WO2003104200A1 - N-mercaptoacyl phenyalanine derivatives, process for their preparation, and pharmaceutical compositions containing them - Google Patents

Abstract

The invention relates to compounds of formula (I); wherein R¹ represents C_1-6alkyl; R² represents pyrazole or pyrimidine; 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

N-MERCAPTOACYL PHENYLALA INE DERIVATIVES, PROCESS FOR THEIR PREPARATION, AND PH ARMACEUTICAL COMPOSITIONS CONTAINING THEM

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 vasoconsthction 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 natriuresis. Compounds with inhibitory activity of the neutral endopeptidase (NEP) enzyme are useful as vasodilators. Both ACE and NEP are responsible for the degradation 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.

Rt

R— C— C— CONH C— COOF

H₂ H | ^{* 2}

C— R,

H₂ (A) wherein:

R is a mercapto group or a R₄COS group convertible in an organism to a mercapto group; Ri is a straight or branched C₂-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, C C₃ 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_rC alkyI or a benzyl group; R 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 heterocydic groups optionally substituted with one or more substituents, the same or different, as indicated for R^

R₄ is a straight or branched Cι-C₄alkyl 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, genehcally 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 C^alkyl;

R² represents pyrazole or pyrimidine; 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.

As used herein, the term "alkyl" means both straight and branched chain saturated hydrocarbon groups. Examples of alkyl groups include methyl, ethyl, propyl and butyl groups.

Preferably, R¹ represents C^alkyl, more preferably C₃.₄alkyl, most preferably isopropyl.

R² represents pyrazole or pyrimidine. The pyrimidine is C-linked to the phenyl ring. The pyrazole can be N-linked or C-linked to the phenyl ring. Preferably, R² represents pyrazole. Most preferably, R² represents N-linked pyrazole.

It is to be understood that the present invention covers all combinations of suitable, convenient and preferred groups described hereinabove.

The compounds of the present invention exhibit improved inhibitory activity against human plasma 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-(mercaptomethyl)-3-methylbutanoyl]-4-(1 H-pyrazol-1-yl)-L-phenylalanine; N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]-4-pyrimidin-5-yl-L-phenylalanine; N-[(2S)-2-(mercaptomethyl)-4-methylpentanoyl]-4-(1 H-pyrazol-5-yl)-L-phenylalanine; N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]-4-(1 H-pyrazol-5-yl)-L-phenylalanine; N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]-4-(1 H-pyrazol-4-yl)-L-phenylalanine; and pharmaceutically acceptable derivatives thereof.

Particularly preferred compounds according to the invention include and may be selected from the following: N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]-4-(1 H-pyrazol-1 -yl)-L-pheny!alanine;

N-[(2S)-2-(mercaptomethyl)-4-methylpentanoyl]-4-(1 H-pyrazol-5-yl)-L-phenylalanine;

N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]-4-(1 H-pyrazol-5-yl)-L-phenylalanine;

N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]-4-(1 H-pyrazol-4-yl)-L-phenylaIanine; 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 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 an 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 glycerides.

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:

(111)

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-(dimethyIamino)propyl]-3-ethyl carbodiimide hydrochlohde, 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. Alternatively, the reaction may be carried out by activation of a compound of formula (III) with thionyl chloride, followed by reaction of the activated compound of formula (III) with a compound of formula (II) in a suitable solvent e.g. DCM, ethyl acetate, preferably ethyl acetate, in the presence of a base e.g. K₂CO₃. The reaction is followed by deprotection under standard conditions, for example, when P¹ represents dialkyl, removal of the protecting group may be effected by NaOH in a solvent e.g. THF (tetrahydrofuran) or suspended in MeOH. Removal of the disulphide may be effected by treatment with tributyl phosphine. The compound of formula (I) may then be precipitated by treatment with acid e.g. HCI.

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

Alternatively, compounds of formula (III) may be prepared from compounds of formula (XI):

R¹ (XI)

by reaction with an acid e.g. HCI, suitably in the presence of a solvent e.g. toluene or DCM.

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

by reaction with ephedrine in the presence of a solvent e.g. isopropyl acetate.

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

by reaction with thioacetic acid, in the presence of a suitable catalyst e.g. Cs₂CO₃, K₂CO₃, Na₂CO₃, preferably Cs₂CO₃, and in the presence of a solvent e.g. MIBK (methyl isobutyl ketone).

