¹A - I - 2%EtOH/DCM) gave the title compound (97 mg, 85%). LC/MS [M+NH₄]⁺

410.

Step c) 5- { 1 -azido-2-r4-cvclopropyl-3-(3-methoxy-propoxy)-benzyloxyl-ethvU -3-ethyl-dihydro- furan-2-one (l ie)

The alcohol 1 Ib (97 mg, 0.25 mmol) was treated with Ph₃P, DIAD and DPPA according to the method described in Example 10, step c, which gave the title compound (70 mg, 68%). LC/MS

[M+NH₄]⁺ 435.

Step d) 5-Azido-6-[4-cyclopropyl-3-(3-methoxy-propoxy)-benzyloxyl-4-hydroxy-2-ethyl- hexanoic acid [l-(4-fluorobenzylcarbamoyl)-2-methyl-butyl] -amide (l id) The lactone l ie (50 mg, 0.12 mmol) was reacted with the amine Ig according to the procedure described in 1 step h which gave the title compound (55 mg, 70%). LC/MS [M+H]⁺ 656.

Step e) 5-Amino-6-[4-cyclopropyl-3-(3-methoxy-propoxy)-benzyloxyl-4-hydroxy-2-ethyl- hexanoic acid [l-(4-fluorobenzylcarbamoyl)-2-methyl-butyl] -amide (l ie)

The azide 1 Id (55 mg, 0.084 mmol) was ) was reacted according to the procedure described for

Example 10, step e, which gave the title compound (47 mg, 89%). LC/MS [M+H]⁺ 630. Purity

>95%.

Example 12

12d, R = N₃ 12e, R = NH₂

Step a) 5-(1.2-Dihydroxy-ethyl)-3-propyl-dihydro-furan-2-one (12a)

The procedure described for the preparation of intermediate 1, step e was followed but using 1- iodopropane instead of 2-iodopropane, followed by removal of the benzyl groups as described in intermediate 1 , step f, which gave the title compound.

Step b) 5- (2-[4-cyclopropyl-3-(3-methoxy-propoxy)-benzyloxyl- 1 -hydroxy-ethyll -3-propyl- dihydro-furan-2-one (12b)

The lactone 12a (54.6 mg, 0.29 mmol) was alkylated with the bromo derivative Id according to the procedure described in Example 1 step e. Silica gel column chromatography (gradient 0 - ¹A - 1 - 2%EtOH/DCM) gave the title compound (62 mg, 53%). LC/MS [M+NH₄]⁺ 424.

Step c) 5- { 1 -azido-2-r4-cvclopropyl-3-(3-methoxy-propoxy)-benzyloxyl-ethvU -3-propyl- dihydro-furan-2-one (12c)

The alcohol 12b (62 mg, 0.15 mmol) was treated with Ph₃P, DIAD and DPPA according to the method described in Example 10, step c, which gave the title compound (28 mg, 42%). LC/MS [M+NH₄]⁺ 449.

Step d) 5-Azido-6-r4-cvclopropyl-3-(3-methoxy-propoxy)-benzyloxyl-4-hvdroxy-2-propyl- hexanoic acid ri-(4-fluorobenzylcarbamoyl)-2-methyl-butvH -amide (12d) The lactone 12c (28 mg, 0.065 mmol) was reacted with the amine Ig according to the procedure described in 1 step h, which gave the title compound (28 mg, 55%). LC/MS [M+H]⁺ 670.

Step e) 5-Amino-6-r4-cvclopropyl-3-(3-methoxy-propoxy)-benzyloxyl-4-hvdroxy-2-propyl- hexanoic acid ri-(4-fluorobenzylcarbamoyl)-2-methyl-butvH -amide (12e)

The azide 12d (28 mg, 0.042 mmol) was reacted according to the procedure described for

Example 10, step e, which gave the title compound (40 mg, quant.). LC/MS [M+H]⁺ 644. Purity

>95%.

