WO2003104189A2 - Compounds - Google Patents

Abstract

The invention relates to compounds of formula (I) wherein R¹ represents benzyl; R² represents pyrazole; or a pharmaceutically acceptable derivative thereof. The invention also relates to pharmaceutical compositions containing compounds of formula (I) and to the use of compounds of formula (I) in medicine, particularly in the amelioration of a clinical condition for which a ACE and/or NEP inhibitor is indicated.

Description

Compounds

Field of the Invention

The present invention relates to thiol derivatives with metalloprotease activity, more particularly N-mercaptoacyl phenylalanine derivatives with mixed ACE-NEP inhibitory activity, to pharmaceutical compositions containing them and to their use in medicine.

Background of the Invention

Angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) are two zinc metalloproteases involved in the metabolism of a variety of regulatory peptides and particularly those involved in the control of blood pressure and fluid homeostasis (Fournie- Zaluski et al. (1994) J. Med. Chem. 37:1070-1083). ACE, a zinc-containing carboxydipeptidase, converts the inactive precursor angiotensin I into angiotensin II, a peptide which promotes vasoconstriction and sodium retention and thereby leads to an increase in blood pressure. Compounds with ACE inhibitory activity are useful in the treatment of hypertension, heart failure and post-infarct. NEP (also called 'enkephalinase') is a zinc-containing endopeptidase that is found in high concentration within the brush border region of the kidney. NEP inactivates the atrial natriuretic factor (ANF). ANF is a hormone secreted by heart which increases the vasodilatation and, on the renal level, increases diuresis and nathuresis. Compounds with inhibitory activity of the neutral endopeptidase (NEP) enzyme are useful as vasodilators. Both ACE and NEP are responsible for the degredation of the vasorelaxant peptide bradykinin at its endothelial and epithelial sites of action respectively. Therefore, as ACE and NEP exert their action on the cardiovascular system with different mechanisms of action, compounds with mixed ACE-NEP inhibitory activity are generally used, alone or in combination, in the treatment of hypertension, renal failure, congestive heart failure and ischemic cardiopathologies.

WO 97/24342, herein incorporated by reference, discloses certain N-mercaptoacyl phenylalanine derivatives of formula (A) which have mixed ACE-NEP inhibitory activity and are useful in the treatment of cardiovascular diseases e.g. hypertension and congestive heart failure.

H

R— C— C— CONH C— COOR,

H₂ H

C— R,

H₂ (A) wherein:

R is a mercapto group or a R₄COS group convertible in an organism to a mercapto group; R-₁ is a straight or branched C -C₄ alkyl group or an aryl or arylalkyl group having from 1 to 6 carbon atoms in the alkyl moiety wherein the aryl is phenyl or a 5 or 6 membered aromatic heterocycle with one or two heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur, optionally substituted with one or more substituents, the same or different, selected from the group consisting of halogen atoms, hydroxy groups, alkoxy, alkyl, alkylthio, alkylsulphonyl or alkyloxycarbonyl groups having from 1 to 6 carbon atoms in the alkyl moiety, CrC₃ alkyl groups containing one or more fluorine atoms, carboxy groups, nitro groups, amino or aminocarbonyl groups, acylamino groups, aminosulphonyl groups, mono- or di-alkylamino or mono- or di-alkylaminocarbonyl groups having from 1 to 6 carbon atoms in the moiety;

R₂ is a hydrogen atom, a straight or branched C C alkyl or a benzyl group; R₃ is a phenyl group substituted with a 5 or 6 membered aromatic heterocycle with one or two heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur, being the phenyl and the heterocyclic groups optionally substituted with one or more substituents, the same or different, as indicated for R^

R₄ is a straight or branched d-C₄aIkyl group or a phenyl group; the carbon atoms marked with an asterisk are stereogenic centers; and pharmaceutically acceptable salts thereof.

Surprisingly, it has been found that compounds according to the present invention, generically disclosed in WO 97/24342, and having a specific substitution pattern, exhibit improved properties over those compounds specifically disclosed in WO 97/24342.

Summary of the Invention

Accordingly, the present invention provides compounds of formula (I):

wherein R¹ represents benzyl; R² represents pyrazole; or a pharmaceutically acceptable derivative thereof. Further aspects of the invention are:

-A pharmaceutical composition comprising a compound of the invention together with a pharmaceutically acceptable carrier and/or excipient. -A compound of the invention for use in therapy. -Use of a compound of the invention for the manufacture of a medicament for the treatment of a patient suffering from a condition susceptible to amelioration by an ACE and/or NEP inhibitor.

-A method of treating a patient suffering from a condition susceptible to amelioration by an ACE and/or NEP inhibitor comprising administering a therapeutically effective amount of a compound of the invention.

Detailed Description of the Invention

The compounds of formula (I) contain chiral (asymmetric) centres (marked ^*). The individual stereoisomers (enantiomers and diastereoisomers) and mixtures of these are within the scope of the present invention.

R² represents a pyrazole group. The pyrazole can be N-linked or C-linked to the phenyl ring. Most preferably, R² represents N-linked pyrazole.

The compounds of the present invention exhibit improved inhibitory activity against human ACE in addition to good inhibitory activity against NEP and therefore achieve greater efficacy in man.

As used herein, the term "mixed ACE-NEP inhibitor" means a compound with both ACE and NEP inhibitory activity. The term "dual" has been more commonly used in the literature. For the purposes of this patent application, the terms mixed and dual are to be considered equivalent.