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

wherein A is C_halky!, by hydrolysis of the ester groups under standard conditions e.g. using NaOH, followed by reaction with formaldehyde in the presence of a secondary amine e.g. dimethylamine (Mannich reaction), followed by neutralisation with an acid e.g. HCI.

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 incorporated 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):

(V)

wherein P¹ is an oxygen protecting group e.g. methyl, SEM is 2-(thmethylsilyl)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 incorporated 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 pyrimidine 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 HCl in a solvent such as dioxane under nitrogen at room temperature.

Compounds of formula (VIII) may be prepared from compounds of formula (IV) by reaction with 5-bromopyridine in the presence of a base e.g. potassium carbonate, and a metal catalyst e.g. PdCI₂. The reaction is carried out at elevated temperature, preferably at 30- 100°C, more preferably at about 50°C.

According to a process (E), compounds of formula (II) wherein R² is C4-linked pyrazole may be prepared from compounds of formula (IX):

wherein P¹ is an oxygen protecting group e.g. methyl, and P² is an amino protecting group, such as Boc, by reaction with HCl in a solvent such as dioxane under nitrogen at room temperature. Compounds of formula (IX) may be prepared by reaction of a compound of formula (IV) with a compound of formula (X)

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 and under nitrogen. Preferably the reaction is carried out at 30-100°C, more preferably at about 70°C.

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

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

(XV)

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

Compounds of formula (XV) may be prepared by reaction of compounds of formula (XVI):

(XVI) 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 (XVII):

R — H (XVII)

wherein R² is pyrazole or pyrimidine, preferably pyrazole, 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 (XVII) are known in the art and are commercially available.

Compounds of formula (XVI) may be prepared from compounds of formula (XVIII):

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 (XVIII) may be prepared from compounds of formula (XIX):

(XIX)

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

Alternatively, compounds of formula (XVI) may be prepared from compounds of formula (XIX) 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 2:

Methyl N-(tert-butoxycarbonyl)-4-(1 H-pyrazol-1 -vD-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 -yl)-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 4:

Methyl N-(tert-butoxycarbonyl)- 4-pyrimidin-5-yl-L-phenylalaninate

The boronic acid^* (1 ) (3.0g), 5-bromopyridine (1.77g), PdCI₂[dppfj (0.39g)and potassium carbonate (5.1g) were mixed in degassed DME (45ml). The mixture was heated to 50°C for 3 hours, then cooled to room temperature. The mixture was diluted with saturated aqueous ammonium chloride (25ml) and concentrated in vacuo. The residue was partitioned between water and ethyl acetate. The organic portion was dried over sodium sulphate, and solvent evaporated in vacuo. Silica gel chromatography (ethyl acetate/cyclohexane 1 :10) gave the title compound (1.13g).

LCMS RT 2.92 minutes MH⁺ 358

M.E Jung, T.I. Lazarova; J. Org. Chem., 1999, 64, 2976 Intermediate 5:

Methyl 4-pyrimidin-5-yl-L-phenylalaninate

The carbamate (4) (1.12g) was dissolved in dichloromethane (20ml) and treated with 4M HCl in dioxane (10ml). The mixture was stirred at room temperature for 18 hours. Solvent was evaporated in vacuo, and the residue co-evaporated three times with dichloromethane (20ml) to give the title compound LCMS RT 1.89minutes MH⁺ 258

Synthetic Intermediates for Examples 3 and 4:

Intermediate 7: 5-lodo-1 -IT2-(trimethylsilyl)ethoxylmethyll-1 H-pyrazole

To the protected pyrazole* (6) (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 8:

Methyl N-(tert-butoxycarbonyl)-4-(1-{[2-(trimethylsilyl)ethoxylmethyl)-1 H-pyrazol-5-yl)-L- phenylalaninate

To a degassed solution of the pyrazole intermediate (7) (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 9: Methyl 4-(1 H-pyrazol-5-yl )-L-phenylalaninate

To the phenylalanine derivative (8) (0.38g, O.δmmol) was added ethanol (6ml) and 2N HCl (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

Synthesis of intermediates for Example 5:

Intermediate 11 : 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 12:

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

To a mixture of bromopyrazole (11 ) (1.0g, 2.56mmol), boronic acid (1 ) (0.7g, 2.14mmol), and potassium carbonate (1.5g, 10.7mmol) in degassed DME (10ml) was added PdCI₂[dppf] (0.088g, 0.1 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 4.1 1 min, [CPh₃]⁺ 243

Intermediate 13:

Methyl 4-(1 H-pyrazol-4-vn-L-ohenylalaninate The carbamate (12) (0.74g, 1.26 mmol) was taken up in 2M HCl 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