Example 13

13d, R = NH₂

Step a) 5- (2-[4-Cyclopropylethynyl-3-(3-methoxy-propoxy)-benzyloxyl- 1 -hydroxy-ethyll -3- isopropyl-dihydro-furan-2-one (13a)

A mixture of a bromo compound 2c [250 mg, 0.55 mmol], a 70 % solution of cyclopropylacetylene in toluene (250 μl), cesium carbonate (50 mg) and dichlorobis(tricyclohexylphosphine)palladium(II) (125 mg) in DMSO (3 ml) was heated at + 100° C for 24 hours. Thereafter the reaction mixture was lyophilized and the residue was dissolved in ethyl acetate and washed with brine. The organic phase was evaporated and the residue purified on a silica gel column eluting with hexanes 3 and ethyl acetate 2 which gave the title compound (183 mg, 76%). LC/MS confirmed the structure with a major positive peak at 448 (M+NH₄).

Step b) 5-{l-Azido 244-cvclopropylethvnyl-3-(3-methoxy-propoxy)-benzyloxy|-ethvU-3- isopropyl-dihydro-furan-2-one ( 13b)

A solution of the alcohol 13a (183 mg, 0.43 mmol) and triphenylphosphine (147 mg, 0.56 mmol) in THF (10 ml) under nitrogen was cooled to -15 °C. Diethyl azodicarboxylate (120 μl, 0.6 mmol) was added and the mixture was stirred at -15 °C for 15 minutes wherafter the temperature was allowed to rise to 0 °C and diphenylphosphoryl azide (130 μl, 0.6 mmol) was added. The temperature was allowed to reach room temperature and stirred over night. The reaction mixture was evaporated and the residue purified on silica gel using hexanes 3 and ethyl acetate 1 followed by a final purification on reparative HPLC to give 56 mg (29%) of the title compound. LC/MS confirmed the structure with a major positive peak at 473 (M+NH4).

Step c) 5-Azido-6-r4-cvclopropylethvnyl-3-(3-methoxy-propoxy)-benzyloxyl-4-hvdroxy-2- isopropyl-hexanoic acid ri-(4-fluoro-benzylcarbamoyl)-2-methyl-butvH -amide (13c) A mixture of compound 13b (17 mg, 0.0374 mmol)], the amine Ig (50 mg), 2-hydroxypyridine (50 mg) and DIPEA (150 μl) was heated at + 70° C for 3 days. The reaction mixture was evaporated and the residue purified by preparative LC/MS which gave 18 mg of the title compound. LC/MS confirmed the structure with a M+l ion at 694.

Step d) 5-Amino-6-[4-cyclopropylethynyl-3-(3-methoxy-propoxy)-benzyloxyl-4-hydroxy-2- isopropyl-hexanoic acid [l-(4-fluoro-benzylcarbamoyl)-2-methyl-butyl] -amide (13d) To a solution of compound 13c (18 mg, 0.026 mmol) in THF (3 ml) and methanol (1 ml) was added water (3 drops) and triphenylphosphine (8 mg, 0.03 mmol). The reaction mixture was then stirred at room temperature for about 1 week. The reaction mixture was evaporated and the residue purified on preparative HPLC using a Phe column which gave 5 mg (29%) of the title compound. LC/MS confirmed the structure with a M+l ion at 668.

Example 14

Step a) 5-Azido-6-r4-cvclopropylethvnyl-3-(3-methoxy-propoxy)-benzyloxyl-4-hvdroxy-2- isopropyl-hexanoic acid ri-(4-cvano-benzylcarbamoyl)-2-methyl-butvH -amide (14a) A mixture of example 13b (21 mg, 0.046 mmol)], amine 9a (50 mg), 2-hydroxypyridine (50 mg) and DIPEA (200 μl) was heated at +70° C for 3 days. The reaction mixture was evaporated and the residue purified by preparative LC/MS to afford 10 mg of the title compound. LC/MS confirmed the structure with a M+l ion at 701 and a M-I ion at 699.

Step b) 5 -Amino-6- r4-cyclopropylethvnyl-3 -(3 -methoxy-propoxy)-benzyloxy1 -4-hvdroxy-2- isopropyl-hexanoic acid ri-(4-cvano-benzylcarbamoyl)-2-methyl-butvH -amide (14b) To a solution of compound 14a (10 mg, 0.014 mmol) in methanol (2 ml) was added water (2 drops) and triphenylphosphine (4 mg, 0.015 mmol). The reaction mixture was then stirred at + 50 °C for about 24 hours. The reaction mixture was evaporated and the residue purified on preparative HPLC using a Phe column to give about 1.1 mg (11%) of the title compound. LC/MS confirmed the structure with a M+l ion at 675.