As used herein, the term "pharmaceutically acceptable" means a compound which is suitable for pharmaceutical use.

As used herein, the term "pharmaceutically acceptable derivative", means any pharmaceutically acceptable salt, solvate, or prodrug e.g. ester, of a compound of formula (I), which upon administration to the recipient is capable of providing (directly or indirectly) a compound of formula (I), or an active metabolite or residue thereof. Such derivatives are recognizable to those skilled in the art, without undue experimentation. Nevertheless, reference is made to the teaching of Burger's Medicinal Chemistry and Drug Discovery, 5^th Edition, Vol 1 : Principles and Practice, which is incorporated herein by reference to the extent of teaching such derivatives. Preferred pharmaceutically acceptable derivatives are salts, solvates and esters. Particularly preferred pharmaceutically acceptable derivatives are salts and solvates.

Examples of pharmaceutically acceptable salts, are the salts with alkali or alkali-earth metals and the salts with pharmaceutically acceptable organic bases. Reference is made to Berge et. al., J. Pharm. Sci, 1977, 66, 1-19, which is incorporated herein by reference.

Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". Solvates of the compound of formula (I) are within the scope of the invention.

Salts and solvates of compounds of formula (I) which are suitable for use in medicine are those wherein the counterion or associated solvent is pharmaceutically acceptable. However, salts and solvates having non-pharmaceutically acceptable counterions or associated solvents are within the scope of the present invention, for example, for use as intermediates in the preparation of other compounds of formula (I) and their pharmaceutically acceptable salts and solvates.

As used herein, the term "prodrug" means a compound which is converted within the body, e.g. by hydrolysis in the blood, into its active form that has medical effects. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference. Esters may be active in their own right and /or be hydrolysable under in vivo conditions in the human body. Suitable pharmaceutically acceptable in vivo hydrolysable ester groups include those which break down readily in the human body to leave the parent acid or its salt.

Preferred compounds according to the invention include and may be selected from the following:

N-[(2S)-2-benzyl-3-mercaptopropanoyl]-4-(1H-pyrazoI-1-yl)-L-phenylalanine;

N-[(2S)-2-benzyl-3-mercaptopropanoyI]-4-(1 H-pyrazol-4-yl)-L-phenylalanine; N-[(2S)-2-benzyl-3-mercaptopropanoyl]-4-(1 H-pyrazol-5-yl)-L-phenylalanine; and pharmaceutically acceptable derivatives thereof.

The compounds of the invention are mixed ACE/NEP inhibitors and are thus of use in the treatment of conditions ameliorated by an ACE and/or NEP inhibitor, e.g. cardiovascular diseases, renal disease. The compounds of the present invention show advantageous properties, they may be more efficacious, show greater selectivity for the target enzymes, have fewer side effects, have a longer duration of action, be more bioavailable by the preferred route, or have other more desirable properties than similar known compounds.

The invention therefore provides a compound of formula (I) or a pharmaceutically acceptable derivative thereof for use in therapy, in particular in human medicine.

There is also provided as a further aspect of the invention the use of a compound of formula (I) or a pharmaceutically acceptable derivative thereof in the preparation of a medicament for use in the treatment of conditions susceptible to amelioration by an ACE and/or NEP inhibitor.

In an alternative or further aspect, there is provided a method for the treatment of a mammal, including man, comprising administration of an effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof in particular in the treatment of conditions susceptible to amelioration by an ACE and/or NEP inhibitor.

It will be appreciated that reference to treatment is intended to include prophylaxis as well as the alleviation of established symptoms. Compounds of formula (I) may be administered as the raw chemical but the active ingredient is preferably presented as a pharmaceutical formulation.

Accordingly, the present invention further provides a pharmaceutical formulation comprising at least one compound of formula (I) or a pharmaceutically acceptable derivative thereof, thereof in association with a pharmaceutically acceptable carrier and/or excipient. The carrier and/or excipient must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deletrious to the receipient thereof.

In another aspect, the invention provides a pharmaceutical composition comprising, as active ingredient, at least one compound of formula (I) or a pharmaceutically acceptable derivative thereof in association with a pharmaceutically acceptable carrier and/or excipient for use in therapy, and in particular in the treatment of human or animal subjects suffering from a condition susceptible to amelioration by a ACE and/or NEP inhibitor.

There is further provided by the present invention a process of preparing a pharmaceutical composition, which process comprises mixing at least one compound of formula (I) or a pharmaceutically acceptable derivative thereof, together with a pharmaceutically acceptable carrier and/or excipient. Thus compounds of formula (I) may be formulated for oral, buccal, parenteral, transdermal, topical (including ophthalmic and nasal), depot or rectal administration or in a form suitable for administration by inhalation or insufflation (either through the mouth or nose).

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycollate); or wetting agents (e.g. sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions or they may be presented as a dry product for constitution with water or other suitable vehicles before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g. almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavouring, colouring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

For buccal administration the compositions may take the form of tablets or lozenges formulated in a conventional manner.

The compounds according to the present invention may be formulated for parenteral administration by injection, e.g. by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g. in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.

The compounds according to the present invention may be formulated for topical administration by insufflation and inhalation. Examples of types of preparation for topical administration include sprays and aerosols for use in an inhaler or insufflator. Powders for external application may be formed with the aid of any suitable powder base, for example, lactose, talc or starch. Spray compositions may be formulated as aqueous solutions or suspensions or as aerosols delivered from pressurised packs, such as metered dose inhalers, with the use of a suitable propellant.