Synthesis of Intermediates for Examples 1-5:

16 17

Intermediate 15:

(R)-4-Benzyl-3-(2-hvdroxymethyl-3-methyl-butanoyl)-oxazolidinone

4-Benzyl-3-(3-methyIbutanoyl)-oxazolidinone (14) ^* (26.1g, O.lmol) was stirred in dry dichloromethane (400ml) at 0°C under nitrogen as titanium tetrachloride (1.0M solution in dichloromethane, 105ml, 0.105mol) was added and the mixture, which contained a yellow precipitate, was stirred a further 15 minutes then diisopropylethylamine (19ml, 0.11mol) was added dropwise, maintaining the temperature below 5°C. The resulting purple solution was stirred for 75 minutes then 1 ,3,5-trioxane (9.9g, 0.11mol) in dichloromethane (60ml) was added, and after a further 10 minutes, titanium tetrachloride (1.0M in DCM, 105ml, 0.105mol) was added. The mixture was stirred for 2.5 hours at 0°C then quenched by the addition of saturated ammonium chloride (500ml). Water (100ml) and dichloromethane (100ml) were added, the aqueous phase extracted with a further 2 x 100ml dichloromethane, the combined organics dried over Na₂SO₄ and evaporated. Recrystallisation from 30% dichloromethane /petroleum gave 18.7g (64%) of the title compound as a white solid. LCMS RT=2.94minutes MH⁺ 292

Similarly prepared was (R)-4-benzyl-3-(2-hydroxymethyl-4-methylpentanoyl)-oxazolidinone. Synth e.g. D.A. Evans et al., Tetrahedron, 1988, 44, 5525 Intermediate 16:

(R)-2-Hvdroxymethyl-3-methylbutanoic acid

To oxazolidinone (15) (23.4g, 80.4mmol) in THF (300ml) at 0°C was added 30% hydrogen peroxide (90ml, O.δmol). Aqueous lithium hydroxide (1.5M, 107ml, 160mmol) was then added dropwise and the mixture allowed to warm to room temperature and stirred for 3 hours. Potassium hydroxide (9g, 160mmol) was then added and the mixture heated at 60°C for 30 minutes then cooled to 0°C. A solution of sodium sulfite (100g) in water (400ml) was cautiously added, then the mixture was concentrated to two-thirds volume and partitioned between water (200ml) and chloroform (500ml). The aqueous phase was extracted with a further 2 x 200ml of chloroform then acidified with 5M HCl (200ml). The product was extracted with ethyl acetate (1 x 400ml, 2 x 250ml), washed with brine (500ml), dried over Na₂SO₄ and evaporated to give a solid (15g). This was redissolved in THF (400ml), potassium hydroxide (2 equivalents) added and the mixture heated at reflux for 3 hours. The volume was reduced to 1/4, water (300ml) added and the mixture washed with chloroform (3 x 300ml). Acidification and extraction into ethyl acetate (5 x 300ml) followed by drying and solvent removal gave 8.1g (76%) of title compound.

Similarly prepared was (R)-2-hydroxymethyl-4-methylpentanoic acid Analytical details were in agreement with literature values

Intermediate 17:

(S)-2-Acetylthiomethyl-3-methylbutanoic acid

To triphenylphosphine (32g, 122 mmol) in dry THF (300ml) at 0°C was added, dropwise, diisopropylazodicarboxylate (24ml, 122 mmol), giving a white precipitate which was stirred a further 10 minutes. A mixture of (R)-2-hydroxymethyl-3-methyIbutanoic acid (δ.09g, 61 mmol) and thiolacetic acid (13.1 ml, 183 mmol) in THF (100ml) was added dropwise to the Mitsunobu reagents. The mixture was allowed to warm to room temperature and stirred for 2.5 hours. The mixture was concentrated in vacuo to one third volume and partitioned between ethyl acetate (400ml) and aqueous sodium hydrogen carbonate (200ml x 3). The combined aqueous extracts were washed with chloroform (2 x 300ml) and cautiously acidified with 5M HCl. The product was extracted into DCM (3 x 300ml), the solution dried over Na₂SO and evaporated to give an orange oil (9.6g, 83%).