Biological Examples

To evaluate the enzymatic inhibition of renin exhibited by the compounds of the invention, an assay using Fluorescence Resonance Energy Transfer (FRET) to generate a spectroscopic response to peptidase cleavage, was used. The activity was measured by a continuous detection of increased fluorescence intensity exhibited by the clevage product (peptide-EVANS). The enzyme used in the assay was recombinant human renin (supplied by Proteos), the substrate consisted of a peptide which in one end is linked to a fluorophore, 5-

(aminoethyl)aminonaphtalene sulphonate (EDANS), and in the other end to a non-fluorescent chromophore, 4'-dimethylaminoazobenzene (Dabcyl), typically Arg-Glu(ED ANS)-Ile-His-Pro- Phe-His-Leu-Val-Ile-His-Thr-Lys(D ABCYL)- Arg (Sigma-Aldrich). The cleavage site by human renin is the peptide bond between Leu and VaI. The compounds were tested at a range of concentrations whereas the enzyme and substrate concentrations were fixed. The assay used employs the enzyme at a concentration of 6.25nM in an assay buffer consisting of of 0.1 mM Tris-HCl, 0.05 M NaCl, 0.5 mM EDTA, 0.05% CHAPS at pH=7.4. The substrate was prepared at a 20 μM stock solution in DMSO. To each well of a 96-well polypropylene plate was added the enzyme containing assay buffer (90.0 μl) and inhibitor of different concentrations (1 μl). To controll wells were added DMSO (1 μl) instead of inhibitor. The renin enzyme was preactivated by incubation at 37 ⁰C for 20 min whereafter the reactions were started by addition of substrate, 10 μl/well, thus giving a total volume of 100 μl/well and a substrate concentration of 2 μM. The assay was performed during 20 min at 37 ⁰C. The total concentration of DMSO was not above 1 %. Product fluorescence (emission filter 340 nM, excitation filter 500 nM) was monitored with a Thermo Labsystems Fluoroskan Ascent plate reader. The Ki was determined by Prism Software. Activity of the inhibitors was determined by measuring the fluorescence at λg_X 340nm and λ_em 500nm. Percent inhibition is calculated as follows: % Inhibition is equal to the (Fluorescence^, inhibitor - Fluorescence_&aC£gr<w); divided by the (Fluorescence _{mmus m}hώιto_r - Fluorescence_&aC£gr<w);

For example, Table 1 shows the Ki- value expressed in nM for a representative selection of compounds according to the invention when tested in an renin enzyme assay such as the one described above. Category A indicates < 0.1 nM inhibition, category B indicates 0.1 - 1 nM inhibition and category C indicates > 1 nM:

Selectivity for/against other related enzymes, such as cathepsin D or BACE is determined with the corresponding FRET assays for the respective recombinant enzyme, which are commercially available and extensively described in the literature.

All references referred to in this application, including patent and patent applications, are incorporated herein by reference to the fullest extent possible.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the following claims:

Claims

Claims

1. A compound according to formula I

wherein

Q is trifluoromethyl, Cs-Cβcycloalkyl, phenyl, p-fluorophenyl, pyridyl, thiazolyl, furyl, thienyl or Cs-Cβcycloalkylethynyl;

R³ is Ci-Cealkyl;

R⁴ is Ci-Cealkyl;

W is cyano or fluoro; or a pharmaceutically acceptable salt, hydrate or N-oxide thereof.

2. A compound according to claim 1, wherein Q is p-fluorophenyl, 2-pyridyl, 5 -thiazolyl or cyclopropyl.

3. A compound according to any of claim 1 or 2, wherein R³ is ethyl or isopropyl.

4. A compound according to any preceding claim, wherein R⁴ is sec. butyl.

5. A compound according to any preceding claim, wherein W is fluoro.

6. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, as claimed in any one of the preceding claims in association with a pharmaceutically acceptable adjuvant, diluent or carrier.

7. Use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof as claimed in any one of claims 1 to 5, in the treatment or prevention of hypertension heart failure, glaucoma, cardiac infarction, kidney failure or restenosis, preferably hypertension.