The compounds according to the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. containing conventional suppository bases such as cocoa butter or other glyce des.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously, transcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds according to the present invention may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The daily dose of the compound of formula (I) or of a pharmaceutically acceptable derivative will depend on several factors such as the seriousness of the disease, the individual response of the patient or the kind of formulation but it is usually comprised between 0.1 mg and 10 mg per kg of body weight divided into a single dose or into more daily doses.

The compounds of formula (I) may also be used in combination with other therapeutic agents. The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable derivative thereof together with a further therapeutic agent.

When a compound of formula (I) or a pharmaceutically acceptable derivative thereof is used in combination with a second therapeutic agent active against the same disease state the dose of each compound may differ from that when the compound is used alone. The compounds of the present invention may be used in combination with other ACE and/or NEP inhibitors and the like.

The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical composition and thus pharmaceutical compositions comprising a combination as defined above together with a pharmaceutically acceptable carrier or excipient comprise a further aspect of the invention. The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical compositions by any convenient route. When administration is sequential, either the mixed ACE-NEP inhibitor or the second therapeutic agent may be administered first. When administration is simultaneous, the combination may be administered either in the same or different pharmaceutical composition.

When combined in the same formulation it will be appreciated that the two compounds must be stable and compatible with each other and the other components of the formulation. When formulated separately they may be provided in any convenient formulation, conveniently in such manner as are known for such compounds in the art.

When a compound of formula (I) or a pharmaceutically acceptable derivative thereof is used in combination with a second therapeutic agent active against the same disease state the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art. It will be appreciated that the amount of a compound of the invention required for use in treatment will vary with the nature of the condition being treated and the age and the condition of the patient and will be ultimately at the discretion of the attendant physician or veterinarian.

The compounds of formula (I) and pharmaceutically acceptable derivatives thereof may be prepared by the processes described hereinafter, said processes constituting a further aspect of the invention. In the following description, the groups are as defined above for compounds of formula (I) unless otherwise stated.

No toxological effects are indicated/expected when a compound of the present invention is administered in the above-mentioned dosage range.

According to a further aspect of the present invention, there is provided a process (A) for preparing a compound of formula (I) which process comprises reacting a compound of formula (II) with a compound of formula (III), followed by deprotection:

(III)

R'

wherein R¹ and R² have the above meanings and P¹ represents an oxygen protecting group e.g. methyl. The condensation reaction is carried out using conventional techniques of peptide chemistry. Deprotection reactions are carried out using conventional techniques.

Suitably, the reaction may be carried out in the presence of a coupling agent, for example 1- [3-(dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride, in the presence of HOBt (1- hydroxybenzotriazole), in a suitable solvent e.g. DMF (N,N-Dimethylformamide), MeCN, DCM, preferably DMF, suitably at room temperature. The reaction is followed by deprotection under standard conditions, for example, when P¹ represents C^alkyl, removal of the protecting group may be effected by NaOH in a solvent e.g. THF (tetrahydrofuran).

Compounds of formula (III) are known or can be prepared according to conventional methods as described for example, in British patent No. 1576161 and Fournie-Zalusky et al. (1994) J. Med. Chem. 37:1070-1083, each incorporated herein by reference to the extent of teaching compounds of formula (III) and their preparation.

According to a process (B), compounds of formula (II) wherein R² is N-linked pyrazole may be prepared from compounds of formula (IV):

wherein P¹ is an oxygen protecting group e.g. methyl, B represents boron, and P² is an amino protecting group, such as Boc, by treatment with pyrazole in the presence of copper acetate, pyridine and TEMPO (tetramethylpyrrolidine oxide) in an organic solvent e.g. DCM (dichloromethane); followed by deprotection of the amino group under standard conditions.

Compounds of formula (IV) are known in the art, see for example M.E Jung, T.I. Lazarova; J. Org. Chem., 1999, 64, 2976, which is incoporated herein by reference to the extent of teaching compounds of formula (IV) and their preparation. According to a process (C), compounds of formula (II) wherein R² is C-5 linked pyrazole may be prepared from compounds of formula (V):

wherein P¹ is an oxygen protecting group e.g. methyl, SEM is 2-(trimethylsilyl)ethoxy]methyl and P² is an amino protecting group, such as Boc, by reflux in a solvent such as ethanol in the presence of an acid such as HCI.

Compounds of formula (V) may be prepared from compounds of formula (IV) by reaction with a compound of formula (VI)

wherein SEM is 2-(trimethylsilyl)ethoxy]methyl, in the presence of a base e.g. potassium carbonate, a solvent e.g. DME, and a metal catalyst e.g. PdCI₂, at elevated temperature. Preferably the reaction is carried out at 30-100°C, more preferably at about 70°C.

Compounds of formula (VI) may be prepared from compounds of formula (VII)

(VII) by reaction with iodine, in the presence of tetrahydrofuran and n-butyllithium, at a temperature below room temperature, preferably -78 - 0°C.

Compounds of formula (VII) are known in the art, see for example N. Fugina, W. Holzer, M, Wasicky; Heterocycles, 1992, 34(2): 303, which is incoporated herein by reference to the extent of teaching compounds of formula (VII) and their preparation.

According to a process (D), compounds of formula (II) wherein R² is C-4 linked pyrazole may be prepared from compounds of formula (VIII):

wherein P¹ is an oxygen protecting group e.g. methyl, and P² is an amino protecting group, such as Boc, by reaction with HCI in a solvent such as dioxane under nitrogen at room temperature.