Similarly prepared was (S)-2-Acetylthiomethyl-4-methylpentanoic acid

Intemediate 1 δ:

Methyl Λ/-{(2S)-2-r(acetylthio)methyll-3-methylbutanoyl}-4-(1H-pyrazol-1-yl)-L- phenylalaninate To (2S)-2-(acetyIthio)-3-methylbutanoic acid acid (4.37g, 23mmol) in dichloromethane (25ml) was added, dropwise, thionyl chloride (2.2ml, 30mmol) and the solution stirred at room temperature under nitrogen for 20 hours, then concentrated in vacuo, azeotroping with further dry dichloromethane (10ml).

The amine hydrochloride (6g) was suspended in a stirred biphasic mixture of dichloromethane (δOml) and water (50ml) and potassium carbonate (14.5g, 105mmol) was added at 0°C. When complete dissolution had occurred, the acid chloride was added in dichloromethane (30ml) and stirring continued at 0°C for 30minut.es, then at room temperature for 1.5hours. Dichloromethane (200ml) and water (100ml) were added and the aqueous phase extracted with further dichloromethane (200ml). The combined organics were washed with 2M HCl, brine (200ml) dried over Na₂SO₄ and the solvent evaporated. This material was redissolved in dichloromethane, hexane added, and the solution concentrated in vacuo until crystallisation commenced. Further dilution with hexane and filtration afforded 6.6g of the title compound. LCMS RT=3.12minutes MH⁺ 416

Similarly prepared were: Intermediate 19:

Methyl Λ/-{(2S)-2-r(acetylthio)methyll-3-methylbutanoyl)-4-pyrimidin-5-yl)-L-phenylalaninate LCMS RT=2.88min MH⁺ 430

Intemediate 20:

Methyl Λ/-{(2S)-2-r(acetylthio)methyll-4-methylpentanoyl)-4-(1 H-pyrazol-5-yl)-L- phenylalaninate

LCMS RT=3.15min M+ 432

Intermediate 21 :

Methyl Λ/-{(2S)-2-r(acetylthio)methyll-3-methylbutanoyl)-4-(1 H-pyrazol-5-vn-L- phenylalaninate

LCMS RT=2.76min M⁺ 418

Intermediate 22:

Methyl Λ/-((2S)-2-r(acetylthio)methvn-3-methylbutanoyl)-4-(1 H-pyrazol-4-yl)-L- phenylalaninate

LCMS RT=2.93min M⁺ 418

Synthesis of intermediates for Example 1 :

(S)-2-Acetylthiomethyl-3-methylbutanoic acid (+)-ephedrine salt Intermediate 24:

A mixture of diethyl isopropylmalonate (1wt) and 2M aqueous sodium hydroxide (2.2equivalents, 5.45voIumes) was heated at 80 ± 5°C for 2 hours. Upon completion of the reaction the contents were acidified with cone HCl (1.13 wt). 60% aqueous dimethylamine (0.41 Wt) was added followed by 37%wt/wt aqueous formaldehyde (0.49 Wt) and the reaction was then stirred at 90±5°C for 16 hours. The solution was cooled to 20+5°C and cone HCl (0.83 Wt) and MIBK (4volumes) were added. The layers were separated and the organic layer was washed with water (1 volume). The MIBK layer was then concentrated under reduced pressure to about 1.4volumes. Cesium carbonate (0.039wt) was then added and the mixture was heated at 40+5°C. Thioacetic acid (0.36 Wt) was added and the mixture was stirred for at least 12 hours at 40±5°C. The reaction mixture was cooled to 20±5°C and 20% potassium bicarbonate (2.25volumes) was added. The layers were separated and the organic layer was washed with 20% potassium bicarbonate (0.9volumes). The combined aqueous layers were adjusted to pH 1 with cone HCl (1.18 Wt) then the acidified layer was extracted with isopropyl acetate (2x2voIumes). The combined organic layers were then washed with water (Ivolumes) and the mixture concentrated to 2volumes by vacuum distillation then isopropyl acetate (4.9volumes) was added.

(+)-Ephedrine hydrochlohde (0.86 Wt) was suspended in isopropyl acetate (4.2volumes) and water (0.7 volumes). 10.8M sodium hydroxide (0.58 Wt) was added and stirred to give a biphasic solution. The phases were separated and organic phase was washed with water (0.35volumes) and the solvent reduced to 3.5 volumes by vacuum distillation. 40% of the ephedrine solution (1.4volumes) was added to the racemate solution. The crystallisation was seeded and stirred for an hour at 20°C, then the remaining ephedrine was added over 2 - 3 hours. The crystallisation was cooled to 0 ± 5°C and stirred for at least 2 hours. The slurry was filtered and the filter cake was washed with isopropyl acetate (2x2.8volumes) and dried in a vacuum oven at 50±5°C.