Compounds of formula (VIII) may be prepared by reaction of a compound of formula (IV) with a compound of formula (IX)

in the presence of a base e.g. potassium carbonate, a solvent e.g. DME, and a metal catalyst e.g. PdCl₂, at elevated temperature and under nitrogen. Preferably the reaction is carried out at 30-100°C, more preferably at about 70°C.

Compounds of formula (IX) are known in the art, see for example J. Elguero, C. Jaramillo, C. Pardo; Synthesis, 1997, 563, which is incorporated herein by reference to the extent of teaching compounds of formula (IX) and their preparation.

According to a process (F), compounds of formula (II) may be prepared from compounds of formula (X):

(X)

wherein P² is an amino protecting group e.g. COH, and R² is as defined above, by deprotection of the amino group under standard conditions e.g. by treatment with MeOH in the presence of HCI, followed by protection of the carboxylic acid group under standard conditions e.g. by reaction with MeOH in the presence of HCI.

Compounds of formula (X) may be prepared by reaction of compounds of formula (XI):

(XI)

wherein P² is an amino protecting group e.g. COH or hydrogen, and X is a leaving group e.g. halogen, preferably iodine, with compounds of formula (XII):

R — H (XII)

wherein R² is as defined above, in the presence of a transition metal catalyst e.g. Cul and a base e.g. Cs₂CO₃, K₂CO₃ , preferably K₂CO₃, and a solvent e.g. NMP (n-methyl pyrrolidinone), 1 ,4-dioxane, DMF, preferably NMP.

Compounds of formula (XII) are known in the art and are commercially available.

Compounds of formula (XI) may be prepared from compounds of formula (XIII):

wherein P¹ is an carboxylic acid protecting group e.g. methyl and P² is an amino protecting group e.g. COH or hydrogen, and X is a leaving group e.g. halogen, preferably iodine, by deprotection of the oxygen group under standard conditions e.g. by reaction with NaOH in a suitable solvent e.g. MeOH.

Compounds of formula (XIII) may be prepared from compounds of formula (XIV):

by reaction with X, e.g. I₂, in the presence of peracetic acid and an acid e.g. H₂SO and, followed by protection of the carboxylic acid group under standard conditions, e.g. by reaction with MeOH in the presence of an activating agent e.g. SOCI₂ and a solvent e.g. toluene, optionally followed by protection of the amino group under standard conditions e.g. by reaction with HCOOH in the presence of an activating group e.g. acetic anyd de

Compounds of formula (XIV) are known in the art and commercially available.

Alternatively, compounds of formula (XI) may be prepared from compounds of formula (XIV) by by reaction with X, e.g. I₂, in the presence of peracetic acid and an acid e.g. H₂SO₄ and, followed by protection of the amino group under standard conditions e.g. by reaction with HCOOH in the presence of an activating group e.g. acetic anydride.

Compounds of formula (II) are novel compounds and hence form another aspect of the invention.

Those skilled in the art will appreciate that in the preparation of the compound of formula (I) or a solvate thereof it may be necessary and/or desirable to protect one or more sensitive groups in the molecule to prevent undesirable side reactions. The protecting groups used in the preparation of the compound of formula (I) may be used in a conventional manner. See for example Protective Groups in Organic Chemistry, Ed. J.F.W. McOmie, Plenum Press, London (1973) or Protective Groups in Organic Synthesis, Theodora Green, John Wiley and Sons, New York (1981 ). Examples of suitable amino protecting groups include acyl type protecting groups (e.g. formyl, trifluoroacetyl, acetyl), aromatic urethane type protecting groups (e.g. benzyloxycarbonyl (Cbz) and substituted Cbz), aliphatic urethane protecting groups (e.g. 9-fluorenylmethoxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl) and alkyl type protecting groups (e.g. benzyl, trityl, chlorothtyl). Examples of suitable oxygen protecting groups may include for example alky silyl groups, e.g. trimethylsilyl or tert-butyldimethylsilyl; alkyl ethers e.g. tetrahydropyranyl or tert-butyl; or esters e.g. acetate.

The following examples illustrate aspects of this invention but should not be construed as limiting the scope of the invention in any way.

Examples:

Synthesis of intermediates for Example 1 :

Intermediate 1 :

Methyl N-(tert-butoxycarbonyl)-4-(1 H-pyrazol-1-yl,-L-phenylalaninate

A mixture of the boronic acid^* (1 ) (1.0g, 3.09mmol), pyrazole (0.2g, 3.09mmol), copper acetate (0.82g, 4.64mmol), pyridine (0.5ml, 6.18mmol), TEMPO (0.482g, 3.09mmol), and 4A molecular sieves (1/8", dry) were stirred in dichloromethane (100ml) at room temperature for two weeks. The mixture was then filtered through hyflo and solvent evaporated in vacuo. Purification via silica gel chromatography (dichloromethane/cyclohexane 1 :1 , to dichloromethane/ethyl acetate 1 :1 ) gave the title compound (0.675mg) as a yellow solid. LCMS RT 3.16min MH⁺ 346 *M.E Jung, T.I. Lazarova; J. Org. Chem., 1999, 64, 2976

Intermediate 3:

Methyl 4-(1 H-pyrazol-1-vO-L-phenylalaninate

4M HCL in dioxane (10ml) was added to a solution of the carbamate (2) (675mg, 1.96mmol) in dioxane (10ml). After 4hours at room temperature, solvent was evaporated, and the residue co-evaporated with ether (2 x 30ml), to give the title compound. LCMS RT 1.97min MH⁺ 246 (as free base)

Synthesis of Intermediates for Example 2:

Intermediate 5: 4-Bromo-1 -trityl-1 H-pyrazole 4-Bromopyrazole (1.0g, 6.8mmol) and triethylamine (0.90ml, 6.5mmol) were stirred under nitrogen in DMF at 0°C. Trityl chloride (1.81g, 6.5mmol) was added, and the mixture was stirred for two days at room temperature. The mixture was then diluted with chloroform (10 ml), and washed with water. The organic portion was dried over sodium sulphate and solvent evaporated in vacuo to give the crude product. The resulting solid was washed with di-isopropyl ether to give the title compound (1.55g)

LCMS RT 4.09min, [CPh₃]⁺ 243

Intermediate 6: Methyl N-(tert-butoxycarbonyl)-4-(1 -trityl-1 H-pyrazol-4-vD-L-phenylalaninate

To a mixture of bromopyrazole (5) (1.0g, 2.56mmoI), boronic acid (1 ) (0.7g, 2.14mmol), and potassium carbonate (1.5g, 10.7mmol) in degassed DME (10ml) was added PdCI₂[dppf] (0.088g, O.l mmol). The reaction mixture was then heated to 70°C under nitrogen for 24 hours. Solvent was then removed in vacuo, and the residue was partitioned between ethyl acetate and water. The organic portion was dried over sodium sulphate and solvent evaporated in vacuo to give the crude product. Purification via silica gel chromatography (ethyl acetate/petroleum ether 40-60 1 :4) gave the title compound (0.74g) LCMS RT 4.11 min, [CPh₃]⁺ 243

Intermediate 7:

Methyl 4-(1 H-pyrazol-4-yl -L-phenylalaninate The carbamate (16) (0.74g, 1.26 mmol) was taken up in 2M HCI in dioxane (15ml) and the reaction stirred for 24 hours at room temperature under nitrogen. Solvent was evaporated in vacuo and the mixture purified via SCX-2 ion exchange chromatography eluting with 1 :9 ammonia;methanol to give the title compound (0.2g) LCMS RT 1.87min, MH⁺ 246

Synthetic Intermediates for Example 3:

Intermediate 9:

5-lodo-1 -rr2-(trimethylsilyl)ethoxylmethyll-1 H-pyrazole

To the protected pyrazole* (8) (1.0g, 5.04mmol) in dry tetrahydrofuran (20ml) under nitrogen at -78°C was added n-butyllithium (5.29mmol) in hexanes, and the mixture was stirred for one hour. Iodine (1.27g, 5.04mmol) was added as a tetrahydrofuran solution (10ml), and the mixture was stirred at room temperature for one hour. Water (50ml) was added to the mixture, which was extracted with ether (2x 50ml). The organics were dried over sodium sulphate, and solvent evaporated in vacuo to give an oil. The crude product was purified by silica gelchromatography (ethyl acetate/petroleum ether 40-60 1 :19) to give the title compound as an oil (0.88g)

LCMS RT 3.64minutes (blue), MH⁺ 325

* N. Fugina, W . Holzer, M. Wasicky; Heterocycles, 1992, 34, 303

Intermediate 10: Methyl N-(tert-butoxycarbonyl)-4-(1 -{ ,2-(trimethylsilyl)ethoxylnnethyl)-1 H-pyrazol-5-yl)-L- phenylalaninate

To a degassed solution of the pyrazole intermediate (9) (0.5g, 1.54mmol) and the boronic acid^* (1 ) (0.415g, 1.28mmol), potassium carbonate (0.89g, 6.43mmol) in DME (5ml) was added PdCI₂[dppf] (53mg, 0.06mmol). The mixture was stirred at 70°C for 16 hours. After cooling to room temperature, solvent was removed in vacuo, and the residue was partitioned between ethyl acetate and water. The organic portion was dried over sodium sulphate, and solvent removed in vacuo to give the crude product. Silica gel chromatography (ethyl acetate/petroleum ether 40-60 1 :4) gave the title compound as an oil (380mg). LCMS RT 3.89minutes (blue), MH⁺ 476

M.E Jung, T.I. Lazarova; J. Org. Chem., 1999, 64, 2976

Intermediate 11 :

Methyl 4-(1 H-pyrazol-5-yl)-L-phenylalaninate To the phenylalanine derivative (10) (0.38g, O.δmmol) was added ethanol (6ml) and 2N HCI (12ml). The reaction was stirred at reflux for 2 hours. The mixture was then cooled and ethanol removed in vacuo. The residue was neutralised with potassium carbonate (saturated aqueous solution) and extracted with chloroform. The organic portion was dried over sodium sulphate, and solvent removed in vacuo to give a gum. Purification by ion exchange chromatography (SCX-2) eluting with 10% ammonia in methanol gave the title compound (0.075g) LCMS RT 1.97minutes MH⁺ 246

Intermediate 12: Methyl A/-{(2S)-2-r(acetylthio)methyll-3-phenylpropanoyl}-4-(1H-pyrazol-1-yl)-L- phenylalaninate