Expected yield: 33% theory; 58% w/w.

HPLC (2minute method) RT 0.86minute 27.1%area, 1.34minute 71.9%area

N-Formyl-4-iodo-L-phenylalanine methyl ester Intermediate 26:

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) 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 1 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 had 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 volumes) 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 (0.86wt, O.δvolumes) was added at 10-15°C, and the mixture stirred at 10- 15°C for a further ΘOminutes. 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 1 h 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 (δvolumes) and washed sequentially with water (4volumes) which was adjusted to pH9 by the addition of 0.880 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 1 hour (seeded if necessary) and then to 7°C over a further 1 hour. The crystallised product was aged for at least a further 1 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 minutes N-formyl-4-iodo-L-phenylalanine Intermediate 27:

N-Formyl-4-iodo-L-phenylalanine methyl ester (1wt) was suspended in methanol (δvolumes) and water (δvolumes) and treated with 2M sodium hydroxide (1.73 Wt) at 22±3°C. This 5 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 Methano 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 0 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, 2voIumes) at δ ± δ°C followed by water (2 x 2volumes) at δ ± δ°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. 5

Expected yield: 86-90% theory; δ2-δ6% w/w. HPLC (2minute method) RT 1.31 min

0 N-Formyl-4-(1 H-pyrazol-1 -vD-L-phenylalanine Intermediate 26:

To a solution of N-formyl-4-iodo-L-phenyIalanine (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 5 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 12δ ± 3°C, and the reaction stirred for at least Iδhours. The brown suspension was sampled and analysed by HPLC to ensure <3% SM remains.

0 After cooling to 3δ + 3°C, water (l Ovolumes) was added, followed by DCM (δvolumes). Charcoal was added and the mixture stirred for 1hour 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 HCl (1.δvolumes) over about 4δ minutes then aged for Iδminutes. The remaining δ concentrated HCl (0.3volumes) to bring the pH to 1 was added over 10 minutes. 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 70-80%th, 57-6δ%w/w. 0 HPLC (2minute method) RT 1.13 min 23

Methyl 4-(1 H-pyrazol-1-yl)-L-phenylalanine hydrochlohde Intermediate 29:

N-Formyl-4-(1 H-pyrazol-1-yl)-L-phenylalanine (1wt) was suspended in methanol (lOvolumes) and stirred at 22 ± 3°C. Acetyl chloride (0.94 wt, 3equivalents) was added δ 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. When complete (ie. <3% amino acid) 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 concentrated in vacuo to about δvolumes at <30°C. Isopropyl acetate (lOvolumes) was again added and the mixture 0 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 3voIumes) and dried in a vacuum oven at δδ ± δ°C. Expected yield: 8δ-9δ% theory; 92-103% w/w. 5 HPLC (2minuite method) RT 1.03 min

1 H NMR (d6-DMSO) 3.15, 3.23 (ABX, J 6, 7Hz, 2H), 3.70 (s, 3H), 4.32 (t, J 6Hz, 1 H), 6.5δ (dd, J 2, 2.δHz, 1 H), 7.38 (d, J 8.δHz, 2H), 7.74 (d, J 2Hz, 1 H), 7.81 (d, J δ.δHz, 2H), 8.δ0 (d_. J 2.δHz, 1 H), 8.70 (br s, 3H). 0

Methyl Λ/-{(2S)-2-r(acetylthio)methyll-3-methylbutanoyl)-4-(1H-pyrazol-1-vn-L- phenylalaninate Intermediate 30:

δ 24

Preparation of Acid Chloride (method 1):

A suspension of (S)-2-Acetylthiomethyl-3-methylbutanoic acid (÷)-ephedrine salt (1.31wt) in toluene (lOvolumes) was treated with aqueous hydrochloric acid (1 M, 6. δvolumes) and 0 stirred vigorously for 1 δ minutes. The phases were separated and the (upper) organic layer was washed with water (δvolumes). The phases were separated and the organic layer was concentrated in vacuo to 2.9 volumes. The solution was treated with thionyl chloride (0.29volumes, 1.1 equivalents) and warmed to about 40°C for 3 hours. The mixture was sampled (1 drop was dissolved in MeOH (1 ml), aged for 20minutes and analysed by HPLC (2minut.es) RT: 1.33minutes acid, 1.61 minutes Me ester/toluene). The acid chloride solution was cooled to 20-2δ°C and used directly in the next step.