To a solution of (2S)-2-acetylthio-3-phenylpropionic acid (85mg, 0.35mmol) in acetonitrile (3ml) was added water soluble carbodiimide (75mg, 0.39mmol), hydroxybenzotriazole (53mg, 0.39mmol) and diisopropylethylamine (0.126ml, 0.7mmol). After 15 minutes, methyl 4-(1 H-pyrazol-1-yl)-L-phenylalaninate hydrochloride (100mg, 0.35mmol) and diisopropylethylamine (0.063ml, 0.35mmol) was added. The resulting mixture was stirred for 24 hours. Solvent was evaporated and the residue partitioned between dichloromethane and aqueous sodium bicarbonate. Chromatography (SiO₂, dichloromethane:methanol 97:3) to give the title compound (94mg, 57%). LCMS RT 3.34min MH⁺ 466

Similarly prepared were:

Intermediate 13: Methyl Λ/-{(2S)-2-r(acetylthio)methvn-3-phenylpropanoyl)-4-(1 H-pyrazol-4-yl)-L- phenylalaninate

LCMS RT 3.13min M⁺ 466

Intermediate 14:

Methyl Λ/-{(2S)-2-r(acetylthio)methyll-3-phenylpropanoyl)-4-(1H-Pyrazol-5-yl)-L- phenylalaninate

LCMS RT 3.1 δmin M⁺ 466

N-Formyl-4-iodo-L-phenylalanine methyl ester Intermediate 16:

Glacial acetic acid (2.73wt, 2.62volumes) was stirred at 20°C and concentrated sulphuric acid (1.34wt, 0.73volumes) added whilst the temperature was kept below 45°C. The mixture was cooled to 20°C, and iodine (0.77wt, O.δequivalents) was added with stirring. L- Phenylalanine (1wt) was added. The mixture was heated to 55±5°C with pumped agitation and 40% peracetic acid (about 0.75wt) added over 2 - 4hours. The reaction was then checked for completeness by HPLC (<5% area phenylalanine remaining - typically, 0-5%). Absence of oxidising species was confirmed by checking with Merckoquant peroxide test strips. A saturated solution of sodium metabisulphite in water was added (minimum volume, 0.5-1 volumes typically) to convert any remaining iodine present to iodide prior to distillation. The mixture was cooled to 35°C, vacuum applied, and the batch volume reduced to about 4volumes by distillation. Methanol (2volumes) was added and the distillation continued until the batch volume was again reduced to about 4volumes. More methanol (2volumes) was added and the distillation continued until the batch volume was again reduced to about 4volumes. Finally, methanol (2.5volumes) was added and the mixture cooled to 20± 5°C. The mixture was used directly in the next step.

The suspension of crude iodophenylalanine (approx 1.8wt, in methanol, 2wt approx) from step 1a was heated to 50°C and thionyl chloride (1.02wt, 0.63volumes, 1.42equivalents) was added over about I hour and at such a rate that the temperature did not exceed 60°C. The mixture was then heated to 55± 5°C, and held at 55°C for a minimum of 2hours. The reaction was then checked for completeness by HPLC (<5% area iodophenylalanine remaining, typically 2%).

The temperature of the mixture was adjusted to 40°C, and distilled under vacuum until the volume has been reduced to about 3volumes (a minimum temperature of 40°C was maintained during this operation). The mixture was then diluted with toluene (1 volume), maintaining the temperature at a minimum of 30°C, followed by water (2.5volumes). The mixture was cooled to 20°C and 0.880 ammonia (4volumes, 3.52wt) and toluene (1 volume) added whilst the temperature was kept below 35°C. The mixture was then allowed to settle. The toluene layer was removed and the aqueous layer extracted with further toluene (1 volumes). The organic layers were combined and distilled under vacuum to give a mobile oil. Toluene (O.δvolumes) was added and the organic solution was used without further treatment in the next step.

Formic acid (1.02wt, 0.84volumes) and toluene (O.δvolumes) were stirred at 12±3°C and acetic anhydride (0.36wt, O.δvolumes) was added at 10-15°C, and the mixture stirred at 10- 15°C for a further 60minutes. The toluene solution of crude iodo methyl ester from the previous step was then added at 10-15°C, and the resulting orange-brown mixture was stirred for a minimum of I hour at 12± 3°C. The reaction was then checked for completeness by HPLC (<3% area iodo methyl ester remaining, typically <2%).

The mixture was concentrated under reduced pressure to about 2volumes. The oil was diluted with dichloromethane (5volumes) and washed sequentially with water (4volumes) which was adjusted to pH9 by the addition of 0.380 ammonia solution and then water (δvolumes).

The dichloromethane solution was then diluted with toluene (4volumes) and concentrated at atmospheric pressure until a batch volume of 6 - 7volumes was achieved, typically with the solution at 65-70°C. The solution was then cooled to 20°C over about I hour (seeded if necessary) and then to 7°C over a further I hour. The crystallised product was aged for at least a further I hour and then filtered in a pressure filter. The cake was washed with cold (5-10°C) toluene (2 x 2volumes), pulled dry, and the product was then dried under vacuum at 50°C.

Expected Yield: 35%th (70%w/w) HPLC (δminute method) RT 4.30 min

N-formyl-4-iodo-L-phenylalanine Intermediate 17: N-FormyI-4-iodo-L-phenylalanine methyl ester (1wt) was suspended in methanol (5volumes) and water (δvolumes) and treated with 2M sodium hydroxide (1.73 Wt) at 22±3°C. This mixture was stirred for about 4hours until complete by HPLC. The reaction mixture was heated to 31 ± 2°C then 2M hydrochloric acid (0.41 Wt) was added at 31±2°C over 20 - 30 minutes, then N-formyl-4-iodo-L-phenylalanine seed (0.001 wt) was added as a slurry in 1 :1 Methanol:water. Further 2M hydrochloric acid (1.34Wt) was added over 1 - 2 hours at 31 ± 2°C then the pH checked (target pH 1). The white slurry was cooled to 0 ± 3°C then stirred for at least 30minutes. The solid was collected by filtration taking care to suck the liquors only to the surface of the cake. The cake was washed with methanol/water (1 :1 , 2volumes) at 5 ± 5°C followed by water (2 x 2volumes) at 5 ± 5°C then sucked dry. The wet solid was dried in a vacuum oven at 50-60°C to give N-formyl-4-iodo-L-phenylalanine as a white powder.

Expected yield: 86-90% theory; 82-δ6% w/w.

HPLC (2minute method) RT 1.31 min

N-Formyl-4-(1 H-pyrazol-1 -yl )-L-phenylalanine

Intermediate 18:

To a solution of N-formyl-4-iodo-L-phenylalanine (1wt) in NMP (2. δvolumes) stirred at 20°C was added anhydrous potassium carbonate (1.52wt, 3.5equivalents) in portions. The batch temperature was increased to 40°C. Pyrazole (0.26wt, 1.2equivalents) was added, followed by copper (I) iodide (0.015wt, 0.025equivalents) and racemic trans-1 ,2-Diaminocyclohexane

(0.036wt, 0.1 equivalents). The batch temperature was increased to 125 ± 3°C, and the reaction stirred for at least 15hours. The brown suspension was sampled and analysed by

HPLC to ensure <3% SM remains.

After cooling to 35 ± 3°C, water (lOvolumes) was added, followed by DCM (δvolumes).

Charcoal was added and the mixture stirred for Ihour then the solution was filtered. The phases were separated and the aqueous phase was washed with further DCM (δvolumes).

The aqueous phase was cooled to 3 ± 3°C, and acidified by slow addition of concentrated HCI (1. δvolumes) over about 45 minutes then aged for 15minut.es. The remaining concentrated HCI (0.3volumes) to bring the pH to 1 was added over 10 minutes (check pH). The solid was collected by filtration and the filter cake slurry washed with water (4volumes) and then displacement washed with water (4volumes) followed by toluene (3volumes). The resulting solid was dried in a vacuum oven at 50-60°C. Expected yield 70-δ0%th, 57-6δ%w/w. δ HPLC (2minute method) RT 1.13 minutes

Methyl 4-(1 H-pyrazol-1-yl)-L-phenylalanine hvdrochloride

Intermediate 19:

N-Formyl-4-(1 H-pyrazol-1-yl)-L-phenylalanine (1wt) was suspended in methanol 0 (lOvolumes) and stirred at 22 ± 3°C. Acetyl chloride (0.94 wt, 3equivalents) was added dropwise at 20-δO°C over about Iδminutes. The resulting solution was warmed to δO ± δ°C and held for at least 14hours. The mixture was analysed (Reaction complete when <3% amino acid). When complete the solution was cooled to 22 ± 3°C and concentrated in vacuo to about δvolumes at <30°C. Isopropyl acetate (l Ovolumes) was added and the mixture δ concentrated in vacuo to about δvolumes at <30°C. Isopropyl acetate (lOvolumes) was again added and the mixture reconcentrated in vacuo to about δ volumes at <30°C. Isopropyl acetate (lOvolumes) was then added and the mixture was cooled to 0-δ°C. The product was collected as an off-white solid by filtration, washed with isopropyl acetate (2 x 3volumes) and dried in a vacuum oven at δδ ± δ°C. 0 Expected yield: δδ-9δ% theory; 92-103% w/w. HPLC (2minute method) RT 1.03 minutes

1 H NMR (d6-DMSO) 3.1δ, 3.23 (ABX, J 6, 7Hz, 2H), 3.70 (s, 3H), 4.32 (t, J 6Hz, 1 H), 6.δ5 (dd, J 2, 2.δHz, 1 H), 7.33 (d, J δ.δHz, 2H), 7.74 (d, J 2Hz, 1 H), 7.δ1 (d, J δ.5Hz, 2H), δ.50 δ (d, J 2.5Hz, 1 H), 3.70 (br s, 3H).

Examples

(2) (3) 0

Example 1 : N-[(2S)-2-benzyl-3-mercaptopropanoyll-4-(1 H-pyrazol-1-yl)-L-phenylalanine 5δmg (O.H mmol) of methyl Λ/-{(2S)-2-[(acetylthio)methyl]-3-phenylpropanoyl}-4-(1 H-pyrazol- 1-yl)-L-phenylalaninate was dissolved in THF (1.5ml) and the mixture was degassed with nitrogen for 0 minutes. After cooling to 0°C, 2N NaOH (6 equivalents) was added dropwise. After stirring for a further 15 minutes at 0°C, the mixture was allowed to come to room δ temperature and was stirred for 4 hours. The mixture was then acidified to pH 4 with 2N HCI, and extracted with ethyl acetate. Evaporation of solvent gave the title compound (27mg, 63%). LCMS RT 3.22min MH⁺ 410