δ Preparation of Acid Chloride (method 2):

A solution of (S)-2-Acetylthiomethyl-3-methylbutanoic acid (3.7δg) in iso-octane (10ml) was treated with thionyl chloride (1.6ml) and warmed to approximately 40°C for 3 hours. The mixture was sampled (1 drop was dissolved in MeOH (1 ml), aged for 20 minutes and analysed by HPLC (2minutes) RT: 1.33 minutes acid, 1.61 minutes methyl ester). The acid 0 chloride solution was cooled to 20-2δ°C and used directly in the next step.

Amide Coupling (method 1 ):

Methyl 4-(1 H-pyrazol-1-yl)-L-phenylalanine hydrochloride (1wt) was suspended in ethyl acetate (δvolumes) and treated with 2M K₂CO₃ (10 volumes) then stirred at 20-2δ° until all δ the solids had dissolved (up to one hour). The phases were separated and the lower aqueous layer extracted with ethyl acetate (δ volumes). The combined organic layers were treated with charcoal (0.2δwt) for about 2 hours at 20-2δ°C. The charcoal was removed by filtration and ethyl acetate (3 volumes) was used to wash through the charcoal bed. The filtrate was treated with potassium carbonate (2M, 10 volumes) and the rapidly stirred 0 biphasic solution was treated with the toluene solution of the acid chloride (prepared above) over 20minut.es keeping the temperature below 2δ°C. The acid chloride was rinsed in with toluene (0.1 volumes). The solution was stirred rapidly for 30minut.es and the phases separated. The upper organic layer was washed with water (δ volumes) and concentrated to about δvolumes by atmospheric distillation. The solution was held at 7δ-80°C and slowly δ diluted with 2,2,4-trimethylpentane (TMP, iso-octane, 1 δvolumes) over about 30minutes and held at 70-30°C for I δminutes to allow crystallisation to develop. The thin slurry was cooled to 0-δ°C over at least 2hours. The solid was collected by filtration, washed with cold (δ°C) ethyl acetate/TMP (1 :3, 4volumes) then cold TMP (4volumes) and dried in a vacuum oven at δδ°C to give the title compound as an off-white solid. 0 Expected yield 74%th, 110%w/w. HPLC (2minute method) RT 1.64 min

Amide Coupling (method 2):

Methyl 4-(1 H-pyrazol-1-yl)-L-phenylalanine hydrochloride (δ.Og) was suspended in ethyl δ acetate (7δml) and treated with 2M K₂CO₃ (33ml) then stirred at 20-2δ°C until all the solids had dissolved. The rapidly stirred biphasic solution was treated with the solution of the acid chloride (prepared above) over 20 minutes keeping the temperature below 2δ°C. The solution was stirred rapidly for 30 minutes and the phases separated. The upper organic layer was washed with 1 M HCl (33ml) and water (33ml) then concentrated to approximately 0 3δml by atmospheric distillation. The solution was held at 7δ-δO°C and slowly diluted with 2,2,4-trimethylpentane (TMP, iso-octane, δδml) over approximately 30 minutes and held at 70-δ0°C for 15 minutes to allow crystallisation to develop. The thin slurry was cooled to 0- δ°C over at least 2 hours. The solid was collected by filtration, washed with cold (δ°C) ethyl acetate/TMP (1 :4, 2δml) then cold TMP (30ml) and dried in a vacuum oven at 5δ°C to give δ the title compound as an off-white solid 6.4g 37% yield . HPLC (2minute method) RT 1.64 min

Examples

(3) (4) (5)

0

Example 1 : N-r(2S)-2-(mercaptomethyl)-3-methylbutanoyll-4-(1 H-pyrazol-1-yl)-L-phenylalanine (method

Ω 5 Methyl A/-{(2S)-2-[(acetylthio)methyl]-3-methylbutanoyl}-4-(1H-pyrazol-1-yl)-L- phenylalaninate (3.17g, 19.6mmol) was dissolved in a mixture of THF (75ml) and methanol

(75ml) which was deoxygenated with a nitrogen stream for 40 minutes. Similarly degassed

2M sodium hydroxide (98ml, 196mmol) was added dropwise at 0°C. The reaction was stirred at 0°C for 20 minutes, then at room temperature for 2 hours. The mixture was re-cooled to 0 0°C and the mixture was acidified with 5M hydrochloric acid (42ml), and partitioned between ethyl acetate (800ml) and water (400ml), the organic phase separated, washed with brine

(400ml), dried over Na₂SO₄ and the solvent removed to give crude product. This was purified by Biotage, eluting with a gradient of 1% to 3% methanol in dichloromethane, containing 0.5% acetic acid, to give 6.67g of pure material. LCMS RT 2.93minutes, MH⁺ 362