0 Similarly prepared were:

Example 2

N-r(2S)-2-benzyl-3-mercaptopropanoyll-4-(1 H-pyrazol-4-yl)-L-phenylalanine from Methyl Λ/-{(2S)-2-[(acetylthio)methyl]-3-phenyIpropanoyl}-4-(1 H-pyrazol-4-yl)-L- phenylalaninate 5 LCMS RT 3.04min MH⁺ 410

Example 3

N-r(2S)-2-benzyl-3-mercaptopropanoyll-4-(1 H-pyrazol-5-yl)-L-phenylalanine from Methyl Λ/-{(2S)-2-[(acetylthio)methyl]-3-phenylpropanoyl}-4-(1 H-pyrazol-5-yl)-L- 0 phenylalaninate

LCMS RT 3.02min MH⁺ 410

LCMS data was generated on a system as characterised below Column: 3.3cm x 4.6mm ID, 3uιn ABZ+PLUS δ Flow Rate: 3ml/minutes Injection Volume: 5μl Temp: RT UV Detection Range: 215 to 330nm

Solvents: A: 0.1% Formic Acid + lOmMolar Ammonium Acetate.

B: 9δ% Acetonitrile + 0.0δ% Formic Acid 0

Gradient: Time (minutes) A% B%

0.00 100 0

0.70 100 0

4.20 0 100 δ.30 0 100 δ.δO 100 0

Biological Data Inhibitory Activity Against ACE

Inhibitory activity against ACE was determined via the following protocol, following the rate δ of cleavage of the substrate MCA-Ala-Ser-Asp-Lys-Dpa-OH, resulting in an increase in fluorescence at 320nm excitation/400nm emission.

1μl of the test compound solution plus TCEP (1 :2. δ compound:TCEP) in acetonitriie/water (1 :1 ) was mixed with 15μl of the substrate solution (176μM in 10μM TCEP solution) and 15μl 0 human ACE in buffer (200pM in δOmM HEPes pH 7.4, 1δ0mM NaCl soln, 1 μM zinc acetate, pH to 7.4 using 1 N NaOH) with 10μM TCEP (final concentrations were approx. 100pM human ACE and MCA substrate at δδuM). After 60 minutes incubation, fluorescence was read using a Tecan SpectraFluor Ultra fluorescence plate reader or equivalent at 320nm excitation/400nm emission. δ

Note: Human ACE refers to human kidney ACE. For the human plasma assay and rat plasma assay, the assay used was identical to that described above except that human/rat plasma was substituted for the buffered human ACE.

0 Inhibitory Activity Against Rabbit NEP

The assay was carried out as above, with the following modifications. Recombinant rabbit kidney NEP was substituted for human ACE and N-Dansyl-D-Ala-Gly-pNitroPhe-Gly was used as substrate instead of MCA-Ala-Ser-Asp-Lys-Dpa-OH. 5

Biological data (pKi) for selected compounds are shown below.

*Compound X: N-(3-mercapto-2-phenylmethylpropionyl)-4-(2-thiozolyl)-phenylaIanine 0 *Compound Y: N-(3-mercapto-2-phenylmethylpropionyl-4-(δ-pyrimidinyl)-phenylalanine Note: Compounds X and Y can be prepared according to processes provided in WO97/24342 2δ

Values in brackets represent values obtained upon repeat testing.

The Compounds of the invention (1-3) show increased Human ACE pKi and Rat plasma ACE pKi compared to Compound X and increased Human ACE pKi compared to Compound δ Y. This surprising potency indicates improved ACE-NEP inhibitory.

Note: Compounds may be tested for ACE inhibitory activity using tests for inhibition of Angiotensin I conversion. The conversion of Angiotensin I to Angiotensin II mediated by ACE was measured by using purified human ACE enzyme. The ability of the compounds of the 0 invention to inhibit this conversion is calculated from the altered ratio of Angiotensin I to Angiotensin II.

Claims

Claims:

1. A compound of formula (I)

0 wherein:

R¹ represents benzyl;

R² represents pyrazole; or a pharmaceutically acceptable derivative thereof.

5 2. A compound according to claim 1 selected from:

N-[(2S)-2-benzyl-3-mercaptopropanoyl]-4-(1 H-pyrazol-1-yl)-L-phenylaIanine;

N-[(2S)-2-benzyl-3-mercaptopropanoyl]-4-(1 H-pyrazol-4-yl)-L-phenylalanine;

N-[(2S)-2-benzyl-3-mercaptopropanoyl]-4-(1 H-pyrazol-5-yl)-L-phenylalanine.; and pharmaceutically acceptable derivatives thereof. 0

3. A compound according to claim 1 or 2 for use in therapy.

4. A pharmaceutical composition comprising a compound according to claim 1 or 2 together with a pharmaceutical carrier and/or excipient. δ δ. Use of a compound according to claim 1 or 2 for the manufacture of a medicament for the treatment of a patient suffering from a condition susceptible to amelioration by an ACE and/or NEP inhibitor.

0 6. A method of treating a patient suffering from a condition susceptible to amelioration by an ACE and/or NEP inhibitor comprising administering a therapeutically effective amount of a compound according to claim 1 or 2.

7. A process for preparing a compound of formula (I)

δ comprising reacting a compound of formula (II) with a compound of formula (III), followed by deprotection:

(i i)

(III)

R ' 0

wherein:

R¹ represents benzyl; R² represents pyrazole; P¹ represents an oxygen protecting group.

δ. A compound of formula (II): 2δ

(ii)

wherein:

R² represents pyrazole; δ P¹ represens an oxygen protecting group.

0