N- (2S)-2-(mercaptomethyl)-3-methylbutanoyll-4-(1 H-pyrazol-1 -vD-L-phenylalanine (method 2) δ N-[(2S)-2-(acetylthiomethyl)-3-methylbutanoyl]-4-(1 H-pyrazol-1 -yl)-L-phenylalanine methyl ester (33. δg) was charged to the vessel and de-oxygenated by 3 vacuum/nitrogen cycles. The solid was suspended in a mixture of water (3 volumes, 100ml) and MeOH (δ volumes, 270ml) and stirred at 20°C under nitrogen. 32%w/w (10.8M) NaOH (3.δequivalents, 0.78 volumes, 26ml) was added over 10 minutes and the mixture stirred at 20°C for 60 minutes. 0 Once all the starting material had been consumed, tributylphosphine (0.02equivalents, 0.012 volumes, 0.4ml) was added and the solution stirred for 60 minutes. The solution was line- filtered into a new reaction vessel and line-washed with water (3 volumes, 100ml). The clarified solution was acidified to pH 4.9 by addition of 36%w/w (11.6M) HCl (2.7equivalents, 0.δ7 volumes, 19ml) over 1δ minutes and aged for 15 minutes to allow crystallisation to 5 develop. The slurry was acidified to pH 1.2 by addition of 36%w/w (11.6M) HCl (1.9equivalents, 0.39 volumes, 13ml). The slurry was cooled to 0-δ°C and aged for 30 minutes then the solid collected by filtration, washed with cold (0-δ°C) 1 :1 MeOH/water (2 x 3 volumes, 100ml) and dried in a vacuum oven at δ0°C to give the title compound as a pale yellow to brown powder. 28.04g, 96.7% yield. 0

Similarly prepared were:

Example 2:

N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyll-4-pyrimidin-δ-yl-L-phenylalanine δ from Methyl Λ/-{(2S)-2-[(acetylthio)methyl]-3-methylbutanoyl}-4-pyrimidin-δ-yl)-L- phenylalaninate LCMS RT 2.66minutes, MH⁺ 373

Example 3: 0 N-F(2S)-2-(mercaptomethyl)-4-methylpentanovn-4-(1 H-pyrazol-δ-yl)-L-phenylalanine from Methyl Λ/-{(2S)-2-[(acetylthio)methyl]-4-methyIpentanoyl}-4-(1 H-pyrazol-5-yI)-L- phenylalaninate

LCMS RT 2.94minutes, MH⁺ 376

δ Example 4:

N-r(2S)-2-(mercaptomethyl)-3-methylbutanoyll-4-(1 H-pyrazol-δ-yl)-L-phenylalanine from Methyl Λ/-{(2S)-2-[(acetylthio)methyl]-3-methylbutanoyl}-4-(1 H-pyrazoI-δ-yl)-L- phenylalaninate

LCMS RT=2.79min M⁺ 362 0

Example δ: N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyll-4-(1 H-pyrazol-4-yl)-L-phenylalanine from Methyl Λ/-{(2S)-2-[(acetylthio)methyl]-3-methylbutanoyl}-4-(1 H-pyrazol-4-yl)-L- phenylalaninate

LCMS RT 2.72minutes, MH⁺ 362 δ

Alternative process for Example 1 :

Methyl Λ/-{(2S)-2-[(acetyIthio)methyl]-3-methylbutanoyl}-4-(1 H-pyrazol-1 -yl)-L- phenylalaninate (1wt) was charged to the vessel and de-oxygenated by 3 vacuum/nitrogen cycles. The solid was suspended in a mixture of water (3volumes) and MeOH (δvolumes) 0 and stirred at 20°C under nitrogen. 32%w/w (10. δM) NaOH (3.δequivalents, 0.73volumes) was added over δminutes and the mixture stirred at 20°C for δOminutes.

Once all the starting material has been consumed, tributylphosphine (0.02eq, 0.012volumes) was added and the solution stirred for 30-60minutes. A sample was analysed by HPLC to δ confirm most of the disulfide was reduced back to starting material.

The solution was line-filtered into a new reaction vessel and line-washed with water (3volumes). The clarified solution was acidified to pH 3.0-3.6 by addition of 36%w/w (11.6M) HCl (3.4equivalents, 0.70volumes) and aged for lOminutes to allow crystallisation to 0 develop. The slurry was acidified to pH 1 by addition of 36%w/w (1 1.6M) HCl (1.2equivalents, 0.24volumes). The slurry was cooled to 0-δ°C and aged for 30minut.es then the solid collected by filtration, washed with cold (0-δ°C) 1 :1 MeOH/water (2 x 3volumes) and dried in a vacuum oven at δ0°C to give N-[(2S)-2-(mercaptomethyI)-3-methylbutanoyI]- 4-(1 H-pyrazol-1 -yl)-L-phenylalanine as a pale yellow to brown powder. δ Expected yield: 92 - 9δ%th, δO - δ2%w/w

HPLC (2minute method) RT 1.45 min

1 H NMR (d6-DMSO) 0.81 , 0.86 (2d, J 7Hz, 6H), 1.58 (dd, J 7, 9Hz, 1 H), 1.72 (octuplet, J 0 7Hz, 1 H), 2.15 (m, 1 H), 2.42-2.59 (m, 2H), 2.91 , 2.94 (ABX, J 10, 14Hz, 1 H), 3.11 , 3.14 (ABX, J δ, 14Hz, 1 H), 4.δ9 (m, 1 H), 6.δ1 (t, J 2Hz, 1 H), 7.38 (d, J 8Hz, 2H), 7.72 (m, 3H), 3.26 (d, J δHz, 1 H), δ.43 (d, J 2Hz, 1 H), 12.7 (br s, 1 H). Analytical Methods 8 Minute Method

2 Minute Method

LCMS data was generated on a system as characterised below

Column: 3.3cm x 4.6mm ID, 3um ABZ+PLUS Flow Rate: 3ml/minute Injection Volume: δμl δ Temp: RT

UV Detection Range: 215 to 330nm

Solvents: A: 0.1% Formic Acid + 10mMolar Ammonium Acetate. B: 95%o Acetonitrile + 0.05% Formic Acid

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

0 Biological Data

Inhibitory Activity Against ACE

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

1 μl of the test compound solution plus TCEP (1 :2.5 compound:TCEP) in acetonitrile/water (1 :1) was mixed with 15μl of the substrate solution (176μM in 10μM TCEP solution) and 15μl human ACE in buffer (200pM in 50mM HEPes pH 7.4, 1δ0mM NaCI soln, 1 μM zinc acetate, 0 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.

Inhibitory Activity Against Rabbit NEP 0 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. 3δ

Biological data (pKi) for selected compounds are shown below (compound identifiers as above, except for compound X)

(i)

(ϋ)

^*Compound X: N-(3-mercapto-2-phenylmethyIpropionyl)-4-(2-thiazolyl)-phenylalanine 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.

Tables (i) and (ii) represent values obtained upon repeat testing.

The Compounds of the invention (1-5) show increased Human ACE pKi, Human plasma ACE pKi and Rat plasma ACE pKi compared to Compound X. This surprising potency indicates improved ACE-NEP inhibitory activity. 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 is measured using purified human ACE enzyme. The ability of the compounds of the 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)

wherein: 0 R¹ represents C_halky);

R² represents pyrazole or pyrimidine; or a pharmaceutically acceptable derivative thereof.

2. A compound according to claim 1 wherein R¹ represents C^alkyl. 5

3. A compound according to claim 1 wherein R¹ represents isopropyl.

4. A compound according to any of claims 1 to 3 wherein R² represents pyrazole.

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

N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]-4-(1 H-pyrazol-1 -yl)-L-phenyIalanine;

N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]-4-pyrimidin-δ-yl-L-phenylalanine;

N-[(2S)-2-(mercaptomethyl)-4-methylpentanoyl]-4-(1 H-pyrazol-δ-yl)-L-phenylalanine;

N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]-4-(1 H-pyrazol-δ-yl)-L-phenylalanine; δ N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]-4-(1 H-pyrazol-4-yl)-L-phenyIalanine; and pharmaceutically acceptable derivatives thereof.

6. A compound according to any one of claims 1-δ for use in therapy.

0 7. A pharmaceutical composition comprising a compound according to any one of claims 1-δ together with a pharmaceutical carrier and/or excipient. 33

δ. Use of a compound according to any one of claims 1-5 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.

9. 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 any one of claims 1-δ.

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

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

(II)

(111)

wherein:

R¹ represents d-₆alkyl;

R² represents pyrazole or pyrimidine; and P¹ represents an oxygen protecting group.

11. A compound of formula (II):

(ii)

wherein:

R represents pyrazole or pyrimidine; and P¹ represents a protecing group.